AU1947900A - Cloning, expression and uses of a novel secreted protein F-spondin - Google Patents

Description

Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

(ORIGINAL)

o go o* o *o r Name of Applicants:

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Actual Inventors: The Trustees of Columbia University in the City of New York, of Broadway and 116th Street, New York, New York 10027, United States of America JESSELL, Thomas M KLAR, Avihu DAVIES COLLISON CAVE, Patent Attorneys, of 1 Little Collins Street, Melbourne, Victoria 3000, Australia "Cloning, expression and uses of a novel secreted protein, F-spondin" Address for Service: Invention Title: The following statement is a full description of this invention, including the best method of performing it known to us: P:\OPER\MRO\2266018.CLM 25t2/00 1A CLONING. EXPRESSION AND USES OF A NOVEL SECRETED PROTEIN,

F-SPONDIN

Background of the invention Throughout this application various references are referred to within parenthesis. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the sequence listing and the claims.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and :°oooo 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.

As used herein, the term "derived from" shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily •*go been obtained directly from the specified source.

The early development of the vertebrate nervous system is S" 25 controlled by local cell interactions that determine the identity of specific neural cell types and the pathways of growing axons. One of the first cell types to differentiate within the embryonic nervous system is the floor plate, a small group of epithelial cells located at the ventral midline of the neural tube (Schoenwolf and Smith, 1990). The differentiation of the floor plate is induced by local, possibly contactdependent signals from the notochord (Figure 1) (van Straaten et al., 1988; Placzek et al., 1990c; Hatta et al., 1991). Signals that derive from the floor plate have been implicated in the P:\OPER\MRO\2266018.CLM 25/2/00 1B control of cell identity in the neural tube and in the guidance of axons (Figure 1) (Jessell and Dodd, 1991).

Evidence that the floor plate is a source of polarizing signals that control cell identity and pattern in the neural tube has come from experiments in chick embryos in which floor plate cells grafted next to the neural tube of host embryos give rise to additional ectopic motor neurons and to other ventral neuronal types defined by cell specific antigenic markers (Yamada et al., 1991; Placzek et al., 1991). Inversely, preventing floor plate differentiation by removing the notochord leads to the formation of a spinal cord that is devoid of motor neurons and other ventral neurons. These grafting experiments suggest that the floor plate has a central role in establishing the identity and pattern of neuronal cell types present in the ventral spinal cord. The floor plate also has limb polarizing activity when grated into the chick wing bud, possibly through the release of morphogenically active retinoids (Wagner et al., 1990).

After the identity of spinal cord neurons has been established, the floor plate appears to provide both long-range and local guidance cues that promote the 15 growth of axons to and across the ventral midline of the spinal cord. First, the floor plate secretes a diffusible chemoattractant which can orient the growth of axons of commissural neuron in vitro (Figure 1) (Tessier- Lavigne et al., 1988; Placzek et al., 1990a; Tessier- 20 Lavigne and Placzek, 1991) and may account for the homing of these axons to the floor plate in vitro (Weber, 1938; Placzek et al., 1990b; Bovolenta and Dodd, 1991; Yaginuma and Oppenheim, 1991). Second, the floor plate may contribute to the change in trajectory of commissural axons from the transverse to the longitudinal plane that occurs immediately after crossing the ventral midline (Figure 1) (Holley and Silver, 1987; Dodd et al., 1988; Bovolenta and Dodd, 1990). In support of this proposal, genetic mutations in mice and zebrafish that result in the absence of the floor plate during embryonic development lead to errors in the pathfinding of commissural axons at the midline of the spinal cord (Bovolenta and Dodd, 1991; Bernhardt and Kuwada, 1990).

Third, the floor plate may promote the fasciculation of commissural axons that occurs after they cross the midline of the spinal cord (Holley and Silver, 1987) by regulating the expression of glycoproteins of the immunoglobulin superfamily (Dodd et al., 1988; Schachner et al., 1990; Furley et al., 1990). The specialized role of the floor plate in vertebrate neural development has parallels in invertebrate organisms in that cells at the midline of the embryonic drosophila and C. elegans central nervous systems have been implicated in neural patterning and axon guidance (Klambt et al., 1991; Nambu et al., 1991; Hedgecock and Hall, 1990).

To identify molecules that may mediate the diverse functions of the floor plate during early neural development, subtractive hybridization techniques have .:15 been used to isolate cDNA clones expressed selectively by 15 the floor plate. Thr characterization of cDNA clones encoding a novel secreted protein, F-spondin, that is expressed at high levels by the rate floor plate during embryonic development is described here. The predicted 20 amino acid sequence of F-spondin reveals that the protein contains domains similar to those present in the thrombospondin and other proteins implicated in cell adhesion and neurite outgrowth. In vitro assays show that F-spondin promotes neural cell adhesion and neurite outgrowth suggesting that the secretion of this protein by the floor plate contributes to the growth and guidance of axons in the developing CNS.

Summary of the invention This invention provides isolated vertebrate nucleic acid molecule encoding F-spondin. The isolated nucleic acid may be cDNA or RNA. The isolated vertebrate nucleic acid may be derived from human, rat, chicken or Xenopus.

This invention also provides a nucleic acid probe comprising a nucleic acid molecule of at least nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a F-spondin. The nucleic acid probe may be DNA or RNA.

This invention provides the method to obtain F-spondin 15 nucleic acid molecule. In an embodiment, a rat F-spondin gene is isolated by substractive hybridization. In another embodiment, a chicken F-spondin gene is isolated by screening a chicken cDNA library using a rat F-spondin probe. In a further embodiment, a Xenopus F-spondin is also isolated.

This invention further provides a host vector system for the production of a polypeptide having the biological activity of F-spondin.. The isolated vertebrate F-spondin 25 nucleic acid molecule is linked to a promoter of RNA transcription and then to a plasmid. The suitable host is a bacterial cell, insect cell, or animal cell, depending on the type of promoter and plasmid used. This invention also provides a method of producing a polypeptide having the biological activity of F-spondin, which comprises growing the selected host vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.

This invention further provides purified vertebrate Fspondin. Such purified F-spondin will be useful for adhesion and outgrowth of axon. This invention provides a method of attaching nerve cells to a matrix comprising contacting the matrix with nerve cell and purified Fspondin at a concentration effective to effect attachment of the cells to the matrix. This invention further provides a method of stimulating growth of a nerve cell comprising contacting the nerve cell with purified Fspondin at a concentration effective to stimulate growth of the nerve cell. This invention provides a method of regenerating nerve cells in a subject comprising administering to the subject purified F-spondin at a concentration effective to regenerate nerve cells in the subject. Finally, this invention provides a pharmaceutical composxtion for stimulating nerve cell growth comprising a pharmaceutically acceptable carrier and purified F-spondin at a concentration effective to stimulate nerve cell growth.

2 Brief Description of Figures Figure 1.

Figure 2.

Figure 3.

**so a *0* as 4 a 0 S. 0 6 00 4* S 5* *5 Diagram shoving the induction and proposed functions of the floor plate during early spinal cord development. For details see text.

Schematic diagram of the subtractive hybridization protocol used to identify floor plate specific cDNA clones.For details see text.

Expression of F-spondin mRNA. Total cellular RNA or poly RNA was isolated from different tissues and separated on 1% agaroseformaldehyde gels and blotted to nylon membranes. The blot was analyzed with cDNA probes derived from the F-spondin 3' noncoding region labelled by random priming.

A. Preferential expression of F-spondin mRNA in E13 (embryonic day 13) floor plate compared with E13 dorsal spinal cord at adult spleen. Two transcripts of 4.5 and 4.7 kb are detected in floor plate RNA.

B. NCAM, Neural Cell Adhesion Molecule, mRNA is expressed at approximately equivalent levels in E13 floor plate and dorsal spinal cord and PO (postnatal; day 0) brain.

C. F-spondin mRNA is detected in blots of total RNA adult kidney and brain but not in adult liver or sciatic nerve.

Figure 4. Restriction map of the F-spondin cDNA. The arrow indicates the direction of translation.

The restriction sites are indicated above the cDNA.

Figure 5. cDNA and predicted amino acid sequence of Fspondin.

A. Nucleotide and amino acid sequence of rat F-spondin determined from cDNA clones.

The numbering of amino acids starts at the first methionine. Underline NH 2 terminal residues indicates the putative signal sequence. Potential sites of N-linked glycosylation are indicated by double lines.

B. Analysis of the hydrophobicity of the predicted F-spondin amino acid sequence.

The plot was generated using the parameters given in Kyte and Doolittle 15 (1982). The NH 2 terminus of the protein is to the left. Negative values indicate hydrophobic residues.

Figure 6. Alignment of the carboxy terminal domain F- 0. spondin and homology to thrombospondin type one repeats in other proteins.

A. Schematic representation of the domain structure of F-spondin. The black box represents the signal sequence. The hatched box represents the thrombospondin type 1 repeats (TSRs).

B. Alignment of the six repeats motifs in Fspondin which occupy residues 440-807 of the protein. The position of the first and last amino acids of each repeat is shown on the left. Numbers over each repeat refer to the position of residues.

Positions in which there are four or more identical residues are enclosed in boxes.

C. Comparison of the conserved F-spondin motif with the conserved TSRs found in thrombospondin I, thrombospondin II, region II of the plasmodial circumsporooite (cs) proteins thrombospondin-related anonymous protein (TRAP), properdin and in the N-and Cterminal regions of the complement proteins C6, C7, C8a, C8b and C9. The number at the right of the figure indicates the number of TSR domains that contain VTCG sequence as a proportion of S* the total number of TSR domains.

Figure 7. Localization of F-spondin mRNA in the 15 developing spinal cord.

A. Autoradiographic localization of F-spondin mRNA in the hindbrain of a day 10 rat embryo by in situ hybridization using an antisense RNA probe. Intense hybridization is detected at the ventral midline of the neural tube and possible also in the axial mesoderm underlying the neural tube.

B. Localization of whole mount in situ F- 25 spondin mRNA by Ell (embryonic day 11) rat embryos hybridization histochemistry using digoxigenin-labelled antisense probe.

Hybridization is detected in the floor plate of the midbrain, hindbrain and spinal cord (arrow heads).

C. Bright field micrograph showing localization of F-spondin mRNA in E12 *b.

9 (embryonic day 12) rat spinal cord. The floor plate is intensely labelled.

D. Dark field micrograph of a similar section showing a low level of hybridization is in the ventral horn in addition to intense labelling in the floor plate.

Hybridization is also detected in the ventral root.

E. Dark field micrograph showing the floor plate and the ventral ventricular zone of E13 spinal cord express high levels of Fspondin mRNA.

F. Bright field micrograph of E16 (embryonic day 16) spinal cord showing that F-spondin mRNA levels are still high in the floor plate and the ventral ventricular zone.

G. Dark field micrograph shoving that by E16, significant hybridization is also detected in ventral and intermediate regions of the spinal cord.

H. Dark field micrographs shoving a uniform distribution of F-spondin mRNA.

Scale bar: A=100 Mm; B-350 gm; C-80 mm; E-100 Mm; F-170 gm; G-170 gm; H=120m.

F-Spondin my c is secreted by cos cells and associated with the cell surface.

A. Position of insertion of an oligonucleotide encoding for a 10 amino acid region of the c-myc oncogene ligated into unique NcoI site or Spel sites within the F-spondin cDNA.

B. Immunoprecipitation of conditioned media obtained by exposure of 40h to cos cells transfected with pFP5myS, pFPSmyN and to Figure 8.

Figure 9.

S

S

S

mock transfected cells. Both constructs generated a single protein band at 116 kDa.

C. Phase contrast micrograph showing a small group of transfected cos cells.

D. Immunofluorescence micrograph showing the localization of F-spondin m y on the cell surface. Immunoreactivity is detectable at much higher levels at cell-cell rather than at cell-substrate contacts.

Scale bar in C, D 204m F-spondinmyc promotes the extension of neurites from DRG neurons in vitro. F-spondinye protein obtained from transfected cos cells supernatants was affinity purified and analyzed by SDS-PAGE(8-25%) and silver staining. (A) Two stained bands are observed, which may reflect differences in the glycosylation of Fspondin. Neural cells isolated from E14 rat dorsal root ganglia were plated on F-spondin or on cos cell-conditioned media or BSA (not shown) substrates for 14h and then fixed and labelled with MAb 3A10 and visualized by indirect immunofluorescence. The length of the longest neurite of each 3A10-positive neurons was measured (or recorded as 0 mm if no neurite was seen). The percentage of neurons (ordinate) with neurites longer than a given length in gm (abscissa) is plotted. Similar results were obtained in 5 experiments. Only non-fasciculated neurites were included in the plots shown in D. Scale bar in B and C 100 4m.

Figure 10.

F-spondin promotes the adhesion of dorsal spinal cord cells. A single cell suspension of E13 dorsal spinal cord cells (106 cells/35mm disk) was plated on, Fspondinmy c on BSA and on Fspondinmyc substrate in the presence of heparin (1 for lh. Cells were then washed in PBS, fixed and counted.

E. Box plot shoving dose-dependent adhesion of E13 dorsal spinal cord cells to different amounts of Fspondin my e substrate. Each box represents cell counts from different fields. Similar results were obtained in 3 separate experiments.

F. Box plot showing inhibition of the adhesion of E13 dorsal spinal cord cells to F-spondinYC in the presence of different concentrations of heparin and chondroitin sulfate.

The inhibition at all concentrations of chondroitin sulfate and heparin is significant (p<0.001; Ttest).

Scale bar in A, C, D 200 gm, B tm Box plot: The box enclosed 50% of the population with the median marked as a bold line and the mean as a dot. The range of the data is indicated by the extent of the lines. Each plot represents determinations form one of three similar experiments.

12 Detailed Description of the Invention This invention provides isolated vertebrate nucleic acid molecules which encode F-spondin. As used herein, the term F-spondin encompasses any amino acid sequence, polypeptide or protein having the biological activities provided by the F-spondin.

In one embodiment of this invention, the isolated nucleic acid molecules described hereinabove are DNA. In other embodiments of this invention, the isolated nucleic acid 1 molecules described hereinabove are cDNA, or RNA. In the preferred embodiment of this invention, the isolated nucleic molecules are cDNAs as shown in sequence ID numbers 11 and 13) This invention also encompasses DNAs and cDNAs which encode amino acid sequences which differ from those of Fspondin, but which should not produce phenotypic changes.

Alternatively, this invention also encompasses DNAs and cDNAs which hybridize to the DNA and cDNA of the subject invention. Hybridization methods are well known to those of skill in the art.

The DNA molecules of the subject invention also include DNA molecules coding for polypeptide analogs, fragments or derivatives of antigenic polypeptides which differ 25 from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (deletion analogs containing less than all of the residues specified for the protein, substitution analogs wherein one or more residues specified are replaced by other residues and addition analogs wherein one or more amino acid residues is added to a terminal or medial p6rtion of the polypeptides) and which share some or all properties of naturally-occurring forms. These sequences include: the incorporation of codons "preferred" for expression by selected non-mammalian host; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences that facilitate construction of readily expressed vectors.

The DNA molecule described and claimed herein are useful for the information which they provide concerning the amino acid sequence of the polypeptide and as products for the large scale synthesis of the polypeptide by a variety of recombinant techniques. The molecule is useful for generating new cloning and expression vectors, transformed and transfected procaryotic and eucaryotic host cells, and new and useful methods for cultured growth of such host cells capable of expression of the 15 polypeptide and related products.

Moreover, the isolated nucleic acid molecules are useful for the development of probes to study the neurodevelopment.

F-spondin may be produced by a variety of vertebrates. In an embodiment, a rat F-spondin nucleic acid is isolated.

A restriction map of the cDNA of rat F-spondin is shown in Figure 4. The Xhol-Dral fragment of rat F-spondin was 25 excised from the F-spondin cDNA. The Xhol site was blunt-ended with T4 DNA polymerase, and Bgl2 linkers (12 mers) was ligated. The fragment was subcloned into BamH1 site of pBluescript SK (Strategene). The 5' of the gene is located near the T3 promoter. The resulting plasmid, pFP5/KS, encoding the rat F-spondin was deposited on March 19, 1992 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, 14 U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure.

Plasmid, pFP5/KS was accorded ATCC accession number 75215.

In another embodiment, a chicken F-spondin cDNA was isolated (Seq. ID No. 11). The translation initiates at nucleotide position 136. In a further embodiment, a partial Xenopus F-spondin was isolated (Seq. ID No. 13).

Throughout this application, references to specific nucleotides are to nucleotides present on the coding strand of the nucleic acid. The following standard abbreviations are used throughout the specification to indicate specific nucleotides: C=cytosine A-adenosine 15 S: T=thymidine G=guanosine .i For the purpose of illustration only, applicants used a o substractive hybridization techniques to isolate and characterized F-spondin cDNA clones in rats. Similar substractive hybridization techniques are applicable to isolate and characterize the F-spondin genes in different vertebrates.

Alternatively, the F-spondin genes may be isolated using 25 the probe generated from the rat F-spondin gene. The chicken and Xenopus homologous F-spondin genes have recently been cloned by applicants. These genes are extremely conserved and share 90% homology at the amino acid level and about 70% homology at the nucleic acid level. The chicken gene was isolated by low stringency screening of embryonic spinal cord cDNA library whereas the Xenopus F-spondin gene was isolated by low stringency screening of the whole embryo cDNA library, both using probes from the coding region of rat F-spondin.

For the human F-spondin gene, it is conceivable that the degree of homology between rat and human would be even greater since both rat and humans are mammals. Human embryonic brain cDNA library, available from Clontech, and human genomic library may be used for such screening.

Duplicated filters of human libraries may be screened with radiolabelled probe derived from the rat F-spondin.

The probe may be encompassing the coding region, since the homology of F-spondin across species is through the 0 whole coding region. The filters containing the human libraries will be hybridized with the probes at low stringency (Sambrook et al. 1989) and positive clone will be further analyzed by DNA sequencing techniques which are well known to an ordinary skilled artisan.

This invention provides a nucleic probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a F-spondin, for example, with a coding sequencing included within the sequence shown in Figure 5 and Sequence ID number 9. As used herein, the phrase "specifically hybridizing" means the ability of a nucleic acid molecule to recognize a nucleic acid sequence 25 complementary to its own and to form double-helical segments through hydrogen bonding between complementary base pairs. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary greatly in length and may be labeled with a detectable label, such as a radioisotope or fluorescent dye, to facilitate detection of the probe.

DNA probe molecules may be produced by insertion of a DNA molecule which encodes F-spondin into suitable vectors, such as plasmids or bacteriophages, followed by transforming into suitable bacterial host cells, replication in the transformed bacterial host cells and harvesting of the DNA probes, using methods well known in the art. Alternatively, probes may be generated chemically from DNA synthesizers.

The probes are useful for 'in situ' hybridization to locate tissues which express this gene, or for other hybridization assays for the presence of this gene or its mRNA in various biological tissues.

Vectors which comprise the isolated nucleic acid molecule described hereinabove also are provided. Suitable vectors comprise, but are not limited to, a plasmid or a 5 virus. These vectors may be transformed into a suitable 15 host cell to form a host cell vector system for the production of a polypeptide having the biological activity of F-spondin.

This invention further provides an isolated DNA or cDNA molecule described hereinabove wherein the host cell is selected from the group consisting of bacterial cells (such as E.coli), yeast cells, fungal cells, insect cells and animal cells. Suitable animal cells include, but are not limited to Vero cells, HeLa cells, Cos cells, CV1 25 cells and various primary mammalian cells.

This invention provides a method to identify and purify expressed F-spondin proteins. A myc-epitope was first introduced into the F-spondin protein. This F-spondin carrying myc-spondin may linked to an expression vector.

Such vector may be used to transfect cell and the distribution of F-spondin in the cell can be detected by reacting myc antibodies known to be reactive to the introduced myc-epitope with the transfected cells which is expressing the F-spondin carrying myc-epitope. Taking advantage of this myc-epitope, F-spondin may be purified by an antibody affinity column which binds with this mycepitope.

In one embodiment, myc-epitope is introduced in the Ncol site of the rat F-spondin. After that the smal (125), Dra (2731) fragment of the rat F-spondin was isolated.

Bgl2 linkers were added, and the fragment was subcloned into BamH 1 site of pcDNA neo (InVitrogene). The of the gene is located near the T7 RNA promoter. The resulting plasmid, pcFP5.myn, was deposited on March 19, 1992 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A.

under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. Plasmid, was accorded ATCC designation number 75216.

S The above uses of the myc-epitope for identification and purification of F-spondin should not be considered limiting only to the myc-epitope. Other epitopes with specific antibodies against them which are well known to an ordinary skilled in the art could be similarly used.

Also provided by this invention are F-spondin complete protein sequences (seq. ID Nos. 10, 12). In an embodiment a complete rat F-spondin protein sequence is disclosed (Seq. ID No. 10). In aother embodiment a complete chicken F-spondin protein sequence is provided (Seq. ID No. 12). In a further embodiment a partial Xenopus F-spondin protein sequence is also provided (Seq.

ID No. 14).

Further provided by this invention is a purified, Fspondin polypeptide. As used herein, the term "purified F-spondin" shall mean isolated naturally-occurring Fspondin or protein (purified from nature or manufactured such that the primary, secondary and tertiary conformation, and posttranslational modifications are identical to naturally-occurring material) as well as non-naturally occurring polypeptides having a primary structural conformation continuous sequence of amino acid residues). Such polypeptides include derivatives and analogs.

Such F-spondin will be useful for adhesion and outgrowth of axon. Therefore, this invention also provides a method of attaching nerve cells to a matrix comprising contacting the matrix with nerve cell and purified Falo" spondin at a concentration effective to effect attachment 15 "0 of the cells to the matrix.

Methods to determine such a concentration are well-known in the art. The effect concentration of F-spondin may be determined by using different concentrations of purified F-spondin to the matrix and the nerve cell. The concentration in which attachment of the matrix and the nerve cell is observed is the effective concentration.

N

This invention further-provides a method of stimulating 25 growth of a nerve cell comprising contacting the nerve cell with purified F-spondin at a concentration effective to stimulate growth of the nerve cell.

This invention also provides a method of regenerating nerve cells in a subject comprising administering to the subject purified F-spondin at a concentration effective to regenerate nerve cells in the subject.

Finally, this invention provides a pharmaceutical composition for stimulating nerve cell growth comprising a pharmaceutically acceptable carrier and purified Fspondin at a concentration effective to stimulate nerve cell growth.

For the purposes of this invention "pharmaceutically acceptable carriers" means any of the standard pharmaceutical vehicles. Examples of suitable vehicles are well known in the art and may include, but not limited to, any of the standard pharmaceutical vehicles such as a phosphate buffered saline solutions, phosphate buffered saline containing Polysorb 80, water, emulsions such as oil/water emulsion, and various type of wetting agents.

This invention will be better understood from the i S. SExperimental Details which follow. However, one skilled "*in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

.i Experimental Details Experimental Procedures Library Construction and Screening Directional cDNA libraries were constructed in Lambda ZAP* II (Stratagene®) from embryonic day 13 floor plate and dorsal spinal cord poly -selected RNA. The ends of the cDNA inserts were located downstream of the T3 RNA polymerase promotor, and the 3' ends downstream of the T7 RNA polymerase promotor. DNA was prepared from the library using the plate lysate method (Sambrook et al., 1989). The DNA was linearised with XhoI and RNA was transcribed with T3 RNA polymerase (Stratagenee). RNA from the dorsal spinal cord library was transcribed in the presence of UTP-biotin (Clontec) diluted 1:10 with UTP. First-strand cDNA was transcribed from the T3 floor plate RNA using an oligo dT Xhol linker (Stratagene*).

Solution hybridization of first strand floor plate cDNA and the dorsal T3 biotinylated RNA was performed as described by Sive and St. John (1988). Approximately 1 g of cDNA was hybridized with a 30-fold molar excess of RNA. The nucleic acids were dissolved in 10 Al of hybridization buffer containing 50 mM HEPES (pH 7.6), 0.2% SDS, 2 mM EDTA, 500 mM NaC1, and incubated at 680C.

Under these conditions, CoT values greater than 100 were obtained. The hybridization mixture was diluted to 60 Al with hybridization buffer without SDS, and 10 Mg streptavidin was added. The cDNA/biotin RNA hybrids were removed by phenol-chloroform extraction. The remaining single strand cDNA was isolated and hybridized with a 300 fold excess of biotinylated RNA as described above.

About 10% of the starting cDNA was recovered in the first hybridization and about 15-20% from the second hybridization.

The subtracted cDNAs were subjected to 20 cycles of a PCR reaction using oligo dT XhoI linker primer and SK primers (Stratagene*). The products of the PCR reaction were cut with EcoRI and XhoI, the primers and the flanking sequences were removed with sephacryl S-300 spin columns (Pharmacia*). The inserts were cloned into Lambda ZAP II arms.

Duplicate filters of the subtracted floor plate library were screened with radiolabelled first strand cDNA derived from floor plate and dorsal spinal cord. 100 ng of mRNA was incubated in 20 Al of 50 mM Tris pH 8.3, M MgCl 2 150 mM KC1, 1.0 mM dGTP, 1.0 mM dTTP, 100 MCi[32p]dATP (3000 Ci/mmol), 100 MCi[ 32 PdCTP (3000 Ci/mmol), 100 mg/ml oligo dT, 10 mM DTT, 10 U of RNasin 15 (Promega), 20 U of MulV reverse transcriptase (BRL), for min in 37°C. 4x10 3 recombinant phage were plated and screened. Hybridization and washes were performed at high stringency (Sambrook et al., 1989). The floor plate cDNA probe hybridized selectively with 24 phages. Cross 0 hybridization analysis revealed that these corresponded to three difference cDNAs designated FP2, FP5 and FP24.

The pattern of expression in the spinal cord was determined by in situ hybridization. FP2 and FP5 are expressed selectively- in the floor plate while FP24 is 2 expressed in the floor plate, roofplate and in the ventricular zone of the spinal cord. The degree of enrichment as determined by screening the floor plate enriched library and floor plate library with FP2, and FP35, which is expressed selectively in the floor 30 plate (McKanna Cohen, 1989) is about RNA Transfer Analysis Total RNA was prepared from various tissues using the RNA Azol method (Biotex Laboratories) and then enriched for poly containing transcripts by passage over an oligo (dT) cellulose matrix. RNA transfer was performed as described by Thomas (1980). Probes were labelled by random priming (Feinberg and Vogelstein, 1984) and hybridized under standard conditions.

DNA Sequencing and Analysis cDNA inserts were excised directly as Bluescript plasmids (Stratagene®). The nucleotide sequence of the inserts were determined by the dideoxy chain-termination method (Sanger et al., 1977) using both double-stranded and single-stranded DNA as template for T7 DNA polymerase (Sequenase, United States Biochemicals). The nucleotide sequence of the entire coding region was determined by 15 sequencing both strands. Sequences were assembled on an Apple Maclintosh computer using MacVector (IBI) program.

.go. In Situ Hybridization In situ hybridization was preformed as described previously (Wilkinson et al., 1987) using a T3 or T7 RNA polymerase-derived 5 S]UTP-labelled single stranded antisense RNA probe which encompasses a region of the 3' untranslated region of F-spondin (nt 3359-4029), or the .:TSRs (nt 1545-2626). Exposure times range from four to 25 fourteen days. Sense probes were used as controls.

For whole mount in situ hybridization, Ell rat embryos were fixed in 0.1 M OPS, 2 mM EGTA, 1 mM Mg S04, 3.7% formaldehyde for 2 hours. In =it hybridization was preformed essentially as described by Harland (1991), with a few modifications: anti-digoxygenin antibody (Boehringer Mannheim), was preabsorbed to E14 rat acetone powder (Harlow and Lane, 1988) before addition to the hybridization mixture. The chromogenic reaction was carried out for 1-2h.

DNA Constructs The myc epitope was introduced as follows: Two partially complementary ol igonuc leot ides with the sequence:

CTAGCGAGCAGAGCGATCTCCGAAACCA

3 (Seq. ID No. 1) and 51CATAGCTCCGAACGTCGTG3 (Seq. ID No. 2) were annealed to obtain a double-stranded

DNA

fragment coding for the c-myc proto-oncogefle epitope EQKLISEEDL (Seq. ID No. 3) f lanked by a SpeI site. the fragment was cloned into a unique SpeI site (nt 1365) in F-spondin. The same epitope was also introduced into a NcoI site (nt 1575) using the oligonucleotides:

CATGGGAGCAGAAGCTGATCTCCGAGGAGGACCTCG

3 (Seq. ID No. 4) and 5' -CATGCGAGGTCCTCCTCGGAGATCAGc~rCTGCTCC- 3 (Seq. ID No. The tagged F-spondin DNA was subcloned into the expression vector pMT2l (provided by Genetics Institute), or pcDNA-I (InVitrogen) cos Cells Transfection a a a. a.a a. a a a 20 Cos cells were transfected f ollows: 80% conf luent transfected with 5 gsg DNA, DE.AE Dextran (Pharmacia9), After 6h cells were washed serum, 0. 1mM choloroquine incubation in DMEM 10% isolation of F-spondin the (BRL), and the cells were by the DEE-Dextran method as overnight cultures were per 100 mu dish, in 250 Ag/ml 100 mX Tris pH 7.3, in DMEM.

and incubated in DND( 10% calf (Sigma) for 2.5h, followed by calf serjm overnight. For medium was changed to OPTI-MD( incubated for 48h.

metabolic Labeling of Cos Cells and Immunipreci~itatiofi Transfected cos cells were -preincubated in methioninefree DMEM (BRL@-GIBCO). After 1h at 37*C, 25OpCi/al( 3 5

S)

methionine (NEN) was added, and the cells were incubated for an additional 3h. The medium was collected and incubated with anti-myc antibody (MAb 9E10) for Ih. The immune complex was precipitated with fixed Staphylococcus aureus (BRL*) for 1h. Pellets were washed three times with PBS, before resuspension in lx sample buffer. 35

S-

labelled immunoprecipitated proteins were visualized after electrophoresis on 10% SDS-polyacrylamide gels.

Immunocvtochemistrv F-spondin tagged with the c-myc epitope was detected with MAb 9E10 (Evan et al., 1985). Fluoresceinated isotypespecific second antibody (Boehringer* Mannheim; goat antimouse IgG) was used at a dilution of 1:100. For Immunofluorescence labelling (Dodd and Jessell, 1985), cultures were washed once at 22*C with L15 and then incubated with primary antibody for 30 min at 22C.

15 Cultures were then washed twice in L15-1% normal goat 15 serum (NGS) and incubated with secondary FITC conjugated isotype-specific antibody diluted in L15-1% NGS for min at 22°C. Cultures were washed twice and fixed in 4% paraformaldehyde in 0.2 M phosphate buffer (PB) for min, rinsed in 0.12M PB and coverslipped in 0.05% paraphenylenediamine (Sigma) in 0.2 M sodium carbonate (pH glycerol Cultures were viewed on a Zeiss Axioplan microscope under epifluorescence optics.

25 Cell Culture Spinal cords were dissected from embryonic day 13 rats and placed into L15 medium at 4*C. The dorsal region of the spinal cord were dissected and incubated with 0.05 trypsin (Gibco) for 20 min in a Ca 2 +/Mg2+-free modified essential medium (S-MEM) (Gibco) supplemented with 8 mg ml'1 glucose. The tissue was then washed with S-MEM and triturated to give a single cell suspension.

Spinal cord cells were plated in 35 mm tissue culture dishes on appropriate substrates and grown in Ham's E12 medium (Gibco) supplemented with N3 additive (F12-N3) (Romijin et al., 1982) at a density of 106 cells/dish in a 5% C02 humidified incubator at 376C. Dorsal root ganglia were dissected from E14 rats and treated as described above. Cells were incubated with 0.1 trypsin, and plated with F12-N3 supplemented with 100 ng NGF at a density of 4xl0 4 /dish.

Neurite Outgrowth Assays 5x10 10 cos cells were transfected with pFP5myN and 1 conditioned medium was collected. F-spondin my c, was affinity purified on a monoclonal anti-myc (9E10) affinity column. Affinity purified F-spondin my c l/ml) was absorbed onto nitrocellulose (Lemmon et al., 1989). For controls, parental cos cell conditioned 15 medium was purified on the same column and used as a 15 substrate on nitrocellulose. The nitrocellulose was then blocked with bovine serum albumin (10 mg/ml) which provided a further control for background neurite outgrowth. E14 dorsal root ganglion (DRG) neurons were 2 plated on immobilized protein substrates at a density of 2-l0x104 cells/35 mm tissue culture dish (Nunc, 35 mm diameter) and grown for 14h. Cultures were then fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and stained using MAb 3A10 (Furley et al., 1990; available from Developmental Studies Hybridoma Bank), which recognizes a neuronal filament-associated protein and serves as a marker for fine neurites. Neuronal cell bodies and neurites were visualized by indirect immunofluorescence on a Zeiss Axioplan microscope.

Neurite lengths were measured as the distance from the edge of the soma (sharply defined by 3A10 fluorescence) to the tip of its longest neurite. Neurite lengths were only measured if the entire length to the neurite could be unambiguously identified. About 25 neurites were measurable within each protein-coated area (3-4 mm 2 Adhesion Assay Dissociated E13 dorsal spinal cord cells were plated on immobilized protein substrate at a density of 1o6 cells/35 mm tissue culture dish (Nunc, 35 mm diameter).

After one hour the cultures were washed twice with PBS and fixed in 4% paraformaldehyde. Cells were counted on a Zeiss Axioplan microscope at 400x magnification. Ten independent counts were taken from each experiment. The floor plate is a transient neural cell group implicated in the control of cell pattern and axonal growth in the developing vertebrate nervous system.

Experimental Results 15 Identification and Seauence of a Floor Plate-Enriched cDNA Clone Cellular assays have revealed that the floor plate has several specializing signalling functions during the embryonic development of the spinal cord. Floor platederived signals are likely to be encoded by proteins whose mRNAs are restricted to or are highly enriched in the floor plate. In order to identify such molecules subtractive hybridization techniques have been used to isolate cDNA clones that are expressed by the floor plate 25 but not by the dorsal spinal cord in embryonic day 13 rat embryos (see Figure 2 and Experimental Procedures).

One cDNA clone identified in this screen, designated contained a 0.5 kb insert which hybridized to two major transcripts of 4.5 and 4.7 kb in poly (A)-selected RNA S derived from E13 rat floor plate (Figure 3A). Very faint hybridization to the same two transcripts was detected in RNA derived from E13 dorsal spinal cord (Figure 3A) and post-natal day 0 brain (Figure 3C), whereas no hybridization was detected to RNA derived from adult liver and spleen (Figures 3A, The specificity of expression of FP5 transcripts within E13 rat spinal cord was confirmed by in s~i hybridization histochemistry which showed that FP5 mRNA is expressed at very high levels in the floor plate but is undetectable in the dorsal region of E13 rat spinal cord (see below). These studies indicate that FP5 transcripts are highly enriched in the floor plate.

Screening of an E13 rat floor plate cDNA library with the kb cDNA insert from the FP5 clone identified several additional cDNA clones of which clone FP5-9 contained a 4 kb insert. The FP5-9 cDNA contains a single long open reading frame that starts with a methionine codon at nucleotide 226 associated with a conventional translation initiation sequence (Kozak, 1984) and ends with a TGA 15 stop codon at nucleotide 2646 (Fig. 5A). No in-frame oeoo methionine codons were found upstream of the putative translation initiation site and sequences 5' of the initiation site contain stop codons in all three reading frames. Sequencing of several other independently isolated FP5 cDNA subclones spanning the entire coding region did not reveal any differences in the nucleotide sequence of the open reading frame.

Translation of the open reading frame FP5-9 predicts a protein 807 amino acids with a molecular mass of 90,766 daltons, and N-terminal hydrophobic leader sequence (Figure 5A; Seq. ID No. 9) with a consensus signal peptide cleavage site (von Heijne, 1985). No other long stretches of hydrophobic residues were observed (Figure suggesting that the protein does not possess a transmembrane spanning domain. The amino terminal domain of FP5-9 contains a region of clustered basic residues (residues 138-142) which could represent a site for proteolytic processing by mammalian subtilisin-like cleavage enzymes (Steiner, 1991). In addition, the predicted protein contains three N-linked glycosylation sites (Figure 5A). Collectively, these features suggest that the FP5-9 cDNA encodes a secreted protein.

The Protein Encoded by the FP5-9 cDNA has Structural Features of Cell and Substrate Adhesion Molecules Analysis of the predicted amino acid sequence of the 9 encoded protein reveals that it is separable into two major domains (Figure 6A). The NH 2 -terminal domain of 440 residues contains 10 cysteine residues and exhibits no sequence homology to other proteins in the Genbank database. The COOH terminal of the protein extends from residues 441-807 and contains six repeats of a domain 59 amino acids in length which can be aligned on the basis of conserved cysteine, tryptophan and arginine residues (Figures 6B, C).

Similar domains are present in a small number of proteins (Patthy, 1988; Smith et al., 1991). In particular, the adhesive glycoprotein encoded by the thrombospondin I and II genes (Lawler and Hynes, 1986; Bornstein et al., 1991) each possess 3 of these domains which have been designated thrombospondin type 1 repeats (TSRs) (Lawler and Hynes, 1986) (Figure 6C) Two TSRs are found in protein C6-C9 of the alternative complement cascade, one at the NH 2 -terminal and one at the COOH-terminal of each protein (Haefliger et al., 1989; Smith et al., 1991).

Moreover, the complement-binding protein properdin contains 6 TSRs which comprise 80% of the protein (Goundis and Reid, 1988). In addition to these vertebrate proteins, the central core of the TSR is similar to region II of malarial circumsporozoite

(CS)

and other plasmodial proteins (Figure 6C) (Rich et al., 1990; Robson et al., 1988) which appear to mediate the binding of malarial sporozoites to host cells in the early stages of parasitic infection (Dame et al., 1984).

Finally, two TSRs are present in the C.elegans gene Uncwhich appears to regulate axonal pathfinding in a subset of neurons (Hedgecock et al., 1990; Culotti et al., 1991). The organization of cysteine and tryptophan residues in the TSRs of the FP5-9 encoded protein is not similar to that of the NH 2 -terminal TSRs of the C6-C9 complement proteins (Figure 6B). However, the core region of the TSRs in FP5-9 (residues 14-19) is most similar to that of thrombospondin, properdin and the malarial CS proteins (Figure 6B). We have named the 9 gene F-spondin to reflect its high level of expression in the floor plate (see below) and the presence of the TSRs.

S .15 The TSRs in thrombospondin promote the adhesion of a variety of different cell types (Prater et al., 1991).

Similarly, the TSR core region of the plasmodium vivax CS protein promotes the attachment of human hematopoietic cell lines in vitro (Rich et al., 1990). The amino acid sequence VTCG which is contained within this common motif appears to be critical to the cell adhesive properties of the CS proteins. A VTCG sequence (Seq. ID No. 6) is also 25 present in the two TSRs of thrombospondin that promote cell adhesion (Prater et al., 1991). Strikingly, there is a VTCG in the fourth TSR of F-spondin and the second and third TSRs of F-spondin contain sequences (VSCG, Seq.

ID No. 7; ATCG, Seq. ID No. 8) that vary by a single conservative substitution (Figure 6B). These observations raise the possibi: -ty that the TSRs in Fspondin mediate cell adhesion. A search of the Genbank database for other proteins implicated in cell adhesion and recognition that contain a VTCG sequence identified V-CAM1 (Hession et al., 1991) and the VLA4 integrin r subunit (Takada et al., 1989).

Analysis of the predicted amino acid sequence of Fspondin reveals several other structural features that may contribute to the functional properties of the protein. The charged region that is interposed between the fifth and sixth TSRs contains the sequence LRE that has been shown to function as a neuronal cell attachment site in the extracellular matrix glycoprotein S-laminin (Hunter et al., 1989a, The first, third, fifth and sixth TSR's of F-spondin contain clusters of basic residues that have been implicated in the binding of proteins to heparin and other sulfated glycosaminoglycans "(Cardin and Weintraub, 1989). The first, fourth and fifth TSRs of F-spondin also contain a WSXWS sequence 15 (Figure 6B) which is present in the variant fibronectin type III repeats found in the receptors for several growth and differentiation factors, including ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF) and the interleukins (ILs) 2-7 (Bazan, 1990; Davis, et al., 1991; Patthy, 1990). The function of the WSXWS motif is unclear although mutation at this site in the IL2 receptor blocks transmembrane signalling (Miyazaki et al., 1991) Expression Pattern of F-SPondin mRNA Northern blot analyses of E13 embryos indicate that Fspondin is expressed at much higher levels in the floor plate than in the dorsal spinal cord. More detailed information on the distribution of F-spondin was provided by localizing its mRNA in developing rat embryos by in situ hybridization. F-spondin mRNA was first detected at E10.5 in cells located at the ventral midline of the neural tube at the level of the prospective midbrain, hindbrain and spinal cord (Figure 7A). At this stage, cells at the ventral midline of the neural tube have acquired floor plate-derived chemoattractant activity (Placzek, et al., 1990c) although no antigenic markers of floor plate differentiation can be detected. The expression of F-spondin mRNA therefore provides an early molecular marker of floor plate differentiation.

The expression of F-spondin mRNA is maintained at high levels in E11-E12 floor plate (Figure 7B) whereas other regions of the spinal cord and hindbrain exhibit undetectable levels of hybridization at this stage. By E12-E13 low levels of mRNA are detected in the ventral horn although there is still no detectable mRNA in the dorsal horn (Figures 7C, In addition, the ventral ventricular zone immediately above the floor plate begins 15 to express high levels of F-spondin mRNA (Figure 7) whereas hybridization to cells in the ventricular zone in the dorsal half of the spinal cord is not detectable (Figure 7E). Thus, expression of F-spondin mRNA reveals 2 a molecular difference between ventricular zone cells in the dorsal and ventral spinal cord. Recent studies have suggested that the ventral ventricular zone is the site of origin of oligodendrocyte and astrocyte precursors that subsequently migrate laterally and dorsally to populate the remainder of the spinal cord (Miller, 1991).

F-spondin mRNA levels remain high in the floor plate and ventral ventricular zone at E16 and by this stage significant hybridization is also detected in cells in the ventral and intermediate regions of the spinal cord (Figures 7F, By PO, the levels of F-spondin mRNA in the floor plate have decreased and there is an increase in hybridization to other cells in the spinal cord, resulting in an uniform expression of F-spondin mRNA (Figure 7H). F-spondin mRNA is also preferentially expressed in the floor plate of the Ell-E16 hindbrain and midbrain and becomes more widely expressed in the braii at later embryonic stages (not shown).

In addition to the expression of F-spondin in the embryonic CNS, from Ell-E12 onwards hybridization is also detected in association with sensory and motor nerve branches that project into the periphery (Figure 7D).

The association with peripheral nerve branches suggests that F-spondin mRNA is expressed in Schwann cells. The expression of F-spondin mRNA in association with peripheral nerves persists till E16, but appears to decrease at later stages, and by PO, little or no hybridization is detected in peripheral nerve (Figure 3C). These results provide evidence that over the period 15 of initial outgrowth of central and peripheral axons, Fspondin mRNA is expressed predominantly by the floor plate with lower levels of expression in cells of the *peripheral nerves, probably Schwann cells.

F-spondin mRNA is also expressed outside the nervous system. In particular, mesodermal cells underlying the ventral midline of the spinal cord express low levels of F-spondin mRNA from Ell (Figure 7D). In addition, S: embryonic and PO kidney (Figure 3C), lung and condensing cartilage (not shown) expresses F-spondin mRNA.

Expression of mRNA in the CNS, lung and kidney persists post-natally and in the adult (not shown).

Secretion and Cell Surface Association of F-Sbondin To determine the cellular localization of the F-spondin protein when expressed in mammalian cells, two epitopetagged derivatives, F-spondin l y were generated, each of which contain a 10 amino acid insert derived from the human c-myc proto-oncogene that can be detected by MAb 9E10 (Evan et al., 1985) (Figu-- 8A). The cDNAs encoding F-spondinmy c were cloned into a mammalian expression vector and transfected into cos cells. To examine whether F-spondin m ye is present in medium conditioned by transfected cells, cos cells were labelled with 35

S-

methionine for 3-4h and the released proteins were immunprecipitated with MAb 9E10. Immunoprecipitates from cos cells transfected with two different F-spondin my constructs revealed a single major band of -116 kDa that was absent from mock-transfected cells (Figure 8B).

Immunoprecipitation of proteins extracted from the cos cells indicated that the amount of F-spondin recovered from the medium was similar to that associated with the cells (not shown). Thus cos cells release a significant fraction of synthesized F-spondin y c. Other myc epitopetagged proteins, for example the drosophila wingless 15 protein, are synthesized by cos cells but are not detected in the medium Basler, Personal communication) suggesting that the presence of Fspondinmy in the medium does not result from leakage from damaged cells. Thus, under these in Yitro conditions

F-

20 spondinmyc is secreted from cells. The apparent molecular weight of F-spondin determined by SDS-PAGE (-116kDa) is significantly greater than that predicted from the amino :acid sequence (-90kDa) This difference in molecular weight may derive, in-part, from glycoslyation of the core protein.

The cellular localization of F-spondinl

Y

c in transfected cos cells was also determined by immunocytochemistry.

High levels of immunoreactivity were associated with the cell surface (Figures 8C, D) with both F-spondin m ye constructs (Figure 8A). No immunoreactivity was detected on the surface of untransfected cos cells (not shown).

The absence of a membrane spanning region and the presence of multiple heparin attachment sites in Fspondin suggests that the cell surface association of Fspondin my c involves the binding of the secreted protein to the cell surface or extracellular matrix. In support of this, F-spondin" m y present in the medium removed from transfected cos cells was found to bind to the surface of untransfected cos cells in vitro (not shown).

F-Spondin Promotes Neural Cell Adhesion and Neurite Outgrowth in vitro The structural features of F-spondin combined with its secretion and association with the cell surface raise the possibility that F-spondin can promote the adhesion of neural cells and the outgrowth of axons. Since F-spondin is expressed at highest levels in the floor plate, the 15 effect of F-spondin on the adhesion and outgrowth of dorsal spinal cord cells to include the population of commissural neurons that project to and across the floor plate was examined. In addition, the expression of Fspondin mRNA in peripheral nerve suggested that the S: 20 dorsal root ganglion (DRG) neurons might adhere to and S" extend neurites on F-spondin.

The F-spondinmye protein was purified on a MAb 9E10 affinity column from medium exposed to transfected cos cells (Figure 9A) and 'immobilized onto a nitrocellulose substrate (Lemmon et al., 1989). The ability of Fspondinmyc to promote the outgrowth of E14 DRG neurons was compared with that of MAb 9E10 affinity-purified proteins secreted from untransfected cos cells and BSA. Outgrowth of DRG neurons on EHS laminin was used as a positive control. Over 80% of DRG neurons extended neurites on Fspondin (Figures 9B, D) and the length of DRG neurites that extended on F-spondin was similar to that on laminin (not shown) and significantly greater than that on parental cos cell proteins and on BSA (Figures 9C, D).

Similar results were obtained with both versions of Fspondin m yc (not shown). In addition, the number of DRG neurons that adhered to a substrate of F-spondinlyc after 18h was about 3 fold greater than that to BSA and parental cos cell proteins, and similar to that on laminin (not shown). These observations provide evidence that F-spondin can promote the adhesion of DRG neurons and the extension of neurites in vitro. The expression of F-spondin by peripheral nerve cells in yiv2 occurs before many sensory neurons have extended peripheral projections and could therefore contribute to the growth of developing sensory axons in the peripheral nervous system.

15 The ability of F-spondin 0 e to promote the adhesion and outgrowth of dorsal spinal cord cells was also examined.

We found that dorsal spinal cord cells adhered well to Fspondinmyc. Within 60 min (Figures 10A, E) the number of o cells adhering to F-spondin was 10-15 fold greater than that to MAb 9E10 affinity-purified proteins secreted from untransfected cos cells or to BSA (Figures 10C, The majority of the adherent cells are neurons as determined by detection of the polysialic acid side chain of NCAM with MAb 5A5 (not shown; see Dodd et al., 1988; Karagogeos et al., 1991)-. Moreover, many adherent spinal cord neurons extended short neurites during this time period (Figure 10B). To examine further whether Fspondin promotes the outgrowth of spinal cord neurites the neurite length of adherent spinal cord neurons after 3 1h in vitro was determined. The length of spinal cord neurites on F-spondin my c had increased by 18 hours; however neurites outgrowth on purified cos cell proteins and on BSA has also increased significantly and was not detectably different from that on F-spondinyc. Thus it remains unclear whether F-spondin promotes extensive neurite outgrowth as well as the adhesion of spinal cord neurons.

The adhesion of a variety of cell lines to TSRs or to peptide derived from these repeats has been shown to be inhibited by glycosaminoglycans and other sulfated glycoconjugates (Roberts, 1988; Bernfield and Sanderson, 1990; Prater et al., 1991). Moreover, heparin sulfate proteoglycans have been suggested to function as cell surface receptors for thrombospondin (Holt et al., 1984; Sun et al., 1989; Bernfield and Sanderson, 1990). It is possible therefore that the interactions of neural cells with F-spondin may be inhibitable by addition of soluble glycosaminoglycans. It was found that adhesion of dorsal spinal cord neurons to F-spondin was markedly inhibited •by heparin, dextran sulfate (not shown) and to a lesser extent by chondroitin sulfate (Figures 10D, F) To control for non-specific inhibition of the interactions of spinal cord cells with all adhesive substrates, the 20 spinal cord neurons adhere well to fibronectin was determined and it was found that their adhesion is not significantly affected by concentrations of heparin that block adhesion to F-spondin (not shown). Heparin also reduced to near background levels the adhesion of DRG neurons to F-spondin (not shown). It was not possible to determine whether the outgrowth of neurites from DRG neurons is also blocked by addition of glycosaminoglycans because heparin caused the detachment of virtually all neurons from the F-spondin substrate, even when added to DRG neurons that had been permitted to settle on Fspondin for 2-3h.

ExDerimental discussion Floor plate cells are located at the ventral midline of the developing nervous system and have been implicated in the control of neural cell identity and in the guidance of developing axons (Jessell and Dodd, 1991). In order to identify genes that might contribute to the functions of the floor plate, subtractive hybridization techniques have been used to isolate cDNA clones encoding a novel protein, F-spondin. F-spondin mRNA expressed at high levels by the developing floor plate and at low or undetectable levels in other regions of the embryonic spinal cord over the period that axons first extend. The predicted structure of F-spondin together with its biochemical properties indicates that it is a secreted glycoprotein with homology to other proteins that mediate cell adhesion and neurite outgrowth. F-spondin promotes the adhesion and outgrowth of axons from embryonic 1: neurons in vitro, suggesting that it may contribute to the growth and guidance of commissural axons at the ventral midline of the spinal cord and of sensory axons in the periphery.

SLocalization of F-Spondin Several lines of evidence suggest that the F-spondin protein may be associated with the extracellular matrix.

First, F-spondin has several clusters of basic residues that function as glycosaminoglycan binding domains in other secreted proteins. Second, F-spondin is associated with the surface of cos cell transfectants. Third, the complement binding protein properdin which consists almost entirely of 6 TSRs has been shown to bind sulfated glycoconjugates (Holt et al., 1990).

The restricted distribution of F-spondin mRNA in "ne embryonic nervous system contrasts with that of other secreted glycoproteins which promote neural cell adhesion and neurite outgrowth. For example, the expression of Fspondin mRNA is more restricted than that of thrombospondin I (O'Shea and Dixit, 1988; O'Shea et al., 1990) and of tenascin/cytotactin (Wehrle and Chiqet, 1990) which appears to be widely expressed in the embryonic central nervous system. Similarly, laminin and fibronectin are expressed in many regions of the developing peripheral nervous system (Sanes et al., 1990). One glycoprotein which has a restricted distribution during nervous system development is laminin, an isoform of the laminin B chain (Hunter et S al., 1989a).

The TSRs of F-SDondin may be ResRonsible for Neural Cell Adhesion and Axon Extension The domains of F-spondin that mediate neural cell adhesion and neurite extension have not been mapped although several indirect lines of evidence suggest that the TSRs may be involved. First, proteolytic fragments of thrombospondin which contain the TSRs promote the adhesion of melanoma cells and antibodies directed against the TSRs domain block cell adhesion (Prater et Sal., 1991). Second, both native thrombospondin and a 140 kDa proteolytic fragment which includes the TSR domains promote the outgrowth of neurites from central and Speripheral neurons in vitro (Osterhout and Higgins, 1990; Osterhout at al., 1992; Neugebauer et al., 1991; O'Shea et al., 1991). In addition, antibodies directed against the TSR domains block neurite outgrowth on thrombospondin (Osterhout and Higgins, 1990; Osterhout et al., 1992).

Third, the plasmodial CS proteins, which contain the core domain of the TSRs also promote the adhesion of a wide variety of mammalian cells (Rich et al., 1990).

The adhesive properties of the CS proteins have been mapped to the VTCG sequence (Rich et al., 1990). In addition, the two peptides derived from the TSRs in thrombospondin that are potent attachment factors for melanoma cells also contain the VTCG sequence whereas the peptide derived from the third TSR which does not contain this sequence is not adhesive (Prater et al., 1991).

Thus, the presence of a VTCG in the fourth TSR of Fspondin suggests that this domain could be involved in the adhesive properties of F-spondin. Nevertheless, other domains within F-spondin may be involved in neural cell adhesion or neurite outgrowth. For example, the region interposed between the fifth and sixth TSP-1 1 repeats of F-spondin contains an LRE sequence that mediates the neuronal attachment properties of S-laminin (Hunter et al., 1989b).

The ability of neural cells to adhere to and extend 15 1 neurites on F-spondin suggests that there are neural receptors for this protein. The inhibition by heparin of the adhesion of dorsal spinal cord cells and DRG neurons .to F-spondin suggests that proteoglycans may constitute neuronal F-spondin receptors or may regulate receptor 20 function.

The conservation of TSRs in F-spondin and thrombospondin also raises the possibility that receptors for the TSR domains of thrombospondin may interact with the related domains of F-spondin. There is evidence that the TSRs of thrombospondin can interact with 3 distinct classes of cellular receptors (Frazier, 1991). First, thrombospondin and a VTCG-containing peptide from the TSR core region can bind to an 88 kDa membrane glycoprotein, GPIV, or CD36, which is present on many cell types (Asch et al., 1990, 1991). Second, thrombospondin can bind to sulfated glycoconjugates including the heparin sulfate proteoglycan syndecan (Roberts, 1988; Sun et al., 1989; Holt et al., 1989; Bernfied and Sanderson, 1990). In addition, the adhesion of cells to VTCG-containing peptides derived from the TSR domains of thrombospondin and plasmodial CS proteins can be inhibited by heparin and other glycosaminoglycans (Holt et al., 1990; Prater et al., 1991; Rich et al., 1991). Third, antibodies against integrins block neurite outgrowth on thrombospondin (Neugebauer et al., 1991). Since antibodies to the TSR domains of thrombospondin block the outgrowth of neurites on thrombospondin (Osterhout and Higgins, 1990; Osterhout et al., 1992) it is possible that sequences within the TSRs interact with neuronal integrins.

Possible Functions of F-Soondin in Neural Development 1 The most prominent expression of F-spondin in the 15 embryonic nervous system is in the floor plate, an epithelial cell group that has been implicated in several aspects of spinal cord development. Midline neural plate cells that give rise to the floor plate undergo marked cell shape changes during the closure of the neural tube.

Thus, one possible function of F-spondin could be to Smediate adhesive interactions between floor plate cells Sthat maintain the integrity of the floor plate during the formation of the embryonic spinal cord. The expression of F-spondin mRNA in floor plate cells is highest at the time that the floor plate has been suggested to have roles in the chemotropic (Tessier-Lavigne et al., 1988; Placzek et al., 1990a) and contact (Dodd et al., 1988) guidance of commissural axons. It is found that recombinant F-spondin"c secreted from cos cells does not mimic the ability of the floor plate derived chemoattractant to promote the outgrowth of commissural axons from dorsal spinal cord explants (Klar, Placzek, 41 Tessier-Lavigne, Dodd and Jessell, unpublished observations). This suggests that F-spondin may not be involved in the long-range guidance of commissural axons to the floor plate, at least through chemotropism.

F-spondin could be involved in the contact-dependent guidance of commissural axons once they reach the ventral midline of the spinal cord under the influence of chemotropic guidance cues. The growth cones of commissural neurons cross the midline by growing between the basal surface of floor plate cells and the underlying 1 basal lamina (Kuwada et al., 1990; Yaginuma et al., 1991). F-spondin secreted by the floor plate may accumulate at high levels in association with the basal surface of floor plate cells or with the underlying basal lamina thus generating a difference in adhesive properties of the floor plate and the lateral 15 neuroepithelium. The growth cones of commissural neurons may adhere preferentially to F-spondin, prompting them to change trajectory at the boundary of the floor plate and lateral neuroepithelium. It is also possible that Fspondin has a more active signalling role which induces changes in the properties of commissural growth cones eO that permits them to respond to other midline guidance cues. Several proteins are expressed selectively on the surface of floor plate cells at this stage of spinal cord 2 development (Dodd and Jessell, 1988; Chuang and Lagenaur, 1990) and could provide cues that contribute to the guidance of commissural axons at the midline.

F-spondin mRNA is also expressed by cells in the peripheral nerve, presumably Schwann cells, from Ell to 3 E16 over the period that motor and sensory axons project to their peripheral targets. Non-neuronal cells in peripheral nerve are known to secrete a variety of 42 extracellular matrix glycoprotein, including laminin and fibronectin that can promote the growth of developing axons. Antibody inhibition studies have provided evidence for the existence of additional molecules that mediate neuronal outgrowth on peripheral nerve substrates (Tuttle et al., 1989). The ability of recombinant

F-

spondin to promote the outgrowth of embryonic sensory neurons in vitro suggests that the protein may be released by non-neuronal cells in the peripheral nerve and could contribute to the initial outgrowth of sensory axons in vivo.

Taken together, the present studies identify F-spondin as a novel secreted protein with potential roles in neural cell adhesion and neurite outgrowth in vivo. The development of antibodies that recognize native F-spondin will be important in determining the localization of the protein within the nervous system and in assessing its function in more detail.

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54 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Jemuell, Thomas M Klar, Avihu (ii) TITLE OF INVENTION: CLONING, EXPRESSION AND USES OF A NOVEL SECRETD PROTEIN, F-SPONDIN (iii) NUMBE oF SEQUENCES: (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Cooper Dunham STREET: 30 Rockefeller Plaza CITY: Now York STATE: New York COUNTRY: USA ZIP: 10112 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: 13B4 PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOYNwARE: Patontlr& Release 01.0, Version #1.25 (vi) CURRENT APPLICATION

DATA:

*15 APPLICATION NUMBER: US FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGEN'T

INFORMATION:

MANE: White, John P REGISTRATION NUMBER: 28,678 REFRE=NCE/DOCKET NUMBER: 40028 (ix) TELECOIMMUNICATION

INFORMATION:

TELEPHONE: (212) 977-9550 TELEFAX: (212) 664-0525 TELEX: 422523 COOP U! S. INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: COMA (xi) SEQUENCE DESCRIPTION: SEQ ID No: CTAGCGAGCA GAAGCTGATC TCCGAGGAGG ACCTCA -36 INFORMATION FOR SEQ ID No:.2: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA SEQUENCE DESCRIPTION: SEQ ID NO:2: CTAGTGAGGT CCTCCTCGGA GATCAGCTTC TGCTCG 3E INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Glu Gln Lys Lou Ile Ser Glu Glu Asp Lou 1 5 INFORMATION FOR SEQ ID NO:4: Wi SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear *fee 20 (ii) MOLECULE TYPE: cDNA S (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CATGGGAGCA GAAGCTGATC TCCGAGGAGG ACCTCG 3E INFORMATION FOR SEQ ID NO:S: Wi SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPS: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NOsS: CATGCGAGGT CCTCCTCGGA GATCAGCTTC TGCTCC INFORMATION FOR SEQ ID 110:6: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUNNCE DESCRIPTION: SEQ ID 110:6: Val Thr Cys Gly 1 INFORMATION FOR SEQ ID 110:7: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: mingle TOPOLOGY: linear (1i) MO0LECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 110:7: Val Ser Cy. Gly INFORMATION FOR SEQ ID 110:8: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids ~20 TYPE: amino acid STRANDEDNESS: single TOPOLO0GY: linear (1i) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTI-ON: SEQ ID 110:8: Ala Thr Cys Gly 1 INFORMATION FOR SEQ ID 110:9: SEQUENCE CHARACTERISTICS: LENGTH: 4029 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE I NAME/XEY: CDS (1B) LOCATION: 226. .2647 (xi) SEQUENCE DESCRIPTZION: SEQ ID NO:9: CCCTCCCTCT T~CGGCTCCT TCGCCACCGC CCGcCCCCTCA GC TCCGC TGC TCGGCTCcGc TCAGMGCAGC GCAGCTC0GC AGCCAAAGCG AGGcGGGCTC GGGCTCCCCA CCGCCAGTGc 120 CACCCGGGCT CCTCCAGCTT TCGCCTCTGC AGCTCCCGTC ACTTGGAGTA AAAGTGTCCT 180 GACAGGGGTC TGCAACATCA GCAGAAAGTT GGGAGGTCCT CGAGA ATG AGG CTA 234 TCT CCC GCG, CCC CTG AGG CTT MGC COO GGT CCC- GCG CTG CTG GCC CTG 282 Ser Pro Ala Pro Lou Arg Lou Ser Arg Gly Pro Ala Lou Leu Ala Lau 10 GCG CTG CCC Crc GCC GCA OCO CTC OCT TTC TCG GAT GAG ACC CTG GAC 330 Ala Lou Pro Lou Ala Ala Ala Lou Ala Ph. Ser Asp Glu Thr Lou Asp 25 30 AAA GTG GCC AAO TCG GAG 0CC TAC TOC AGC CGC ATC TTG CGC GCC CAG 378 Lys Val Ala Lys Ser Glu Gly Tyr Cys Sec Arg Ile Lou Arg Ala Glra 45 GGC ACA COG OT GAG OGA TAC ACA GAG TTC AOC CTC CGC GTG GAA GGC 426 *Gly Thz Arg Arq Glu Gly Tyr Thr Glu Ph. Ser Lou Arg Val Glu Gly 55 60 GAC CCT GAC TIC TAT RAG CCA OA AOC MGC TAC CGA GTG ACA CTC TCG 474 Asp Pro Asp Ph. Tyr Lys Pro Gly Ser Ser Tyr Arg Val Thr Lou Ser 75 *GCT GCC CCT CCC TCC TAC TTC MGA 0CC TTC ACG TTA ATT OCT CTC AAA 522 Ala Pro Pro Ser Tyr Ph. Arg Gly Ph. Thz Lou Ile Ala Lou Lys 85 90 GAG AAC CGC GAK GGC GAT AAM GAA OAR GAC CAC OCIG GCC ACC TIC CAG 570 Glu hun Arg Glu G2ly Asp Lys Glu Glu Asp His Ala Gly Thr Ph. Gln :100 105 110 115 *ATC ATA GAT GAK CRA GMA ACC CMG TTT ATG AGT AAC TOT CCT GTO GCA 618 Ile Ile Asp Glu Glu, Glu ThrGlrI Ph* Mat Ser Aun Cys Pro Val Ala 120 125 130 GTC ACT GAA MC ACC CCT COG MGG MGG ACA COG ATC CMG GTG TIT TGG 666 Val Thz Glu Bar Tbxr Pro Arg Arq Arg Thr Arg Ile Gln Val Phe Trp, 135 140 145 ATA GCG CCA CCC'ACA GCC ACA GGC TGT GTG ATI CTG AMG 0CC AGC ATT 714 Ile Ala Pro Pro Thr Gly Thr Gly Cys Val Ila Lou Lys Ala Ser 119 150 155 160 GTA CAG MAA CGC ATT ATC TAT TTT CAA GAC GAG 0CC TCC CT.G ACC MAG 762 Val Gln Lys Arg Ile Ile Tyr Phe Gln Asp Glu Oly Ser Lou Thr Lys 165 170 175 AAG CTG TOT GMA CMG OAT CCC ACA CTT GAT OA OTO ACO GAC MGA CCG 810 Lys

ATC

TAT

Tyr

OCT

Ala

OTC

Val OCT4 Ala 260 Lou Lou Gly

AAT

Anni

CTGO

Lou 245 2AA clu Cyl

GAC

Amy

AAC

Ann

CAC

His 230

TOG

Trp

CTT

Lou 58 Giu Gin Asp Pro Thr Lou Asp Gly Val Thr Asp Arg Pro 185 190 195 TGC TGC OCC TGC OGA ACT GCC AAG TAC MGA CTC AcC. ?T eye Cy. Ala Cy. Oly Thr Ala Lys Tyr Ar; Lou Thr Phe 200 205 210 TOG TO GAG AMG ACT CAT CCA AMG GAT TAC CCT COT OG Trp Sor Giu Lys Thr His Pro Lys Asp Tyr Pro Ar; Ar; 215 220 225 TOG TCT GCC ATC ATT GOC GGA TCC CAC TCC AMG AAC TAC Trp Sor Ala Ile Ile Gly Gly 8cr His So: Lys Ann Tyr 235 240 GAG TMC OGA 000 TAT 0CC MGT GAA 000 GTC AMG ChA OTT Olu Tyr Oly Oly Tyr Ala So: Glu Gly Val Lys Gin Val 250 255 0CC TCA CCA OTA AAA ATr. GAG OAA GAA AT? COA CAA CMG Oly So: Pro Val Lys Hot Oiu Glu Giu Ile Arg Gin Gin 265 270 275 b MGT OAT GAA OTC So:

TOG

Trp

GAC

Asp

CC?

Pro

TOT

Cys 340 0cc dly

CC?.

Pro

COT

Pro

G=C

Val

GAC~

Asp 420 Amp Cho Gin

MG

Arg

GAC

Amp 325

CC

Oly

ACO

Thr Cho Gin

ITT

Pho Ou Val CCT OTC Pro Val 295 ACA CC Thr Arg 310 TOG AAC Trp An TOG OTC Trp, Val GC MGC Amp Bar GAA A Oiu Lys 375 TAT GAC Tyr Asp 390 CTC ACT OTC ATC AAA CCC AAA 0CC CAG TOG CCA TCC Lou Thr Val Ile Lys Ala Lys Ala Gin TZ-p Pro Ser 280 285 290 AT OTO AGA GCA GCA CCC TCA GCC GMA TTC TCA OTG Ann Val Ar; Ala Ala Pro Sor Ala Giu Phs So: Val 300 305 CAC TTO -ATG TCC TTC CTA ACC ATC ATO GGC CCC MT His Lou Not Sor Pho Lou Thr Hot Not Gly Pro So: 315 320 GTO 0CC CTA TCT GCA GMG GAT CTO TGC ACC MOG GAG Vai Gly Lou Bor Ala Giu Asp Lou Cys Thr Lys Oiu 330 335 CMG AMA OTO GTO CMG AC CTA AT? CCC TOG OAT OCT Gin Lys Vai Val Gin Amp Lou Ile Pro Trp Amp Ala 345 350 355 000 OTO AMC TAC GAG TCA CCA MAC AMG CCC ACA ATT Oly Val Thr Tyr Clu Sor Pro Amn Lys Pro Thr Ile 360 365 370 ATC OGA CCC CTG ACT MGT CTG GAC CAT CC? CMG AGT Ile Arg Pro Lou Thr Sor Lou Amp His Pro Gin So: 380 385 COO GMA GOT 000 TCC ATC MCA CAA OTO 0CC MGA GTC Pro Ciu Gly Oly So: Ile Thr Gln Val Ala Ar; Val 395 400 858 906 954 1002 1050 1098 1146 1194 1242 1290 1338 1386 1434

GAG

clu

OTO

Val A ATT 0C= CMG AM OA GMA CMA TOC MAC ATT OTA CC Arg Ile Ala Ar; Lys Oly Giu Gin Cyn Ann Ile Val Pro 410 415 OAT OAT AT? OTA 0CC GM CTO OCT CCR GMA GAG AM GAT Amp Amp Ile Val Ala Amp Lou Ala Pro Giu Giu Lys Amp 425 430 435 1482 1530 59 GAL GAT GAc AOC cOT GAP Acc TGC ATC TAC TCC AAC TOO TCC CCA TOG 1578 Giu Ap Amp Thr Pro Glu Thr Cyn Ile Tyr Ser LAn Trp 5cr Pro Trp 440 445 450 Too GCC TOO AGC TCT TCC ACT TOT GAA LAG GOT AAG AGO ATO COO CAA 1626 Ser Ala Cys 8cr Ser Ser Thr Cys Glu Lys Gly Lys Arg Hot Arg Gin 455 460 465 OC ATO CTG AAG GCA CAG CTO GAC CTC POT GTC CCC TOT CCT GAC ACC 1674 Ar; Not Lou Lys Ala Gin Lou Amp Lou Ser Val Pro Cys Pro Ap Thr 470 475 480 CAG GAC TTC CAG CCC TGC ATO 0CC =C 0CC TOO AGO GAT GAA GAT 0CC 1722 Gin Asp Ph* Gin Pro Cys Hot Gly Pro Gly Cys Ser Asp Glu Amp Gly 485 490 495 TCC ACC TOT ACC ATO TCIG GAG TOG ATC ACC TOG TCA ccc TOC LOT GTC 1770 Scr Thr Cys Thr Met Ser Glu Trp Ile Th: Trp Bar Pro Cyrn Ser Val 500 505 510 515 TCO TOT GCC ATG OCT ATO, AG TCC CGO GAG LOG TAO GTO LAG CLO, TTC 1818 Ser Cys Gly Hot Oly Met Arg Ser Ar; Glu Ar; Tyr Val Lys Gin Ph.

*520 525 530 COG GAA GAC 0CC TCG GTO TOO ATG CTO CCC LOG GAL GAG ACA GAG LAG 1866 *Pro Glu Asp Oly Ser Val Cys Hot Lou Pro Thr Giu Giu Thr Giu Lys 535 540 545 *TOO LOG OTC ALC GAG GKO TOC TCT OCT AGO AGC TOO CTO OTO ACT GAG 1914 Cys Tb: Val Man Giu Giu Cys Pro Bar 5cr Cy. Lou Va1 Thr Glu 550 555 560 TOG GOT GAG TOC GAT GAC TOO AGO 0CC ACC TOT OCA ATO 000 ATO LAG 1962 Trp Gly Giu Trp Asp Asp Cys Ser Ala Tb: Cys Gly Met Gly Met Lys 565 570 575 LAG COG CAC CGO ATO, GTC ALO ATG AGC CCC 000 GAC 000 TCC ATG TGC 2010 Lys kr; His Arg Not Va1 Lys Met 5cr Pro Ala Asp Gly Ser Met Cys .:580 585 590 595 LAG GOG GAG ACT TOG CLO GOO GAG AAA TOO ATG ATGOCCT GAG TOO CAT 2058 Lys Ala Glu Tb: Ser Gin Ala Glu Lys Cys Met Met Pro Giu Cys His 600 605 610 ACC ATC COG TOO ?TT CTG TOT COT TOO TCO GAG TOG AGO GAC TOT AGO 2106 Thr Ile Pro Cyn Lou Lou 5cr Pro Trp 5cr Glu Trp 5cr Asp Cyn 615 620 625 GTG ACC TOT 000 LAG 0CC ATO 000 A00 COOC CAG OG ATO, CTC LAG TOT 2154 Val Tb: Cys Gly Lye Gly Met Arg Tb: Arg Gin Arg Hot Lou Lys Ser 630 635 640 CTO WAP GAM CTO 000 GAC TOT PAT GAG GAT cTO GAG CAG 000 GAG LA 2202 Lou Ala Giu Lou Gly Asp Cys AMn Ciu Asp Lou Oiu Gin Ala Giu Lys 645 650 655 TOT ATO CTG CA GAG TOO Ccc ATT aAO TOO GAm cTc AGT GAG TOG TOO 2250 Cys Met Lou Pro Glu Cym Pro Ile Ap Cys Giu Lou 5cr Glu Trp, 660 665 670 675 CAG TOO TOT GAL TOT LAC AG TOO TOT 000 ARA GCT CAc ATo AT? COP 2298 Gin Try, 5cr Glu Cys AMn Lys 8cr Cys Oly Lys Gly His Hot Ile Ar; 680 685 690 ACC OG ACA ATC CMA ATO GMA CCT CAG TTT GGA GOT GCA CCC TOC CCA Thr Ar; Tb: Ile Gin Not Giu Pro Gin Phs Gly Gly Ala Pro Cys Pro 695 700 705 GAG ACT GTG CMA CGC MG MAG TGC COT GCC COG AM TOO CTT COC AGC Glu Thr Val Gin Ar; Lys Lys Cy. Ar; Ala Ar; Lys Cys Lou Ar; Sor 710 715 720 2346 2394 CCA TCO Pro Sor 725 AG? GAG Sar Glu 740 coo Arg

ATG

met ATC CMG MG CTO OC TOG MGG GAG GCC OA GAG MrC AGO, AGO 110 Gin Lys Lou Arg Trp Arg Giu Ala Ar; Glu Bar Ar; Ar; 730 735 CMG CTG MGG GMA GAG TCA GAT OGA GMG CMG TTC CCA GGC TOT Gin Lou Ar; Giu Glu S.r Asp Gly Giu Gin Phe Pro Oly Cys 745 750 755 COO COG TOG ACA 0CC TOG TCA GMG TOO ACC MAA CTO TGC OGA Arg Pro Trp Tb: Ala Trp, So: Giu Cys Thr Lys 'Au Cys Gly 760 765 770 ATC CMA GM CGO TAC ATO ACT OTO AM MO AGO TTC MAA AGO Ile Gin Giu Ar; Tyr Not Tb: Val Lys Lys Ar; Ph. Lys Bar 775 780 785 TTT ACC MGC TOO A GAC MGQ MG GMG ATC MGA 000 TGC MAC Ph. Tb: 8cr Cys Lys Asp Lys Lys Giu Ile Arg Ala Cys Asn 790 795 800 CCT TOT T MGTMGGGTT CRMCTCOCA GGTOCATT CCMGATTCTA Pro Cys GOT GGO Gly Gly TOO CMG Ser Gin GTG CAC Val His 805 GTCACMATG OTTGGGTGGT GTATTTGCTT GTTTAMGATG ATTTAMRTTG TGTCCACATG ATT TACCOGTOTG GTTTOCC TMGTCT GAGGCGAGG GACATCTTGT CTGAMTAC?1! CTTGGTOMGT CMCAGCGGGCATCT TOTCOCMAG CGCCATCTTC cTOCACTGAG TYTGMO TTGTC7MC TTOGTACTMA ACTGAATCGT OTCOCTCTG AGCATCOOT GGTCAMOMA GOGOAGACT TTOGCCATCC ACAAPGGAA OCAACCMGGA TGOCCAT~OC GGMAACACA CATTAATT GCAAAMGACAGTCCTCTCCOT A CTCTTAC74A AACOTOTTTG TCCCCTCCT 11"1TTATTTA GCACAACTCC MGGCATCTOQ GTAMCTCTCC MOGOTCATOCG G??CTTCGOT GOCTOAMGO

AMOCTO

MGGTGAMGTO OOTTA CAACC AATACTGCTT TACTGGCATC ACAAGGTCAG CAGGTOATOA TOCACATTTCATTOT OMOCOTOAT TTCOOTTGMG TTrOATT TGGTOOCATA AATCTCCTAM GATOCTGAC GCACATCA t1ibCAGATCTTC TTTOAGCMA TOTAGACAGT AMOCTUOCA CTOGTTCCAA AMCCAACTTA AAATCTTCCT ACACATATCC AGACC1T1TTTAGTT CAMACTTCCT TMGAATMMA CATTTTAGCT CTGAGAACTA CTTGATAMCT CTGCCAGGAA OCCMAGT CAATTCTTCA ACAAAAATAC 2442 2490 2538 2586 2634 2687 2747 2807 2867 2927 2987 3047 3107 3167 3227 3287 3347 3407 3467 3527

TATCTTCCCT

AATCTATTCC

CCTGACTTCT

CCACTGACTC

AGGATGTTTA

ACCACCACAT

A.AGATCGACA

TTTCCTGCCA

TGTTCAATTA

ACTTAA-r171

CATGACTAAG

GGACCAGGCC

CAAGCCATCA

TCTCAGCTAT

CCTTTTGCAA

CAGTGCTATG

CATCTATGTA

AAAAAAAAAA

TTTTAAGTCA

ACACAAACCT

CCTGATTTTC

GCAGCACCCA

GAGCTCA?!TG

GTCTGTTTAT

AATACAGTAC

CAACCCCTGA

AA

TGATATTT'TA

ACAAGAAAGG

ATGGATAGTC

AACCCAGGAG

GCAGGTTGTA

TACCGCTTCA

TTTAAGGTCT

ATATGTATT

TAGTTAGAGG

GTTACTCAGT

CAAAGGAAGG

CAACAA.ATAT

CTCATGCATC

TCCAAATACA

GCATAAACA

TTCCTTAACA

AGAGAGAGAC

CAAGCCTGTG

CCAGGGGTC

TCAGAGAAAG

TGTTAAAAGC

TTTTGTGGTC

CATCAGAATA

CAAGAGAGCC

358 364 370 376 382 388 394' 400' 402', INFORMATION FOR SEQ ID 140:10: SEQUENCE CHARACTERISTICS: LENGTH: 807 amino acids TYPE: &mino acid TOPOLOGY: linear (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID 14:10: met 1 Lou Thr A.rg Lou Ser Pro Ala Pro Lou Ala Lou Ala Lou Asp Lys Lou Pro Lou Ala Arg Lou 10 Ala Ala 25 Glu Gly Val Ala Lys Ser Arg Gly Pro Lou Ala Phe Tyr Cys Ser Thr Giu Phe Arg Ile Lou Ser Lou Arg Ala Lou is ~35 Arg Ala Gin Gly Thr Arg so Val Glu Arg Phe Giu Gly Tyr Tyr Lys Pro Gly Asp Pro 65 Thr Asp 70 Pro Gly 75 Arg Ser Tyr Arg ValI so Lou Ser Ala Ala Pro 8cr Tyr Pho 90 Gly Phe Thr Lou Ile Ala Lou Lys Thr Phe Gin 115 Pro Val hl^ 13 C Glu 100 11e Asn Arg Giu Giy 105 Glu Lys Glu Glu Asp Ile Asp Glu Giu 120 Thr Thr Gin Ph. met 125 Thr His Ala Gly 110 Ser Asn Cys krg Ile Gin Val Thr Glu Ser 135 Pro Arg Arg Axrg 140 Cys Val Ph. Trp Ile Ala Pro Pro Thr Gly 145 150 Thr Gly 155 Val Ile Lou Lys 160 Ala Ser Ile Val Gin Lys Axq 110 Ile Tyr Phe Gin Asp Glu Gly Ser 165 170 175 62 Pro Thr Lou Asp Lou Thr Lys Lys 180 Lou Cys Glu Gin Asp 185 Gly Val Thr 190 Asp Leu Pro 225 Lys Lys Ar; Trp Phe 305 Gly Thr Trp Pro Pro 4 385 Ala Ile Glu Ser Hot 465 Pro Arg Thr 210 Arg Au n Gin Gin Pro 290 Sor Pro Lys Asp Thr 370 Gin Ar; Val Lys Pro 450 Ar; Asp Pro 195 Ph.

Ar; Tyr Val Gin 275 Ser Val1 Ser Glu Ala 355 Ile Ser Val Pro Asp 435 Trp Gin Thr Ile Tyr Ala Val1 Ala 260 Ser Trp Asp Pro Cy s 340 Gly Pro Pro Val Asp 420 Glu Ser Ar; Gin Lou Gly Asn Lou 245 Giu Asp Gin Arg Asp 325 Gly Thr Gin Phe Ile 405 an Asp Ala Met Asp 485 Asp Asn His 230 Trp Leu Giu Pro Thr 310 Trp Trp Asp Giu Tyr 390 Glu Val

ASP

cys Le~x 470 Ph.

Cy s Trp 215 Trp Glu Gly Val1 Val 295 Ar; An Val Ser Lys 375 Asp Ar; Asp Thr Ser 455 Gin Cy 5 200 Ser Ser Tyr Ser Leu 280 Ann His Val1 Gin Gly 360 Ile Pro Ile Agp Pro 440 Ser Pro Ala Cys Glu Lye Ala Ile Gly Gly 250 Pro Val 265 Thr Val Val Ar; Lou Met Gly Lou 330 Lys Val 345 Val Thr Arg Pro Giu Gly Ala Ar; 410 Ile Val 425 Giu Thr Ser Thr Gin Lou Cyn Met 490 Gly Thr Ile 235 Tyr Lys Ile Ala Ser 315 Ser Val Tyr Lou Gly 395 Lys Ala Cyn Cyn Asp 475 Thr His 220 Gly Ala Met Lys Ala 300 Ph.

Ala Gin Glu Thr 380 Ser Gly Asp 1is Glu 460

LOU

Ala 205 Pro Gly Ser Glu Ala 285 Pro Leu Glu Asp 5cr 365 5cr 11.

Glu Lou Tyr 445 Lys 5cr Lye Lye Ser Giu Glu 270 Lys Ser Thr Asp Lou 350 Pro Lou Thr Gin Ala 430 Ser Gly Val Tyr Asp His Gly 255 Glu Ala Ala met Lou 335 Ile Ann Asp Gin Cys 415 Pro Ann Lys Pro Ar; Ty r Ser 240 Val Ile Gin Glu Met 320 Cys Pro Lys His Val1 400 Ann Glu Trp Ar; Cys 480 Gly Pro Gly Cyn 5cr Asp 495 Giu Asp Gly 5cr 500 Thr Cys Thr Met 5cr 505 Giu Trp Ile Thr Trp 5cr Pro 510 a a.

Cys Lys Thr 545 Val Oly Ser Giu Ap 625 Lou -Ala met Pro 705 20 Lou Ser Pro ser Gin 530 Glu Thr met met Cys 610 Cys Lysa Glu Trp Ile 690 Cys Arc; Arc; Gly Val 515 Phe Lys Glu Lys Cys 595 His 5cr 5cr Lys Ser 675 Arc; Pro Scr Arc; Cym 755 5cr Pro Cym Trp Lys 580 Lys Thr Val Lou Cym 6 6C Gin Thr Glu Pro Ser 740 Arc; Cys Glu Thr Gly 565 Arc; Ala Ile Thr Ala 645 met Trp, Ar; Thr 5cr 725 Glu Gly Asp Val 550 Glu His Glu Pro Cys 630 Glu Lou Ser Th~r Val1 710 Ile Gin met Gly 535 Asn Trp Arc Thr Cys 615 Gly Lou Pro Glu Ile 695 Gin G In Lou Gly 520 5cr Giu Asp Met Scr 600 Lou Lys Gly Giu Cys 680 G I r Arc; Lys Arg Met Val Glu Asp Val 585 Gin Lau Gly

ASP

Cys 665 Aen met Lys Lou GIlu 745 Arc; Cys Cys Cys 570 Lys Ala Scr met Cys 650 Pro Lys re Iu Lys Arg 730 Glu Scr met 5cr 555 5cr met Glu Pro Arc; 635 Awn Ile Ser Pro Cys 715 Trp Ser ALrc Lou 540 Pro Ala 5cr Lys Trp 620 Thr Glu Asp Cys Gln 700 Ar; Arc; Asp Glu 525 Pro 5cr Thr Pro Cys 605 5cr Arc; Asp Cye Gly 685 Pho Aia Glu Giy Glu 765 Arc; Thr 5cr Cys Ala 590 Hot Glu G&in Lou Glu 670 Lys Gly krc; Ala Giu 750 Cys Tyr 0 iu Cys Gly 575 Asp Not Trp Arc; Giu 655 Lou Gly Gly Lys Arc; 735 Gin Thr Val1 Glu Lou 560 Met Gly Pro Ser Met 640 Gin Scr His Ala Cys 720 Glu Ph.

Lys Met Arc; Pro Trp 760 Thr Ala Try 5cr Lou Cy. Gly Gly Gly 770 Ile Gin Giu Arc; Tyr Hot Thr Val Lye Lye Arc; 775 780 Phe 785 Aia (2) Lye Ser 5cr Gin Ph* Thr 5cr Cys Lys 790 Cyg An Vl His Pro Cys 805 IMFOR)AT!oN FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 3226 base pairs TYPE: nucleic acid Asp 795 Lys Lys Giu Ile 800 64 sTRAJIDIDNESSI single TOPOLOGY: linear (ii) moLECULE TYPE: cDMA (iix) FRATURZ i MAIU/UlYs CDS LocLTIOIIZ 136. .2S43 (xi) S3QUNCI DISCRIPTION: SZQ ID NO:11: GTGTCCCTCC CTCCCT rccTCTCTC TUSc..isiCTC cGCCTGCC CCCTCCCoGC E& CCTCTCCCG cGCCOCLGCC TC ccccGC cOCCcGGC TGCCCGAGCT GTGCGGGCGC CGAGG ATO GCA CO CO CTG COG CCC CTG GCC CTG COG CTG Not Ala Ala Ar; Lou Ar; Pro Lou Ala Lou Arg Lou 1 5

C

CTO OCO COC Lou Ala Ar; is CTG GAG AA Lou Glu Lys GCC CAA GCC Ala Gin Gly 45 GAG GGC CAT Glu Gly Amp CrT TCT OCT Lou Ser Ala CTG AMG GAA Lou Lys Glu TTT CMG ATC Ph. Gin Ile 110 CTC GOTT0? Val Ala Val 125 TTC TOG ACA Ph. Try, Thr MGT ATT GTO Sax Ile Val ACC TTC CCC Thr Ph. Pro GCC GCC AAM Ala Ala Lys ACC MGG MG Thr Arq Ar; 50 CCG GAL TTC Pro Glu Ph.

65 0CC ACT CCT Ala Thr Pro s0 GGA AAA GMA Oly Lys Olu ATA GAT GAL Ile Asp Glu ACT GAG MGC Th: Glu Ser 130 OCT CCT CCT Ala Pro Pro 145 CAG LAG COC Gln Lys Arvj 160

TTO

Lou Tcc 35 clu

TAC

Tyr

CO

Ala

COT

Cly

GAL

Glu 115

ACA

Th: 20 Glu

G

Oly

AMG

Lys

TAC

Tyr

GAT

Amp 100

GAG

Clu

CCT

pro Ala Ar; CCC TAC Gly Tyr TAC ALT Tyr Asn OCT OCC Pro Gly 70 T1"T OA Ph. Ar; 85 AAA GAG Lys Clu AO CMG Tb: Gln MGA MG Ar; Ar; Gly Ph* TOC MGC Cys 8cr COAL T!? Clu Ph* 55 MhC MGT Awn Ser OGA TTC Oly Ph.

CAR GAC Ciu hop TTC ATG Phe Hot 120 MGG ACA Ar; Thr 135 GCG MGG GCC TTC TCC Ser

COG

Ar;

AGC

Bar

TAC

Tyr

ACA

Thr

CAT

His 105

AC

Bar

COC

Ar; GAC GAG ACC Asp Glu Thr ATC CTG OL Ile Lou Ar; CTG MOG GTO Lou Arg Val OGC GTO ACO Ar; Val Thr TTG ATT'GCT Lou Ile Ala O OGA ACT Ala Cly Tb: MAT TOT CCC Ann Cym Pro ATC CMG GTC Ile Gin Val 140 CTG AMA 0CC 120 171 219 267 315 363 411 459 507 555 603 651 ACT OT ACO Tb:

A??

I10 Cly

ATT

I10 Thr

TAT

Tyr 165 OwC Gly 150 Ph.

TOT OWC A?? Cym Val 11.

CMG GAC GAG Gin Asp Clu Lou

GGT

Gly 170 155 TCT CTC Ser Lou ACC AAA AGA ATC ACC LA M ATCTOT GAL CAL OAT TCA 0CC TCT GAA CCT OTG ACT CAC Thr Lys Arg Ile Cyn Giu Gin Asp Ser Ala 8cr Glu Gly Val Thr Asp 175 180 185 AAA CC& ACA TTA GAT TGC TOT 0CC TOT GGA ACT 0CC AAA TAC AGO CTA 747 Lys Pro Thr Lou Asp Cys Cys Ala Cys Gly Thr Ala Lys Tyr Az; Lou 190 195 200 ACO TTT TAT OCA AAT TOG TCG GAA AAA ACA CAT CCC MAA GAC TTT CCT 795 Thr Ph. Tyr Gly hAn Trp, Ser Glu Lys Thr His Pro Lys Asp Ph. Pro 205 210 215 220 OG0 OC ACC MAC CAT TOO TCT GCO ATC ATT OGT hC TCT CAC TCA MOG 843 Ar; Ar; Thr hAn His Trp Ser Ala Ile Ile Gly Bar Bcr Him Bar Lys 225 230 235 MAC TAC ATC CTT TOG GAG TAT OGA 000 TAT OCT AG? GMA GOT GTC MAG 891 hAn Tyr Ile Lou Trp Ciu Tyr Gly Gly Tyr Ala Ser Giu Gly Val Lys 240 245 250 CAG GTT OCA GAO CTO OA TCC CCA GTC MAG ATO GMA GM GMA AT? CGA 939 GIn Val Ala Glu Lou Gly Ser Pro Val Lys Hot Giu Giu Giu Ile Ar; 255 260 265 *CAA CAA hOT GAT GAG GTT T AU. GTC ATC MG WCA MAA OCA CAG TOG 987 Gin Gin 8cr Ap Giu Val Lou Thr Val Ile Lys Ala Lys Ala Gin Trp 270 275 280 CCT CC TOG CAG CC? CTG MAT GTO AGA OCT OCT CCC TCT OCT GAG TTT 1035 Ala Try, Gin Pro Lou Anr Val hrg Ala Ala Pro 8cr Ala Giu Phe, 285 290 295 300 *TCT OTT GA? CGC CAC COG CAC CTG ATO TCC TTC CTC ACC ATG CTG 000 1083 Ser Val Asp Ar; His Arg His Lou Not Bar Pb. Lou Thr Hot Lou Oly 305 310 315 CCC AG? CCC GAC TOG MAT GTG OC CTO TCT OCT GAG GAC CTC TOC ACC 1131 Pro 5cr Pro Asp Trp hen Val Gly Lou Ser Ala Olu Asp Lou Cys Thr 320 325 330 MAG GAC TOT QOC TOG OTT CM MAA GTC OTO CAG GAT TTA ATC CCC TOO 1179 Lys Asp Cys Gly Trp Val Gin Lys Val Val Gin Ap Lou Ile Pro Trp 335 340 345 G AT 0CC OGC ACA GAC MT GGC OTC ACC TAT GAG TCA CCC M~C AAA CC? 1227 *Asp Ala Gly Tb: Asp Bar Gly Val Tb: Tyr Glu Bar Pro hen Lye Pro *:350 355 360 ACA OTT CCT CMA GAG MRG AT? MA CCA CT? ACA MGC Th GAT CAC CCT 1275 Thr Val Pro Gin Ciu Lye Ile. Arg Pro Lou Tbr Bar Lou Asp His Pro 365 370 375 380 CAG AG? CCA TTT TAT GAT CCA GMA GGA OA ?CT ATC hAG CT? GTh 0CC 1323 Gin Bar Pro Phe Tyr Asp Pro Giu Gly Gly Bar Ile Lys Lou Val Ala 385 390 395 MGA GTC OTO CT? GM AGA AT? GCA CG MAG 000 GAG CMG TG MhC TC 1371 Arg Val Val Lou Oiu Ar; le Ala Arl Lys Gly Glu Gin Cys han Ph.

400 405 410 0Th CC? GA? MAC ATA GhT CAT AT? OTO OCA GAC CTA OCA CCA OAA GMA 1419 Val Pro Asp ken Ile Ap Asp Ile Val Ala Ap Lou Ala Pro Giu Giu 415 420 425 66 AM GAL GAA GAT OAT A=C C= GAG A=C TOC ATA TAT TCA AMC TOG TCC 1467 Lys olu Glu Asp Asp Thr Pro Glu Thr Cys Ile Tyr Ser Len Trp Ser 430 435 440 CO c TOG 1 TC G TGC MGC TCC TCT ACC TOT GAG AMG GOC AMG AGO ATO 1515 Pro Try, Sor Ala Cys 8cr Ser Sar Thr Cys Glu Lys Oly Lys Ar; Met 445 450 455 460 AGO ChoGAGA ATG CrT AAA OCT CAG CTO GAC CTC AGT GTG CCC TOT CCT 1563 Ar; Gin Ar; Hot Lou Lys Ala Gin Lou Asp Lou Ser Val Pro Cys Pro 465 470 475 GAT ACC CAA GAT TTT CMG CCL TOC ATO GOT CCL GGC TOC ACT GAT GMA 1611 Asp Tb: Gin Asp Ph* Gin Pro Cyl Met Gly Pro Oly Cys Ser Asp Glu 480 48S 490 GAT GOT TCA ACT TOC ATO ATG TCT GMC TOO ATT ACA TOO TCC CCC TOT 1659 Asp Giy Ser Thr Cys Met Met Bar Asp Trp Ile Tb: Trp Ser Pro Cys 495 500 505 LOT OTT TCC TOT GGA ATO OA CG, CGA TCT AGA GAG AGA TAT GTA AM 1707 Ser Val Ser Cym Oly Met Gly Tb: Arg Ser Ar; Glu Ar; Tyr Val Lys *9*510 515 CMA TTC CCC GMA OAT CCPC TCT ATO TOC AAA OTO CCT ACT GMA GMA ACT 1755 *Gin Ph. Pro Olu Asp Gly Bar Met Cys Lys Val Pro Tb: Olu Glu Tb: S 530 535 540 :GAG AAA TOT ATT OTA ART GAG GAA TOC TCC CCT MGC MC TGC CTT GTC 1803 Glu Lys Cy Ile Val Ann Giu Glu Cys Bar Pro Ser Bar Cys Lou Val 545 550 555 ACC GMA TOOGOA GMG TOG GAT GMA TOC MCT OCT MGC TOT 0CC ACA OCA 1851 Tb: Clu Trp Oly Glu Try, Asp Olu Cym Sor Ala Bar Cy. Oly Tb: Oly 560 565 570 ATG MAA AGO OGA CAC MGA ATO ATC AMG ATO ACT CCT OCT OAT OCA TCT 1899 Met Lys Ar; Ar; His Arq Met Ile Lys Met Tb: Pro Ala Ap Oly Bar 75 580 585 99ATG TOC AMG OCA GMA ACT ACA GMG OCA GMG MA TOC ATO ATO CCC GMA 1947 Met Cys Lys Ala Olu Tb: Tb: Olu Ala Glu Lys Cys Met Mest Pro Oiu 590 595 600 TOC CAT ACT ATT CCC TOC CIT CIA TCC CCA TOG TCT GMA TOO ACC GAC 1995 .*Cys His Tb: Ile Pro Cys Lou Lou Bar Pro Trp Bar Olu Trp Ser Asp 610 615 620 TOO ACC OTO MCA TOT 000 AMC OGA ATO OGA AC CCG CM MGO ATO CTO 2043 cyl Bar Val Tb: Cys Gly Lys Gly lst Ar; Tb: Ar; Gin Arg Met Lou 625 630 635 AA TCI GCA OCT GMG OTT OCA GAC TOC ART GMG GMA CTG GAO CMR OCA 2091 Lys Bar Ala Ala Oiu Lou Oly Asp Cys m Ge lu Glu Lou Giu Gin Ala 640 645 650 GG MAA TOC ATG CIA CCI GMA TOC CCC ATT GMC TOT GMG CIA ACO GMG 2139 Giu Lys Cys Met Lou Pro Giu Cys Pro Ile Asp Cym Clu Lou Tb: Oiu 655 660 665 TOO TCC CMG TOO TCC GMG TOC ALT AMC TCC TOT 000 AMG 0CC CMC ATO 2187 Trp Bar Gin Try, Bar Glu Cys Aen Tb: Bar Cys Gly Lys Oly Big Met 670 675 680 ATC MGA ACA MGA ATG ATC AAA ATA GAA CC MTT1 =GA OGA ACA GCA 2235 Ile Ar; Th~r Arq Hot Ile Lys Ile Glu Pro Gin Ph. Gly Gly Th: Ala 685 690 695 700 TGC CCA GAA ACT OTC CAA OT ACT AAA TOT COA OTA MGG AAA TOC CTG 2283 Cys Pro Glu Thr Val Gin Arg Thr Lys Cys Ar; Val Ar; Lys Cys Lou 705 710 715 MGA GOC CCA GGT ATG GAA AAG MGG OT TOG AMG GAG GCC COG GAG AAA 2331 Ar; Gly Pro Gly Hot Glu Lys Arg Ar; Trp Lys Glu Ala Ar; Glu Lys 720 .725 730 MGA AGA MT GAR. CAA OCA AAA AAA AAT ATT GAT AhT GMG CAA TAT CCA 2379 Ar; Ar; Glu Gin Ala Lys Lys Awn Ile Amp AMn Giu, Gin Tyr Pro 735 740 745 GTT TOT MGG CTG AAA CCA TOG ACT GCT TOG ACA GAA TOT TCT ACA CTC 2427 Val Cys Ar; Lou Lys Pro Trp Th~r Ala Trp Thr GJlu Cys 8cr Thr Lou 750 755 760 TOT GGA GOT OGA AT? CMG GMG COC TAG ATO ATO OTA AMG AMG MG TCC 2475 Cya Gly Gly Gly Ile Gin Glu Ar; Tyr Hot Ho~t Val Lys Lys Arg 8cr 765 770 775 780 AAA MGC ACT CMG TTT ACT MGC TG AAA GAC ARA AMG GAG CTA MGA OCA 2523 Lye Ser Thr Gin Ph. Tb: Sor Cys Lys Asp Lys Lye Glu Lou Ar; Ala *785 790 795 TOT AAC GTT CAT CCT TOT TA GGAAAACACA A'GI~ T .c cTOATOCACT 2573 *Cys Aen Val His Pro cyc 800 CTGAGCTATA MGGAAARGTCA ACCTTGGTT GGTITTrTAAA ACAAACAAAA GTATAAAGTG 2633 a a a.

TATATTAGTT TTT-1 CATGTGGTT A4.4:TATAAA TATTTCCTCC GATTAATCTA CTGCATAAAA ATMGTAMGTC ATTGTGMGTC CATGGAATMo ~CATATAGAA ATACTACTTG AAcTAATfl!G AAOTGACATOG TTTCATATOT AATCCAAMGC AOTGCTATG TGATTATACA GGTTCAATAA TATTAAAIGGT GCATOTTTAT GAAATAATTA COCTACATAC TTTTOTTCAC TGCTTTGTcTTGCTGGTO CAAMAAATAT GGTAKACTTT GATGCTAC CTAGCCCTTA ATTTAACTGA MTAcAGACA TATCTGTOGA TAAMaACATG GOATOCATOC ATATTAACAT GGGAMGATTT CTCTCTTGAT TTGATTTAAA ACTATOCCAA GGMAAATTT CMGTAATOCT CTTTACAA TATT TA MOGATAGTT ATGGATOCTG coTTccATGC AAAATCATCT 2693 2753 2813 2873 2933 2993 3053 3113 3173 3226

TTOTTTCTCA

ATAAChChMT AATAGCA TACTTAAATA ATCTOTOCAG CTCAA3TAGT ATGTCAGCCC CACACAACAAAMACATGT GCT=ATCACA OTACCTOTCA CTG INFORMATIOK FOR SEQ ID N10112: Mi SEQUENCR

CSARACTERISTICS:

LENGTH: 802 amino acids TYPZ: amino acid 68 TOPOLOGY: linear (ii) M4oLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Met Ala Ala Arq 1 Ph.

Ala Arg Giu Thr Lys Asp 15 Giu Pro 145 Lys 20 Cys Asp Ann His 225 Trp Lou Glu Pro Lys Arg so Phe Pro Glu Glu Ser 130 Pro Arq Glu Cym Trp 210 Trp Glu Oly Val Lou Ser Glu Tyr Ala Gly Glu 115 Thr Thr I le Gin Cym 195 Ser 8cr Tyr 5cr

LOU

27S Val Glu Cly Lys Tyr Asp 100 Glu Pro Gly 110 Asp 180 Ala Glu Ala Oly Pro 260 Thr Lou Al a Gly Tyr Pro Ph* Lys Thr krg Thr Tyr 165 5cr Cym Lys Ile Giy 245 Val Arg Tyr Ann Gly 70 Arg Glu Gln Arq Gly 150 Ph* Ala Gly Thr Ile 230 Tyr Lys Gly Ph.

Cym 8cr 40 Glu Ph.

55 Ann Ser Gly Ph.

Glu Amp Piac Het 120 Arg Thr 135 Cym Val Gin Asp Ser Glu Thr Ala 200 Him Pro 215 Gly Ser Ala Ser Not Glu Ser 25 Arq 5cr Tyr Thr Him 105 8cr Ar; Ile Glu Gly 185 Lys Lys Ser Glu Glu 265 Asp Ile Lou Arg Lou 90 Ala Ann 110 Lou Gly 170 Val Tyr Any Hio Gly 250 Glu Glu Lou Arg Val 75 110 Gly Cys Gin Lys 155 5cr Thr Arg Ph.

8cr 235 Val 110 Arg Pro Lou Ala Lou Ar; Lou Lou Ala Thr Ar; Val Thr Ala Thr Pro Val 140 Ala

LOU

Asp Lou Pro 220 Lys Lys Arg Trp Lou Ala Glu Lou Lou Ph* Val 125 Ph* 8cr Thr Lye Thr 205 Ar; Ann Gin Gin Pro 285 Giu Gin Gly 8cr Lys Gln 110 Ala Trp Il1e Lys pro 190 Pho Arg Tyr Val Gift 270 Aia Ar; Lys Gly Asp Ala Glu 110 Val Thr Val Ar; 175 Thr Tyr Thr 110 Ala 255 5cr Trp Thr Ala Thr Pro Ala Gly 11.

rhr Al a Gln 160 Ile Lou Gly Ann Lou 240 Glu

ASP

Gln Val Ile Lys Ala Lye Ala Gin 2 so Pro Lou 290 Amn Val Arg Ala Ala 295 Pro 5cr Ala Giu Ph* 300 5cr Val Asp Ar; *9 His 305 Trp Trp Asp Giu Tyr 385 Glu Ile Asp Cys Lou 465 Phe Cys 20 y Asp Val 545 Glu Arg Asm Val1 Ser Lys 370 Asp Axrg Asp Th: 5cr 450 Lys Gin Hot met Gly S30 Ann Trp His Val Gin Gly 355 Ile Pro Ile Asp Pro 435 5cr Ala Pro met Gly 515 5cr Glu Asp Lou Gly Lys 40 Val A.rg Giu Ala Ile 420 Giu Sor Gin Cym Ser 500 Thr Not Glu Glu Not Lou 325 Val1 Thr Pro Gly Arg 405 Val Thr Thr Lou met 485 Asp Arg Cys cys Cym 565 Ser Ph* Lou Thr Hot Lou Gly Pro Ser Pro 310 Ser Val Tyr Lou Gly 390 Lys Ala cys Cys Asp 470 Gly Trp Ser Lys Ser 550 5cr Ala Gin Glu Thr 375 Ser Gly Asp Ile Giu 455 Lou Pro Ile Arg Val 535 Pro Ala Giu Asp Scr 360 5cr Ile Glu Lou Tyr 440 Lys Scr Gly Thr Glu 520 Pro Sor Scr Asp Lou 345 Pro Lou Lys Gln Ala 425 Ser Gly Vai Cys Trp 505 Ar; Thr 5cr Cys Lou 330 Ile Aen Asp Lou Cys 410 Pro Amn Lys Pro Ser 490 5cr Tyr Glu cys Gly 570 Cym Pro Lys His Val 395 Asn Giu Trp Ar; Cys 475 Asp Pro Val Giu Lou 555 Thr Thr Trp Pro Pro 380 Ala Ph* Glu 5cr met 460 Pro Giu cys Lys Thr 540 Val Giy Lyn Asp Thr 365 Gin Arg Val1 Lys Pro 445 Arg Asp Asp Ser Gin 525 Giu Th: met Asp Ala 350 Val Ser Val Pro Glu 430 Trp Gin Thr Gly Val 510 Ph* Lys Glu Lys Cys 33 S Gly Pro Pro Val Asp 41S Glu 5cr Ar; Gin 5cr 495 Ser Pro Cys Trp Ar; 575 Asp 320 Gly Thr Gin Phe Lou 400 Aen Asp Al a Mot Asp 480 Tb: Cym Glu 110 Gly 560 Arg His Ar; Met Ile Lys Met Thr Pro Ala 580 585 Asp Gly 5cr Hot Cys Lys Ala 590 Giu Thr Thr Glu Ala Giu Lys 595 Cys Hot Hot Pro Glu Cys His Tb: Ile 600 605 Pro Cys 62S 610 Gly Lou Lys Lou Gly Ser met Pro Ar; 630 615 Thr Ser Arg Glu Gln Trp Arg Ser Met 635 620 Lou eye Lys 8cr sor Val1 Ala Tb: Ala 640 Glu Lou Gly amp Cyf Ann Giu Glu Lou Glu Gin Ala Giu Lys Cys Met 645 650 655 Lou Pro Giu Cys Pro Ile Asp Cys Giu Lou Thr Glu Trp Ser Gin Trp 660 665 670 Ser Olu Cys Ann Thr Ser Cys Gly Lys Gly His nt Ile Arg Thr Ax; 675 680 685 Met Ile Lys Ile Glu Pro Gin Ph. Gly Gly Thr Ala Cys Pro Giu Thr 690 695 700 Val Gin Arg Thr Lys Cys Arg Val Ar; Lys Cys Lou Ar; Gly Pro Gly 705 710 715 720 Met G1u Lys Arg Arg Trp Lys Glu Ala Ar Giu Lys Arg Arg Ser Giu 725 730 735 Gin Ala Lys Lys Ann Ile amp Asn Giu GIn Tyr Pro Val Cys Arg Lu 740 745 750 Lys Pro Trp Thr Ala Trp Thr Glu Cys Ser Thr Lou Cys Gly Gly Gly 755 760 765 Ile Gin Giu Arg Tyr Met Met Vai Lys Lys Arg Ser Lys Ser Thr Gln 770 775 780 Ph. Thr Ser Cys Lys ap Lys Lys Giu Lou Arg Ala Cys Ann Val His 785 790 795 800 Pro Cy.

INFPORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 1816 bae pairs TYPE: nucieic acid STRANDEDNESS: single 20 TOPOLOGY: linear (ii) MOLECULE TYPE: cONA (ix) FEATURE: JNAN/KZY: CDS LOCATION: 2..1705 (xi) SEQUENCZ DESCRIPTION: SEQ ID NO:13: T TCA GOT GAR TAT OTT CTT TOG ACT ATC AGA CAA 0CC ACT GAT GGT 46 St Giy Glu Tyr Val Lou Trp Ser Met Arg Gin Ala Ser Asp Gly 1 5 10 GTC AAA CAA TA OCT GAG TTG GGT TCT CCA GTC AAA ATG GAA GAA GAA 94 Val Lys Gin Val Ala Giu Lou Oly Ser Pro Val Lys Net Giu Glu Glu 20 25 ATT CGA CAG AG GGA GAT GAA GTT CTA ACA GTA ATC AAA GCC AAA OCT 42 Ile Arg Gin Lys Gly Amp Giu Val Lou Thr Val lie Lys Ala Lys Ala 40 CAG TOG Gin Trp GAG TTC Glu Phe ATG GOT Met Gly TOT ACC Cys Thr CCA TOG Pro Trp AAG CCC Lys Pro CAC CCA His Pro 145 OCT CGA Ala Arg 160 AT'? ATA Ile 110 GAG AAA Glu Lys Pro

TCT'

Bar

CC'?

Pro

AAA

Lys

GAT

Asp

ACC

Tbhr 130

CAA

Gin

GTT

Val

CCC

Pro

GAC

Asp 71 GCC TOG CAG CCC CTC AAT GTG AGO GCC 0CC CCT TCA OCT Ala Trp Gin Pro Lou Ann Val Arq Ala Ala Pro Ser Ala 5s GTG GAC AGA GC CT CAC CTG ATCO TCA TTT CTG GCC ATG Val Asp Arg Ser Arg His Lou Met Ser Ph. Lou Ala Met 70 AGC CcA GAC TOG AAT GTA OA CTC ACC TCC GAG GAT CTC Ser Pro Asp Try Anfl Val Gly Lou Th~r Ser Glu Asp Lou 85 90 GAG TOT GGC TOG OTT C.AG AAG GTO GTC CAG OAT TTG AT'? Oiu Cys Gly Trp Val Gin Lys Val Val Gin Asp Lou Ile 100 105 110 GCA GGC ACT GAC hOT 000 GTh ACC TAC GAG TCT CCA AAC Ala Gly Tkir Asp Ser Gly Val Tkir Tyr Glu Ser Pro Ann 115 120 125 AT'? CCC CAG GAT AAA ATC CGA CCT CTG ACA AGT CTG GAT Ile Pro Gin Asp Lys Ile Arq Pro Lou Thr Ser Lou Asp 135 140 AGC CC'? TCT ATO ACC AGA GOT 000 CCA ATC ATA CC'? ATA Sor Pro 8cr Not Thz Arg Oly Gly Pro Ile Ile Pro Ile 150 155 OTC AT'? GAK AGO AT'? 0CC AGG MhG OA GMA CAG TOC MAT Val Ile Oiu Arq Ile Ala Arg Lys Oly Olu Gin Cys Ann 165 170 175 GAC MAC OTG OAT GAC ATA 0TA GCA OAT CG GOTA AG GMA Asp An Val Asp Asp Ile Val Ala Asp Lou Val Thr Oiu 180 185 190 OAA OAT OAT ACC COG GAG ACC TGC ATA TAT TCC MAC TOG Oiu Asp Asp Thz Pro Giu Thr Gys Ile Tyr Ser Ann Trp 195 200 205 fee* 4 0 00.0 0 *0000 *0*6 .00.

190 238 286 334 382 430 478 526 574 622 670 718 766 814 862 TCC CCC Ser

ATG

Hot CCh Pro 240

QAC

Asp

TOC

Gym

MAG

Lys Pro

A

Arg 225

GAC

Asp

GMA

Giu hoc Bar

CAG

GIn TOO TCG GCC TGC AGC TCG=C A=C TGC GAC MRG GOC MAG CG Trp Bar Ala Cym 8cr Sor Ala Thr Cys Asp Lys Gly Lys Arg 210 215 220 GAG GC ATO TTA AMG OCT CG T'TA OAT CTC AGT OTT? CC TGC Gin Arg Not Lou Lys Ala Gin Lou Asp Lou Sar Val Pro Gym 230 235 ACT CMG GAC TI? GMR CCC TGC ATG 000 CC GOC TGC hOC OAT Thr Gin Asp Phe Giu Pro Gys Hot Oly Pro Oly Gys Ser Asp 245 250 0CC TCT ACC TGC ATO ATG TCA GMA TOG ATC ACC TG TCG CG Ala Ser Thr Gyn Hot Hot Ser Glu Trp, Ile Thr Trp Ser Pro 260 265 270 GCC TCC TOC 000 ATG OGA ATT GAG GTC AGO GAG MGA TAC GTC Ala Ser Cys Oty Met Gly Ile Giu Val hrg Glu Arq Tyr Val 275 280 285 TIC CCA GAA OAC GOT TCC TTG TOT MAA GTC CCA AG GMA GM Ph* Pro Giu Asp Gly Sa: Lou Cym Lys Val Pro Thr Oiu Giu 290 295 300 ACT GAG AAA TGC ATT GTC AAT GAG GAG TGT GAG CCA AGC AGC TOT ATA 958 Thr Glu Lys Cys Ile Val Amn Giu Glu Cys Glu Pro Ser Bar Cys Ile 305 310 315 GTC ACG GAA TOGOCA GAG TOG GAG GAG TGC AGC GCT ACA TGC OG ATG 1006 Val Thr Giu Trp Ala Giu Trp, Glu Glu Cys 5cr Ala Thr Cys Ar; Met 320 325 330 335 GOT ATG AAG AAG COG CAC AGG ATG ATA MAG ATG ACT CCA GCO GAT GGA 1054 Gly Met Lys Lys Arg His Arg Not Ile Lys Met Thr Pro Ala Asp Gly 340 345 350 TCT ATO TGC AAA 0CC GAC ACA ACA GAG OTT GAG AAA TOC ATO ATO CCC 1102 Ser Met Cym Lys Ala Amp Th: Thr Glu Val Glu Lys Cys Met Met Pro 360 .365 GAA TOT CAT ACC ATC CCO TGC oTG TTG TCC CCT TOG TOT OAA TOG AG? 1150 Glu Cys His Thr Ile Pro Cys Val Lou 5cr Pro Try, Bar Glu Trp 8cr 370 375 380 GAT TGC AGC GTT ACC TOT GGC AAM GGC ACC AGA ACC AGA CAG AGA ATO 1198 Asp Cys 5cr Val Thr Cys Gly Lys Gly Thr Arg Th~r Arg Gin Ar; met *0*385 390 395 TTG AAG TCC CCO TCT GAA CT? OA GAT TOC AAT GAG GMA CTO GAM CTO 1246 Lou Lys 5cr Pro 5cr Glu Lou Gly Amp eye hAn Glu Giu Lou Giu Lou *400 405 410 415 AAA CAA OTO GM AM =O ATO CT =C GM OCCC ATA AOC TOT GMR 1294 Lys Gin Val Giu Lys Cys Met Lou Pro Giu Cys Pro Ile 5cr Cys Glu 420 425 430 TTG ACA GAG TOG TCT TAC TOG TCT GAG TOT AAC MAA TOC TO GGC MAG 1342 Lou Thr Glu Trp 5cr Tyr Trp 5cr Glu Cym Asn Lye Cym 5cr Gly Lys 435 440 445 *.:GGT CAC ATO ATT OT ACC COA ATO ATC ACA ATO GMR CCA ChG TTT OGA 1390 *Gly His Met Ile Ar; Thr Ar; Met Ile Thr Met Giu Pro Gin Ph. Gly 450 455 460 GGA 0CC OTC TOT CO GMA ACC GTG CAA COC AAARMA TOC C"A TEA COT 1438 Oly Ala Val Cys Pro Olu Thr Val Gln Arg Lys Lys Cys Ar; Lou Ar; *465 470 475 *AMA TOT CMA AMA AG? TCC GOG hT GAG OA AGO CAT TTA MAG OAT GCC 1486 Lys Cys Gin Lys 5cr 5cr Giy, Awn Glu Arg Arg His Lou Lys Amp Ala 480 485 490 495 C"A GA AAG AG.A AGO AG? GMA AMA ATA AAO GMA GAT TCA GAT OGA GMA 1534 Arig Giu Lys Arg Arg 5cr Oiu Lys Ile Lys Ou amp 5cr Amp Gly Giu 500 505 510 CAG TAC CCT 0Th TOT ARA ATO AMA CCA TOG ACT OCA TOO ACG GMh TOT 1582 Gin Tyr Pro Val Cya Lys Met Lys Pro Trp Thr Ala Trp, Tb: Glu Cys 515 520 525 ACC AMA TTC TO OT 000 GGG ATA ChA GAG OOG TTC ATO ACT OTG MRG 1630 Tb: Lys Ph. Cys Gly Gly Gly Ile Gin Glu Arg Ph* Met Tb: Val Lyu 530 535 540 73 AM AGA TTX AAA MT TCT CMG TTC ACC MGC TGC AMG GAC AAM AM GAG Lys Ar'; Phe Lys Ser Ser Gin Phe Thr Ser Cys Lys Asp Lys Lys Glu 545 550 555 ATC CGG GCT TOC AAT QTC CAT CCA TGT TAACCTGCCT

GAAAAGAGGG

110 Ar'; Ala Cys Awn Val His Pro Cy.

560 565 ATTGACACTA CAATCGCAAC MGAAGTCAAT CTTTATTAGA TATTITTTAT

CATAGAATAT

ATACATGTGC TTTCATTTTG CATGTACTTT

T

INFORMATION FOR SEQ ID NO: 141 SEQUENCE CHARACTERISTICS: LENGTH: 568 amino acids TYPE: amino acid TOPOLOGY: linear (1i) MOLECULE TYPE: protein 1678 1725 1785 1816 a a *aa.

a. a.

an.

a.

Ser Lys Ax'; Trp Phe 65 G ly Thr Trp (xi) Gly Giu Gin Val Gin Lys Pro All 50 Ser VaJ Pro So: Lys Gil Asp Al~ 11: SEQUENCE DESC Tyr Val Lou Ala Giu Lou 20 Gly Asp Glu Trp, Gin Pro LAsp Ar'; Ser 70 r Pro Asp Trp 85 a Cys Gly Trp 100 a Gly Thr Asp 5 TrF 3or Met Gly Ser Pro 25 Val Lou Thr 40 Lou Ann Val 55 Ar' His Lou an Val Gly Val Gin Lys 105 8cr Gly Val -120 Arg Val Val Ar'; Hot Lou 90 Val1 Thr Pro Gin Ala Scr Asp Lys 110 Ala Ser 75 Thr Val Tyr

LOW

Pro 155 Met Glu Lys Ala Ala Pro Phe Lou Ser Giu Gin Asp Giu Ser 125 Thr 8cr 140 Ile l1e :RIpTIoN: SEQ ID NO:14: Glu Lys 8cr Ala Asp Lou 110 Pro Lou pro Giy Giu Ala Ala met Lou Ile Awn Asp 110 Val Ilie Gin Glu met s0 Cy.

Pro Lys His Ala 160 Pro Thr Ile Pro Gin Asp Lye Ile Ar'; 130 135 Pro Gin 5cr Pro 8cr Met Thr Ar'; Gly Gly 145 150 Ar'; Val Val Ile Glu Ar'; Ile Ala Arg Lys Gly 165 170 Glu Gin Cys Awn Ile 175 Ile Pro Lys Asp Asp Glu 195 180 Asp Asp Asp Thr Asp Pro 1wYl 1Ala. hop Lou 185 Glu Thr Cys Ile Tyr 200 Vali ser 205 190 Ann Trp 8cr 74 Pro Tz-p ser Ala Cys Ser 5csr Ala Thr Cys Amp Lys Gly Lys Ar; Met 210 215 220 Ar; Gin Ar; Not Lou Lys Ala Gin I4eU Asp Lou 5cr Val Pro Cys Pro 225 230 235 240 Asp Thr Gin Amp Ph* Glu Pro Cys Met Gly Pro Gly Cym 8cr ASP Amp 245 250 255 Giu Ala Ser Thr Cys Met Met Sor Glu Trp Ile Tkhr Trp Ser Pro Cym 260 265 270 5cr Ala 5cr Cys Gly Met Gly Ile Glu Val Arg Giu Ar; Tyr Val Lys 275 280 285 Gin Phe Pro Giu Amp Gly Ser Lou Cym Lys Val Pro Thr Glu Giu Thr 290 295 300 Giu Lys cy. Ile Val ken Glu Giu Cyn Glu Pro Ser 5cr Cy Ile Val 305 310 315 320 Thr Giu Trp Ala Giu Trp, Giu Glu Cym 5cr Ala Thr cys Ar; Met Gly 325 330 335 Met Lys Lys Ar; His Arg Met Ile Lys Mot Thr Pro Ala Asp Gly 340 345 350 Mot Cys Lys Ala Asp Thr Thr Glu Val Giu Lys Cys Met Met Pro Giu *355 360 36S Cys His Thr le Pro Cym Val Lou Sor Pro Trp 5cr Giu Trp Ser Amp *370 375 380 *.Cys 5cr Val Th: Cym Gly Lys Gly Thr Ar; Thr Arg Gin Ar; Met Lou 385 390 395 400 Lys 5cr Pro 5cr Giu Lou Gly Asp Cys Ann Giu Giu Lou Glu Lou Lys 405 410 415 Gin Val Glu Lys Cys Mot Lou Pro Glu Cys Pro le 5cr Cys Giu Lou 420 425 430 Thr Giu Trp 5cr Tyr Trp, 5cr Giu Cym hen Lys Cyn 5cr Gly Lys Gly 435 440 445 *.:His Met Ile Ar; Tb: Ar; Mot Ile Thr Hot Giu Pro Gin Ph* Gly Gly .450 455 460 Ala Val Cys Pro Giu Thr Val Gin Ar; Lye Lys Cys Ar; Lou Arq Lys 46S 470 475 480 Cys Gln Lye 5cr 5cr Giy Amn Giu Ar; Ar; Him Lou Lys Asp Ala Ar; 485 490 49S Giu Lye Ar; Ax; 5cr Giu Lys Ile Lye Gila Asp 5cr Asp Giy Giu Gin 500 505 510 3' Tyr Pro Val Cys Lys Mot Lys Pro Trp Tb: Aia Trp Tb: Giu Cym Th: 515 520 525 Lys Ph. Cys Gly Gly Gly le Gin Giu Ar; Phe Met Tb: Vai Lye Lye 530 535 540 Ar; Ph* Lys Ser Ser Gin Ph@ Thr Ser Cys Lys Asp Lys Lys Glu Ile 545 550 55560 Ar; Ala Cys Aen Val Him Pro Cyi 565 INFORMATION FOR SEQ ID 140:15: SEQUENCE

CHARACTERISTICS:

LENGTH: 59 amino acids TYPE: amino acid STRANDEDNESS: singie TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Thr Cy Ile Tyr Ser Aen Trp 5cr Pro Trp Scr Ala Cys Ser Ser Thr Cys Giu Lys Gly Lys Axg Mot Ar; Gin Ar; Hot Loeu Lys Ala 25 Gin Lou Asp Leu Ser Val Pro Cys Pro Asp Thr Gin Asp Ph. Gin Pro 40 15Cys Met Giy Pro Gly Cys Ser Asp Glu Asp Gly *50 INFORMATION FOR SEQ ID NO:16: SEQUENCE

CHARACTERISTICS:

LENGTH: 56 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) M4OLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NOzl6: Ser T1hr Cym Thr Met 5cr Giu Trp Ile Thr Trp Ser Pro Cy. Ser Val.

1 5 1 10 1 Sec Cys Gly Not Gly Met Arg Sec Arq Giu Arg Tyr Val Lys Gin Ph.

25 Pro Asp Gly Sec Val Cys Met Lou Pro Thr Glu Glu Thr Giu Lys Cys 40 Thr Val Amn Glu Glu Cys Ser Pro 500s INFORMATION FOR SEQ ID NO:17: SEQUENCE

CHARACTERISTICS:

LENGTH: 56 amino acids TYPES amino acid STRANDEDNESS: single TOPOL40GY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 110:17: Ser Ser Cys Lou Val Thr Glu Trp Gly Glu Trp Asp Asp Cys Ser Ala 1 5 10 Thr Cyu Gly Met Gly Not Lys Lys Arg His Arq Met Val Lys Not Ser 25 Pro Ala Asp Gly Ser Hot Cys Lys Ala Glu Thr Ser Gln Ala Glu Lys 40 Cys Met Het Pro Glu Cys His Thr 50 INFORMATION FOR SEQ ID 110:18: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 53 amino acids TYPE: amino acid STRANDEDNE.SS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 110:18: Ile Pro Cys Lou Lou Ser Pro Tr-p Glu Trp Ser Asp Cy. Ser Val Thr 1 5 10 *20 Cys Gly Lys Gly Met Ar; Thr Arg Gln Ar; Hot Lou Lys Ser Lou Ala 25 Glu Lou Gly Asp Cys Asn Glu Asp Lou Glu Gin Ala Glu Lys Cys Het 40 Lou Pro Glu Cys Pro INFPORMATION FOR SEQ ID 110:19: SEQUENCE CHARACTERISTICS: LENGTH: 56 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 110:19: 77 Ile Asp Cys Glu Leu Ser Glu Trp Ser Gin Trp Ser Glu Cys Asn Lys 1 5 10 1 Cys Gly Lye Gly His Met Ile Arg Thr Arq Thr Ile Gin Met Glu 25 Pro Gin Ph& Gly Gly Ala Pro Cys Pro Glu Thr Val Gin Arg Lys Lys 40 Cys Arg Ala Ar; Lys Cys Lou Arg INFORMATION FOR SEQ ID 140:20: SEQUENCE CHARACTERISTICS: LENGTH: 55 amino acids TYPE: amino acid sTRA.NDEDRESS: single TOPOLOGY: linear MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 140:20: Pro Gin Cys Arg Met Arg Pro Trp Thr Ala Trp Sec Glu Cys Thr Lys 1 5 10 Lou Cys Gly Gly Gly Ile Gin Glu Ar; Tyr Met Thr Val Lys Lys Ar; 1520 25 Phe Lys Ser Ser Gin Ph. Thr Ser Cys Lye Asp Lys Lys Glu Ile Arg ~*35 40 Ala Cym Ann Val His Pro Cys

Claims (31)

1. An isolated nucleic acid molecule encoding vertebrate F- spondin having the nucleotide sequence selected from a group consisting of: the sequence set forth in Seq. I.D. No. 9; the sequence set forth in Seq. I.D. No. 11; the sequence set forth in Seq. I.D. No. 13; and a sequence complementary to a sequence which is capable of hybridizing under low stringency conditions to the sequence of either or o

2. An isolated nucleic acid molecule encoding vertebrate F- spondin having the amino acid sequence selected from a group consisting of: the sequence set forth in Seq. I.D. No. the sequence set forth in Seq. I.D. No. 12; the sequence set forth in Seq. I.D. No. 14; and a sequence complementary to a sequence which is capable of hybridizing under low stringency conditions to the sequence of either or

3. The isolated nucleic acid molecule of claim 1 or 2, wherein the nucleic acid is DNA.

4. The isolated nucleic acid molecule of claim 3, wherein the DNA is cDNA. The isolated nucleic acid molecule of claim 1 or 2, wherein the nucleic acid is RNA.

6. The isolated nucleic acid molecule of claim 1 or 2, wherein the vertebrate is a rat. P:\OPER\MRO\2266018.CLMN 25/2/00 79

7. The isolated nucleic acid molecule of claim 1 or 2, wherein the vertebrate is a chicken.

8. The isolated nucleic acid molecule of claim 1 or 2, wherein the vertebrate is a Xenopus.

9. A nucleic acid probe comprising a nucleic acid molecule of at lest 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of the nucleic acid molecule of claim 1 or 2. S' 10. The nucleic acid probe of claim 9, wherein the nucleic acid is DNA.

11. The nucleic acid probe of claim 9, wherein the nucleic acid is RNA.

12. The isolated nucleic acid molecule of claim 1 or 2 operatively linked to a promoter of RNA transcription. S*

13. A vector which comprises the isolated nucleic acid molecule of claim 1 or 2.

14. The vector of claim 13, wherein the isolated nucleic acid molecule is linked to a plasmid. A host vector system for the production of a polypeptide having the biological activity of F-spondin which comprises the vector of claim 13 in a suitable host.

16. The host vector system of claim 15, wherein the suitable host is a bacterial cell, insect cell or animal cell.

17. A method of producing a polypeptide having the biological P:kOPER\MRO\2266018.CLM -25/2/00 activity of F-spondin which comprises growing the host vector system of claim 15 under conditions permitting production of the polypeptide and recovering the polypeptide so produced.

18. A polypeptide encoded by the isolated nucleic acid molecule of claim 1 or 2.

19. A method of attaching nerve cells to a matrix comprising contacting the matrix with nerve cell and the polypeptide of claim 18 at a concentration effective to effect I attachment of the cells to the matrix.

20. A method of stimulating growth of a nerve cell comprising contacting the nerve cell with the polypeptide of claim 18 at a concentration effective to stimulate growth of the nerve cell.

21. A method of regenerating nerve cells in a subject comprising administering to the subject the polypeptide of claim 18 at a concentration effective to regenerate nerve cells in the subject.

22. A pharmaceutical composition for stimulating nerve cell growth comprising a pharmaceutically acceptable carrier and the polypeptide of claim 18 at a concentration effective to stimulate nerve cell growth.

23. An isolated nucleic acid molecule encoding human F-spondin having the nucleotide sequence selected from a group consisting of: the sequence set forth in Seq. I.D. No. 9; the sequence set forth in Seq. I.D. No. 11; the sequence set forth in Seq. I.D. No. 13; and P:\OPER\MRO\2266018.CLM 25/2/00 81 a sequence complementary to a sequence which is capable of hybridizing under low stringency conditions to the sequence of either or

24. An isolated nucleic acid molecule encoding human F-spondin having the amino acid sequence selected from a group consisting of: the sequence set forth in Seq. I.D. No. the sequence set forth in Seq. I.D. No. 12; the sequence set forth in Seq. I.D. No. 14; and a sequence complementary to a sequence which is capable of hybridizing under low stringency conditions to the sequence of either or

25. Purified, human F-spondin polypeptide.

26. A purified human F-spondin encoded by the nucleic acid molecule of claim 23 or 24. S* 27. A method of attaching nerve cells to a matrix comprising contacting the matrix with nerve cell and human F-spondin at a concentration effective to effect attachment of the cells to the matrix.

28. A method of stimulating growth of a nerve cell comprising contacting the nerve cell with human F-spondin at a concentration effective to stimulate growth of the nerve cell.

29. A method of regenerating nerve cells in a subject comprising administering to the subject human F-spondin at a concentration effective to regenerate nerve cells in the subject. P:\OPER\MRO\2266018.CLM -25/2/00 82 A pharmaceutical composition for stimulating nerve cell growth comprising a pharmaceutically acceptable carrier and human F-spondin at a concentration effective to stimulate nerve cell growth.

31. An isolated vertebrate nucleic acid molecule encoding F- spondin.

32. Purified, vertebrate F-spondin polypeptide.

33. A method for attaching nerve cells to a matrix comprising contacting the matrix with nerve cell and purified F- spondin at a concentration effective to effect attachment of the cells to the matrix.

34. A method of stimulating growth of a nerve cell comprising contacting the nerve cell with purified F-spondin at a concentration effective to stimulate growth of the nerve cell.

35. A method of regenerating nerve cells in a subject comprising administering to the subject purified F-spondin at a concentration effective to regenerate nerve cells in the subject.

36. A pharmaceutical composition for stimulating nerve cell growth comprising a pharmaceutically acceptable carrier and purified F-spondin at a concentration effective to stimulate nerve cell growth. DATED this 25th day of February 2000 The Trustees of Columbia University in the City of New York By it Patent Attorneys DAVIES COLLISON CAVE