CA2324167A1

CA2324167A1 - A membrane-bound netrin

Info

Publication number: CA2324167A1
Application number: CA 2324167
Authority: CA
Inventors: Shigeyoshi Itohara; Toshiaki Nakashinba; Toshio Ikeda; Kei Tashiro; Tasuku Honjo
Original assignee: Individual
Current assignee: RIKEN Institute of Physical and Chemical Research
Priority date: 2000-05-19
Filing date: 2000-11-20
Publication date: 2001-11-19
Also published as: JP2001327289A

Abstract

It is an object of the present invention to identify unknown members of the UNC-6/Netrin family that are predicted to exist and analyze their function in organisms, for the purposes of elucidating the function of Netrin molecules in the brain of vertebrate animals, providing suitable treatment for brain damage caused by disease, and allowing the promotion of normal neural pathway formation during transplant treatment and the like.
Specifically, the present invention provides a membrane-bound Netrin having a hydrophobic region at its C-terminus, wherein the hydrophobic region is able to bind to the cell membrane by means of glycosylphosphatidylinositol (GPI)

Description

A MEMBRANE-BOUND NETRIN
FTRLD OF THE INVENTION
The present invention relates to a novel member of the Netrin protein family having nervous system axon guidance function, which differs structurally and functionally from known Netrins.
HACRGROUND OF THE INV.NmTnN
In the process of development of the nervous system, growing axons are guided appropriately to correct targets to form precise wiring of intricate networks. The molecular mechanism of axon guidance is being clarified through the identification of a number of gene families encoding the keys to the guidance of axon growth cone and cell migration.
These genes include members of the immunoglobulin super family, ephrins, semaphorin, slits and Netrins (Tessier-Lavigne and Goodman, Science 274: 1123-1133, 1996; Chisholm and Tessier-Lavigne, Curr. Opin. Neurobiol. 9: 603-615, 1999). A number of these are membrane proteins that have been identified as contact-repulsive molecules or contact-attractive molecules while others are secretion proteins that have been identified as diffusible chemotropic signals.
Netrin-1 and Netrin-2 are chemo-attractive factors, first identified through the biochemical formation of molecules having commissural axon outgrowth-promoting activity (Serafini et al., Cell 78: 409-424, 1994; Kennedy et al., Cell 78:425-435, 1994). UNC-6 is a homologue of Netrin found in C.elegans. In vertebrate animals, Netrins play a role in attracting the lamina alaris axons running along the rostrocaudal axis, toward the tabula or the ventromedian area (Kennedy et al., Cell 78: 425-435, 1994;
Shirasaki et al., Neuron 17:1079-1088, 1996), and in the repulsion of the trochlear motor axons away from the midline (Colamarino and Tessier-Lavigne, Cell 81: 621-629, 1995).
From gene analysis in nematodes and, in single cell analysis in Xenopus laevis spinal neurons, it was concluded that these dual effects (attraction and repulsion) were transmitted by a single ligand via two types of receptor sub-family belonging to the immunoglobulin super family (de la Torre et al., Neuron 19: 1211-1224, 1997; Hong et al., Cell 97: 927-941, 1999). The attraction effect is transmitted by means of UNC-40 in nematodes, frazzled protein in Drosophila, DCC (Deleted in colorectal cancer) in vertebrate animals, and a DCC sub-family receptor to which neogenin belongs. (Chan et al., Cell 87: 187-195, 1996;
Keino-Masu et al., Cell 87: 175-185, 1996; Kolodziej et al., Cell 87: 197-214, 1996; de la Torre et al., Neuron 19:
1211-1224, 1997). In contrast, several of the repulsion effects of Netrin require members of the UNC-5 family in addition to the members of the UNC-40/DCC family. (Hedgecock, et al., Neuron 2: 61-85, 1990; Hong et al., 1999).
In mammals, three types of UNC-5 receptor family member, UNC5H1, UNC5H2 and UNC5H3/RCM (rostral cerebellar malformation) have been isolated (Ackerman et al., Nature 386: 838-842, 1997; Leonardo et al., Nature 386: 833-838, 1997). The members of both receptor sub-families all bind to Netrin.
It had come to be thought that Netrin and UNC-6 constituted a phylogenically conserved small protein family.
(Chisholm and Tessier-Lavigne, Curr. Opin. Neurobiol. 9:
603-615, 1999). It differs from other axon guidance molecules, in other words, semaphorin (Dodd and Schuchardt, Cell 81: 471-474, 1995) and ephrin (Flanagan and Vanderhaegen, Annu. Rev. Neurosci. 21: 309-345, 1998), and, in the types that have been studied up until now, at most only two types of UNC-6/Netrin family members have been isolated. However, as is the case with other families, the existence of further members is predicted. The high level of variety within the semaphorin and ephrin families possibly reflects their contribution to the formation of complex and high level unified neuron networks in vertebrate animals.
Therefore, to further clarify the function of Netrin in the brains of vertebrate animals, and to provide suitable treatment for brain damage caused by disease, and further to allow promotion of normal neural pathway formation during transplant treatment and the like, there is a need to identify various unknown members of the UNC-6/Netrin family that are predicted to exist and to analyze their function in organisms.
HREIF SUMMARY OF THE INVENTION
Therefore, the present inventors, as a result of focused examination directed to discovering a novel member of the UNC-6/Netrin family in mice, discovered a membrane-bound Netrin differing structurally and functionally from previously known Netrins, thereby completing the present invention. The novel Netrin of the present invention differs from classical Netrins, is primarily bound to the plasma membrane by means of a glycosylphospha-tidylinsitol (GPI) lipid anchor, and has numerous isoforms most likely formed by alternative splicing. Further, it has become apparent that the Netrin of the present invention does not bind with classical Netrin receptors, and exhibits no duplication of function with Netrin-1.
In other words, the present invention provides the following (1) to (18) (1) A membrane-bound Netrin having a hydrophobic region at its C-terminus, wherein the hydrophobic region is able to bind to the cell membrane by means of glycosylphosphatidylinositol (GPI).
(2) The membrane-bound Netrin according to (1) above which comprises an amino acid sequence indicated by SEQ ID
NOS: 8, 10, 12, 14 or 16.
(3) A polynucleotide encoding the membrane-bound Netrin according to (1) or (2) above.

(4) The polynucleotide according to (3) above comprising the nucleotide sequence indicated by SEQ ID NO: 7, 9, 11, 13 or 15.

(5) A protein or fragment thereof, according to either (a) or (b) below:
(a) A protein comprising an amino acid sequence indicated by SEQ ID NO: 8, 10, 12, 14, 16 or 18; or, a fragment of said protein.
(b) A protein comprising an amino acid sequence derived from an amino acid sequence indicated by SEQ ID NO: 8, 10, 12, 14, 16 or 18, by deletion, substitution or insertion of one or several amino acids, or a fragment of said protein, wherein said protein or fragment has Netrin function.

(6) A polynucleotide according to either (a) or (b) below, or a fragment thereof:
(a) A polynucleotide comprising a nucleotide sequence indicated by SEQ ID NO: 7, 9, 11, 13, 15 or 17; or, a fragment of said polynucleotide.
(b) A polynucleotide comprising a nucleotide sequence derived from the nucleotide sequence indicated by SEQ ID
NO: 7, 9, il, 13, 15, or 17, by deletion, substitution or insertion of one or several nucleotides, or a fragment of this polynucleotide, encoding a protein having Netrin function.
The term "several" in (5) or (6) above refers to a number of mutations able to be induced by site specific mutagenesis, for example, and means a number within a range such that pre-mutation function is not lost.
Further, a reference to the term "fragment" herein includes, but is not limited to, each of the domains of the protein of the present invention as described herein and the corresponding polynucleotides that encode them.

(7) An expression vector comprising the polynucleotide according to (3), (4) or (6) above, or a fragment of this polynucleotide.
A vector able to comprise the above-mentioned polynucleotide can be any usable vector known within the relevant field, and if suitable for expression of the protein encoded by the above-mentioned polynucleotide, is not particularly limited. However, the following can be used: pcDNA4Myc/His (Invitrogen), pGEX-2T (Amersham Pharmacia), pET-32a (Novagen), Retro-X System (Clontech), and the like. Further, as is well known in the relevant field, a regulatory sequence such as promoter, enhancer or the like, able to control expression of the above polynucleotide can be included within the expression vector where thought suitable.
Further, the protein to be expressed can be just the protein of the present invention or a fragment thereof or, as required depending on one's purpose, for example, fusion proteins with glutathione S-transferase and green fluorescent protein, or with tags such as (His)6or myc, etc., may be expressed.

(8) A host cell transfected with the expression vector of ( 7 ) above .
As a host cell, any cell which can express the above polypeptide is acceptable including animal cells, plant cells, bacteria and the like that are known to be usable in this field. In particular, the use of E. coli, HEK293 cell, COS7 cell or the like is preferable.

(9) A method for producing the membrane-bound Netrin according to (1) or (2) above, or the protein or fragment thereof according to (5) above comprising culturing the host cell according to (8) above.

(10) A primer used in the amplification of the membrane-bound Netrin according to (1) or (2) above, or the protein or fragment thereof, according to claim 5.

(11) The primer according to (10) above, which corresponds to the nucleotides encoding amino acids 353 to 359, and amino acids 520 to 526 in the amino acid sequence indicated by SEQ ID NO: 8, or a portion of said nucleotides.

(12) The primer according to (10) or (11), wherein said primer comprises the sequences indicated by SEQ ID
NOS: 19 and 20.
In (11) and (12) above, a primer including forward and/or reverse primers) either individually or in combination, is used.

(13) A probe specific to the membrane-bound Netrin isoform according to (1) or (2) above, or the protein, or fragment thereof, according to (5) above.

(14) The probe according to (13) above having a sequence corresponding to nucleotides 1959 to 2261 of the nucleotide sequence indicated by SEQ ID NO: 7, nucleotides 1959 to 2084 of the nucleotide sequence indicated by SEQ ID NO: 13, or nucleotides 2262-2403 of the nucleotide sequence indicated by SEQ ID NO: 7.
In (13) and (14) above, the probe may be either DNA or RNA, and may include either a sense sequence or an antisense sequence. Further, the probe can be labeled with radioactive indicators, or non-radioactive indicators such as fluorescent indicators.

(15) An antibody having specificity against the membrane-bound Netrin according to (1) or (2) above or the protein, or fragment thereof, according to (5) above.

(16) The antibody according to (15) above having specificity against specific isoforms of the membrane-binding Netrin according to (1) or (2) above.
The "antibody" of (15) and (16) above, may either be a polyclonal antibody or a monoclonal antibody. A polyclonal antibody can be obtained by immunizing an animal by a typical method with the above-mentioned membrane-bound Netrin or a polypeptide segment thereof as an antigen.
Further, a monoclonal antibody may be obtained from a hybridoma obtained by fusion of an antibody producing cell and a myeloma cell. Preparation of monoclonal and polyclonal antibodies can be performed according to procedures known within the relevant art.

(17) A transgenic animal into which is introduced a gene encoding the membrane-bound Netrin according to (1) or (2) above or the protein according to (5) above.

(18) A homozygotic or heterozygotic knock-out animal in which a gene encoding the membrane-bound Netrin according to (1) or (2) above, or the protein according to (5) above, has been inactivated.
This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No.2000-148843, which is a priority document of the present application.
BREIFDESCRIPTION OF THE DRAWINGS
Figure 1 indicates expression of 7D5 in the brain:
(A) Indicates the expression profile of 7D5 in the development process of the cerebellum according to northern blot analysis.
(B) Indicates tissue distribution of 7D5 in an adult mouse . -actin was used as a control for RNA loading.
Arrows (A) and (B) indicate 7D5 signals.
Figure 2 indicates the alignment of the putative amino acid sequence of Netrin-Bla (7D5) with vertebrate Netrin.
Amino acid residues identical as between Netrin-Bla and other Netrins are shown in negative (white print on black background). The arrows above the sequences indicate domain structures of Netrin-B1a (domains VI, V1, V2, V3 and C'), defined by homology with other Netrins. The * symbol indicates the putative N-glycosylation site of Netrin-Bia.
Figure 3 is a schematic representation and hydrophobicity plot of Netrin-B1 and other UNC-6/Netrins.
(A) is a schematic representation of the domain structures of members of the UNC-6/Netrin family.
Homology(%) between the amino acid sequences of homologous domains is represented. Netrin-Bla is bound on the cell membrane by a GPI-linkage whereas Netrin-1 and Netrin-3 are secreted. The C domain of classical Netrins is highly charged ( indicated by + -I- ) .
(B) indicates a Kyte-Doolittle hydrophobic plot of the amino acid sequences of Netrin-Bla and mouse Netrin-1.
Figure 4 indicates the structure and expression of Netrin-B1 isoforms.
(A) indicates the sequences in the vicinity of the variable regions of six types of isoform, Netrin-Bia-Blf.
Netrin-Bia most resembles Netrin-1, Netrin-2, and Netrin-3 in respect of domain structure. Netrin-Blb and Blc has no domain V2 (EGF2) and no domain V2 and V3 (EGF3), respectively. Netrin-B1d has an unknown domain (Ukd) insert between domain V1 (EGF1) and C' whose sequence is shown in negative. Netrin-B1e has a shorter Ukd. All of these five types of isoform (Netrin-Bla to B1e) have domains VI, V1 and C'. Netrin-Bif lacks domains V2, V3 and C'. This isoform, exceptionally, has no C-terminus hydrophobic stretch. Absent sequences are indicated by "~" and the "-" at the ends of a sequence indicates that the sequence continues.
(B) is a schematic representation of Netrin-B1 isoforms. The hydrophobic stretches of the N and C termini are indicated by shading, and the Ukd domains of Netrin-Bld and Ble are indicated with dots.

(C) indicates an RT-PCR analysis of 5 types of isoform in adult whole brain and various regions of PO brain.
RT-PCR is indicated by an arrow in Fig. B. RT-PCR was performed with primer sets corresponding to the nucleotides of EGF1 and domain C', respectively. Further southern blot hybridization was performed using the isoform specific probes) indicated in the lower portion of Figure B.
Isoforms are indicated with an arrow at the right.
Figure 5 indicates the PI-PLC sensitivity of recombinant Netrin-B1a and Netrin-Bld.
Figure 6 indicates the regional distribution of Netrin-B1 transcription products in mouse brain analyzed by whole mount in situ hybridization.
PO mouse brain parasagittal sections (A, B, D, E, G and H), and P2 mouse brain coronal vibratome slices (C and F) were hybridized with digoxigenin labeled antisense cRNA
probes specific for Netrin-B1 (A-F excluding B) or Unc5h3/RCM (G and H). The sense probe for Netrin-B1 show no signal under the same conditions as those for the antisense probe (B). As can be seen in the lateral view of the cerebral hemisphere, the distribution of Netrin-B1 (E) and Unc5h3/RCM (H) is complementary, and delineates a boundary between the neocortex and allocortex as indicated by an arrow. The bars in the figure indicate 1mm (A, B, E, and H), 0.85mm (C), 0.65mm (F), and 0.8mm (D and G), respectively.
Abbreviations are as follows: deep cerebellar nuclei (DCN), external germinal layer (EGL), hippocampal formation (HP), inferior colliculus (IC), inferior olive (IO), mammillary body (MB), olfactory bulb (OB), piriform cortex (PR), red nucleus (RN), retrosplenial cortex(RC), superior colliculus (SC), and thalamus (TH).
Figure 7 indicates an ontogenic analysis of Netrin-B1 expression.
Figure 8 indicates Netrin-B1's lack of affinity to Netrin receptors.
Unc5h3/RCM whose cytoplasm domains were replaced by ECFP
were expressed in COS7 cells. Expression of the receptor protein was detected as ECFP fluorescence (blue signal) (A, C and E). DCC was expressed in 293 EBNA cells, and detected using mouse monoclonal anti-DCC antibody and Alexa 488 conjugated anti-mouse IgG (green signal) (G and I). In the same field, binding of myc-tagged chick Netrin-1 (Netrini/myc) on the cells was detected with immunocytochemical methods using monoclonal anti-myc antibody (9E10) and Alexa 546 conjugated anti-mouse IgG(B) in the case of Unc5h3/RCM, and rabbit polyclonal anti-myc antibody and Alexa 546 conjugated anti-rabbit IgG (H) in the case of DCC (red signal). However, with the secreted forms of myc-tagged Netrin-B1a (sNetrin-B1a/Myc) and myc-tagged Netrin-Bld (sNetrin-B1a/Myc), there was no indication of binding on the cells expressing both receptors in high concentration (D, F and J). A similar result was obtained with cells expressing Unc5h1 or Unc5h2 receptors. With both sNetrin-B1c and sNetrin-Ble, there was no indication of binding to cells expressing either of the receptors investigated (data not indicated). Figure K shows the relative concentration of the tested ligands used in the binding experiment as clarified by anti-myc immunoblotting.

The figure shows representative results obtained from one of three independent experiments. The bars in the figure represent l0,um.
Figure 9 indicates Netrin-B1's inability to complement Netrin-1 activity in collagen gel cerebellar plate explant.
Cerebellar plate (CP) obtained from E12 mouse were co-cultured in collagen gel with an aggregate of HEK293T cells expressing Netrin-1 (A), secretion-type Netrin-Bla (sNetrin-Bla)(B), secretion type Netrin-Bld (sNetrin-Bld), and mock transfected cells (D), respectively. Cells expressing Netrinl attracted axons from the CP (A). However, neither sNetrin-Bla nor sNetrin-Bld could elicit CP axon growth (B
and C) (n~8). The bars in the figure are 100,u m (A-D).
Figure 10 indicates the phylogenetic relationship between Netrin-Bl and other members of the Unc-6/Netrin family.
A phylogenetic tree was constructed based on the amino acid sequences of C.elegans Unc-6 (P34710), chick Netrin-1 (Q90922), chick Netrin-2 (Q90923), mouse Netrin-1 (AAC52971), mouse Netrin-3 (AAD40063), human Netrin-1 (NP004813), human NTN2L (NP006172), zebra fish Netrin-1 (AAB70266), zebra fish Netrin-1a (AAC60252), Drosophila Netrin-A (Q24567), Drosophila Netrin-B (Q24568) and Netrin-B1a (AB038667, SEQ
ID NO: 8) using CLUSTAL X program. The figure indicates that Netrin-B1 has evolved at a great distance from other members of the family, and that Netrin-B1 may become the prototype of a novel sub-family.

Divergence of UNC-6/Netrin Familv Members of the UNC-6/Netrin family are laminin-related small proteins. Laminin is a heterotrimeric extracellular matrix protein of a , a and y chains, the N-terminal portion of each chain is constructed from a single spherical domain (domain VI) and several EGF-like repeats (domain V) continuing therefrom. (Sasaki et al., J. Biol. Chem. 263:
16536-16544, 1988; Beck et al., FASEB J. 4: 148-160, 1990;
Timpl et al., Matrix Biol. 14: 275-281, 1994). Members of the UNC-6/Netrin family have the characteristic that domain structure is conserved from domain VI of laminin to V (the third EGF-like repeat) (Ishii et al., Neuron 9:873-881, 1992; Serafini et al., Cell 78: 409-424, 1994; Engvall et al., J. Cell Biochem. 61: 493-501, 1996).
We screened the cDNA fragment of secretion protein and membrane protein predicted by signal sequence trap method, and identified a novel member of the UNC-6/Netrin family.
The overall homology level of the Netrin (denoted Netrin-B1) of the present invention to known UNC-6/Netrin family members is low. However, its domain structure matches the structure of classical members which provides the molecular basis for including this novel gene in the UNC-6/Netrin family. As with other UNC-6/Netrin (Ishii et al., Neuron 9: 873-881, 1992; Serafini et al., Cell 78: 409-424, 1994), Netrin-B1 is related in respect of the y -chain more than the ~ -chain of laminin (with 32~ homology to mouse y chain and, 27% homology to the ~ -chain), further supporting this conclusion. Since in all types that were examined, chick (Serafini et al., Cell 78: 409-424, 1994), fly (Mitchell et al., Neuron 17: 203-215, 1996; Harris et al., Neuron 17: 217-228, 1996), mouse (Puschel, Mech Dev 83:65-75, 1999; Wang et al., J. Neurosci. 19: 4938-4947, 1999), and human (Van Raay et al., Genomics 41: 279-282, 1997), only two types of gene were isolated, the UNC-6/Netrin family was thought to be a small family. The Netrin-B1 of the present invention, is a third member of this family in mouse. The present inventors suggest the possibility that the UNC-6/Netrin family has, like other axon guidance molecule families, such as semaphorin (Dodd and Schuchardt, Cell 81:471-474, 1995) and ephrin (Flanagan and Vanderhaeghen, Annu. Rev. Neurosci 21:309-345, 1998), diversified during the course of evolution more greatly than had been predicted up until now. From a phylogenetic analysis (Fig. 10) of the gene family, it is suggested that Netrin-B1 evolved independently from classical Netrins, and that Netrin-Bl may form the prototype of a new sub-family.
Netrin-B1 has several characteristics that differ from classical Netrins. One characteristic of Netrin-B1 can be seen in its C-terminal sequence. Domain C of Netrin-1 is rich in lysine which is a basic residue and functions as a heparin-binding site (Serafini et al., Cell 78: 409-424, 1994). This basic C domain can bind to the cell surfaces via negatively charged molecules such as proteoglycan. Therefore, it is thought that the diffusion range of Netrin is determined by the expression level of Netrin relative to the binding site concentration in the environment. The C-terminal sequence of Netrin-B1 (domain C') is not rich in lysine, rather it encodes a signal for the GPI anchor (Fig.
3). From expression in HEK293T cell and PI-PLC processing, it became clear that Netrin-B1a and Bid were bound by means of a GPI linkage on cell surface. This characteristic of Netrin-B1 contrasts with diffuse classical Netrin. Therefore, it is thought that Netrin-B1 has specialized in order to play a role in a narrower region. However, this does not exclude the possibility that Netrin-B1, will under certain conditions, disperse afar. It has been reported that GPI
anchor molecules such as axonin 1/Tag1 may be released from the membrane, by internally expressed glycosyl PtdIns specific phosphalipase D, and that this oxygen activity was detected in posterior root ganglion neurons and in the brain (Lierheimer et al., Eur. J. Biochem. 243:502-510, 1997).
From broad-ranging cDNA analysis, it became clear that Netrin-B1 included at least six types of isoform (Fig. 4).
The longest isoform most resembling classical Netrin in respect of domain structure was named Netrin-Bla. Netrin-B1b and Blc are respectively absent 1 unit and 2 units of EGF-like repeat. Since it is thought that the EGF-like motif functions in relation to protein-protein interaction, there is the possibility that these isoform differ in their binding specificity for related molecules. In the nematode, C.elegans, a mutant strain lacking domain V2 of UNC-6 exhibits selective loss of function. A null mutant exhibited loss of both dorsal and anterior guidance in relation to nearby axons (Hedgecock et al., Neuron 2: 61-85, 1990), however a V2 deletion mutant strain exhibited loss of dorsal guidance only (Wadsworth, et al., Neuron 16: 35-46, 1996) suggesting that the existence of this domain was important in some types of expression forms. Netrin-Bld and Bie are absent two EGF-like repeats but have an unknown domain insert between domain V1 (EGF-1) and C'. It is possible that through this insert, the binding specificity of these isoforms for their partners has been further altered. Since Netrin-Blf is absent a C' domain, this isoform diffuses and like classical Netrins can influence distant targets.
From Southern blot hybridization of genomic DNA and partial sequencing through genomic cloning, it was suggested that these isoforms are derived from a single gene, and are likely formed by alternative splicing (Data not indicated).
This data suggests that the exon-intron structure of Netrin-B1 also resembles that of other UNC-6/Netrin family members (Ishii et al., Neuron 9: 873-881, 1992; Harris et al., Neuron 17: 217-228, 1996). The Ukd domain found in Netrin-Bld and B1e is thought to encode a novel extra exon that was obtained during the process of evolution. Interestingly, from semi-quantitative RT-PCR (Fig. 4C), it was suggested that alternative splicing appeared to be regulated in a regional and developmental stage specific manner. It is likely that alternative splicing further diversifies the function of Netrin-B1. It is not known whether classical Netrins having slicing-mutant forms or not.
Netrin B1's Lack of affinity to Netrin receptor The binding ability of isoforms of Netrin-B1 to Netrin receptors was examined. To assess any functional duplication, the binding ability of Netrin-B1 isoforms to Netrin receptors was examined. Unc5hl, Unc5h2, Unc5h3/RCM, DCC and neogenin are receptors for classical Netrins in vertebrate animals (Keino-Masu et al., Cell 87: 175-185, 1996; Leonardo et al., Cold Spring Harb. Symp. Quant. Biol. LXII: 467-478, 1997; Ackerman et al., Nature 386: 838-842, 1997). Classical Netrin can bind with all of these receptors.
The binding ability of Netrin-Bla-myc, B1b-myc, Bld-myc, and B1e-myc to Unc5h3/RCM expressed in COS7 and HEK293T
cells, and other Netrin receptors was examined. Netrinl-myc used as a positive control bonded to these test receptors, and exhibited functional expression of the receptor molecules but not any of the test ligands exhibited detectable affinity for these receptors even at a concentration which was 100 times higher compared to Netrinl-myc. Detection of bound molecules, and determination of their relative concentrations was conducted using anti-myc antibody. It is possible to obtain a false negative result due to contamination of an excessive amount of degradation product lacking the myc epitope tag.
Consequently, the quality of the test ligand was evaluated and this possibility was excluded by Western Blot analysis using a polyclonal antibody for Netrin-B1 domain VI. Netrin-Blc and Blf were not examined. However, it was not considered possible that these isoforms having less similarity to classical Netrin would exhibit affinity for these receptors.
To further evaluate the possibility of interactions the naturally expressing Netrin receptors of Netrin-B1, an in vitro explant co-culture experiment using a collagen gel matrix was performed. It has been well clarified that commissure axons and cerebellar efferent axons express Netrini receptors, and that Netrini promotes the growth of these axons (Serafini et al., Cell 78: 409-424, 1994;
Shirasaki et al., Neuron 14: 961-972, 1995). Unlike Netrin 1, the soluble form of Netrin-B1a demonstrated no detectable effect on these axons. The results of the explant co-culture experiment and the results of the receptor-binding assay suggested that Netrin-Bl may have a function differing from that of classical Netrins. It can be contemplated that Netrin-B1 possesses specificity for receptors differing from known Netrin receptors. However, the possibility remains that Netrin-B1 interacts with previously known Netrin receptors in the presence of co-factor.
ession and sredicted function of Netrin-B1 As for the predicted function of Netrin-B1, there are thought to be two possibilities. Firstly, based on the structural similarity to classical Netrin, it can be predicted that Netrin-B1 plays a chemical attraction and/or chemical repulsion role in axon growth, and the movement of cells that express the putative receptors for Netrin-B1.
Alternatively, there is the possibility that GPI anchor type Netrin-Bl plays an independent role in transmits a signal from the extracellular environment such as that seen in the CNTF a receptor (Davis et al., Science 253: 59-63, 1991).
The ephrin/Eph receptor system is proposed to involve directional signal transmission (Araujo et al., Development 125: 4195-4204, 1998; Bruckner et al., Science 275: 1640-1643, 1997; Holder and Klein, Development 126: 2033-2044, 1999; Davy et al., Genes Dev. 13:3125-3135, 1999).
Expression of Netrin-B1 was detected in the mid- and hind-brain regions up to E12 (No data indicated). Thereafter, Netrin-B1 expressed in many isolated regions in the CNS.
Expression was limited to the CNS during the developmental process. Therefore, it was thought that Netrin-B1 is a specialized molecule having a role in the CNS. This is in contrast to Netrin-3 in mice. Netrin-3 is suggested be a factor specialized for the peripheral nervous system (Puschel, Mech. Dev. 83: 65-75, 1999; Wang, J. Neurosci. 19:
4938-4947, 1999).
Expression of Netrin-B1 is most prominent in the thalamus. Neurons of the thalamus include mammillary bodies also expressing Netrin-B1 interrupting afferent signals from various regions of the nervous system to the cerebellar cortex. Conversely, neocortical neurons in the V layer project axons toward the thamalic nuclei through the cortico-thalamic pathway. This projection is thought to be influenced by Netrin-1 extruded from the ganglionic eminence which is an intermediate target (Metin et al., Development 124:5063-5074, 1997; Richards et al., J. Neurosci. 17: 2445-2458, 1997). There is the possibility that Netrin-B1 performs an attraction and/or target selection influencing this projection beyond the ganglionic eminence toward the thamalic nuclei.
Expression of Netrin-B1 in the cerebellum is limited to the deep cerebellar nuclei. The deep cerebellar nuclei receive the projection of three types of afferent fibers, i.e. mossy fibers, climbing fibers, and Purkinje cells, and extend efferent fibers to the red nucleus and other brain regions. Interestingly, Netrin-B1 is also expressed in the red nucleus which is a target for efferent fibers from regions about deep cerebellar nuclei such as the inferior olive nuclei, which is the origin of climbing fibers and from the deep cerebellum. There is the possibility that Netrin-B1 plays an important role in the formation of functional pathways for the regulation of movement activity.
Further, the deep cerebellum nucleus is thought to regulate movement or development of granular cells and Purkinje cells.
The deep cerebellar nucleus is believed to be one site that can be used in the future evaluation of the functional importance of Netrin-B1.
Expression in the olfactory bulb was detected up to E14 and high level expression was maintained up to adulthood.
This expression appears to be restricted to Mitral cells and Tufted cells (Fig. 7D and G). Olfactory receptor neurons project to the mitral cells at around E14 and can regenerate throughout the life of the organism. Further, granular cells are continuously supplemented even in adults from the subventricular even in adults. Therefore, olfactory bulb needs to continuously renew the structural connections between cells. It is possible that the continuous expression of Netrin-B1 supports the maintenance of circuitry in the olfactory system.
The complementary expression of Netrin-B1 and Unc5h3/RCM
in the cerebral cortex is interesting. These genes are potentially useful as marker genes of the allocortex and neocortex.
Examples:
~inle 1' A novel member of the UNC-6/Netrin familv:
Tso~at~on of Netrin-B1 (7D5) The date of birth was designated 0 days post-natum (PO).
To analyze the molecules involved in intercellular communication during development of the cerebellum, a signal sequence trap cDNA library was constructed from a 21 days post-natal (P21) mouse cerebellum. The signal sequence trap method is conducted with invertase-defective yeast strain (Jacobs et al., Gene 198: 289-296, 1997), however it enables selective analysis of cDNA encoding secretion and membrane proteins.
The total RNA of a P21 mouse cerebellum was extracted by the guanidinium thiocyanate technique (Chomczynski and Sacchi, Anal. Biochem. 162: 156-159, 1987). Poly(A)+ RNA was selected on a QuickPrep mRNA purification kit (Pharmacia).
Following the Yabe et al. procedure (J. Biol. Chem. 272:
18232-18239, 1997), a DNA library was synthesized from 3,u g of Poly(A)+ RNA. To explain simply, a first cDNA chain was primed with 40pmo1 of XhoI unidirectional primer . 5'-GAGACGGTAATACGATCGACAGTAGSTCGAGNNNNNNNNN-3' (SEQ ID NO: 1).
After dA tailing, a second cDNA chain was synthesized using l0pmol of EcoRI linker primer . 5'-CCGCGAATTCTGACTAACTGATTTTTTTTTTTTTTTTTNN-3' (SEQ ID NO: 2).
Duplex cDNA was size fractionated by agarose gel electrophoresis (300-800bp), and amplified by polymerase chain reaction (PCR) using external primers . 5'-GACGGTAATACGATCGACAGTAGC-3' (SEQ ID N0: 3) and 5'-CCGCGAATTCTGACTAACTGATT-3' (SEQ ID NO: 4). The PCR product was digested with EcoRI and Xhol, re-fractionated by agarose gel electrophoresis, and unidirectionally ligated to the EcoRI and XhoI of a pSuc2t7F1 o n vector (Yabe et al., J.
Biol. Chem. 272: 18232-18239, 1997). cDNA was inserted into this vector upstream of the invertase gene lacking a signal sequence and, downstream of the yeast ADH1 promoter for effective expression.
To screen cDNA having a signal sequence, this library was inoculated into invertase-deficient yeast strainYT455 (Suc2D9, ade2-101, ura3-52) that is unable to reproduce on a raffinose plate (Jacobs et al., Gene 198: 289-296, 1997) . A
transformant that was able to export to the exterior of the cell the invertase for fusion having a cDNA-derived signal sequence, was able to reproduce on this plate. The inserted cDNA was amplified using the following primer set: 5'-CAGGAAACAGCTATGACCCAAGCATACAATCAACTCCAAGCTC-3' (SEQ ID NO:
5) and 5'-TGTAAAACGACGGCCAGTACTCCTCTGAAATTAATACGACTCAC-3' (SEQ ID NO: 6). The amplified DNA was spotted on nylon membrane in duplicate. These membranes were hybridized with 3zP-labelled cDNA directly synthesized from PO or P21 mouse cerebellum mRNA. The sequence of the clones indicating differential hybridization signal was determined by an automated sequencer (ABI Prism 377) employing BigDye Primer Cycle Sequencing method (Applied Biosystems). From among these, clone 7D5 which exhibits low homology to UNC-6/Netrin was isolated.
To obtain a full-length cDNA clone, 2.5 X 106 phage plaques of PO and adult mouse brain cDNA libraries (Stratagene) were screened with 'ZP-labelled 7D5 cDNA
fragment isolated by the signal sequence trap method. The phage insert was excised in vivo according to the manufacturer's manual.
Fig. 1 indicates the expression profile of clone 7D5 according to Northern blot analysis. Poly(A)+ RNA of PO and P21 mouse cerebellum was extracted as described above. 2 ,u g Poly(A)+ RNA was subjected to electrophoresis in 1% agarose gel (2,u g each lane), and transferred to a nylon membrane (Hybond N Filter, Amersham). This filter was hybridized with 32P- labeled cDNA probe of clone 7D5. The filter was washed twice with 2 x SSC and 1% SDS at 65 'C for 30 minutes, and then washed twice with 0.2 x SSC and 0.1% SDS. The membrane was then analyzed using Image Analyzer (BAS5000, Fuji Film).
As a result, a single stranded band approx. 4.5kb in size was detected. 7D5 expressed strongly in cerebellum at PO and was down-regulated by P21 (Fig. 1A). Further, when an adult mouse multiple tissue Northern blot (Clontech) was hybridized with the above probe, 7D5 specifically expressed in the brain and no signal was detected in various mouse tissues other than the brain (Fig. 1B).
To isolate the full-length cDNA clone encoding the 7D5 gene, a cDNA library prepared from an adult and PO mouse brain and primed with oligo-dT was screened using the trapped cDNA fragment as a probe. The 39 overlapping clones were isolated, and their sequences determined. The longest cDNA fragment was 4090 nucleotides long (SEQ ID NO: 7). The predicted open reading frame (ORF) of this clone consisted of 539 amino acids (Fig. 2, SEQ ID NO: 8). The sequence of the trapped cDNA fragment (7D5), corresponded, as was expected, with the sequence of 180 amino acids of the N-terminus. Signal peptides are commonly situated at the N-terminus.
2: Comparison of the sequences of Netrin-B1 and known Netrins The putative amino acid sequence of Netrin-Bl (SEQ ID
N0: 8) exhibited low homology to chick Netrinl, Netrin2, and mouse Netrin-1 and Netrin-3 (31%, 30%, 29% and 29%, respectively) (Fig. 2 and 3A). The predicted domain structure of this protein resembled that of the UNC-6/Netrin family, in other words, in respect of the laminin globular domain (domain VI) and three epidermal growth factor (EGF)-like repeats continuing therefrom (domains Vl to V3) (Figs.
2 and 3A). The Cys residue was conserved in these domains of the 7D5 gene which is a residue that is phylogenically conserved among the UNC-6/Netrin family, further evidencing the structural similarity of 7D5 to UNC-6/Netrin. Two thirds of the N-terminus side of UNC-6 exhibited homology to the N-terminal domain (domain VI and domain V) of laminin ~ and chains which constitute the extracellular matrix molecule of the large hetero-trimer. For this reason, UNC-6 was firstly identified as a laminin-related protein. The 7D5 gene also possesses this characteristic. As with UNC-6 and Netrin, 7D5 within these domains, has greater homology in respect of the Y chain more so than the ~ chain of laminin (homology of 32%
to mouse Y -chain, homology of 27% to the ~ -chain). From these facts, it was concluded that 7D5 is a novel member of the UNC-6/Netrin family and it was named Netrin-B1.
Characteristics of Netrin-B1 which differ from other family members, could be found in the C-terminal sequence.
The C-terminal sequence (domain C) of Chick Netrin-1 and Netrin-2 is rich in basic amino acids consisting of lysine residues whereas the corresponding domain of Netrin-B1 is not rich in lysine residues. When a Kyte-Doolittle hydrophobic plot was conducted on the amino acid sequences of Netrin-B1a and mouse Netrin-1 described in Example 3 (Fig.
3B), two hydrophobic stretches were observed in both the N-terminus and C-terminus of Netrin-Bla. Among UNC-6/Netrin family members, a C-terminal stretch is particular to Netrin-Bla.
The hydrophobic stretches of Netrin-Bia are thought to be a signal for the signal peptide and GPI linker predicted from ~ and ~ +2 rules (Von Heijne, Nucleic Acids Res. 14:
4683-5690, 1986; Gerber et al., J. Biol. Chem. 267: 12168-12173, 1992). Thus, this domain is referred to as domain C'.
It was predicted that this hydrophobic stretch of the C-terminus plays a role as a signal for the GPI link to the membrane, and further, it was considered that there was the possibility that Netrin-B1, in contrast to diffuse classical Netrins, was restricted to the membrane surface.
xamg~P 3' Netrin-B1 isoforms From a comparison of the sequences of 39 cDNA clones, it became clear that there existed six types of isoform probably formed by alternative splicing. These isoforms are schematically represented in Fig. 4B. The isoform having the longest amino acid sequence (Netrin-Bia, SEQ ID NO: 8), most greatly resembled the UNC-6/Netrin family in respect of its domain structure. Of the 39 types of cDNA clones, seven encode this isoform. Netrin-Blb (whose nucleotide sequence is indicated by SEQ ID NO: 9, and whose amino acid sequence by SEQ ID NO: 10) and Blc (whose nucleotide sequence is indicated by SEQ ID NO: 11, and whose amino acid sequence by SEQ ID NO: 12), do have deletions of one or two EGF-like repeat units (EGF 2 or EGF 2 to 3), respectively (Fig. 4A
and B). From the 39 clones, 1 and 3 of these mutants, respectively, were obtained. Netrin-B1d (whose nucleotide sequence is represented by SEQ ID NO: 13, and whose amino acid sequence by SEQ ID NO: 14) and Bie (whose nucleotide sequence is represented by SEQ ID N015, and whose amino acid sequence by SEQ ID NO: 16) possess an insert of 42 amino acids and 22 amino acids, respectively, between domain EGF1 and domain C' (Fig. 4 A and B). These isoforms were indicated in 8 and 5 clones respectively. Half of the N-terminal side of the insert in Netrin-B1d and Ble was identical, but Netrin-Bld was just 20 amino acids longer than Netrin-Ble. In a GeneBank data base search, the inserted domain did not exhibit homology with any known gene, so it was denoted (Unknown) domain (Ukd). The shortest isoform, Netrin-Blf (whose nucleotide sequence is indicated by SEQ ID NO: 17; and whose amino acid sequence is indicated by SEQ ID NO: 18) was found in only one clone, domains V2 (EGF2), V3 (EGF3) and C' were absent (Fig. 4A and B). The remaining 14 clones partially possessed coding sequences, and it was impossible to determine to which of the isoforms these clones belonged.
The above results show that the isoforms of Netrin-B1 had variety in their C-terminal structures but all isoforms comprised N-terminal domain VI and EGF1. The nucleotide sequences of Netrin-Bla and other isoforms were registered in the GenBank database (Accession Numbers: AB038667, AB038666, AB038665, AB038664, AB038663, AB038662).
ale 4: RT-PCR analysis To confirm the expression of the isoforms thought to be formed by alternative splicing, and to examine their regional and temporal distribution, RT-PCR was performed using a primers set of nucleotides corresponding to EGF1 (amino acids 353-359 of Netrin-B1a) and domain C' (amino acids 520 to 526 of Netrin-Bla)(Fig. 4B).
Total RNA from various regions of the mouse brain were extracted. For RT-PCR, 5,u g of DnaseI processed total RNA
was reverse transcribed with Superscript II (Gibco), and 1/50 volume of the reaction product (total RNAl00ng) was subjected to PCR. For confirmation of the isoform mutants, the following primer set was used: 5'-CCTGTATCCCCAGCATTTCC-3' (SEQ ID NO: 19) and 5'-AGCAGCAGTGCTGGGGAGCC-3' (SEQ ID
NO: 20). These primers correspond respectively to the nucleotide sequences of EGF1 (+1058 to +1077 of Netrin-Bla) and domain C' (+1558 to +1577 of Netrin-Bla), and amplified bands of 520, 352, 217, 343 and 283bp respectively were formed from Netrin-Bla, Bib, Blc, Bid and Ble. Reaction conditions were 96~C for 3 minutes, followed by 96'C for 1 minute, and 60 ~C for 1 minute and 72 ~C for 1 minute for 33 cycles, and finally 72~C for 7 minutes.
To identify the products, the isoform specificity of the amplified fragment was confirmed by southern hybridization using an isoform specific internal probe. (Fig. 4B, C). For Netrin-Bla and Blb, the nucleotide sequence of EGF-2 and 3 corresponding to (a+1087 to +1389nt of Netrin-B1) was used as a probe. For Netrin-Bid and Ble, a fragment corresponding to the Ukd of Netrin-B1d (+1087 to +1212nt of Netrin-B1d) was used. For Netrin-Blc, the internal sequence of domain C' (+1390~'+1531nt of Netrin-B1a) was used.
From the probe corresponding to EGF2 and EGF3 (amino acids 363-463 of Netrin-B1a (SEQ ID NO: 8)), the existence of Netrin-Bia and Bld was confirmed. From the probe of Domain Ukd (amino acids 363-404 of Netrin-B1d (SEQ ID NO:
14)), Netrin-B1d and B1e were detected. The shortest fragment as clarified by the C' probe (amino acids 464-511 of Netrin-Bla (SEQ ID NO: 8) was identified as a product of Netrin-Blc. Since there is the possibility that the amplification efficiency of these isoforms differed, it was not possible to evaluate the relative amounts of these isoforms precisely. However, these results suggested that all isoforms were being formed at the perinatal stage and that Netrin-Bla and B1d appeared to be the main isoforms in the brains of adults.

5~ Construction of a mammalian exsression vector The full coding sequence of Netrin-B1a and B1d was ligated to pcDNA4Myc/HisA (Invitrogen) at the EcoRI and PmeI
sites and synthesis of an intact protein was conducted. In these constructions, the synthesized protein is not fused to the myc and His-tagged peptide. The obtained constructions were used in the PI-PLC processing experiment.
To maximize extracellular secretion of Netrin-Bla, Blb, Bld and B1e in the mammalian cell system, the coding sequences of these isoforms excluding the C-terminal sequence were also sub-cloned in pcDNA4Myc/HisA. In other words, the sequence corresponding to the 26 amino acids of the hydrophobic C terminus of Netrin-B1 was substituted by a spacer sequences comprising an ApaI site, ligated to EcoRI/ApaI site and fused to a Myc/His-tagged sequence at the C-terminus. These vectors included the sequence from 80nt upstream of these isoforms' initial ATG to the 5'-terminus of the C-terminal hydrophobic domain. The linking amino acid sequence between Netrin-B1 and myc/His was Gly-Gly- Pro- Phe.
P 6- Construction of a Netrin-B1a domain- protein ~~rP~~;nn vector and expression in a host cell The sequence corresponding to domain VI (amino acids 44-259) of Netrin-Bla (SEQ ID NO: 8) was sub-cloned in pGEX-2T(Amersham Pharmacia) and fused with the glutathione S -transferase (GST) gene. The fusion protein was allowed to express in E.Coli by isopropyl-b-D-thiogalactopyranoside induction.

Further, (His)6-tagged domain VI protein was synthesized by cloning the corresponding sequence in pET-32a (Novagen) and expressing in E. coli.
.xamnle 7: Preparation of an antibodv The Netrin-Bla domain VI-GST fusion protein allowed to express in Example 6 was purified using a glutathione column.
A rabbit was immunized several times by typical techniques using purified antigen and Freund's complete and incomplete adjuvant. Antiserum was affinity purified against (His)6-tagged domain VI protein (amino acid 66-116) that was synthesized in Example 6. Affinity purification was conducted by purifying (His)6-tagged domain VI protein expressed in E. coli with a Ni-NTA column (Qiagen). The purified fusion protein was coupled to a HiTrap NHS-activated affinity column (Amersham Pharmacia).
E~gIP R- PI-PLC Treatment and Western blot analysis A hydrophobic plot of Netrin-Bla indicates that Netrin-Bl differs from Netrinl and has two hydrophobic sequences at both termini (Fig. 3). The sequence at the N-terminus supports the possibility of operating as a signal sequence in secretion of yeast invertase but this activity has not been confirmed in mammal cells. This signal sequence is observed in common between members of the UNC-6/Netrin family. However, the C-terminus sequence is particular to Netrin-B1, and is thought to be a GPI lipid anchor.
To confirm this possibility, recombinant Netrin-Bla and B1d were expressed in HEK293 cells.

HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) to which 2mM of L-glutamine and 10% f, et al. " calf serum (FCS) has been added. The cells were inoculated onto l0cm tissue culture plates at a cell concentration of 4 x 106 cells per plate. After 24 hours, cells at 80% confluence were transfected with 20 ~ g of expression vector (with no epitope fusion) obtained in example 5, using a CellPhect Transfection Kit (Amersham Pharmacia). After 40 hours, the transfected cells were incubated in 8m1 OptiMEMI(Gibco) at 37'C for 2 hours, both together with and without 100mU/ml PI-PLC (Sigma). The media from the PI-PLC processed and unprocessed cultures (1.5m1) were centrifuged at 2,OOOg for 10 minutes then 60,000 for 100 minutes thereby effecting clarification. These supernatants were TCA-precipitated, and pellets dissolved in an SDS-sample buffer. The cells cultures without PI-PLC were dissolved mRIPA buffer 750 ~ 1 (50mM Tris-HC1 p H7.5, 150mM
NaCl, 1% Triton X-100, 1% cholic acid, 0.1% SDS and 20mM
EDTA). These samples were fractionated with 10% SDS-PAGE, and electrotransferred to a PVDF membrane (Millipore).
Recombinant Netrin-B1 was detected using the affinity purification rabbit anti-Netrin-B1 antibody and as a secondary antibody, HRP-conjugated anti-rabbit IgG (Zymed), and visualized using an ECL System (Amersham Pharmacia).
60 hours after transfection, the transfected HEK293T
cells were incubated in Opti-MEM, both together with 200mU/ml PI-PLC, and without PI-PLC at 37 C for two hours.
The supernatant was clarified by centrifugation, and precipitated with TCA. A cell lysate was obtained from the cells untreated with PI-PLC. These samples were fractionate by SDS-PAGE, then from immunoblot analysis with affinity purified anti-Netrin-B1 polyclonal antibody for Netrin-B1 common domain (domain VI), each of the isoforms were detected in the cell in the lysate (Fig. 5), indicating that synthesis of the recombinant protein was successful. The transfected cell was exposed for 2 hours to phosphatidyl inositol-specific phosphalipase C (PI-PLC) which specifically cleaves the GPI anchor linkage protein from the membrane surface. As a result the recombinant protein was released into the supernatant (Fig. 5). In contrast, there was substantially no release of the recombinant protein with the same processing conducted without PI-PLC. From these results, it was indicated that at least in respect of these isoforms, and in all likelihood all isoforms with the exception of Netrin-Blf are primarily bound to the cell membrane by means of a GPI anchor. However, when the transfected cells were incubated for one day in a standard growth medium, a detectable amount of recombinant protein was release into the supernatant without any evidence of degradation (Data not indicated).
The calculated values of the molecular weights of nascent Netrin-B1a and Hld were 60.5kDa and 53.9kDa respectively. The cleavage site for GPI anchoring was predicted from ~J and c.~ +2 rules (Gerber, J. Biol . Chem. 267 12168-12173, 1992) (Fig. 2). The calculated values of the molecular weights of these cleaved proteins were 58.0 and 51.4kDa, respectively. The size as evaluated from immunoblot analysis was fractionally larger than the above-mentioned calculated value (Fig. 5), suggesting that other post-translational modifications were involved. The putative N-glycosylation site is indicated in Fig. 2. These modifications were confirmed by N-glycosidase F treatment (Data not shown). In Fig. 5, the recombinant protein released by PI-PLC processing, traveled more slowly that the product derived from the cell lysate. There is the possibility that these electrophoresis speeds were influenced by lipid elements bound to the recombinant protein.
P 9' Expression of Netrin-B1 in the Central Nervous Svstem (CNS) To examine the regional distribution of Netrin-B1 in detail, the N-terminal coding sequence was used as a probe common to all isoforms, and in situ hybridization in mouse cerebella was conducted.
First, to prepare a Netrin-B1 probe, the signal sequence trapped cDNA sequence of 7D5 clone was transferred into EcoRI and XhoI sites of pSP72 (Promega). Antisense and sense ribo-probe (corresponding to 867-1052nt of SEQ ID NO: 7) were each labeled with digoxigenin dUTP from a linearized template using SP6 and T7 RNA polymerase (Boehringer Mannheim).
For whole mount in situ hybridization, mouse brain was dissected in phosphate-buffered saline, and immobilized overnight in 4~ paraformaldehyde (PFA) PBS solution. The immobilized brain was rinsed three times with PBS, and after dehydration, rehydrated with a MeOH gradient, and co-incubated with 10 ,~.~ g/ml of proteinase K at room temperature for 40 minutes. Next, the brain sample was once again immobilized in 0.2% glutalaldehyde and 4%PFA PBS solution at room temperature, rinsed with PBS, then hybridized with l,u g/ml of riboprobe for 16 hours at 65~C. The hybridized brain was washed with 2 x SSC/0.1% CHAPS three times, at 65~C for 30 minutes and 0.2 x SSC/0.1% CHAPS three times at 65~C for 30 minutes. This was blocked with a 10% heat-inactivated sheep serum and 2%BSA TBT (50mM Tris-HC1 pH7.5, 150mM NaCl and 0.1% Triton X-100) solution, followed by co-incubation overnight at 4~C with alkaline phosphatase conjugated anti-digoxigenin Fab fragment (1:2000 dilution; Boehringer Mannheim). After washing thoroughly with TBT, the hybridized probe was detected using BMPurple as a phosphatase substrate.
In the case of coronal vibratome slices, slices of a thickness of 300,u m were prepared. The following procedures are the same as those described above except that the duration of incubation with proteinase K was ten minutes.
For in situ hybridization of the thin sections, the embryos were fixed in the same manner as above. PO and P21 brain were perfused with 4% paraformaldehyde PBS solution, post-fixed overnight in 4% PFA, then submerged in 30%
sucrose PBS until the brain settled to the bottom. The sample was embedded in O.C.T. compounds, and sliced to a thickness of 20,u m using a cryostat. The sliced were re-immobilized with 4% PFA, co-incubated with 2 ,u g/ml proteinase K for 10 minutes at 37 C, then after acetylation, the slices were further co-incubated with the above-mentioned riboprobe for 16 hours at 55~C, and then wased at 55~C with 50% formamide/2 x SSC/0.01% Tween-20 solution.
This was then processed with 10~ g/ml RNase A at 37~C for 30 minutes, and then at 55~C , washed once with 2 x SSC/0.01%
Tween-20, then washed twice with 0.1 x SSC/0.01% Tween-20.
The hybridization signal was detected as described above using anti-digoxigenin Fab antibody and BMPurple.
PO mouse brain parasagittal slices (A, B, D, E, G and H), and P2 mouse brain coronal vibratome slices (C and F) were hybridized with Netrin-B1 (A-F excluding B) or Unc5h3/RCM (G
and H) specific digoxigenin labeled antisense cRNA probe (corresponding to the antisense sequence of 867-1052nt of SEQ ID NO: 7). As a result of this, it was indicated that Netrin-B1 was regionally limited in its expression (Fig. 6A
- F). The strongest expression was detected in thalamus (Fig.
6A and D). In the olfactory bulb, inferior collicus and superior collicus, red nuclei, mammillary bodies, deep cerebellar nuclei and inferior olive nuclei a medium level expression was detected (Fig. 6A and D). In P2 brain coronal vibratome slices, strong expression was detected in the thamalic nuclei (Fig. 6C), and intermediate expression detected in the inferior collicus and cerebellar deep nuclei (Fig. 6F). Further, weak but clearly discernible expression was detected in the piriform cortex, the posterior area of the splenium of the corpus callosum, the granular cortex, and the hippocampal formation from CA1 to CA3 (Fig. 6C).
In several regions of the brain, expression of Netrin-B1 was in contrast with expression of Unc5h3 (Fig. 6G and H).
For example, Unc5h3 was strongly expressed in the external embryonic cell layer of the cerebellum and the Purkinje cell layer (Fig. 6G, and Ackerman et al., Nature 386: 838-842, 1997) but Netrin-B1 strongly expressed in the deep cerebellum nucleus (Fig. 6D). Like Unc5h3, other Netrin receptors are all reported to express in the external embryonic cellular layer and the internal granular cell layer (Leonardo et al., Cold Spring Harb. Symp. Quant. Biol.
LXII: 467-478, 1997). Among Netrin receptors only DCC is known to express in the deep cerebellum nucleus (Livesey and Hunt, Mol. Cell Neurosci. 8: 417-429, 1997). Interestingly, in cerebellar cortex surface, Netrin-B1 and Unc5h3 expressed respectively in the alhocortex and the neocortex (Fig. 6E
and H), and this expression delineated the boundary between the allocortex and the neocortex.
~X.~.m,Dle 10 ~ Exsression of Netrin-B1 in the ontocrenesis process Ontogenetic expression of Netrin-B1 in the representative region was examined by in situ hybridization (Fig. 7) .
With the exception of B, expression of Netrin-Bl during ontogenesis was examined by in situ hybridization using frozen parasagittal slices. Digoxigenin-labeled antisense cRNA to Netrin-B1 was used as a probe (corresponding to the anitisense sequence of 867-1052nt in SEQ ID NO: 7) . At E14, expression of Netrin-B1 was initially detected in the accessory olfactory bulb (AOB), restricted to the mitral cell layer (ML) and tufted cell (TF) at PO and persisting to P21. In the thalamus, expression at E14 was seen in the ventral thalamus (VT), dorsal thalamic nuclei (DT), and peretectal area (PT), reaching a peak at birth and being down-regulated during postnatal development. In the cerebellum, expression of Netrin-Bl at E14 was detected, however this was restricted to the deep cerebellar nuclei (DCN), and in contrast to the olfactory bulb, this expression was down-regulated at post-natal P21.
In the deep cerebellar nuclei, expression of Netrin-B1 was detected at E14. (Fig. 7C), sustained until PO (Fig. 7F), but was down-regulated up until P21 (Fig. 7I) . This matched well with the results of Northern analysis (Fig. 1A). Even in the inferior collicus, post-natal expression of Netrin-B1 followed a similar course over time (Fig. 7F and I).
At E14, expression of Netrin-B1 was limited to the ventral thalamus and dorsal thalamic nuclei, and the peretectal area of the mesencephalon (Fig. 7B).
Interestingly, this expression was regulated layer-specifically in the superior collicus (Fig. 7E and Fig. 6A).
Expression of Netrin-B1 in the thalamic region is thought to follow the same course over time as in the cerebellum (Fig.
7E and7H).
Expression of Netrin-B1 in the olfactory bulb was detected at E14 (Fig. 7A), which increased until PO (Fig.
7D), and was maintained at a high level to P21 (Fig. 7G) and into adulthood (data not indicated).
Expression of Netrin-B1 up to E12 in the mesencephalon and rhombencephalon regions was detected (data not indicated). From the whole-mount hybridization, a signal was obtained from either region in E10 (data not indicated).

Example 11: Recegtor bindinaa exseriment Until now, all of the members identified as belonging to the UNC-6/Netrin family were all previously known Netrin receptors, that is, they bind to the Unc5 family (Unc5hl, Unc5h2, andUnc5h3/RCM in mammals) (Leonardo et al., Nature 386: 833-838, 1997) and the Unc40 family (mammalian DCC and neogenin) (Keino-Masu et al., Cell 87: 175-185, 1996). To examine whether Netrin-B1 is a functional enhancer for UNC-6/Netrin, Netrin-B1's binding activity toward Netrin receptors was investigated.
Myc-tagged Netrin-1, and myc-tagged soluble forms the recombinant proteins, Netrin-Bla, Blc, Bld and Ble, were yielded as the culture supernatant from stable transformants cultured overnight. The mass and amounts of fusion proteins in the culture supernatant were examined by Western Blot analysis using anti-myc antibody (9E10) and HRP conjugated mouse IgG. The relative concentration of the fusion protein was measured with Image Analyzer (LAS100, Fuji). The minimum concentration of Netrin-1 binding with the receptor under experimental conditions was determined by, continuous dilution of the culture supernatant. To concentrate the soluble forms of Netrin-Bia, Blc, Bld and Ble, a Centriprep-30 Concentrator (Amicon) was used. This processing triggered no aggregation of secreted Netrin-Bl.
The cDNA of the Netrin receptor was obtained from Marc Tessier-Lavigne (Unc5hl, Unc5h2 and DCC; pCEP4-DCC) and Susan Ackermann (Unc5h3/RCM). The cDNA of chick Netrin-1 (pGNetlmY°) was also obtained from Marc Tessier-Lavigne. The coding sequence of the cytoplasm domain of Unc5hl, Unc5h2 and Unc5h3/RCM was substituted in frame, by an ECFP coding sequence, and a fusion protein comprising the receptor and ECFP-derived extracellar and transmembrane domains, was obtained. To construct an expression vector, a l.7kb NaeI
fragment of Unc5hl, a l.7kb Eco47III/SmaI fragment of Unc5h2, and a 2.2kb EcoRI/BalI fragment of Unc5h3/RCM was subcloned in pECFP (Clontech) at the SmaI site. An expression vector of the Unc5 family was transfected into a COS7 cell, and pCEP4-DCC was transfected into a 293EBNA cell (InVitrogen).
48 hours into transfection, myc-tagged recombinant protein was added to a medium to which 10% FCS and 20 ,u g/ml heparin (Sigma) had been added, and was incubated for 90min at room temperature. After washing one or four times with PBS, the cells were immobilized in MeOH for 5 minutes then in 4%
paraformaldehyde PBS solution for 15 minutes. In the case of the Unc5 family, binding of the recombinant protein was detected using monoclonal anti-myc antibody (9E10) and Alexa 546 conjugated anti-mouse IgG (Molecular Probes). Cells transfected with the receptor were visualized by CFP
fluorescence. In the case of DCC double staining was performed with monoclonal anti-DCC antibody (Oncogene Science) and affinity purified rabbit polyclonal anti-myc antibody. The binding of these primary antibodies were visualized using Alexa 488 conjugated anti-mouse IgG
(Molecular Probes) and Alexa 546 conjugated anti-rabbit IgG
(Molecular Probes), respectively. Fluorescent images were obtained with a CCD camera (Prinston Instruments) having MetaMorph software (Roper).

The C-termini of extracellular and transmembrane domains of Unc5h3/RCM, Unc5h1 and Unc5h2 cells were fused with ECFP
which is a mutant green fluorescence protein, and this was expressed in COS7 cells. Expression of the receptor molecule was monitored by the fluorescence of the membrane, These cells were co-incubated with myc-tagged Netrin-B1 and myc-tagged chick Netrin-1 protein collected as the supernatant from stably expressing cell. To maximize secretion, the hydrophobic C-termini of the Netrin-B1 isoforms were removed.
In a culture incubated with chick Netrin-1, Unc5h3/RCM
expressing cells able to be recognized by ECFP fluorescence were labeled by anti-myc antibody on their cell surface (Fig.
8 A and B), and this was consistent with previous reported results (Leonardo et al., Nature 386: 833-838, 1997). In contrast, no signal was detected in the culture treated with the series of test ligands (myc-tagged Netrin-Bla, Blb, B1d and B1e) (Fig. 8 D, F, etc.). The relative amounts of proteins used in these binding assays were evaluated by immunoblotting using anti-myc antibody. A representative result is indicated in Fig. 8. These test ligands exhibited no binding toward Unc5h3/RCM even at concentrations of 100 times greater than with chick Netrin-1. Therefore, Netrin-B1 is apparently not a ligand of Unc5h3/RCM. Similarly, Netrin-B1 did not exhibit binding to Unc5h1 and Unc5h (data not indicated). DCC belonging to another receptor family was also expressed in COS cells, and binding experiment with Netrin-B1 was conducted. In this case, the expression of DCC
was detected by immunocytological techniques employing DCC-specific monoclonal antibodies (Fig. 8, G and I). Cells expressing DCC exhibited binding toward chick Netrinl, However binding toward Netrin-B1 was not exhibited even at concentrations of over 100 times greater (Fig. 8 H and J).
Example 12: Ex 1 To further confirm the functional differences between Netrin-B1 and Netrin l, 3-dimensional collagen explant simultaneous culture experiment using dorsal spine and cerebellum plates was conducted.
The procedures followed for an explant in collagen gel were in accordance with the procedures described in Shirasaki et al. (Neuron 14: 961-972, 1995). An E12 mouse embryo was dissected in a DME/F12 medium (Sigma) to which glucose had been added. Cerebellar plate explants were obtained from open-book preparations of the hindbrain. An aggregate of the cell strain (293T cell) stably expressing Netrin-1, and the secretion form of Netrin-Bla and Netrin-B1d was prepared as described above (Kennedy et al., Cell 78: 425-435, 1994) . The explant and 293T cell aggregate was buried within a collagen matrix prepared from rat tail tendons. The medium that was used was DME/F12 to which was added 3.85mg/ml glucose, lOmg/ml streptomycin, 100 ,1.~ g/ml transferin, 5,u g/ml insulin, 5.29ng/ml sodium selenite, 16.4 ,u g/ml putrescine dihydrochloride, 6.29ng/ml progesterone, 7.40ng/ml hydrocortizone, and 10~ fetal calf serum.
As described above (Serafini et al., Cell 78: 409-424, 1994; Kennedy et al., Cell 78: 425-435, 1994; Shirasaki et al., Neuron 14: 961-972, 1995), Netrin-1 secreted from the transformed cell exhibited growth promotion and attraction activity toward commissure axons and cerebellar efferent axons (Fig. 9A). However, Netrin-B1 did not elicit the growth of these axons (Fig. 9B and C), consistent with the results of the binding assay.
These results strongly suggested that Netrin-B1 was not a ligand to any known Netrin receptor.
ple 13: Creation of a transgenic animal Transgenic animals can be produced by known techniques within the relevant field. For example, a transgenic animal may be produced by injecting the fertilized ovum of an animal with a vector comprising the Netrin-B1 gene of the present invention and regulatory sequences such as a promoters and an enhancer to control expression of the gene.
This is then transplanted to a surrogate parent, and the desired transgenic animal can be obtained by analyzing with PCR whether or not the progeny have the introduced gene. In this case, it is also possible to produce a chimeric animal by injection of pluripotent embryonic stem cell (ES cell) into the animal blastocyst.
Example 14: Creation of a Knock-out Animal A knock-out animal can be produced by using known standard genetic recombination techniques (gene targeting for example, Methods in Enzymology 225: 803-890, 1993), for example, as follows.
First, a targeting vector is produced by substituting the sequence of the isolated Netrin-B1 gene of the present invention by, for example, marker genes such as neomycin-resistance gene, and further, adding to the terminal portion of this gene, for example an MC1 promoter controlled diphtheria toxin fragment A gene without a PolyA signal.
This targeting vector is introduced into the ES cells of a mouse or the like, cell are selected in which homologous recombination of the target sequence or the mutant sequence it contains, into the cellular genomic DNA of the same gene.
The selection of such a gene recombinant cell can, for example, be conducted by adding 6418 to the cell culture, thereby allowing for removal of non-recombinant cells not having a marker gene. Random recombinant cells are excluded by their expression of fragment A of diphtheria toxin. The gene of the selected gene recombinant cell, is a .mutant sequence into whose coding sequence a marker gene has been introduced, and is unable to produce Netrin-B1.
Next, this gene recombinant ES cell is injected into a mouse early embryo (blastocyst), and this embryo is allowed to develop into an individual in the body of a female mouse, and a chimera mouse is allowed to be born. Then, this chimera mouse is mated with a wild type mouse and allowed to birth mice progeny, and by selecting mouse individuals having the mutant sequence in one or both allele from among the mice progeny, it is possible to obtain a knock-out mouse having no ability to produce Netrin-B1 or who produces an amount of Netrin-B1 that is relatively low compared to the wild type. A knock-out animal includes homozygote in which both alleles are substituted by mutant sequences ( - / - ), and heterozygote in which only one allele is substituted by a mutant sequence (+/-) .

As described in detail above, UNC-6/Netrin family is highly conserved in the process of evolution. However, in mammals, these family members are thought to be more branched than was expected. Molecules that are highly branched from previously known UNC-6 such as Netrin-B1 may play a particular important role in the particularly highly organized cell structure of vertebrate animals. The present invention makes possible the further clarification of the function of Netrin in the cerebella of vertebrate animals, provision of suitable treatment for brain damage caused by disease, and the promotion of normal neural pathway formation during transplant treatment and the like. A mouse with increased or defective function is effective for examining the role of Netrin-B1, and it is thought that useful information regarding candidate receptors can be obtained.
Further, the Netrin-B1 protein and fragment thereof, and the polynucleotide encoding this protein or fragment, being the subjects of the present invention, is a potentially useful medicaments in the treatment of patients required an increase in Netrin-B1 protein expression, particularly in cases such as where regeneration of neural circuit formation is required. On the other hand the antibody against Netrin-B1 protein and a fragment thereof, is a potentially useful medicament in the treatment of patients requiring a reduction in the expression of Netrin-B1 protein. Further, the primer and probe of the present invention, apart from the above-described analysis of function, are expected to be useful in the treatment of the above diseases, their diagnosis and in the production of medicaments.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: RIKEN
(ii) TITLE OF INVENTION: A MEMBRANE-BOUND NETRIDT
(iii) NUMBER OF SEQUENCES: 20 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: P.O.BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONTARIO
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE:
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,324,167 (B) FILING DATE: 20-NOV-2000 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: JP 2000-148843 (B) FILING DATE: 19-MAY-2000 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SMART & BIGGAR
(C) REFERENCE/DOCKET NUMBER: 72813-131 (ix) TELECOMMUNICATION INFORMATION
(A) TELEPHONE: (613)-232-2486 (B) TELEFAX: (613)-232-8440 (2) SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 (B) TYPE: nucleic acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid Synthetic DNA
(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

(2) SEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 (B) TYPE: nucleic acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid Synthetic DNA
(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

(2) SEQ ID NO: 3 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 (B) TYPE: nucleic acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid Synthetic DNA
(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

(2) SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: nucleic acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid Synthetic DNA
(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

(2) SEQ ID NO: 5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 (B) TYPE: nucleic acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid Syntheaic DNA
(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:

(2) SEQ ID NO: 6 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 (B) TYPE: nucleic acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid Synthetic DNA
(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 6:

(2) SEQ ID N0: 7 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4090 (B) TYPE: nucleic acid (ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 873..2492 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

a AGGATTATTG GATGTACAAC GGGAGAGCCG TCACTTTGCT AAAT'CATTAT CTGCTCCTGG 540 ACATCCCTGG ACATCTTTCA CAAAAGTCAA ATAGATATGT TCTAC:GAGGA GAAATGGCTG 600 TAGAAGACAC GAAGATTGCC AAGATTTTAG AG ATG TAT TTG 7.'CA AGA TTC CTG 893 Met Tyr Leu :3er Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val Ser Ser Val Met Gln Pro Tyr CTT TTC GTG TGG GGA CAT TAT GAT GTA TGT AAG AGC C;TG ATT TAC ACA 989 Leu Phe Val Trp Gly His Tyr Asp Val Cys Lys Ser Leu Ile Tyr Thr GAA GAA GGC AAA GTT TGG GAT TAC ACA GCC TGC CAG C;CG GAA TCC ACG 1037 Glu Glu Gly Lys Val Trp Asp Tyr Thr Ala Cys Gln Pro Glu Ser Thr GAC ATG ACC AAG TAT CTG AAA GTG AAA CTG GAC CCT C;CG GAT ATT ACC 1085 Asp Met Thr Lys Tyr Leu Lys Val Lys Leu Asp Pro Pro Asp Ile Thr TGT GGA GAC CCT CCA GAG TCC TTC TGT GCA ATG GGC F~AC CCT TAC ATG 1133 Cys Gly Asp Pro Pro Glu Ser Phe Cys Ala Met Gly F,sn Pro Tyr Met Cys Asn Asn Glu Cys Asp Ala Ser Thr Pro Glu Leu P,la His Pro Pro 90 95 1.00 GAG CTG ATG TTT GAT TTT GAA GGA AGA CAT CCC TCC A,CA TTT TGG CAG 1229 Glu Leu Met Phe Asp Phe Glu Gly Arg His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys Pro Leu Gln V'al Asn Ile Thr CTG TCT TGG AGC AAA ACC ATT GAA CTC ACA GAC AAC A.TA GTT ATT ACC 1325 Leu Ser Trp Ser Lys Thr Ile Glu Leu Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Pro Asp Gln Met Ile Leu Glu Lys Ser Leu Asp TAC GGA CGA ACA TGG CAG CCC TAT CAG TAT TAT GCC A.CA GAC TGC CTC 1421 Tyr Gly Arg Thr Trp Gln Pro Tyr Gln Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met Asp Pro Lys Ser Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu Glu Tyr Ser Thr Gly Tyr Ser ACG AAT AGC AAA ATA ATC CAC TTC GAG ATC AAA GAC F,GG TTT GCG TTT 1565 Thr Asn Ser Lys Ile Ile His Phe Glu Ile Lys Asp F,rg Phe Ala Phe Phe Ala Gly Pro Arg Leu Arg Asn Met Ala Ser Leu Tyr Gly Gln Leu GAT ACA ACC AAG AAA CTC AGA GAT TTC TTC ACT GTC P.CA GAC CTG AGG 1661 Asp Thr Thr Lys Lys Leu Arg Asp Phe Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly Glu Ile Phe V'al Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile Ser Asp Ile Lys Val Arg Gly AGG TGC AAG TGC AAC CTG CAT GCC ACT TCG TGT TTG T'AT GAC AAC AGC 1805 Arg Cys Lys Cys Asn Leu His Ala Thr Ser Cys Leu T'yr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Gly Thr Ala Asn Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Asn Cys Glu Cys Phe Gly His Ser Asn Arg Cys Ser Tyr Ile Asp Leu Leu Asn Thr Val Ile Cys Val Ser Cys Lys His Asn Thr Arg Gly Gln His Cys Glu Leu Cys Arg Leu Gly Tyr Phe Arg Asn Ala Ser Ala Gln Leu Asp Asp Glu Asn Val Cys Ile Glu Cys Tyr Cys Asn Pro Leu Gly Ser Ile His Asp Arg Cys Asn Gly Ser Gly Phe Cys Glu Cys Lys Thr Gly Thr Thr Gly Pro Lys Cys Asp Glu C-ys Leu Pro Gly Asn Ser Trp Tyr Tyr Gly Cys Gln Pro Asn val Cys Asp Asn Glu Leu CTG CAC TGC CAG AAT GGA GGG ACC TGC CAG AAC AAT C~TG CGC TGC GCG 2333 Leu His Cys Gln Asn Gly Gly Thr Cys Gln Asn Asn ~Tal Arg Cys Ala TGC CCA GAC GCC TAC ACC GGC ATC CTC TGT GAG AAG C:TA CGG TGC GAA 2381 Cys Pro Asp Ala Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg Cys Glu Glu Ala Gly Ser Cys Gly Ser Glu Ser Gly Gln Gly Ala Pro Pro Arg Gly Ser Pro Ala Leu Leu Leu Leu Thr Met Leu Leu Cily Thr Ala Gly CCC CTG GTG TTC TAG GGGTCACACC CAGCCCTCCG ACAGGCC',TGT GCTGTGGGGA 2532 Pro Leu Val Phe AGCAAACACA ACCCAAGCGA TTGCCACTGA CATAGAAAAC ACGCP,CACCC ACTCCAACAC 2592 AGTGTATAAA AGAAGAGGGC CTAACTGAAC TAAGCCATAT CTCTC'.AGAAC CGGACAGCAC 2652 ATCGCACATC GGAGTTGAGA CTGTTCATCA TTGACTCCAG AGGAA,TTGGC AGCTGTTGCT 2712 GAGCGCCCCC AAGAGGAAAG ACGGAAAACA AACTGATCAA CCAAC'.CTAAA AACATTCGCT 2832 CATTCTTCGC TGTCAGGTGC ATTGTGGGTA TAAGGAAATC TGTTp,CAAGC TGCCATATTG 2952 GCCTGCTTCA GTCCCCCCGA CCCCCAAATC CCTTCCAACC TGTGC'.TTTAG TGAACGTTGC 3012 CAGCCCCCTC CAAAGCGCAA GCCAGTCATA CCCCTGTATA TTTTP.GCAGC ACTGCGGTCC 3132 CAGTGCCAGC TCACGTCCAC TTCACAAGAG TGGTTAGAGG AAAAG'AGAAA GTGTATCTAT 3192 CCTTTTGTAT TCAAATGAAG TTATTTTTCT TGAAATAATG TAATA.TGTAG ATTTTTTGTA 3252 TTCTCTGTAA GGGCAACGAG CGTGCTGGCA TCAAAGAATA TCGGT'TTACA TATAGCAAGT 3432 GAACACAGTC AGCTGACAAC TTTAATAACC AGGAAGACGG ATTGA.TGGTC ACTAGCTTGG 3672 (2) SEQ ID NO: 8 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 539 (B) TYPE: amino acid (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 8:
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu T:rp Val Thr val Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val Cys LysSerLeu IleTyr ThrGluGlu GlyLysVal TrpAsp TyrThr Ala CysGlnPro GluSer ThrAspMet ThrLysTyr LeuLys ValLys 10Leu AspProPro AspIle ThrCysGly AspProPro CTluSer PheCys Ala MetGlyAsn ProTyr MetCysAsn AsnGluCys AspAla SerThr Pro GluLeuAla HisPro ProGluLeu MetPheAsp PheGlu GlyArg His ProSerThr PheTrp GlnSerAla ThrTrpLys CPluTyr ProLys 20 115 120 1.25 Pro LeuGlnVal AsnIle ThrLeuSer TrpSerLys ThrIle GluLeu Thr AspAsnIle ValIle ThrPheGlu SerGlyArg FroAsp GlnMet Ile LeuGluLys SerLeu AspTyrGly ArgThrTrp GlnPro TyrGln Tyr TyrAlaThr AspCys LeuHisAla PheHisMet AspPro LysSer Val LysAspLeu SerGln HisThrVal LeuGluIle IleCys ThrGlu Glu TyrSerThr GlyTyr SerThrAsn SerLysIle IleHis PheGlu 40Ile LysAspArg PheAla PhePheAla GlyProArg LeuArg AsnMet Ala SerLeuTyr GlyGln LeuAspThr ThrLysLys LeuArg AspPhe Phe ThrValThr AspLeu ArgIleArg LeuLeuArg ProAla ValGly Glu IlePheVal AspGlu LeuHisLeu AlaArgTyr PheTyr AlaIle Ser AspIleLys ValArg GlyArgCys LysCysAsn LeuHis AlaThr Ser CysLeuTyr AspAsn SerLysLeu ThrCysGlu CysGlu HisAsn Thr ThrGlyPro AspCys GlyLysCys LysLysAsn TyrGln GlyArg Pro TrpSerPro GlySer TyrLeuPro IleProLys GlyThr AlaAsn a Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Asn Cys C~lu Cys Phe Gly His Ser Asn Arg Cys Ser Tyr Ile Asp Leu Leu Asn Thr Val Ile Cys Val Ser Cys Lys His Asn Thr Arg Gly Gln His Cys Glu Leu Cys Arg Leu Gly Tyr Phe Arg Asn Ala Ser Ala Gln Leu Asp Asp Glu Asn Val Cys Ile Glu Cys Tyr Cys Asn Pro Leu Gly Ser Ile His Asp Arg Cys Asn Gly Ser Gly Phe Cys Glu Cys Lys Thr Gly Thr Thr Gly Pro Lys 435 440 9:45 Cys Asp Glu Cys Leu Pro Gly Asn Ser Trp Tyr Tyr Gly Cys Gln Pro Asn Val Cys Asp Asn Glu Leu Leu His Cys Gln Asn Gly Gly Thr Cys Gln Asn Asn Val Arg Cys Ala Cys Pro Asp Ala Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg Cys Glu Glu Ala Gly Ser Cys Gly Ser Glu Ser Gly Gln Gly Ala Pro Pro Arg Gly Ser Pro Ala Leu L~eu Leu Leu Thr Met Leu Leu Gly Thr Ala Gly Pro Leu Val Phe (2) SEQ ID NO: 9 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1452 (B) TYPE: nucleic acid (ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1452 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr s o GCC TGC CAG CCG GAA TCC ACG GAC ATG ACC AAG TAT C:TG AAA GTG AAA 192 Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Cilu Ser Phe Cys Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys Asp Ala Ser Thr Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp Phe Glu Gly Arg CAT CCC TCC ACA TTT TGG CAG TCT GCT ACT TGG AAG CiAG TAC CCC AAA 384 His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys C9lu Tyr Pro Lys 115 12 0 1.2 5 Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu ACA GAC AAC ATA GTT ATT ACC TTT GAA TCG GGG CGT C'.CA GAC CAA ATG 480 Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Faro Asp Gln Met ATC CTA GAG AAA TCT CTC GAC TAC GGA CGA ACA TGG C'.AG CCC TAT CAG 528 Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln TAT TAT GCC ACA GAC TGC CTC CAT GCA TTC CAC ATG G'~AC CCG AAA TCC 576 Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met P.sp Pro Lys Ser GTG AAG GAT TTA TCT CAG CAC ACG GTC TTG GAA ATC A.TT TGC ACG GAA 624 Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu GAG TAC TCC ACT GGG TAC TCC ACG AAT AGC AAA ATA A.TC CAC TTC GAG 672 Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg Leu Arg Asn Met Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys Leu Arg Asp Phe Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr TCG TGT TTG TAT GAC AAC AGC AAA CTG ACA TGT GAA 7.'GT GAG CAC AAC 960 Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu C:ys Glu His Asn ACT ACA GGT CCC GAC TGT GGG AAA TGC AAG AAG AAC 7.'AC CAG GGC CGA 1008 Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn 7:'yr Gln Gly Arg Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys CTly Thr Ala Asn Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Lys Cys Tyr Cys Asn Pro 355 360 3.65 Leu Gly Ser Ile His Asp Arg Cys Asn Gly Ser Gly Phe Cys Glu Cys AAG ACT GGA ACA ACA GGG CCT AAA TGT GAT GAG TGT C'TG CCA GGA AAT 1200 Lys Thr Gly Thr Thr Gly Pro Lys Cys Asp Glu Cys heu Pro Gly Asn TCC TGG TAC TAC GGC TGT CAA CCT AAT GTC TGC GAC P,AT GAG CTC CTG 1248 Ser Trp Tyr Tyr Gly Cys Gln Pro Asn Val Cys Asp P,sn Glu Leu Leu CAC TGC CAG AAT GGA GGG ACC TGC CAG AAC AAT GTG C'GC TGC GCG TGC 1296 His Cys Gln Asn Gly Gly Thr Cys Gln Asn Asn Val A.rg Cys Ala Cys CCA GAC GCC TAC ACC GGC ATC CTC TGT GAG AAG CTA C'GG TGC GAA GAG 1344 Pro Asp Ala Tyr Thr Gly Ile Leu Cys Glu Lys Leu A.rg Cys Glu Glu GCG GGC AGC TGT GGC TCC GAA TCC GGC CAG GGA GCA C'CC CCG CGG GGC 1392 Ala Gly Ser Cys Gly Ser Glu Ser Gly Gln Gly Ala Pro Pro Arg Gly TCC CCA GCA CTG CTG CTG CTG ACC ATG CTG CTG GGG A.CT GCC GGT CCC 1440 Ser Pro Ala Leu Leu Leu Leu Thr Met Leu Leu Gly Thr Ala Gly Pro Leu Val Phe (2) SEQ ID N0: 10 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 483 (B) TYPE: amino acid (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val a Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys P,sp Ala Ser Thr Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp F~he Glu Gly Arg His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Fro Asp Gln Met Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met A.sp Pro Lys Ser Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg Leu Arg Asn Met Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys Leu Arg Asp Phe Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Gly Thr Ala Asn Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Lys Cys Tyr Cys Asn Pro Leu Gly Ser Ile His Asp Arg Cys Asn Gly Ser Gly Phe Cys Glu Cys Lys Thr Gly Thr Thr Gly Pro Lys Cys Asp Glu Cys Leu Pro Gly Asn Ser Trp Tyr Tyr Gly Cys Gln Pro Asn Val Cys Asp Asn Glu Leu Leu His Cys Gln Asn Gly Gly Thr Cys Gln Asn Asn Val Arg Cys Ala Cys Pro Asp Ala Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg Cys Glu Glu 435 440 4:45 Ala Gly Ser Cys Gly Ser Glu Ser Gly Gln Gly Ala Fro Pro Arg Gly Ser Pro Ala Leu Leu Leu Leu Thr Met Leu Leu Gly Thr Ala Gly Pro Leu Val Phe (2) SEQ ID NO: 11 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1317 (B) TYPE: nucleic acid (ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1317 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys CTG GAC CCT CCG GAT ATT ACC TGT GGA GAC CCT CCA G.AG TCC TTC TGT 240 Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys GCA ATG GGC AAC CCT TAC ATG TGC AAT AAT GAG TGT G.AT GCG AGT ACC 288 Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys Asp Ala Ser Thr CCT GAA CTG GCA CAC CCT CCT GAG CTG ATG TTT GAT T'TT GAA GGA AGA 336 Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp P:he Glu Gly Arg CAT CCC TCC ACA TTT TGG CAG TCT GCT ACT TGG AAG CiAG TAC CCC AAA 384 His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys C~lu Tyr Pro Lys 115 12 0 1.2 5 Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu ACA GAC AAC ATA GTT ATT ACC TTT GAA TCG GGG CGT C'.CA GAC CAA ATG 480 Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Pro Asp Gln Met ATC CTA GAG AAA TCT CTC GAC TAC GGA CGA ACA TGG C'AG CCC TAT CAG 528 Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met P.sp Pro Lys Ser GTG AAG GAT TTA TCT CAG CAC ACG GTC TTG GAA ATC P.TT TGC ACG GAA 624 Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu GAG TAC TCC ACT GGG TAC TCC ACG AAT AGC AAA ATA A.TC CAC TTC GAG 672 Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu ATC AAA GAC AGG TTT GCG TTT TTC GCT GGA CCT CGG C'TA CGA AAT ATG 720 Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg Leu Arg Asn Met Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys Leu Arg Asp Phe Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Gly Thr Ala Asn Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Thr Asn Val Cys Asp Asn i Glu Leu Leu His Cys Gln Asn Gly Gly Thr Cys Gln F~sn Asn Val Arg Cys Ala Cys Pro Asp Ala Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg TGC GAA GAG GCG GGC AGC TGT GGC TCC GAA TCC GGC C'AG GGA GCA CCC 1248 Cys Glu Glu Ala Gly Ser Cys Gly Ser Glu Ser Gly Gln Gly Ala Pro CCG CGG GGC TCC CCA GCA CTG CTG CTG CTG ACC ATG C'.TG CTG GGG ACT 1296 Pro Arg Gly Ser Pro Ala Leu Leu Leu Leu Thr Met Lieu Leu Gly Thr Ala Gly Pro Leu Val Phe (2) SEQ ID NO: 12 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 438 (B) TYPE: amino acid (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 12:
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys Asp Ala Ser Thr 50 Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp Phe Glu Gly Arg His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys T:hr Ile Glu Leu Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg P:ro Asp Gln Met Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp G.ln Pro Tyr Gln a a Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met P,sp Pro Lys Ser Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile I:le Cys Thr Glu Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg L~eu Arg Asn Met Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys L~eu Arg Asp Phe Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Gly Thr Ala Asn Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Thr Asn Val Cys Asp Asn Glu Leu Leu His Cys Gln Asn Gly Gly Thr Cys Gln Asn Asn Val Arg Cys Ala Cys Pro Asp Ala Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg Cys Glu Glu Ala Gly Ser Cys Gly Ser Glu Ser Gly Gln Gly Ala Pro Pro Arg Gly Ser Pro Ala Leu Leu Leu Leu Thr Met Leu Leu Gly Thr Ala Gly Pro Leu Val Phe (2) SEQ ID NO: 13 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1443 (B) TYPE: nucleic acid (ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1443 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
ATG TAT TTG TCA AGA TTC CTG TCG ATC CAT GCC CTG T'GG GTG ACA GTG 48 Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val TCC TCT GTG ATG CAG CCC TAC CTT TTC GTG TGG GGA C'.AT TAT GAT GTA 96 Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly H:is Tyr Asp Val Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr GCC TGC CAG CCG GAA TCC ACG GAC ATG ACC AAG TAT C'TG AAA GTG AAA 192 Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr L~eu Lys Val Lys Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys A.sp Ala Ser Thr Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp Phe Glu Gly Arg His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Pro Asp Gln Met Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln TAT TAT GCC ACA GAC TGC CTC CAT GCA TTC CAC ATG G.AC CCG AAA TCC 576 Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met Asp Pro Lys Ser Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu GAG TAC TCC ACT GGG TAC TCC ACG AAT AGC AAA ATA A'TC CAC TTC GAG 672 Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu ATC AAA GAC AGG TTT GCG TTT TTC GCT GGA CCT CGG C'TA CGA AAT ATG 720 Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg L~eu Arg Asn Met GCT TCC CTC TAT GGA CAG CTG GAT ACA ACC AAG AAA C'TC AGA GAT TTC 768 Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys L.°u Arg Asp Phe TTC ACT GTC ACA GAC CTG AGG ATC AGG CTG TTG AGA C;CC GCC GTT GGG 816 Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile TCA GAC ATA AAG GTG CGA GGA AGG TGC AAG TGC AAC C".TG CAT GCC ACT 912 Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu C",ys Glu His Asn Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Lily Thr Ala Asn ACC TGT ATC CCC AGC ATT TCC AGT ATC GGT ACT CCT C'.CA AAG TTT AAT 1104 Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Thr Pro E~ro Lys Phe Asn AGG ATA TGG CCG AAT ATT TCT TCC CTT GAG GTT TCT p,AC CCA AAA CAA 1152 Arg Ile Trp Pro Asn Ile Ser Ser Leu Glu Val Ser Asn Pro Lys Gln Val Ala Pro Lys Leu Ala Leu Ser Thr Val Ser Ser v'al Gln Val Ala AAC CAC AAG CGA GCT AAT GTC TGC GAC AAT GAG CTC C'.TG CAC TGC CAG 1248 Asn His Lys Arg Ala Asn Val Cys Asp Asn Glu Leu L~eu His Cys Gln AAT GGA GGG ACC TGC CAG AAC AAT GTG CGC TGC GCG T'GC CCA GAC GCC 1296 Asn Gly Gly Thr Cys Gln Asn Asn Val Arg Cys Ala C'ys Pro Asp Ala TAC ACC GGC ATC CTC TGT GAG AAG CTA CGG TGC GAA G'~AG GCG GGC AGC 1344 Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg Cys Glu G'~lu Ala Gly Ser Cys Gly Ser Glu Ser Gly Gln Gly Ala Pro Pro Arg Gly Ser Pro Ala Leu Leu Leu Leu Thr Met Leu Leu Gly Thr Ala Gly Pro Leu Val Phe TAG

(2) SEQ ID NO: 14 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 480 (B) TYPE: amino acid (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: No {iv) ANTI-SENSE: No {vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus {xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys P,sp Ala Ser Thr Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp F~he Glu Gly Arg His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Fro Asp Gln Met Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met A.sp Pro Lys Ser Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg Leu Arg Asn Met Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys Leu Arg Asp Phe Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn n Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys C~ly Thr Ala Asn Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Thr Pro Pro Lys Phe Asn 355 360 _I65 Arg Ile Trp Pro Asn Ile Ser Ser Leu Glu Val Ser Asn Pro Lys Gln Val Ala Pro Lys Leu Ala Leu Ser Thr Val Ser Ser Val Gln Val Ala Asn His Lys Arg Ala Asn Val Cys Asp Asn Glu Leu Leu His Cys Gln Asn Gly Gly Thr Cys Gln Asn Asn Val Arg Cys Ala C;ys Pro Asp Ala Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg Cys Glu G~lu Ala Gly Ser 435 440 9:45 Cys Gly Ser Glu Ser Gly Gln Gly Ala Pro Pro Arg Gly Ser Pro Ala Leu Leu Leu Leu Thr Met Leu Leu Gly Thr Ala Gly Pro Leu Val Phe (2) SEQ ID N0: 15 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1383 (B) TYPE: nucleic acid (ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1383 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
ATG TAT TTG TCA AGA TTC CTG TCG ATC CAT GCC CTG T'GG GTG ACA GTG 48 Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu T'rp Val Thr Val TCC TCT GTG ATG CAG CCC TAC CTT TTC GTG TGG GGA C'AT TAT GAT GTA 96 Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly H:is Tyr Asp Val Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys m a n ° a Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys Asp Ala Ser Thr Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp F~he Glu Gly Arg His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys 115 12 0 1.2 5 CCT CTC CAG GTT AAC ATC ACT CTG TCT TGG AGC AAA P.CC ATT GAA CTC 432 Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu ACA GAC AAC ATA GTT ATT ACC TTT GAA TCG GGG CGT C'.CA GAC CAA ATG 480 Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Fro Asp Gln Met ATC CTA GAG AAA TCT CTC GAC TAC GGA CGA ACA TGG C'.AG CCC TAT CAG 528 Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met P.sp Pro Lys Ser GTG AAG GAT TTA TCT CAG CAC ACG GTC TTG GAA ATC P.TT TGC ACG GAA 624 Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu GAG TAC TCC ACT GGG TAC TCC ACG AAT AGC AAA ATA A.TC CAC TTC GAG 672 Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu ATC AAA GAC AGG TTT GCG TTT TTC GCT GGA CCT CGG C'.TA CGA AAT ATG 720 Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg L~eu Arg Asn Met GCT TCC CTC TAT GGA CAG CTG GAT ACA ACC AAG AAA C'TC AGA GAT TTC 768 Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys L~eu Arg Asp Phe TTC ACT GTC ACA GAC CTG AGG ATC AGG CTG TTG AGA C'CC GCC GTT GGG 816 Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile TCA GAC ATA AAG GTG CGA GGA AGG TGC AAG TGC AAC C'TG CAT GCC ACT 912 Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr TCG TGT TTG TAT GAC AAC AGC AAA CTG ACA TGT GAA T'GT GAG CAC AAC 960 Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg a i Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Gly Thr Ala Asn ACC TGT ATC CCC AGC ATT TCC AGT ATC GGT ACT CCT C'.CA AAG TTT AAT 1104 Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Thr Pro Faro Lys Phe Asn AGG ATA TGG CCG AAT ATT TCT TCC CTT GAG GTT TCT F,AC CCA AAA CAA 1152 Arg Ile Trp Pro Asn Ile Ser Ser Leu Glu Val Ser P.sn Pro Lys Gln GCT AAT GTC TGC GAC AAT GAG CTC CTG CAC TGC CAG P,AT GGA GGG ACC 1200 Ala Asn Val Cys Asp Asn Glu Leu Leu His Cys Gln P.sn Gly Gly Thr Cys Gln Asn Asn Val Arg Cys Ala Cys Pro Asp Ala T'yr Thr Gly Ile CTC TGT GAG AAG CTA CGG TGC GAA GAG GCG GGC AGC T'GT GGC TCC GAA 1296 Leu Cys Glu Lys Leu Arg Cys Glu Glu Ala Gly Ser C'ys Gly Ser Glu TCC GGC CAG GGA GCA CCC CCG CGG GGC TCC CCA GCA C'TG CTG CTG CTG 1344 Ser Gly Gln G1y Ala Pro Pro Arg Gly Ser Pro Ala L~eu Leu Leu Leu Thr Met Leu Leu Gly Thr Ala Gly Pro Leu Val Phe (2) SEQ ID N0: 16 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 460 (B) TYPE: amino acid (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu T'rp Val Thr Val Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys Asp Ala Ser Thr Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp Phe Glu Gly Arg a m ~ ~ a His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys 115 120 1.25 Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Fro Asp Gln Met Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met P,sp Pro Lys Ser Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg L~eu Arg Asn Met Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys Leu Arg Asp Phe Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Gly Thr Ala Asn Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Thr Pro Pro Lys Phe Asn Arg Ile Trp Pro Asn Ile Ser Ser Leu Glu Val Ser Asn Pro Lys Gln Ala Asn Val Cys Asp Asn Glu Leu Leu His Cys Gln Asn Gly Gly Thr Cys Gln Asn Asn Val Arg Cys Ala Cys Pro Asp Ala Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg Cys Glu Glu Ala Gly Ser Cys Gly Ser Glu Ser Gly Gln Gly Ala Pro Pro Arg Gly Ser Pro Ala Leu Leu Leu Leu a m ~ s Thr Met Leu Leu Gly Thr Ala Gly Pro Leu Val Phe (2) SEQ ID NO: 17 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1095 (B) TYPE: nucleic acid (ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1095 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 17:

Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val TCC TCT GTG ATG CAG CCC TAC CTT TTC GTG TGG GGA C'AT TAT GAT GTA 96 Ser Ser val Met Gln Pro Tyr Leu Phe Val Trp Gly H:is Tyr Asp Val Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val T'rp Asp Tyr Thr Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys Asp Ala Ser Thr Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp Phe Glu Gly Arg His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys Pro Leu Gln val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Pro Asp Gln Met ATC CTA GAG AAA TCT CTC GAC TAC GGA CGA ACA TGG C.AG CCC TAT CAG 528 Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln TAT TAT GCC ACA GAC TGC CTC CAT GCA TTC CAC ATG G;~1C CCG AAA TCC 576 Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met A;sp Pro Lys Ser m s Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg Leu Arg Asn Met Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys Leu Arg Asp Phe Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Gly Thr Ala Asn Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Ser Lys (2) SEQ ID NO: 18 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 364 (B) TYPE: amino acid (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr m Ala CysGlnPro GluSer ThrAspMet ThrLysTyr LeuLysVal Lys Leu AspProPro AspIle ThrCysGly AspProPro GluSerPhe Cys Ala MetGlyAsn ProTyr MetCysAsn AsnGluCys AspAlaSer Thr Pro GluLeuAla HisPro ProGluLeu MetPheAsp PheGluGly Arg His ProSerThr PheTrp GlnSerAla ThrTrpLys GluTyrPro Lys Pro LeuGlnVal AsnIle ThrLeuSer TrpSerLys ThrIleGlu Leu Thr AspAsnIle ValIle ThrPheGlu SerGlyArg ProAspGln Met Ile LeuGluLys SerLeu AspTyrGly ArgThrTrp GlnProTyr Gln Tyr TyrAlaThr AspCys LeuHisAla PheHisMet A.spProLys Ser Val LysAspLeu SerGln HisThrVal LeuGluIle IleCysThr Glu Glu TyrSerThr GlyTyr SerThrAsn SerLysIle IleHisPhe Glu Ile LysAspArg PheAla PhePheAla GlyProArg LeuArgAsn Met Ala SerLeuTyr GlyGln LeuAspThr ThrLysLys LeuArgAsp Phe Phe ThrValThr AspLeu ArgIleArg LeuLeuArg ProAlaVal Gly Glu IlePheVal AspGlu LeuHisLeu AlaArgTyr PheTyrAla Ile Ser AspIleLys ValArg GlyArgCys LysCysAsn LeuHisAla Thr Ser CysLeuTyr AspAsn SerLysLeu ThrCysGlu CysGluHis Asn Thr ThrGlyPro AspCys GlyLysCys LysLysAsn TyrGlnGly Arg Pro TrpSerPro GlySer TyrLeuPro IleProLys GlyThrAla Asn Thr CysIlePro SerIle SerSerIle GlySerLys (2) SEQ ID NO: 19 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 I'.

~e; a (B) TYPE: nucleic acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid Synthetic DNA
(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 19:

(2) SEQ ID NO: 20 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid Synthetic DNA
(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 20:

Claims

1. A membrane-bound Netrin having a hydrophobic region at its C-terminus, wherein said hydrophobic region is able to bind to the cell membrane by means of glycosylphospha-tidylinositol (GPI).

2. The membrane-bound Netrin according to claim 1 which comprises an amino acid sequence indicated by SEQ ID NO:
8, 10, 12, 14 or 16.

3. A polynucleotide encoding the membrane-bound Netrin according to claim 1 or 2.

4. The polynucleotide according to claim 3 comprising the nucleotide sequence indicated by SEQ ID NO: 7, 9, 11, 23 or 15.

5. A protein or fragment thereof, according to either (a) or (b) below:
(a) A protein comprising an amino acid sequence indicated by SEQ ID NO: 8, 10, 12, 14, 16 or 18; or, a fragment of said protein.
(b) A protein comprising an amino acid sequence derived from an amino acid sequence indicated by SEQ ID NO:
8, 10, 12, 14, 16 or 18, by deletion, substitution or insertion of one or several amino acids, or a fragment of said protein, wherein said protein or fragment has Netrin function.

6. A polynucleotide according to either (a) or (b) below, or a fragment thereof:

(a) A polynucleotide comprising a nucleotide sequence indicated by SEQ ID NO: 7, 9, 11, 13, 15 or 17; or, a fragment of said polynucleotide.
(b) A polynucleotide comprising a nucleotide sequence derived from the nucleotide sequence indicated by SEQ ID NO: 7, 9, 11, 13, 15, or 17, by deletion, substitution or insertion of one or several nucleotides, or a fragment of said polynucleotide, encoding a protein having Netrin function.

7. An expression vector comprising the polynucleotide according to any one of claims 3, 4 and 6, or a fragment of said polynucleotide.

8. A host cell transfected with the expression vector according to claim 7.

9. A method for producing the membrane-bound Netrin according to claim 1 or 2, or the protein or fragment thereof according to claim 5 comprising culturing the host cell according to claim 8.

10.A primer used in the amplification of the membrane-bound Netrin according to claim 1 or 2, or the protein or fragment thereof, according to claim 5.

11.The primer according to claim 10, which corresponds to the nucleotides encoding amino acids 353 to 359, or amino acids 520 to 526 in the amino acid sequence indicated by SEQ ID NO: 8, or a portion of said nucleotides.

12.The primer according to claim 10 or 11, wherein said primer comprises the sequences indicated by SEQ ID NOS:
19 or 20.

13.A probe specific to the membrane-bound Netrin isoform according to any one of claim 1 or 2 or the protein, or fragment thereof, according to claim 5.

14.The probe according to claim 13 having a sequence corresponding to nucleotides 1959 to 2261 of the nucleotide sequence indicated by SEQ ID NO: 7, nucleotides 1959 to 2084 of the nucleotide sequence indicated by SEQ ID NO: 13, or nucleotides 2262-2403 of the nucleotide sequence indicated bay SEQ ID NO: 7.

15.An antibody having specificity against the membrane-bound Netrin according to claim 1 or 2 or the protein, or fragment thereof, according to claim 5.

16.The antibody according to claim 15 having specificity against specific isoforms of the membrane-bound Netrin according to claim 1 or 2.

17.A transgenic animal into which is introduced a gene encoding the membrane-bound Netrin according to claim 1 or 2 or the protein according to claim 5.

18.A homozygotic or heterozygotic knock-out animal in which a gene encoding the membrane-bound Netrin according to claim 1 or 2 or the protein according to claim 5, has been disrupted.