AU780316B2 - Vertebrate homologues of UNC-53 protein of C. elegans - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 DIVISIONAL APPLICATION NAME OF APPLICANT(S): Janssen Pharmaceutica N.V.

ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street Melbourne, 3000.

INVENTION TITLE: "Vertebrate homologues of UNC-53 protein of C. elegans" The following statement is a full description of this invention, including the best method of performing it known to us: Q\OPER\JEIApr\252473 I 109doc 19/4/02 VERTEBRATE HOMOLOGUES OF UNC-53 PROTEIN OF C. ELEGANS The present invention relates to vertebrate homologues of UNC-53 protein of C. eleaans 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 2 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 eleaans designated "unc-53" has been previously identified and cloned (Abstract, International C. eleaans 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. eleaans (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 3 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. elegans 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 sem- 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 4 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 crosslinking 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 5 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. eleaans 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. elegans 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. eleaans 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. eleaans, 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.

6 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, 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 lld 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. eleaans, or a functional equivalent derivative, fragment or bioprecursor of said homologue according to the invention. Preferably, the cDNA comprises a sequence of nucleotides encoding an amino acid sequence as illustrated in figures 9b or lld 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 lld. 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, 8 green fluorescent protein.

The present invention, therefore, also further comprises a transgenic cell, tissue or organism comprising a transgene capable of expressing a vertebrate homologue of UNC-53 protein of C. elegans 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.eleaans 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 9 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. elegans or the cDNA or genomic DNA encoding it or a functional equivalent, derivative, fragment or bioprecursor of said homologue, may advantageously be used as a medicament, or in the preparation of a medicament to 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 10 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 11 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 12 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 B-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 neurodegenerative 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 13 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. eleaans. For example, the method of action of the compound may include direct interaction with the vertebrate homologue of UNC-53 protein, interaction with processes for regulating phosphorylation or dephosphorylation of the vertebrate homologue of 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 14 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, B-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: contacting said compound with a transgenic cell according to the invention as described above, 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 15 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 neurodegenerative 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 16 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; contacting an extract of said cell with an antibody to the vertebrate homologue of UNC-53 17 protein or a functional equivalent, fragment or bioprecursor of said protein, identifying the antibody/vertebrate homologue of UNC-53 complex, and 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: forming an antibody to the first identified protein bound to the vertebrate homologue of UNC-53 protein in the method as described above, contacting a cell extract with the antibody, identifying the antibody/protein complex, analysing the complex to identify any further protein bound to the first protein other than the antibody, and optionally repeating steps to to identify further proteins in the pathway.

According to this aspect of the present 18 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: contacting an extract of the cell with the vertebrate homologue of UNC-53 protein of c. eleans or a functional equivalent, fragment or bioprecursor of said homologue, identifying the vertebrate homologue of UNC-53 protein/protein complex formed and 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 above not being a vertebrate homologue of UNC-53 protein and repeating steps and 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 19 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. elegans 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 20 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 21 molecule such as 8-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 22 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 Spodontera fruaiperda 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 23 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. elegans, 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 24 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. eleaans or a functional equivalent, derivative, fragment or bioprecursor of said homologue.

25 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 16'64CB (or 3595). Also provided are plasmids pLM1 deposited under Accession No. LMBP 3762, pLM4 (LMBP 3763), pEGFP72 (LMBP 3764) and pCB501 (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. eleaans 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 26 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 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 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 screening for the localisation of said reporter molecule as compared to a cell according to step which has not been contacted 27 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 28 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 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 29 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,deposited under LMBP Accession No. 3486.

Figure 2 illustrates the full-length UNC-53 protein from C. elegans.

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. elegans 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-53/1 hu-UNC-53/1 forms a unique pair with Ce-UNC-53 (cosmid F45E10) compared to the rest of 30 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 EcoRl 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, pLMI, 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, phhl5, the position of the reverse primers HU53rvl, HU53rv2, HU53rv3 and HU53rv4, the position of peptides B72628 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 lla is an annotated sequence listing of the insert of clone gbH09036 (EST46037) of Hu-UNC- 31 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 lla to an ORF from position 18 to 1793.

Figure lic summarises how the 3' and 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 llc.

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 32 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 ORP 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).

33 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 1p34 and figures and 25b indicating the chromosomal location of hu-UNC- 53/1 in 1q31. Essentially the same techniques were used to assign the gene hu-unc-53/2 to chromosome locus 11p15 (25d and e) as illustrated in micrograph 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-Unc53/1 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 34 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 ecl 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-unc53/1 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 plusend 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 35 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 pEGFPecl, 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.

36 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, 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(n152), anterior axons are the shorter, stop ahead of the vulva region and form numerous collateral branches towards the dorsal cord; c) unc-53(n152),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 Am.

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 The cDNA is under the promotor region A 37 (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(n152); unc53 (n152),pA/unc-53; unc-53(nl52),pA/unc-53-Hl. The observed phenotypes are put in three classes hsauvagel, the axon is straight, unbranched, and migrates until the head; vulveT, the axon is straight, unbranched, and stops in the vulva region; hmutantl, 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-H1) 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 (H1).

Figure 52 plasmid Figure 53 plasmid pLM6.

Figure 54 plasmid pLM1.

Figure 55 Figure 56 Figure 57 Figure 58 Figure 59 Figure 60 Figure 61 is an illustration of the sequence for is an illustration of the sequence for is an illustration of the sequence for sequence sequence sequence sequence sequence sequence sequence map map map map map map map plasmid pCB251.

plasmid pNP0O.

plasmid pCB501.

plasmid pTB115.

plasmid pPD95.75.

clone X16.

plasmid pLM3 38 DEPOSITED MATERIALS Deposit pCB2O1 plasmid DNA in E. coli Lambda clone 3B encoding hu-unc-53/1 MCF-7 clone z4 (mock) MCF-7 clone (pCB2Ol) NIH-3T3 mock NIH-3T3 pCB2Ol pLM1 pLM4 pEGFP72 pCB5O1 SAC clone comprising fragment of huunc53/2 gene Worm strain with chimeric C.elegans/human unc-53 gene Date 3 December 1996 3 December 1996 3 December 1996 3 December 1996 3 December 1996 3 December 1996 13 November 1997 13 November 1997 13 November 1997 13 November 1997 15 November 1997 15 November 1997 Acc. Nr LMBP 3594 LMBP 3595 LMBP 1600CB LMBP 1601CB LMBP 1602CO LMBP 1603CB LMBP 3762 LMBP 3763 LMvBP 3764 LMBP 3765 LMBP 3773 LMBP-1 663CB The above plasmids and cell lines were deposited at the Belgian Coordinated collections of Microorganisms (BCCM) at laboratorium voor moleculaire biologie plasmidencollective (LMB.P) 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 homoloque of the UNC-53 protein of C.el1egans.

Extensive searches with the ce-UNC-53 sequence C(Figures 1 and 2) against the public domain databases 39 (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 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. 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 pCR231 and was used as a probe to screen cDNA libraries.

40 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 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 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).

41 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 Ceunc-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, 42 HeLa S3 and Promyelocytic Leukemia HL60) was probed using the whole insert of pHH3b. No or weak expression was detected in the Burkitt Lymphoma DRajii, the Leukemia Molt4 and the Promyelocytic Leukemia HL60 cell lines. Five different transcripts are detected in the remaining cancer cell lines: transcripts I 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 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-Unc53/1.

A Northern blot of poly-A+RNA from normal 43 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 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-UNC53/1 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 44 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 Nuoleotide 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 pCB201 (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 Betagalactosidase, was used as a control vector (mock transfection). The differences between mock and 45 transfected N4 and MCF-7 clones were analysed using phase-contrast and Nomarski microscopy coupled with time lapse analysis, phagokinesis and immunocytochemical characterisation of the F-actin.

Phenotyvic changes in mouse N4 neuroblastoma cells N4 neuroblastoma cells were stably transfected with control construct pCDNA3.1 and the C. eleans 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 46 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 Factin 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 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 47 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. eleaans 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 MCP- 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 FITCor 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 48 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.

49 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).

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 50 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" stressfibres).

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. eleoans and vertebrates.

These experiments identify homology domain E (predicted nucleotide binding domain) of UNC-53 as the 51 "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. elegans 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 pCDU3) and negative 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 52 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-53

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 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 53 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 nontransfected 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 reuronal 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 54 (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-UNC53/1 mRNA pattern in cancer cell lines, moved us to investigate if whether hu-UNC53/1 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).

55 Tnhlp I Fnr; fnrmatinn iinNITH-IT rplk stabhly tranncfjrtFrd with prR l mock poB201 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 56 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 Iq, and that corresponds to band 1q31. At total of 80 metaphase cells were analyzed with 72 exhibiting specific labeling (Fig. 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 lq31-q32 appears to be involved in the pathogenesis 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 1q31 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 llplS.l 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-unc53/1 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 57 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 llp, an area which corresponds to band 11pl5.1. A total of 80 metaphase cells were analysed with 72 exhibiting specific labeling.

Chromosome 11pl5 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 11p15 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, 58 10080-10096 (1991)). Chromosome transfer studies have shown that chromosome 11 can suppress tumorigenicity of both human breast cancer (Negrini et al., Cancer 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 11p15 (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 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 SEOUENCE 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 1q31 being a locus linked to three diseases, suggests the potential implication of huunc-53 genes in oncogenesis. By using the complete 59 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 phenotvyic 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 2 17 92 0.84 37 83 0.69 16 27 62 0.70 71 0.78 13 85 0.87 Clones were passaged thrice, frozen and stored.

60 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 ruffling 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.cmu.edu/CLMIBhp/Imggallpg/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 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 61 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 visualisina unc-53-induced F-actin recruitment in MCF-7 cells 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 62 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 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 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' 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.

63 Table 3. Analysis of motility in phagokinesis assays Track morphology: fraction linear tracks plasmid clone round linear 1/r Mock 24 18 13 0.42 17 11 0.39 22 12 0.35 pCB201 Clone 2 16 9 0.36 13 13 7 8 0.53 9 9 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 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 64 affect microtubule 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 (colocalisation) 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 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 colocalisation, 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.

C.eleaans-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- 65 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, antitubulin 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 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 toxicity of Ce-UNC- 53 from activation of motility and microtubule binding from the activation of motility.

66 Analysis of the microtubule association of the C.eleaans 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 67 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.

2.

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 30h. 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 plusend 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 68 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 colocalise with the microtubules staining (figure 39).

Putative Assay Mammalian cells, in this case N4, 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 24and/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 microtubuline 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 pEGFPecl coding 69 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 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 Nterminal 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 70 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 pEGFPecl, 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.

71 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 plusends.

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 72 expressed by pEGFP72 shows microtubule plus-end association c) GFP-C.elegans UNC-53-(deletion variant) fusion proteins expressed by plasmids pEGFPsma and pEGFPecl show microtubule association.

d) GFP-C.elegans-UNC-53-(deletion variant) fusion proteins expressed by plasmids pEGFPsma and pEGFPecl 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.

73 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 CAAGTCAGCACCCCGAAGCAGCTCT-3'. Human genomic DNA was used also as template (100ng/reaction). The amplification conditions were as follows: 1 min at 940C, 30 sec at 55 0 C, 30 sec at 72 0 c, then 35 more times and a final extension of 20 min at 72 0 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 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 pgr 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 0 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 74 method. The screening produced several positive clones in each library called respectively XHH3, XHH4, 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 XHH3b 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 Xgtl0rv primer 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 plKlenTaq 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 0 C and 3 min at 72 0 C and a final extension of 10 min at 72°C, using HU53rvl and Agtl0rv as primers. 0.4 pl of this primary PCR product was amplified using HU53rv2 and XgtlOrv as nested primers with the following parameters: 3 min at 94°C, 38 cycles of 1 min at 94 0 C, 1 min at 52"C and 3 min sec at 72 0 C and a final extension of 10 min at 72 0

C.

The second nested PCR reaction was performed on 0.4 p1 of a 1/50 diluted purified 2.4 kb fragment using HU53rv3 and XgtlOrv as primers: 3 min at 94°C, cycles of 1 min at 94°C, 1 min at 56°C and 3 min sec at 72 0 C and a final extension of 10 min at 72 0

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 75 extends 699 bp in 5' direction (see fig 9).

4. 5' extension using PCR Primer HU53rv4 ctg-g-3') was designed on the 5' end of clone pCB210- 14 and was used together with Xgtl0rv 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 0 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).

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 XDNA (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 XHH3b 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 76 isolated using standard protocols (Qiagen plasmid DNA isolation kit), the sequence of the insert was determined.

3' extension of BST 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 ix KlenTaq PCR buffer, 0.2 mM of each dNTP, Ix advantage KlenTaq polymerase mix (Clontech), 0.15 mM AP1 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 0 C for 30s and 70*C for 4 min, cycles of 94 0 C for 30 s and 68 0 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 pCRF2.1 (TA cloning kit, Invitrogen) and the sequences of the inserts were determined.

gene specific primer (EST46037-F1) nested gene specific primer (ES46037-F2) 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 77 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) S'ACCTGAGTCCTTTCTTAGGCAAAGTGTTCC Marathon cDNA library human placenta (fragment B2.1) human HeLa S3 (fragment D2.1) human colorectal adenocarcinoma SW480 (fragment H2.1) PCR fraaments E2.3. C2.3 EST 485068 is similar to but not identical with the 5'end of HU-Unc53/1. A primer pair consisting of one 3' EST 485068 primer and one 5' HU-Unc53/2 primer were used to PCR amplify those fragments. IgtlO 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) 78 and 1 ml template. The cycling parameters were minutes at 95°C, 35 cycles of 45 seconds at 94 0 C, 45 seconds at 65 0 C and 2 minutes at 72 0

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) (97012802) 5' CGCTCCCCATCAGATGCAGGCCGG PCR fragment E1.3-3 EST 01222 is homologous but not identical with the 5'end of HU-Unc53/1. 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 MgC12, 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, cycles of 45 seconds at 94°C, 45 seconds at 65C 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.

79 RACE fragiments A2.2-2. B2.1-4. D2.1-5: extension Method as described previously.

gene specific primer (97041701)

'TATGCTACGGCCACTCATCTCCGTGG

nested gene specific primer (97041702)

'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 Method as described previously.

gene specific primer (9708080)

'TCGGTTGTTAGCAGTAGTTGACCCTCC

nested gene specific primer (97080804) 5 'ACCTGAAAGTCTGGACTGCATTTCA~C Marathon cDNA library human colorectal adenocarcinoma SW480 (fragment D4.1-1) gene specific primer (97080801) 5' ACAACCTGGATkATCTGGGCCAGGAGG 80 nested gene specific primer (97080802) 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.lxSSC:0.2% SDS at Also from Clontech, a poly A RNA blot from human cancer cell lines (melanoma G361, lung carcinoma A549, colorectal adenocarcinoma SW480, Burkitt's 81 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-NarI 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-NdeI 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 ApaI-StyI fragment from pTB72, the latter restriction site made blunt with Klenow, into pcDNA3 digested with EcoRV and ApaI. pCDU4 encodes for the homology block

E.

Expression of a domain of the human UNC53 in eukaryotic cells 1. pCB201: 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 open reading frame by amplification with hhl5fw primer 5'AGAGCGGATCCATATGCCTCCTTGCCGTCAAGGTG-3' and M13rv primer (5'-cag-gaa-aca-gct-atg-ac-3'). The amplified fragment was then moved to pCDNA3.l.His-A-Vector 82 digested with BamH1 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 XhoI. A BamHI and Xbaal restriction site were created on the pHH3b open reading frame using U3-Bfw ggg-atc-cat-gca-aat-gag-g-3') and U-rv tct-cta-gag-gag-gcc-agt-3') as primers. This amplified fragment was then moved to pBluescript KS, digested with BamHI 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 pLM1 (see below) (using NarI and XbaI as enzymes) into pCB300 digested with the same enzymes. The phage HH3b fragment of this plasmid, named pLM6 (fig 53), was then removed using BamHI and XbaI, 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-Unc53/1. Plasmid pLM5 was 83 checked by sequencing and used on transient and stable transfection experiments carried out in N4 cells. The plasmid pLM1 was created using a PvuII and partial BamHI 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 pLM1 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 XhoI. A BamHI and XbaI restriction site were created on the pHH3b open reading frame using U2fw ttc-ggt-cag-gat-cct-tc-3') and U-rv 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 BamHI 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-Unc53/1. 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 84 pLM1 was digested with EcoRV and XbaI. 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-Unc53/1 available at this moment.

pLM3 was used on transient and stable transfection experiments carried out in N4 cells.

pLM4: the partial transcript of HU-Unc53/1 cloned in an eukaryotic GFP expression vector pLM1 was digested with Clal and XbaI. This fragment was cloned in pEGFP-cl, digested with AccI and XbaI. 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-Unc53/1. 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/l glucose, 862 mg/l L-Alanyl- L-Glutamin, 110 mg/l Na-pyruvate; GibcoBRL) supplemented with 10% foetal calf serum (FCS; GibcoBRL), and 100 U/ml penicillin (GibcoBRL) and 100 pg/ml streptomycin). The culture was grown at 37 0 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 85 with 10% FCS was added to the cells. The culture was further incubated for four hours at 37'C in standard sterile air. DNA-CaClI was meanwhile prepared by mixing 30 g 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./HEPES (1.25 M CaC1 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 NaC1, 0.01 M Kcl, 1.4 mM NaHPO,, 0.01 M glucose, pH 7.05) while pipeting a sterile airflow through the latter solutions. The DNA-Ca.(POj) precipitate was then placed at 37'C for ten minutes. The DNA-Ca 3

(PO),

2 precipitate was vortexed and added to the cells, together with 100 pl of a 0.01 M chloroquine (Sigma) stock in H.O. After four hours of incubation at 37 0

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 0 C in a 10% CO. atmosphere. After 48 hours of incubation, the cells were harvested, diluted and cultivated under selection (600 pg/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% 86 NaCOj, 2 mM glutamine, 100 U/ml penicillin, and 100 ug streptomycin). The culture was grown overnight at 37 0 C in a 10% CO 2 atmosphere. Transfection mixture was prepared by adding 12 Ag DNA in 600 gl optimem 1(GibcoBRL) to 36 1l LipofectAMINE (GibcoBRL) in 600 gl 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 CO.

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 pg/ml G418) for two weeks prior to clone selection.

Fixation of cells for Immunofluorescence Medium was removed from the 9 cm- wells containing the coverslips. A 4% solution of paraformaldehyde (Sigma) in PHEM (1 g/l glucose, 0.4 g/l Kcl, 8 g.l NaC1, 0.06 g/l KH PO 4 0.0475 g/l Na:HPO,, 0.35 g/l NaHCO,, 1.51 g/l PIPES, 0.76 g/l EGTA, 0.19 g/l 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-X100 (Serva) for 30 minutes, after which th slide was washed again for three times 10 minutes with PHEM. The coverslips were then placed 87 under PBS (0.14 M NaCI, 2.7 mM Kcl, 10 mM NaHPO,, 1.8 mM KH 2

PO

4 pH 7.3) containing 0.2% Tween (Sigma) for at least one hour at 4 0

'C

Immunofluorescence stainina The coverslips were inverted on 35 il 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 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 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 88 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 SCILTM software.

C. eleaans-UNC-53 preferentially binds microtubule plus ends or GTP-tubulin 1. Cloning of C.elegans cDNA in pEGFP-C1 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 89 (ata gcc aga tct acg tca 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 BglII- 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 Cterminal fragment of C.elegans Unc53 was cloned in plasmid pEGFPsac (Figure 29), digested with the same enzymes, resulting in plasmid pEFP72 (Figure Plasmid pEGFP encodes GFP in fusion with the full length C.e. Unc53.

c) Construction of N-terminal deletions of GFP- C.eleaans UNC-53 fusion protein, other than DEGFPsac: pEGFP72 was digested with SmaI. 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 Ecl136II and SmaI, the resulting plasmid after ligation and transformation in E.coli of the 6.7 kb fragment was designated pEGFPecl (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 SmaI and XbaI. The latter site was made blunt with Klenow polymerase. The resulting fragment 90 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 hul-UNC-53-GFP fusion, and a deletion derivative The 5.4 kb hul-unc53 fragment was isolated as ClaI-XbaI fragment from pLM1 (Figure 54), and cloned in pEGFP-C1 digested with AccI and XbaI. pEGFP-C1 was isolated from E.coli GM41 (Hfa H, dam-3, thi-1, rel- This makes the XbaI 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 91 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 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(n152) alleles: In wild type worms the two pairs of ALN and PLN neurones each send an axon in a straight line longitudinally from the tail to the head (see 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(n152) worms.

3. The minigene pA/unc-53 rescues the elongation defect of ALN and PLN neurones: 92 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 HpaI site, position 29800 on the genomic of unc-53, and replaced by the equivalent domain of the human-1 gene (unc-53H1) (see fig. 51).

The resulting clone is named pA/unc-53H1. 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.

93 The degree of rescue was analyzed quantitatively and summarized in Figure 51b: The four strains compared are: wt; un-53(n152); unc-53(n152),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 "gaaaggatccgtacgataaggtattttgtgtcgg" on the plasmid pPD95.75(Figure 59) so as to be inserted at the 94 position in fusion into the exon 12 of the unc-53 gene at a single restriction site SplI and contains its stop codon at 3' plus one polyadenilation site. The PCR amplification product is directed by HindIII and BamHI, 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 SplI 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 XhoI of pBS.

b) pAB/unc-53 (pNP8 Figure The promoter region AB of the X16 clone (between PstI and SplI) has been inserted in the clone pTB115 (Figure 58) in which the region between the sites PstI and SplI, containing the promoter of the gene mec-7 and the start of the gene unc-53, has been removed.

c) pA/GFP (pNP10 Figure 56) The promoter region A has come from the X16 clone between the sites PstI and NheI and integrated in the vector pPD95.75 containing the GFP in the sites PstI and XbaI.

d) pA/unc-53 (pNP9 Figure 44) The promoter region A has come from the X16 clone between the sites PstI and BstXI and is integrated into the clone pTB115 in which the region between the sites PstI 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) 95 The clone pA/unc-53 (pNP9) has been deleted from the region 3' of the gene unc-53 of the nematode between the sites HpaI and NcoI. The 3' region of the Hlunc-53 gene has been amplified by. PCR with the oligonucleotides U4Afw ctt-gaa-gc-3') and Urv agt-3') and digested with HpaI 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 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 P %OPERUEH\Res ClhU004OW-\152J7J cms doM-0311 L -96- 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 bandpass filter, emission through a 590 nm long-pass filter.

The image acquisition is effected by means of a CCD camera and an NIH image program using a Macintosh computer. The images are processed using the Adobe Photoshop program.

15 The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of o: suggestion that that prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which follow, 20 unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

S**o 25 oooo EDITORIAL NOTE APPLICATION NUMBER 35574/02 The following Sequence Listing pages 97 to 183 are part of the description. The claims pages follow on pages 184 to 204.

97 Seauence 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.

Seauence 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.

Seauence 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 pLM1 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 Seauence ID No 9 is a nucleotide sequence of plasmid pCB50l 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.

GA TATC -rCLGAAT c6GGcrTTGAGCA.IGTTCA3 CC TGG TTAAG rCC.A CTGAAT TCCGGGGAAA&6CC6AGCCGr.Arc~c rc~eAZcc FArc C 7ATAGACGTCTTAAGCCGAAGAAAC TCGTTCAUG TCGGACCAATTCAGG TTCGACTTAGGCCCC T TTCGGC rcGGCCrAGGGAGC TCC rGG6G.%ACC, U* PCRZI fi- !a ilO 11 primner EOORI -su-s spect sequetic ILnker? ~LPHH14j-3 I SA E F G6FF E0V C P G V 0A E F R G KP S RI PA 9;P y G-6rGTAAGCCGCTCAGCA.%GGCGCC FGA.-6CCCTGGCGAAATGCAATCGACACGAGCTGCTCTCCAGCAAGCCAAGGCCAAAGAGCT C r: CAGT TCGGC GAGrC GT TCCGCGGAC TTCGC:GGCAC YCGCT TC TACCG TTTACC T, GCTCGACGAGAGGTCG T CCGG T CCGCG TTTTC TC.4 pHH14-3 V K P L S K A PE AA V SC E 00GK S5D0 EL L S SK A K A OK S C 7E *6CC FG TCcC CTGCCAAGGGCCAGGAG. AGCaCGCCTTCCTCAAGG TGGACCCCGAGCTGGrGG FOACCGTGC T660AGACC T6ACAC rGC GACCCGGACAGGGGAGACGGTTCCCGGTCC TCC TCGCGCGGAAGGAGTTCCACCTGGGGC TCGACCACCACTGGCACGACCC rCTGGACCTCGTCGACG..

piH14-3 S GP V P S A 9 G 0 E; A F L K V 0OP E LV V T V L 0 OLE 0 L L C TTCAGCCAGATGC TG5ACCCAGAG TCCCAGAGAAA~AGACAGTGCAGAAFGTCCTGGATC TCCGGCAGAACCTGGAAGAGACCATG.TCCAGCCTGCG.% AGTCGGrCACGACCTG6GT:TCAGGGTCTCTTTCTCCTGrCACGTCTTACAGGACCTA.6GCCGrcrTG6ACCTTCTCTGGTACA' GTCGGACGC.r pHHI 4.3 C)RF (1-5S?1pi pLM?1 OARF full availa~e OR HU-jnd53Jl pLMI 0FI F S 0 M L 0 P S 0 ZK R T V 0 N V L 0 L Q Q N L E E T M S S L 9 3 7 CCAGr.T2AC tCA:A: T:C: :7'---ACG-%CAGCGATSATGCCA;.ZCCACGCAGCGTCTCCAGCCCTCTCC*c CCCCT: 5 r:c,: pHH14-3 O'FF pL%4g u~7 OVF 2a-4ariaveOAIF HLU-UncdI -LMI OR s S: v r 3 3 L f 0 S 0 D A P P 5 3 L S .1 c 3 5 F pHHIA*3 -I3,v.a:1A CrI. HJ-U.-C3;* i t I E* Tuesday. IS Novem~ber 1997 10:33 f, c Hu-UncS3iI seq (I 6013) Site "n Se(IuOnC /-1l 7 Page 2 CC %CGGC GAACGiGGCCCAC TACTCCCACACCAT ,CC:A tGCGCAGECCCCAGCAAGCrCAGCC A TATE TCCCGCC tGGA-;C TGG TCGAArTC TG. C ;-GC CGCT GC CCccio TGATGAGGG TG TGG TACGGG TACGCG TCGGGG TCG TTCGAG rC GGr TAAG AGGGCGGACC TCGAC C ACT TAGGGACC TGArG: DMM I *j OM. C Z1-57 00)= Ft.N47?O-- Sl available ORF HtU.U~ICS3/1 pLM I OR H GE R AN H Y H TM P P4R 5SP 5K L S H (S R L E L V E S L1D3 GAT,GAGG TGGACC TCAAGTCCGrCrACATGCAGCGACAGTGACCTCATGGGCMAGACCATGACGGAG6A TGA TGACATCAC TACCGCTGGGATGA~Cz, C -,AC tCCiC C TGG&G T TCACGCCGA16 TAC TCGC GTCACTGGAGTACCCGTTCTGG TAC TGCCTCCA TAC T G T.GTGATGGCCGAccc rAC TTTr,;.

pHH14-3 .4 kS7 fl~I NPF i-57-7ow 4 W ORF Pil available ORP HU-tUndJSl jPLMI OR C E V 0 L XS G Y MS D S 0 L M GK T M E 0 00 1T T G V E S GC TCCATCAGTAGTGGACTCAGCGATGCCTCAGACAXTC TCAGTTCAGAAGAATTC-AATGCCAGCTCCTCACTCAACrCCC TCCCAAGTAC TCCCACTG:- TGAGTCGCCYCGGAGTCTGTTAAGZTCAAGTC TYC TTAAGTTACGGTCGAGGAGTGAGT TGA6GGAGGG TTCATGAGGGTGZC:; pHH14-3 pC8212 .A-(-57thol DIM? OF :ull available OAF HU-Unc3/1 pl-MI OR 3S SI S S GL SO A S~ L S S E EF N ASS S L N S L P S T PT 7 TCGC AGGAACTCAACAATAGTGC TACGCACAC-' TCAGAGA6ECGCTCAC TGGCAGAAAGTGGGCTG&kGC 166 TT TAGTGAATCAGLGAGA-Gi: ASCSTCCTTGASTGTTATZACGATGCG1SaC SAS;TCTC-, TCGCC.AG IGACCGTCTTTCACCCC.ACTCGACC-Z=ATC"C ITAGTC TCCTCTTr VCGS pHH14-3 pumzlz %jI avaiatem ORF HU-LYK53/1 pLPAI OF; PI S( T 7V. L 3 E K R L A E S GL S'd S E 5 r- E ,t C: -AAAAAC TGGA'.TACGA*CAGTGGTA5CC.Sl, :AS-:AACC T.GGAE TTCTAAGTGGCGGACGGrAGCSGCCTGAGAGC TGTGATG47TCATCCE aK114-3 0L;t1z1z L E S Ks E, c '3 S 100 Tuesday. IS Novem~ber 1997 10.33 page3 H*u.UncS3/1 seq( >i 80 13) Silo artdSoquerce 7 CATGAA* TAAAGCCCA TCAGCC TGGCCACC 7TTCCC GAAAAGGGCAAGACCCCCC r~rcrGr. T .AC T CCCCCA TCZC TCA:A.

z:C rCT rGacrrT rCCG~AG TCGGACCCGGTGG:;ACCAAGGGAC TTC T rCCCGTrc rGGGGTGGACACCGCATrTGAAGGGGGTAG TGAGTTG r.

p1*114-3 PCB2 12 lull avaJlable OAF HUULn3/I p)LM1 OR G EL K K P I L G H P G S L K K G KT P P v A v tSp I TNHT t pH-H14-3 pC821 *full available OAF HU-UncS3/I pLM I OR S A L X VA G K P EG K A T 0 KG K L A V K N T CL 0ORS S S D GCTGIC6GGGACCGCC fGAGTGATGC TAAG.IAGCCC:CCCTCGGGCATTGC rCGCCCC FCCAC TTCGGGAFCCT TTGGC TACAAGAAGCC TCC TCCTGCC..

C CGACC AGCC C TGCGGAC TCAC TACGA T TC TTCGGGiia AGC CCG TAAC GAGCGGGGAGG TG AAGCC C T ACAAACCGA TGTC TC ?GG AGGAGaACCG 7 pHH414-3 pCBZ I? %il a,ala OAF HUUMCS311 PLMIl OR AG 0 A L SOD A K K P DS G I A P P S T S G 5F6 G Y K P P P 4 -G TGTCGGTGA,-±G TCG TT GACC -CA,:,CcA7:TGGAGTCGT TC TAGGrc t CAGG GTCCGTACC;GACAGT TCGG TCATT ACCCGCGT TC T:pHH14-3 pCB2 12 Vu1 availatle ORF 1-11ULnc ;J1 pl-Ml OR T A T v .4 0 T G G S T L 5 K 1 0 K S S G I P V K P V NH :431 r rrCA~z3rC TCC.6T-:;AA: :A:cCGAGGACCICGGCCCAAGArT3TAG'CA7CGCrGGACGGGCCGGTCGrA.C* ;ull 13.IIatqd ORF rlU UfIc5311 =PLIVI OP C' S I FAr i I L Tuesday. 18 November 199 7 10 33 faHu4JncS:JI1 seq (I 6013) SaIe and Sequence Page rC rArGAGC., TGACCGGCGGCG6GG rGGACC TCGCCCTGTGACAGCAGC AT TACCCCAGTC TCCTACACACCCA;C7.-CG:TrC.

A.;A rAe TCGCAc rGGCCGCCCGCCCCACCTGGAGCGGS.&CAC TCGTCG TCGT AAC TCGGG TCAGAGGAG TCGrGGTTCGrccc rcCGC~Gr~r,~pH 14-3 pCB212 %Jl avadiable ORF HU-Unc53/1 oLM I OR S M S V T G G R G G P R P Vs SS S lo0P S LL S T K 0G G L Tp P G TG A G G AGC C T AC C AA GG TA GC C A GG 666 CG GAC C AC TCCAGC CC C TG rC AA TCAGAC AG ATC GGG AAAA GGAA AA. G CC AA AUC AA.GGCAG TG G: C TGAC TTCC TCGGA TGG 7TCCA TCGC6TCACCCGCC TGG TGAGGTCGGrAC ATTAG TCTCTCTAGCCC rT rCC TCT7CCGG TT MCG TCCG TC ACC pHH14-3 pC8212I pCS2IO-14 Uif avaati, OAF HU-Unc~i. pLJM OR P L K EP T K V A S G q7 P AP VN 0 TODR E K f K A K A K A V A C T-.GaAC rCAG CAACATC TCC TTGAAGAG TAT7GGC TCC CAGAAAG TAC TCCCUGAACCAM CAAGCCACCCCACAGCCACCAAGC TrGCAGAGC T-- G AAcc rGAG TCrTGTTAGAGGAAC T T rCA TACCGAGGG TCTTTC ATG AGG6TTCG TCG CTCG TGGG6TG TCGGTGG TTCGACCGTCTC:6A.

pHH14-3 nCrR21l~t !uji avagala~ OpF: HU-Unc3i~ *pLMI OR L 0 S 0 N 1 3 L X S G P E S T P K M 0 A S M P I A f K: L A E L Cc-ACCAACCCCTCTCA:GCCACAGCGAAAC-,7;:AACCACCCTCACTAGCCAArCr1GAICAA;Gr:AACTCCAACAC r.;7_ra,,rAreA- C6G TTGO6AGAGTCCCGGTGTCGCrTCTCr..AAC47TTTGGTGGATATCGGTTAACTG,'I-CA, TTGAGTTGtCA5CSC;AA.-G!T~aT.4 .WW IA.1 nCrR21m-1 juo a.&Lta~aORF HU-UncS311 nVMI OR 2 P L Q a T a K 3 F 'iK P P 3 L A 'i L 0 K I -N S hI S L 0 L P S Tuesday. 18 Navemnber 1997 10:33 9 Page S, CC AG T A tACCACCCA TGC T TCAA.A G TCCCA -C TC AGC TAC AAGC TCAGCATC FGc5.3GccC Ic ?cCC rccc c G T rCACCCAGTC:.G~cIC GGTCACTAT66TGGGTACGAGTTTCCAGG'CrAGACG rAC6AgTTCGGTCGTAGACCCCCGGGGAGGGGACAGTGCGTCAGGCCGTGI, PC5210-14 NOf avaftia ORF HU*Unc3/l pLMI OR S S DT T 4 AS X V P 0 L HA t SS AS G GP L P Sc C F p T P c A rcc rc AA TA TTAAC TCA GCC AGC T IT TCCC !GC C TGGA GC T AA TGAG TGG TTTC AGTG TGCC AAAAGAGAC CCGCA TG TACCCCAAACTC G AGGTTATATT ~TG CAGGGTCCGC CArTCTACAGTAAG ~CT GGGAATCG.TAA C- DH?114-3 oC8B210-14 N.fl av~aiable OAF H1-Un lj pUWl OR IL N I N S ASF S 0G L EL M SG FS VP K E T RM YP K L S G C TGCACAGGAGCATG ;AGTCCCTCCArGATGCCAAT-GA.5CCTCCCCAGTCC TCCCCAGCAGTACTCCCGTCCCCACCCCACC oCTCCCeCrCTGCTCT GAC GTG TCC T CG TAC CTCAGGGAGGTC T AC GG T-ACTCG GAG GGGTCAC GG AAGGGG rCG TC ATGAGGGC AGGG66TGGGG TGGACGAGGGGGACCACG 43 pHHl14-3 nlcR71O-l4 ;-;[avaiata OAF: HU.U~nc31 pLM1 OR H a S .4 ES3 L C H P S L P S A F P S S T P V PT P P A P P A A TTGG-':CSAGCCCCAGAGCTGGCAAC JGGA: AGTAATCAGCGGGATCGGAACAC TCTTCCCAAGAA r* GGr 7f TC TT T TC TC T CC TTC TCGAC TG ACc- rAC T TCGGGG TC TC GAC CCGT TGACC TSTC AT TAG TCGCCCTAICCT TG TGAGAAGGG TTCT TT': p1*13b pC8210-14L n-la~a~latq OAF HU-UK3i1 =pDLMI OR W,1 I~-r E E r s G Q SL 0 3 N 0 R 0 P H T L P K r r 103 Tuelay. 18 Novembow 1997 10.33 Jpage LHU-UICS3/1 sag QI 6013) Site anid Sequence GC. CArTACCAGrC cA rCCCAGGAGGAGACCAXAGA (CCGACA TTCCCATACCAT TGGCT~GCCTGAArCCGA rGACCAG TCAGAGCT CC cG rcc l.;GGTCGAAGTCA6GCCcCCTCTGGrrCC TCTCCGCTGTAAGGGTATGGTIACCACCCG&CGGAcrT.*CCCTAC rGGTCAGrTC6CG.Gi PHH 14-3 pHH~b ktA availab~s ORF MULkc3ii pLM I OR L R i Y a E E T K E RA S H T I G GL P E S 0 0 O0S E L F TC CcCCCAC TC CCA TGTC TC TG A G TGC AAA GG;C CAAC T TACC AAC A TAG TGAG TC CCAC TGCGC CA CCACGCC AAG AA TC ACCCG CTCC AAC 4 AGAGGGA TAAGGTACAGAGAC TCAC G TTCC C GT T GAAGG TGTA TCAC TCAGGTGACGCCGGTGGTGCGG TTCT TAG T GGGC G.AG TTG T pH14-3 Wtj avaiiale OAF HU.4Jrc5W.' "ILM OR S P P AL P M S L S A~ K G0L T N I V SP TrAA 1T T P A I T P S N ocA recC:ACCCACOAaGcGcc 1'TCGAGC TOTACAGCGaCTCCCAAATGGGGAGCACCC TGTCCC TGGCCGAGAGACCCAAG6GAA TGATTCGGTCAG3- CGTAGGGaT6GGOTGCTCCGCCOGAA6CTCGACA-G TCCCCGAGGGTTTACCCCTCGTGGGACAGOGACCGGCTCTCTGGGTTCCCTTACTAAGCCAGTCZp1*114-3 oHH3b .uiI available OAF HU-LncS3/1 -piMI OR 31P T H E A A F E L. e S G S 0 M C S T L S L A E P K G M I R S si pHH14-3 -I pH-H3b %:11 a,,a~iatie OAF HU-4Jnxfi3I pL-.A1 OR ~R O P TO0 C V M L L A 5 SA SS T Y 3 5AC E E IM 3 PHH3b 3.:at- CAF HI*UfiC5311 p.Ml OR.

0 R. 9 E L S E X V A T L Fi L S N N L V 104 *Tuesday. 18 Novuniber 1997 10:33 vPage7 a Mu.UnCS3/1 seq f I 6013) Silo an~dSe~uenice r T tGA6:A5AGCC TGG rGAA TA TGACA TCCCGCC ACACC TGGCAGAG-CGGCCGAGGAGAAGGACAc rGAGC GCc TAr rrTGa4.;AALC. rt.

AAC TC 7,TC TCGGAC CAC TTATAC TG TAGGGCGGAC CC TG rGGACCG TC TC TGCCGGC TCC TC TTCC TGTGAC TCGACGACc rTAACGCTC TTrGGT,'7C7 L2 GP.F= PC651 OR,= !ulavaiia~e OAF HU).UncS3I pLMI OR F E 0 S L V N m S L 2 H L A E A EE K D T EL L DLR E T I C T TC FGAAGAAAAAGAAC TCT(GAG6CCCAGGCAG 7C AT TCAGGGAGCCC TTAATGCCTCAGAAACCACACCCAAAGAAC TTCGGATCAAGAGACAAZ G AA ACAC T TC TTT TTC TTGAlGAC TCC GGG T CC GrC A3 TA AG TCC CTCG GAAT TA CCGAG TCTT TGG TG TGGG TT TC TTGAAGC C TAG--TCCTGFT T T pHHab .WI avaiaao ORF HU-Unr11= p4M I OR F L K K K N S E A 0 A V I Q G A I W A S E T T P K E L R I R 0 Ni FCC TC AA AFAGC ATC TC AGCCTCAACAGC ATCAC T 3CCA TTCCAGCATCGGCAGC AGCAAI GATGCTA TCGAAAAAGAAGAMAAuAGAGT TGGG FATCGTAGATTCGGAGrTT6CGTAG.-GATCGA.&GGCGTACCGCGTCGTCCTACGACTACGCTFVTTCTTCTTTT.-TTCTCAACCr pHH3b avai~a~e CRF HU-Unc53/t =;LMA OF; S S 0 S IS SILNS51 S H S S I G S S K 0A 0 A KK K K K K S -;A.^CCGAACC FTCAAGGAA, TTZ;TT TCGC-*A. ATAT T TT TCCCCGGTTCAG TCGAAGGAGTA GAGCC T ATCTCCTCT;CGATGG;rpHH3b .'-jil availabl CRF HU-Unx:3/l pLM i OR Y E LI S S F N K A 7S I K K G P K S A S S fS 0 i E I A F F D.

C v I :AGCCC--TCAFCCCCC4AAC TAAC- 5 TAC~A7,C-.GC rrCA&CCC TCC-±TCAAGT:CF-CCACCT ;7::TCCGTZ* 4cTs I CAF:T2- U2 OF; PC22-! p ammZ~b i. I;IatbaORC HU*L,531 piN41 OR S P K L 1 YE 7 S S SI w S 3- F L S S. V r Tuesday. IS November 1997 ia:33 fir *-unc53It sag (I W 13) Site and Sequence P*Pt ACCL XCC TGC 17CACC CAGCCCCCC ACAC TAGGC TG TTCCATGCAAA TAGAGAGAGCCAGA6AGAAGAG 6 A rC-7G 4C 7CGC TC TGA, Tr CcrCCCc666ACGAGTGGGTC666666TGTGATCCGACAA6GrACGTTTACTCCTCC tCCTCGGTC TC rrcT rccTCCA -,GCCTCA rCGAGACT1cT4- L2 ORF pCGS. ORF oHH3b lud availa~le ORF HU4IrS3I1 p)LM I OR r E G P A HP A PH TR L FH A NE E EEP E K KE V S E L A S E TAT TGGGA GAAG AAA rGAA GC T TAC AGACATC C.C T TGG AGCC CTCAAC TC TGCCCACC AAC TGG A TC AG CT TC GGGAG AC CAT GC AC AAC ATGC AG T Aa CC CTCTTCC iT r TAC rTCGA ATGTCTG TAG GCGaACC TCCGGGAGTTGAGACGGGTGG TTGAcc rAGTCGA AG CCC TC rGGTACGTr,-.TG TACGTCA.

ful avash. OAF IJ.LhnCS3/ OWI4 OR LW OAF pL.MS OAF L V E K E M K L T 0 1 A L EA L N S A H 0 L 0 0 L R E T A4 H PI M 0 L GGA. rGGACC i6C TGAAAGCAGA6.IATGACCGAC T:;AAGG TAGCC CCAGGCCCC TCA TC AGGCTCCAC TCCAGGGCAGG rTCC TGGATCATC TGCATT.; CC TCCACC TGGACG AC T TTCG iC re TTACTGGCTG A: TTCCA1'CGGGG TCCGGGGA TAG TCCGAGTGAGTCCCGTCCAZGACC TAGTAGACGYAAuZ F; PC2eoHH3b fjfl avaaiIaL~ OAF 11U-Unc53/1 pLM1 OR U3 OAF pLMS OAF E v 0 L L K A E 1i 0 1- L v p A p GPS S G S I P G 0 1 P G j S A tCr CCArC5TCTGCT3A74C--TCTCGCCGCTCAAAACI CCCC.,G A.:~rcr A-AIGGGCCGGC CGA::T;--,5AAGGAAGCCGGGTCAGAACGTCTGTGTC TGGACAGTGGGTACC rACC~Gr i:ATG-'±.Cg: U2 CRF.;- PCBZ! F nI.4-4pl 3aaal1 OAF HU.Unc-&V/l oLM I OR U3 OAIF pLJhlS OAF S S 0 R S L GL A L 7~ S FGP SL A C TODL 5SP MHOG TC 106 Tuesday. IS November~ 1997 10:34 i 5 'u-UrcS3I1 seq (I 60 13) Sit@ and Sequjene Page? G r: %.%A.SGAGGAAG rGACCC TCCGGGrTGGTGG. A.z rGcCCCCGC AGC AC ATCA C AA GGGGAC T rG AAXAGCA~.r,.T T C rT C I GGGCTTA3 ;T 1CC TCC TTCAC TGGGAGGCCCACCAICCAC CC TACGGGGGCG TCO TAGTAG T TCCCC rGAACTTCGTCGT:C rraAG~ACq;c:-. y L12 OPF p=5 CfiF 3#4 b tug Aasilable OAF HU-UnS.WI )LMI OR U3 OAF PLMS OAF G P K E E V T L A V 7 V R M P 9 0 HI I K G 0L X 0 0E F F L G C S TGA1AAG r6ACGAAGATGC T- ATGAAGC TGTTTTCCAAGTGTTCAAGGAC TA IAT T TC *;7.-CCAGTCACCrTTCAACTGACCTTC TACGACCtTCGACAAAAGGTTCACAAGTTCC TGArT. AAAGA TTTTACCTGGGTCGGAGTGGrCCCT U2 OF PCEL-1 OPF

I

HH3b h~iI 144 LI I~~~iI4 L14 hal avatlable OAF MLLLkwg3fl Ml OR U3 OAF pLMS6 OAF K V S GK V D VK L 0E A V F V F K 0YI S K M0P AS TL G TCT4CGGAGG CGGCA'-.C C~rTA.

U2 OFF i OPF pHH3b LU4 QPR- pC=320Q OF :At avi.Latlo OR= HIJ-Ur~c53;. I OR U3 OAF P~LMS ORF S T E S I H G V 5 1 3 A K R V L 0 A E PPE M P PC c ;Z e,V n rT'GS -3~rcr.%-GC7 C TG C -AC rcz Tr G T!

-W

U41 ORF p CE2Oi OR.= 3i.a:iat,*tCAF HU.Uic53.:I D LA r, UJ3 ORF oLM5 ORF pH~i-t QII I IL I S L V F E L P t' L 107 Tueiay. I8 Nvtb"~ 1997 10 34 k lPage 16 JuUflS/ seq W, 301 Sie an'd Sequ~me CC: r:G..MGCACCGGCGCCTC6 cTC TCGGCCCCAGCGGCACGGGCA.3ACCTACC TGAICCAA TCGc rrczG.GAc: rs5T?;':aG.TCG: rc CAC T7CGTGGCCG:GGAGCAGAG ACCCGGGG CGC rGCCCG TrCGGATGGAC G rAGCGACCGGC tC4GGeCCA.C T:GCGAGAc: U2 ORF CCBZS I CAP 004p.m U4 OAF pCE12OI ORF -am avabe OPP HU-tUnc3I oLMI OR U3 OAF PLM5 OAF pHH L L KH A R L VL S G P 5 G T G KC T Y L T N A L A E V L Z~ R S 13 G-.aC r:CsGTGCrcTCCC TACAGrCGTGG.IAGTTGTACGTGGTCGTCAGAACGT TCCTAGACGTTGArATAGAAAGG TTGGATCGGG 6TC TATCTGZ U2 ORP PCSZ5I OASF nwwIh U4 ORF PC8201 OAF %AU avaiable OAF HU-4jndJ/l pLMI OR U3 OAF pLMS OAF 7 V T FN MH Q00 S C K L Q L Y L S h L A 11 0 1 pHM3b U4 ORP pCS2Ol ORF .aalu ORF HU-UL~nC311 pLAtI OR U3 OAF pLM5 OAF r 0 3 D L 5 E 5 E L I rc 108 Tuesday. 18 November 1997 10:34 Page tII tcL 4-Unc531It Seq I >6013) Ste S eque ne U2 0 p AA-rccPC 8 Z 5 i c R .MKIh U4 OP.' pCOM20 OA IiI avaiade UN,- HU-ULCS31 PLiM1 OR 113 ORF .pLM5 OF pHHlIS Y H K C P Yv Ii G T T N 0OP V K T P M H GI L I H SF R M1 LT F i AACAXACG TGGAGCCACCCAATGGC TTCC 7GGT TCG TT4CC TGAGGAGGAAC TGG TAG AG TCAGACAGCGACA TCAITOCCAACAAGGAAGAGC rGC Tr;TCCTGTSTACAGACACAGA CTCTTGCAT CA CTTG GTGTTCGTG C T CA"- UJ2 ORX pc-6251 op.

aHH3h U4 U)Hl pCO2Ol OF Il available OAIF HU-Ufld3/l DLMI CA U13 OA F pLMS OAF pHH IS E P A :1 G F L i R I R R K L V E S D S 0 1 N1 A N K E IC I L 7:;-.CTr zc GTA:C:AA~c r7:ZT ':AcAcc rrc,. TTAGAAGCACAGCACC TCA Z,4TTCCTC4.:GCCC TTGC7-TCTrTCT L.2 0 K -OR pHH3b U4I OFR ;C0201 Op= a. ail1 OF HU-U~ncS3.11 pLIi OA U13 OAF pLM5 OAF L D v x- L W fL K h. 3 -3 C F L P C L 109 ruosey. 1e November 1997 10 34 Page it- U9_. jUfCS3It Sag (I ral 3) Site and Secuence G 7T3 GC A aAGGACTTCCGGCC.-ST-T~ CC rr:,r G CAACAAC rrccc T C aar.--CGTA'C TCCTGAACCCTG6ACCAA TAACTGGAC ACCr Ta TG GA TAGrAAGiA7A.:A r:rCc rrT:C rC3arr T r: op; PC 6:61 CI ONmmJ U4 OPF PC8201 ORF tufl avwIable OAF HU-4.ki53I1 PLMI OR pHHlS C P i G I E 0 F A T 'i F 1 0 L V N N S I I P Y L 0 E 5 A k 3G I i 3 TCCATGG. CAAAAGC 1GCTT6GGAGGACCCA3TG3AAFGGGTCCGGGACACAC UCCC TGGCCA rCAGCCAACAAACCAArcAACTGr.;CCC: C ~..GTACCTG tCTTrCGACGAACCTCCTGGGTCACCTTACCCAGGCCCGGAA6GACCGGr~CCGTGTCTGTATTrr Aarr7GG-., U2 ORFPCSIS I S pHH3b jP rsailable ORF HW4JriS3/t pLM I OR U3 ORF p4J.5 OAF X a E DP; V :t I 0 T L D 5 A '1 13 as 0 L V 14 ':CC -C-AccG T :-GcCC ACAGCAT :G:CI T:A:C TCCCGAGGAT AGGACAG TCA A -CAGC;,c::CAAG TTC rC I SGAC1CAGAC: C M L U2 ~JI~P pCaZE C V~ r'I-JwI% U4 ORF= PCB2Oi OFF !ul 3r.aPable 0;;F HUJ.Unrv3.'1 0,11I CA "I lCC I PAS ORCFli p p r v G *j p -l s I a S P E 0 R T VI. k 0 S 3 3 3 0 3L Tue4day. IS November 1997 10:34 f!S. lu-Urtc53/I seq 6013l Ste a,.d Secuence Page i3 CZ-*G r~c.AfcGTGAA67tCrc CACGGTTr A 7G1AACTCAGAGGTC rtGc rcrTGrtAGAC7GGGGrrG- AC;.CGTG r. rATCCC;, V12 O;W -PGI-25 OP.F pnnJG U4 ORiF pC8B0l ORF full available ORF HU-UttcSNI a pLJ611 CA U3 OR4F "S.1 ORFpt* 57:025F.

AfMIL L K LO E A AN Y I ES P DRET I LOP NL O0A TL G F G.GCAATCAC TGTCACCC CC GGACAGCAGAACGCTCGC ATCAGCTATCT TAGCTCC TCC TC rC CC CTCTCCTCTTC.ICAGCACTGGC TC ICCAGCCCC A ccG rtAG TGACAGTGGGCCTGTCGTC TTGCGACC TAGTCGATAGAATCGAGAGGAGAGGGAAGAGrAAAG CTCGGACCGAGAGGTCGGGC pHH3b N H C H rAE R 4 0L S L LL S PL L F 0 S T G 3P AP G Aa Gj G A GG A-~7T 7TG GCTG TACCTTT AGAAC T T C TAGGAAGGAA rG TCG T rroHH3b SE IjE~ G S j C C r F E F1V L G Q 11 G G -;'-TiTCCC C*A TA TGC -:-7AG*TTGGAA.'.T -TC T- TTC T.IltC tT~'4-4At* CAA CG; GATtT GTAA G ZArTA GAA CCTTITCTCTA-GCCCA AGGA. GAs%- A ;AaHH3b pHH I C P L M I F T G L L V G~ K D D S G S F P L L S I T ft .;'.;.cCCCAAA.a,,cCc rcZCCAAG TCTT:.:'T;G.GCICAG4TA .11r7T,.~,:;CCCCr-. 4 :CCpN-f3b i A F w r H r S S NI. E TuoesdaY. 18 NOvembow 1997 10:34 7 fig 14u-Und53Il seq O> W6I 3) Site anid SeqUenCO0 .A rc tGGGGGA GGAAC TC TCAGA tr 7-t Ac A~jcCc r rCCAA TTC CATCAC A T.aCCAAC AA: C rCz:c CAGGA T,_t r.G T~ C AGACC CC CTCCG TC C TTC GAGGA G TrCT AAASG AG rG M T GGGA AG GG rTA AG G TAG G TG AC GGrTr G6AGAGG G rc rc r A .1A c jC 7 ElI ~G R 0 A P 0 1: S 0 TL P N 3 T T A N N S 9 RO A G 4 CCAGAAAAAGAAGCArG TGGTT rAAAATGTT7AAA'CAATC TGTAAAAGGTAAAAAtGA UCA AA AGCAAACAAACAACAA r"!A.

G.3rTCTrTCTTCGTAC~AATrTTTrACAATrAGrTAACATTTCCATTTTACrTTr6TT..rrrrGTCGrTTGTrrG rTrrTrGTACCTr7 0 K K K N V V X M F K S I C X R K .K N K N K 0 T N K K~ 0 I ~C AC rTCGACC TCTC TCCTG CAG -cAr CCAGGGGAGrA..CA GArCCT TAG r rar TC TGC:GACCiG T..

V Q E Q m 0 L P R R A A R S C P L 0 0 1 3 0 1 N K T A i rGAGAAG ICACC AAACCACAAAAATAACCTTACAGCC TTCAGGGAAAGAC IACCAGC TCTGTCTTTCTACCCTCTAA? TTAACAA TGCACCGGAATTC L R S~l M rrTT r L S SGK 0 y 0L C LS5T L .F N N A E c r7GGAcrFAAcC G..%CC TGAAr IGG 0 L T ~I21f Ni 0047Kw hh2~N~A2..97l4oo Ah2U2~S)..C71L0O 13/25/97 02:119" hh2tWSJtlj 120 I 2,01, O2~47P I AI~aC"c, 0~0PACMY ACRJA7M0 04)4J.A~ 01A~A IA CTA?4I CoA~fl~ Y7Y1C~ CVACIA WCVCTC I7VrIA! GICCOACAG2 A A N N I A C K 4 0 F L P P A 4 24CMOAIAMCMA TCA, A MUDAOC AAWovIYM cvtcACMAICCA AC0 ~~1R~~~YMAM K2wii C tAAcA1#Ak o:CMccC7A rmvAI? AOT=5~ MWCC0A 2~ IC L4. Li hA INC A K PD 0 34YCAGAAG ~ci7 CC%0Qy CVC 0AVCA MCOMM C0CYCA 1 0 44OACCCMA OCO* CtpACIA CC4.UAXt4 0TC0CCT CACeAC"AC COCAC07A OCATCA)AAA MOMAT WCUIYMCAC OUMA0'41A DOOMOM Co-gJMcA G7CICOM C W4ACCr cCAIW 414 PAAA.AIMAOM0T CACACI~A AY CrAO00AIC CUI"AWO AGA00CA PAAMCO 1 1.11.CCCMATAG OGCCC 1VCIXWCOU TM? C 44A7Ay CAACA Ak CACA CCAOOC1Ty AACA.K7AIU AAAAICMM ACCACICACI 74CCCCACCCC CIC ICCCIAA 7T7rUATAC TArr.470 ~CrACMWA 1 CACC=CAAO CAOCCOCCAC AA2.NCCrT lTGATCM AOCTftx1cC C:LXCV YUAOVowAA kAcclwt C1=02MI C7CU31WC TICCAA CCCMKAAG 2CMAOUM 01072X= ACWUCa"I7 ACVTOACO 2; 1 4. I I GC S C£ T P T N C S T s I A I p 9 f 0 A A 1IC770aCA AMCC ItO~C? AC7AMCA CIACCCCIC MICCCO Ch4 4.C0?7p CAeCCACCAA AAAIAC7V'r CWTOMMC4 CA71O~A ICAII'AACC? CA14KOAOOAG C(UITAC CM ~AC P 4 L v V S 'V V A C P 1 All~br4 V~AT4C WA7CA AACTICC A'C0.VAIt4AM 117TtVM T CP.OrXItr MtC.

S1TMAGA AAIV4XAMr C"VMiACM nX%7h.AP4 AL4LACA4FCA CMC4-IA(%A LLXkALTtC *IAPrCACAVWAA -rAICA. rMr re* 1u.-1-1, .VI .4 a 0 C N a A c 1 0 P 0 No: s c C 1 E 9 T' L P S 964 A-TNAA= TICAAG A07MOC CtCCtM RACCC C1U24MWM C7ACW 1CAIfrt 4044 %CkQACA CAOW W= lt~lAC IC C CACCc1M CWtC1 CAOMYC AA A11WAC ACOcm2Vo CICCICUNCC 2CCWo= AOCACrAC 70720ACC COX.AMO 0PUY cIA.C 141 A r IPIa A L 7 N 9 N S 2LK 9 M 0 9r 440 IAA0ft? 1WMGAA ACrrriPMC O=ACAC CAACAAGA CTM7CM4A AAXAIA CAAG AO.1,11. A NOCICW CA~T CCCCICACT GTCTIC TC4.ACT~e lVCCC7AC TTICT R I a 0 0N IAAIINNA L A A A C V? I a L 0 I0 CAAM CAACQAGMMAM WAYNAG AGAANCAM~ ACt24~tAA GAG ACC? CIACWrA 0OlU.NCV? CirnY~irM 7mmcV~rw mCATrW -11-C4 IgARC.4? CTCWCCCA CACIOCCC CCCACC1=k S Y 121; x g g P I 9 9 F A 0 A A V P t N P 1 K A A a 1 A 3 r 42CAMW CCAAAAO ACMCAMW AMCCO10l CCCUAA1M C-MAAI C1YMA'T CCCMIA 0 7CICCIC WlIl 12CC ICOCCACAC OICMU C22IA CTA M -A~ *141 vK aa 0 4 w 2A K 9S N fA P CII T' rPA1 P024 1341 I=M.AAW OXAAGMI AACAC4ICC ANC*AG CA17JCCCK CCCA1- A114AA CTaA.O N~TcattC-flCAG I LCACQ= IcIrcCYC CTActm..A ACIMICAC C1IA= 7fl It"A- .41 RA N P 0 9 1 P 3 A P A P A 9 I 3 C t 3 C K L, S 144, &MAAIA cCOMAC CCeCTICC CCAIC,? CAACOU OGA0.AAC C0.A110MA AOWCSAIM' 72tICPAco oxcc~l7C GTCAC= GOICCOAA GGICliII OCWCC CCICACCCI leCACGAG '1 0 0 0 a p P L 0 A NL A 3 5 a 21 AAMCCTC CAOCACAAIC CCCAC A COACCAC I3'CAGC4 C T 4CCAhC T V-?CCA CJ~AAA; ICV1AM UCICCW =MCC7 IC0PWm AWIC,'A oATT400 ccICAIAGI crm44I4.' 1ANI MCCACrAW C.CCSCCCI0 MACAC ICACCAICCA& CjaC1CA=OW 07ICIC OCA~CA GACCAAOVA C007CCWC CYOWIUAC K1T2401C0 AMCaO~7 CIWAAJIA CCCACAAX MCw17r-T Cry.T4W1- S4 A N I 04t 0 T KP4 A T V A P P 1603 AWrAATACCO CAMIMA ITACCICAG CCCCACAOC AATA.C ICCACI frT7MYL.WTr CA-144'.. T ,IMTI CMCAM C AIWAMI GOlOCVC0 rfAIO1UIT A006 rNOA C~I'MICAAC 1.T'1 W1 I.2 0 1 A T a it I C A r 1 5 0 V A W I74 CCOICI CAGACICACA. CTCA=A IGIIACTICC G&GACPC 4.ACA~pPiV GAU'41.14 C4VA'A4AI CATOICCAGA CIC7OCCU CACTI CIM ACM.ACACCO MAGWTt4. 94TCVC CMY0ACLIrC .7I4,i I404 r C T00P A L t C 4.1 0 9 AP t A AP L I v r. 1 1441 ICACCANCAC COCC ICVCIVXrAAO ASC CTOO &%AAC? GW23=COr 0CTCC'TAC AlrA0 ;AC AC7OIVCTU WCTVOCI CCAACCIIC 11010114CC Mrf-2MA CICCGAGCCO CCGACC20C ?CA-MrrT4 .A N V A A 1 4424A7C1ICIC 7~.A.AAIP4AOWA ACCASUPCA Gr AO. AACAG 4 AVWA CA14.41NA IADOCACIAG CCCAJtIP A&ACCKCrT= -ItCJ~ 4tM.1.A lo~ CA P A IN- V I tK "CIG S I L 4. LI T G P1 P 4 202CCCITICAC ACCAIUOA CCACOAAT CAC=CCT MAorACI7 A txrAJI A' GCcpA C,r. r1:1 CTAAA.C2 PITOACACIJ C CWALtrT O I Mn.AC C(.Am1v CImtQlfl4r ,A.-IA 4 Cc"ICV~ -74AX~ Ar.4G -rr, n f..G 'I A 1. 0 P 1 Si p R 0 0V 7. A P M M f" 4AllA mu .,Afl **874 ;r,.Pnf A' ~TV~ IA C G A AM C ,AA 70 .A A4 -1M V C N A A MI A A P1 A3- 1 2 V 204?GUT1Cr CAICIU.AC 7CTCACACLA tCIA 4.AI GAO4ACr V07AY17A? rAICAV7AC (r--74? A7CACAACCG CIACACC2M AACT"V" CCIRT CI ICCIV; AXt11%4.41A t- I V4..A -1 H NA DOAV L 310 IN 001 VI AH TOG N D C'.

0044 ACAIOAOCA I4O)=704 C'WAAMS ACAIC AC ICAIGACAfi ACAICCIA ACA7IM.A W14..W7 WPACIVOL? ACrCCIACA CCIYOT 70"02Y0 ICIAC10AA 10"12CT IXIACCCACI ACCACCIIAA 24 1 0 TP T N 0 L N N L. P A C N A V V P R T L.V 1 QXA4.T1 A~ASl C41UflCAO AGACTACAC ACCCACACCG II~'A AC.TP IA'l L.

21 a04.0 D AI 05WAD aN 2 vAS1 21 2 f D"L F 2442 -r C14CAO A C T OTACAC. OCATCA'rUA CWAAIA.A Wel-vtA CCCCACC~T C4AIcT c14? AC CACKOACCC CUCAC74CCT 'P.A4117AT.

11,21197 01:00pH A0C331CPg *t~a D t I SSsIa It A A P ASI aa av COB eSATCACA CMPCM TCCWCAICA rIMfAft CMACACACTT QtCE71VW CMAAACT GcAC7lA CACI1CSVA @flCIQMv AOaAOPAg CCACAAC CrV7IM CWAWACAC LTI17TA CCMCACS 14I A1. AflXI A'2W=AC1 ACACC70AC AGAAIWVC VIfToA)v lEAP as-IC A-AMW5Af AL-IA lXl 1EAC ACCAC WIWCTVAC IVMCACC7C lCrffAC ICACICAICC AL7AL-tACAIJ flrrflml mxOcC7CCA III;' ACAAWWMC ATa..lCA*GA7M CAACOGR CrMr;AA T CW.flflflc ICACACWCC CACAAjAScA I -JTCOCCG ?u1TI7 TC WaCAAoG CTCCACCR'C oC7OA AL'TACAAflWV CN5VGfl,5 .klse c"""V=AA CMACCT CITAMtWC AOACAOMW AtCMVMA OOACACW CiTCa07r CAOCACrAIC Va.Qmtv CI'VITA CAUTPCAO nmyOCCoW ?ACC=rr ccmgnscM OAOICCACCC OTan=AC PO a SBA PAQ* A La? A? 1 ta A 9AA VS MP)CAA5oAACA ArIOW CC5.AOWC &A&CA ClECAQAAC 1 AAACAA CACCACwrAA A5Cfl7VIA OlnnryTW WScmAWWO mCUVCCC cCTCcact nnxrcrnu Accrrrm-t~ CIucmcmlC ?CtAcACACT 39*1 A A L A a A A S Q V P V 7 3 ft a&_LLL t a S a 0 P a Jill C CCAJCAtA TTCRO= CTAcrmtCCA C? .47 ~cACC LPAP.A cCICAWOU S I ISA Ot A I TDO A0 A C aaarc ta A 04 AC CaCA tACCrmm= mccOA~CA CAG C7 CAC Cg ?rvcTTAC A CICACA CASC OCC1 SCI rRmE5CIA AAACWCCWTKM ICAC ACTWC AAAC.A AC-1U2 ACAAPTI A C 7 A N a I I A S a V S A a s A c p p a s a a P 5 a I IA! ArTstArA mAtACTC CAIWO AMO CQ X AflCtcn AGQCAA.A CCAW 57CAA.AA( IWIV2T~cV CCTTACO TCtCTKAVt CC1AC AC W 1MAC5UAC 5CCClt5.A OCASTl A29 1 1 I S P a It V 3 a R a a A a s a 0 0 a P A IL JA AXftM WIVKIACM CASCTICTOT AACA CATOAIAIr5. AIA1VWU AICGAATCC 5A577 ITUDAICCO ICACCACT CCACCA CflUCII O C AICCC 7lTATCOAC 7M57VE1W CCAAC ItA TC of a L a PA AQt9 P 2VSRW S o PAQ A 0 PC A 372£ CfTLXtcOa AAAtwA c~TkMOrr O(CAOOCCa IhTkrcAfC "CCrAA ACTC'TxtA ICCICAS C&ACA1?X CAVOGACMt ACI AA CCIPCCOG ACAAQIMA0 11,AC1TICCCACW CITCCAWC a P P a v A IN pI a a a P 3 A 11'; OAC ACACA OAC ACTAr~C ACICTVMA ACACI'T CATWCAf CfltK t A~C.TAltC atwym 1C&,AA ATC AAdU IWYCA'?CVTA?t TC'krCCCACOO C5 ACCCCAC: CIC5I WTITAC IT?"~tAC CATAAVr CIACWTAA CWTAC7A( AACICVICC A4II:AI01G A1"CAC ISA I Cyurr 7A CCWCVQr~? TC PAr5AGCC'fC CWAVIrAAT aflT2)Cfl5A CAarWCA &TAWWCC CCICACCrt G&CICA rtT CAMA OCM A OWCTTCIA CACC'CAOT TWtACMDVI CCACssCC S1A P A N 5 A PV oo 1- A C T t A S I A C CAL, AG= RCCCAMCCA'I @XCCCrrCt AACArCA cc IflYAC CMCCAA! AriAC3A.A VCCCCCAC CCCAAACA GwACKCTC OCAIWT7 PXACALIA SAAl tsA, D.4JI)SA15 'I V P N a A 3 IT 7 L 1 A I I A A f. 1. ;T P r P kI C i B0a1 ICCCCACCA UATCTI'WC ATnWC,,A w'ECC.r c7CAAOC WC-CV C ,)risCAM 'A it A T L B It A, 1. A S P A A a Pt a r c I S 7 L P I BO)ClIUXAACC CAC~TUACA AMCcCW IVCCWG CTCAXCC 7AACTIIA AIAATACI tvcrtCAC't CACMi'?AC MSUACoICT 1IAAG AOA A OACCACPC ATTCAM'?CUi Ir'ATGCA2 ACQC71C7'? B1B1 ACAtA.ACA£'? GACCCOCACC MCAT A Aj A'J CATA'W f~lA1ll I W' Vr,r 1SACVTCCA CVOOOIO AACTA ANW ;;flfAIA L BC) 41~m CCrArMVTX AWAW:Cn CAtzACACI" c. sar.C(crtirrr W m TOCCA WCTAAGACC 7ClC TCCNC~XA GC l .ACClA.AAI&.

4121 IcTgaTCCA GCCTCCTC 1CCyCCC AckuarcAIC ACTTIWVA ACr'SCCAU C)XCc -I 5tA).CA r? ACACCCAW CQCAC7ULACG AOMAOC WCI'TTAAT 1C&A.P.T CCAACOMIA AW?52.7CAC AGWSCA-T 'A C L I p P T7o a I to B C I B" A 0 a 9 1 A P I I a I Beat CAOCCTC AACCCCACCA lACCAMCAC TCACACCCI! SVCAAC C ACOrACTA TUAJCCATAC AVIEACA5'.C ClCAACAGG flvaclW1 xcawCTCCT AuonaQWoC LamACCWr ?CCCC7CA? ACrACCATC PWS.-Mllj .19 tA a A PIO ft P 1I TBRB A S 0 PA 0 GI BAI1 ACTCCCAA 1AOCTCCAin ?CCV'rAIU AGMOAOCAC kaICAUAACCSTTr CCCrA7n'I;A CUOACT ATCCAWTCC AOMKACC TfVCIT= TWA CITC5 crCC1CA GCTA.AGCCCT ACAAA.tP 'I to V II G C S T C I V I C: 1 T V V C1 P..V LOsA CAAfl TCAtUi GkASWACT t'TCrL'IMft? ?CCArftt SSAXA'l'17TA CnN AN A AAA Ad' AL CTCAACTAC CTAGGAOUMA MAODACCAA AWT7CC 57A &AACAA.At MAlA'. U CTMrTSIIA IQV.rAP;.

.1 1 3 9 f I a A C A AA I C a a 1 1 T A to A BAA) ILAY'17.11N rsr apI AAC- SI'r t'7Sc5'AsAA A'~l'TC 1 I A, P.A. A A l' PAA5JSNL~SY SSIThA~V~l EAC~fI'fl'AAN'IAAVI' A r. isA- Ao C A 1. A I A LI 472; f I r a 1 N C C 7 A P 5 P B 1 ma-AAIACAACCC UCCSw~A"C At~'A7CIkiMCCN17C? A I WCASM TII IVIC ACSC C CCS'SACCI tC 'l'A CC IAA CAC5.'TSIOVAAt. A.IC~ ttlS f IAC A V I A A IA 7I I I ICI 1 1 A AT A ?I Aa a a a A A a C A 9 C A-Z 3 .aAA P P C A SOB MMAAA ITO.1 A.IC T~mtAACGC c r C MCA..C CAS5 ClI.TAT'.A VICTTC7CCP TCI'TCACCA tCCCAC ACfCSXCG 11ALLACECIT GCCtM5A Cow (M1TAAA<. SAO;AIAP I. 12S19,100P. 333323ICS3.971120 .V .3J~ rM t 9 It T T 0 S 8 U P Al 6 P R Lb 7 N P 0 8 T 0 5 1 033OAQATAII GkAAA CC=T1C TIUt9v= 1CACCDAACT 7.3CL=ACMA IDOWCACA ICAt AACAT06PA .:CI3 C7A AAAMA MMIQCIIA 471~VI3 WCVO707CA PKS P X3CTQM CMTTC7CAC ICCWC? ?AArrmcr3A A1SXAT3IWAT AtMAW3Y0 MLACCOWIAT LXAM'V.I!A .mTAACC CPAAGA93y AaarCAaAc ATMAr? AcATAcL7A wt-T ,.iA 31A3T 0 33* SM.. LI t T7 05£i.- L A L A w 0L b 0 933A~04=~ A1GACA TWAGAOM0 AATAW00 CrPAO.M(~7 ACA17.Alco 233 OW~ C~C0T CTTCTAA IQtTACIC1~TOMCCCO AOOC0CTCCA 172W7CM CCAOCACA0C '52; L. P L I t a T S L 0 II 3 L 0 D T C C C S A P It 9 C U 933 7WAxcCA C AACOCMCc cAac73cA0, A101'YocmA AirAc3.Cm r1'3aAICI wioAw A3mA3AF HA MT1T7A ATAICA CtA ~AMA TWANoA ArIOCACACC .ACACCO0177 CTA1OXr OCTM~l. A1ACI QWCVTO ACCAC. C~ AAACTAATAAC.AT ;3 I OP CQ 1ALP90 0 0 0V.L 0 LAV V A A L IS r P SIB3 34VTAC* IAAATAVA7 AkcTV 1AMAAAt7V3T AWWA1"A.3 CACAA3A C0AATA' $1 -MAAOXTOV?U~TAGACCA MA)CC A TAAC3 rm-rrITkA cTATAArmI TAckACA w-m-1C T 9 93 L LP CC IL V I 530 lASvT L 581 rACCAAAIO C0IUMCC5 773 1lT 3TCCTT AAT-T 107MAWCA ACtCVAC MA C~9 A3373W COIOOV..A A F. P1 5 L0 3 F 7' I 5V V 'S 0 5 5 0 0 2 3 INS1 CW.~AfAO 9C(IIOT IlOCr3ACW hn7A1OTKT ArCAAIX. T 113 A(OATM CAMAAC tTSS3C3.

S lL A0Tt~kkVYC CMCOIIG WACVA03AA 63 ACA1.110A.11,,. T 17CM l lolt CA1V OMM OAGA CA CWCC TO MTC= ACCAAAAAC A -1 Q P L 9 Af L A 0 Q P N a. p. v A V 0 W P L C I t L I 6161 AMCA1AA CAACV7W GCCACMCA ACCS AS lt=0001ACWAA13 CA33 CCAAAC AA=,CT A0CAAGA CAMCTTAG AA ~CC17 ACGCIOT7 1CTCA UDUOAAA C9711AACC .37 1. R 0 g30pL0m L I T A vT .AVkr3 453 CGAUCI A3AoaC C3VV.3,,3A WC1MWW3 O73L-KAWC GAAAlA .A fm7V-co C TTCV0AAAO C3A.CIWT IOSACICWC CIRAAATc CrtVV707 3.VAV C A.

6.0I Alvr.A r TAAAAAWAI ~'1II-W rl-YIV VAV' ,.3V 3 AV TA3013At ATI7T7CSA AC70AC3T 3A^ WX~mm 33L1VAl3.S M.3 1.O 5319ACMTACC A7.C 000 OOCTOTIOT 3.7CA1'mlm A7O3ATr-.; AtAX-WI ASTTVMV.3l3 .1 1, w y S 3 P, V t. L. 1 A V I 6 1 9 L I I 3 A 3 P 4433 a.A=lACC CAITATVC 07101 AAVCCW VCMA ACMt .3A1 .3 0, P A or V N 0 T7 r w A A I P 0 Li 33 B. 33 I- P 3.L 6723 CAICCI7TA AQTOUCAT a.AChCATAI CCA1O~r-. t(7033CA7A RCAYTACCA3 TePX'XC' :V .A 3.~0 31 3MT A7 V M03O3AA' AA~7cftw7 wloA017 CCCATWC7 3 9V.Al" AT C, VAA, 33 ~C 7 O t A~c i.,s o -1 A SO 0. D PL L 0 P WA M' A 4' 3.F:A CV AWIAA C XTAATU A CATM-0A 1150XX17A OW0S"*-I ~oT: V Q.AV.3* CT3'3.

GAWAC ODCACAC r=AACqVtg 0AAAC CC7CT' TAI'A3f E T! 37Y3 505 3A4CCAACA MAACCCA TCKtCAt AVrr.3*C? CMI.177A crtVc,,L.T rtS AA.,10 73V.~3IT CT700rTAayorrmW TAmACOCcA ctj3A.cc7 CoA N3JA.VV, CCLXZ f T30 P P 3 V L L3 L. r A P 0. P f t. 5 Q I r .31 7 A L5A HA0 PPP OP 3 P53L. PL A t; I P I 3L 121 CCACV.7C-rr ACAC~ ,,CACEGAO VCCCCCy C1TA-.-1 I3C12Cl AATV. 0MtGA.A 1CMt0JCMC CT3TVT3t I900AC CA1701.L CAAACCCAC UMLVT)OX M-1 .3 A A 50 A r 3. P 0 P I. L IS T L 3. 3. v P II V P A t t.

1303 Lx.r' k- IcC V1WIC (3CAA 3-T.nAAf: nrArmnrl Soo 1-, '3AV-lf 11?. WMIAA. K-K ftV T WAL3TAnl. AVMV 3AAAS*3AVA AAA A 33AAAAAAAAA AAMA S O.ID 0 t'I"4r'C, 9( 4 C C EC a hF pa-I 7771rc rGCAGAAT TCGGC irC rGAG CA ~AC CC G rc TAAGTCC A AC tG A rr-C-5-GGAAZC6ACCGGA C~C rC 4;',;CCC r~ rGC: C %-ACAC G TC? T AAGC CG AAGAAAC TC GT TCAAG ?C GGAC C AATTCAGG T rC GAC T TAGGCC CC r TTEGCC TC GGC C TAGGGAGC rCCrTGGG.%Ar. t L....pCA2. Weru. tamtda gtIO prim~er EcoRl wisect sequence Iiksm?': I=8 S AE F GF F E 0 vO PG v0A E F Q G P S R I P R G-:3G CAAGCCGC TCAGCAGGCGCCTGAG~C C TAiGAAGCA~CGCAGIC~CCCACAGCAGCCLA4 C C&GTTCGGCGAGTCGTTCCGCGGACTTCGCCGCACCGCTCTACCGTTAGCC GCTCTCcaGCGGGGTCGTTCCGGTCGCGrTTT~cG pHHl4-3 i CP L SK A PE AAV S E C KS0 0ELLIS S K A K A 0 K S C 'lCC TGTCCCCTC TGCCAAGGGCCAGGAC.%C-CGCGCCT ICC ICAAGG IGGACCCCGAGCrGGTGGTGACCGTGCTGGGAGACCTGGAGCAGcTGC7 GACCCGGACAGGGGA6ACGGTTCCCGGTCC TCC ICGCGCGGAAGGAGTTCCACCIGGGGC ICGACCACCACTGGCACGACCTCTGGACCTCGTCGACGA' *pHH14-3 G P V P S A K G 0 r Z R A F L K VO0 P E L V V I V L G0 OLE Q L L C ?ICAGCCAGArrGCTGG 1CCCAGAGICCCAGAGJAAAZAGGACAG IGCAGAAIG ICCTGGATC ICCGGCAGAACCTGGAAGAGACCAIGTCCAGCCTGCGA GAAGTCGGTCTACGACCTGGGTCICAGGGTCTC7TTC TCCTGTCACGTCTTACAGGACCrAGAGGCCGTCTTGCACCTTCTC rGGTACAGT3CGGACGCr ORF (1-57201D pl..I? full available OFR HU-Unc53,l pi.M1 OR F S 0 M L 0 P E S 0 K P T V 0 N V L 0 L 9 3 M L E E T M S S L A r~c ccc A tI T: G CCC TC 5AC G A T Gc0ArGTC C-cIC G5Y GcA: TcC GC'-rC CAG T CAA TG.:r:GCC CV: OFI-iCm-= vL 147 Cr-F a, ~ia~a OAF HU-n5Zi tL% OP 3~ 3 L 3 E Y 0OS DO D S S 3 LIS k 13S P 7 -5 T-1CC G;CA1 C c n CC--zT Ac :C T CCC- coCcri TG C Gcc ACI C GcCC:C T.C t a l.a Unci!- e:= Tuesday. I a Novembef 199?7 1:33 I. 7 cPg f, c, u.Unc5311 seq (I -6013) Stamn .**Alc@ ,.:"CAAACGGGaCCCAC rAC TC~CACAC CA TZcCC:ArGCQAGC CC CAscAAGC rCAGCC A tA TC M:CC rGA7C ;aTCAA rCC rrI-r :;-CC~CG t TGCCCGGG rGAT.GGG r T GG TA:66 ra TACC CGGGG TCGT TCGAG TCGG rA TAGAGOCGaACC TCSACCA3C T F G ACGG pHH14-3 rd IA? I*flC x 4-1 available ORP HU.Unc31 a ;)LM i OR .G ER A HY S M T PM R S P S K L3H I(SARL EL V ES LOS C 7.c rcc ACC TGGAa T TCAGC% TcG TrAC TCG.: GTACTGGAGTACCCG TTCTGG rAC T6CC TCC TAC TAC TG TAG TGATGGCCGACCC TAC TTT~i,7 a HHI4.3 oR.F (i-s7so) rLM7 OAF tNU avaie ORp HU-Ur45wj pLMIl OR SE V 0L K SG Y mSD 5S0 L M 6 K! 7mT E 0 0 0 I T G v 0E S GC TCCATCAG TAG TGG AC TCAGCGATrCC TCAGACAA tc rCAGTTCAGAAGA.AYTCAATGCCAGC TCCTCACTCAACTCCC rCCCAAGrAC TCCCA: TG: C.:AG. TAGTCATCACC rZ GTCGCTACGGAG7CTG 7T-%AGTCAAGTC TTC TTAAGrTACGG TCGAGGAGTGAGTTGAGGGAGGG pHH14-3 pC8212 57fo2o PLW7 OFF Ja-,ailablo ORF HLU.UncSW1 PLMI OP 331 3 5 S 0A S L S S E E F N1 A 3 S 5 L N S L P S T P T :CGACTs%. 7:.:AAA'CCCCGCAAATGCGi TGG TT AGrTGAA~c~~~4Ac T.:-C3FT7GA: I T-.Ar -aA CTC TCC,. GCGCrT~CCA C.C-ACC A% ccT nI4H IA.

pC82l2 'Llavailable CRF HU*LnSWI -PLMI OR f4 3 Z 3 E K A S L A S G L S 'd F S E 3 :?4~AA±ACTGGA~r cTA crCA GGCG GGGAZC 6,i--AGTF,-;T;F:F:C.; 14HI.3 ocu;z .2,jatlat:P 2 1HU-Unr-S3; pLM*l* OP.

E. C r.

Tuesday. 18 Novinbef 1997 10:33 lHj-UncS3I1 seg (I 6013) Sdo at A ue TCrAA.AAG~CCCATCAGCC r:-GCCACC:SZ T rCCC-,.AGAAGGGCAAGACCCCACC rG rssGc rG rAi.c r r.C:CC:ATC.: TCA:A:,;- :c rc T TGAC rT Tr C6Gr ArG~cc G 66CC, ACCAAZ6GAcr FTc rTCCCG rGGGG TGGAC ACCGACAT rGAAGGG6GTAG rGAGrrrpHH 14-3 PC0212 MlU availa~o ORF HtJnJfl .94 0F O G E LK K S L S R GS L K K G K T P P V A V T S Pt I T C:-AGAGTCCCTCAAATCCAGCAACCTG66AACTACAAAA TAAGCTTGCAGTGAAGAATACTGGCTCCACCTCCTCCTCTGA7 :.e7TACG,SIAGT'-TCAGC6TC6 3ATC3TTGTTTTCCATGAGCCTCTTACGGrFCAGGAA flHH IA-3 full avada~lo ORF HU-Und53/1 pU.4I OR A L K V AG K P E G6K A T 0K G KL AV K N T GL R S SS 0 SC 766 TCG6ACCGC: rGAGTCATGC TAAGAAGaCC:CCTCGGGCATT6C TCGCCCCTCCAC TTCGGATCCTTTGGC TACAAGAAGCC ICC TCCTG--CA: C,.CACCGCGTATCATTCGZGACCG CA~,GAGGACCAGACGTrcTCGGAGAGACG66 pI*414-3 eCN212 %A avaa OFIF llU-Unc53r PLMI OR R C R L SO0 A K KX '5 G A A P S T S G SF6G Y K K P P P A nH1114-3 Lu a:vjaate OAF HU-Lgnc311 =pLPA I OR 7a T 4T V M C r G 1 A T L 5 K 1 0 K S S G I P VKVP 21 7 PHH 24*3 ).la~l.309' U-Unc53:l p.MI OR.

I N I S L Tuesasay. 16 Novembe, 1997 10 33 Ht.-UncS3/1 sag (I 60 13) SOO. 4, .e0Ueftcs pie r A,;A TAC TCGCAc rGGCCGCCCGCCCCACCGGA~CGG7ACAC TC~rCG rcG rAAc YGGGGC.%rGA.GAGTG TGG TTC-G rCCC rcrCAG~rCGGA,! nH4l 43 fDa.lzIz PC82lO.14 Nil) avIalat OFIF HU-UneSW3I LM I OR i M S V T GG R G P P 'VS S S1 0P S L L 5 TK 0 ISG L TrPS G4::Z TaAAGGAGCC TACCIGG TAC.CCAG rOG CWGG.ZCAC rCCAGCCCC TG TCAA TCAGAATCGGAAGGAGAAGCCACCAACs rG: pI 9 114- 3 pC82 pCBl-il Wu availata ORF HU-UncS-i pLIA OR P L K E Pr TK V A S T P A P V N0 T 0 R E K E K A K A K A V A C r7G3;AC TCAGACAiACATC TCC TTGAAGAG TA T-CGC :CCCAGAAAa TACTC AAACACAGCCCCCGTAcAcGGCA3ZAG-T': GACCTGAGTCTGTTGAGAGGAAC TC rCA"C33GGGTCTTTCATGAGGTTCTTGTTCGTTCGGTGGGGTTCGGTGGrCGACGtCTCGAZ .HMIA.i OC19210-14 uava.-latle ORF HU-U-nc563I PIJAI OR L 3 0 Pi 1 3 L -3 S P E S T P K I 43 A S N 9 T P E L cA:ACCCrTCTCSGCCAZ;.SCAA-'c-7; -cAccc rC A:TACCCA~rc rr P acA P.3.ATC cC.- G-7.TGG3AGAGr:CC6GT3ITCSCPTCTC..%LGZrPP6GGTGAGTATCGGtP±ACP.-GT:CATGAGGT7GCAAC7AZA.-;.3:T. t4 OH14-3 pCO210-14 9 s. 3,!a*,IeOFI HU.Unc5311 OLMA; 0= r P -L I A P S L A N 3 <i SN 5 L P Tuesday. 18 Novemlbef 1997 10*33 1-1 Pasis rK. rjnc.jI seq (I .o-j 603Slo. ,lCQUeflt rZA ACCACC1T^C T1CA.LZG T:CC.%A..C r:CAT6CrAcAzc rcAccArc :;I~aCCC r: t:crrcCT.r.i: r.C:L ;TAC TrTFCCAG7C TAOACG TACGA TGT TCG.%GTC tASCCCCGGGAGGAAGGAC:AAZ. C 4GCrr 1 PHN14-,3 PC0210-14 lull avaiabl OAF HU-Unc53ll pLA It OR S S 0T T A AS X V P D LN A T S SA S GG PL P 3C F T P S 0 A F CArcE TCAATATTAAZ tCAUCCAG4 c rCTCC~C-'aCC rGGAGCTAATGAGTGGT TATTCAAGGCCCAGACCACCCa, G r AGA T TATAA T G %CiT CGTCG AAG AG" 7: C CGACC T CGA TTACTIC AC CAAAG TCAC AC GG TT t TC TGGGC~ G A GCG.TGZ GT.

nMH I -I PC821D-14 Mi available OAF HU-Und3/l pLMi OR I L N I N SA S F S 0 G L E L M S GF SV P XE T PM YP K L 3 i c r;c ACA3AGC ATGGAGTCCrccAGA TGCCAA 1TG.IGCC TCCCCAGTGCC TTCCCC AGC AG rAC TCCCGTCCCCACCCCACC TGC TCCCCC TGC TGC: I: GACGTriTCCTCGTAC~ FCAGGGAGG IC ACGG 7-AC TCGGAGGGTrCACGGAAGGGG TCGTC.AGGCAZ;GTGGC TCGACC:AGGGGGACGACG6A: pHH14.3 PC8210-14 a-ihla.4a ORF HU-Unc5311 PUMI OR.

4 z- ES L C r SLP S A F P S ST 2 V P T P P A P P A A CC A C .AA ^A 5.%AG C TAC TT G r AA ZCC CC AGAG CT 5GG C AC TGG ACAGrTAA T -AGCG GA TC GGAACA:'TC I 7CCC AAGAAAG T CTT- T C E IT 5Z CT TC T: GA,: T GAAC r 7 T C IG G GG TC TCG.%C CC G TTG A CC 73 TC A Tr A TCCCC CT AG CC TTrGTG AGAA GGT C T TlC pHH 14-3 pHH3b pC32 10-i4 iia;laba OAF HU-Un=3/, ;L.%V1 OR 4e pr.f HU 120 Tuet-lay. 10 NOV4flibif 1297 10:33 ah N% u.ut'c531 S"q (I >80 13) Site ar,. .&quemle C.z7Ci .r,&cAGC TTCAG TcCC ;AG.L, A::A-:;A;AGCCACairTCCCA rcc ArTar~~GGGC TGCc r ;s rCCG. rGAC,:.G.C:ACC c .rcc AGGrCGAAGCTCAGGG rcc TCCTC ,Gr! rC CTCCGC rGTA GGGTATGGTA4CCACCCGACGGAC r TI6GC TAC TGGTcAGrTC TcA-cG p14114-3 ,lull vaidaki ORF HU-Unc3Ii pLAI1 OR L 4 0 L 0S 0E E K EA R H S H rI GG L P E S 0C O SE L P 7,-.CC rGCAC TTCCCATGTc rC TGA35TGC ±'AAG :5CAACTTACC CATAGTGAGTCCCACTGCGGCCACCACGCCAG&A TCACCCGC TCCA.C.

AG3GGAGGAGTCGGCTAZ7CrGTGAGTGATATAGTAG GTGGGTrC TTAGTGGCGAGGTT. pHH3b lull avajiatle ORF HU-Urc5W/l -pLMI OR S A P A 1 PM S L S A K G O0LT N I V 3 P T A A Tr PRA I T R S M rcCC:ACCACGAGGCGGCC TTCGAGCT,'.% ACAG:3'C TCCCAAA rGGGGAGCACCC TG TCcc rGGCCGAGAG.*CCCAAGGGAATGATTCGGTCAGZ C.;TAGGG:TGGGTGCTCCGCCGGAAGCTCGACA3-G 7:SCGAGGGTTTACCCCTCGTGGGACAGGGACCGGCTCTC TGGaTrccc VTAC TAAGCCAGTC: pHH14-3 14 available ORF HU)-Urc5WI piM I OR SIP T HE 1 A FE L-S a3 0 M GS TL SIL A E P K G M I It S G TCTGZ33TGCCT3G TAAZG-.TZ~tA:G 'A3GACCGGAGGTCACG~A.%GrTCt ATGo :TCGA'-TCCTCTC'-T,.CGT TA.-! pHH3b '.adoal OFIF H4u-12ncs31 PJAI OP.

P O P T Cv L 3 L A S AS S T y S A E E R M 0 3 DHH3b -~3I~'H'J(nc53;I CUM; OP i tS. Ii Tuesday. 15 Novembor1IM7 1033 r uI I Hu-Jr-C53/1 Se" 80 13) Site an!d Secuence -'Page rT',Aa:A:;AGcc rGG rGAAr~a TSAC. rCCCGCCr,:-3AC4CC tGCA jGCGGAIAATAC TGrC T5G.a r~ZCA;%AC r.

AACr rc~ r:r--GGACCACTTATAC TG raGGCGGACrc t6 TGGICCG TC rC rGCCGGc rcc rc Trcc TGrGACTCGACGAcc rAAACGC 7,crr TGT- U2 OPF -pC-S25l ORF tug ORF HLPUncS3Jl a pLM i OR F EQ0 S L V N M r 5 R L A H L A E TA E E K 0 7 E L L0 L R E ~r i C T T T TGAA GAA AA AGAAC TC TG AGGC C CAGG C AG C AT TC A GGGAGC CC T TAA TGC CTC AGAAAC CACAC CC AAAGA AC T TC GG ACAA G GAA- G AA AG A rTC TT T 7TC T G C TCC GG T CC G -,15TAAG TCCC TCGGGAA TTACG GAG TCTT TGGTG TGGG T TTC TTG GC C r A 7C TC TG tTT T- -U2 a~ PC8251 OF -3 nfjg m NJul ir-il OFIF NU-UnCSI -pL"I OR F L K K K N S E A 0 A V 1 0 G A L N A S E T r P K E L R I X R 0 ti TC CrC AG A AGC ATCTC AACC TCAACAC ATCA C T G ATTCG T GACACAG G G GGAAGAAAAAAA aG AGG AG TC TA TCG TAGA T TC GAG TTG TCG TAG 1TCGG TA AGG TCG TAGCC GTCG TCG TTCC TACGAC TAC GC T T T TrTC T TT T7T TTC TCA-%CZ U2 O Ce~sC:.,r wI~nh ftjl availale OR HU-tjnc53Jl pLM1I OR S S 05 S 5S L N S T SNM S S I G S SK 0DAC A K KX K K K S a 5A CT CG A TCTT A -AG CZ- t~z AG3G CAGrCACT CC CATA C G A G GGGATTG A A nHHih ~aVailale OAF -t-Un:3/1 *LMI OR EL q S S~ A I K G PK S A 3SY S 0 1E El i r 7 o A: rA -G 7.CC TA Z C -OHH3b 33 Pc a. 3jIlaw C, z ?LUL531; Mi OR r 3'SI L S S T r Tuesdtay. 1S November 1997 10:33 *+-UncS3/1 sq (I 80 13) Sfesbm eco '4 -5 5 P140f AMC. CCCTGCTCACCCAGCCCCCCACAC.A,; TG GTC iTGCATGAGGAGGAGG CCAG. JrAG4,G7.rZ3; 7, cr~r3,j,rGc rCCCGGGCGAG6GGGTCGGGGGG rGT CC:;CAA&GGTACGTtTT1CTCCTCC TCCTCGGTC TC TTC T ?CCTCC17i.',5C TC4,ACZCGAG.± T.C:- U2 OPP -pC'625 OAF .VI aradatts 0A,= H-U-UncS3il =pLMI OR r E G P A HP A P H I QL F H A M E E E EP EK K E VS EL TArGGGAGAAGGMAATGAAGCTTACAGAZ:ATC~rC TTaGAGCCC TCAAC CGCACATGTCGTCGAACTGAACTCLT ATACCCTCTTcc rrTACTTCGAATGTC rGT±-G 2z CAACC CCGGGAGTTGAGACGGGTGG TTG CC TGTGACtCGT: ~~Tc C A TAG U21-1 op

PNMJ

tuII1 avwjla~I OAtF HU-Unc5,3/1 o4MI OR U3 OAF pLM5 OAF L V E K E M KL r o I I L E AL N S A QL D0 L RE r M H.N M 0L rGAG, TGGACCTGC rGAA GCAGAGAAr~::AC C-.AG3G TAGCCCCAGGCCCC TCATCAGGCTCCAC TCCAGGGCAGGTCCC rGGA-CATc TGCAT7.; 'C:CcCACCrGGACGACTTTCGTC TC rr- :TG..-:.-rCcATCGGGGrCCGGGrGGTAGTCCGAGGrGATCCCGTCCAAGG' CTASrAGA,-GAA- 0A'F. p cB e -pp Wl4qh '-Ia i£daf AF HU-Unx&11 PLMI OR U3 ORF pLMS OAF E L L K AE N 0 L i P G P S S G S T P 6 Q .1 4-A GG-ZGCGGCG Z 4TCC G 1 AAGGAG-C:;ZG.5 TC AGAACG I--TGTG-.C I SACAr6TGG T-C: tArsc A L; PC= OF= 3,alav- OAFli HU*U.%-c5311 =o~l-1 OP 'S S A' S L G L 4- -U3 OFF pLNLS OAF S F Z' S L A C r S P 0 123 Tuesday. IS November 1997 10:34 A ~j 7 a.

u-Lhi053I 30q (1 21 6013) Site and Sequene Pg 3 -IAGGCAGGCCTCC GTGZZ rGCCCC.'CAGCACATC C A .3,3CCr TGAAZCAGC.~z,-Arr; Tr.:CT~- ra- z c r rrc Tcc rC TTC AC TGGG~ A CC CACC Ac C YC A CG GGGGC G rC G r GTA GTA G r rT cCCc r.3AAC TCTCG r: C r ri .33AC C GA -A T- Ul prCS251 I O; pHH3b ?uII avablabie OAtF HU-U~cS3JI -AMI OR W OAF pLM5 OAF G3 P K E E V ?I L A V v A -4 P P 0 H I I K G 0 L K Q 0 E F F L G C 3 rCTCACGGAAAA0 TTGAC rGGAAGATac 3AT3AC rGT rTrCCAAGTG AG;CAATCAATGACA-C--~cT3 3 rTCCAo CACCTrTCAACTGACC TTC TAC3.Z:C 7A. r GACAAAAGGrrCACAAG rCC TG.*ATArAGATTTCCTGGGTCGGAIAG.3ACCCT 1.12 OFF ;CK-5I OP piHt3b IuJJ avaiaLeoOAF HUUnc5311 PLM I CI U3 OAIF e pIMS OAF K VS5G K V 0VK X PL E AV F 0V FK D YI S K M0P AS 7AA:CA:7,;AGCCArCAT.CACACC*-C.I.C:A: 3TGAAACGa3TO rTGGAGCA%;GCCCCC:GAACTCCTrGCCTCr0 'TTCAAT;.

Va:It 0.9F HJU.ic53;', Xuhil OR U3 ORF LM5 OAF S I E S I H G Y 5 V K P V L 0 1 Z E M P P C A .1 A r r 5 5. T A CG! j; 6.2 Qr- iO.

nHHlh U4a OFF C 2 20 1 OR' R 1 7 1 GP Q3 OW~ PVLMS OPF 7 Lr VI %eioday. 18 NOveffier 1997 10.34 Pitgs Ti !2jUnc5311seq ft .6ol31 Sitsmr.sequw.'c TC~G~c~crcG rc r: CC:C GCCGGC.-Z;ACC racc r:.ICCAA~c ;c r 7GcaAic 751 Lac:~r G-4Z;3 T CGrGGCCG:GGAGCAGGAGAGCCCGG TCGCCGTGCCCGTTCTGGATGGAC TC6 rTAGCGAACCGGC rCA %7G~ACCA,.C:C .G4C U2 GPF 2S A mmmmmmmmm

PMKM

U14 ORF pCB2OlI OAF riliable OFIF HL$tMJcY1 .pLM1 OR U13 OAF pLMS OF pHHIS L L K H A R L V L S G P 3 G T G KC T Y L T N R L A E Y L V R S 6 GAG~G rc.CACAACCCATCGTCACCA'C TCIACAGCACCAGCAG rc TTGCAAGGATCTGCAAC TG TATC rrTCCAACC TACCCAACCAGAAaAC: r3c4c-,trTCGACGCTGATGAGGTGCGXT TAAGTAAAAAGTGGTG G CAC U2 OF F -CS2Sh ORF pmimfl U14 OAF DC8201 OFF !No avmiable OFIF HL'RCS~l PLIi OR 113 OAF pLMS OF pHiH I Mh H 0 0 aS C K 0L 0 L Y L S L Al ti 1 0 :rTr.:r rGCCcTG-C-AGrGCC:AC3r%.:C..4TrAiT U2 rZ:. -CH251 CI .WUWih U4 OAF C820I OFF '.IavathEJ. CAP HU-jn3/1 OW OR U13 OAF .pLUS ORF pHIS L E L G£ 3 E L r cr Tuesday. 18 Novemb1er 1997 to034 1-Ig 1 -Iu.UncS/I Seq (I S0l3 Site Sequence .rG r ara~r TrAGG raccAc~aAr:icc MT6.%- rflAcAcccAAcc.%rGCc t. C:c rrGj..:7rC:A r.j c.

7. r: -TACaG azrATAAT~rCCATGGG.,Z- A'TCGGACArrr.T-.CrGTGGGTra TrACCG AArG-- t.2 OP S C2AP

L_-

INUMEMEN

nHwllh U4 OPF: pCE120I ORF MlJ available ORP HU-LnvC3/1 PLMI OA U3 OAF a pLMS ORF pHH Y K C P I f GT T N a P V K M T P Nf 01 G L S F R ML Tr 3 ,&ACG rGGGCACA rGGCr rcc TGGI.TC.G TTACCTGAGGAGGAAGCTGGTAGAG TCAGAC.1G:GACA rCAATGCCAACAAGGAAGAGC TCDCTT: ,7TG-CCGCTACAGACZATGCCTCTCACTTArTTGTTGl- GTTCTCC U2 ORF pCe92- OP. F

.HH~

U4 OFF PCS2Ol OAF k.i flO 14t US in&lJi a Ala flfl a ai L13OAF .pLkd ORF V E I 0 1 R 2 K L V E S 0 S 0 1 M A N K E E L L U4 OSF :C8:01 OFF wat4w OSF MU-UnCS3Il PLM I ORi U3 ORF PLNIS OAF L1 E K N, 3 7 c 3 I P L TueSe4y. 18 NOvember 1997 10 34 j~s it 4J-UkC53/1 seq (I )6Go13) S4oe aft acuenco 17r-CCA-.T AGGAC rTC CG- ACC Ga7.7:Ar -c c mr7 SA zAAAc r: rcc ra :Ac r, f4~: rCC ccar.-z T :GA~CCTG.GACCAi.GA-%TGGACACC rrIGT TGA-;ArArAGGaA-A ;CC~r:CTr:rrf, ~A C m U4 OP' PCB2OI ORP jll arn UE9 OAF HUUnd3t* LM I OR U3 OAF pLM6 OAF pMS I G I G E D F R T VF 1 0DL v N NS I I P Y L 0EG A 2 I CCITG7AC AGAAACC TrC TTGGG AGGACCC AZ TGGAl TGG TCCGGGAC AC AC T TCCC TGCCATCAGCC CACAAGACC ricT.;,-C cAzGrTACTGTCTTTCGACGAACCCTCCTGGGTCACC TTACCCAGGCCCTGTGTGAAGGGACCG6TA CC rGTTCrGGrrAZr T7C-CAGS.,; U2 OPF OC5.'5 I O-; nW~lI~ ut 0RF oCB201_ OF avaiLable OAF MULUnc&Vl a-M I OR U3 OAF PLM5 OAF A A DV'q 0 T L P V~ 2 S A 2 1 D 0 5 9 L 4 HI C -:A:CrCCCGAG~GAGCAGtCAA-5CAGC4C:C.A;CTCGG-^CTC-AC:-Cri sr~- ~csz~i ~m pHJD L4 OR! pC820I OFF O. LM? OR U1OFF aLMS OAF pHM IS s P P E 0 R 7 p K 0 3 7P 3 3 'L 0 Tue-day. IS Novwnber 1997 10:34 g 1 3 ~mi EiC AA-UnC5-Vl sqa(1 >G013) Sites a equuicepaei cG i:cAr:ca.c.AAAC TrCAAGA CTZCCA.L- 7A: At rG Crcc~. G :cc C cc 7 I:5CAiC 7 rr.a"ZG-r: Ac..3Ac rTTZLA~A~rTC TTCGAG G-T:A7G TAAC TCA0c.4r rc r~cC rC 7~ TZ5FA CC 7-;5G r-G..A3 -CCGT r. A-TCC.

U12 CAF S 1 IOP pHH3b

A-

114 ORF PC8201 CRF fll available OAF HU-.UncS3il pVA I OR tI m e)AR pI.MS ORF A MIL LK L 0 EA A NY I E S P ORPE T I L O P N L 0 A T L G F C C3 T TAG TG ACaG T GGG GGCC I GrECGr TTTG C GAC C G tAGTC GATA GA AT CGAGCGAG A C AAG GG A hA rc TC G rG ACCGAG AGTcC GGG t: pHH-3b 2 1PR r A E A 0 L S L L L S P L L F 0 S 6 G P A P r~rC C CCC rAc rrTC7*::-: r7 rCCAACA Ai:.4-':TC 3!.A.5'Arcc T-CCTTACCACCCrCCCAAhC: nI.wl4h _NN IS E a: E T3 G S 'd c F E ft F L G R h G G! A F '7:j GT TAr- r,%C GACCCC T7r A.-IX1 -C.-C:AC~A-C2AAZAAAGt.'.1 -QT 7TL UwWIP nHWI t. L C L h T F r I 3 0 3 3 S F I. L I F pHH~b I 128 rW~sday. 18 Novea i997 10:34 PgI fig I *j-i 5l sag (I 6013) Site and Sequence I iPg A.;A TC TGGGGGAGGCAGGAAGC TCC TCAGA TT T-C TCACAGACCC T TCCC Ai rTCCA TCA:C AC ?iCCAAC AAC T.c C CCCAG4 CTG GA; rt~ AGAcccccrcCTrCTCAGGAGTCTAAAAGAGTGrC TGGGAAGGGrrAAGGTri;GGrGAC6Gt G TGAGGAGGI 1C CTA*;% ACCC ElI WGR OEA P 0 1F S 0 T L P NS t T A R N S S PR 0 L A G 0 KK K V V .K M FK S IC K R. K X NK N KQ TN K K 0 rC ,AC TTCGACC TC TC TCTCC T TGG TCAACGG TTCCA Tc TC TCGACGGGCG AGGACGGGAG ACC TAC TG rA TCCCC TG TAG T TG T TC TGCCGAC7,G TTiZ Id R E R N 0 L P R R A A R S C P L 0 0 1 G D I M K T A A ti T,5AGAA6 TCACCAAACCACAAAMATAACCTTACAGCCTTCAGGGAAAGAC TACCAGC rcrG rcr rC TACCCTCTAATT.c rGCACCGGAATTCA ACTCrTTCAGTGGTTTGGTGTTTATTGGAAGTCGGAGTCCTTTCTG4TGGTCGAGACAGAAAGTGGGAATTAAATTGrTACGTGGCCTrAAGT L Q Sn H r 0 TTK IT L 0P S GX 0 y 0 L C L S T F NN A PE F.

C r7GiGACTTAACC 6013 G4ACCrGAATTGG zfinker L 0 L T llSlmKl 02.5mw m2m.mci)Klll2O K.g. 2 m A .3 H V L. T K 1 ml am L I A b L 0 0 II V V A -j I I v v v A U c K a K: K I d u C 1, KA iCA AA....AAAZA 1AlC.AA M0A CAGA.AGAACA U A K A b I m 1 A A K A K A 1, L LtlC i A r s L K 9 Q 0C 0J~ ml A I-A v mU I~n Im, m J b m C ma A v.t m r e m j 1' A. K 0 K ma U D V i A m.*.AA IKA.XAAMVQm CiAIJJAAA mA: Mml1- 3m m~mm .V .I.A.mnm.I m AKCA .IAt I .ImAI fl A A A S S S t K 3m K A G S c A K 1 I ACAiCA ACCVACXAQ MC"SA2.M MAZAAC7A*SG ATA06AA ACACICACC ICCCACCCC AmmA m.I AE-m.AVATAC 1?ATT406,11K TVAIIAAAI A=.A2JlmZ .3 P, mm V L AL K C tA A 9 K K KF 0 0 A A* A C lCAA .AS A.AAAA.1 IAJA NiA CKVWC CACC.A. lU VAmf'CMA Ac.7mA.7 .1 m~rIC .11K .,mm CAAAAO A Ci ICCA~W *m L I A I I I I I A I C I T K S A I K 0 F G A A T -Am lnACL-tl ACTAA

M

TKXA -ACMVW mXC CACCI0 CAMcCO

M

CAGCAC.AA

Am mi~mAmmA.AA~~mm IA&jlAKAC~7 AUTC aVA -X mta iTO C CAAOKCOn

M

T OAXLCCALM.

I~m K A A ma v U K K C A A mA A KA A 0 L 6 L K Km L r N n. .2AmIAW .A.A:LLt CAAL TklAMMW AAACCA A1MCAA1 Vll-~m. C. lAi LA .C7 t (CmAmt~lTi nCW mAG7 inC!t mAITnltAC.M *AWAmCA Il K C 1 9 L X A A S K1 K L T T V A F A 5 S S P I I A L~ A..AAmAjaAEA-IUA ASOCGCCAG VCWA1I5I ACCACC0IM OCC1WM C AAOCVC AA"A ~C VmimIKj.At- Vm11~mKWn KAWT,&C I ,XmiWALC LX)OAGAA0 UnAALM.X V1VMCG T" A n~AX I..Mi A. IAA.

m AA..A lW~A .2l~~A 1, Aw AmaA WhAZ tIfl W"CAtmAAAC &kA,:AIC QAmt6ALAAC A A K KI K K A A a a A A ma I K L o A.m-C.AAXA.AZA m mTAAW AAA.AACAACC ACC"=~AA "A"CIKT 4'T0A.ACA 00IS;GCT ICmCLWl CCTrAVVCCIS ?1KZTAA ICmAAJAXC C~T CAA CPA1LCC CCGZ7 r Ac- f-D SAVLL-: Ic At( o A AAC.1 C- -J

I

.j K 1 a F K a a K s a A A ma K 9 K p K K S S K 3 A 3 r 1211 CA"AG&CrA CCAA.AA0 ACCCCAC=K AWACIUlTm CCCUAA0AIn CLAAAAAaIC CI m CAASA17 GCAGIIC SIICrCTe A41VrlCVS IAlAVACC WCICCAAC 4XXXICbAO 0%K inWnA LiACmAm~ m AA Cr.O1CS-A4? FI 9 GAO SA K AK 9 K 1 KTNA P KOO ZAK S ASK& P 13SK1 7CCVCAAAW 03OAMGM AACAOMCCA AlMCGA= CAmaQOCCr CVCACACSV SAAACCAAA ACCA.AIC Aarwrmnc ccCCr m TK~ VKWCAC6L? 7CinCQnV GTACCOG0AGG79CAC CrI1?rnI TcTC7Clr Al K a 9 F 2 A A P 1 A A K 9 P K K 2 K 0 A C S SA ~Tw CC=AAVC CCCAACVCC CCAAXCT (C*KGCAAW2 O CQA C.ahaloToA Aa m

IKAWC

77IMrnOACS GCCCTTTA OGGYMCS G=WWcc... WCmIm.CC CCWmT~Ce,. OCCTACCC? WSGACWow mSl 0 C P CO 0 K r 0 L K0 9 K N S S K S S C, A K S K 0 K IK IAO.IALCVCCC CAWAGAAW CCCACVMA CQAAC.AC ICCACMC C7?CCAGCC7 GcUKCCIA AAI.C OiTtuA ~W inCIA GGOKmACCIP OLCI01 AAnCAAm I QAWMGZAA CL.CAMAtCn C51 uI PA r al KU KI 80 mam Ko GOV aal amaGmam1T -K AVM GACCACCATA AJ.CCACAQCA I AAD.CA GACTIVMMW OKS'lVCU CA.MCAIXCA CACCACAC-.

ml A1 K V ma Vm 0 L. P U 0K S V ma Ko K K ma Kt ma V A r r SAKS AWMKT"%XO WIV IM m.UICTAOCCAJ CLmCAm AATACAACCA MCCAACAC7 OXAAJI VACTnClVm Wu.VTAl"Aw WAmLC:AA'm CA10W JLAKMV IrAIUR

M

IS* 1515273m CO1VCAAC VMl 2

IA&CM"

ml v a K 0 T 0 ma 9 KO K V A k S K 5 ma A G K V K f S K 1mam1 CTACA15.' CA0A AA AAQA TOTTACASAC AAAAAV AACASWMg CI lA CCA"ACr C .701ccA 0SVT=IU GACrWCA ACAA13mACO LniCAaIIA CrtKICCACA m 1ACACLM G0?VW0 mI1 K F T09P ACK L 5.7 T0 9 I PD A KAKA Laa T VIt lbS I IVCKI.AAC YCG4ACACC? amnCwAwA0 AC7CAC WAIccT GAWCOO m WC O= avckhAl:A AWVYCTO ACCK0CW CGACACTP 77%OTAC CCTICA0A C0 AC ICAvcST115 7~0OAGM ITPWAAOrAA ACCAVCCC arTiTA. A.ACT~TCC~ CfITWQAIA CACWTTKW ACCAA?0Ma CChOWAa 0MMWM~ LAIACICA OMM0ACAM 0AW=ACA CCsoCWKe, T aMAAM MCAPrCAMCU1C CWVVW~a MAGC 2091 WAGACIWA CCACCC= CCYCQ0 C) AUXAW.A CQCCWA AIQCAA? G WATWMIC IGAOCC C~lmfI.%CCC CMAGCfM 0 WAODCAQ T1TMCCVT %9WA TACCCOTTKC CCAmaA""CQ ACMIWK ml A K K K 2 K 9 S a KA I 1 K A C L 0k 0 L A 2a A 0 1 _1b~ AM~ TCA2CAAC SOAMIAn COLAagT0 W1VCWCC IVKO0M" CACIMOC1 CCOMWAl COMMMCA WAOMM ACICAOTCCA OAIACACA ?SADX= A0ACICT=T 0TCCACC~k WCCCCK K C A KV.. V K K A .1 K K. K 5. 0 I K N. K A A 2241 mACVQClW CAIVIK.ACC I

TM

AACAM OIXAKWA GVAG CVU 2OAA=At CA=IAtCAC OCCCCrT ACAGAAO IATACACV55C AG4ASCIVO't CCOlOILMV C CACt.= ACCrKCCOIA& UfMC0C~q CQCA .1 KO TK 0.1 2~m 0O ?KC KID tN 1 Sa11 0 2221 ACAIGAM& 10K1017 CIGACAAA ACAVCCQAAC C0AAA7T ACAACGA? ACAVOA-CIA ?WZW9ACT

IACIXIZ

m ACCCCTACAA GACTCgSK7 M0AUVIS. AC7AAT7m 1Ug m =CrA IWmAMSACT ALTACCICI.

LAI 'C2VA?A CCCOmTCV 0-AICCC m 1C CC VA* ?TGMV1W AC003ACA C m IWAAC.AA mATACC m

ICCCT

VCWMIKTA7 I*ALCAr.A CrITOCCAO WA3ACAC? ACACACCA lGCCTcIV GACST*IC17 ?A7CC;AwA 2455 G. 4 D A s w 0 a s 0 a L 11,121 O UK2s1A2120 g V 4 V I, 1 11 1 5 1 A S t 12 1 P A IS I A 9 L P. V 0 X *1 A1WX.. A V I If a K A 0 v 9 a a s 2. w S a L. 0 V 9 9 9 S U C V* 71 2.4 AXt AC1 ACMAAJ2VAC ALX TC' 1fIAal,?WA ?2~rAAANC MAP.S1CAL "AhOWATC I VAA.AAV. ~AAca* tr A1'V1I C AAAM.'21 crlC Wc AQV tAX CArAA p .lV1A. I .MfMU UMCILIXT*C1M2.VC GAIt1CA h"TOXL"M CTAC2'.Afl* 9 N. p. v 1K A 9 V r V fI £M .2V P V A V V T K K P T V CV QK 0 A I L. 1. T A 5 C V -r V T 5 A S A T L G K 1 P 0 5 G2CA.2..X.SAC* ,212zACA5AA2 AC:ACIG AAWASCC CA.AS£ICAIC l.2.I4~V.1A IWA2.V tlAC A.ST W*C2..2CA4I WIVA.IA 2.WItrcrM CM7CACTAA 2 A A A G A K l a C A Q K Q 0 D G I L. A 2 UAI ,2.5M V42l.JTII A1WII ,J ~*AAVAAIAAW AG*WAQSA VAAC2WA?CTLXPA A20 TK M AL 0 0S6L PI0P 5 LVaI a5K CA a ias .;12 CMVC,-t...C .ACMTCAC TACCVAC1Y IOCCGMWC CVMMkka LACCCA ACOOZI GAACAMUT AA*.C2 i'~A2JczrI~.~C& Ac(lmxvW cm WA1A Tcc a2.t we~0C~ cIVC CAOA .2 I 2 2. L P 5 Ka A A L 2. v V 0 L 2. K K S K 22. cc"V.CA.kC .XT"?w'2 :Ikllkx:A. UX*A&.JKL.v tm>Al2Q2. 14.1S=CAA CrjLUflMX CT2'XA*AC 2422 AAXCI UA. MTAC2 ACv2.1WT r.ACICA CC AAAA.AL ACA1CW" AC GA07AJ1CTC .2 SS2 V V t G L A OP VI K V A L 0 144; A L I T D 9 a K K 1. 5 A s AAA(A,.C~ A2.2.~1V.1~A2.ACC O..CACA SA14MAIGA AAWA2.CK ACAO1C&WCA IVUACK 12AA.2 2 2214 .pA V V 2A PP AM 0C T s L0 SPA04 i~r, A ,rUM-WSA2r AIACI*Cr O.rTC AV2UI1.A2U A2.AACC CA2~l6.2CA2.AM2.222. C.2.00'X A2..AAA OTCAT2CVA CACIMAC WM*AG CAO AC P.s. 4 1 1A 5 A K S A P 8 K 5 2. V W 0 1 K A S 9 9 K A V 5 32 ACAACAGCCCACVC CCC AACAaXCIA CCIVA2=AC CA&C2CCA0C AWCICC CAC2TOAA UKAGCG .tIA CCQ~MJ ?2QVCGAAI GAC*COM 7702ACCO ?COACGA= fCCKJMPTU .k K, C L 0 IF 0 S V 2SK L K T S C K K 1 D 2 K L. S 3 a C 31 OrA1~I 03ICAOV CTUICAUCAO C TCCACALC AO C1CAC? CCAICCACT C7CCV10T WVAG cCTCCA CCG&AACA GAcAOVcCICA~~aOO IcCACPAcIA oqr&C2 QC4=h9ICQ ICA~C0V0cc .V V KK rVO 9T9L VtAxs S A D o A L ?P r va VI 40KV ICCCA00A CAT7VTVV ACS=WIC 1TClTW cM54.*CCAC 1T2IGACV CCT77TVCAI AACVACACI CTTAV.A 1QC0mA07 ACG G GM27CV AG&GACA ~A CAGIC M ATIC2CA .2 V K, T T L S I I V L. S S P A A s P V r c A S I 2.PL 4582 2J2CArSCCA CACVUNCA AlIOLVC 7CTICCCtVG CTW-TAQCCC 7SOT1 ACAhACIC1AG fl2. 2r.! m,;.CAin%'T 71CU.4A hAM0C4AC GA.CCAIC2WA ATICS.CACC ICIICAIUAC &coomc2 422A*. aCVU 4wCCACACC T a2 9 L A S V A a a L 0 K A A N S r r. A 4.31 A.AMVA Qu1kA.AGAA VVMC~SVr ACAAGATTA W21TTVCC C&OWWAGT ClCACACV ICCC""WA .2 G A. 2 V K I P A 0 2 T P N F 1 C L A S P V V K 9 4122 CTT CA AACCCACVS 7ICCW IACAGTCC &CrV.AICYV GA1IQSTA CGACCAMIAC WA"A C5ACAGO 2ACfCAG 4CGACT ACrMC~t 12r4CAACW ?CCCC*TA ACIACOTA CKCVOVTCP .0t~ K V K CC~~ L SC 2. A 2. 0 0 1 P. T VT 0 S 4012 GAAGVAC WAVVCA 7CCAIr.V OCCATAA W IACAT2T X2TAOCAarA CAAGAAAAT AC1CACG cl rAAMACG CT~1A C CCTAatTcoVV AOACA AACA1COA CIV T2A TUAQ2VKC2V R1 P K K A K VN A 0 5 V V S A 5 G V C 2. V A P A 4241 CACOG&A CnV AA1CCCCPMT AQ AAAOIVVCAL CP2IGCA CACMCGWA TGAAA1ct ICOAACO ACCOA.2AC ACVC2TAAC TCTTUVMI T?!ACTC OCACOA C AOCMI AC 1 .2 Kt I V A K A S L K 1 9 2 L S. 2. V S K A A K 9 C A 8 SKISCAIVAWM "55KACTV 4'.GAACV A1VJC VA.AGAV.A SAACAM? CAAAAA WCAUAIC C?.AJIMATC CKAG7ACVCA GtC2YIII AWICC AT?2CWVVJ CT2QWA CMITCCTTA C .2 I A K A 11 K L. K A 1 0 K A 1 AC A A I T. K 2 K K C K 4141 VO?CAA2SA CArCCICK 1AC1A AfCAA0O= AC2OMCAQT CITTGCAC CCACW OCAAVW 4252 2. 07W WACM. I 0AWV2M A£GOGISVIM AAJCAILWK GAAAC*O 2.2CA.VO AC4C AACS1C CVCACA 1V A S V 2 2. A .A N L V A K p t. g s L a w N A P I K 0 1 K T a T A S S 2142 2 WAOOC AOCrAAI CAWMM TKAAAACJ UT2CAA CAO&OCWA1 CCACUAC CCICMVC VaWCW6C V LKOC" M ICCWW A AASmT C4.C VPICAh7 OWAVMAC ~AC?C 11135191 01:00r" l.l.4W02,l .113 3:0 p-ISO II L c r l.S L. k1 S 0 L P 9N G St T CST -m Li-m o lAlAol ,!SI0 007VcA-wr i lJrllsA *.AM lV~ 0.01 uA S,.SII.A vi.

L1;, L. l.A 1l L1AA.. ll. 1 K D C.A. It1151)1 T *.VlAtfL5, .n A AM~tAl IAT 1 IV V LMMXTAA1CAZ 500~~ A1 0 1 0 10 OA o LS D Q g A I!:i S OWil SIl iA 7lvj..lASI .c.h.A ,l-l7 I. I I I I I A 0 0 0 I V I i 0 .AS...IV II II T V t M f~l A sA Q 1 A. K Nb W WA KV C OL 001&P iL 1 0 .0 11, 10 5 IS 0.0. 1 lfllfbA V C O V C L 0 V1 .I I V S I 1 0 W I A0 0 6 t. u 1. L L G 9I V Al;. I. r 4aN.M m .A4MM cl M) xj nc rV ltlOIfL7 ACIrth1m%3_=rloaj.; I11 L0A L V I t S LI V MTP I S.00 I VS 1LL L rrl.s la. M. 53 S a.J u~ 1 U SI L V 1 L v P r L 0 a v v L g V1.10 A. 1.;ACA CIW lASSj AI'LIXNOC M-.I.,o*,IZ0J0 A WA.jl tCM1C M0TA~TA .3 0 P I~ I L b V li V T A 7 P I L A0 0 L I I I 033 MACZIVII CSACCI7 GALCACV 41ACCCkC TAC WAAA GC OA1IAA C0rfAc 1.ZAIGAWC 0C CCA AC~l75.AC C 9TT0CSgACC0T!S5 AGCG AM G I5 M 0 v I II i V L A 0 W p3 o' a U t 0. 0 A I 0 0 V I 0 01 .20 0.AI~jAC GSrWACCA CIYCCV 4V~h lCC&SALl!OI fZ37?0C~C 01.r 00 tl lVC.A I~ C N ,lla I.1.Il C GA C W A G O0- C O A W -13T0 P V 9001 LOOP& L 0 LW N 70 0009Z5 VOPW 64301 C06AGMU CV0AOV C7IGOCCCA TCW3 GAAWICA10 W.MACAGAA 7WAL.S4A QIU.1XAT MXv1Csr~iA CTII.CCMAC GAACCWS AGGACWCCI CTTCGOOVC 0h 03C V7T I 3VALC5 CCVC00nA .0 IN 2 L. V 0 1 1 0 W I P 0 V W 0 L 0 o F L 0 W 0 1 04.0 A1VC0CC 705XAAIC*? OACISCAI CCCOAOCC OXA1V4CI7 CAAOVIA75 MXGG ICST A0UTVc0 7WC7S.CC ArrMfAD1 AC7GCAA (IWIIZCOA I.'lVTAUOIA CII=flAAC GACVI'XVrU G SCAAG4 .k 0 V 7 I G r a L P L 0 c P I 0 V 0 a 0 a V N P 7 0 L.

0101 GIAC=ACC AICC40CCC ODVI C? CW*T S115SA10IW ACWMV!AO AMOtUM1 ACCChACV .1 OV 0311 I V LLI AVOk SOL QLIG SBA pW0 6001 L"AMIC C01VS1CCC 7ATCY0C O AAWCJ3IAG AGAACLACIC CAGCIVVATQ CShCl&W MCn=1 ormiAThSO GAATWM0O AT700&(W !WC&LI. VL.700T GI5O300ArA7AC CI IXUQ.~ Q~rCUC .L U1 P A 0 MV V 07D V P W A F 9 g 01 0 W P1 P L L 0 6720 CA7VCfVOCA 001W200 O=AATA7 CrAt0W~A (tA1X0C0CS A00DMC000 IIAAXIVC 7I7TTr .1 01 P 0 V 0 P 00 a v 0 Pt 0 1 2' S I S it Q ft P P p 4001 ACUVTI) CA107I!A0T I.0IQTA MltCA1IAVT LC0AWA7 CCACAMX.M 0.MMIIXX CtCCMAW 1ACL o7CMrCGA &OM~~0A? GII!CTACW. (,XCWrAO Clflt0ITc C~flIrAO1.cO tMATC .1 02000 P L N 0. V 00LK L0 00A0 MVI T OVQS 1000 6081 Clr-AG1MA CVCM0QQ 0AS1A OA 7l3A7VCA Gk9CAQC 1A0740II!A QQ0LX. CTSIACAA.C GACTTrUACP GODwVAC norpCoott o"CCGKy M" WA Wrcaam~ csr YF 7i -77 .1 S S 5 V 0 L S L S 7 L 0 G P G GWMC~CAWACACC IALAA&CAVMCAC? CCIVI1*IA Q300C3V3.- &0O. 0CMOACCA IL VP~ L L 0.r 0 LOP .O rP 00 Po FLI OI 7050 0500VI.V 0TT7070 000SCA CL3V1AWM CCV0*1050 500*57C Cl U304AC CAOMMV5* Ca05AMM *000005047. AMU II.QT00W CVOZ0C 00S7015001IACT 00*5*570 SC CT .LA T8,L LO 1 0000AD Pl 2 SL. OLG0A QI P 9 L 7121 C5C0ho17 I5A~1vu05 CAACAS50*5 WCCCOWI C1,CAGWW OALC53050I CA15TCC GOC557O 05TOVCOCOA 1VI01SC CTIlg 9II! 70000CO 000075050 CTOGACCCA5 OIC70TAWO CC56110* .t P A 0 a P L. F 0 a 2 L. 0 V L L L v P x I C I A L. L L.

11 CC7MA050C QLYM5CYC ACADGGA C0OCeV0CC MITM0A CPPTAATSA7 II7IIOCC 03010 C.OAUMOO.6CAWA5000 707505157? 005676010 AAGACA1517 WAOTAAVA ASAAACOOA ACM055*5 .0 P P 0 9 T a V I 0 0 A I A V a a a 0 I It I 0 5 0 7501 ALV70LVTAA WACA01 A700I1V15 0500AI AIW= 13MAAIGAAAAA &.AAS0000A OOM0AMA 1200 AAMAAAAAAA AAA

.EM"

132 COAATTCCCGGRGMtCfl~r=AtG"tCICCCAGBGCTGGCACatGGSCZTM VCAOCSrCAttGGRACitCtCCatG**ASGCICM loo GC1CL TAO SPA BG~ OS L OB a *aL PIa x6LAR ?ACC AC TCG MCC C AGUWCBCC fGGAGGIC*TA TCCCTCCBI M tsoraocte GRAPCCCA tGKC 4rEACrMMC bucrrCrr Zoo L 050 C C f11 E ft1BA "T 1 SOG LPCE S OCI0 PIP EL0 C TGCC itCCCBOTC TC ?.;G,'GCMBOCGCCC~aCCPACAtKG TrGOCCCBC TGCGCCBCCACCCCASGFFC*CCCZC ccmOeSNeeC zoo DBAtP n SLOS 508G t r t V S P TA Bi Al 0 11t IA I P CRICeeAeCAGrGcr: flC;zG OCGCC5( TCCCAMATOOOOAGOCCCTO CCcC i reoACteCC rGJT~ClfZt3GSTCrCC ASO T H 0Rf C t S G S0 RGS3T L StBA C0CP GA I IS GSPf COBOXCCCpCGGBCGBS?%3tjCOC TCA;iGGC IOTCCCTrOCCTCCMkTO(CTCCtCCacCIBCTCC TCRC7 AGC3AGX-C;CIt(ACC 100 4 OP TO 0 vAO S VtL S L A SS ASS 3 TI S aC C aft001(C0 rCCC GSMC "CG TAG "MC'P~ t, T*CCSGAAAQTGCCACC C C TCACT ree irGCCAAtGC tIRK tcO:;W -GC iTTTOBCR 600 At LA Ac ft Vt Ri itLt S 0t S A SL V aaiF CO0 SLtYA0R I IaL AW0A LBC1 10LOLA ?O t .0.0mY flm A aaBRAAcRACTCCiGz:;zn.:RC GACsoCeCimartCCW MfCCBCACCCABBGC4tWATCSGt;CArcXMR ltCTC74 @CO (cI o S C 32 1 06 A L MA4 5CT? P 0C ELA I I ASS440 010"ly OW. A ATAGC TCCACC CCOr Z C :eo::BrCCCA TCGGCCBCCAGCMOUl M tr~alCCCa ~mAMABBa xn~G agflwGrTA ICA Moo CC reoGAG rTCr 94c BC nto FMRAAOOZC CCRROfC AGC t 1CC iCATAc TCOOBATA T*CBOGOI TSC7T.ACACCCOBITCi7TEA 1000 tA0S SF 4 c( Sli O KaP K SA4 5 5v S 0I C CIATPOS CCC CCCCCCCAC TEA t~ 202ACPBGC tbC tTUPCCC TCB 7CMAr tCCPACflritCirTCTGG;CAC70AirGVCGM 1)00 OAGGBMTCB*GC flCSP;CS ,:C?2CCCCAAC1C ICCCBCCSBCTOOBTCAcCfrCCACGerATrRBA.tCb~rte&Mi 1300 K ER t 1.f3 1St A LAS A tOOL LA I t A 1 OL C V SACC T C TVA aGCAOBZf AaCtOCCeCGCCCCTtCA TC1 CCaciaC TCeAO PGCCC70Mr. C?1C CeTTflAr fCCC BOB0 3t L o L R a .LV POPaF S SOGS I PCO0 VG SA :azcCcCcCriAG::: I:.fecC~cC TTGCAGCaCAGCCGCSCCCTOCACCSC:C )OO0CCASM ISO AB tO S L GI O S PSLAS T 3t S P 0M)0 G S COGPS9 E t Vt tI L 01 .i P 1 1 G t u 0 0Cfr FL C S 9 AC ?GG*SA1CT ?GSC T: G ff* fO tfCC-.UC-- GTTCMCCC r:2'r !tC aa:cAcccac::r 7: 'z UZAC tBAGC2l I jS 1 v0. 1V 1v S K ns 30 a GL S tO) 1 E a OtI C aP a I ,ol

S

S

9 134 ruwsday. f0 Nov.eY*b. 107 13:57 'Q 'cC 'b A. 6 fig 54 P.MI (i 02& 34*ad SuWmece Enavmm 7 2 of 046 ouzy,. IF4ww) .dblQs I 9 cllul 6aftin S~M Stagndad Cc" G r GC AC I ITCGGGGAAA G rCGoCCCCrA r r rr~rc. r rATTTFC Ar CAAAr£16 T c cGC rC NAG&CAA r'AACCCrGA rAAA r c~i:CG rAAA3GCCCC FT ACACGC6CC tV0.4 rAAAj..&TAAA0£r rAr rtA.;i.taAAAT AGCGAG A CTr A rw;ArAr G 'GF 36' k VA VP I 01C L rFrF I M S N M P 13340 0. 3j CIAA Gr TA rTA TAAC T rTYTCC TC rCA rACTCAruAff T TAA.;GCAC AIC000AAT. rAc UAAAACGCCC rA AA0rAAGGAcAAcGA6 0 t.1K tS63 1 0 4F A V AL 1 F fA A FC t Pv fA H Pc rL VlCVK 04E O G( 00 A£ SVG l I D NS4 ILES1 t?*'3CCCCG*A0AACGtTTccAAl0.ATGA0CjC r'r FAA..;rtCG TrGGGTrACCGATAICrCAACTCGGT P 3p P E RF Pi M 5 ofS F K VI ILC G A V LSO 1 0 AG0 E *3L G GGrTGAG1.ACTC.AC'3TCACAGAAA.rCA IC TACGOAtC0CArGACAGrA6AAA1TA4GCA6TGCTGC A I ofV S Q M0L VE YS PV T E H f 0 4M rV 4 LCS3A a ArACTAT~fAA CGCA~TCTTAACQ~CGA30ACCGAAGiCACC"TTTTtTCArhACATGGGGQrAT67AA rF TOOfAC TCACtATFGVGACGCCGGTTGAATOA&CACTGTIGC VAGCCTCCrG0CTTCCTC1ATrI00CGAAA ACGV0?GTACCCCCTAGTACITT I r mS 0N TA &4L L Lr TII G 6P K E T 4F L X G00 C-CCCTTOArCGr004.AACC30A'C TGAATGAAGCCA IC.ACGAC0.AGC6TGAC.ACC4CGATGCCT70 AOCAJ rBGAACAAC3.TTGC6CLAACT ',A0iCGGACrACAACCCTTGGCCTCGACFTACr 'CGor£;c'r FrrrorCCCTT.rCT£CGAC0 O ArCCayi.~CWr6yocICrr AT. ACGFCGAACTACrTACrCfACrCC:3CA.U- ZATrTGG.G.TAGTGAGCACTICCCGOcr T4 T; rrT AGAGAACAA=3TrA. FCTAr~CCFCTCC;CCzAfrCAACGTrCcG13GAc0C0.aCC0GGAGGCw t.I S GE L r. IL A 2 0 JL1 0 V .4 A VE V A GOP 113 S l 4 V :1~AA ~£~cotrk .CAAGFAAC.'CTGACCCCGGTCfAcCCACGGA

:CCATAGCA'C

I AJ 8 0K S5G AG _IGS4 GC A301 VAL GP 0 ;K SO :kIv 0C~cG0'3G~cG~c..~V A0AACAA0V..:.AA~zcTGGA:.~FCCrCACIG16r5A-A TTG Ta.AZ.:ICAGACCA r r r r. S a A N t 3 S 0 t C 3 F 0 1 A E 1 '3 4 S L I K 14 v L 0 0 r*C.4Ar6 AAC4AT04V.VAACIA~r CFVAr.AA TAAGACAG!A'cICCG.-A~CAGCAA~ S I I L 1 0 L 4 L F K V 1 I L F 0 11 L M I I P E F S F N A 50 P 1 E 4 1 c GS S 0 0 F L13 V I C C 1 0 L K 4 V C P F L F P K V T 0 F 3 3 A I P .i A L I. AL. 1 >1 AA 135 ZZ6~LM la 825 t 8*Uowlbnc rGC~GrGrGCGA FAAGTCGTrCM rTACCGrA TGGCtCA&%4CGA fhG; rTACCGGA rTu"CGCArCGGTCGGrC rZAACGGGG rsr-c Ac&~C CrA rCACCOC TA rCAGC~cAG"M c A. C TA4 T f GC I A rCAA rGCC TATITCC GCGTCGCC&GCCrOACTTGCCCC AGCACSTGVG3TCG A S G 0 K C L r 6L D 3 R L PO0K A 134 G TG G C TO0 CC AGC rGO A GCG ACGACC TAC ACC GAAC T.7AGAI AC C I AC AGC TGArCTA TOAGAAAGCrGCCACGCT1 CC GAGGG &GAUAG6C GAC 4G rA TCC ,C ~;aC TC TGC GGArG TGGC ITG AC TC AT 4C C Ac CGA TAC rc r r CGC GGTrC SA &GG C ITC ccI c c T~G rTc c A r E R TYTI T L A I L QE L .ES3A IL PE G QK AO V Ip GG TA AGCGGC AGGG TC V.ACAGGAGA C GC AC G ArGA rTCC AGGGr4AAC G'a TA TC TAAG TC C VG TCGGG I TCGCCA CC1'C TGACTT CA V rcVCGCTCCC&GCC rTGTCC TC TCGC G7GC TC CC; CA-AGG TCCCCC IT TG CGGACCA rAGAA.TA TCAGGACAGC CCAAAGCGGGAC TVA I7Wo 'j 3 G q vG rG E41 T L P G4ItA VY L 1 SPYP V F9M L L G~~CATtGGTCCGCGG: GAC kGGAACCACAGGC TrrACGGTtccrGGCCTTTTCT~TTTG a CT,,C&GCA&AACACTACGAGCAGVCCcCC:CGCCTC'GTAC: TTGCT:TGGCGAArGCAGC~,AACACGAA E 0F .Cs5 S G GR S L VK4AAS MA A FL AIL A rC VP F CT'CACArGTTCTTTCCTGCTATCCCCTGATCTGGATACCGATAcCGCcTTTGAGGAGCTATACCGCTCGCCGCACCAACGACCGAGCG 1900 G4rGTAA4G .ACG.ChAVAGG4A CCTTGC~ArGGAACCCCAC VGGGG=ZGCT GGC ICGC A Mr F P A L P 0 V0NA I ITA fEf. A 01 A RR S A Tr R CAGeGAOTCAGTGAGCG.AGGAAGCGGAAAGCCCCAATC C AAkCCGCC IC rcCCCCGCGCG tsG6AT CAT TAAGCArC TGGCACGAC AGG ITT 2000 GTCGcrcAGTCACTCGCTCC TrCGCCTTcrCGcG6TTAT CGV V G2CGG-A AGGGCGCGCAACCGGTAAGTAATTACGTCGACC6VGCVG TCCAAA 3 CS V3 E EAE ER P I Q K P PL P A IVP 1 1. C S V H 4F CCC:ACGAAGCGGCATGACGCAACGCAArT&AV2 AGT A2CrCACTCATTAGGCACCCCAG2CTTTACACTTTATGCrTCCGCrcTGATr.T r-aTGACCTTCGCCCG VCAC VCGCGTTGCG-TT AVCAC.'CAArC0AGTGrAArccrGrGGGTCCGA.ATGTGAAAVC6AGCcGAWArACA D 0'd A G SE RN0 A 1 4 S L TM F G0&L 0 FRNI P AR M A.:AZACCT VAAC( CrCCArTGTTAAAG V;?GT:CT7T;7-2A ZTGGTACTAATGCGGTTCGCGcGrT&TTGGGAGT6ATTTCCCTOJTrICrA L CG I V 36G. 0 R F T G x5 10MG V1A IQA I NPMH R E 0KL ;',,-CCSGGC.CCCCTCAGTCGICC!TAtCGA:A:: *2.A ITCGA&VTCCTGCJGCC:CV6CtcTTcArccAGATGCGGACCCAGATCCCAG CC 'r rZ 4=CACGC TAAT:;..V 4-CtTAACACCTCGCGACGAGAGTCraVCrCACCTGGGTCTCAGVC 1 -WPFLMi- V GO L 0P EV 0 1 0 0 E rV 0 O L LF 3 2NI M V L SO !3 rCr, rC CC rTCACGtCVT TAGGACC TA GCC5 :T jC: TC TC GGTACAGGCG.SCGCTCC AGGTCCAC TGAGIGTCGAIGGACC IC; 2'C)~ ORF oLM1 0 Mr V LVI LA a 1 4 EIm 5 S LI R6GS 0 1 Tm SO5L E rUACC 'C TA ACA2CGArA IGCCAAC:CACSCJ,ClT::A:::TCTVCCACccCGT~CcrcCT.:tCATCCTA7CCA~fCCAGT,CGCG 'I D S D 0 A P P .1 S i j S N R 5 3 P L 3 2 1 T I I S 3 P 6 T.asda. 18 No-wnee 197 13:57 Pp ~C TCrAcc~ GGCGGGSCCCCGCA C C =CCTCCC CGCGACT ~GrCGTrCCG rG ATGAGGT6CCATA L04A6 0 A 0'3 V4 S 05 EGT Wor ~I vM 4GE HI 3 M ~S T ArCCCAT4CGCAGCCCCArCAA4C fCAGCCATArcTc :TlIAC 1CSTCGA1'CCC tGG~CCCArGAGC GGACCTCAA6TcCGGCTAtr.

r4CGS6T&CGCGrCGGGG1C~tTCGAG rCGGrh AAGGzAC CTCGACCAGC VTA(GGACCTGAGCCTACTCCAccrrGAG ?TCAGGCCGATyAC ho~wt p~Ll ORF pMI P 3P 3 9 S LS3H 1 S3t E L V S LOS0 EV 0 LK 3 T M Ot2.t.CArGACC TCAT .GSCAArACCATGACGGAGGAr i;A.CA 'CACACCGCTGGrr6G&AAGCAGC rCCArCAGrAGr6GACTCAGCGATGCCTC wmawl poLl OAF puLl ."nS PLP I ORF St~l i S 4 3. E5 C F 4A 555 3 St Pf3L 5 T lA 4A N 3TIi V L I .CiiJA sIATGAGA4-G.C.:. TTAG1ZAArCAGAGGAGAA&GCCCCTAAAAACTGGAGTACGACAGTGGTAGCC oDAF "I.1 E KIt3 lA( f 6 1 5 S EE A P l KKL T 0SS G r2;&rcA..-A~CrGG44TTCTAA6 rGCcc.AZ' TGAt6ArTCA,'CCa*6ct TGGAGAAC 164444. GCCCA!CAr.CCYSGG OAF p~Ll L. 4 -P G T Si 9~ w R'CS CO0OS S t 6 G LKI P 1S3IC :z-::tr,,clAGA .CACCCAz, :::.:rr:cccAtCACTCACc.cAGCCCAroGCC tCAAAC CAGCCAAACC? -nAF DL~i LO St K XG~r K S P I r T A403 A I. v A G6K P 14 iC:LAocT: AC A. ACA%7,'G ACCTCC TC: TCrGA rGCT" rca&&cC2CrC 7GAOGMA tC rAAG4 :AF cL2.tI 1 3 )v A S SO~ D LS '4 A Tws"Y. IS No-ow 1997 13:51 Pao. IT TheidMI IS S w s 99 l.5 pt.

A.Z:ZCCCC rCGrGCAT rGCrCCCCCC TCCACTTrCGCcA7.:: :ACAAGcC~tCCTCCGcCACAGGCACAGCCAcrrCArCA4CTGfGG a3400 rCSGGGGGACCCGTAACGAGCGGGGacOTGAAGCCC ,A4 AAU~CCSAT6IC7TcCa4AGGGGrcccra TcGG GACAGrACGTSACACC OPF "MI s r s FR PS vSS 7 K0P P A Tr 4 A T V m r TGG rrTc.IGCCACrCCGAGrC rCCGC.CCZ.AGCGAArGCC~.C4CTCrfrCAACCGGC A4a;rCcGTGAGAGrcTCTAGGTCrT~CAOoTCC;Ta ZU& ~trcGGICATP1ACCCoCaCrCTATCoAArCTACAA 4TcrcOCT ORF "I SA r. L SK 1o 0 K S G1lO j JR V N G Q T LO 0 5 5 C P f3AEI C: IAAGGACCGAWGACC1C=GGAAGAT TG TAGOTCA= ;C;,ACGGGGCC6GTCGGrTC ACT CAAGATAC TCGC&C TGGCCCCGCC =CT maidpLMI G FLA P 0A R 34 1 01 4 S LP 9P A 9SS5SIMO5V TG04 ItG GAGCGGGA. AC ICG YCG rCG TAAcTGCGGTCAGGAZ-:: =T t.OTCCCTCCrGAAIGCGGAACGTC TGAC TTCC1C4GGTCCATCGOTCACC -man Oli ORF Ami P0 v551S1 0 P 3LL S I (@GGL 41.TPS AL K EP TItV A 3G rCCAGCCC TG rCAA rCAGc AGA rCOI"4;A-:AAGGCCAAA4CA0GCAGTGGCC rTGGAC CAGACAACA TC fCCt TGAAGAGr 3800 rGT4Wr~GACGIGI'T fTC=: :TCCGTTTCGGTTCCI TCACCGGAACCTCGTC TGTTGTAGAGG4ACT1CTCA Kr? v N1 or 05 a E< KA K A K A V AL 0S X 135 KS C7CCCA-.AGAC TAC rCCCAAG AZAGCA.:.:::.%~rCACCAA~cTGcCzA"rCGCC~cACAcCCCITrCAGGCCACCGAAGA 3900 3 C S ITP K '1 0 A S ~IA I X L ACE L PPT P 1. RAT1 4 K :CA.cccccr,,Ac rAccAA-.:ttz; AM -::ACAG:CGGArCTAC.ACATCCAGTAfCCACCCATGCC rAGCCCc ZS4 1 F guLl 3 L 1 A 'I I 0 -1 L I 5 I 0 I. P D f I H 1 A 3 K V P rA :::':TCAC:CCATCcACAtCTCAATATTAACrCA~ccA:::rcfTCC U c00 .4144 o0J.I r A S I? 3 A O I L -3 1 4 A Twmsday. IA NOmwvaw 197 13:57 a sdaaruda.,c.

CCGGCCGGAGCTAAT6GYGGTITCAGTGCCAA.;4:::-.ATGACCCCAACTC rCAGGCCrGCAC-rA'CAr44.IG;:.CTCc,.ArGC G rCC GUACC ICU ATAC ICACCAAAGTCACACGG? r r rc rcr GCG ACATGGGGtI I AGAGAGCCGGACaTGTCt rCGT ACCTC AGGAG IC rAca mus l"I CA~pL j L E LIPSOGP' S JP P IC? (LSG0L H tS n(ES L aN CA tGACC GCi6 tc~GCC rrCCCArAGrACTCCC~rCC:AC.CCACCGC lCCCCCTGCTGCTCCCACA6LAGAAGAGACGG'A-IGCT..r"o 43W GrYAc TCGGAGGGCACGGAAGGGrGCGCAt6AGGGCC~GGGTGACGAGGrGGAcA~cGAGGGP6TcricT'ccrGCCrTCCGACTGAC wmot 0LU1 OF pLd i A M 5 L P S A F P s s r I PT P A P P 4A P I tE EC EC CL V IAGTGGAAGCCCCAGAGC TGGGCAAC TGrACAO TAA7'CAC-:A TCGGAACACTC T CCCAAGAAAGWZCICAGGTACCAGCT1CAGTCCCkVGGGAG CrC-ACCTTCGGGICTCGCCCGTrOACCTGTCA:A.CT.:CCCTGCCT6TGAGAGGGTrcTccGATCCAtGGCAOTCAGG:CCCCTC ORF pLIA I S G SPA 0 A 0 SL o1 sN m 'oasorL P C KGL Rv t 1 sL o 0 EC AccAAGGAGAGrcCAcArTCCCATACCATGGTGGGCTGcc T6AAccGrGACAGAGCTGCCTCTCCCCCTGCACTCCArCtTCrSA6TG TG ;TTCCTCtCCGC1GTAACGGrAtGGTALACCACCCACZACT7ArACTGGTCAGTCrCGACGAAGGGGGAC fGAArOGGIIcArAGACTCAC OAF oLN I CAAA&r6GCCAIC I ACCAACArAGTrGrcccAc r3.GCC A.:CcCAAAAGACACCCGCTCCACAGCA rCCCACACCCAGAGGCGCC TCAOCT 77*'TCCCG.; I GAA TGGT IrMrA rCACTCAG7G. :TCATGGGCGAGTr?cOtA4GGTGGTGC CCCCGAAGc rcG Wtsen PLI OAF "Il LG I m I V S P A AT I I A II A S 16 3 PT 1 E A AF CL V&ACIC MCCaArTOCGAGC&CCC tGt.cCC::A t:.:CAGAATGATTCGOTCAGGATCCTTcCIAGACCCChc.J,.0ATGrTCAC SO G S SG t L S 4 15 RC 30 IA G F A00P7 O V m T:A.ST rZTccc T,;GCC tCCAS I:C: -A:7::::.CCTGAGAAArGATcIGAZAAATCCSAGCT-:CaTA: GAkcGG ;A'lz: ltrACCC CTCTACTI 4ACTGT~AUCTTCAAGAT CTT ACO ,AF .U, L 5 A 5S a S 5 5 A sE EC 4' 0 15C 1 4 L ECL 7 4CCC 0tT C C~cP -A.C A (CG;GCTcT.T.GA G CA%--4~C~ r CAA ;4t 1:r~rG .ion PLM I 'I A I 1 AP F EI C Tuesday. IS NOW.Ns 00 W 13:5? CC fOGCACC i6GCAG*GC3GCCCGAGGAGGWAC 7 CC IGGA f I TGCGAG AACCA fAGAC r r iC tGAAGAAAAAGAAC 1C rGA4GCCAG GG cCCTG fGGACCGCrCTCCGGC fCC TTCCTG rGAV 7:ACGACc TkAACGC TC FT YOG f C rGG~c rTC rt tC TGAGX CCGGGC ORF "LIt LAmP"L aC r AE EKD 0 T L LDL 2C r7 I .L X K K" PS A 0 GcAr rca! rC&666aGCCCT FA rCCTc' ACCACA:: :zoGAAcTCGGA!CAAGAGACAAAACTCC TCAGATAOCATC rCAccrC~CTCA ORFpLmI A V1 GA L NAS 5 E r E LAR I K A 0 145Q St03 S 51 L P 5200 AGrGACGGTrAA0tc~rrccGTCGTCGrC.,TACGAC?;::, rTytc rrcrrt rrCTCAACCCAGArACTCGAAGCrr1'AAGA1TTGtTTC6 AG" ;Lml OA~pLMi I SPISS 1 S3310 ADAK 111(1 SVVYELRS 59.1* 6! TCAGTATAAAAA1GGG0CCUCLTGCTTCCCA1'AC,;ATAAA6GAATTGCTAACCCGC TC FCAGCCCCCTCATccccCAAACr*AA mw0 CAG rCAAT rrrrccccGGG I CAGTCGAAGGAG t";A ZTArAC rCC rC TAACGATGtGGGCTGAGAAGrCG6GGGGAGVA"GGGTTTrc was"l "I F StIC GA PI S ASSS 1 D I 0 1 IA T P 05 S AP S S P It L 0 *:-47GrCCAAAGC rrCACCc'TC:I7CAAtCCT::.:: 76CCICC6G6GGCACGATGTCACC0A-G 0CCTGCTCACCCAGCCCCCCAC 5400 17CAGsr GUarCA.,-:C~AGGAGCCCCGGACTACA6T0CTCCCGGGACGAGTGGICGGGGGTGt 011m1 PLM I 1 rC iAS3 S S V S~ G T 0 I GP A 14 AP H C IGr f rC 1 ;!ACGG GCGC rCTGAGC I ktGAGAAGG~AAA16AAGC11~-ACAT ;A a~c rnc. rrrTACecC rtcrc: 71. -:C.ArAGCC CCACGCGAZ:JC TCGA rAccc rc rrcC f rAC rC.AArrCC GTA ORF KLm I f EL VS (LA RSC L *CEK tM K Lr 0 1 r;A~CC f_ &AIC ,cCCAAL 7 :7 ZACA tGcCAACArGCAC r [IA" rGGACC tGC TOAAGCACGAATGAC 00 si- fl::I pLGAC ircrACCCACGAGLCTCTT~A tl .Il L. E A 3 A r r .4 4 I Pl_ t M (LEJO LL A A! E O LCCCTA .:7:::-::Z;CC.a7ACC~ft:T:CAGCCCCAl'T L nsen 5700 140 11, 1.8 177= 599 &93.37 rcaCCCAr rC: TGCEArcrCGCC- CT .:I:.:Ai6aArGGCcAG rictrrr ccAAAG,;;rA~. ~C..CCGGtrr ',.tGG~tAAGGAAGCCCCGG rCAGcTCTCYrr. 4 %GGGA(C IrACCGTA4 TCA GAAc~t !Grrcc TccrTc&c rG~ASCCCACCA s OflFpMi L rn S9 r G L A 0101t SI,'M 0 Gi I e S C;P K~ S IvT 1. 4 v CC.9C TCCTACSGGGGGrccGC1 AG rT CCCC TGW'.sc.c TTAAGACWt~TC~C r r rcAACTGACT-TCTAC R M P P 0N1 M I I D 1 L 9 0Sf F F L G C S t V S G 9 V 0 K C rrAGAAGCTGTCCAAGrtcAAa~ rArA TTT:7AA.&AT%5ACCCAC rACCCTSC*GAC TAAGCACTACCATCCACGCTACAG"A 6W GAC TCTTCGACAICGT rC~ArrcTTCCTGrATA r~TACCTGGCGGAS&TGGGACCTGArCGrGACTCCG TAGS ?ACCO&ATrTCGT ORF ptLUI L. 0f A V F VF r K r H P A E TL G L T! S 3 A 1 IST TCCCCrAAC rTG~~AGccccAA.CCtCC rr5CC6 TCGAG6TCAA r"At.ATACAG cTTCCC TCA.AGSCTGAAGGA AGTrcraGTCTrrrTCcCcAcGrc cG=C:-.-A;WGCG CAIT~r-TrCAGJGGSAGTTTccAiccrtCCT mM pINSI -OAF pLUS 7 T.CCCTrACCfTC:- :Z-&C7CT4"rC TG~tSTArG13 l!ZGACTTC:T:ZCCCAGCASGS.AGC AiwI PLAI C,hF PLUSI LC V 0 S L VF 1 .4 M AYE S 1 L L Q LV VL L S GCGCACGGOC.A2~. CA.A~ c' C EACSGrGAcCGC TCGCc: !'AG M- 1~cc:; csrc:XACGACAC 0O;r pUSS 3' G 1 4~ F Y. t -f t I LV C z 5 G I tvIS I1 5 T 7CA. TA 'eA 4T r FC tC: T ITTCC T~ A::CC CA:;!cfACCA 0.*TF cPVUS 1LA 3 14 r~ 1 1 V L

I

L I 141 TUesSQy BG IS 7 1357? Pao. CAGCCTGfAAAAATGACACCCAC:Ar0C t S.C CrArTGC T CACC G.=ACArCTC rtr r&OrcG6.AcAtJ rrTACrGT6GrGGGACCGAAC3,-GAC TCG.A6TCCtACAACTGGA66t r6 rr~cAcctc rcGGtrTACCGAAG4ACC I V K N r P M Of G L A S F A M L 1' F 3 9 M v E 0 A hi G F L r r:G TACcr ,AGA1GAAGCrrA~CGCA AA.cr~cxA AG~tGcrrCGGGTGCTCGAC fG6TACCC~AGC7t~rTA AA~iC A tGGACrCC rccTT CGACCArCTCAcTe1'6TCGcTrTA-fl rAcGGrGTTCC TTCTCGACGMCCCCACGAGC r6ACCCATG6TTCGACAA r7 -ORF pM I V S LA Q A L V C SO 0 I 3 A1M A C C EL L Q V LO VY P KAL W V Aeco *l t~tAGC.6G TGGAAOOAAc rc TCGrT1CGTO GTCrM0A0GTAGCCGGGAAACAAA0ACACAAGGTACCGTAAC rCC r&AoocCroo OAiF PLuli AL r F L II K i3TSOF L I GP P FF L3C P I16 I C D F A r',;TC~A~r GGACCCATATCTCACG--G

CAGAIITAGTCTGC"ACGTGGGM

~69W OFWm LMI C1 0 L V P 4 P 1 1 0 0- 2 5 A L 0 A I P p p G 0 P A A V I C4.,ZAGGCZZCCCrCrGCTAZ A.AUCACAGTrCACGCCACATGGCC I CACYGCAT r! o4o.-rT r CCAG CCCrofGAGrrcCO :TG AGT fCGAA rhCC rAC G cOTGG TrOM6CGGTrrC r IIAUI PLM I OAF PoJJI A SP PCO A T V X 0 S T 3 3 LO0S O PL MA 4 i L CAL 0C E A OAF pLIA E s S 0 A E t L 1 A T L G F NHC E L I. L L S P AP i E I C Tuesday. 18BNOVwftw~ 197 13:57 fig S1 "LM Ii P AWas) 54. mwd SagmePg 4A GACArGftCTTGaGCtGTaccTrrsGAA r-,cc r--44G-AAGGrGGGG fGGcGTrG.QAACTTGrGcccccrAAACACArTTAr55c74 TC: ?CCCT4tCCAA4AACCACOACATGGAAACTCrtTGAAGGTccrrcc1rAcCACCCcACCCAAC&ACCTTGACG44GfiATT1GTGTAAUrACCG 4:f G S V C C TV F E f 'L G 0 9G G V A r61N1t C P LIM TV FTI C.,:CrctAArGAcrT rGGGAA&GArGArrcTGG.T:'TTZCcTtGAcrTr6TTCAArTACAACCCCGGCTTTGGGGAGGGCSTCAGAAAA G A =AGA T TAC TGAAACCCCrr TTC rAC TAAGACCCA A AAC GAAGAAC AAA rTAA T T GA*CCAAAACCCCC CC CAATC FITT L L VG O S K GS5 V 5FP .L L V 5 T hS3V A FVVGQ6V 0 CA TC AACAC TGCACArt CC C GAA fTCA GC T r; ;AC rTAA CCAG4C M A TCCAAGACC TT ACCCTQGCGrA T K C S S S 0 E r S L 0 L T4L t. LI L CR3 IF A y VRD L L VCAAGATC TCGCCGGCGGIGGCGCCACCTCQGSrT TAA.GrGGATATCAC CTrIITrGGACCrCAATG=GGItI. tAGCrGGACS TC S TarS"6'I CGGTChCCATcGC ttCTCCGGGCSTGGCTAGC6GGAAGCGTG- E XP GV T 0 N AL A A "P P VFASV 0NS3E 9A 0TDK RPSO TrCCAGCTGAAGGCGAATGGaACGCGCCC IG.iZCG CAtAAGCGCGCGGGGTGTGGTACCGCArCGTGACCCTACACTCAr.

LA QS3L N G E V OA C I G AL S A AG V 6 TDS R3v TM ATL AS3 C;CCTAGCGCCCGCTCCTT,^Cr-CTtC:TT:::: 'CrC~~TCC~CACrTLTG"CCC~AG A A 0 A P VAV FfP S 'L A tF AG V 5 0 QA L N 56. LPL G F CSACGGCT-l-CCAAA TATSGtSTCGTAC4GTAGCCATCCCCTAAGACCSGTTTCGCCCIIIGA a ico .1 V S ALK It4L 0 P K 0. G 3 R SO S G P 3V P S ?74ZAG TCCAiCG FTCr1 r TTAFAGf .5C IC? I5 G7::z AZ A.AACAc rcAAccc rATCTCGZTCFAT C rTTrGATTIATAAGQStTITGCC Sri..CC ICAGGTGCAAOGAAtrtCACCTAGAACAA;*, r5AZ5CTTGTTGTGAGTTGGGAIAA4CCAGAAAGAA.51AAAATCCCTAALCGGa r L E 5 tV F r 3 .1 5! L P F -S r TL M P IS~ S F O f L GI L P z, sAT Acc a, T7T.A: 4 AAA'-- 7 7 ;AA frcc rrAT TG T TATArrCiATT TAA7C :Sa A I V C hEL F ftA 1IF MX I L T L TI 143 Tuesday, 18 Novemntif 199711 .:4a rig 34 pLM4 (I 10070) Site and Sequence ag Enzymnes: 100 ad 146 enZymes (FiJtffsdl '1 Senings: Liear. Cafi~in sims Only Stan~dard Genetic Code 4 FTArT AAYAGTAArCAATTACGGG6 TCATTAG TCA TAGCCCA TA rA TGGAGTTCCGCG rTACA TAAC TTACGGTArGCCCGrCc~ r:.cTA--- L LI( VI1N4Y G V I S S PlIy Gy VRy I T Y G K V P A "L T CCCAACGICCCCCGCCCAT TGACGTCAAFAAGAC GTATGT TCCCATAGTAACGCCAATAGGGAcrTATGACGTCAATGGG TGGAGATTTACG.7 GGG TTGC TGGGGrGCGGGTAAC TGCAG T TATTACT6CA TACAAGGGTATCATrGCGGTTATCCCTGAAAGGTAT6CAGTTACCCACCTCATATGCC.; A0 A P P P 1 0 V N N 0 V C S H 5 N A N R 0 F P L T S N 6 G V F T v rT GACGGG ;GAACC G TCA GTAG T C AA TA7 ACG GT TCA TG CG GG GGATAAC TGC AG TTACTGCCA T7 ACCGG GC C G TA ATACGGG TCA n C P L G S T S S V S Y A K Y A P I R 0 N A R L A L C 9 v C ATGACC TT ATG GG AC TTT CC TAC TTGGCAGTAC ATC TACG TAT TAG TCA TCGC TATTACCA TGG TGATGCG6 TT TT6GCAG TACA TCAA TGGGCG TGG G r AC TGG AA TAC CC TG AAA GG ATGAAC C G CATGTA3A T GC A TAA TC AG TAGC6A TAA TGGTACCAC TACG C C C CG TC A TGTAG T AC CC GCCCT 0 L M G L S Y L A V H L R I S M A Y V M 6 0 A V L A V H 0 lJ A w 745CGGT FIGAC TCACGGGGATTTCCU35TCTCC AC_-C AT IGACO TCAATGGGAGTTTGTT TTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGT A TCGCCAAAC TGAG TGCCCCeTA.AGG T TC GAG rGZ 3G TAAC TGCAG TT ACCC TCAZAAAAACCG TGGT TT TAG TT CC TGAAAGGT TT ACAGC AT rA V L TIG I S K S' 4 OVE F VILA P K S T G L S K M S ACAAC TCCGCCCCCATTG ACGC AAA tGGGCGG 7 GrCST 3 TACGG TGGOAG6TC TA TATAAGCAGAGC TGG6TTAG TGAAC6ICAGTCCGC TAGCGCT.; ~7 AG GC GGG GT AAC GC TT T AC C ZCCA T :C C AAT GC C ACCC TC CAG A TAA T TCGTC TCGAC CAAA TCAC TTG GC AG T CTAGGC C aTCG A ,I L R P I 0 A N G 6 A C T V G G L Y K 0 S V F S E P S 0 0L A L CCZ6 TACGGC~ACCA CTACG CGC GCCC .;:cACCGGCCCCCCGrGCATC GCTCGACC TGCCGCTCAGTGCCA~tC.ATCr.

VA T .4 S K G E E L G 6 V P I L V E L 0 G 0 V N 6 H K F S ,GGAGG CGA~GCGA.G 6CcACZT-'Ic-L :c rGAccc -GAAGTTCATCTGCACCACCGGCAACTCCCG6TGCCC TG6CCCACCCT:6GTGA: S G E G E G 0 A T Y L T L K F (C T T G K L P V P v p r L 'J T rG Ac tAC' CGTGA G T G: r Ac c c: :G c A r.A :C -1 CAC GAC T TC TTC A6GTCC ZC C A GC CC GA TA: T GG :AC r6GA7GCCGCAC:CACGAC T;l-%T:TG~ -GAAGTA;CGTCGCTr:C:A; C c:::x C C o m m i. O H F A 14 0 E .F t. S o. E~~ TueSday. I aNovefrtlw 1997 11:40 ps 't fig 34 pLM4 ri 10070) Silo Mwd S~uo CGCACCA Trc rTCAAGGACGACGGC&AC TACAAGAZCCGCGCCGAGG TGGT TC6AGC CCC rGGA -'ccGcA CAGC TAAG..C.TCi GCS rGGr.GAAGAM6TTcc rGcrCGccGA-GrrC TGCGCGGC TCCAC TTCAAGCrCCCGC TGT.3GACCAcTTGGC~rAGC TCGAcrTccGT~r A T I F F K 0 0 G N Y K T R A E V K F E G 0 r L V hl A I E L. K Ca I AC T itAAGGAGGACGGCAACATCC TGGGGCACAAGc r~zA TACAAC TACA.1CAGCCAC1ACG TCTATATCATGO.:CGACAAGCAGAAAACGGCArCA., TGAAG TTCC TCC TGCCG TTGTAGGACCC CGTGTTC GA~C TCA TGTrGA TG riG TCGG G orroCAGArA TAG ACCoCGTG TTCGTcCrrC ;GTAGT-.

0 F K E 0 GN I L G H K L E Y N Y N S M M V Y IM A 0 K Q K H G I TGAAC TTCAAGATCCGCCACAACA TZGAGGACGGCAGCGTGC AGC rCGcC CACCAC TACCAGCAGAACACCCC. CCGCGACGGCCCCGTGCTGC .1 cC A TTAAG TTCTAOGGG Gr rTAGCTCCT3cCCG c GACG TCGAGCGC TGGGrGATCTC TTGTGGTAGCCGC TCCGGGC ACCCG.

V N FK I FtH I E 0OGS VOL AO0H 7 Q 0 T P I6 G0GP V LL CCC GACAACCAC TACC TCAGCCCCATCCCCCTAGCAAAGACCCCAACGGAAGCGCA TCACA T6OTCC TGC TGGAT TCG TGACCCCGCCGGA GGC TOT TGGTGATGGACTCG TGGGTCAGGCGGGAC TCG TT iT TOGO G TTGCTCTTCGCGC TA TTACCAGOCGACTCAAGC AC TGCOGGCCCT P H Y L S T 0 SA L S KG0 P RNE K AG 0 HMV L L E F V T A A G TACTC TCGGCATGGACGAGC TOTACAAGTCCGIAC TCAGATCTCGAGC TCA C TTCGAT TTCAGTCGA TAAGC TTGATATCGAA TTCC TGcAncr AG TGAGAGCTACC TCCGATG TTCAG6CC TGAGTC TAGAGC TCGAG TTCGAAGC TTAAACGTCAGCTATTCGAACTATAC TTAGGAC rCG I TL OMO fL Y K S. 5 S R A0A S NS A V 0K L 0 E F LO0P CCTGCTCtTCA6CCAGATGCGGACCCSA it:CA-.AAACAGGACAG iGAAATGTCCTGGATCTCCGGCAO..CTGGAAGAGACCATGTCCAG.

GGACGAGAAGTCGGTCTACGACC TGGGTCICAG.30TZTZ iTCTCC TO TCACGTCTTACAGAccrAGAGCCTCTOACCTTCTCTGTCAGTCO; ORF pI.Mi L L. F S 014L 0 P'ES OARK A T V 0N V L DILR 0 NIL E Tm 5 3 rC7 CGAGGGTCCCAG6GGCTCAkCAGCTCCC TaGAAACC TGCtACGCAACGATGATGCCAACCCACGCAGCGTGTCCAGc crCCA~Accc rc-; ifisen pLM I OAF PLm I v r H S S L 7- T C Y 0 S 0 G A N P R S V 5 3 ?1 I R S rc::c TCT3TCATG3CGCTA iGGCCAO TCCAG7:-333-: TrCAGGC rG3TGACGCGCCCTC T3TGGGTS5GGAGC iGCCGC TCGCAGGGC A-:CCCGCC T- ,;-AAA ACCA:CGCGTAC-C-.A CC ACC'GCGGGGA3ACACCCACCC TCGACG.rCG gCC TCCr-.13CG6GC3.± insert pM I OAF pL M I L, A GO A PS, 3 1: 9 E G Tuesday. IS November 1997 11:48 bg 34 OLPA4 10070) Site ana Sequae Pipe I -acArGCAC6GCGACGGCCCLC TAC !CCCACAC At:CCCATGCGC 3CCCCA:,CAAGc rcA-ccs TAC ticccc cc C~ 7GtACGTGCCGC TTGCCCGGG TGA TG.1GG6T3 T6G TA'C6G3 TACCG TCGGGG TCG- rcG AG TC GG TA TAGAZ3;GCGGACC TCGAC--A:;C TTAG Amnr pLM I M G E A AM H V H T M P M R S P S K L SH I SARL E L V ES L G:TC GG ATGAGG rGGACc rCAAGTCCG3C TACATGAGCGAC AG3 TGACCTCATGGGCAAGACCA TGAGAGGATGATGACATCACTACCGCTGGATI.

C 75 AGCC TAC TCCACC IGG AG TTCAGGCCGArTTCrTCGCTG TCAC TGGAG TACCCG T C TGG TAc TGCC TCC TAC TAC TGTAG TGATGGCCGArCC TAZ ~Isefl pLMI OjiF pLMI S 50 EV 0DL K S G YMKS o So0 L M G' K T MT ED 0 CI1T t G VO A GCAGC TCCATCAG TAGrTGGAC TCAGCGATGCC TCAGACAATCTCAGT TCAGAAGAAT TC AATGC CAGC rCC TCkC TCAAC TCCC TCCCAAG TACTCC TTCG TCGAGG TAG TCATCACCTGAG TCGC TACGGAG TC TG T TAGAGTCAAGTCTTC TrAAG TTACGGTCGAGGAG TGAG T rGAGGGAGGGS IrCATGtGGO isert pt.MI OAF oL~i E S S 5 1 S 56G L S 0 A S 0OM L S S E EF N A SS S L N L S T P C T~ TTCGAGGACTCAAC ATAG T&C TACGCAC AGACTCAGAGAA.CGC TCAC TGGCAGAAAG TGGGC TGAGCTGG rTTAGTGAATCA2ASGAC G 7Z C GAAGAGC GTCC TTG AG TTG TTATCACGA 7GC 3 TG TC T AG Tc C TTCGCGAG TGACC GTC T TTC ACCCGAC TCGACCA AA TCAC T rAG TCTCC TC insert pLM I OAF Pull1 A 3 A A N S T I V L R T D S E K~ A S L A E S G L S V F S f 5 E E insert pLMI ORF p0.41 DK K LE Y C S G 3 L K ME e -G I SK V~ P. E FtP C D DDS insert pLM I OAF O±M1 E L K K 3 S P 5 L K <r 3 *Tuesday. ISNovember 1997 11:48 tig 34 pLM4 (I 10070) Site and Sequwtice Page L CICAGCCCAGAG rGCCC TC AAAG rCGCAGGCAAAC C TSAGGGCAAAGC TACAGACA.'GGG rAAcr rGC TGA- TAC VGC:r.-. CGC -C GT~ rCGorC tCAC666AGTTTCAGCGTCCGTTT6GA: rCCCGrrrCGATa TCTG TTCCCAF TCGAACG TcAc rrCTrArcAC:Cc-TC i4.: IflSWM UJI A 03 A L K V A G K P EG K Ar T K G K L A V K N r G L 0 R s3i TC TGATGCr6GTCGGGACCGCCTGAGTGATGCTAAGAACCCCrCGGGCATTGCTCGCCCCTCCACTTCGG6ATCC TTC6GCrACAAGA.AGCcC 1: AG Ac TAC GACC AGCCC TGGCGGAC TCAC TACGA rTC T TCGGGGGGAGC CC GTAAC GAG CGGGGAGG TGAAGCCCrAGG AAGC CGA TGTT TGAGA OAF pLM I S 0 A G R D R L SO A K K P P S G I A R P S T S G SF G Y K K P P CrGCCACAGGCACAGCCAC GACGG1TcTCCGTCGGTGACAO TACGTTTGACCACCAAGTCGTGAGAGTCGTTCTAGG TC TTCAGGA6TCCGTAGGGACA6 TTCGCATTTACCCG:irtsefl pL~L4 OAF pU~i P ArTc T A TV HOM T G G SA T L S K i0 K S 536 I P V K F V NOGT CAAGAC TAGC TTAGArO IT TCCAACAGCGCAGAGCCASGATTCCTGGCTCC TO AGCCCGTTCTAACATCCAGTACCGCAGCCTGCCCCG3C CACAAi G; IC IGA TCGAATC TACA.A-GGTTO TCGCGTCTCGGTCC TAAGGACCG GACCTCOG:AAGATTGTAGGIC ATGGCGTCGGACGGG3C:GGTCG3.7T.

inun pLMI ORF pIMI KT S L 0 V S N S A EP G F LA P GOAR S N 1 0 Y R SL F A I 7*~TtC T,!T GCGTG :CGZC;- CGGGGT! C-CCCC TG TGAGCAGCAGCA7 TGACCCCAGTCTCC rCAG.CCC AGC~C.z: TTAlCr AA TAC: TCGC AC JGGCCGCZCGCCCC:Cr T4G-A.2CGGGACAc rc6T tCG7AC OGGTCAGAOAGIC-;ZT:TCG mnseti pUAI OAF P.M I S S S m S V7 -1 6 R -1 G Q P V S S S 1 0 P S L L S T K 0 G LI C rG;.Ac±G: rGCC ZGACCA: t.CCiGCCCC T. CAACAGACAGAT:666GA-,GAGA.CAA6C.I6i G. 4C'ib- rGAc-CCI: -GICCATCGZZ,:CGGiG GGTC6GG6ACAGTTAAGrTCTTCTGACGCCTTICCTCTCCc6. TTCG'TTCC risert PLM I ORF oLM I Tuesday. 1S November 1997 11:48 Page fig 34 pLM4 01 10070) Sk*. and SequenceU AG -CGCC F TGGcACCG4AACcArT CCTTGAAAGTAT TGGCrCCCAGAGAG ATCCCAAGAACCA CA-CCACCCA -C-"c.CACAAGCTCGC,! 7ACCGCG.ACCTGAGTc rGTTGTAGAGGAACTTC TCA TAACCGAGGGGTC TCTCATGAGGGTTTrGGTTCGT rCaGrGCGTZTCG-.-7GT rCG6.C,;r Wmsef PIM I 041F pLM I A AL D S N I S L K S I G 5 PCE S TP K NO A SH P TA T K L A tTTGTCAAACCACCCTCACTAGCCAATC ?TGACAAGGTCAAC rCCsAcrrC TGGATCT*A:: C TCGACGGTGGTTGGGGAGA6 TCCCGGTG TCGCTTCTCGAAACAGTTTGGTGGGA6TGATCGGTTAGAACT6TTCCAGTTGAGGTTGTCAGACCT.O.T..

isen PLM1 OAF pIMI E L P P T PL R A T A K S F V K P P S L ANIL 0 (CV N S NS L 0 L CATCArCCAGTGArACCACCCATGCTTCAAAG6TCCCAGATC TGCATGCTACAAGCTCAGCArCr06GC~CrcTTCCCTTCCTGCTTCACCCCCAGTCC C TAGTAGGTCAC TATGGTGGGTACGAAGTTTCCAGGGTC TAGACGTACGATGTTCGAG TCGTAGACCCCCGGGAAGGGAAGACATGGGGTCA, insert pUM I ORF pLMi P S S s 0 r T H A S K V P 01L1 8 TSS5 A S 6 G PL P S CF T P 3 p GGCACCCATCCtCAATATATCTCAGCCAGCT IC CCCAGGGCCTGGAGC TAATGACGGTTTCAGTGTGCCAA A6rAGACCCGCATTACCCCAA I: CC-- TGaTAGGAGTTATAATTGAGTCGG TCGAAGAGGGTCrCcOACCrCATTACTCACCAAAGTCACACGTTTTCTCTGGCGTACA TGGGr T rGAz onset! pLIi OAF PLMI A P I L N I N S A 3 F S 2 G L E L M4 S G F S V P K C T R M4 Y P K. L :ACGCC TG;CACAGG-'C-'TGGAGTCC:- TCCACG-CCs'ATG 3GCC rCCCAGTGCCTTCCCCAGCAGTACTCZCGTcCCCCzC ,AcC;GC TCCCCC-T:- ;..;3CCGGACG rGTCCr GTACC-.CAGAGGC 7ACGGT TAC rCGGAGGGG rCACGGAGGGGTCGCArGAGCAOGGGGrGZGTAAGGO~tA: irten pLMi OAF pUMl G L H R S P. E 3 L 0J M S L P S A F P S S T 2V P T P p A p p 'c rc CCCAG,-~A -ACGGAAGAX :TGG..AaCCCCAGACC rGGGCAACTG.3ACAGTAA TCAGC-GCGATCZ :AACAC IC insert pLM I ORF pLM I 148 Tuesday. IS Novemnber 199711:4a page L fig 34 pLM4 (I 100701 Site and Setqugrm GAA~aC: 7CAGGrACC.%GCTTCAG TCCCAGG GAGACCAAGGAGAGCSACATTCCCATACCA r TGG rGGGCc.,;CC TGAA r~c:ATCA.-'A6 rcAG~_.

C7.TTCCCGAGTCCATGG rcGA~AG ?CA6GGTCC TCC TCTGGTTCCTC TCCGCTGTAAGG6TATGG T ACCAzCCG.CG.;sC rTAZGCTrC r C4Tc r: onset pLMI OAIF pLAl K G L P Y 0L 05S0£E E T K E RRH S H T I G GL P E£S000 oS E C 76CC T: TCCCCCTGCAC TTCCCATGTCTC fGAG TGCAAAGGGCCAACT fACCAACATAGTGAGrCCCAC TGGCCACCACGCA4GMTCACCCGc; GAC GGAA7.GGGGACG rG AAGGGTAC.IGAGAC TCACG T TTCCCGG T GAA TGG TTG TATCAC TCAGGG TGACGCCGGTGG TGCG GT TCT TAG TGGGC G isnflPLM? OAF pLM I L P S P P A L P m S L S AK G01L T N I V S P T SA T P ArI T P C CAACAG CA TCC CC ACC CACGAGGCGGC CT TCGAC C G TACA GCGGC TCC C AAA TGG AGC ACC C TGTCCC TGGCCGA AG AC CCAAG GAATGA TTC.Z G6 T TG TC GTAGGGG TGGG TGC TC CGCCGGAAGC TC GAC ATG TCGCCGAGG6 TT TACCC cc CG TGGGACAGGGACCGGC TC TC TGG GT TCCC T TAC TAA6:insert p1N41 OAF pLM I N S IP T ME A A F EL Y S G S QM 6S TL S L A ER P M I R G6 7AGGA rCC TTCCGAGACCCCACGACGATGTTCACGGCTcAGTGCTGTCCCTGGCC TCCAGTGCC TCCTCCACCTACTCC TCAGCTGAGGAGAGGA M' CA:;TCCTAGGAAGGCTCTGGGGT6CCTGCTACAAG TGCCGAG TCACGACAG6ACCGGAGGTCACGGAGGAGGTGGATGAGGA6 TCAC TCC TCTCC fTA.

-isert PLM I OAF pLMI 36GS3F A DP T D00 V 65G V L 5 L A SS A S 5sT Y S5 S~ A E Rt C TCTG-'.3CAAATCCG6 .CCTTC6TAZGGAA -GGAA TCATCCCAGGAAAAA6G6GCCCACCTTGLCG T CT 56C 1C TSCCAAT6C--TATCG7.L* Inset? pLMi OAF pl. A 2S C I R K L A R E L 7- S S 0 E K v A r L T S 0 L S A H A N LI C..ZC-*AA-'TGTCT: C:7:C.C TTA 7 5 6TTA.-;GC7.CGCT(i? G6ACCG tCTCTGCCGGCTCC TCT7CCT37G-CT,'CG6SCTASAAG: ;CTfi imnf pLMl OR F P.M I F s .4 m r~ s~ I L R M E 7A E E K Y T£ I L C E r 149W.

Tuesday. 18 Novemer 19971 11:4a Pag jig 34 pt.M4 (I 10070) SiO* and SequerCe rc%!AGAC r 7TCTGAAGAAAAAGAACTC TGAGGCCAZSGCAGTCAT rCAGGZAGCCC T TAA rGCC TC AGAAACC ACACCCAA-'7AAC ITC-;GA TCAAUG4 G7 %C IG-.AGACTICTTI rTCTTGAGACTCCG4G I:CGCAGTAAG rccc rcGGGAAITACGGAG IcrTG GGGGGIr TTGAGC -AGrTCrC insefl pLMI OAIF pUMA 1 OtFL K K XN S E A A V OG AL NAS E TI7P K E L I K C,"AACTCCTCAGAAGCATCTCAAGCCTC GTT rTGAGaAGTCTATCGTAGAG TTCGGAG TTG7CG TAG GA TCGG AAGGCGACG TCG TCGT cc ACGACTACGCTTCTCTTTTCT insen oLMI OAF pLM i e N S SO0S I S 5 L. NS I T S M S S I G S S K 0 A 0 A K K K K K k 6TGGGTCTATGAGCTTCGAAGTTCCrTCAACAAAGCGTTCAGTATAAAAGGGGCCCAAG ICAGCTTCCTCATACTCGGArATAGACG;AATTGCTAZ CAACCCAGA TAC TCGAAGC TTCAAGGAAGTTGTTCGCAAG ICATATTTT I ICCCGGGITCAGTCGAAGGAGTATGLAGCCTATA rCTiTCc TAACGAT3 insert pLMI OAF pLMI li V Y E L R 5 5 F M A F S 1 K K G P K S A S S If S 0 I E E I A T CTTCAGcCCcCcrArccCCCAAA-C TACAGCATGGTTCCACAGAGACTGCTTCACCCTCCATCAAGTCCTCCACrGTCCTC:GTGGGCACT 7G-i rGA.CAAGTCG6GGrGAGTAGGGGG rTTTGA7GT TACCAAGTGTCTCTGACGAAGTGGGAGGTAGTTCAGGAGGTGG.AAGGAG3-CACCCGTGj insert pLMI ORF pLli1 P 0 S S AP S S P K L o' MG S TE TAS P SI1 K 53 ITL S S V G T .rCA:-CrAGGGcC CCTGCCACCCAGCCCCC:ACA: AGGC IGTT I rCA~G3AGACCC AG-GA I4GATCG;--AGC GCG.

C C TG-CTCCCG GACGAGTGGTCGGG"G-G>T:- ICCaACAAGGTACGTTTACTC, ftC TCCTCGG IC T TTCTTcc IccAIACC ICG4CGCG inse1 pLM I OAIF pL.Al v E G P A H P A P M I R L F H A U E E r- E K K 5 E L R OAFe pLMI T t. L 7- A L N S A H '0 L 0 1) L E f 'I -f r Tuesday. 18 November 1997 11:48 Page f Tuesday. IONovei ibaf 199711:48 Page Gcz.iT rG34,;GrGACC TGC rGAAG~AGGAA-rGAZ::AC TrAAG~tAG-CCCAG-Scccc rCATCAC;C rCCACTCC."Z:AGCrC.:crGATC_-,.:7 ~C;AACC 7 CC ACC TGGACGACT rTCGl'CTCTT.1C TGACTTC CA TCGGGTCCGGGAGTZG rCCGACGG TGAGG TCC:G TCCAG:-!zCC rG a OAF pLMl L E V 0 L L KC A E M4 D R L K V A P G P S S a 5 T P 6 0 W G S .3 GCATTATC -TCCCCACGCC CTCCCTAGGCCTGGCACTCACCCATTCC TTCGCCCCAGTCTTGCAACACAGACCTGTCACCCATGGA7GC~ATCAGTA.

CG3rAATAGA.AGG6GGTGCGG6GAGGGATCCGGACC6TGVG TGGGTAAGGM~rCG6GGTCAGAACGTC:TGTGTC TOGACAG TGG~tACCTA!CGTA6 TCA7 isert PLMI OAF pLIt A L5 5 P P S L G L AL T H S F GP S LA 0 TODL S P m 0G IS 6A.ICACCAGG TT TCCTCCT rCACT6GGAGGCCCACCACCACTCCTACGGGG-SCGTCGTGTA6 rAGTTTCCCCT6AACTTCGTCGTCC T Onsfl piAt T C G P K E E V T L 4 V V VA R P P 0 H IICK 60L K0 0 E F FILG C rG TAGCAAG.3FCAGTGGAAAAG TTGAC TGGAAr.ATGCTGGATGAAGCTGTTTTCCAAGrGT rCAAGGAcrATATT TC TAAA TGGACCC-A3CC rC rCC inset "Ii OAF pUhi1 S K 4 S G K V 0 V 4 H L 0 E A V F 0 V F K D Y 1 5 K M 0 ;A S T r TGAST CATCCATGGC T;CaGCA% AGCCACGTAA'ZACG T -M;G rGcAGAzCCCCCCGA6 ATGCC TCCTTC:Z TCQAaLGT; T,--TGs: r:A-:GTaGGTACCGA.,:%GT-%3 rCGGCACTT T':TAACAGCCGGGTTA:SGZ;*!-r;GT-Ainsert pLMI OAF pAtI L T E 3 1 H G Y S I S H V K R V L D A E P P E M P CP P -1 TrV .AAZ.r±-CAGTC 7C:C-TZAAAG3STCT .4GG .'-UTCTGC- rTZTTC IG-. TGATCCCCAAGCC64r GAT.3c'AZcACT-ACA inser p.M I OAF pIMII 1 I S I 6 C V 0 3 L V F E LIC P K 2 s4 11' 4 Y I 151 TUesday. 10 NOVefMiOe 1997 11:48 Page 4 fig 34 pLM4 (I 10070) Sit@eantdSequenice -CZ Fcc GCT6AAGCACCGG.6CC,-CGTCC T: CZOGCCCCAGCGGCACGGGCAAG6CrA rsc ACCATC6C TrGCCz4~ r~cCTr:; r-Z';aAGGACGAC rTCGTGCCGCo6AGCAGGAC;A.rCCGGrC~GrGGCCCGrrC TGGA TOGAC TGGrT.16CG:AACCG6.TAGC rc:Cr.. wtset PLM I OAF pIM I SIL LL KN A 9L Vi L 6 P S Gr TGK I Y L r N R L A E Y L V E P rC 7GGCCGTGAGG FCACAGAGGGCATCGTCAGCACCTTCAACATGCACCAGCAGTCTTGCAAGGATCTGCAAC TGTATCTTTCCAACCTAGCCA~C,L.4 A4GACCGGCAC TCCAGTGTC FCCCGTAGCAGTCGTGG.A6TTGTACGTGGTCGTCAGACO TCCTAGACGTTGACA TAGAAAGGTTGGATCGGTGGinsert PIM I OAF pLMi S RE v T E G6 VS T F N MN m 0 S5C K 0101 TI SN" L A N C :ACCGGGAAACAGGAATTGGGGATGTGCCCCTGGrGArTC TAT TGGATGACCTGAG TGAAGCAGGCTCCATCAGTGAGT'GGTCAAFGGGGCCC rCA: 7ZTGGCCC TTTGTCC rTAACCCCrACACGGGCACCAC FAAGATAACC TACTG6ACTCACrnCGTCCGAGGTAGTCACTCAACCA6TTACCCCGCGAGT:- 6 insert pLM I OAF pLMI D R E T G I G 0 V P L V, I L L 0 D L S E A G 5 1 S E L V N G A L T C :-CAio TATCATAAA tGTCCC TA TAT TATAGG-IACC ACCAA TCAGCC TG TAAAAAFGACACCCAACCATGGC TTGCACT TGASC T-TCAGGATG TC: Z.:GTTCAtAGTATFACA6O.GATATAATATCCAG~iTGGTTAGTCGACATTTTACTGTGGGTTGTACCAACGTGAAC rCGAAGTCCTACAACTG: risert p.M I OAF pIM I X Y N K C P Y I G T N 0 P V K M T P1 4 N 1 HLS F 2 M LT :cCA.CGGSACAGCCAA.-0:C TTC TCGT T.CC rGAGGACGA.IGC TOGTA1GAGTCA6ACAGCG-CATCAF CC CAAiAG6AU insert pIM I OAF pL?A I S N N V E P A N 6 F L 'I Q T L R A K L V E S 0 S 0 1 N AL M K E E insert pLI OAF pLMI L H L V i T F L E K N S T 5 D F F F 152 I uesaY. IS NOvomw 1997 11:4a fig 34 iptj4 (I >100701 Site and Sequencg Page Ic -r CaTGTCCCATTGGCT TGAGG;AC TTCCGGACZ TGc;T CA ITaACC rGTG6AACAACIC TATCA Irrccc ATr:A&Aa~j'AZ LG3ACAGCACAGGGTAACCG rAAC TCC TGAAGGCC TGGI*CC*A6IAACTGG4CACC Trr 110* TAG TAAG5GGAA6A TG ICC TTCCTCM TTC:Tr: insert PLM I ORF PLMlI L S CP I G E C F R 7 'dF E OL VN NS I I YL0 E.GA K 0r AT AA.%GG TC CA rGGAC AGA AAGC roeT rt. AG a AC AcG.1TGAAraaar C CG AC ACAC Tc TCCC C ATCACCCAAC AAGACCAA CAA3G .T.

TATTCCAGGTACCTGTCTrTCGACGACCCTCCTGGGTCACCTTACCCAGGCCCTGTGTGAG4ACCGGrAGTC GIGCG~ATrG insert pLMl ORE ptM I I K V H G00K A A 'dE 0 P VE V VA 0 i T v L 5VP *A 0 0 DQSk I AC:ACCTGCCCCCACCCACCG TGGGCCC TCACAG;CATTGCC TCACCTCCCAATA66.ACACAAAA~CAGCCCCCAAGC

TCTGGACTCAGATC:

TGTGGCGGGGGTGGTGACCCGGAGTGTCG TAACGGAGTGGAGGGC !CCTArCCTGTCAGTTTC r6TCGrGGGTTCAAGAGACCTGAGTTaAGJ.

wngest PiMI OAF pIi H L P P P T V GP H S I A S P P E 0 T VK 0 S T P S L0 SODP 'C GATGGCCATG C GTGAAAC TTCAAGAAGCTGCCAACTACA TTGAGTC TCCAGATCGAGAAACCATCC TGGACCCCAACCTTCAGGCAACACTTTA4 TcACCGGTACGACGAC TTTGAAGTTCTTCGACG TTGATGTAAC TCAGAGGTCTAOcCT ITGGTAGGACCTGGGGTTGGAAGTCCTTGTAATT insert PLMi -ORF pU~i L NRA M L L K L 0 E A AN Y I E S P0R E T I L D PN L 0 ArT.

insert PiM I GN M CIN P ART A E 4 v H 0 L S L L L S P LI L F S TOG S P c:-AzcIAG A.%-GGO G fC TTGGroCTGA r IGGA7C SrC Tc Tc r GT:ccrcc. rc: cTA. :TC^.Ccr :CAASAACCACGAcA rGGAAAc rc TrGAAG.SATCC ICTA:CC~* insert pLM I I G G E 0 E G G G 0 E C T. G 3 1 C C T F E N F L G 4 N G G Vi .SUGA.CTTCIGCCCC:TAAA -ACTAC-.:: r:C r:AA'AC TTGGCAAAIAGAtTC rGGCrrCIIccrTAT--7.I,.ITC.,T.

insert pIMI1 7I 1 I'4 G 1 00D S G 3 L 153 I uesday. 18 NOVfeibf 1997 11:£8 Page It tig 34 piMA (I 10070) Site anid Sequemse ACaAACTCC TGGGC rr rCTGGCGAGGGG TTCAGAAAACA TCAAAACAC TGCAGCAGTTCCCCGGAArTCAGCr rGG.'C T r ACcC7G-AAC Ir rGC m. 7 TGAGGACCCGAAAGACCCC rCCCC AAGTCT TT TGTAG T TTTG TGACG TCGTCAAGGGGCC TTAAG TCGAACC TGAAr TGZ T:C.;ACT TGA ACG,: G r.m S aF'.V G G V 0 T SK M C SSS P EF S LODL T RL NLL AAAiGAAGCCGAATTCCAGCAC AC rGZCGGCCGTTAC TAG rTC TAGATAACTGATCATAATCAGCCATACCACA IT TGTAGAGG TT FTAC TTGCFFTA~..a T FTCTFCGGC ITAAGGTCG TG FGACCGCCGGCAATGATCAAGATC FATTGACTAGTATTAGTCGGTATGGTGTAAACAFC TCCAAA- rACGA13FrTnmserl pLMl I xKA SR IP A H V Q P L L V LO0 N .S S A I P H L F IL L AA;3CCTCCCACACCTCCCCC FGAACCTG-AAACAT%AAAT6AATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTAAAGGTTCAAATAAAZ;CAAT.

TTGGAG6GGFGGAGGGGGAC TTGGACTTTGTATTTTACTTACGTAACAACAACAATGAACAAA FAACGTCGAATATTACCAA ThTT FATTTCGrTAr T S1 F S P TF. NI K MO 0 iLLi L T C L L Q L I M V T N K AlI CGtAG FG FTAAAGTG FTTAT TTCGTAAAAAAG TGACGTAAGATCAACACCAAACAGGTrTGAGFAGTACATAGAA TTGCGCATTTAA CATTCGCAAT A S 0 1SO 0I KH F F HC ILiV V vVC PN SS M YL NA. L VS V rTTTTTAAAArTCGCGTAA17 FFTTGTTAAA TCGTATTTACAAGCAACGCAACCTTAtAAGAAA: TATAAAACAATTTTAAGCGCAAF FALAAAACAA- TAGTCGAGTAAMTTGGTTATCCGGCTTTAGCCGTTTTAGGGAATATFAGFTTCTAC

TG

L iL K F A L N F 5 S F F N 0 A E I G K I P Y K S K E T C-;',rA-GGTTGAGTGrTC;7 T:A;TGA:AA.CATTAAACTGCCACTA6GGGAAC T-7TAz~G.

T ATCC CAAC TCACAA.-AAGGFc AA.%CC IITT TC TC AGG TGATAAT TCTTGCACCTGAGG TTGCAGFTT7CCCGC TTT TTGGCAG;. TAG TC CCGC TA E I G L S V V P V *4 U K S P L L K N V 0 S N V K G R IC I V 0 G 0 G';*CCAC IACGTGAACCAt:Accc rAATFCAiA. TTT FOGGGFCGAGGTGCCGTAAAGCACTA.AATCGGAACCC 7A-AGAGCCC:-CAFT tAc,.; T-, CCGGTGATCACTGGTAarGGGAT F T.%iC'-A.AAAACCCCAGCTCCACGZCATTTCGTGATTTAGCC TTGGGATTTCCCTCGGCG AAFCT:-GA L Q E EP 3 P.-S S F L G S R C R K A L N RN P4 K G S P P F R A GZG±ACCCGZGT4GSAS:; A.AG.AAGCGAAAR3GAGCGGGCGC TAGGGCG- TGC :,TAGCGGF:ACCC .TGCG:GTAA' C 7-CCCC TTTCGGCCiZ -7SAACcCTZ TTTC-CC: tTCT FCGCTTTCCTCGCCCGGATCCCGCGACCG T A--CATCGCCA5TG-ACGCGiC.T T- AG tK P A N ii A K X A K G A G A Q A L A S v A V T L A Ji T P A A L NI A) i G G r F R G n G T C 154 rusday. 10 NOvwnbgr 1997 1 1:4a Page I fig 34 pLM4 (I 10070) Site and Souence CA T TCAAA TATG TATCCGC TCArGAGACAA r~AAcCc rGA TAAATGC TTC.IA rAA rATTGAAAAGAAGAGTCCT6AGGCGGAA GAACCAGCTTrA G rAAG TT tArACATAGGCATACTCTGTTTGGGACAT ACGAAGTTATTAA rrT TCCTC TCAGCTCCGCCrTCTTGGTGAcCCT- S SNM Y PL M R0 P. M L 0 .1Y. K R K S P E A EA T SC G TGTGTGTCAGTTAGTGTGGAAAGTCCCCAGGC TCCCCAGC AGGC AGAAhGTATGCAAAGCA TGCATCTCAAT TAG rCAGCAACCAGGTTGAAGrc: ACACACAGTCAATCCCACACCTTTAC.GGTCC3AGGGGrCGrCCG

TCTTCATACGTTTCGTACGTAGAGTTAATCAGTCGTTGGTCCACACCTTTCAG

m C VS G vE 5 P 0 A P 0 A 5V C K AC I S I S Q0P GV E SP CCAGGCTCCCCAGCAGGCAGAAGTA TGC AAAGCATGCATCTCAATTAG TCAGCAACCA TAG TCCCGCCCCTA.ACTCCGCCCATCCCGCCCC TA.ACTCcrG: A GGTCCGAGGGGTCGTCCGTCTTCATACG rTTCGTACS TAGAG TTAATCAG TCGTTGGTATCAGGGCGGGGATTGAGGCGGrAGGCGGGATTG.::-.

0 A P Q OA EV CK A C ISI S 0 0P S RP LARP SARP.- L P.

CCAGTrcCCCCATTCTCCGCCCCATGGCTGACTAArTTTTTTTATTTATGCAGAGGCCGAGCCGccCGCCTCTAGCTATTCCAGAAGTAT,Zl.i P V PP I L RP HA 0 F F L F MO RP A P P A PL S Y SA SS E AGr.CT" TTTGGAGcCTAGGCTTTGCAAAGACGA Tc AAGAC AGGA GAGGATC GTTTCGCATATTGAAAAATGATTGCAC GCAGG TTCTCC- TCCGAAAAAACC TCCGGATCCGAAAACGTTTC TAGC T AG T TC rc TG TCCTACTCC TAGCAAAGCGTACTAACTTGTTCTACC TAACGTGCG TCCAAGAGS E A FL E A. AF A K (0 0C E 6 G. GS FR M I E 00G L HA GS P GGCCGCT TGGGTGGAGAGGC TA TTCSGC TA TGAC TGGGCAC AACAGACAA TCGGC TGC TCTGATGCCGCCGTG TCCGGC TG TCAGCGCAIGGGCGCCC: SI1 C CGGCGAACCCACC TC TCCGATAAGCCG ATAC TGACCCG TGTTGTC TGTTAGCCGACGA6AC TACGGCGGC AC AAGGCC6ACAGTCGCGTCCCCGCGGG: AM N4.

A A 4 V E A L F G Y 0 V A Q Q T i G C S D A A V F A L S A 0 G R P rj7 cT TIr 7G TCAAGACCGACC rGT:CGG TGCc: TGAArTGAAC TC ACGAGGCAGCGCC TA TCGGC TGCCACGA GGCGTCC r-scGcAj 321C L F V K T 0 L 3 G A L I E L 0 0 E A A R L S V L A T T G *4 P C A C :rCTCe CGTTGTCAC TGAAGC -r2AAG-5CT65C TGCTATTG6;6CGAAGTGCC6CGGGCAGGATC1'CCTGTCATCTCA*CCT rGC7CC TGC6GA6A.

ACGA C TGCAACAG rGtCTTCG:.CC TTCCC.-GACCACGATAACCCGC TTCACGGCCCCGTcc TAGAGGACAGTAGAGTGGAACGAGGACGSC TCT7 4 VL 0 V v T E A G 4 -1 V IL L EV P G00D L L S 5 H L AP A E ;.C66:GA t-:AAGCCCTCTTGTC-A':AIGGAT: .A7- 6ACG.AAA:ATCAGG6CCTCGCGCCACCCGAAc TGTT:-GCCA 'GC TCAACi GA CC* TG 7-:oCrcrCGCGCG 4,c- 4' rc C:.rrcZACCGCGTGC-GGTCiTC"CCTC- E A V C 23 0 0C E r (1 G L A oAC: F A Q L P 0 1155 TuesdaY. IS NOVOSTbeK 1997 11:4a page 11 fg 34 pLM4 (I 10070) Site and Sequence CCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCC TGCT TGCCGAATA ?C*?GGrGGAAAATGGCCGC TYTrrCTGGATTCaTCGAC 76 TGGCCGGC- G.,C TrCCGCTCC TAGAGCAGCAC TGOGTACCGCTACGGACGAACGGCTTA TAGTACCACC TTTTACCGGCGAA~AA 'CAAGCTGLZACCGGCCG4 P 0G EO0L V V TM H G A C L P N I M V E NG RAF G~ F I 0 C GARL G.GrGTGGCGGACC GC FATCAGGACA TAGCGT T.SGC TACCCG TGATAT TGC TGAAG&GC TTGGCGGC GAA rGGGC TGACCEc rTCCTCG TSC TTTACGG- CCCACACCGCCTGGCGATAGTCC TG TATCGCAAC CGATGGGC AC TA TAAC GAC TTCrCcLAACCGCCGCT TACCCGAC T6CGAGGCA.CZCAATGCCA G V A -0 R Y 0 0 1A L A T R 0 1 A E E L 6 G E V A 0 R F L V L Y G A TCGC CGCTCCC GATTCGCAGCGCA TCGCC TTC TA TGCC TTC TTGAC GAG TTCT C TGAGCGGGAC TCrTGGGGT TCGAAAt3ACCGACCAAGCGACGCC TAGCGGCGACGGcrAAGCGTCGCGTAGCGGAAGATAGCGGAAGAAC TGCTCAAGAAGACTCGCCC rGAGACCCAAGCTTTACrGGCTGGTTCGCTGCGi I A AP O S RI A F Y A LL D E F F. A C L W GS K P T K R R CAACCTGCCATC ACGAGATTTCGATTCCACCGCCGCCTTCTA TGAAAGGT TGGGCTTCGGAA TCGTTTTCCGGGACGCCG6C !GGATGATCCTCCAGCG: GT TGG ACGG TAG TG CTC TAAACTAAGG TGGC GCCGG AAGA TACT TTCCAACCCGAAGCC TTAGCAAAMGGCC CTGCGGCCGACC TACT AGGAGGTCGCG P T CNHHE I S I P P P P S M K G VA SE SF S T P AG .5 S S A GGGCGATC TCATGCTGGAGTTC TTCGCCCACCCTAGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGA.AGGAACCCGCGCTATGACGGCAATMA CCCCTACGAGT*CGACC tCAAGAAGC6GGTGGGA7-CCCCC TCCGATTGACT TTGTGCCTTCCTCTGTTATGGCC TTCCTTGGGCGCGATACTGCCGT TAT7 G61 S C V S SS P TLG G G L K H G AQ 0Y R KE P AL AG0.

AAAGACA AATAAAAGCACGGTGTTGGGTCGT77GT TCATAA.ACGCGGGGTTCG6TccCAG6CTGGCACTCrGTCGATACCCCACCGAGaCCCCATT6- K 0OR K A T VIL GARL F IN A G F G P A A G T L SI P HA DP I GGUCcAA TAC GCCCGC GTT TCT TCC TTTTC CCACcC:AcCC CCAAG TTCGGGTGAAGGCCCAGGGCTCGCA6CC ACG TCiGCGCGCAG6CCTGC CC GG rTA TGCGGGCCAAAGAA5 AAAAG 6G7GGG6T GGGGGT TC AAGCC CAC T TCC GGGTC CC GCG 7CGGT TGC AG C GCC6 T CGGACG'- G A N I P A L P F P P T P 0 V R V K A 0 G 5 0 P T S G R Q A L F A7 GC C TC AG T AC TCATAATAC T TT AGAT T 5A TT TA A.AAC TTC A TTT TTAA TT T AA6GA TC TAGG TGAAGATC C tT T T7ZAT AAT: rcTAG ACC~ .PO0v TNm I vF P L I NFlI F N LK G SA R SF L II S F A cTT A;IC GTG 5T r FC:;TT:C'- C GA A ACC CC GT AGA.AG A T CAA AGGA ICIT C TTG AGA KCCT 7TT TtT: T :cCG6T A ATC TGC T SIL N V S F AS 3 T A E R P. KR S< OI LL El IL F F C A .S A ouI, f A v C A I K S 1 0 L F F Q P 3 A~ E R 156 Tuesday. IS Novemiber 1997 11:48 ae.

fig 34 PLMA (I 0070) Sit. and SoQuemenag i ArAcCCMATACTGTCCTC TAGTGTAGCCG TAG TTA6GCCAC CAC TTCAA6SAAC TCTG TAGCACCGCC TACArACIC tCG: TC TGC rAATCC TGTTACCA-- TrA tG TTTATGACAGGAA6.kTCACA TCGGCATC AA TCCGG TGGT GAAG, r TC TTGAGACATC G TGGC GGA TG TA TOAGC SAGACGA T T ACAATGGT..

pIJ(. W, a L 5 F C S R S A T T S R T L .H R L H T 5 L C S C V rGGCTGC TGCCAG TGGCGA TAAG TCG TG TC TTAC CGGG I TGGAC TCAAGACGA TACTrACCGGA TAAGGCGCASCG CGGGC TGAACGG0GGr TC TS ,7 A C CGAC GAC GGTCACC GC TAT TCAGC AC AGAA TGGC C C AACC T GAG T TC T GC TA TCAA T GGCC TA rT C CGCG T CGCCAGCC C GAC TTGC CC CCC A C A-- V L L P V A 1 SRV L P GV T700D0 SY R I R A SG RA E RGV R GTGTGrCGGGTCGAACCrCGCTGCTGGAGGGCTrGACTCTArGGATGTCGCACrCGAACCTTCGCGrGCAAGG6CrTcCrCT TTCCGCCTi 9c.

A H S P A V 3 RP T P N .0 T 'VS V S Y E K A PR F P K G EL AT AGG TA TCCGG TAAGCGGCAGGGTCGGAACAGGAG AGCGCACGAGGGAGC TTCCAGGG G6AAACG CC TGG TA tC TT TAtAG TCC TGTC GCGT T TC GCCACC rCCA rAGGCCAr TCGCCGTCCCAGCC TTTCCTC TCGC GC TC CC TC AGG TCCCCC TTGCGGACC ATAGATA TCAGAC AGCCC4AACGGTG:; G INR. AA GS E QE S A R G S F0 G6E T P G 1 F I VL S G FA T rC TGAC T TAGCGTCGATT TTTGTGATGCTCGTCAGGGGGGCGGAGCC TATGGAAAAACGCCAGCAACGCGdCC.TTTTTACGGTTCCTIIXC TTTTGC T AGACTGAACTCGCAGCTAAAAACAC TACGAGCAGTCCCCCCGCC TCGGATAkCCTTTTTGCGGTC6TTGCGCC6GAAAAATGCCAAGGACCGG.JCGA: SO 0 S V O F C A R 0006G A Y G6K T P A TR NPF Y GS~ P F A ,;CC TTTTGCTCACATGTTCTTTCCTGCGT 7CCCT:ATTCTGTGGATAACCG TAT TACCGCCATGCAT 100"3 C G ACGAGTGTAAAGrGACGC AATA.GaaCTAAGACACCTAT TGGCATAATGGCGGTAG TA I L LIT C S F L Y P I- ILV I T V L P P C I Tuesday. IS November 1997 10:34 Fog 3pEGFP72 (I >9697) Site arid SOQUOCS Enzymes: 72 ol 148 enizymes (Filtered) S-ealngs: inear. Cetai Sitas Onl. Standard Genetic Code I gI

I

TPG TTAT rAA TAGTATCAA TTACGGGG MAT TAG ITCA TAGCCCATATATGGAGTTCCGCGTTACA TAAC 7TACGG TAAATGZ;CCC GCC TGCGcc: ArCAATAATTATCATTA%7 TAArGCCCCAGTAATCAAG rATC6GGTATA;CCTCMGGGCAATGATGAAGCCATTTACCGGGCGGAcCGAC TG3: LIL IV tIN YVGV I1S5S. P I Y GV P R y I T Y GK V P Aj W CCCAACGACCCCCGCCCT tGACGTCAA rAAFIG.%CIATGTTCCCATAGTAACGCCAATAGGGACTTCATGAC6TC&ATG6GI.G;ITrAG CGGTTGC TGGGGGCGGGFAAC TGCAG TTATTACTGCATACAAGGGTATCATTGCGGTTATCCCTGAAAGGTAACTGCAGTTACCCACCTCA TAAA7GC.., A 0 a P P P 1 0 V N N 0 V C S H S N A N R 0 F P LIT S M G G v F T v 891Nd.:I Aa 11 rgII AAAC TGCCCACTTGGC AG? ACA TCA G TGTAYCA TATGCCAAGTACGCCcCCCTATTGACG TCAA7GACGGTAAATGGCCC 6CC TGGCAT TA TGCCCAGT.

TTTGACGGGTGAACCG TCA ?GTAG ITC ACA TAG TA TACGG TTCA TGCGGGGGATAACTGC AGTTACTGCCATTACCGGGCGGAC CG IAAACGGGTCAT N C PILG S T S S V S Y AK V A P Y.AR Q RA. M AR L A L C P V Iaa IPca CArGACC TTATGGGAC TTTCC TACT ?GGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG TTTGGCAGTACATCAATGGGCGTGGA G tAC TGG AA TAC CC TGA A.AG ATGAACC GTC A? I AGA IGC ATAA TCAGTAGCGATAA TGG TACCAC TACG CC AAAACCG TC ATG TAG TTACCCGC Acc r NM 0 MG L1S L* V H A R C S H Y Y V GO0 A VILA V H 0 A V TA%:CGGTTTGACTCAGG.ATTTCCAATC7C:AC:-:CATTGACGTCATGGGGTTTGTTTGGCACCAAAATCAACGGGCTTTCCAAAATGTCGT; A7-CAATATi CTASTCGa3GG:G TAACTGCAGTTACCCTCAAACAAAACCGTGGTTTTAGTTrCCC rGAAAGGTTTTACAGCAT A V L T SI S K S 'PH A a 'd E F V I A P K 3 T G 1 S k. M S.

Nhel Ec47 .;'ATCCCATL tC:".TACGGT6GGGAGGTC TATArAAGCAGAC-CTG~rTfr.TGAACCGTCAGATcCCI:r-AGCGCT.; T.;TTGAG6CGGG6TAA: rG:GTTTA~C:SC C r::G::ATGCCACCC TCCA4GATATATTCG rc TCGACCAAATCACTTGGCA:;TCTAGCSCATCGCGi- L R P 1 0 A N G R A C I V G G L Y K 0 3 V F S E P S 0 P I A I C: ZGTCtC AC TG3I AGC GA1 TGG TAc.,A:;:GTtCCCC: TCC-C- ACAA3 rGGCCCCACCACGGGTAGGACCAGC TCGACCtGCCGCTG;CA rTTCCCGGrGT rC~A GC~i: 9GFP.C.e.ur453 *j t 3 K C E T G V V P I L V E I 0 G 0 V N G H K F eGFP.C e~urc53 w ;C E G 0 A r L T L F I C I T G K I P P r L1 V 158 Tuesday. 18 Novem~ber 1997 10:34 Paw% IIg 30 pEGFP72 (I 9697) Site and Sequence C .ICCr G-±CC fACGGCOTGC.AGTGC TTC.AGCCOC TACCCCGACCACA TGAAGCAGCACGaC c rrcrcAAGTCC:C' CC: 3AoGC.,:-,C'iL G tGOOAC TGGA TGCCGCACZTCACGAAG TCGGCGATGGGGC roo TG TACT TCG TCOTGC TGAAGMOG rTCAGGCGG TACGGGC rTCCGA TSC AOOTCC r*_ eGFP.C.*.unc53 T L T f GV 0C F S R Y P MM .K H 0F F K S AMP E y GYV a E rspl COCACCA TCT TC VrCAAG6ACG.%CGGCAAC TACAAGACCCOCGCCGAGGTGAAGTTCGAGGGCGACACCCrGG AACCGCA VCGAGC VOAAGGGCATCG eGFP C.a.unc53 A T I F F K 00 OGN Y K T P A E V K F E G01' L V N R I E L K G0I A.C TtCAAGG GGACOGCAACATCCTGGGGCACAAGC VGGAGTACAAC tACAACAGCCACAACGTCTATATCATGGCCGACMAGCAGAA0, 4CGGCATC 4-: TGAAGTTCCTCCTGCCGTTGTAGGACCCCOTGTTCGACCTCA VO1'TGA1'O V1GTCGG TGTTGCAr.ATATArOTACCGGCTGTTCGTCTTCTVGCCGTAGrr 0 F K E 0 G N I L G N K L E Y N Y N S H N V f' I M1 A D KC 0 K N 6 I h.

GO rGAAC TTCAAGATCCGCCACAACATCGAGGACGGCAGCGTCAGC'CGCCGACCAC TACCAGCAGAACACCCCC-rCGGCGACGGCCCCGTGCTOC r; CCACTTGAAGTCTAGGCGGTGTTGVAGC TCC TGccG TCGCACG TCGAGCGOC TGGTGATGOTCGTCTTGTGG6OOTAGCCGCTGCCGG6,CACGACGAZeGFP.C.o.urcS3 v N F K I RNHNf I E OGS V Q L A 0 HY0 Q N T P I 0G V L L CC: GACAACC AC VACC TGA5CLZ CCAGV :CGCCZ -GJLC AAAGACCCCAACGAGAAGCGCGATICACATGGTCCT1GC TGGAGI eGFP.C.o.ur453 0 N HNY L 5ST 0 5 A L SK 0OP N E K R D 4 V L L E F VT A A G Asu 11 PspM 11 Sgi 11 EcON I -Ce. urtc53 r L G mI 0 E S G S T S N V E L I P I Y T 0 V A N H. N L Nru I EcoR I t-GFP+C eurcS3 Cea unc53 :1 U. F C- t L 1 :1a I V I PI E 159 TuoSdaY. 18 Novembter 1997 10:34 Page EGFP72 (I >9697) Site and Seqljer"e T -TCCCTGCA TTCACGAAACGTTT6GCAJAAATCACATCGAACC rGGATGGCC TCGAAACGTG TC TCGAC TACC FGA.AAArc rGGGTC6G T6C T A4,AGCGGACGTAAGTGCTrTGCAAACCGTTTTTAGTGTAGC TTGGACCTACCGGAGC TTTGCACAGAGCTGA TGGACTT IT AGACCCAGAGCTGACGA &GFP.C.aurc3 C.s. unc53 F 5 P A F T K RIL A K IT S N L DG L E TrCL 0 Y L K N L 6 L 0 Ear I EcoR V P'vu 11 ?Csp6321 klind III CGAACTCACCAAAACCGAATCGACAGCGGAAACTTGGGTGCAGTTCTCCAGCTGC

TCTTCCGCTCTCCACCTACAAGCAGIAGCTTCGGCAACTGAA.

GCI TTGAGTGGTTTTGGCTATAGCTGTCGCCTTTGAACCCACGTCAAGAGTCGACGAGAAGGACGAGAGG TGGATG TTCGTCTTCGAAGCCGTTGAC TT &GFP.C.e.uncS3 Ce. wi- K L T K T 0 1 0 S G N L G A V L 0 L L F L L S T Y K 0 K L R 0 L 1: PmsCl SetiIl PmaCl T T rc TAG TC TTC T TTAACCTCG TTGATGGGTGTA6GG TAATACGGTGGGCGCCAAGATTTAATGGGAGCGGTCACAGCGG TGCAGTC GT TGGCGAAGT eGFP.C..unc53 Cea. isncS3 K 0O0K K L E 0 L P T S I M P PA VS K L P S P R VA T SA TA S GC'AC TAACCCAAATTCCA-CTTTC CA.AATGCL1C rCC-'GGC TCAGACTCCACAGTCA6AATATCGA-*,ATTGATTCATCAAAGATTGGTA-TCA eGFP.Ce a urx53 Ce. uncS3 r .4 P N S t1 F P Q 14 S T S R L 0 T P 0 S R I S K 1 0 S S K I 6 I ;-!:-'AAAGACGTCTGG6A:1 rAA-CC-TA:T4%C*TCtAAATCArCrCGCG-GGCrC-GG:A eC.FPC.s.uncW Cis. unfCS3 L K P r S G 5 S r T S S N N T toi 3 F P P i S Z 3 S G 4 i 11 160 Tuesday. 18 NOVOmOW 1997 10:34

P~L

fig 30 oEGFP72 (I 9697) Site and SeuerCG ag FcoR V 1IKsp6321 IASU 11 TG-.rGGC TCGACGATATCCACATCTGCGLAGAGC ?TAGAATCATCATCAACC;TACAGC TCTA'TTTCGAATC TAAACCGACC TACc TCCC.AAz rCCA-* AC AACCGAGC TGCTATAGGTGTAGACGC rTCTCAAT AG rAGTAG TTGC ATGTCGAGA AGC Tr4ArGTGC TGGATGGAG~r iT.GGrTT 9GFP.C Ce. ua,53 v G S T IS T S A KS L ES S S TY S S 1 NIL N RP T S39 L 9 9 )Cba I Nho I CCTrC TAGACCACAAACCCACTAGTTCTGTCTAAACTCAAAATCGGAAGC TCAAAGCTAGCCG TCCGAGCCGTGGCAC..,AACtT GGAGATCTGGTGrTTTGGG CGATCAAGCACAACGATGtTGA TGTT rT TAGCC TTGG TGTGCAGTTGCCCT-+,

TTA

eGP+C.e.unc53 C o. unc53 R AP TO0L VAR V AT T T K I G655 XLA A P KAV S TP K L I sm

I

C ',7C TGTGAAG;AC TAT TCGAGCAAAACAAGAGC:CGA rAACAGCGG TGG TGGTGGTGG TGGAATGC rGAAA1'TAAAGTTA tTC AG; TAGC,"AAACCCATC GACAA TC.:ATACTG TT CTGG TTT CC CACCACC AGCTTAr CAAZTAC T TG'A eGFP+C e uflcS3 ufncS3 A S V K r I G A K 0 0 .4 5 G G G G G G MI L K L K L F S S N: N .P T r:ATCGAATAGCCCACAAzCTA'AGAA. 3GC~:CGGCGGTGCC TC*AICACTGCAATGTGC

GTAAGGCTA~

eGFP C-.a~u,~c53 Ce. uncS3 I'SS S P 0 P 7 K A A A V P 0 0 0 T L S K I A A P V 11 c G L I BstX I Hind III eGFPC a urxS3 C.e+ uncS53 r S~ L G S A T S S K L C T P K VI S Y A r o A 2 1 G.

161 Tuesday. 18 NOVOffbef 1997 10:34 Page A42 3o pEGFP72 (I 9697) Sime and Sequen~ce Ear I Jgup& 3 21 BspM 11 I I AC TCGAAACGATGCTCAAAMAGCAG TGAAGAAGAGTCCGGATACGC TGGATTCAACAGCACGTCGCCAACO TCArCATCGACGGAAGTTCCCcTAGCA- TGAGc rT IGC TACG AG TTTCTCG TCAC T XTTC TCAGGCCTATGCGACCTAAGTTGTCG TGCAGCG6 TTGCAG FAGTAGC TGCCT TCCAAGGGATTCGT; eGFP.C.o.urc53 0 S K Q C S K S S EE E S G6 tAG F NS T SP T S S TE G S L S V Barn I Sph I IAva III G~c 'TCCACATC TTCCAAGiAG TTCAACG TCAGACGAAAAGTC TCCG TCATCAGACGATCTTACTC TTAACGCC TCCATCG TGACAGC TA-7CAGACAGCC' CGTAAGGTGTAGAAGGTTC TCAAGTT6CAG TCTGC TT TTCAGAGGC AG TAG TCTGCTAGAATGAGAATTGCGGArGGTAGCAC TGTCGATAGTC TGTCGGC SGFP.C.o.umcS3 C.9. urc53 H 5 T S S K S S T S D E K S P S S 0 0 T LIt A S I V T A I R 0F psp I AAGC CGCAACACCGG TTTC TCCAA TA TT ATCAAC AAGCC TGT TGAGGA AAAACCAAC AC TGG CAGTG AAAGr.A6TGAAAAGE ACAGC GA AAAAAGA TC TA7CGGCGTTGTGGCCAAAGAGGTTTATAATAGT TGTTCG6ACAAC TCCTTTTTGGTTGTGACCGTCACTTCCTCACTTTTCGTGTCGCTTTTICTAG &GFP.C.e.uncS3 urcS3 A A T P Vs 3 Nt IIN K P v E E K P T L AV K G V K S T A K K 0 pmaCl Pvu 11 PmaCI jEroR V C.-:CrCCAGC GTCC-C-GIACAC:a-IACGGTTATCJTAGCCTAA-GTAAAGCi'TAACk.

rG.SGTCGG;TiTAGCCTCAACAATCAGG1TAATACCGTGTi~rTC T7CAACTGTTTACTGGGGCAC TATAG~reGFP.C.veunc53 C.o. unc53 'P A v A 2 0 T 3 1 G v v S P M A H K K LI N 0 P V I S E Atwrn I eGFP.Ce.urc53 Ce0 unC53 SEVL S 7 T 0 V P P P 1 IV P L. ti1 Tuesday. is November 1997 io034 ae hg 30 EGFP72 01 >9697) sit* wd s qi e Mae I pip A TCCGACAACCACCAACSTACGATG TTC TTCTAAAACAAGG-AAAATCACATCGCCTG TCA.AG1'CGTTTGGA1'ATGA'GCAGTCGTCCGCGTC TGAAGACT eGFP.C.o.ur.c53 urc53 I A OP P PT 0 V LL K Q6K I TS P V K S F G Y E aS S A S E 0 C-"A.TTGTGGCTCATGCSTCGGCTCAGGTGAC 7CCGCCGACA.-AA-C TTCTGGTAATCATTCGCTGGAGAGAAGGATGGGAAAGAATAAGACATCAGAATZ GGrAACACCGATACGCAGCCGAGCCACTGAGGCGGCTGTT TTTGAAGACCATTAG TAAGCGACC rCTCTTCCTACCCTTTCTTATTC TG rAGTC TTA..: eGFP.Ce.unc53 C.o. uncS3 S I V ANH A S A V T P P TE K T S G MN S L E R R MG K N KT S( ra C,3CGGC TACACCTCIGACGCCGGTGTTGCGATC TGCGCCAAAA TGjGGGAGAAGCTGAAAGAATACGATGACATGACTCGTCGAGCACAGAACGGCT.7 G T:GCCG.ITGTG6ACACT6CGGCCACAACGCTACACGCGGTTTTAC TCCC TCTTCGAC TTTC TTATGCTAC TGTACTGAGCAGCTCGTGTC rTGCC6. T4 eGFP.C aeuncS3 C.e. urx:53 S G YT SDA GV A MC AK mR E KL K E Y 00 O T 4 A 0 NG V Asu 11 OS51I 11MI CC -GACAAC TTCGAAGA.'-STTCCTCCfTG TCGrTC-~GGATA TCCGA TA-*CAACGACTCGACGACATATCCACGGACGATTTGTCCGAAACATi eGF13Cew~uc53 C.e. u*x53 0 .4 F E CS3 S S L S S a I S 0 N N EL 0 0 1S tO0L 5 G VON0 GCr rCA7Ct3GAGG TT TT CGC TGATAAGGG7- A: AAGCGGTAGG TGCAG ACAAGGAGTT TCGGGGC TCAGGCCGTC AGCCA~rA -T:.C e^GFP.C e urcSM Coe. uncS3 V VA S SO0Y S v VP H P r S 3 SS3 K P R Vp PRS S T S 163 Tuesday. IS November 1997 10:34 'Page r fig 30oaEGFP?2 (I 9697) Sito and Sequence ho~g I CGTTTG CtCGAGCAGAACAGGAGAATG T AC AAAC T tc TG TCCCAGT6CCGAACGGCCAACG TGGCGCCGC TGCCAC: TCAACC TTGA,-; G: TAAGAGc rAGAGC TCGTCTTG rcc rTTTCA:&TGTr TGAAGACAGGG TCACGGC rTGC TCGGT TGC,&CCGCGGCGACGG T:;AGTTGG"GCC TGTeGFP.C.@.umcS3 unc53 0 S R S R A E 0 E N V T K L L'S Q C R T S 0 Q G A A1 A r S T F G Q ~SflI PV1SalI CAT TCGC TAAGArccCCCGGGATACCATCCTATTCTCCACAC TTArCAGTGTCAGCTGATAAGGACACAATG TCrTGCAC ICACAiAC TAG tCGACGAZ G TAAGCGAT TCrTAGGGGCCCTATr.AGTAGGA TAAGAGGOTGTGMATAGTCACAG TCGAC TATTCC TG TGTTACAGATACG I GAGTG TC TGA TCAGC TGCT:; eGFP.C.&ur53 Ce. ur=3 HI S LA SP GV S S Y S P HL S V S A 0KO0TNSM S OMH5 TSR R C rC TTCACAAAAACC AAGCTATcAGCCcAATTrCATTCAcrC TATCGT.%ATCCrAAGAGT TCACACCACCAGCACAGAATGGCGGCTC- GAAIGAATGTTrrrGGTTC0ATAArCCGTTAAGAA?6AACAGCATTACGTGGAAtTTCrCAAGTGTAG0rGGCCGGCrTACCGCGAG.; 90FP.C.a.uwc53 C.o. uncS3 S' S 0 KP S Y S6 3F St L 0 R KC H L E F T S TE H R NAA L Samn HI C 7 T 11-%C CCGAGACGG TGC CGAAC TCGATGC 3 AAA TArGA T C TC AGGATCC T&C CGCGCGTC CCAGG TGAAGC TC IAC TGGTA A GG4.

'4-IC TCTGCCCACGGCrTGAGC rACAG: 7TArACTAAGAAZTCCTAGGATGAGCCG.CGCAA:IGG: rCCACC F CGsArACCATA3AkTACrC- .GFP.C.*.uncS3 C.S. urc53 L S PR A V P N 5 11 3 K v D S S G S Y S A R 5 R G G3 S S T G I Y Barn HI NI'e I 1d .GFP.C a utc53 wic53 T L H P L SOD E tS 3 As .1 A K 5 E 5) 0 3 L !3 7 1 164 Tuesday. 18 November 1997 10:34 Page fig 30 pEGFP72 (I .9697) Site anld SqueriCS ,arGGATC tC TCAATGAGAAGTACGAACAT6C TAT TCGGGACA tGGCAC GTACTTGGAGTT TACAAGAA.CAc rG TCGAC PCAC T CZG.A G; rACC TAG AGAGTTACTC TTCA TGC TTGTACGATAAGCCC TGT-CCG TGCAC TGAACCTCACAArGT TCrTG TGACAG4 fGAG TGATG tT T rrTG rCCeGFP.C.e.urvc53 uncS3 Y G S LN EK Y ENH AtI Q 0 MA R L E C Y~ KN T V 0SL T K K0E EarlI fund IIII KSPS321 GAACTArAGCATTTTGATCtTTTTGAGCAAAAGCTAGAAACTCACTCAACACATTGATCGATCCAACTTGAGCCTGAAGGGCA~TAG-I 7 r C rTGArACC TCGr&,ACAAACTAGAAAAACTCGTTTCAATCTTTTGAGTAGTGTGTCAGCAGGTGACTCGGACTCTCGrATGCrzf.; aGFP.C.o.ur;cS3 N Y G ALPF D LF E 0 KLR K L T N 0 O 0R S NL KP E EA I Q F AGGCAGG-ACA~T GCTC ATTTGAGGGATATTAGCAATCATCTTGCATCCAAC rCAGCTCATGc rAACGAAGGCGC rGGTGAGC rrC rjCc TCAACCArCT: ?CCGTCC TG TAACGAGTAAAC TCCC TATAATCGTTAGAGAACGTAGG TTGAGTCGAGTACGATTGC TrCCGCGACCACTCGAAGAAGCAG TTGGTAGA'- *GFP.C.s.ur53 0 C I A ML ARC S MNHL A S N S A H ANE GA GE L LR 0 P Ear I Clal I Clal I st I Ksp6321 AArC'G TTGCATCCCATCTCATCTATGT:AC :TCGTCGAAAACCAGCAAGCAGGAGAAGATCAGC TTGAGC,-CcTTTGGCAAGAA:AAaA.3A: -IC: T TAG Y:AACGT GT AGC TAG ATAC AC ACAC TTTTCG TCGTTCGTCc rC TTCTAG'.-C3AAC TCAGCA-ACC 5T ?C T TCT &GFP.C.a.ur453 Ce. unc53 L _7S VA S H S S MS S S S< S SK 0 EK 1 3 L S F GP i jam Hl Nd. I flspM 11 C ,ATCCr- i-Cc rCAC rc TCCAA3 -CACCA.A:AGAACAAGAACTACGACGAAGCACATATGCZA'AAT T TCC-5GATCrC r:TMr~ eGFP.C e C e. unc53 3 y 0 E A Hi M SI3 rL 0 Tuesday. IS November 1997 10:34 fig 30 PEGFP72 (I 9697) Sde anldSequence Page 4 ISst I 1 a A-':ArrG-,rG rGArTGAGT rGAAGC AAACTCA A1ACGCATAG 6C C T TACGAAG rCCGCC r GA1CAATC TGGA -CCCC 4-GTTA rAAc TACAC TAAC TCAAC T TCG Trc rCGAGT T C TTGCGCTAPCACG TGAAA rGC rCAGGCGGAAC TG T AG -CC TAGAC0GiC-3: rA:A -eGFP.C a uvx53 C.9. urc53 NC (0V I E L K 0C ELK E R 5A L Y E V RL 0 NIL 0 q A R E V 0 T TC TGAGGGAGACAG raAACAAG TTGAAAMCCGAGAACAAGCAATTAAAGAUAGAAGTGGACMAC TCACCAACGGTCCAGCC -XC TCGTGTTPCrTCCc: A.AGAC rc~CTCrGTCAC TTGT TCAACTTTTGGCTCTTG TTCGTPAATTlc TPTCTTCACCTG rTTGAGTGGTTGCCAGGTCGGTrGACACS.A.AAGG eGFP.C..ur4c53 C.a. uncS3 V L A E T V N K L K r E M KC 0 L K K E V 0 K L T N G P A P A S S P JKspl asrl u1 C GCC TCA.'.T rCCAG TrATC TACGAC GATGAGCATG TC TA TGATGCAGCGTG TACCAG TAC ATCAGC TAG TCA T rICfGACZ; TCC TC 5GC TGCAAZ GCGGAGTPTAAGG PCAATAGATGCTGC YACTCGTACAGATACTACGTCGCACATCGTCATGTAGTCGATCAGTTAGA~thC r rrac PGGAGA;:CGACGTT: r-FP C a w~53 Ce. uncS3 A S I P V I DOE HV Y 0 A A C S S T SASO0S S K R SS 3 C 14 Hpa I IEcaR

V

7-:A rCA-lGGT.tAC TGA' CTA-CC-GZ- T GTGT TACCGCA(AAAT~~ PAGT ATA.TrCCT .GGICCTATAGTGAATGCTTTTtT.G-TCAAACG~ATG1 &GFP.C..wfS C.e. unc53 I P V 'I V C I A G E I S S I V N P 0 K C V G Y L 4 r S T CIA C .GFP.C a ur;453 LuvcS3 Tuesday. IS November 1997 WAS3 Page pEGFP72 if 3..9897) Site and Sequnce GrrCT-TCCTGGCATCAAATTGGTGAACTrCGACGGcTCATrGGAGACTCCACAACCATGAACCAGCCArCCrAcATTCTTACT,-cCAtC AC VAAGArAGGAACCGATAGTTTAACCACT rGAAGC TGCGCAGrAACC TC TGAGGTG TTGGTACTATT6GTCGGrAGG TTGACrGTAAGATAGAeGFP.C a unc53 Ce. unc53 0 S I L G YO 0 G EL R AV GO0S T T F41T S HP Toa I L AC TACAA TCCGAATGT TCA TGCACGG TGCCGCAC AGAG TCGCG TAG ACAG TC TGG TCC T TGATATGCTTCTrTCCAA.*GCAAATGATCTCCAAC TCGTC..

TGArGTTAGGCT rAcEAAGTACGTGCCACGGCGTC TC TA.GCGCATC TO CAGACCAGGAAC TATACGAAGAAGGTTTCGTTTAC TAAGAGG TTGAGCAGeGFP.Csunc53 Cse. unc53 r T I R mFM H GA A 0S R VO S5L V L 0OMLL P K QM I L 0L V rat 110SrI Sri Au AGTCAATTrTGACAGAGAGCTCTGGTGTTAGCTGGAGCAACTGGAA TTGGAAAGAGCAAACTGGCGAAGACCCTOGGC erTA TGTATCTATTCG..A T CAGT A AAAC T GTC T CTCTGC AGAC CACAA T C GACC TC G T TGACC tTAA CC TTT CTCG T TTGAC CGC TTC T=GAC CGAC ISA ATIAC ATAG ATA4GC T T eGFP.Ce.unc53 C.a. w'ieS3 K S I T E A L V L A G A T GE IGK S K L A K T L A AY V S I RAT AATCAA T-C AAGATA35TTGTz&ArATCAG-A.-TCCTGA ACAATAAAGAAGAATTGCrTCAAGiG~iAACGACGCCTG AGTCT4U; eGFP.C, a unc53 C.e. unc53 N0 S E D 5 V I4 1 1 P E N3 N K E E L L 0 1/ E R R L E 9 1 L R 3 Ava III NsiI I e-3FP.C e uicS3 C a. uncS3 E S I L C 1 3? I A F V V S A \I D cIt tI 167 Tuesday. IS Novembf 1997 10:3S wP~eI fig 30 pECIFP72 (I -9897) Ste &Md EooR V CAT r rGVrTArG.VCACATCCCGATATCAAATCCC TGAGCTTCAAArTCACCACAArVTCAAAA GCAGTA&VGrCGAI% TCG TCVCGz4GArrCA GTVAAACA rCA VACGrTCAG TTGGC rArAGTTTAGGGACrCGAAGTTrMrGTGTTTAGrTTACArCA rTACAGC TrAGCAGAGCTTCC TAAGTA; eGFP.C.o uncS3 P F V V C T V N A Y 0 1 9 E L 0 1 H N N F K H S V M S N A L E G FI Ear I ,Sst I Ksp6321 CC TACGT VACC TCCGAICGA.GGGCGG rAGAGGATGAG VA1'CG rC TAAC VG VACAGATGCCATCAGAGC TC TTCAAAATCArrGAC rTCrC::CAATAG- GGArGCAi rGGAG6GC GC GCCGCC ATCCTAC TC AAGCAGAT TGACAV6TCTACGGrTAG TCrCGAGAAG rTTTAGTAAC rGAGcALZ T A rcrs .@GFP.C..urc53 C.O. unC6J LA RYL R RR AV E E Y RL T V MP S EL F K I 1 OFF P I A Ear I IKspa321 EcoR I Spt 1 84rn Hl C-T TCA6GCCGTCAVAA VT r*TGAGAAAACGAA-rCTGTTGATGiTGACAGiTTGGTCCAAGAGCATGCTrGAACrGrCC TC VAACTGTCCAVCGA rCCC G. rGCC*;GG6TTAT VAAAATAAC TCTTTTGCTTAAGACAACTACACTGTCAACCAGGTCCGACGAACTGACAGGAGATGACAGC VACCTAG-.2 eGFP.C.a.urnc3 C.s. urc53 L 0 A. V N N F I f K T N 5 V 0 V T V G P R A C L N C P L T 'V 0 6 S G3.urG.GTCATVCIATrTrG2AArGAGAA'C T-.CATT 7 rCATATTTGGAACGTGTGCTA 3AaATGGCAAAAAAACCTTCGG rCGCTrC.A:T7CTCC C 4: T AC: riAAGrCTVAAC: TAc rCT TGA.AG 7AATATAAACC TGCACAC TC:T~CTICG T TrT TT GGAA6C3. A~cACG T:AA6"C 7 .GFP.C o urx53 C.s. unc53 REv F I I L1 N E NE F :P y L E A V A A 0 G K K T F G P C T 3 F E Bam HIi TlhI Tth ~c AGG CVZATr TVACCG6:ACZ: CTACCACVVTTGGCCVCTraA CG GTTTGCA:;AAGV IGA7TCTGG:.:GA eGFP.C-.*.unc-53 C.e. uw-S3 168 Tuesday. 18 November 1997 10.35ag1 fig 30 pEGFP72 (1 3-9897) Site and Sequwrce I apM I IXho I Sphl I cc IGCCAACTCATCCCGACAACACTTCAATCCCCTCGAG rCGrTGATCCAAT7GCAGCrACCAAGCArCAGACCATCGACAACArTAAGCr ~GrACGGT TGAGTAG6GCTGTTGTGAAGTTAGGGGAGC TCAGCAACTAGGTrAACGTACO* VGGTTC TAkGrCTGGTAGCT6TTGTMA.CTrGTCTTcrTGA @GFP.C.a.unc53I C.a. urxlS3 P A NS5 SR0HF N PL E SIL I Q L HA TX M T I DON I. TE 0 IA sp 718 G A TTAGAAG AGAGC GGAGA GG6GGCGAAAGGA AT AGAAG CA TGG CC ATGG AC TAC TAAGGGG TAAAAGGGGGA AAAGGGG GG T TAAAGGGTC T TGAG.~.

SNML L S P LP P FP y L T G T F P f F PLF PP tISO NILL Xma I jS I a p a I ~nI GT TCCC TTTG TTCC TAG TCCTCCCGGGTGCCGACGCCGAAGC GATTTAAAAACCTTTT TC T TTCCGAAACA T TCCCATTGC TCA TTAA TAG TCAAAtTG CAAGGGAAACAAGGATCAGGAGGGCCCACGGC TGCGGC TTCGC TAAArTTTGGAAAAAGAAAGCTTTGrMAAG.GTAACGAGTM TTATCAGTTTAA;- F P LF LVIL P GA 0 A EA I. KP FSFR N I S HC SIL IV KIL Xma I )(ho I B ami M ba I O I AATAAACAG TG TAT ACiTAAAAAAASAAAAAAAACACAGGGGGUGGCCGGATCCACCGATCTAGATAACTGATCATAATCAGCCATACA&I TrATrTGTCACATACAT5.4TTTTTTTTTTTTT7T.-TTGAGCTCCCCCCCGGCCCTAGGTGGCCTAGATCTATTGACTAOTATTAGTCGGTATGGTG-i N K 0 C M Y L K K K K K (IL E G GP G I M R I. I To0 H NO0 P Y H JDra I "am Ia C P GF T CF K K p D P pp E PE T N EC N CC C I V Y C 8s, I CTTArAATGGTTACA-'ArAAAGAATACATC4' A.-T rc.ICAAATAAACATTTTTTTCA:TGCATTCTAGTTGTGGTTrGTCCAAACTcATCAA-, LI LJ 01 K 0 H H K F H K S I F F T A F .1L V F V 0T H C MIU I Ssp I 47TTAACGCGTAATT:1±."GCGT TAaTAT7T G TtA:*A4TTCGCGT74ITTTTTGTTT4A.TCAGCTTTTT,aACCaaTGGCCGAATCGC-.4 7ZZ.,rZC aTa%,F-~r...TtArT-C A rrAAAAC4.!TrAGTCGr.%:rr:trTAcTAC~CTrTCC.;T- I r T K L ALlI F C ts S :Z IF K S 4 I FL TN 'i P K S A 169 Tuesday. IS NOVwmbce 1997 10:3S Pagi, I I ng 30 pEGFP72 (I 9697) Silo and Sequence A A rCC T TA TAAArTCAAAAGAA TAGACC GAGATAGGG TT6GAG TG TTG T TC CAG T TTGGAACA AGAG tCC AC TA T TAAAGAAC GrGGAC T.CCAACGTCAAA T TAGGGAA TA T TTAG T T TC TTATC T6GC TCTATC CC AAC TCACAACAAGG TCAAAC CT TGT TC TCAGG rGATAA TTGCACCTGAGGrGAG T T K S L IN OKXN A P RA. G V L F O F 6 TRyV H Y W T P 7SI GGGCGAAAAACCGTCTATCAGGGCGATGGCCCAC TACG TGAACCArCACCC TAATCAAGTrTTTTGGGGTCGAGYGCCGTAAAGCACTMAATCGGA4CC CCCGCTT TTTGGCAGArTAG TCCCGC rACCGGG TGATGCACTTGG TAGGGGAT TAGT TCAAAAAACCCCAGCTCCACGGCATTTCGTGATTTAGCC TTr G E K P 531 R A M A HY V N H H P N 0V F W G A G A V K H I1G T e TAAAGGGAGCCCCCGATTTAGAGC rTGACGGGGAAAGCCGGCGAACG TGGCGAGAAA6GAAGGr.AAGAALAGCGAAAGGAGCGGGCGCTAGGGCGC TGi.-: GATTTCCCTCGGGGCTruATCTCGAACrGCCCCTTCGCCCTTGCACCGCTCTTTCCTTCCCTTCTTTCGCrTTCCTCGCCCGCGA:.CCGCGACCG L K G A P D L E 1 0 G E S R R TyVP E R K G A K A K E R A LGA V AAG rG TAGCGGTCAC6CTGCGCGTAACCACCACACCCCCGCGCTAATGCCCGCTACAGGGC6GCGCAGTGGCACTTTTCGGGGAAATGTGCCGG4 7 1 TTC ACATCGCCAGTGCGACGCGCArTGGTGGTGTGGGCGGCGCGAATTACGCGGC.A TGTCCCGCGCAG TCCACCGTGAAAAGCCCCTTTACACGCGCC I 0 V A S A C A P P H P P A L M R R Y R A R 0 V A L F G E M C A E rpH I ?Ss' 1 p 3 2 1 ACCCC TAIT TGT TTATTIT'TC TAAATACATTC AAAA TGA TCCGC TCATGAGACAA TAACCC TGA TAAATGC TCAATAATATTAAAAAGGAAGAGT: TGGGATAAACAAATAAAGA TTTTG TAAG TTATACATAGGCGA TAC TC TGTTA TTGGACAT TTACGAAG TAT TATAACTT P L F V YF 5 K Y1 01 C I R S5. D NN P0DK C F N N I E K GAR V Pph I Ava If0 OxaN I PvU 11Ii L Q QK E P AV E C V S VAR V V K V P R PS Q 0K Y AK H AS Pph I I Ava III Nsi I TA&G tCAGCAACCAGTG G:AAAGTCCCCAGGC7-CC:zAGCAGGCAGAAGATGCAAAr.CATGCATCTCAATTATCAGCACCATAG.C::GCCCT4 L v S N 0 4 K V P 2 P S A 0 K Y A K H A S 0 L V S 4~ H S A P I AsiCi I A H P A 2N S 4 a F A P P S A P) V L r N F Y L C ;G P L 170 Tuesday. 18 NOVOinOW 1997 10:35 ld 6~g 30 pEC3FP72 (13 9697) Ske and Se*,.dca .Stur 11 Ca I C TC rG AG C rA TTCCAG AAG TAGr TGA GGC TTT TT TGGAGGCC 1'AGGCT T TTGCAAAGA TCGA TCAAGAGACAGGA 6AGG A M3 T T CGCA r3JTTG.A GAoGAC TCGATAAGG TCTTCATCACrCC TCCGAAJAAAACC TCCGGArCCGAAAACGTTTCrAGC TAG TTC TC TG tcCY T.cCc rAGCAAACG TACT!,I 7 L .A IP EV V AR L F WA P RL LOR S I K q 0O0E 0ORF A.

BSPM I Cma III ACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTAT TCGGCTATGACTGGGCACAACArACAATCGGC TGc rCTGArGCCGCCGIr- TTTCTACCTAACGTGCGTCCAAGAGGCCGGCGAACCCACCTCTCCGATAAGCCGATACTGACCCGT6TTGTCT6T rAGCCGACGAGACrACGGCGGCAC tj K m 0 C T 0 V L A P L G V R G Y S A Mi T G M N Q 0 S A A L M P P C T TCCGGC TGTCAGCGCAGGGGCGCCCGGTTCTTTTT6 TCAAGACcGACCTGTCCGGTGCCCTGAATGAACTGCAAGAC6AGGCAGCGCGGC rATCGTG AAGCAATGGCCGGGCAGAAATCGCGACGCAGGCTCT TC TcrCG TCGCGCCGATAZ;C.CCZ, G C 0R RG AR F F LS RP TC PV P M NC K TA9 ORG ITRG Sail fSO TI G3aCCACGACGGGCGTTCCrT~rGCAGC TGTGCTCGACG TYGT1CAC TGAAGCGGGAAGGGACYGCTGCTATTGGGCGA.IGT6CCGGGGCAGGATC TCC-7 ACCGG TGCTGCCCGCAAGGAACGCGTCGACACGAGC TGCAACA6TGACTTCGCCCTTCCC TGACCGACGAnAACCCGC TTCACGGCCCCGTCCTAG.%.A a PF A L AO0L C S TIL S L KRA E G T GC Y VAK CRP GA R 1 G:ArCTCACCrTGCTCCTGCCGAGAAAGTATC:A-,CATGGCTGATGCATGCGGCGGCTGCATACGCTGACCGGCTACCGCCCATCGACCACCAA

CA

3 TAGAGTGGAACGAGGCGGCTC'.TT:ATAG37STACCGACtACGTTACCCGCCGCGTATGCGAAC TAGGCCGTGGACGGGTAAGCTG3T~T- C m L T L LIL P A K Y'S V L .4 0C G G C I A L I PAL P A HS T T I Ear I IKspS32t 3 C TITT CG AGC ACG7CTCG GAAGCCGIAC IT CGACAG CGAG TCC TACGAGC TGC C TAGTGCCCC C-G.: AN I A S S E H V L G V K P V L S I A M I V T K S I A G 5 R 0 P if Pph I tdc I ?CAAG C5AGCAI-C C AGATC TCGTCGTGACCCATGGCGATGCCTGC tTTCCGAA,. rCAT3GTG.AAATGGCG-T7 .1':4AGCGGTCC-GAC;TTICG:TC:;TA.Z33C TGC:-:C :c IAGAGCAGCAc TGGGIACCGC IACGGACG ACGGC!T *A~iTACC.CCTITTACC6GC..:GA S 5 D ASRR A C P T 1 IS S P M A MP A C A I S V K M L Ear I (ag I Rs, 11 :Ksp6321 2A: C rTAAG T GC TG AC-:CGZCC I 5rCSA TAG T^C T3 TATC5CAACC GA TGGGC AC T.1 TA AC.j: T TC% i:A.:,CGC CS:T r-zC:, F I- D 3 s t I Q T .A W L P *4 1I L 4 3 L A I l 171 Tuesday. 10 November 199 7 10:3S5~ Mg930 pC-GFP72 (I 9697) Site and Squence Gc rGACCGC T TCCTCG TGC rT TACGG TA TCGCCZC TCCCGA T rCGC AGCGCATCGCC r rc rA rC Gcc T TC r GACGAG r rc ri TC GAGCG.GGAC rC i CGAC TGGCGAAGGAGCAC6AAA tGCCA rAGCGGC GAGGCTA.GCG rC GCG TAGCGGAAGArTAGCGGAAGAAC TGC TCAAGAA6AC TCGC~C TGaGACC: L. TA .S SC F TV S PL P1 a I SASPS I AFLI TSS3ERDSG Au 11 Bopt I GTTCGAAAT GACCGACCAAGCGACGCCC AACC TGCC.%TCACGAGATTTCGATTCCACCGCCGCC TTCTATGA,%AGGTT'GGGCTTrCGGAACGTTTTCCGG cAGC rT TAC TG GC TGG TTCGC TGCGG6 TTGGAC GGOTAG ;GC tCTAAAGC TAAGG TGGCGGCGGAAG.ATAC TT TCC*ACCCGAACCTTAGCA&ArGCC V A N 0 A PS 0 AQPa A T RF A F H QR LL K V G LR A F P Na. I Ks3PI Ar 11 I I

I

GACGCCGGCTGGATGATCC TCCAGCGCGGGGATC TCATGCTGGAGTTCTCGCCCACCCAGGGA6GCTACGAAACACGGAGGAGACAATACCG.

c rGCGGCCGACC TAC TAGGArGGTCGCGCCCC TAGAGTAC SACCTCAAGAAGCGGGTGGGATCCCCC TCCGATTGACTrTG TGCCTTCCTC TG TrATGGCC GA RALO0 OPP AR G Sm GV L R PP .G E A N T E GO0N T G r apt T TCCT TG GCGC GA TAC rGCCGTTAT TT TTC TGTC TTATTTTGCGTGCCACACCCAGCAACAAG TAT TTCGCCCAAGCCAGGGTCCCGACG

TGAG

A NP RY V GN K K T E .mA R C VVV C S T R GSV P G A L rGTCGATACCCCACCGAGACCCCAT

TGGGGCCATAGCCCGCGTTVCTTCCTTTTCCCCACCCCACCCCCAATTCGGTGGGCCCAGGCTCGA

ACAGC TATGGGGTGGCTCTGGGG TAACCCCGG TTATGCGGGCGCAAAG AGGAAAAGGGGTGGGGTGGGGGGTTCAAGCCCACTCC66TCCCAGGT y C R Y P T E T PILG P 1 RP RF F L FP T P P P K F 6 RP RA R 'wn I OxaN IF prI fra I 3 .:CAAGTCGGGCGGCAGGCC:'TGCCAAGCCA':TTACrCATATATACTTTAGATTGATTAAMCTTCATTTTAATTAAAAGATCTAGG.G 3Q a RA G GARP Cx H i Si L ALL (YT L D. F K T SF LI. K 0 L6 E pX I 0 P F .S H DO0 N P L T ,V F V PL SV A P R RK 0O A I F IA ArZAUAAAGACCC A TTAGACG ACG ACG T7.5 T rT TTTGG TGGC A TGG TC C CACC AC AAAC GCC TAG TTC TC A TGT AG A.AAGG S F FS A R N L L L a K T T A T S G G G a 3 A A T N s F S Ssrl M 4 L o a 0s A o r P 5 S A v v P 0 P L 2 E L C 3 T I Tuesday. 1O November 1997 10:35 Page tt flg 30pEGFP?2 (I 969 7) Sile WW SeQuencs ACC rCGCTCTGCTAATCCTGTTACCAGTGGCrGC TG,:CAGTGGCGATAAGTCGTGTC TTACCGGGTTGGAC TC34GACGA TAG rTACCGGATAMGGCGCA T GG AGCGAGACGATTAGGACA ATGG rCACCGACG ACGG TCAC CGCTA TTCAGCAC AGAATGGCCCAACC TG AG r lC TGC TArC AA TGGC C TA TCCGCG P A S A N P V T S 6 C C 0 W R V V S Y A V G L K T I V T G G 4 ApaL I G CGG TCGGGC TG AACGGGGGG T CG TGCAC ACAGC CC AG C TTGGAGCGACGACC TACACCGAC TGAG A TC CrACAGC GTG AGC TA TGAGAAAGC6CC CGC C ACCCGAC T rGCcCCC CAAGC ACG TG TG TCGG TC GAACC TCGC TTGCTGGA TG TGGC TTGAC TCTArG GA TGTCGCAC TC GA TAC TC T TTCGC A V GL NG G FV H T A 0LG A N 0LhR TElI P T A. A MRK A AC GC TTC CCGAAGGGAG AAAGGC GG ACAGG TATC C GG T AAGCGGC AGG GTCGGAACAG GAGAGCGC ACG A G.GC TTCC AG GGGGA AACiC C TGG TA T T GC GAAG GGC TTCC CTC TTTCCG CC TG T CC ATAG GC C AT TC GCC GTCCCAGCC TTGTCCTC TCGCG TGC TCC C TC GAAGG TC CCC CT TTGC GGACC A Ta T TT A TAGTCC TG TCGGG6TTTC GCCAC CTC TGACT TGAGC GTCGA TT TTTG TGA TGCTC GTCAGGGG GC GGAGCC TATGG AAA AACGC C AGCAACGCGGZ AAA TA TCAGGAC AGCC CAG CGGGAGAC TAAC TCG CAG C TAAACAC TA6AGCAG TCC CC CCG CC TC GGTACC T T TTTGCGG TC G T TCGCCS; L S C AV S PPL T. A S (F V ML V RGA EP ME KQ 0RG Ave III N4si I C TTT TACGG TTCC TGGCC T TTTG C TGGCCTT TS7CT :AC AT T TC TTTCC TGC GVTA TCCCCTG ATTC TGTGGATAACCG TA TTACC GC C ATGCAT G AA A ATGCCAG CCVAAA ACG A CGA;T AC AG A AAGGACGCAT?6G GG ATAXGCAC C AT T GC AA ATGGC G GTACG TA L F T V P6 G ILL A F C S M V L S C V I P F C G6. P Y Y H A \4orday. I December 1997 14:12 PijI 2. 0 0 buJ IGAIl ACC ,'.AAl CTT(3C -G-CTGC.AGGAATCGATATC7AAGiCTTAMZGATACCGrC*GAC(:T .,A(6ATCACAACAAATTCACACA.7C:!ACA rCCA r rA m*CCAMGCCGv r r R: r'AAG rG Ai; rr rAA lI TTr r r rACGAr rAr.4AAATr-W7TC rTTAATAACTATC TrCGACTTGAGrTA rC TG ATCACT V' U Ar, T7rTTGAGTGA7TT7TCA7TGA0,.Ai5 TATTAAAAGGAAZA rTA r IrAC rrTcc r rArr GCCCc TAAC 280 r, r GA7TTACiTT rrTTc.Ar CAAC TAGA7:rrACAAAAc-TriCAATACAAT rCCArrTTCACArTAC(Cr C 36A) 360 3?o 360 390 400 (40 420 L 'C AC GT G CGC CACG-C A GCA.'AC C G' T',AGC AAc r.A C C AAA,' rCCAAC 1'1 rCCAC AAA I Gi IVA ACA 4120 \C AGGCcT-1A Ar-TcCCAC ACTCAA GA Arc:GAAAATrG GrAGAAr r -A rr r r GAcr:rCAAAc r r(;r I C1 7T- rACGG CTT 1 A,7C CA4 TT TC-:AG T T A TC AAA AAA r r r %CcAC f4 A( A A Arrs A(AAA AG r 61w.

I I fX AC AAA I< 1* ICAG r fGA'X -I TT G A AAGA TCC AA A TG:AAC PV. AA,'C'rTTAA ,cO 710 720 7.30 74I0 MC 7W0 770 G G 4AUL AG'-G CAAG I*C A IAC I Zi rA T G4 AA T CrT (T 5A4, TT C A T-GT TG AAC-A-r r-.r C AA G CC ACT GT ^AAAC C TT TT(. TArc.A AAAG TAA 1 1 d40 A,'AAoC TAUTTC AAA:C C CACCGZ A rc G -r r:A AC TCTTA A TTT TA AAA A 7 T.GCkATTTAC 410 -1a *C 6Ct:1 C I' yrG 'GAAAAG: CeA-CAA At- CAATT Tzr CG.,-C TTC A rA AGAc r r r rAAA r r pt *G "G CA CA; G- AA AA 2-A A G A G TC r, r r: 10!;0 *C0 1070 10 1 OSA 1100 11 10 1:20 G :C sCC crCc:T C:c' rC. A r c rcc at-AzQAAC AA -TCZ AA TTTC:G rT M.A rr rA rA- I I M Sr;. rt.; A 3tA. CTs-C,\C: -34Ar..GT~tATI.1 rG(AA IG A2 -AC 7jrcrr CC7 CACC rijA CC TC T C ATCATGAC AtT TAA TG TCGG rTrT TorG 1330 1C ~T ,T-C -A C:G CC AC T A. 7 rrTAC AAAAc. r 33A.AAC A I II AC TA r C*(AAGCC 1 4rX' r r T.1 Trrrrc\T A r r'AA r ~GCA r4C"(,A TAA AC AC GAC v rAAAAGT T TA rTTA'AAAA 1'4 ArT7T u grTT7A'AA" I. !GA A ,,T7CAAAAAA7*AA rAAA Ar ir AACAAA TTGIA.TCGl. 1 tj(4j I AAAA riI' *1f' 4 G .C C Tr4 AIG I' TA 7 7TC CTC, A C'-TG r '16 GTC.l:AT-Tl'CCAT7,CTArGAA r*CGC rACTCAGCACATATCC/AA,\ 16Nl0 1 T A,7 rA r Ic AG TAAT AAAG E' tnA A IAGAAT rTT.AA TTC AAG~ 1760 107 70 AIM I -TidZ,V, TTC AA l;::,AAATTCAcCCAG3TT-,-'AZAA f Ii I CCA TGCTTTTTGG. C I S3A r. cAC c;CAS C7 A C*A TAAAAA~GTTV:C AAAAGCC- CUAC t- TT7-A;AAACtAArA. .'-ciCGi GGACc'CT-TCATCTC:G V VCG cr j~ rc,-4 Lx~; I ~G~I TA~~C~kiA~TTAGT ArATTTA -iACC f IA 'TC I TrATrjCAATACATA 2 iGOX* 21:0~ 12 N 2 I~ 2 WO .117o a~W eAlArAGG AZCG3C(A.-A TAT~ r IrAAACG. r r cAAGAT^T*TAGG:.A !\Tr:,'GcLk ;c ACTATATC'fA .T rACATCT A;TACAI.1' AACCAAGCA W7tM' r rtcuArF I A' 8:10 .1I ~C A'ACA G ~w:.~VCAA'CAGA.-AGATTTCCA-lPA(. 1..AA! I ,2 Monday, I OCCCmt>e- 1997 14-12 0 z 2480 2470 2180 2 (1 O 2600 2510 256120 A AArCGTT TCcALNTrrc r rtir lACrfiAAA6.rTTAAT( (.,CTGTC,A7GCCAAAATCGTrATCCCTTTC'r. -C CAT A ACCAC rTCAmr,.AAA f AA A.:AAA -!CATTCCC TCCG TCGCOCCTTCGAGTGGrC AATAATA/' T 26W3' TGC'~rTCCACCrATATCrACATCTGCGAAGAGCTTAGG TATCCGATCCTTCCGGCT TC TTTTAGAAAT rr ,)rx -\7ArrATTCAGAATCATCATCAACGTA(.Aci, rc rA7TTrGaATCTAAACCCACCTACrif.CrAA' *rCCA 2MI~ 2810 2820 2630 20'40 2850 2860 28170 aAAcc r rc I.AG;ACACAAACCC (7CTA:3TTCGTGT7GCTACAA(-AAAMTCIGAi!.CTCAAAGCTA 2 J/Q' .:'-GC-cCAAGCCGTGAGCA-CCCA,'AMCT TGCT.-C rG-rGAAGAc1Ar F G(SAGZ AAAAC AAGACrC(2G C 4 T A Ar 3CGGTGGA,\ccrTC GAA-Gc (iGTGAA T( T~rA rcAArACAAAr~ r TGAA r-c c V Jof1 3150 3170 :11 w 3I1Y) 3200 :3210 :22 ArtCAGrG AA-A TC:.GT"J^rCATTC-AAC ACCACGTCrGCCAACGjTCATCATCC.ACGCAJ\ 3 29W Tcc TICfA6~ rCcAA T Cr 7C AA GA GTT,- AA ZGT C AGACAAAT TCGATA. T 7A TC AACAA C C CT GCCAAAAAC Z AAC Ac rGi;C AG *G AAAGGAj fG AAAAW ACA GC GAAAAAA :3500 'J620 ~33 3A'4O 3550 35GO 1611 A, rcc Ac c T-CCAG C TC GC'C AC.CYA.' A C Ar.(CC AA'Ab TC GGAGT T"rc TTCTC AA T rATC,C(: C A T a(AG-T'C AA4-G AC CC CGTGA TAT:7G AA AAA iC AG AAC C TGAA A ArC rC CA ATC AA 7rAic AT M6ilk AA:3 G~2TC VAAAA iA.aTTTCCCTAAAAATGACTT,^AATCCGI\ '31) AT CCGCG-- C7AAGA: TCCA-: 76CCT.2'GCGTA-G~A!CrC GAAAAAA. lAFO UK) lrj87G 3660J 3800 300 :1(1 Ir)1 -1) I I I I .1 Li. I I I I. SL.-I II L L~s. L% L L L .L rrc' rC -TAArCATT(rt:- 7CGACAt, AAAGAAl TAAGAC.TC4GAATCCAJ3i-GGC TA\CACCrCT 3,;2 C .5c r' r r GA i; G GC C CAA A AT( Ak- CAGAA GTAAAG. A A ;A r GA'-*A ;.IA C I C G ICGA G 3GD A A G A ACG GC TA TC C rT71CGMA AG c T-:C T -G TC -aTC -C G AA-A T: CU. TA ACAAC C A *lk~iz C GA -G A CAA TC C(A C. ,AT 7 Vr C C GSAG TAGC AT GG-,A A^,AG T C GCC r C ZAA.AA I'AG, G AC 4 tX TCC CAC TTV GT rC(*C; (A 1*C:::ACGCT 7:TTCC C. A A AC CCCGAG 7LCC(.AC t. GG T'C rCC A$ 1~> 4260l 4280 42711) TAAAT GC~ G VC AU.AGCCA L*2 4 iGG1'-CGCCGC TGCCAC.TCAACC 'TCCLC AACAV rrz ;eTAA(,ATC(:CGCXC.MAACTCA--CCTA.'T rr 1 YIO 4 CA(: (AGATC 7CACC.T^IA. A -17AC %CAATGTC TA 7CCACTCACAGAC7AG -t:IACjACC I'c I I !:o~1 ,:AaAA1-CAC. TCAG ,CCA-TTTC .ArrCACTTC-\-CGTAAATGCC^A:CTTCAAGA~ F cA(:A L 41G C rr A G.C( :CIc r..C T T jA2 CGA GAC G3GG 3 C C C AAC r C' C TC G A AA T C'T 1 I J~

I

7:A GC A TCC C r. cI r VC i~ AA Tr.TA rc t. ri, r 1r A CA I I C IA cf ~I I A, A t T r.A r A Trr AC C-r-:AAAT CC CAA!, r. r'1: Azr(: C^CA~!A;T,'(IC ~aT(,-CAAGTAG.rjAAF.cjr rArrc~jGc. A(tTCGCAC, 7CACj rC, 7~ 4 rc C7C7~ C.Ar AG CAACTA- CACC A r rc- r I s I i I AAA AG1: 7TAA6. A f. Ac T C A r r (,AT CGA TtC\AjA ZT AC C -:AAAt;: AA VA'1 *AVI Monday, 1 December 19.97 14:12 Page -7 4910 11W 49,K 49 1950 '.C4 1970 A r*cAGGCAGGACATTCCrCAT 'Tt1A6i36 rTAGCAATCATCT -7GCArCCAAC TCAGCTCF TCC TA4AC 4911() rAGGCGCr3GTGAG3CrrCrrCGCAA CA!'C fCTGCAATCAGTTGCATCCCATCSATCATCGATG TCAT 50,11) ;rcrrcAAAA(;CAGCM.GCAGGC#AArc AGiCTTr3A~c rcc; r r r(-GCAAGAA,:AA,,4fACA(;(: rGGA I 5-11C, f:rIGTCCTCAC'lCTCCAA lCACCAAAAAGAAAACAAV.-ToAGACUjAACAArArLcccCAMT 5180 ccGArCTCAAGCAAC i C I IGACAACd TTGATGTGA7TGAGTTGAAGCAA.GAGC CAAAGAACGCGA *A 52 6260 6270 2 X) 5290 530 5io, 631 tGCACTTTAC'GAA3 CCGCC fF GACAATTCr ATCG(,,SCCCGCGAAGTTGArG r rc rGAGGrGA(:ACAG r t:AXAMAt2l I CgALt1 (,\IJTACIC5ir~~ A 33( ;G (TCrT7CTTCCCC.CGCCTCAA TTCC*A 2TA rc ACG--ACGATG.W.CAr,TCTATGATGCAGC~TTGC r 6') *CC TCTGGC TGCAACTCAATCAACGTTAcrGTAAACGT;GA 55x0 ilt I CGi I TAACAACAGTCGAC(3 I'CArCCCGATCTn(C,'C 1.16X) 51) 5620 C-630 416C 560/00~ 1 AAG(J CAG rG .iAAAAGf ,'GACTGGAA ,-TCCGATGAANjGCTGTTTTCCAAGTG r rAAGGA: rA tAI [C 66~70 CTAAAATGrGACCCAGCCTCTACCCTCZ";C TAAGCAC iGAG I*CA r(CrA rGtCTACAG, ATCAGCCA-G7 h 4 GAMACGAMT; TTGC.AT\^C AGAC CCC M:GA2A TGc c rcc r rlac:cci~r I'G I CA\APAc-ArArC AzrC 5610i Tt:CC:TCAA.ACGTCTGNC,'.,AGAAA. rCC TC GA AGCcC.C TUTCG5AC GCrGATcCzCiAAGCCGA rGA 5880O 69860 5970 F!(E 5990 6C'0 80 ;0 602 i;ArtTACCT',ACCAATCGCTTGGCCC..ZA:-c ru; rc;GACccC.C TGGrCCTGAGi TC\AAGGGCL\T-C AG(:Acc r rCAACA rG5ACC AC-C A.G TCT TC- riA.CATC rGcAACTGTA'rc r r '.r.ATAGACCG3GAAACAGGAAT rc-sG3c-, r(,c cCCCT .T ATTC-AT~ATGACc rGAG r AAGCAGG 6 tCCATr:AGT,.AGTTGC-Tc-AATGGG*"CC:C rC-ACCr(; AcATACA'M iC.TsF C_ r 2C SrACC I*G AAA AATC.ACAC'lCCAACATGGCr r~iAC! fiAGC !TCAG -Gr- 610- 630 6320 6350 6-1. t., -AACG T5GAGC CAGCC .ArG'3C T-Cz7-. rcs r Gr AC r-TGAGG4GGAAC; C TGCAG AGr CAIJACA~C!i ,A A A 'CAAT('CCAAC AAGG AAG AGt: rGc: r TG rA-7GTC-C~rCT GTT AT.

,;A~vr2AGGACr7C.Z3GACCTGGV C ('IA::CYC TG'AAZAACTrA 6r,1 fr~AGA~~'80 A C AAGGA rcC- GATNAGcG A i AA AA 4GZCr~ T 5GC~GAGGACC C AG i1G XATGGG .CGCCAC 63 E660 66 0 1 F 6700 V 7? AZ'\ACTTCCCT-GCA'rCAIc-CAAjCVs53 'CCA4 CCAAACCTGTACCAk--r:CC i CCCA.CGTGI-LL. 0 7 2 0 I CACAGCArrrCCTCACcrc ,CTT [3CAL-A A- "'X -TC rjCcA rG,.\AC TCA %A A(C GCCA\C TA:ArTG AGTC7C~CGA !C'AGAAACC C,8 'X rcT!C-(CACC'AA(r2TTr.Cr,tA.A:- rAA G-.rTT-G-QC~aATkATJ rG-ACCC-CCGGt4CAZCACA-\:- li Gr.. IGCCATcAGCTA 7CT rTC7-CCCCTCTCCTCTTCAriACCAC~IL-(/:C;*IrC A(:CC'CAc.,- 701o0 /2 1; 70C !C.LI0 7050 Ot ?7 C r I C f i TGC TrG-4cc rTTC- AA A;% r 'G:\AC.A rGTGGTC A ACTTGTGXcccc FAAACA-47TTA* .c.cc TU r^ A r: 'i j-CAAAACATliAC f TTc:GA:IaIGT~TLAC~G

I

7 V~GGACZ~C7TCAAAAAA CAa~A ~C GCAC A(:,TCCCCGGAAITCA'C fA 7:07 -Monday. I December 1997 14:12 Fq g PC8S01 736C -7370 7200 7090 74e00 A1) 7n A: 1 (;crcccc rrGTCATCAGATCGCCATZTCGZGCCCGTGCCTc rGACTr(: rAA:; rcCAA r "Ac tTTCAAI" 7'12:) .4c,"CTACATGCTCT rx rcc:c rc i i~f I'CC.:ACCCCTAT rTTTG-.TATTATCAAAMMAC TTCTTC rTA T) AA!CG IAA r*AAAAACTCGAAAAAAATG7'3 C 7CCCTCCC CCC.ATTTAATA ATT A TC CC AAAATC T /6:9) A ACAATGT rCrG rci'rACAC rrc r i A i',r rTTTTTACTTCTGA.AAATTTTTTTTAAACATCA AGAA 77%^W) 7710 7720 '1~K h/U0 77WC i/iUo A I I I. I I I1 I I L I&AIU.

4AAAG CACACAAAA TACCTTATC.4ATG T ACG TTrCAG rTTArTGACC GCAAT rA T r Tr T-CCA 777r, 'c rGGGC CTC TCATGACGTCAAA T: AThCT!C'ATCGTGAAAAAGI TTTTGGAG TA7TT TTC-G &A TTT7C MU'; A rI*IC G A.",Gacr,r c, r rrTr(: MICTA.AA CACAAG TATTGATGAG CA:GAT 3CAA~ AAAGANT:G%^ 71:^70 ;AGG T T T TG AGi;,:TC-. -Y .AAGG :AG r AG;AAG i mrrA Ar ff~iAAA.; I G.o I fa; G 8 0 aodo 8090 SIXc 8!0 3!20 rACGGGCCC TT 'CGT.: CGCGCG 77rG C7.'AT GCG 1 A GAAC T~AAAIAGT''= I q) -A(U FCACA~3c r;rG rc rGi rAA~jGCGA I C GGGAGiCAG ACAAGCCCGTCA-3GrC 3CGT: AGjCCGC VT fk260 rTG TC GCT:,(CTTAAr- TICGG CATCAGAGCAG a-TG rAC rCACK; r(cc-.CA rA rCcGG 8117 TGAAATACCGCACA~iATGC GAA .AAAAATAC,:C(cA rcAGC*;..G--C rAAG;G:iC.r r'A;c; n ur( WO E20 81';-40 A4. WOi2 84S 7.A(G(CiGGAAC:,.CTATTTCITTATT7hTAAATACATTC AAA rAT3'ArcuCc r: 4 IGA ACAA 1 AACC 05354) -Tr,.7AAATGTCAA TCA::AAA1I rA GTA7-T' CAT7T,:,T ~Gr:GCCCTT tW A A CrTr5 GrTGC. A CG AG Tc: 7 -C 4A C ATC C A A-tG C G CTM AP; ra.-TG AGAG r r r 57-r V360 ~3770 2780 8790 88T, 68'10 882 .A(;CCGAG3AACIV rr:AA C:IG I:TCOrlicrAAtGCIM 7t ,j ACAC TCCGCCAAC TrAC T7 'rGA t.CGATCGZAGGACC-CGAA(:Cl')A~C rAc(.rrr7TGCAC. rrACACCACGA I rAG~a. .GGCAACAACG r r'GCGC'C-AAA ATTtACTGGJCGAAc r r,^c 2170 1. :AGCTTCCG rAAAG I iGCAGlGACCAC-TzT'Cr-CI; 2' r,:CT-cC^ t7!rCG:rrATTGC 'AA!,Tc rG.;C~ ~r:s( r (,AC.-AAA AC A0A 1A p T AGC:T Gi~c r; AC i G A r, AAGCA-TGG3,AACTl AA 9~7QG500 I f r rAc rAAAAATA TTM3AT T TCAAI:

T

TC r r A ATT T AA A AG:,AT c r AG G A ASA rcr ~1 TC7C C S~:-TA Ca CC C ~CAA~S T-TCTTT rGAG.A I CC* T rrrTC tCGAT TTCTT AAA 'A.'.AAAA:(.:At s~cTCCA2~cr:;r T$T~cc.(.Ac,1A~W.CCA..T.TT~C Au 'JO

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1 C(2~4 A.~TCTC rc..~A'~rA3. :r3 ''A~:A2AC rcA~A c r 1.180 Monday, I December 1997 14:12 fig PC8501 oo010 94120 98400 OAR) ORO W AGCACCGCCTACA I ACC (CCGCTCTGCTAMTCCTSTTACCAGTGGCTGCTGCCAGTGZCGA-7AAGTCGTGT 2M7 C I I AC:CGGG TTGGACTCAAGACIArAc- rgIACCGGAAAGGCG.AGcCGTCGGW.(TG dIACGGCUG;( r rcc i S ACACAGCCCAi73c r TC-GAGCcAACGACCTACACCGAAcTGAGATACCTACAGCG-3AGCATTGAGAAAG ICO 1 0 CCCCACCCT TCCCGAAGGGAGAAAC.GCGGACAGr4TA rCCGG rAAGCGGCAGGG TCG AACAGGAGAG.-GC 1(XW; 1C, AC6A GGG AGC TT CC AGGG GGGAA A C CC TGTA TC T rA rAG rc C TG TCGGG T TTCG :C ACC TrC TGAC T TG 100 mc.i 10160 10170 10180 10190 10200 10210 10220 A6CGTCGA r r rr*TG NGA rnCTA GCA30C*GGCGCGGAGCCTA rGGAAAAAC(.*(A;CA~AC(*,'C,((:c: I I I 1I AC (G r rCC IGGCc rTTTCCTGCCCTTTiCTCACACG r rc rr rcc TGCGTTATCCC TGA-Trc TGTGGAT 102Nu AACCGTATrTACCGCC' rI KAG I GA~crGA rACCGCCGCCGCAGCCGAACGACCGA SCGCAC GAG TCAG IOJWiC C,'AGCGAGGAAGCCGAAC.AGCGCCCjAATACGCAAACCCCC ?CTCCCCGCGCGT TGG CGATrCArTilAT rG ('L(:CCCACCACtGCITTCCCGAC7 AAAGCGGGCA~irGA(CCCAACGCAATTAATGTGAGrlAGC IC IMOC' 10510 10620 IOM 10540 10660 1C580o 1 C G/A I I I .1 S-f Is I A" TCATTAGGCACCCCAGGC rT rACAk r rA rGCT-CCGGCTCGTATGrT TTrGG AArr G rGAGCGGA r *lt.( ,A4AAArr FrCACAC'AGGAAACAGCl 178 Tuesday. IS Nvember' 1997 10O.9 Page rhg 13 pCS2O1 (I 5082) Site and Sequenca

C

Enzymes 100 at146 enzyrm (Famed) "I")j Settings: Linear. Certain Sfta Onty. Standari Genetic Cade G;ACGGATCGGG AGATC TCCCGATCCCCTATGGTC6ACTC TCAiGTACAArC TGCTCTGArGccGCATAGr rAAGCCAG7ArC TGC TC:Tcr. TS T6 16?T C T~GCC TAGCCCTCTA.!AGGGC TAGGGGA TACCAC2C TGAGAG TCATG rT AOCGAGAC TA:GGCG TA TCAA T TCGG TCA TACAGACG;CeACACAAx- T 0 A E I S R S P M V 0 S 0 Y N L L C R I V K P V S A P C L C V GGAGTCGC TGAGAG TGCGCGAG AAAATTTAACCTAC AACAAGGcAAGGCT TGACC GACAA TTGC ATGAAGAA TCTC TTAGG TTAGGCG r T T Ta.3 CC TCCAGCGAC TCATC ACGCGC TCG TTT TAAA T7GA TG TTG TTCCGTTCcGAAc TGGC TG TTAACG rAc rTC TTAGACGAArCCCAA rCCGCAAA.r- .S 66 A.R V VARE 0NL S YN K A R L OA ILN E E 5A G. A F C C TGCTTCGCGATG TACGGGCCAGATA TACGCGTTGAC AT TGA TTAT TGAC TAGY TATTAATAGTAATCAAT TACGGGGrC AT TAG TCATAGCCCA y.A TA G.Z GAAGCGC TACA GCCC GG TCT ATArGCGCAAC TGrTAAC TA.ATAAC TGArTCAATAA rTArCATTAGT AAr0CCCCA;TAA rC AG7TCGGGT-A A A sAc r ~G0 1 YA L T LI 110 LL I V 1 N Y G V I S S .P1 TGaAG TTCC GCG TTACATA AC I TACGG rAAA TGGCCCGCC TGGC TGACCGCcCAACGACCCCCGCCCATTGACGTCAA TAATGACGTATGT TCCCA TA.G- ACC TCAAGGCGC AA TG TAT TGAA TGCCATT TAC:GGGCGGACCGAC TGGCGGGTTGC TGGGGGCGGGTAAC TGCAG TTATTAC TGCA TACAAGGGTATCA G V P R Y I T Y G K 4d P A V4 L T A Q A P P P I D V N N 0 V C S H 3 AACGC CA A AGGGACT TTCCA T TGCGTCAATGaG TGGACTA TTTAC6GTAAATGCCCAC T rG6CAGTACATCAAGTG TA TCATA TGCCAAGrACGc: T TGC T ATCCC TGAAAGG TAA CTGCAGT TACC CACC TGA TAA ATGCCA T TTYACGG GTGAAC C GTCA TG TAG TTCACA TAG TA TACG G TTCA TGC GG3 N A N R 0 F P L. r S M G G L F T V N C P L G S T S S V S Y A K Y A C CT A TTG-1C G TCAA TGACGGTA ATGGCCCGC C T.CGAT TA TGCCAG TACATGACC TTA TGGAC T TTCC AC T TGcAG ACA TCTACG TA TTAG -C 4 GGA TA AC TGC AG T TAC TGC CA 7T TAC CGGGCGGA CCS TA ATAC6GG TC ATG TAC TGG AArAC CCTG AAAGGAT GAACC GTC ATGT AGATGC A TAATC;.i P Y AG0. R. 4 A RL A L C P V H 01L M G L 5 Y L A V H L R I S H S.aCA T T ACCA TGG T GATSCX TGC AG TA-AT C AA I GGCG TG GA T ACGT TTGAC TCACGGG GATT TC C G TC TC CACCCC A TTGACG Tr A AZ GAUTGGACCAC TAZGC AAAACCG TCAS T TACCCGC ACC TATCGC CAAAC TGAGTGCCCC TAG TCAGAGTGGGTAAC TGC i T P Y Y Hi G 0 A A V 0 V A V I A V L T G 1 3 K S P P H R '0 rc.,AAACGT TT .%2-TCCT -AGTTrCGA GTTAGZ GTA SS TACCC-;. CTCCGC AC ATGCC 4CcCCT F V L P K 5 T G L S X M S 0 L P 1 0 A N G A A C I VI i; Y V: 0 S S A N H C L L A V A N .Y 0 S L 0 P S w GC 'AAACTT.-C TTACC-'VCGr,.--;TTCCAC--T:ATCATCATCATGG TATGGCT;.,-CTGAC TGAC-':ACC.iLT GCrG.2TCTGTACuA-' C4 I PAT7GA.TCrL CC S NG N 14 14 6 14 GT.GACG :ACGTTTACAGCL1 1 T6 G2 1A6 C Y 1 79 Tusday. IS November 1997 10:09 Page fig 13 pCB201 (I 5082) S4@. and Socuwnce G. 4 GACGATAAGGTAC TAGGATCCArTA TGCC TCC T I3CCG r CGAGGTG IC AArTAACATATC AG TC TCC rCAAAG6I T GAAGGAG.. rGc.G rc,,i G~C TAT TCCA TGGATCC TAGG TATACGGA.ZG AACGGCAGC TCCAc, 3 TArT T rArTAG TC.GAGGGAGT TTCCAG.ICT TCC rC TTI ACGC%: 7r, 2 0 0KV P R I HM P P C A R G V N N I S V S L K G L K E K C V t0 -;CCTGGTGI TCGAGACGCTGATCCCCAAGCCGArG-IGCAGCACTACATA-'CcCTTCTGAGCACCGGCGCCTCGTCCTCTCGGGCCCCAGG;C.

C*aACCACAAGC TC TGCGACTAGGGG TTCGGC T.c rACG TCG TGA TGTAT TCGGAGGACGAC TTCG TGGCCGCGAGCAGGAGACCCGGGTCGC-GT ist U4 U4 ORF 3 L VFE T L I P K P-M H0 H Y I S LL LK N R RL V L S GP So Gr GqCAAGACCTACC 1GACCAATCGC T TGGCCGA5 TACC IGG TGGAGCGCTC TGGCCG TGAGG TCACAGAGGGCATCGTCAGCACC TTCAAc A rGcCCL CC--CGTTC TGGATGGAC TGGTTAGC0AACCGGCTCATGGACCACC TCGCGAGACCGGCACTCCAGTGTCTCCCG TAOCAGTCGTGGAAGIT TCGI,! I: insert -U4 6 K r f L I N R L A E Y L V E R S G R E V T E G I V 3 T F N4 M N rc TTGCAAGGATCT roccrGTATC rrTCCAACZ IAGCCAACCAGATA5ACCGGGAAACAGGAATTGGGGATGTGCCCC rooi'ojArC TATTGGAT:- :AGAACGTTCCTAGACGTTGACATAGALAAGG IGiA ICGTTGGTC IATITGGCCC TTTGTCCTTAhC:-CCTACACGGGGACCAC TA.A:AIAACCT-Z pCB2OI insert =U)4 U4 CRF S C K D L 0 L Y L 5 N L A N 0 1 D R E T G I G 0 V P L V i L L 0 ~AGrGAGCAGCCCATCATG~TT~C~ ;GGGCCC TCACC TCAAGTATCATAATGTCCCTATATTATAGGTACCACi:rACAI Z--lpC8201 imseft .U4 U-14 ORF C- S E A G S 1 S L I 0 A L. T C K T K C P Y I I G T I F oCE1201 insert U4 U4 CRF 'I T. PhHGN~, L 3 F N L F S fl VEC A N 0F I V L Tuesday. 18 November 1997 10.09 Pg 6g~ 13pCB2OI oI 5082) Site and Sequene Pg AGGAAGC rGG TAGAG TCAGACAGCGACA rCAATGCCAACAAGGAAGAGC TGC T rCGGG roCcac TCGao GrACCCAAGCcrG rATCA-.c PC8201 insert UA U4 ORF R K L V E S 3S0 1 NHA NK E ELIL R VLODV vP K L Y H LH T TCC TTGAGAAGCAC AG:-ACCTCAGAC TTCC TCATCGGCCCT TGC TCrTTC TG CGTG TCCC AT TGGCATTGAGGAC TTCCGGACCTGGTTC AT TG -CC AGGAAC TCT TCG TG TC GTGGAGTC TGAAGG AGTAGCCGGGAACGAAGAAAGAC AGCACAGGG TAACCGTAAC TCC TGAAGGCC TGGACCAAG A4C =16-4 pCB201 inseti U4 U4 OF F L E K~ H S T S D F LI G P C F F L S C P IG I E 0 F R T V F 101L G TGGAAC U.C Tc TA TC ArTCCC TATCTACAGGAAGGAGCCAA6GATGGGA TAAAGGTCCATGGACAGAAAGCTGCT G66AGGACCCAG pCEL2OI insert U4 U4 OF NI H S I I P Y L Q E G A K 0 6 I K V H G 0 K A A W f 0 P V E v C :7GACAC;..- T TCCCTZCCCATC ASCCCAACAAGACC AATC*AAGC TG TACC GCCACACGGGCTA.G~T.- CCT-- GC .C TG TGAAGGGA:-CGGTAG TCGGG rTGTTC TGG TTA T TTCG AC ATGGTGGACGGOOOTGGG U4 ORF 0 T L P V P S A 0 00D0 SKIL Y H L P P P T V G P H S I AS P F 4GC A TC A! AAC AG:AZ CC AAG T TZ T Gr;AC TCAG ATC CT C TGA TGG C CATG CT GC 7 GAAAC T TC O-AAGC T GCCAV 7C A r-A TrC: .STCAGTTTZ^TGTCGT- CZTTCAA:,AOACC rGOATCTAGA:CACC5:TACACACTTTG-TTG:TTOA:.TT!7%Ct.-.

PC0201 insert U4 Ua ORF E 0 R I V 0 5 T P S S L 0 S 0 P L M 4 M L L K I 0 E A H fI "Ar AtCGGAACCA.-CCTGC;:CCAACCTTCA3CACAC T7TAAOGTTCGGCA rCACT-CACCCCCGGA CAACGCG-C4:.:;-t gCB201 insert U4 U4 Os= P 09E T I L C't2 N r L5 T Lt -P IH-- P C Eg';I C Tuesday. IS November 1997 10:09 Page fig 13 pC8201 0 W82) Sitand Sequence TCT TAGC TCC TCCTC TCCCCTCTCC TC TTTCAGA6CAC TGGC TC TC CAGCCCCAGGAGGAGAACAGGAGGGAGG AGGAGA TGAAAGAGGGrACAG r- AGA ATC6-GG AG GAGAGGGGAGA6GAGAAAG TCTCGT6ACCGAGAG GTCGGGGTCCTCCTCTTGTCC TCCCrcTCCTCCTTAC T TTCTCCTC-CCTGTCCA. pO8 l Iinet U4 S L L L 5 P L L F 0 S T6G S P AP GG .E 0 E G G QO ER G6G T G CTTGGTGC.-GTACCTTTGAGAACTTCCTAGGAAG6AATGGTGGGGTGGCGTTTG6GAACTTGTGCCCCCTAUCACATTTACTGGCCTCCTCTAATGC- GIACCACGACATGGAAACTC TTGAAGGATCCTTCC TTACCACCCCACCGCAAACCCTTGAACACGGGGATTGTGTAAATGACCGGAGAGATTACTG tmen -U4 C T F E NF L G R N G G V AFG NL C PL NT F T GIL. L TTGGGGAAAAGATGAT TCTGGGTCTTTCCC T7GAc rTCT TGT TTCAATTACAAAC TCC TGGGCTTTCTGGGGAGGGGTTCAGAAAACATCAAAACAC T- AACCCCTTTTC TAC TAAGACCCAGAAAGGGAAC TGAAGAAC AAAGTTAATG TTTGAGGACCCGAAAGACCCC TCCCCAAG TC TTT TG TAG rT TTGTGAc: pCO201 inar U4 V GK 00D S 5F P. L L VS 5 T H VA F VG GV 0K T S K ItC AGCAG TCC TAAATGATTCTCACAAGCAACCCTGAGAGAGACAGTCTTGTGAGGGAGATCTGGGGGAGGCAGGAGCTCCTCAGATTTTCTCACAG~cC:- TCG TCAAGATTTACTAAGAGTGTTCG TTGGGAC TC TCCTGTCAAACAC TCCCTCTAACCCCCTCCGTCC TTCGAGGAG TCTAAA6.G GTC TGG'pCO201 insert Us S s M I LT S NP E R 051 V RE I VG q 0 E AP 0 1 F S Q T T rC CCAA TTCCATC AC CAC TGCC AACAC TCGTCCGGAAT TC TGCAGATAT~CAGCACAGrGGCGGCCGC TCGAGTC TAGAGGGCCCG TTTAAACCCGC TZ AAGGTT AAG GTA TG TGAC GGTGAGCAGCC TT A AGACG TCTATASTC G TG TC ACC cG~CGAGC TCAGA TCT C C6 C G:AAA T TGGGA: -pC8201 insert U4 L P N SI1T T AN T A F C R Y P A0VR P LE S RG P V TA.

A TC AG C TCG Ac TGC C TTCTAG T TGCAGC C ATC T:TTGTTGC CC C C CCCC T GCC TT CC T TGAC CC TGGAAGG TG CCAC TCCCAC TG TC CTTC T AG TC GG AGC TG AGGA AG ATCAAC GGTCGG AG:-A AC AACGGAGGGGCAC GGAAGGAAC TG GAC C T I CCAC GGTG AGGG TGAC AcGGA AAGZ s aS 3 VP S 5 COP S V V C P S P V P SL T L E G T P Tv V L TAA TAAAATGAGGAAA TTGC ATCGC ATTGTC TGAG TAn:G TG TCA TTC TAT TCTGGGGGGTGZGGTGGGGCAGGACAGC AAGG5GGAGGA;. rTGGG&AGA.C.

N E E I A S H C L 5 4 C H S I L 6 G G V G 0 0 S K G E 0 Vs E D ATAGCAGGCATGCTGGGGATGCGGTGGG,'CT-rGGCTCTGAGGCGGAAGAACCAGCTGGGGCCTAGGGGGTATCCCCACGCGCCCT~ rAGCGGCG: TAzTCG TC C;TAC GAC C CC AC GCCACCCAGA AC C 4AC TCCG CC TCGCGACC CCGAGACC CCA TAGGGGGCCGGG:A TcGC CGC Z N S Q H A G D AV G S A S E AE R TS'aG S RG Y PH A P C S 6 T AA T TCGC G CC GCC C ACAC CACC AAT GCGC G TC: CAC 7 5GCGA T GTGA AC 5TCGCGGG ATC GC GGGC GGAACAAAG A AGAAG AAGA G-,i L S AA G V V V 7R 5 V T A T L A S A L A P P F 4 F F P S F L 4 T F A G F P R A L N P S I P LG F R P S AL A L DP V K L 0 182 Tuesday. 18 NovemnWe 1997 10.09 pawe fig 13PC B201 QI 082) &toa Mrd Sequoe rTGrGTTCACGTAGGGCCACGCCCGATGACGG TTrCGCCCT I GACGTTGGAGTCCACGTC rTAATAGTGGACTCT TG TTCCAAACTG_ CA.: TACCAAGTGCATCACCCGGTAGCGGGACTATC TGCCAAI CGGGAAACTGCAACCTCSGGTGCAAGAAATT.'TCACC rG~ssr.ACATTGAC: S0 G S A S GP S P F V RP LI IE S F FN S G L LF 0 T'.G AAZ AACACCAACCCT ATC TCGG TCTATTC TTTTGATTTATAAGGGAT T rGGGGATTTCGG6CC TATTGGTTAAAAAArGAGC TGAT TTAACAAAAAT I I TGTTGTAGTTGGGATAGAGCCAGATAAGAAAAC TAAA TAT TCCc TAAAACCCCTAAAGCCGGArAACCAATTTTTTAC TCGAC TAAATTG TTTA~.- I rIL N P I SV 'V S F 01. S G I6 1S A YVIL K NE L11.0 K F AACGCGAAT TAATTCTG TGGAATGTGrtGFCAGTTAGGGTGTGGAAAGTCCCCA6GCTCCCCAGGCAGC AGAAGTATGCAAAGCATGCATC YCAAT TAG- T T.3CGC TTAA TTAAGAACC TTACACACAGTCAA TCCCACACCTTTCAGGGGTCCGAGGGG TCCGTC CGTC TTCATACGTTTCGT ACGTAGAGTMATC-+ 4 A N F CS G MC V S. G V ES5 P 0 A P 0 A a R S HO0 S M N I N CAC AAC CAGGTGTGGAAAG TCCCC AGGCTCCCCAGCAGGCAGAAG TA TGCAAAGCATGCATCTCAATTAG TCAGCAACC ATAG TCCCGCCCC IAC TC G rCG TTG5TCCACACC TTTCAGGGG rccGAGGGGTCGTCCG TCTTCA TACG IT TCGTACG TAGAGTTAATCAGTC GTTGGTACAGGCGCGGGrATTGAG3, S AT R C G K S P G SP A G R S MO0S MHI N SATI I V PP LI P GCCCA rCCCGCcCCTAAC TCCGCCCAGTTCCGCCC A TC TC CGCCCCATGGCTGACTAATTTFFTTTTATTATGCAGAGGCCGAGGCCGCC TCTGCC TC7 CGaGG AGGC GGGGAT TGAGGCGGG TCAGGCGGGTAGAGGCGGGGTCCGACTGAT TAAAAAAAA TAATACG TCTCCGGC TCCGGCGG GACGGAGA P IP P L TP P S S ANH S P P H G .1L1 F F I1 A E SE AAS A 3 G6GC TAT TCC AGAAGT AG IGAGGAGGCT TTTTTGGAGGC CTAGGC ITT T6CAAAAAGC TCCCGGGAGCTTG TA TATCCATTrTCGGA TCTG ATCAAGAGA C 7 GATA _GG TC 7 CA TCACTCC TCCGAAAAAAC C TCCGGA TCCGAAAACG TTTTTCGAGGGCCCTCGAAC ATATAGGTAAAAGCC TAGAC TAG TTCCFC E L F0.< G GF F G G LGF C KK I P GAC I S I FPG S D 0 C~ -I AC ICC TAGCA-AAGC TAC TAAC ITGT TCACC TA ACG TGCG TCCAGAGGCCGGCGAACCCACC TC TCCGAIAAGCCGATACTGACC CGTG -3 G S F A H I E 0 G L N A G 5 P A A V V E R I F G Y 0 W A 0 AA I,-SC TGCF:T:ATGCCGCCG TGFTTCC GC- TG TC AGCGC AGGGGCGCCCGGT TC ITTTGTC AAGACCGACCTG6FCC GGTGCCC TG AA ICA AC I T TAS:C GAC GA Z IC TA C GGC G GCAC AAG GC'_CAC AG TC G CG T CC C C GrGGGC C AAGAAAAC G ITIC IGGC GG AC AGGC C AC GGG AC TAC 7 TG A, 16C SOD A A V F RIL S AS 0 GR P VIL F V K TO ILS GA&L N E L rG 7:--rC TdCCGTCGCG6CGATAGCACCGAC CGG-C CTSCCCGCAAGGAAC GCG TCGAC ACGAGC TGCAACAG TGAC I CGCCC TTCC CTG.ACGACGIL 1 2C -2A A P L S V L A r G V P C A A V L 0 V V F E A G R 0 V L I Gi~±t G CC3 333CAGGA cC CCTG ICAT: -MACC TTGCrCTGCC 3ArAAAG TATCCArCATGGCTGATGc-*ATGCGGCGS CTGCAAGC T7.

;.C:CGz TT FACGGC C6TCCTAGAGGACAGTAZAGTGGAACGAGGACGGCTCTTTCATAGGTAGTACCGACTACGTTCGCCCGACGTATGCGAC 13 Z V P 50 011 S S HIS l P AtE K V S I MAO0 A M R RIL H I L0 P A r C P F D M 0 A K P I E R A R F A H E A G L V0 0 0 1, 0 E C -:V*AG rCECC 6-:GGGCGGC TTGACAAG*- -TCCGAG FTCCGCGCs TACGGGCTGCCGCTCCTAGAGCAGCZC FGGACG:G.G* C A P A E L F A~ I K A P H P 0 G E 0-I v V F H G 0 A~ C I 183 ruesday. 18 November 1997 10:09 Pg ig 13 pC201 (1 >5082) Site and Sequence C GAA TATCA TGG TGGAAAA TGGC CC T CTGGA TTCA TCGAC TG TG GCCGCTGGG TG TGGCGG ACC GC TA TC A6GAC A TAGCG TTG GCTACccG rGA GCTTATAGTACCACCYTTrTACCGGCGAAAAG.ACC TAA6TAGCTGACACCGGCCGACCCACACCGCCTGGCGATAGTCC TGTA P 141 v VE NG R FSG F I D C G RL GV A DA R QD t A LA T R rAT TGCTGAAGAGCYTGGCGGCGAATGGGC TGACCGC rcc TCGTGCTTTAICGGTATCGCCGCTCCCGATTCGCAGCGCATCGCC

TTCTATCGCCTTCTT

AT AAC GAC TTC TCGAA CCGCC GC rTACC CG AC TGGCGAAGGAGCAC GAAA TGC CA TAGCGGC GAGGGC TAAGC GTCGCG TAG CG6GAGATAGCGGAAGA.; I A EE L 6G E V A RF L V L Y G I A A P0 D O 0R I A F V AQILL GACGAGTTCTTCTGAGCGGGACTCTGr.GGTTCGAAAT6ACCGACCAACGAGCCCAACC TGCCATCACG.AGA TTTCGATT'CCACCGCCGCC YTCTATGA C TGC TCAAGAAGAC TCGCCCTGAf3ACCCCAAGCT TTACTGGC TGGTTCGC TGCGGGTTGGACGGTAG TGCTCTAAAGC TAAGG TGGCGGCGGAAGA TACT 0 E FF .A GL V GS K.PTR R P TA C MH E I S I PP pP S M AA G G TTG GG C TTCGGAA TC GT T TTC CGGA CGCC GGC GGA TGA TC CT C CAGCGC GG GGA TC TC A TGC TGG AG T TC TC G C C CAC CCCAA CT TG rT TA T TTrCCAAC6*GAGCC T AGCAAAG GC CCTGCGGC CGACTAC TA~GG6TCCGCC CC TAG AG TAC GACC TC AA6AAGC6GG TGGGG TTGAAC AAATA A KC G WAS E S F S G TP A G. S S SA G I S C V S S T P T CL L G CAGC TT ATAATGG TTACAU TAAA GCAATAGCATCAC AAAT rTCAC AA AAA4GCA TT T r TICAC TG CA TTCTAG TTG TGG T TTGTC CAAAC TCATCA C GTCGAA TAT TACAG ATCTTTGAT TAGTTTT 2T;AAGTAGAGTACACACGTTA

AT

L I MVT N K A I AS 0 IS 0 K H FF H C I L VV V C P N 3 A TGT ATC TT A TC ATG TC TGtA T ACC GTC GAC CTC AGC TAG AG T TGGC G TAA TC ATGGTC ATA GC TG TTC CTGTGTGAA AT TGTTA TC C GC TCAC AAT 7 ZATGATGTCGA T GCACTGAACG TC TCGAACCGCA TTAGTAC CAG TATCGAC AAAGGAC ACAC TT TAAC AA TAGGCGAG TG TTA M Y L I S v YA RP L ARA A VR N HS C F L C EI V I R S 6 082 F Hl T T Y E P E A .S V P G V P N E A ti S H A V

Claims (3)

184- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. 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. 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, E or F which differ from those blocks of Figure 9a or 12a only in conservative amino acid changes. 3. 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. a 4. 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. 5. A vertebrate protein comprising an amino acid sequence as claimed in any one of claims 1 to 4 which is a human protein or a mouse protein. 6. A vertebrate protein having an amino acid sequence encoded by the nucleotide sequence shown in Sequence ID No. 4. 7. A vertebrate protein homologue according to any one of claims 1 to 6 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. 8. A vertebrate protein homologue according to any one of claims P nPEREH\R$Clms04OC2)52471 clmsdo.Ol IdV0

185- 1 to 6 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. 9. A cDNA encoding a vertebrate homologue of UNC-53 protein of C. elegans according to any one of claims 1 to 8. A cDNA according to claim 9 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. 11. A cDNA according to claim 9 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. 12. A cDNA according to any one of claims 9 to 10 which cDNA comprises at least from nucleotide position 1 to position 6013 of the sequence as shown in Sequence ID No. 3. 13. A cDNA according to claim 9 or 11 which comprises the nucleotide sequence illustrated in Sequence ID No. 4. 14. A nucleic acid molecule capable of hybridising to the DNA sequences according to any one of claims 9 to 13 under high stringency conditions. A DNA expression vector which comprises a cDNA as claimed in any one of claims 9 to 13. 16. A vector according to claim 15 which comprises a promoter of C. elegans UNC-53 protein or a vertebrate homologue thereof P %OPERIEH\Rnc Ctns\r\2 ci\2I2473 I c~ns dm.O3II 04 -186- according to any one of claims 1 to 8. 17. A vector according to claim 16 wherein said promoter sequence is derived from a gene encoding a mouse or human homologue of an UNC-53 protein of C. elegans. 18. A vector according to any one of claims 15 to 17 which further comprises a sequence encoding a reporter molecule. 19. A vector according to claim 18 wherein said reporter molecule is a fluorophore. A host cell transformed or transfected with the vector of any one of claims 15 to 19. 21. A host cell transformed or transfected with the vector of claim 18 or 19. 22. A host cell according to claim 20 or 21, which cell comprises a prokaryotic cell such as a bacterial cell or a eukaryotic cell as a fungal, an animal, a plant or an insect cell. 23. A transgenic cell, tissue or non-human organism comprising a transgene capable of expressing a protein according to any one of claims 1 to 8. 24. A transgenic cell, tissue or non-human organism according to claim 23 which comprises any of a COS cell, Hep G2, MCF-7 cell, N4 mouse neuroblastoma cell, a NIH3T3 cell, a colorectal carcinoma or human derived cells. A transgenic cell, tissue or non-human organism according to claim 23 or 24 wherein said transgene comprises a vector according to any one of claims 15 to 19. 26. A transgenic cell, tissue or non-human organism according to P %OPERVJEHRIs Clml~Ms\OOI4)?nl I 43 d1-O3 104 -187- any one of claims 23 to 25 wherein said transgene comprises a vector according to claim 18 or 19. 27. A transgenic cell, tissue or non-human organism according to any one of claims 23 to 25 wherein said organism comprises any of an insect, a fungus, a non-human mammal, a plant or a nematode worm. 28. 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. 29. A vertebrate protein homologue of an UNC-53 protein of C. eleqans, 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 neurodegenerative diseases or acute traumatic injuries or fibrotic disease. 30. A vertebrate protein homologue of an UNC-53 protein of C. elegans for use as claimed in claim 29 wherein said vertebrate homologue is as claimed in any one of claims 1 to 8. 31. Use of a vertebrate protein homologue of an UNC-53 protein of C. elegans, 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. 32. Use of a vertebrate protein homologue of UNC-53 protein of C. elegans according to claim 31 wherein said vertebrate protein homologue is as claimed in any one of claims 1 to 8. P \OPERUEH\Rn CT-U0000M.1525?473I dn doc03/ I"1 -188- 33. A pharmaceutical composition comprising a vertebrate homologue of an UNC-53 protein of C. elegans, or a functional equivalent, derivative, fragment or bioprecursor of said vertebrate protein, together with a pharmaceutically acceptable carrier, diluent or excipient therefor. 34. A pharmaceutical composition as claimed in claim 33 which comprises a vertebrate homologue of an UNC-53 protein of C. elegans according to any one of claims 1 to 8. A nucleic acid sequence encoding a vertebrate homologue of an UNC-53 protein of C. elegans or a functional equivalent, fragment, derivative or bioprecursor of said vertebrate homologue, for use 0 as a medicament. 36. A nucleic acid sequence according to claim 35 wherein said sequence is a cDNA sequence as claimed in any one of claims 9 to 13 or a functional fragment of said cDNA sequence. 37. Use of a nucleic acid sequence encoding a vertebrate homologue of an UNC-53 protein of C. elegans 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. 38. Use of a nucleic acid sequence according to claim 37 wherein said sequence is a cDNA sequence as claimed in any one of claims 9 to 13 or a functional fragment of said nucleic acid sequence. 39. A pharmaceutical composition comprising a nucleic acid sequence according to claim 35 or 36 and a pharmaceutically acceptable carrier, diluent or excipient therefor. A pharmaceutical composition according to claim 39 wherein P NOPERUEH"R, CmmO.S:0r%2V-24731 11- dw.3'11 14 -189- said nucleic acid sequence is a cDNA sequence as claimed in any one of claims 9 to 13. 41. 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 20 or 22 or a transgenic cell as claimed in any one of claims 23 to 26 and screening for a phenotypic change in said cell. 42. 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. 43. A method according to claim 42 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. 44. A method according to any one of claims 41 to 43 wherein said phenotypic change to be screened is a change in cell growth, or shape or a change in cell motility. A method according to any one of claims 41 to 43 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. 46. A method as claimed in any one of claims 41 to 43 wherein P kOPEPJEI{42 ClmCauD4lU)2rl?3 cInmSdo.031 IM4 -190- said transgenic cell is an N4 neuroblastoma cell and the phenotypic change to the screened is the length of neurite growth. 47. A method as claimed in any one of claims 41 to 43 wherein said transgenic cell is an MCF-7 breast carcinoma cell or an NIH3T3 cell and the phenotypic change to screened is the extent of phagokinesis or contact inhibition. 48. 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 one of claims 23 to 27 or a mutant organism produced according to the method of claim 28 and screening for a phenotypic change in said organism. 49. A method according to claim 48, 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. elegans 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. 9* 50. A method according to claim 49 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. 51. An antibody, antisense DNA sequence or mRNA sequence which is identifiable by the method according to any one of the claims 41 to 50 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 P OPEREI{Rc CIm.,2.Y\Oc04?lr '73 c I do~sd.03/I -191- or wound healing or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease. 52. Use of an antibody, antisense DNA sequence or mRNA sequence which is identifiable by the method according to any one of the claims 41 to 50 as an enhancer of the regulation of cell shape, or growth or motility or the direction of cell migration in the preparation 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. 53. A pharmaceutical composition comprising an antibody, antisense DNA sequence or mRNA sequence identified according to the method of any one of claims 41 to 50 and a pharmaceutically acceptable carrier, diluent or excipient therefor. 54. An antibody, antisense DNA sequence or mRNA sequence which is identifiable by the method according to any one of claims 41 to 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. 55. Use of an antibody, antisense DNA sequence or mRNA sequence according to claim 54 in the manufacture of a medicament for alleviating the spread of disease inducing cells or metastasis or loss of contact inhibition. 56. A pharmaceutical composition comprising an antibody, antisense DNA sequence or mRNA sequence as claimed in claim 54, and a pharmaceutically acceptable carrier diluent or excipient therefor. 57. A method of determining whether a compound is an inhibitor or an enhancer of transcription of a gene encoding a vertebrate P IOPERVEH\R. C! s2400 !OV!21524-31 0, d-03/1 Id!4 -192- homologue of UNC-53 protein of C. elegans, which method comprises the steps of contacting said compound with a cell according to claim 21 or 26 and monitoring the level of said reporter molecule and comparing the results obtained from said monitoring step with a control comprising a cell according to claim 21 or 26, which cell has not been contacted with said compound. 58. A method as claimed in claim 57 wherein said reporter molecule detected is mRNA or green fluorescent protein. 59. An antibody, antisense DNA sequence or mRNA sequence which is identifiable by the method according to claim 57 or 58, as an enhancer of transcription of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans or a functional fragment of said gene, for use in promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease. Use of an antibody, antisense DNA sequence or mRNA sequence which is identifiable by the method of claim 57 or 58, as an enhancer of transcription of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans or a functional S"fragment of said gene, in the manufacture of a medicament for promoting neuronal regeneration, revascularisation or wound healing, or for treatment of chronic neurodegenerative diseases or acute traumatic injuries or fibrotic disease. 61. A pharmaceutical composition which comprises an antibody, antisense DNA sequence or mRNA sequence of claim 59 and a pharmaceutically acceptable carrier, diluent or excipient therefor. 62. An antibody, antisense DNA sequence or mRNA sequence which is identifiable by the method of claim 57 or 58 as an inhibitor of transcription of a gene coding for a vertebrate homologue of an P kOPE JE.H's Cls\20CUO(u\ 25'4131 0- dO/III14

193- UNC-53 protein or 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. 63. Use of an antibody, antisense DNA sequence or mRNA sequence which is identifiable by the method of claim 57 or 58 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. 64. A pharmaceutical composition which comprises an antibody, antisense DNA sequence or mRNA sequence of claim 62 and a pharmaceutically acceptable carrier, diluent or excipient S. therefor. 65. 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 21 to 24 to be contacted with said compound and at least one cell according to :any one of claims 20 to 27 to be used as a control and means for contacting said compound with one or said at least one transgenic cells. 66. A kit for determining whether a compound is an inhibitor or an enhancer of transcription of a gene coding for a vertebrate homologue of an UNC-53 protein of C. elegans or functional fragment of said gene which kit comprises at least one cell as claimed in any one of claims 20 to 24 means for contacting said compound with said cells. 67. 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, P OPERVEMRa Clms\ 4O%0\2S?4731 cmsdo0131 IJ04 -194- 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 28 or a transgenic organism as claimed in any one of claims 23 to 27 and a wild type of said vertebrate mutant organism. 68. 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 the cDNA according to any one of claims 9 to 13. S. 69. A method of identifying a protein which is active in the signal transduction pathway of a cell of which a vertebrate homologue of an UNC-53 protein of C. elegans or a functional equivalent, fragment or bioprecursor of said vertebrate homologue is a component, which method comprises: contacting an extract of said cell with an antibody to the vertebrate homologue of the UNC-53 protein of C. elegans or a functional equivalent, fragment, derivative or bioprecursor of said protein, identifying the antibody/vertebrate homologue complex, and analysing the complex to identify any protein bound to the vertebrate homologue of UNC-53 protein of C. elegans other than the antibody. 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: forming an antibody to the first identified protein P (OPERVEHcsri*ms 0 2 I cndo.O03MI 4 -195- bound to the vertebrate homologue of UNC-53 protein C. elegans in claim 69, contacting a cell extract with said antibody and identifying the antibody/protein complex, analysing the complex to identify any further protein bound to the first protein other than the antibody, and optionally repeating steps to to identify further proteins in said pathway. 71. A method of identifying a protein which is active in the signal transduction pathway of a cell of which a vertebrate homologue of an UNC-53 protein of C. elegans or a functional equivalent, fragment or bioprecursor of said homologue protein is a component, which method comprises: contacting an extract of said cell with the vertebrate homologue of an UNC-53 protein of C. elegans or a functional equivalent, derivative or bioprecursor of said vertebrate homologue, identifying any vertebrate homologue of UNC-53 protein/protein complex formed, and 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. 72. A method according to claim 71 which further comprises contacting a cell extract with any protein identified from step not being the same as the vertebrate homologue of UNC-53 protein used and repeating steps and so as to identify any further protein involved in the signal transduction pathway of said cell. 73. A method of identifying a protein involved in the signal transduction pathway of a cell of which a vertebrate homologue of an UNC-53 protein of C. elegans is a component which method comprises: providing an appropriate host cell having a DNA P OPERUJEWIRnCl.,U2 'O(42ll5 ,31 I IndocMOII 111M4 -196- 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 said host cell a first hybrid DNA sequence encoding a first fusion of a fragment or all of a DNA sequence according to any one of claim 9 to 13 and either said DNA binding domain or the activating domain of the transcription factor, 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, detecting any binding of the protein being investigated with a protein according to any one of claims 1 to 8 by detecting for the production of any reporter gene product in said host cell. 74. 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 with process comprises culturing the cells of any one of claims 20 to 27 and recovering said vertebrate homologue of UNC-53 protein expressed. 75. 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. 76. A nucleotide sequence comprising the sequence as shown in P /OPERVE\Ra CI-VOWOOV524731 Cln do.031/ /04 -197- figure 77. A nucleotide sequence comprising the sequence as shown in figure 16. 78. A nucleotide sequence comprising the sequence as shown in figure 17. 79. 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. 80. A method according to claim 79 wherein said cell comprises a cell according to any one of claims 20 to 27. 81. A method according to claim 79 wherein said cell has undergone loss of contact inhibition. 82. A method according to any one of claims 79 to 81 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. 83. A method according to claim 82 wherein said nucleic acid sequence comprises an antisense DNA or RNA sequence. 84. A method according to claim 83 wherein said mRNA sequence comprises 3' untranslated regions of mRNA encoding for said vertebrate homologue. A method according to any one of claims 79 to 81 wherein said compound to be tested comprises a protein having an amino acid P OPERVEHXR= CtmWO0410aU524731 clmdomiZ3/l IA -198- sequence potentially suitable for inhibiting function of said vertebrate homologue. 86. A method according to claim 83 wherein said protein comprises a protein identified according to any one of the methods of claims 69 to 73. 87. A pharmaceutical composition comprising an antibody, antisense DNA sequence or mRNA sequence identified according to any one of claims 79 to 85 together with a pharmaceutically acceptable carrier, diluent or excipient therefor. 88. An antisense DNA or RNA sequence identified according to the method any one of claims 82 to 84 for use in treatment of loss of contact inhibition or carcinoma which is mediated by the vertebrate homologue of an UNC-53 protein of C. elegans or a functional equivalent, fragment, derivative or bioprecursor thereof. 89. Use of an antisense DNA or RNA sequence identified according to the method of any one of claims 82 to 84 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. An antisense DNA or RNA sequence according to claim 88 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. 91. A NIH3T3 cell line transfected with pcB201 and deposited under LMBP Accession No. 1603CB. 92. A plasmid pCB 201 of Sequence ID No. 10 deposited under LMBP Accession No. LMBP 3594. P %OPERVEHiRm Ch, 20'O .2I52'flI cmmtsm)'I I Ku -199- 93. A MCF-7 cell line transfected with plasmid pCB 201 deposited under LMBP Accession No. LMBP 1601CB. 94. 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 linked to a report molecule, removing any unbound antibody and monitoring for the presence of said reporter molecule. An assay according to claim 94 wherein said reporter molecule is an antibody conjugated with a suitable fluorophore or detectable enzyme. S. 96. A method for detecting for expression of a gene coding for a e* 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 thereof with a cell extract which probe is linked to a reporter and analysing for the presence of said reporter. 97. A method according to claim 95 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. 98. A method according to claim 97 wherein said complimentary sequence is a 3' or 5' untranslated region of said mRNA. 99. A method according to claim 96 or 98 wherein said reporter comprises a radiolabel. P %OPERUE"HR CIr.VaOG%20$24313 C.1 dMOC.J1 I04 -200- 100. A method according to claim 96 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. 101. A method according to claim 100 wherein said reporter comprises an antibody conjugated with a detectable fluorophore or enzyme. 102. Phage Lambda clone 3b of Sequence ID No. 5 deposited under Accession No. LMBP 3595. 103. A method of determining whether a compound is an inhibitor or an enhancer of association of UNC-53 or a vertebrate homologue S.thereof according to any one of claims 1 to 8 to microtubules or plus end regions thereof, which method comprises: 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, screening for the localisation of said reporter molecule as compared to a cell according to step which •has not been contacted with said compound. S: 104. An antibody, antisense DNA sequence or mRNA sequence identifiable by the method according to claim 103. 105. An antibody, antisense DNA sequence or mRNA sequence identifiable by the method according to claim 103 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. 106. An antibody, antisense DNA sequence or mRNA sequence identifiable by the method according to claim 103 as an enhancer PXOPER\E/f\R.C1,2/)4k\Oc/2i'24731 c1..doc0I /1 4 -201- 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. 107. A pharmaceutical composition comprising an antibody, antisense DNA sequence or mRNA sequence according to claim 104 or 105 and a pharmaceutically acceptable carrier, diluent or excipient therefor. 108. 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 one of claims 1 to 8 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 one of claims 19 to 23 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 cell. 109. A composition comprising UNC-53 of C. elegans or a vertebrate •homologue thereof according to any one of claims 1 to 8 linked to an antibody, antisense DNA sequence or mRNA sequence 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. 110. A composition according to claim 109 which further comprises a cell transformation or transfecting agent. 111. 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 P OPERJEHR Clnis200O5'J-\5?4731 do0O71IO -202- according to any one of claims 1 to 8, 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. 112. A method of identifying a molecule which covalently modifies UNC-53 or a vertebrate homologue thereof according to any one of claims 1 to 8, which method comprises: contacting either an extract from a cell expressing UNC-53 or said vertebrate homologue or a mixture of enzymes comprising candidate UNC-53 modifying enzymes in the presence of an indicator of covalent modification of a protein, identifying any covalently modified UNC-53 protein from step 15 S identifying said molecule involved in said modification step. 113. A method according to claim 112, wherein said indicator comprises "p. S 20 114. A method of identifying a compound which alleviates or enhances the toxicity of UNC-53 or a vertebrate homologue thereof according to any one of claims 1 to 8, which method comprises contacting said compound with a cell, tissue or organism according to claim 26, and monitoring for the presence of said reporter molecule adjacent said microtubules or the plus end regions thereof. 115. Plasmid pLM1 of Sequence ID No. 6 deposited under Accession No. LMBP 3762. 116. Plasmid pLM4 of Sequence ID No. 7 deposited under Accession No. LMBP 3763. 117. Plasmid pEGF72 of Sequence ID No. 8 deposited under Accession POPERIEH\Ra CmN0040\2)5271 cl-doc.O311I I -203- No. LMBP 3764. 118. Plasmid pCB501 of Sequence ID No. 9 deposited under LMBP Accession No. LMBP 3765. 119. A worm strain comprising a chimeric C. elegans human UNC-53 gene deposited under LMBP Accession No. LMBP-1663CB. 120. A vertebrate homologue according to any one of claims 1 to 2 which is a mouse homologue. 121. A homologue according to claim 120 having the sequence illustrated in Figure 14. S. 122. A vertebrate protein homologue according to any one of claims 1 to 8, 29 to 30 or 120 to 121, a cDNA according to any one of claims 9 to 13, a nucleic acid molecule according to claim 14, a vector according to any one of claims 15 to 19, a host cell according to any one of claims 20 to 22, a transgenic cell according to any one of claims 23 to 27, a method according to any one of claims 28, 41 to 50, 57 to 58, 68 to 73, 79 to 86, 96 to S101, 103 or 111 to 114, a use according to any one of claims 31 to 32, 37 to 38, 52, 55, 60, 63 or 89, a pharmaceutical composition according to any one of claims 33 to 34, 39 to 40, 53, 56, 61, 64, :oo: 87 or 107, a nucleic acid sequence according to any one of claims to 36, an antibody according to claim 51 or 54, 59, 62 or 104 to 106, a kit according to any one of claims 65 to 67 or 108, a process according to any one of claims 74 to 76, a nucleotide sequence according to any one of claims 77 to 78, an antisense DNA or RNA sequence according to claim 88 or 90, a NIH3T3 cell line according to claim 91, a plasmid pCB 201 according to claim 92, a MCF-7 cell line according to claim 93, an assay according to any one of claims 94 to 95, a phage Lambda clone 3b according to claim 102, a composition according to any one of claims 109 to 110, a plasmid pLM1 according to claim 115, a plasmid pLM4 according to claim 116, a plasmid pEGF72 according to claim 117, a plasmid P IOPRUEW.O.WONW-151731Is do..03/1 1104 204 according to claim 118, a worm strain according to claim 119 substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this 3rd day of November, 2004 JANSSEN PHARMACEUTICA N.V. by DAVIES COLLISON CAVE Patent Attorneys for the Applicant(s) fee: