CA2174025A1 - Agents modulating the response of neuronal cells to inhibition by mammalian central nervous system myelin - Google Patents

Abstract

A method of assaying for a substance which modulates the response of neuronal cells to inhibition by adult central nervous system myelin. Neuronal cells which have a propensity for neurite growth are grown on mammalian central nervous system (CNS) myelin in the presence of a test substance which is suspected of affecting neurite outgrowth. The invention also relates to isolated nucleic acid molecules encoding a novel protein which plays a role in neurite outgrowth. The invention provides for various uses of the nucleic acid molecule and its protein product.

Description

Title: NOVEL AGENTS MODULATING THE RESPONSE OF NEURONAL
CELLS TO INHIBITION BY MAMMALIAN CENTRAL NERVOUS SYSTEM
MYELIN
FIELD OF THE INVENTION
The present invention relates generally to a novel protein, nucleic acid molecule encoding the protein, a novel hybridoma cell line, and particularly to a hybridoma cell line producing monoclonal antibodies against neuronal cell membranes. Also provided are methods for using the protein, nucleic acid molecule, and monoclonal antibodies; methods for identifying substances which modulate the response of neuronal cells to inhibition by mammalian central nervous ~y~lem myelin; and methods for assaying for neurite growth inhibitory activity.
BACKGROUND OF THE INVENTION
A remarkable feature of axons in the peripheral nerves of adult mammals is that after interruption, they are able to regenerate through the distal nerve stump to reconnect with their targets and re-establish function. The same is not true however in the central nervous ~y~lem (CNS).
Axons injured in the brain, optic nerve or spinal cord of adult mammals do not successfully regrow. This leads to an irreversible disruption of neuronal circuits and permanent neurologic disability. The difference in the regenerative abilities of axons in the CNS and peripheral nervous ~ysl~
(PNS) has puzzled clinicians and neuroscientists and challenged them to explore the molecular basis of this phenomenon.
An increasing number of molecules regulating the growth of neuronal processes are being identified. Until recently, only environmental molecular cues exerting positive effects on neuronal growth were known. These positive signals include growth factors such as nerve growth factor, and brain derived neurotrophic factor (Barde YA., [Review]
Neuron 2: 1525-1534, 1989; and Dechant et al. J. Journal of Neuroscience 13:
2610-2616, 1993), extracellular matrix components such as collagen, laminin and fibronectin (reviewed in Goodman and Schatz, 1993, Neuron 10 (Suppl.):77-98) and cell surface molecules such as NCAM (Goodman and Schatz, 1993, supra).
Of considerable interest recently, is the finding that the nervous ~ysl~ also contains molecules which function to inhibit or restrict axonal growth. The inhibitory molecules share the property of causing 5 growth cone collapse. The growth cone is a specialized structure at the distal tip of the advancing neurite and it is primarily responsible for the transduction of environmental signals which modulate growth cone advancement and axonal extension. Some of these molecules are membrane associated glycoproteins expressed early in development. Posterior 10 sclerotomes for example, contain proteins that cause the collapse of chick dorsal root ganglion (DRG) neuron growth cones (Davies et al., 1990, Neuron 4, 11-20). The posterior tectum of chicks has a 33 kDa phosphoglycerol inositide linked protein that causes the collapse of temporal but not nasal retinal ganglion cell (RGC) growth cones (Stahl et al., 1990, Neuron 5, 735-743). A 88 kDa molecule from chick brain, collapsin, causes the collapse of dorsal root ganglion and retinal ganglion cell growth cones (Luo et al., 1993, Cell 75:217-227). Other molecules including tenascin (Lochter et al., 1991, J.
Cell. Biol. 113, 1159-1171) and janusin (Pesheva et al., 1989, J.Cell Biol 109, 1765-1778) found in the extracellular matrix, may be anti-adhesive and play a 20 role in neurite guidance. The function of the inhibitory molecules during development of the nervous ~y~lell~ may be to focus and restrict axonal outgrowth along specific neural projections and towards appropriate synaptic targets.
The adult mammalian CNS also contains inhibitory 25 molecules which may be responsible for the lack of successful axonal regeneration after injury. Two fractions of non-neuronal origin have been identified in adult mammalian central nervous system myelin which inhibit neurite outgrowth. These fractions designated NI-35 and NI-250 (neurite inhibitor; NI) are found in the myelin of the CNS but not that of peripheral 30 nerves (Caroni and Schwabb, J. Cell Biol. 106:1281-1, 1988). The fractions are absent in periods of embryonic CNS axonal outgrowth but are produced by oligodendrocytes immediately before myelination of established axonal projections (Caroni and Schwabb, J. Cell Biol. 106:1281-1, 1988).
Downregulation of these molecules by the irradiation induced arrest of oligodendrocyte production in fetal rodents is associated with an increased propensity for axonal regeneration on the CNS (Savio and Schwab, PNAS
87:4130-4133, 1990). Further, neutralizing the activity of NI-35 or NI-250 usingantibodies transforms the CNS environment from one that disallows to one that is permissive for axonal growth (Caroni and Schwab, J. Cell Biol.
106:1281-8, 1988). It has more recently been shown that in the presence of anti-NI-35 and NI-250 antibodies, a subset of axons interrupted in the spinal cord of adult rats were able to regenerate over long distances (Schnell and Schwab, 1990, Nature 343 (6255)269-72). Further, co-application of myelin neutralizing antibodies with local administration of the neurotrophin NT-3, resulted in even greater enhancement of adult rat spinal cord axonal regrowth (Schell et al., Nature 367:269-272, 1994).
The mechanism through which the adult CNS myelin proteins block neurite outgrowth is unknown. When applied to the tips of dorsal root ganglion (DRG) neuronal growth cones in culture, NI-35 produces rapid and dramatic growth cone collapse (Bandtlow et al., 1993, Science 259:80-83 and Igarashi et al., 1993, Science 259:77-79 ). The inhibitory effects are seen at low concentrations suggesting that signal amplification may be required.
From recent reports there are indications that the growth cone collapse induced by inhibitory molecules from adult rat CNS myelin may be preceded by a rise in intracellular Ca++ levels (Bandtlow et al., 1993, Science 259:80-83) and be dependent upon a G protein pathway (Igarashi et al., 1993, Science 259:77-79). The NI-35 induced collapse of DRG growth cones can be mimicked by mastoparan, a G protein activator and blocked by the G
protein blocker, pertussis toxin. Further, growth cone collapse with the myelin derived inhibitors is specifically blocked by monoclonal antibodies to NI-35 (Bandtlow et al., 1993, Science 259:80-83 and Igarashi et al., 1993, Science 259:77-79)). Thus, CNS myelin-associated growth inhibiting molecules appear to act by triggering an active biochemical response in neurons which may be transduced by binding to a receptor on the neuronal surface.
SUMMARY OF THE INVENTION
A component of adult mammalian central nervous ~ysle (CNS) myelin causes collapse of neuronal growth cones and inhibits axonal growth. This activity may be responsible for the lack of regrowth of axons interrupted in the CNS. The same activity inhibits spreading of a fibroblast cell line (Caroni and Schnell, supra 1988), suggesting that its effects may not be restricted to neural cells. The present inventors developed an in vitro neurite growth inhibition assay which resembles the inhibitory effect of CNS
myelin on neurite growth in vivo.
The inventors used their neurite growth inhibition assay to screen a panel of monoclonal antibodies raised against rat neuronal membrane proteins, for clones capable of blocking the inhibitory response.
One monoclonal antibody, having the laboratory designation 10D, was found to neutralize the inhibition of neurite growth by several neuronal types on CNS myelin substrates. 10D monoclonal antibody when applied to Western Blots recognizes most prominently bands of Mr 35,000 and 33,000 expressed in neuronal and fibroblast cell lines, and in rat brain and liver. Proteins recognized by 10D monoclonal antibody play a role in the interaction between cells and their growth substrates, and are novel candidates for cellular receptors for myelin inhibitors.
The present inventors also screened a cDNA expression library derived from adult rat brain mRNA using the 10D monoclonal antibody. Resulting clones were tested for their ability to modulate neurite growth on an inhibitory CNS myelin substrate when expressed as antisense transcripts in a neuronal cell line. Transfectants containing antisense constructs derived from the clone having the laboratory designation "D1", showed significant enhancement of neurite growth on myelin. Sequence analysis of the partial D1 cDNA clone indicated that it is a previously unreported gene. Probes derived from sequences in the partial cDNA clone were used to screen a cDNA library, and a gene designated "petrin" encoding a protein involved in modulating neurite growth inhibition was identified.
The petrin gene encodes a 60 to 64 kDa protein which is a 5 new member of the protein phosphatase 2C family ("PP2C"). The novel protein has been designated "Petrin". The human petrin locus was localized to chromosome 12. The present inventors have also shown by in situ hybridization that the petrin gene is expressed in neurons in brain tissue, and in particular, in the Purkinje cells of the cerebellum; in the 3rd and 4th layers 10 of the cerebral cortex; and, dispersed neurons in the hippocampus. RNA blot analysis showed that in the rat brain expression was first detectable at embryonic day 13, and increased to a maximum level in the adult brain.
Northern and DNA analysis also showed that the protein is present in different mammalian species such as mouse, rat, hamster, and human.
The biological function of Petrin was investigated using phosphatase assays on immunoprecipitated material, and it was found that Petrin has serine/threonine phosphatase activity and tyrosine phosphatase activity both of which are magnesium dependent. Phosphatase activity was also shown to be highest while NG108 cells are proliferating and growing 20 neurites and was not detected in late growth stages. Serine/threonine-phosphatase and tyrosine phosphatase activities were inhibited by okadaic acid or ortho-vanadate, respectively.
The present inventors also prepared antisense oligonucleotides to petrin and found that they enhanced neurite growth in a 25 functional in vitro assay.
Therefore, the present invention contemplates a method of assaying for a substance which modulates the response of neuronal cells to inhibition by adult central nervous system myelin comprising growing neuronal cells which have a propensity for neurite outgrowth on 30 maInmalian central nervous ~y~lelll (CNS) myelin in the presence of a test substance which is suspected of affecting neurite outgrowth, and assaying for neurite outgrowth.
In accordance with a further aspect of the invention, hybridoma cell lines are provided which produce monoclonal antibodies which (a) immunoreact with neuronal membrane proteins; (b) neutralize the 5 inhibition of neurite growth by mammalian central nervous ~y~Lell- myelin;
and, (c) recognize bands of Mr 35,000 and Mr 33,000 expressed in neuronal and fibroblast cell lines and in rat cerebrum and rat liver. Prere,led hybridoma cell lines are those having the laboratory designation D10. The monoclonal antibodies produced, and the antigens recognized by this cell line are also a 10 part of the present invention. Accordingly, the present invention also contemplates a monoclonal antibody which (a) immunoreacts with neuronal membrane proteins; (b) neutralizes the inhibition of neurite growth by mammalian central nervous system myelin; and, (c) recognizes bands of Mr 35,000 and Mr 33,000 expressed in neuronal and fibroblast cell lines and in rat 15 cerebrum and rat liver.
The invention also provides a method for assaying for the presence of an activator or inhibitor of a monoclonal antibody produced by the hybridoma cell line of the invention comprising growing neuronal cells which have a propensity for neurite growth on mammalian central nervous 20 system (CNS) myelin in the presence of a known concentration of the monoclonal antibody, and in the presence of a suspected activator or inhibitor of the monoclonal antibody, under conditions which permit neurite outgrowth, and assaying for neurite outgrowth.
Another aspect of the invention relates to an isolated nucleic 25 acid molecule which is present in neuronal cells; its expression is required for neurite growth inhibition by mammalian central nervous system myelin;
and it comprises the nucleic acid sequences shown in the Sequence Listing as SEQ. ID. No. 1, SEQ. ID. No. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, SEQ. ID. NO. 6, SEQ. ID. NO. 7 and SEQ. ID. NO. 8 or as shown in Figures 9 and 11 to 14, and 30 21.
In an embodiment of the invention, the isolated and purified nucleic acid molecule comprises (a) a nucleic acid sequence as shown in SEQ.ID NO:l, SEQ.
.NO:3, SEQ.ID. NO:4, SEQ.ID. NO.5, SEQ.ID. NO.6, SEQ.ID. NO.7 and/or SEQ.ID. NO.8, or in Figures 9, 11 to 14, and 21 wherein T can also be U;
5(b) nucleic acid sequences complementary to (a);
(c) nucleic acid sequences having at least 80-90% identity, ~rere- dbly 90% identity with SEQ.ID NO:l, SEQ.ID. NO:3, SEQ.ID. NO:4, SEQ.
ID. NO.S, SEQ.ID. NO.6, SEQ.ID. NO.7 and SEQ.ID. NO.8;
(d) a fragment of the nucleic acid molecule that is at least 15 10bases and that will hybridize to (a) or (b) under stringent hybridization conditions, or (e) a nucleic acid molecule differing from any of the nucleic acids of (a) to (d) in codon sequences due to the degeneracy of the genetic code.
The invention also relates to a nucleic acid molecule 15comprising (a) a nucleic acid sequence encoding a protein having the amino acid sequence as shown in Figure 24 (or SEQ.ID. NO.12);
(b) nucleic acid sequences complementary to (a);
(c) nucleic acid sequences which are at least 80%, preferably 2090% identical to (a); or, (d) a fragment of (a) or (b) that is at least 15 bases and which will hybridize to (a) or (b) under stringent hybridization conditions.
Preferably, the isolated and purified nucleic acid molecule comprises 25(a) a nucleic acid sequence as shown in Figure 23 (or SEQ.ID.
NO. 11), pre~erably from about nucleotides 486 to 1977 as shown in Figure 23 (or SEQ ID NO:ll), wherein T can also be U;
(b) nucleic acid sequences complementary to (a);
(c) nucleic acid sequences which are at least 80-90% identical, 3 0~refelably 90% identical to (a); or, (d) a fragment of (a) or (b) that is at least 15 bases and which -will hybridize to (a) or (b) under stringent hybridization conditions.
A nucleic acid molecule of the invention, or fragments thereof may be inserted into an appropriate expression vector, i.e. a vector which contains the necessary elements for the transcription and translation of 5 the inserted protein-coding sequence. Accordingly, recombinant molecules adapted for transformation of a host cell may be constructed which comprise a nucleic acid molecule of the invention and one or more transcription and translation elements operatively linked to the nucleic acid molecule.
The recombinant molecule can be used to prepare 10 transformed host cells expressing the protein or part thereof encoded by a nucleic acid molecule of the invention or a fragment thereof. Therefore, the invention provides host cells containing a recombinant molecule of the invention. The invention also contemplates transgenic non-human mammal~ whose germ cells and somatic cells contain a recombinant 15 molecule of the invention.
The invention further provides a method for preparing a protein encoded by the nucleic acid molecule of the invention or parts thereof utilizing the isolated and purified nucleic acid molecules of the invention. In an embodiment of the invention, a method for preparing a Petrin protein is 20 provided comprising (a) transferring a recombinant expression vector of the invention into a host cell; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected transformed host cell under conditions which allow expression of Petrin; and (d) isolating Petrin.
The present invention also includes a protein encoded by a 25 nucleic acid molecule of the present invention. Proteins comprise the amino acid sequence as shown in the Sequence Listing as SEQ. ID. Nos. 2 and 10, and as shown in Figure 10 and the amino acid sequence as shown in the Sequence Listing as SEQ. ID. NO. 9; and sequences having at least 80-90% identity, preferably 90% identity thereto. The protein of the invention may be found 3 o in brain, NG108, and PC12 cells.
In an embodiment of the invention a purified Petrin protein -g is provided which has the amino acid sequence as shown in Figure 24 or SEQ
ID NO:12. Proteins of the invention include truncations of the purified Petrin protein and analogs, homologs, and isoforms of the protein and truncations thereof.
The proteins of the invention may be conjugated with other molecules, such as proteins to prepare fusion proteins. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins.
The invention also permits the construction of nucleotide probes which are unique to nucleic acid molecules of the invention and accordingly to a protein of the invention, or part of a protein of the invention. Thus, the invention also relates to a probe comprising a nucleic acid molecule of the invention or a fragment thereof. The probe may be labelled, for example, with a detectable substance and it may be used to select from a mixture of nucleotide sequences a nucleotide sequence coding for a protein which displays the properties of the protein of the invention, or a partthereof.
The invention further contemplates antibodies having specificity against an epitope of a protein of the invention, or part of the protein which is unique to the protein. Antibodies may be labelled with a detectable substance and they may be used to detect the protein of the invention in tissues and cells.
The invention provides a method for assaying for the presence of an activator or inhibitor of a protein of the invention comprising growing neuronal cells which have a propensity for neurite growth in the presence of a protein of the invention, and a suspected activator or inhibitor substance, and assaying for neurite outgrowth.
The invention also provides a method for assaying for the presence of an activator or inhibitor of a protein of the invention comprising 3 o growing neuronal cells which have a propensity for neurite growth on mammalian central nervous ~ysLem (CNS) myelin and which express a protein of the invention in the presence of a suspected activator or inhibitor substance, and assaying for neurite outgrowth.
Substances which affect cell neurite growth may also be identified by comparing the pattern and level of expression of the novel nucleic acid molecule and/or novel protein of the invention, in tissues and cells in the presence and in the absence of a test substance.
The invention also contemplates a method for assaying for a substance that affects neuronal growth comprising administering to a non-human animal or to a tissue of an animal, a substance suspected of affecting neuronal growth, and detecting, and optionally quantitating, the nucleic acid molecule and/or novel protein of the invention in the non-human animal or tissue.
The invention also contemplates a method for identifying a substance which is capable of binding to a protein of the invention, or a part of the protein, comprising reacting the protein, or part of the protein, with atleast one substance which potentially can bind with the protein, or part of the protein, under conditions which permit the formation of substance-protein complexes, and assaying for substance-protein complexes, and/or for free substance, for non-complexed protein.
Still further, the invention provides a method for assaying a medium for the presence of an activator or inhibitor of the interaction of the protein of the invention or part thereof, and a substance which binds to the protein. In an embodiment, the method comprises providing a known concentration of a protein of the invention, or part of the protein, incubating the protein, or part of the protein with a substance which binds to the protein,or part of the protein, and a suspected activator or inhibitor substance, under conditions which permit the the formation of substance-protein complexes, and assaying for substance-protein complexes.
The invention contemplates a method for assaying for a substance that affects the phosphatase activity of a protein of the invention comprising reacting a protein of the invention with a substrate which is capable of being dephosphorylated by the protein to produce a dephosphorylated product, in the presence of a substance which is suspected of affecting the phosphatase activity of the protein, under conditions which permit dephosphorylation of the substrate, assaying for dephosphorylated 5 product, and comparing to product obtained in the absence of the substance to determine the affect of the substance on the phosphatase activity of the protein.
The invention also contemplates pharmaceutical compositions and methods of using (a) the monoclonal antibody produced by 10 the hybridoma cell line of the invention; (b) inhibitors and activators of the monoclonal antibody produced by the hybridoma cell line of the invention;
(c) inhibitors and activators of the expression of a nucleic acid molecule of the invention; (d) inhibitors and activators of the activity of a protein of the invention; and (e) substances identified using the methods of the invention.
15 The present invention also has diagnostic applications.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating ~refelred embodiments of the invention are given 20 by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
DESCRIPTION OF THE DRAWINGS
The invention will be better understood with reference to 25 the drawings in which:
Figure lA shows photomicrographs of representative fields of cultures of dibutyryl cyclic AMP induced NG108 cells plated onto tissue culture plastic coated with poly-L-lysine alone (a), poly-L-lysine followed by 201lg/cm2 bovine serum albumin (BSA) (b), poly-L-lysine followed by 30 20llg/cm2 CNS myelin (c), and (d) shows a single dbCAMPNG108 cell growing on a myelin-free patch;

-Figure lB is a graph showing the proportion of dbCAMPNG108 cells with a process greater than 1 cell diameter after plating on wells coated with bovine serum albumin or extracts from muscle, sciatic nerve and brain;
Figure 2 is a graph showing process bearing dbCAMPNG108 5 cells determined at 24 hrs after plating onto different densities of CNS myelin on poly-L-lysine coated wells;
Figure 3A shows photomicrographs of dbCAMPNG108 cells grown on poly-L-lysine alone or 10 ~lg/cm2 of CNS myelin showing that lOD
antibody reverses the growth inhibitory effect of CNS myelin;
lo Figure 3B is a graph showing quantitation of process-bearing cells grown on CNS myelin for 24 or 72 hours with 5~1 per well of control ascites (filled bars) or lOD ascites (open bars);
Figure 4A is a photomicrograph of cells grown on poly-L-lysine coated glass slides for 48 hours, fixed and processed for immunocytochemistry with control ascites diluted 1:1000;
Figure 4B is a photomicrograph of cells grown on poly-L-lysine coated glass slides for 48 hours, fixed and processed for immunocytochemistry with lOD ascites diluted 1:1000;
Figure 5 is a photomicrograph showing two identical denaturing 13% polyacrylamide-SDS gels loaded with marker proteins and 10 ~g of protein from liver, cerebrum (both from 2 day old rats), dbCAMPNG108 cells and adult CNS myelin and stained for total proteins with Coomassie Brilliant Blue (left), and one transferred to nitrocellulose and reacted with the lOD monoclonal antibody (right);
Figure 6 is a schematic representing the strategy used to characterize clones selected with the lOD monoclonal antibody;
Figure 7 is a graph showing the number of A3 antisense transformant cells and NG108 parental cells which grew processes on PLL, and myelin with and without the lOD antibody;
Figure 8 shows a Southern blot of EcoRI digested genomic DNA from NG108 cells and the transformed cell line A3 probed with the lkb Dl cDNA insert;
Figure 9 shows the nucleotide sequence of a fragment of the cDNA clone Dl which is designated DlT7;
Figure 10 shows the amino acid sequence of a portion of the 5 protein encoded by the nucleic acid molecule of the invention;
Figure 11 shows the nucleotide sequence of a fragment of the cDNA clone Dl which is designated DlT3;
Figure 12 shows the nucleotide sequence of a fragment of the cDNA clone which is designated ML07T3;
Figure 13 shows the nucleotide sequence of a fragment of the cDNA clone which is designated S4T3;
Figure 14 shows the nucleotide sequence of a fragment of the cDNA clone which is designated S5T7;
Figure 15 is a schematic diagram showing the positions of the sequenced fragments of the Dl cDNA clone;
Figure 16 are photographs showing a control (A) and (B) the neutralization of the neurite growth inhibitory effects of myelin on newborn rat superior cervical ganglion primary neurons by lOD ascites;
Figure 17 is an immunoblot showing that the Dl cDNA recognises corresponding human sequences and localizes them to chromosome 12;
Figure 18 is a blot showing that the Dl cDNA recognises corresponding human sequences and localizes them to chromosome 12;
Figure 19 is a blot showing that the Dl cDNA recognizes human RNA transcripts;
Figure 20 is a graph showing % of NG-108-15 cells with neurite extension versus myelin concentration (,ug/cm2);
Figure 21 shows a nucleotide sequence of a fragment of the Dl cDNA clone;
Figure 22 is a schematic diagram having the sequenced regions of the Dl cDNA;

Figure 23 shows the nucleotide sequence of a Petrin protein of the invention; and Figure 24 shows the amino acid sequence of a Petrin protein of the invention and the amino acid sequences of other members of the 5 protein phosphatase 2C family.
DETAILED DESCRIPTION OF THE INVENTION
The following standard abbreviations for the amino acid residues are used throughout the specification: A, Ala - alanine; C, Cys -cysteine; D, Asp- aspartic acid; E, Glu - glutamic acid; F, Phe - phenylalanine;10 G, Gly - glycine; H, His - histidine; I, Ile - isoleucine; K, Lys - lysine; L, Leu -leucine; M, Met - methionine; N, Asn - asparagine; P, Pro - proline; Q, Gln -glutamine; R, Arg - arginine; S, Ser - serine; T, Thr - threonine; V, Val -valine; W, Trp- tryptophan; Y, Tyr - tyrosine; and p.Y., P.Tyr -phosphotyrosine .
For ease of explanation, the description of the invention is divided into the following sections: (A) assay for neurite growth inhibition by CNS myelin (B) hybridoma cell lines and monoclonal antibodies; (C) novel nucleic acid molecule and novel protein; and (D) applications for which the hybridoma cell lines, monoclonal antibodies, nucleic acid molecules, protein, 2 o and the substances identified using the methods described herein are suited. A. ASSAY FOR NEURITE GROWTH INHIBITION
As discussed herein, the present inventors have developed an in vitro method where the limited neurite outgrowth on CNS myelin in vitro resembles the limited axonal outgrowth in the CNS in vivo. The 25 method may be used to assay for a substance which modulates the response of neuronal cells to inhibition by adult central nervous system myelin. The method involves preparing neuronal cells which have a propensity for neurite growth, growing the neuronal cells on mammalian central nervous system (CNS) myelin in the presence of a test substance which is suspected of 30 affecting neurite growth, and assaying for neurite growth.
Neuronal cells which have a propensity for neurite growth which may be used in the method of the invention include the NG108-15 rat neuroblastoma and glioma hybrid cell line induced with dibutyryl cyclic AMP
and fetal calf serum, preferably cells treated with 0.5 to 1 mM, preferably lmM
of dbcAMP, and 5% fetal calf serum, for 1 to 7 days, preferably 2 days. Other neuronal cells which may be used in the method of the invention include PC12 cells which have been induced to grow neurites by induction for 5-7 days with 100 ng/ml NGF (Green), cerebellar neurons (Trenkner, E. in Culturing Nerve Cells, Banker, G., and Goslin, K. (eds) (Cambridge, USA: MIT Press) 1991), and cortical neurons (Baughman et al." in Culturing Nerve cells, Banker, G. and Goslin, K.(eds) (Cambridge, USA: MIT Press) 1991).
"Mammalian CNS myelin" refers to extracts of mammalian central nervous ~y~ myelin containing myelin basic protein and myelin associated glycoprotein. In a preferred embodiment, the mammalian CNS
myelin is a preparation enriched approximatley four fold for the myelin-specific markers, myelin basic protein and myelin associated glycoprotein. This preparation may be obtained from adult rat brains following standard procedures for myelin isolation as described in Norton and Poduslo, J. Neurochem 21:749-758, 1973. The amount of myelin basic protein and myelin associated glycoprotein may be determined by standard Western blotting techniques (Li et al., Nature 369:747-750, 1994). The myelin may be obtained from any mammals, preferably humans, bovines and rats, and ~refeLdbly adult mammals.
In a preferred embodiment, the assay uses human brain-derived myelin as a substrate. The inventors have found that powerful neurite outgrowth inhibitory activity is present in human CNS myelin.
Human CNS myelin strongly inhibits neuritic outgrowth from newborn rat dorsal root ganglion neurons and NG-108-15 cells. The inhibitory activity in human CNS myelin closely resembles the myelin inhibition of neurite growth that is observed with adult rodent CNS myelin. The inhibition of neurite outgrowth by human CNS myelin can be used as a model to develop strategies to enhance neural recovery and repair in the injured Human CNS.

-The mammalian CNS myelin is dried as a suspension on a support. The support may be a solid support such as glass or plastic and it may be in the shape of for example, a tube, test plate, disc, wells etc. The support is preferably coated with a substance which promotes neuronal outgrowth, for 5 example, poly-L-lysine (PLL), fibronectin, and or laminin.
The test substance may be added to the neuronal cells or the test substance may be introduced by genetically engineering the neuronal cells. For example, the neuronal cells may be transfected with recombinant molecules containing sequences encoding the test substance, or sequences encoding a test substance suspected of being required for inhibition of neurite growth in an antisense orientation.
Conditions for carrying out the above described method of the invention may be selected having regard to factors such as the nature and amounts of the neuronal cells, test substance and mammalian CNS myelin.
In a ~referled embodiment, the neuronal cells on CNS myelin are grown in the presence of the test substance for about 18 to 72 hours, preferably 24 and 72 hours at about 37OC and 5% CO2. The concentration of the neuronal cells which may be used in the assay is between 100 and 3000 cells per square cm, ~rererdbly 1000 cells per 0.33cm2.
Neurite outgrowth is assayed by determining the number of neuronal cells with neural processes. This may be determined by counting both the number of cells with processes greater than 1 cell diameter in length and the total number of cells. Neurite outgrowth may also be assayed by measuring neurite morphology (Lochter et al., J. Cell Biol. 113:1159-1171, 1991), measuring biochemical correlates of neurite growth (Goslin and Banker, J. Cell Biol 108:1507-1515, 1989) and using image analysis systems such as the system known as Leica QuantiMet 500 Plus (Leica, Deerfield, Ill).
As a control for the method of the invention, the method can be carried out by growing the neuronal cells on a non-inhibiting substrate using laminin or PLL, or on a neutral substrate using bovine serum albumin.
B. HYBRIDOMAS AND MONOCLONAL ANTIBODIES

The present invention contemplates a hybridoma cell line which produces monoclonal antibodies which (a) immunoreact with neuronal membrane proteins; (b) neutralize the inhibition of neurite growth by adult mammalian central nervous system myelin; and, (c) recognize bands 5 of Mr 35,000 and Mr 33,000 expressed in neuronal and fibroblast cell lines andin rat cerebrum and rat liver. P~ef~.led hybridoma cell lines are those having the laboratory designation D10.
The hybridomas of the present invention may be formed using conventional methods such as those described by Kohler and Milstein, 10 Nature 256, 495 (1975) and in U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993 which are incorporated herein by reference. (See also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring 15 Harbor Laboratory Press, 1988, which are also incorporated herein by rerere~,ce).
Generally, hybridoma cell lines are prepared by a process involving the fusion under appropriate conditions of an immortalizing cell line and spleen cells from an animal appropriately immunized to produce 20 the desired antibody. Immortalizing cell lines may be murine in origin however, cell lines of other mammalian species may be employed including those of rat, bovine, cannine, human origin, and the like. The immoralizing cell lines are most often of tumor origin, particularly myeloma cells but may also include normal cells transformed with, for example, Epstein 25 Barr Virus. Any immortalizing cell may be used to prepare the hybridomas of the present invention.
Antibody producing cells may be employed as fusion partners such as spleen cells or peripheral blood lymphocytes. The animal from which the cells are to be derived may be immunized at intervals with a 30 membrane fraction obtained from neuronal cells such as rat pheochromocytoma PC-12 (ATCC NO. CRL 1721).

-The immortalizing cells and lymphoid cells may be fused to form hybridomas according to standard and well-known techniques employing polyethylene glycol as a fusing agent. Alternatively, fusion may be accomplished by eletrofusion.
Hybridomas are screened for appropriate monoclonal antibody secretion by assaying the supernatant or protein purified from the ascites for reactivity using the method described in Section A herein. The hybridomas are screened for antibodies which would modulate the inhibition of neurite growth by adult mammalian CNS myelin.
Within one embodiment of the present invention a subject animal such as a rat or mouse, for example a BALB/C mouse, is injected with a membrane fraction obtained from neuronal cells such as rat pheochromocytoma PC-12. The membrane fraction may be admixed with an adjuvant such as Freund's complete or incomplete adjuvant in order to increase the resultant immune response. Between one and three weeks after the initial immunization the animal may be reimmunized with another booster immunization, and its serum tested for antibodies which react with neuronal proteins, or for the ability to block neurite inhibition using the assays described herein. Once the animal has plateaued in its blocking or binding activity, it is sacrificed, and organs which contain large numbers of B
cells such as the spleen and lymph nodes are harvested.
Cells which are obtained from the immunized animal may be immortalized by transfection with a virus such as the Epstein Barr virus (EBV) (see Glasky and Reading, Hybridoma 8(4):377-389, 1989). Alternatively, within a ~refelred embodiment, the harvested spleen and/or lymph node cell suspensions are fused with a suitable myeloma cell in order to create a hybridoma which secretes monoclonal antibody. Suitable myeloma lines include, for example, Sp2 myeloma cells (Shulman et al. Nature 276:269-270, 1978).
Following the fusion, the cells may be placed into culture plates containing a suitable medium, such as RPMI 1640, or DMEM

(Dulbecco's Modified Eagles Medium) aRH Biosciences, Lenexa, Kansas), as well as additional ingredients, such as Fetal Bovine Serum (FBS, ie., from Hyclone, Logan, Utah, or JRH Biosciences). Additionally, the medium should contain a reagent which selectively allows for the growth of fused spleen and 5 myeloma cells such as HAT (hypoxanthine, aminopterin, and thymidine) (Sigma Chemical Co., St. Louis, Missouri). After about seven days, the medium in which the resulting fused cells or hybridomas have been growing may be screened in order to determine the presence of antibodies which modulate the inhibitory activity of CNS myelin in the assays described 10 herein.
Other techniques may also be utilized to construct monoclonal antibodies (see William D. Huse et al., "Generation of a Large Combinational Library of the Immunoglobulin Repertoire in Phage Lambda,"
Science 246:1275-1281, December 1989; see also L. Sastry et al., "Cloning of the15 Immunological Repertoire in Escherichia coli for Generation of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region-Specific cDNA Library," Proc Natl. Acad. Sci USA 86:5728-5732, August 1989; see also Michelle Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A
Rapid Alternative to Hybridomas," Strategies in Molecular Biology 3:1-9, 20 January 1990; these references describe a commercial system available from Stratacyte, La Jolla, California, which enables the production of antibodies through recombinant techniques).
The monoclonal antibodies produced by the hybridoma cell lines of the invention are also part of the present invention. The 25 monoclonal antibodies produced by the hybridoma cell lines of the present invention immunoreact with neuronal membrane proteins and belong to the immunoglobulin M protein class.
Monoclonal antibodies which immunoreact with neuronal membrane proteins includes homogeneous populations of 30 immunoglobulins which are capable of immunoreaction with antigens expressed on neuronal cells. It is understood that immunoglobulins may exist -in acidic, basic, or neutral form depending on their amino acid composition and environment, and they may be found in association with other molecules such as saccharides or lipids. It is also understood that there may bea number of antigens present on the surface of any cell and, alternatively, thatcertain antigens on neuronal cells may also occur on other cell types.
Moreover, such antigens may, in fact, have a number of antigenic determinants. The monoclonal antibodies produced by hybridoma cell lines of the invention may be directed against one or more of these determinants.
Any characteristic antigen associated with neuronal membranes may provide the requisite antigenic determinant. It is contemplated that monoclonal antibodies produced by the hybridoma cell lines fall within the scope of the present invention so long as they remain capable of selectively reacting with neuronal membrane proteins, particularly neuronal membrane proteins obtained from neuronal cells such as rat pheochromocytoma PC-12.
Monoclonal antibodies produced by hybridoma cell lines according to the invention were found to neutralize the inhibition of neurite growth by adult mammalian central nervous system myelin. The monoclonal antibody having the laboratory designation 10D was shown to reverse the near-complete suppression of neurite growth exerted by a substrate of 10~lg/cm2 of CNS myelin, on NG108-15 cells, PC12NGF cells and primary SCG neurons. The 10D monoclonal antibody did not increase neurite growth on non-inhibitory (laminin, PLL) or neutral (BSA) substrates.
Further, this growth promoting effect of the 10D antibody was not observed with pure non-specific IgM antibodies nor with antibodies which bind to CNS
myelin such as anti-galactose cerebroside, anti-myelin basic protein or anti-myelin associated glycoprotein, nor is it observed with antibodies which recognize neurons such as anti-NCAM and anti-THY-1; both of which are present on the cell surface of the neurons.
The antigens recognized by the monoclonal antibodies 3 0 described herein are also a part of the present invention. The present inventors investigated the immunoreactivity of the monoclonal antibodies of the present invention with proteins from tissues, for example adult rat cerebrum and rat liver, and cell lines, for example dbCAMPNG108 cells and NIH
3T3 fibroblast cells using standard immunocytochemistry techniques. The monoclonal antibodies were found to be immunoreactive against bands of Mr 35,000 and 33,000 expressed in neuronal and fibroblast cell lines, and in rat brain and liver.
An antigen recognized by a monoclonal antibody produced by a hybridoma cell line of the invention, in particular the monoclonal antibody with the laboratory designation 10D, may be localized to specific neuronal cells in the brain, brainstem and cerebellum using conventional immunocytochemistry methods. In particular, embryonic, newborn and adult Sprague-Dawley rats may be used. Cryostat sections of fixed brain, cerebellum, brainstem or spinal cord may be incubated with 10D ascites at 1:50 to 1:500 dilutions and processed by the avidin-biotin-peroxidase technique (ABC
Vectastain). This will determine which class of cells in the CNS express the 10D antigen. Both neurons and glia may express this molecule. Regions in the CNS that express the 10D antigen may be surveyed. The possible localization of the antigen to a subset of neural paths and the pattern of acquisition of the 10D antigen will provide important insights on the function of the 10D antigen and establish the optimal neuronal population for determining the effects of 10D antigen blocking or overexpression. If 10D
monoclonal antibody binds to the putative neuronal receptor to the inhibitory myelin proteins, then neurons early in development which appear to be insensitive to the myelin inhibitors (Wictorin et al., 1990; Nature, Vol.
347: 556 and Davies et al., 1994) may be negative for 10D staining. The acquisition of the susceptibility to the myelin inhibitors should coincide with the developmental appearance of neuronal 10D immunoreactivity.
The invention also provides a method for assaying for the presence of an activator or inhibitor of a monoclonal antibody produced by hybridoma cell lines of the invention comprising growing neuronal cells which have a propensity for neurite growth on mammalian central nervous system (CNS) myelin in the presence of a known concentration of the monoclonal antibody, and in the presence of a suspected activator or inhibitor of the monoclonal antibody, and assaying for neurite outgrowth. The methods of the invention permit the identification of potential stimulators 5 or inhibitors of neurite growth in the central nervous ~y~le-l~ environment which have various applications as discussed below.
C. NOVEL NUCLEIC ACID MOLECULE AND PROTEIN
The monoclonal antibody having the laboratory designation 10D was used to identify a clone having the laboratory designation "D1".
10 Transfectants containing antisense constructs derived from the D1 clone showed significant enhancement of neurite growth on myelin. The D1 mRNA appears to be an 7kb transcript present in brain and NG108-15 cells.
Sequence analysis of the partial D1 cDNA clone indicated that it is a previously unreported gene.
The present inventors sequenced the D1 clone and found that it includes the nucleic acid sequences set out in SEQ. ID. No. 1, SEQ. ID.
No. 3, SEQ. ID. NO. 4, SEQ.ID. NO. 5, SEQ.ID. NO. 6, SEQ. ID. NO. 7,and SEQ.
ID. NO. 8, and in Figures 9, 11 to 14, and 21. The partial sequences show no sequence identity with previously-reported genes. The location of the nucleic acid sequences shown in the Sequence Listing in the D1 gene is shown in Figure 15. A diagram of the sequenced regions of D1 is shown in Figure 16.
Probes derived from sequences in the partial cDNA clone were used to screen a cDNA library, and a gene designated "petrin" encoding a protein which plays a role in neurite growth inhibition was identified. The sequence of the Petrin gene is shown in Figure 23. The putative initiation codon is at nucleotide 486 to an in-frame stop codon at nucleotide 1977. The petrin locus was localized to chromosome 12.
Therefore, the present invention provides a isolated and purified nucleic acid molecule comprising (a) a nucleic acid sequence as shown in SEQ. ID NO:1, SEQ.
ID. NO:3, SEQ. ID. NO:4, SEQ. ID. NO. 5, SEQ. ID. NO.6, SEQ. ID. NO.7 and/or SEQ.ID. NO. 8, or in Figures 9, 11 to 14, and 21 wherein T can also be U;
(b) nucleic acid sequences complementary to (a);
(c) nucleic acid sequences having at least 80-90% identity, prererably 90% identity with SEQ.ID NO:l, SEQ.ID. NO:3, SEQ.ID. NO:4, SEQ.
ID. NO. 5,SEQ.ID. NO. 6,SEQ.ID. NO. 7 and SEQ.ID. NO. 8;
(d) a fragment of the nucleic acid molecule that is at least 15 bases and that will hybridize to (a) or (b) under stringent hybridization conditions, or (e) a nucleic acid molecule differing from any of the nucleic acids of (a) to (d) in codon sequences due to the degeneracy of the genetic code.
The invention also reIates to a nucleic acid molecule comprising (a) a nucleic acid sequence encoding a protein having the amino acid sequence as shown in Figure 24 (or SEQ.ID. NO. 12);
(b) nucleic acid sequences complementary to (a);
(c) nucleic acid sequences which are at least 80%, preferably 90% identical to (a); or, (d) a fragment of (a) or (b) that is at least 15 bases and which will hybridize to (a) or (b) under stringent hybridization conditions.
Preferably, the isolated and purified nucleic acid molecule comprises (a) a nucleic acid sequence as shown in Figure 23 (or SEQ.ID.
NO. 11), preferably from about nucleotides 486 to 1977 as shown in Figure 23 (or SEQ ID NO:ll), wherein T can also be U;
(b) nucleic acid sequences complementary to (a);
(c) nucleic acid sequences which are at least 80-90% identical, ~reLerdbly 90% identical to (a); or, (d) a fragment of (a) or (b) that is at least 15 bases and which will hybridize to (a) or (b) under stringent hybridization conditions.
The term "isolated and purified" refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An "isolated and purified" nucleic acid is also substantially free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived. The term "nucleic acid" is intended to include DNA
and RNA and can be either double stranded or single stranded. Therefore, the invention contemplates a double stranded nucleotide sequence comprising a nucleic acid molecule of the invention or a fragment thereof, hydrogen bonded to a complementary nucleotide base sequence, and an RNA made by transcription of this double stranded nucleotide sequence.
It will be appreciated that the invention includes nucleic acid molecules encoding truncations of the protein encoded by the Petrin gene, and analogs and homologs of the protein and truncations thereof, as described herein. It will also be appreciated that variant forms of the nucleic acid molecules of the invention which arise by alternative splicing of an mRNA corresponding to a cDNA of the invention are encompassed by the invention.
Fragments of the nucleic acid molecules contemplated by the present invention include the fragments of the nucleic acid molecule are the nucleotide sequences shown in SEQ. ID. NO:1, SEQ. ID. No. 3, SEQ. ID. NO. 4, SEQ.ID. NO. 5, SEQ.ID. NO. 6, SEQ.ID. NO. 7 and SEQ. ID. NO. 8 and in Figures 9,11 to 14, and 21.
It is also contemplated that nucleic acid molecules of the invention will be prepared having mutations such as insertion or deletion mutations, e.g. nucleic acid molecules encoding analogs of the Petrin protein.
Further, it will be appreciated that the invention includes nucleic acid molecules comprising nucleic acid sequences having substantial sequence identity with the nucleic acid sequences shown in SEQ ID NO:1, SEQ. ID. No. 3, SEQ. ID. NO. 4, SEQ.ID. NO. 5, SEQ.ID. NO. 6, SEQ.ID. NO. 7 and SEQ. ID. NO. 8 or in Figures 9, 11 to 14, and 21, or shown in Figures 23 or SEQ.ID. NO. 11, and fragments thereof. The term "sequences having substantial sequence identity" means those nucleic acid sequences which have slight or inconsequential sequence variations from the sequences disclosed in SEQ ID NO:1, SEQ. ID. No. 3, SEQ. ID. NO. 4, SEQ.ID. NO. 5, SEQ.ID. NO. 6, SEQ.ID. NO. 7 and SEQ.ID. NO. 8 or disclosed in Figures 9, 11 to 14, and 21, or Figures 23 or SEQ.ID. NO. 11, i.e. the sequences function in substantially the same manner to produce substantially the same activity in the assays described in Section A herein. The variations may be attributable to local mutations or structural modifications.
Nucleic acid sequences having substantial identity include nucleic acid sequences having at least 80-90%, prefeLably 90% identity with the nucleic acid sequences as shown in SEQ ID NO:l, SEQ.ID. No.3, SEQ.ID. NO.

4, SEQ.ID. NO. 5, SEQ.ID. NO. 6, SEQ.ID. NO. 7 and SEQ. ID. NO. 8 or in Figures 9,11 to 14, and 21, or as shown in Figures 23 or SEQ.ID. NO. 11; and fragments thereof having at least 15 bases which will hybridize to these sequences under stringent hybridization conditions.
Stringent hybridization conditions are those which are stringent enough to provide specificity, reduce the number of mismatches and yet are sufficiently flexible to allow formation of stable hybrids at an acceptable rate. Such conditions are known to those skilled in the art and are described, for example, in Sambrook, et al, (1989, Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor). By way of example only, stringent hybridization with short nucleotides may be carried out at 5-10 below the Tm using high concentrations of probe such as 0.01-1.Opmole/ml.
Isolated and purified nucleic acid molecules encoding a protein having the activity of Petrin as described herein, and having a sequence which differs from the nucleic acid sequence shown in Figure 23 (or SEQ ID NO:ll) due to degeneracy in the genetic code are also within the scope of the invention. Such nucleic acids encode functionally equivalent proteins (e.g., a protein having Petrin phosphatase activity) but differ in sequence fromthe sequence of Figure 23 (or SEQ ID NO: 11) due to degeneracy in the genetic code.

DNA sequence polymorphisms within the nucleotide sequence of Petrin may result in "silent" mutations in the DNA which do not affect the amino acid encoded. However, DNA sequence polymorphisms may lead to changes in the amino acid sequences of Petrin within a 5 population. These variations in one or more nucleotides (up to about 3-4% of the nucleotides) of the nucleic acids encoding proteins having the activity of Petrin may exist among individuals within a population due to natural allelic variation. Such nucleotide variations and resulting amino acid polymorphisms are within the scope of the invention.
An isolated and purified nucleic acid molecule of the invention which comprises DNA can be isolated by preparing a labelled nucleic acid probe based on all or part of the nucleic acid sequence shown in Figure 23 or SEQ.ID. NO. 11, or shown in Figures 9,11 to 14, and 21 (SEQ ID
NO:1, SEQ. ID. No. 3, SEQ. ID. NO. 4, SEQ.ID. NO. 5, SEQ.ID. NO. 6, SEQ.ID.
NO. 7, SEQ. ID. NO. 8, or SEQ. ID. NO. 11), and using this labelled nucleic acidprobe to screen an appropriate DNA library (e.g. a cDNA or genomic DNA
library). Nucleic acids isolated by screening of a cDNA or genomic DNA
library can be sequenced by standard techniques.
An isolated and purified nucleic acid molecule of the invention which is DNA can also be isolated by selectively amplifying a nucleic acid encoding a petrin protein using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic oligonucleotide primers from the nucleotide sequence shown in Figures 9, 11 to 14, 21 or 23, (SEQ ID NO:1, SEQ. ID. No. 3, SEQ. ID. NO. 4, SEQ.ID. NO. 5, SEQ.ID. NO. 6, SEQ.ID. NO. 7, SEQ. ID. NO. 8, or SEQ. ID. NO. 11) for use in PCR. A nucleic acid can be amplified from cDNA or genomic DNA using oligonucleotide primers and standard PCR amplification techniques. The amplified nucleic acid can be cloned into an appropriate vector and characterized by DNA sequence analysis. cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of `- 21 74025 Chirgwin et al., Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesi7e~
from the mRNA using reverse transcriptase (for example, Moloney MLV
reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV
reverse transcriptase available from Seikagaku America, Inc., St. Petersburg, FL).
An isolated and purified nucleic acid molecule of the invention which is RNA can be isolated by cloning a cDNA encoding a petrin protein into an appropriate vector which allows for transcription of the cDNA to produce an RNA molecule which encodes a protein which exhibits phosphatase activity. For example, a cDNA can be cloned downstream of a bacteriophage promoter, (e.g. a T7 promoter) in a vector, cDNA can be transcribed in vi~ro with T7 polymerase, and the resultant RNA can be isolated by standard techniques.
A nucleic acid molecule of the invention including fragments, may also be chemically synthesized using standard techniques.
Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No.
4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071).
Determination of whether a particular nucleic acid molecule encodes a protein having Petrin activity can be accomplished by expressing the cDNA in an appropriate host cell by standard techniques, and testing the phosphatase activity of the expressed protein or the ability of the expressed protein to inhibit neurite outgrowth as described herein. A cDNA having the biological activity of Petrin so isolated can be sequenced by standard techniques, such as dideoxynucleotide chain termination or Maxam-Gilbert chemical sequencing, to determine the nucleic acid sequence and the predicted amino acid sequence of the encoded protein.
The initiation codon and untranslated sequences of Petrin may be determined using currently available computer software designed for -the purpose, such as PC/Gene (IntelliGenetics Inc., Calif.). The intron-exon structure and the transcription regulatory sequences of the gene encoding Petrin may be identified by using a nucleic acid molecule of the invention encoding Petrin to probe a genomic DNA clone library. Regulatory elements 5 can be identified using conventional techniques. The function of the elements can be confirmed by using them to express a reporter gene such as the bacterial gene lacZ which is operatively linked to the elements. These constructs may be introduced into cultured cells using standard procedures or into non-human transgenic animal models. Such constructs may also be used 10 to identify nuclear proteins interacting with the elements, using techniques known in the art.
The nucleic acid sequences contained in the nucleic acid molecules of the invention or a fragment thereof, preferably one or more of the nucleic acid sequences shown in the Sequence Listing as SEQ. ID. NO. 1 SEQ.ID. NO.3, SEQ.ID. NO. 4, SEQ.ID. NO.5, SEQ.ID. NO. 6, SEQ.ID. NO. 7 and SEQ. ID. NO. 8 and in Figures 9, 11 to 14, and 21, or in Figure 23 (or SEQ.ID. NO. 11) may be inverted relative to their normal presentation for transcription to produce antisense nucleic acid molecules. The antisense nucleic acid molecules may be constructed using chemical synthesis and 2 o enzymatic ligation reactions using procedures known in the art. The antisense nucleic acid molecules of the invention or a fragment thereof, may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with 25 mRNA or the native gene e.g. phosphorothioate derivatives and acridine substituted nucleotides. The antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity 30 of which may be determined by the cell type into which the vector is introduced. In an embodiment of the invention the antisense nucleic acid molecule comprises the following sequence: GCT GCC AGC CAT GAT GCC
GCC CAT (SEQ. ID. NO: 13). This antisense sequence enhanced neurite growth in a functional in vitro assay.
Translation of the Petrin cDNA revealed a single large open 5 reading frame from a putative initiation codon at nucleotide 486 to an in-frame stop codon at nucleotide 1977. The inventors have determined the primary structure of the deduced protein and have determined that it has predicted molecular weight of 60 to 64 kDa. The protein has 3 to 4 distinct regions with up to 60% identity with members of the protein phosphatase 2C
10 family ("PP2C") (See Figure 24). Members of the protein phosphatase 2C
family dephosphorylate serine and threonine residues in proteins. (See review articleby Wera, S., and B.A. Hemmings, Biochem. J. (1995) 311, 17-29).
The novel protein has been designated "petrin".
The present inventors have also shown by in situ 15 hybridization that the petrin gene is expressed in neurons in brain tissue and in particular, in the Purkinje cells of the cerebellum; in the 3rd and 4th layers of the cerebral cortex; and, dispersed neurons in the hippocampus. Expression of petrin occurred after embryonic day 13 and increased constantly with the highest expression found in adults. Northern and DNA analysis also showed 20 that the protein is present in different mammalian species such as mouse, rat, hamster, and human.
The biological function of Petrin was investigated using phosphatase assays on immunoprecipitated material and like other members of the PP2C family, it exhibited magnesium-dependent serine/threonine 25 phosphatase activity. The protein was also shown to have magnesium-dependent tyrosine phosphatase activity. Serine/threonine-phosphatase and tyrosine phosphatase activities were inhibited by okadaic acid or ortho-vanadate, respectively.
The present inventors also prepared antisense 30 oligonucleotides and found that they enhanced neurite growth in a functional in vitro assay. Phosphatase activity was also shown to be highest -while NG108 cells are proliferating and growing neurites, and was not detected in late growth stages.
Therefore, the present invention also includes a protein containing the amino acid sequences as shown in the Sequence Listing as SEQ. ID. NO. 2 and 10 and as shown in Figure 10, and as shown in the Sequence Listing as SEQ.ID. NO. 9; and sequences having 80-90% identity thereto. In an embodiment of the invention, the protein comprises the amino acid sequence as shown in Figure 24 (or SEQ.ID. NO. 12).
The protein of the invention may be found in brain, NG108, and PC12 cells.
In addition to the full length amino acid sequence (Figure 24 or SEQ.ID. NO. 12), the ~roLeills of the present invention include truncations and analogs, and homologs of the protein and truncations thereof as described herein. Truncated proteins may comprise peptides of between 3 and 1900 amino acid residues, ranging in size from a tripeptide to a 1900 mer polypeptide. For example, a truncated protein may comprise the regions highly conserved among the PP2C proteins (e.g. amino acids 281 to 324, 411 to 451, 516 to 557, or 630 to 640 in Figure 24 or SEQ.ID. NO. 12). Truncated proteins also include the proteins having the sequences shown in the Sequence Listing as SEQ.ID. Nos. 2, 9, 10, or as shown in Figure 10.
At the amino terminal end, the truncated proteins may have an amino group (-NH2), a hydrophobic group (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy-carbonyl (PMOC) group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates. The truncated proteins may have a carboxyl group, an amido group, a T-butyloxycarbonyl group, or a macromolecule including lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end.
The proteins of the invention may also include analogs of Petrin as shown in Figure 24 (SEQ.ID. NO. 12) and/or truncations thereof as described herein, containing one or more amino acid substitutions, -insertions, and/or deletions. Amino acid substitutions may be of a conserved or non-conserved nature. Conserved amino acid substitutions involve replacing one or more amino acids with amino acids of similar charge, size, and/or hydrophobicity characterisitics. When only conserved substitutions are made the resulting analog should be functionally equivalent to Petrin as described herein. Non-conserved substitutions involve replacing one or more amino acids with one or more amino acids which possess dissimilar charge, size, and/or hydrophobicity characteristics.
One or more amino acid insertions may be introduced into the amino acid sequence as shown in Figure 24 (SEQ. ID. NO. 12). Amino acid insertions may consist of single amino acid residues or sequential amino acids ranging from 2 to 15 amino acids in length. For example, amino acid insertions may be used to destroy the phosphatase activity of the protein.
Deletions may consist of the removal of one or more amino acids, or discrete portions (e.g.amino acids 281 to 324, 411 to 451, 516 to 557, or 630 to 640 in Figure 24 or SEQ. ID. NO. 12) from the Petrin amino acid sequence as shown in Figure 24 (SEQ. ID. NO. 12). The deleted amino acids may or may not be contiguous. The lower limit length of the resulting analog with a deletion mutation is about 10 amino acids, preferably 100 amino acids.
The proteins of the invention also include homologs of Petrin as shown in Figure 24 or SEQ. ID. NO. 12 and/or truncations thereof as described herein. Such homlogs are proteins whose amino acid sequences are comprised of the amino acid sequences of Petrin regions from other species that hybridize under stringent hybridization conditions (see discussion of stringent hybridization conditions herein) with a probe used to obtain Petrin as shown in Figure 24 or SEQ. ID. NO. 12. Homologs will have the same regions characteristic of Petrin and PP2C proteins. It is anticipated that, outside of these regions of Petrin a protein comprising an amino acid sequence which is about 50% similar, preferably 80 to 90% similar, with the amino acid sequence shown in Figure 24 or SEQ. ID. NO. 12 will exhibit phosphatase activity and inhibit neurite outgrowth.

-The invention also contemplates isoforms of the Petrin protein of the invention. An isoform contains the same number and kinds of amino acids as the protein of the invention, but the isoform has a different molecular structure. The isoforms contemplated by the present invention are 5 those having the same properties as the protein of the invention as described herein.
The present invention also includes a Petrin protein conjugated with a selected ~roleil., or a selectable marker protein (see below) to produce fusion proteins. Additionally, immunogenic portions of Petrin 10 proteins are within the scope of the invention.
The protein encoded by nucleic acid molecules of the invention may be prepared using recombinant DNA methods. Accordingly, the nucleic acid molecules of the present invention or a fragment thereof may be incorporated in a known manner into an appropriate expression 15 vector which ensures good expression of the protein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses, so long as the vector is compatible with the host cell used.
The invention therefore contemplates a recombinant molecule of the invention containing a nucleic acid molecule of the 2 0 invention, or a fragment thereof, and the necessary elements for the transcription and translation of the inserted sequence. Suitable transcription and translation elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of appropriate transcription and translation elements is dependent on the host cell chosen as 25 discussed below, and may be readily accomplished by one of ordinary skill in the art. Examples of such elements include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other genetic elements, such 30 as an origin of replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporated into the expression vector. It will also be appreciated that the necessary transcription and translation elements may be supplied by the native gene and/or its flanking regions.
The recombinant molecules of the invention may also contain a reporter gene encoding a selectable marker protein which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention. Examples of reporter genes are genes encoding a protein such as ~-galactosidase (e.g.lac Z), chloramphenicol, acetyl-transferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. Transcription of the reporter gene is monitored by changes in the concentration of the reporter protein such as ,B-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. This makes it possible to visualize and assay for expression of recombinant molecules of the invention and in particular to determine the effect of a mutation on expression and phenotype.
Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation etc. Methods for transforming transfecting, etc. host cells to express foreign DNA are well known in the art (see, e.g., Itakura et al., U.S. Patent No. 4,704,362; Hinnen et al., PNAS USA 75:1929-1933, 1978; Murray et al., U.S. Patent No. 4,801,542;
Upshall et al., U.S. Patent No. 4,935,349; Hagen et al., U.S. Patent No. 4,784,950;
Axel et al., U.S. Patent No. 4,399,216; Goeddel et al., U.S. Patent No. 4,766,075;
and Sambrook et al. Molecular Cloning A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, all of which are incorporated herein by rerer~l-ce and see the detailed discussion below).
Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells, including bacterial, mammalian, yeast or other fungi, viral, plant, or insect cells, preferably neuronal cells such as NG108-derived lines and PC12.
Bacterial host cells suitable for carrying out the present invention include E. coli, B. subtilis, Salmonella typhimurium, and various species within the genus' Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial species well known to one of ordinary skill in the art. Representative examples of bacterial host cells include E.coli BL21, DE3, Streptomyces lividans strain 66. Suitable bacterial expression vectors 5 ~refeldbly comprise a promoter which functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication.
Representative promoters include the ,B-lactamase (penicillinase) and lactose promoter system (see Chang et al., Nature 275:615, 1978), the trp promoter (Nichols and Yanofsky, Meth in Enzymology 101:155, 1983), the tac promoter (Russell et al., Gene 20: 231, 1982), and the phage T3 promoter (Studier and Moffat, J. Mol. Biol. 189:113-130, 1986). Representative selectable markers include various antibiotic resistance markers such as the kanamycin or ampicillin resistance genes. Suitable expression vectors include but are not limited to bacteriophages such as lambda derivatives or plasmids such as pBR322 (see Bolivar et al., Gene 2:9S, 1977), the pUC plasmids pUC18, pUC19, pUC118, pUC119 (see Messing, Meth in Enzymology 101:20-77, 1983 and Vieira and Messing, Gene 19:259-268, 1982), and pNH8A, pNH16a, pNH18a, pCDM8, Bluescript M13 (Stratagene, La Jolla, Calif.), and pET10 (Studier et al., Meth.
Enzymol. 185:60-89, 1990).
Yeast and fungi host cells suitable for carrying out the present invention include, among others Saccharomyces cerevisae, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus.
Suitable expression vectors for yeast and fungi include, among others, YCp50 (ATCC No. 37419) for yeast, and the amdS cloning vector pV3 (Turnbull, Bio/Technology 7:169, 1989). Protocols for the transformation of yeast are also well known to those of ordinary skill in the art (See for example, Hinnen et al., PNAS USA 75:1929, 1978; Itoh et al., J. Bacteriology 153:163, 1983;and Cullen et al. Bio/Technology 5:369, 1987).
Mammalian cells suitable for carrying out the present invention include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC

21 74~25 -No. CCL 2), 293 (ATCC No. 1573), CHOP, and NS-1 cells. Suitable expression vectors for directing expression in mammalian cells generally include a promoter, as well as other transcription and translation control sequences.
Common promoters include SV40, MMTV, metallothionein-1, adenovirus 5 Ela, CMV, immediate early, immunoglobulin heavy chain promoter and enhancer, and RSV-LTR. Protocols for the transfection of ma~nm~lian cells are well known in the art and include calcium phosphate mediated electroporation, retroviral, and protoplast fusion-mediated transfection (see Sambrook et al., supra).
Given the teachings provided herein, promoters, terminators, and methods for introducing expression vectors of an appropriate type into plant, avian, and insect cells may also be readily accomplished. For example, within one embodiment, the nucleic acid molecule of the invention may be expressed from plant cells (see Sinkar et al., J. Biosci (Bangalore) 11:47-58, 1987, which reviews the use of Agrobacterium rhizogenes vectors; see also Zambryski et al., Genetic Engineering, Principles and Methods, Hollaender and Setlow (eds.), Vol. VI, pp. 253-278, Plenum Press, New York, 1984, which describes the use of expression vectors for plant cells, including, among others, pAS2022, pAS2023, and pAS2034).
Insect cells suitable for carrying out the present invention include cells and cell lines from Bombyx or Spodotera species. Suitable expression vectors for directing expression in insect cells include Baculoviruses such as the Autographa california nuclear polyhedrosis, virus (Miller et al. 1987, in Genetic Engineering, Vol. 8 ed. Setler, J.K. et al., Plenum Press, New York) and the Bombyx mori nuclear polyhedrosis virus (Maeda et al., 1985, Nature 315:592).
Alternatively, the protein encoded by the nucleic acid molecule of the invention may be expressed in non-human transgenic animals such as, mice, rats, rabbits, sheep and pigs (see Hammer et al. (Nature 315:680-683, 1985), Palmiter et al. (Science 222:809-814, 1983), Brinster et al.(Proc Natl. Acad. Sci USA 82:44384442, 1985), Palmiter and Brinster (Cell.

41:343-345, 1985) and U.S. Patent No. 4,736,866).
The proteins of the invention, and parts thereof may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem.
5 Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart).
The proteins of the invention may be conjugated with other molecules, such as proteins or polypeptides. This may be accomplished, for 10 example, by the synthesis of N-terminal or C-terminal fusion proteins. Thus, fusion proteins may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of the protein, and a selected protein with a desired biological function. The resultant fusion proteins contain the protein or a portion thereof fused to the selected protein. Examples of proteins which 15 may be used to prepare fusion proteins include neurotrophic factors, such as nerve growth factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), ciliary neurotrophic factor (CNTF), fibroblast growth factor (FGF), and NT-3.
D. APPLICATIONS
The nucleic acid molecules of the invention or fragments 20 thereof, allow those skilled in the art to construct nucleotide probes for use in the detection of nucleotide sequences in biological materials including cells and tissues. Example of probes include the fragments shown in the Sequence Listing as SEQ. ID. NO. 1 and NOS. 3 to 6, 7, 8 and 9. A nucleotide probe may be labelled with a detectable substance such as a radioactive label which 25 provides for an adequate signal and has sufficient half-life such as 32p, 3H, 14C
or the like. Other detectable substances which may be used include antigens that are recognized by a specific labelled antibody, fluorescent compounds, enzymes, antibodies specific for a labelled antigen, and chemiluminescence.
An appropriate label may be selected having regard to the rate of 30 hybridization and binding of the probe to the nucleic acid to be detected andthe amount of nucleic acid available for hybridization. Labelled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.). The nucleotide probes may be used to detect genes, preferably in human cells, that hybridize to the nucleic acid molecule of the present invention preferably, nucleic acid molecules which hybridize to the nucleic acid molecule of the invention under stringent hybridization conditions as described herein.
In accordance with one embodiment of the invention, the D1 or Petrin cDNA (Figure 23 or SEQ. ID. NO. 11) may be used to identify, study and isolate the corresponding human gene. The present inventors have shown that the D1 cDNA sequences from position bp230 to bpl,095 (as shown in the Sequence Listing as SEQ. ID. NO. 7) specifically recognize human genomic DNA fragments similar in number to those recognized in rat and mouse DNA. The present inventors have also shown using a panel of human-rodent hybrid cell lines that all the D1 gene sequences detected in the human genome reside on chromosome 12. The D1 probes can thus be used to determine whether human disorders are genetically linked to the petrin or D1 gene. The present inventors have also demonstrated that the rat D1 cDNA
can be used to detect human D1 mRNA and study its expression in normal tissue and in disease.
The proteins of the invention or parts thereof, may be used to prepare antibodies. Antibodies having specificity for the protein may also be raised from fusion proteins created by expressing fusion proteins in host cells as described above.
Within the context of the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab')2 and recombinantly produced binding partners. Antibodies are understood to be reactive against the protein encoded by the nucleic acid molecule of the invention if they bind with a Ka of greater than or equal to 10-7 M. As will be appreciated by one of ordinary skill in the art, antibodies may be developed which not only bind to the protein, but 21 74~25 -which bind to a regulator of the protein, and which also block the biological activity of the yrole~
Polyclonal antibodies may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as 5 horses, cows, various fowl, rabbits, mice, or rats. Briefly, a protein of the invention is utilized to immunize the animal through intraperitoneal, intramuscular, intraocular, or subcutaneous injections, in conjunction with an adjuvant such as Freund's complete or incomplete adjuvant. Following several booster immunizations, samples of serum are collected and tested for 10 reactivity to the protein. Particularly preferred polyclonal antisera will give a signal on one of these assays that is at least three times greater than background. Once the titer of the animal has reached a plateau in terms of its reactivity to the protein, larger quantities of antisera may be readily obtainedeither by weekly bleedings, or by exsanguinating the animal.
Monoclonal antibodies may also be readily generated using conventional techniques as described above.
Binding partners may be constructed utilizing recombinant DNA techniques to incorporate the variable regions of a gene which encodes a specifically binding antibody. Within one embodiment, the genes which 2 o encode the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using nucleotide primers for the variable region. These primers may be synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources. Primers for mouse and human variable regions including, among others, primers for VHa, VHb, 25 VHC, VHd, CH1, VL and CL regions are available from Stratacyte (La Jolla, Califl.
These primers may be utilized to amplify heavy or light chain variable regions, which may then be inserted into vectors such as ImmunoZAPTM H or ImmunoZAPTM L (Stratacyte), respectively. These vectors may then be introduced into E. coli for expression. Utilizing these techniques, large 30 amounts of a single-chain protein containing a fusion of the VH and VL
domains may be produced (See Bird et al., Science 242:423-426, 1988). In `

addition, such techniques may be utilized to change a "murine" antibody to a "human" antibody, without altering the binding specificity of the antibody.
The polyclonal or monoclonal antibodies and binding partners may be used to detect a protein of the invention for example, in various biological materials, for example they may be used in an Elisa, radioimmunoassay or histochemical tests. Thus, the antibodies may be used to quantify the amount of the protein in a sample in order to determine its role in particular cellular events or pathological states and to diagnose and treat such pathological states.
In particular, the polyclonal and monoclonal antibodies of the invention may be used in immuno-histochemical analyses, for example, at the cellular and sub-subcellular level, to detect a protein of the invention,to localise it to particular cells and tissues, and to specific subcellular locations, and to quantitate the level of expression.
Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect a protein of the invention. Generally, an antibody specific for the protein may be labelled with a detectable substance as described herein and the protein may be localised in tissue based upon the presence of the detectable substance.
2 o Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against the protein encoded by the nucleic acid molecule of the invention.
Where a radioactive label is used as a detectable substance, the protein encoded by the nucleic acid molecule of the invention may be localized by radioautography. The results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains.
The above described methods for detecting nucleic acid molecules and fragments thereof and protein can be used to monitor neurite growth by detecting and localizing the nucleic acid molecule and/or protein -of the invention in organisms, tissues, and embryos.
It would also be apparent to one skilled in the art that the above described methods may be used to study the developmental expression of a protein of the invention and, accordingly, will provide further insight 5 into the role of the protein in neuronal growth in the CNS.
The invention provides a method for assaying for the presence of an activator or inhibitor of a protein of the invention comprising growing neuronal cells which have a propensity for neurite growth in the presence of a protein of the invention and a suspected activator or inhibitor 10 substance, and assaying for neurite outgrowth. The invention also provides a method for assaying for the presence of an activator or inhibitor of a protein of the invention comprising growing neuronal cells which have a propensity for neurite growth on mammalian central nervous system (CNS) myelin and which express the protein of the invention, in the presence of a suspected 15 activator or inhibitor substance, and assaying for neurite outgrowth. The activator or inhibitor may be an endogenous physiological substance or it may be a natural or synthetic drug. Conditions for carrying out these methods of the invention are selected to favour neurite outgrowth and having regard to factors such as the nature and amounts of the neuronal cells and test 20 substance. The methods permit the identification of potential activators or inhibitors of neurite growth in the central nervous system environment which have various applications as discussed below.
Substances which affect cell neurite growth may also be identified by comparing the pattern and level of expression of the novel 25 nucleic acid of the invention or its protein product, in tissues and cells in the presence and in the absence of a test substance.
The invention also contemplates a method for assaying for a substance that affects neuronal growth comprising administering to a non-human animal or to a tissue of an animal, a substance suspected of 30 affecting neuronal growth, and detecting, and optionally quantitating, the nucleic acid molecule of the invention or a protein of the invention in the non-human animal or tissue.
The invention also contemplates a method for identifying a substance which is capable of binding to a protein of the invention, or a part of the protein, comprising reacting the protein, or part of the protein, with at5 least one substance which potentially can bind with the protein, or part of the protein, under conditions which permit the formation of substance-protein complexes, and assaying for substance-protein complexes, and/or for free substance, and for non-complexed protein.
Still further, the invention provides a method for assaying a 10 medium for the presence of an activator or inhibitor of the interaction of the protein of the invention or part thereof, and a substance which binds to the protein. In an embodiment, the method comprises providing a known concentration of a protein of the invention, or part of the protein, incubating the protein, or part of the protein with a substance which binds to the protein,15 or part of the protein, and a suspected activator or inhibitor substance, under conditions which permit the the formation of substance-protein complexes, and assaying for substance complexes.
The invention also contemplates a method for assaying for a substance that affects the phosphatase activity of a protein of the invention 20 comprising reacting a protein of the invention with a substrate which is capable of being dephosphorylated by the protein to produce a dephosphorylated product, in the presence of a substance which is suspected of affecting the phosphatase activity of the protein, under conditions which permit dephosphorylation of the substrate, assaying for dephosphorylated 25 product, and comparing to a product obtained in the absence of the substance to determine the affect of the substance on the phosphatase activity of the protein. Suitable substrates include serine, threonine, or tyrosine phospho-peptides. Conditions which permit the dephosphorylation of the substrate, may be selected having regard to factors such as the nature and amounts of 30 the substance, substrate, and the amount of protein.
Substances which modulate neurite growth identified using the methods of the invention, including the monoclonal antibody produced by a hybridoma cell line of the invention, the nucleic acid molecule and protein of the invention, and the antisense nucleic acid molecules of the invention, may be useful in regulating neurite outgrowth in vivo and may form the basis for a strategy to enhance or inhibit neurite growth/axonal regeneration in the mammalian CNS. For example, the substances may be used to enhance (1) axonal regrowth in the CNS following traumatic CNS
lesions; (2) formation of neuronal connections in neural transplantation therapies; and 3) the ability of surviving neurons to form new connections and thereby take over some of the functions of neurons lost in CNS
neurodegenerative diseases such as Alzheimer's and Parkinson's Disease.
Accordingly, the substances identified herein may be used to stimulate or inhibit neuronal regeneration associated with conditions involving nerve damage resulting from traumatic injury, stroke, or degenerative disorders of the central nervous system, for example Alzheimer's disease, Parkinson's disease, Huntington's disease, demyelinating diseases, progressive spinal amyotrophy, trauma and ischemia resulting from stroke, and tumors of nerve tissue, epilepsy, glaucoma, and neurofibromatosis.
The substances identified using the methods described 2 o herein or antibodies described herein, may be incorporated into a pharmaceutical composition containing the substance or antibodies, alone or together with other active substances. Such pharmaceutical compositions can be for oral, topical, rectal, parenteral, local, inhalant or intracerebral use. They are therefore in solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets. The methods described by Penn et al, Lancet 335(8691):738-747,1990 for intrathecally delivering substances into the CNS
may be particularly useful for administering the pharmaceutical compositions of the invention.
The pharmaceutical compositions of the invention can be intended for administration to humans or animals. Dosages to be -administered depend on individual needs, on the desired effect and on the chosen route of administration.
The pharmaceutical compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable 5 compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
On this basis, the pharmaceutical compositions include, albeit not exclusively, the active compound or substance in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids. The pharmaceutical compositons may additionally contain other agents such as neurotrophic factors, in particular NGF, BDNF, CNTF, T-3 and FGF.
The antisense nucleic acid molecules of the invention may be used in gene therapy to enhance axonal regeneration. For a discussion of the regulation of gene expression using anti-sense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis Reviews - Trends in Genetics, Vol. 1(1) 1986. Recombinant molecules comprising an antisense sequence or oligonucleotide fragment thereof, may be directly introduced into cells or tissues in vivo using delivery vehicles such as retroviral vectors, adenoviral vectors and DNA virus vectors. They may also be introduced into cells in vivo using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into liposomes. Recombinant molecules may also be delivered in the form of an aerosol or by lavage. The antisense nucleic acid molecules of the invention may also be applied extracellularly such as by direct injection into cells. Freed et al., New Eng. J. Med. 327(22):1549-1555, 1992, describe a method for injecting fetal cells into brains of Parkinson's patients. The methods described by Pace et al, Lancet 335(8691):738-747 for intrathecally delivering substances into the CNS may also be useful for administering pharmaceutical compositions containing antisense nucleic acid molecules of the invention. Antisense nucleic acid molecules of the invention may also be introduced using intracerebroventricular administration (See for example, C. Wahlestedt et al., Nature 363:260-263, 1993).
The utility of the substances, antibodies, antisense nucleic acid molecules, and compositions of the invention may be confirmed in animal experimental model ~y~Lems.
For example, the effect of lOD antibody and substances identified using the methods of the invention can be tested in vivo on the regeneration of interrupted neural pathways by CNS neurons in the rat optic nerve (See Thanos. S., and von Boxderg, Y., Metabolic Brain Disease 4:67-72, 1989). Axons from retinal ganglion cells (RGC) project into the CNS
environment of the optic nerve. This projection does not normally regenerate after injury, but the axons will grow into a non-inhibitory PNS
graft, implicating environmental factors. The model can be used to determine whether RGC axons interrupted within the optic nerve will increase their propensity for regeneration in the presence of a test substance and optionally neurotrophic factors. The regeneration of retinal ganglion cells in the optic nerve is a useful model since this discrete axonal projection, entirely within the CNS, is readily accessible and surgical techniques using the optic nerve are known.
Previous experiments with hybridoma implants into the brain have been used to deliver antibodies to the CNS. Using this approach, Schnell and Schwab were able to deliver antibodies against the CNS myelin inhibitors to promote the regeneration of CNS axons (Schnell L and Schwab ME. Nature 343(6255):269-72, 1990). More recently, this group has combined the use of inhibitor-neutralizing antibodies with co-application of neurotrophic factors to produce an even greater increase in the long distance -regeneration of CNS fibres (Schnell et al., 1994, Nature 367: 170-173). FGF, BDNF and NT-3 have been shown to increase RGC survival after axotomy aohnson et al., 1986, J. Neurosci. 6: 3031-3038; Lipton et al., 1988, Proc. Natl.
Acad. Sci USA 85: 2388-2392; Mey and Thanos, 1993, Brain Research 602:304-317). The strategy of using antibodies with or without the addition of neurotrophins thus has a precedent and the potential to yield a positive result.
A specific protocol for the optic nerve model is described in Example 6.
A second model involves examining the regeneration of central processes of dorsal root ganglion neurons (See Carlstedt et al., Brain Res. Bulletin, Vol.22:93-102, 1989). The ability of test substances to modulate regrowth of peripheral axons within the spinal cord can be tested using this model. The model also permits an assessment of the effect of a test substance on the active phase of axonal regrowth in the face of CNS inhibitors. In some experiments, 300-500 ~g of NGF or vehicle can also be injected into the spinal cord at the time of initial surgery. The administration of other neurotrophins (NT-3, BDNF, CNTF and FGF) in combination with a test substance identified in accordance with the present invention can also be studied.
A specific protocol for the regrowth of dorsal root ganglion neurons is described in Example 7.
Other examples of non-human animal models for testing the application of substances identified in accordance with the present invention are models of neurodegenerative conditions, for example, the MPTP model as described in Langston J.W. et al., Symposium of Current Concepts and Controversies in Parkinson's Disease, Montebello, Quebec, Canada, 1983 and Tatton W.G. et al., Can. J. Neurol. Sci. 1992, 19, and traumatic nerve /1amage for example, animal stroke models such as the one ldescribed in MacMillan et al. Brain Research 151:353 368 (1978)). Models for testing the application of antisense nucleic acid molecules of the invention, and in particular, determining the physicological affects of the molecules, are -described in C. Wahlestedt et al., Nature 363:260-263, 1993.
The invention also provides methods for examining the function of the protein encoded by the nucleic acid molecule of the invention. Cells, tissues, and non-human animals lacking in expression or 5 partially lacking in expression of the protein may be developed using recombinant molecules of the invention having specific deletion or insertion mutations in the nucleic acid molecule of the invention. A recombinant molecule may be used to inactivate or alter the endogenous gene by homologous recombination, and thereby create a deficient cell, tissue or 10 animal. Such a mutant cell, tissue or animal may be used to define specific cell populations, developmental patterns and in vivo processes, normally dependent on the protein encoded by the nucleic acid molecule of the nventlon.
The following non-limiting examples are illustrative of the 15 present invention:
EXAMPLES
The following materials and methods were utilized in the investigations outlined in the examples:
MATERIALS AND METHODS
20 Cells Rat pheochromocytoma PC-12 were obtained from the American Type Culture Collection (ATTC NO. CRL 1721, Rockville, Maryland). Cells were grown in RPMI-1640 media (Gibco) with 15% fetal calf serum (FCS). PC-12 cells were differentiated with 100 ng/ml of nerve growth 25 factor (NGF) for 7 days. Cells of the NG-108-15 line were obtained from Dr. G.
Cheng (University of Manitoba, Manitoba, Canada ). The preparation of the cells is described in Nelson et al., Proc. Nat. Acad. Sci. USA 73:123-127, 1976.NG108-15 cells were grown in DME medium with 10% FCS, lXHAT medium (Gibco) and were induced to differentiate to the neuronal phenotype by 30 reducing the serum to 5% and by the addition of 1 mM dibutyryl cyclic adenosine-monophosphate (dbcAMP) (Sigma) for 2 to 4 days. Primary -superior cervical ganglion neurons were obtained from newborn rats and cultured as described in Paterson and Chun, Dev. Biol. 56:263-280, 1977.
Penicillin (25 U/ml) and Streptomycin (25 ~Ig/ml) were added to all media.
Substrate Preparation CNS myelin was prepared from brains of Sprague-Dawley rats (250-300g) using modifications of previously described procedures (Caroni and Schwab, J. Cell. Biol 106:1281-1298, 1988). Homogenization was carried out using 10 mls per gram of tissue of 0.25 M sucrose, 5 mM EDTA, 5 mM
iodoacetamide (homogenization buffer) using a glass homogenizer. The lo homogenate was centrifuged at 2000 rpm in a Sorval HB-4 rotor for 3 minutesto pellet cell debris and nuclei. The supernatant was layered atop 20 mls of 0.85 M sucrose, 5 mM EDTA, 5 mM iodoacetamide in 38 ml SW-28 tubes (Beckman) and centrifuged at 4C and 28,000 g for 1 hour. The interface was collected, kept on ice and washed in 20 volumes of 30 mM Hepes pH 7.4, 5 mM EDTA, 5 mM iodoacetamide. After centrifugation at 28,000 g for 4 hours, the pellet was resuspended in homogenization buffer and layered onto 0.85M
sucrose with protease inhibitor PMSF. The sample was recentrifuged (28,000 g, 1 hour), the resultant interface was again washed, pelleted at 28,000 g for 4hours and resuspended in a small volume of 30 mM Hepes pH 7.4. Western blot analysis using a monoclonal antibody against myelin basic protein confirmed a four fold enrichment for myelin in the brain extract versus total brain homogenate. Protein determinations were carried out using a protein assay kit (Bio-Rad) and bovine serum albumin (Type IV; Sigma) as a standard.
Extracts of rat sciatic nerve, liver, muscle and human corpus callosum were prepared in a similar fashion.
Antibody Production 3 X 107 NGF treated PC-12 cells were homogenized in a glass homogenizer using 10 ml of 0.25 M sucrose, 0.1 mM MgCl, 10 mM Tris pH 7.4.
The sample was centrifuged at 2000 g for 5 minutes. The supernatant was 3 o transferred to 5 ml tubes and spun at 90,000 g for 1 hour. The resultant pellet was resuspended in 10 mM Hepes pH 7.4. Balb/C mice were immunized 5 -times with aliquots of this crude membrane fraction containing 50 ,ug of protein. All mice produced sera recognizing PC-12 membranes in an ELISA
assay. Splenic lymphocytes were fused with Sp2 myeloma cells (Shulman et al., 1978, Nature 367:170-173) following established procedures (Harlow and 5 Lane, Antibodies, A Laboratory Manual (CSH:CSHL, New York) 1988). All fusion products from a single mouse were plated in 96 wells and supernatants tested for reaction with PC-12 membranes in an ELISA assay. Positive supernatants were tested in the in vitro bioassay described below. Hybridoma pools giving positive bioassay results were plated at limiting dilutions to 10 obtain clonal cell lines.
To collect antibody-containing supernatant from cloned hybridoma 10D, cells were grown in Cell/Perfect protein-free media supplement with Cell/Perfect Ab enhancer (Stratagene). The supernatant was collected and used for immunodetection of membrane bound protein, either 15 undiluted or diluted (up to 1:5), depending on the concentration of the Ab in the supernatant.
Ascites fluid was produced to generate high titre antibody solutions. Balb/C mice were given 0.5 ml of incomplete Freund's adjuvant by intraperitoneal injection. The next day, animals were irradiated with 350 20 mRads and injected with 106 to 107 hybridoma cells. After 2 to 3 weeks, ascites were collected by paracentesis. Ascites fluid was incubated at 37OC for 1 hour and centrifuged at 2000 g for 5 minutes. The supernatant was aliquoted and stored at 4C.
Hybridoma supernatants and ascites were isotyped using a 25 commercial kit (Gibco, N.Y.). Antibody concentration was measured using an ELISA assay with commercially available immunoglobulins used as standards (Cedarlane, R.R#1, Hornby, Ontario, Canada).
Screening To test the biological activity of the hybridoma supernatants, 30 poly-L-lysine treated 96 well dishes (each well has a surface area of approximately 0.33 cm2) were plated with myelin proteins. Briefly, 70 ml -suspensions containing 3.3 llg of CNS extract protein were plated onto test wells. After ovemight drying, wells were washed twice with 10 mM sodium phosphate 140 mM NaCl saline (PBS). Dishes were sterilized by exposure to uv light for 20 minutes. 1000 to 2000 PC-12 or NG108-15 cells in 50 ~l were plated onto each well in the presence of an equal volume of hybridoma supematant.
Neurite Oulg~ vlh Assay Assays were done in 96 well dishes (NunC). Substrate testing wells were precoated with 100 ,ug/ml of poly-L-lysine (Sigma). Test wells were in addition coated with bovine serum albumin (BSA type IV;Sigma) or adult rat brain, sciatic nerve, muscle or liver homogenates or extracts. Substrate coated wells were UV light treated and washed with PBS
twice. Assays were carried out using 50 ,ul of hybridoma supematant with an equal volume of cells suspension or using 1 to 10 ,ul of ascites in 100 ,ul of cells. Control hybridoma supernatants from the same fusion as well as hybridoma supernatants producing antibodies to myelin basic protein (MBP), galactose cerebroside (Gal C), tyrosine hydroxylase (TH), and neural cell adhesion molecule (NCAM) served as controls. Ascites produced from the myeloma fusion partner Sp2 (Cedarlane) or directed against Thy-1 (New England Nuclear) were also used in control experiments. 1000 to 2000 cells were plated per well. After 24 to 72 hours of culture at 37 C and 5% CO2, random fields were photographed. The percentage of cells bearing a process greater than 1 cell diameter in length was determined.
In certain experiments the effect of substrate digestion with trypsin was studied. Substrate coated wells were treated with 0.25 to 0.00025%
trypsin (Sigma T-2904) in PBS for 10 minutes at room temperature. Wells were washed twice with 10% FCS containing cell culture medium. Neurite outgrowth was determined as described above.
Immunocytochemistry NG108-15 cells were grown on poly-L-lysine coated multi-chamber slides. Cells were washed in PBS and reacted with ascites at a -1:1000 dilution in PBS with 1.5 % horse serum for 1 hour. Cells were fixed with 4% paraformaldehyde in PBS for 5 minutes. After washing for 10 minutes cells were processed for peroxidase linked immunocytochemistry using diaminobenzidine (ABC kit Vector Labs, Burlingame, CA 94010). In 5 certain experiments cells were fixed before the application of the primary antibody.
Western Blotting Samples were separated and prepared for immunoblotting essentially as described in Ausubel et al., 1993, Current Protocols in Molecular10 Biology, Boston, Current Protocols, with transfer onto nitrocellulose membranes occurring overnight at 25 volts in 20% methanol transfer buffer.
Membranes were preincubated in PBS containing 3% milk powder for 1 hour, then rinsed and incubated in undiluted 10D supernatant for 2 hours, followed by three 10 min. washes in PBS with 0.1% Tween (PBS-T). The secondary Ab 15 (goat anti-mouse IgM, horseradish peroxidase conjugated) was applied as a 1:1000 dilution in PBS-T. All steps were at room temperature. Detection of the Ag/Ab complexes was accomplished by using the enhanced chemiluminescence (ECL) kit (Amersham) following the manufacturers instructions.
20 Library Screening A ~gtll adult rat brain cDNA expression library (Clontech) was screened with 10D following the protocol handbook provided with the library. In brief, the main steps were the following: E. coli Y109Or- cells wereinfected with 3x104 pfu per plate and after 3h incubation at 420C covered with 25 IPTG-treated NC-filters and incubated for another 3.5h at 370C. Filters were removed, rinsed in PBS with 0.1% Tween 20 (PBS-T) and blocked in PBS with 20% fetal calf serum for 2h at room temperature (RT). Subsequently, the filters were incubated in 10D hybridoma supernatant (1:5 dilution) for 2h at RT (hybridoma cells were grown in "Cell perfect protein-free" tissue culture 30 medium supplemented with Ab-enhancer (Stratagene); obtained Ab concentrations were 10 to 20 ,ug/ml). The secondary Ab (goat anti mouse -IgM-HRP conjugate, Biorad) was applied 1:1000 in PBS for lh at RT. Positive plaques were detected by using the ECL chemiluminescence kit from Amersham.
Phage Preparation, Subcloning and Sequence Analysis.
Phage lysates and DNA extracts were obtained following the protocols in the library handbook. For further analysis, cDNA inserts were cloned into the vector pBS-KS+ (Stratagene), and sequence analysis was performed using the AutoRead Sequencing kit and the ALF sequencing ~y~ . (Pharmacia). Primers were fluorescein-labeled T3- and T7 primers.
Sequence analysis and data base search were performed using the GCG
package.

NEURITE OUTGROWTH IS STRONGLY INHIBITED BY CNS MYELIN.
To test the influence of CNS myelin on neurite outgrowth, an in vitro assay was developed. A sucrose density fraction was prepared from adult rat brains following standard procedures for myelin isolation, and was estimated by Western blotting to be enriched four fold for the myelin-specific markers myelin basic protein and myelin associated glycoprotein (data not shown). This material is referred to as "CNS myelin"
below. The inhibitory properties of this material as a substrate for neurite growth were studied primarily with the NG108-15 rat neuroblastoma and glioma hybrid cell line. Cells of this line grow in 10% serum-supplemented medium, and with reduction of serum and transfer to lmM dibutyrlyl cyclic AMP-containing medium, undergo a phenotypic change that includes acquiring competence for activity-dependent acetycholine release (Christian et al, Brain Res. 147:261-276, 1978) and enhanced extension of neurites.
NG108-15 cells that had been induced with lmM dbcAMP for two days (herein called dbCAMPNG108 cells) were plated in tissue culture wells that had been treated with poly-L-lysine (PLL) alone, or PLL followed by an extract of 3 0 CNS myelin proteins.
Figure lA shows photomicrographs of representative fields of cultures of dbCAMPNG108 cells plated onto poly-L-lysine alone (a), poly-L-lysine followed by 20,ug/cm2 bovine serum albumin (BSA) (b) and poly-L-lysine followed by 20,ug/cm2 CNS myelin (c). Panel (d) shows a single dbCAMPNG108 cell growing on a myelin-free patch. The border between the myelin-coated ("cns") and uncoated ("pL" for poly-L-lysine) surfaces is emphasized with small arrowheads.
Figure lB shows the studies where bovine serum albumin or extracts from muscle, sciatic nerve and brain were dried onto poly-L-lysine coated wells at 20 ,ug/cm2. Equal numbers of dbCAMPNG108 cells were plated in each well. After 24 hours, random fields were photographed and the proportion of cells with a process greater than 1 cell diameter was determined.
10 to 16 independent wells were scored for each substrate. The error bars in Figure lB represent the standard error of the mean. * denotes statistically different from poly-L-lysine at p<0.025; ** denotes significant at p<0.01; ***
p<0.0005 using t-test.
As shown in Figure 1 the propensity for neurite extension by dbCAMPNG108 cells was significantly influenced by their substrate. 24 hours after plating onto a poly-L-lysine surface, 64%+5% (mean and standard error) of dbCAMPNG108 cells had neuritic processes greater than 1 cell diameter in length. The fraction of cells with processes was slightly reduced on wells coated with 20 ,ug/cm2 of bovine serum albumin (BSA) or with extracts of muscle proteins or peripheral nerve myelin that had been prepared in the same manner as the CNS myelin. In contrast, on adult rat CNS myelin, the elaboration of neural processes was strongly inhibited, with less than 2% of dbCAMPNG108 cells having significant neurites at 24 hours. Cells on CNS
myelin were generally round 24 to 72 hours after plating. Cells on poly-L-lysine displayed more spreading, more neurites and longer processes (Figure lA). In addition, whereas differentiated dbCAMPNG108 cells could be maintained for several weeks on poly-L-lysine surfaces, there were few 3 o remaining viable cells after seven days on CNS myelin coated surfaces.
The neurite growth inhibition by CNS myelin is contact -dependent. Myelin coated wells often contained a peripheral rim containing patches that were bare of myelin proteins. Cells in such areas displayed interesting properties but they were not scored in the assays. As shown in one such area in Figure lA, the arrest of neurite outgrowth is often limited to 5 those neurites in contact with the inhibitory substrate. Other processes on the same cell are seemingly not affected. This suggests that arrest of neurite advance occurs through a contact dependent mechanism restricted to the process in contact with inhibitor.
Investigations were carried out to determine whether the 10 inhibitory activity of CNS myelin was labile to trypsin digestion. Wells coated with 20 ,ug/cm2 of CNS myelin treated with 0.025% trypsin at room temperature for 10 minutes retained no significant inhibitory activity. On wells incubated with 0.00025% trypsin, approximately 10% of dbCAMPNG108 cells had processes at 24 hours.
Figure 2 shows the results of studies where CNS myelin was plated onto poly-L-lysine coated wells. The fraction of process bearing dbCAMPNG108 cells was determined at 24 hrs. Error bars in Figure 2 represent the standard error of the mean. Each point represents data from 2 to 10 wells.
The neurite growth inhibition by myelin protein enriched 20 CNS extract was concentration dependent. As shown in Figure 2, the fraction of process bearing dbCAMPNG108 cells decreased as the amount of plated myelin increased. The half maximal-inhibition of neurite outgrowth was observed at approximately 5 ,ug of proleill per cm2. This observation indicates that poor neurite outgrowth on CNS myelin is due to a concentration 25 dependent inhibition rather than a lack of trophic factor support. A similar concentration dependent inhibition of neurite growth on CNS myelin was observed with PC12 cells and primary newborn superior cervical ganglion (SCG) neurons (not shown). As discussed below, these results indicate that the in vitro assay detects an inhibitory activity that parallels the 30 previously-described CNS myelin inhibitor of neurite growth.

, AN ANTI-NEURONAL ANTIBODY INCREASES NEURITE OUTGROWTH
ON INHIBITORY CNS DERIVED SUBSTRATE.
To study the neuronal molecules that mediate the inhibition of neurite growth by adult CNS myelin proteins, monoclonal antibodies were generated to neural cell membranes and these antibodies were screened in vitro for their ability to promote outgrowth on this inhibitory substrate.
A panel of monoclonal antineuronal antibodies was produced by immunizing mice with a crude membrane preparation from NGF treated PC-12 cells. Those hybridoma pools which were positive against PC-12 membranes on an ELISA were tested for their ability to promote neurite outgrowth on CNS myelin. To screen the hybridoma library, PC-12 or dbcAMPNG108 cells were grown on 10 ~g/cm2 of CNS myelin in microtitre wells, with a 1:1 mixture of medium supplemented with NGF or dbcAMP, and antibody-containing hybridoma supernatants. Those hybridoma pools yielding supernatants that increased neurite production over control levels were plated at limiting dilutions to generate clonal lines. Ascites fluid was produced with one line called 10D, with the highest neurite promoting activity. dbCAMPNG108 cells were used predominantly in subsequent experiments because of their rapid growth and readily-induced neuronal di~r~llliation with a reliable proliferation of neurites.
Figure 3A shows photomicrographs of dbCAMPNG108 cells grown on poly-L-lysine alone or 10 ~1g/cm2 of CNS myelin showing that 10D
antibody reverses the growth inhibitory effect of CNS myelin. 5 ',ll of control ascites or 5 ',1l 10D antineuronal antibody ascites was added to the wells at the time of cell plating. Cells were photographed after 72 hrs in culture. The totalvolume in each assay was 105 ',ll. Bar = 100 llm.
Figure 3B shows the results of the quantitation of process-bearing cells grown on CNS myelin for 24 or 72 hours with 5,u1 per well of control ascites (filled bars) or 10D ascites (open bars).
Whereas few dbCAMPNG108 cells were able to extend neurites on 10 ,ug/cm2 of rat CNS myelin, antibody 10D was able to reverse this 21 740~5 -inhibition (Figure 3). Only 1-2% of dbCAMPNG108 cells extended neurites at 24 or 72 hours on CNS myelin in the presence of control ascites. In contrast, in the presence of 10D ascites, the fraction of cells with neurites greater than one cell diameter in length after 24 hours increased to 32% (Figure 3A). By 72 5 hours, the fraction of process bearing cells on myelin with 10D was equal to that on poly-L-lysine indicating that the antibody completely overcame growth inhibition on this non-permissive substrate (Figure 3A and 3B). 10D
ascites also neutralized the neurite growth inhibitory effects of myelin on PC-12 cells.
The biological activity of antibody 10D was also observed with primary neurons. Sympathetic superior cervical ganglion (SCG) neurons can be isolated from neonatal rats and maintained in culture in the presence of 100 ng/ml NGF and 10~M cytosine arabinofuranoside (AraC), under which conditions they survive and extend abundant neurites which fasciculate to 15 form bundles connecting aggregates of cell bodies (Hawrot and Patterson, Meth. Enzymol. 58:574-585, 1979). When these cells are plated on 10~lg/cm2 CNS myelin however, the neurons survive as aggregates of cell bodies but neurite formation is inhibited, as shown in Figure 16A. Addition of antibody 10D-containing ascites to a sister culture of SCG neurons on a CNS myelin 20 substrate causes reversal of the inhibition, allowing the formation of bundles of neurites as shown in Figure 16B. Therefore, antibody 10D may be useful to promote the growth of neurites by primary neurons in an inhibitory CNS
environment.
The improved outgrowth with 10D is not due to a 25 non-specific immunoglobulin effect since sister hybridoma supernatants and ascites derived from the same fusion, and control ascites derived from Sp2 cells (the hybridoma fusion partner), did not overcome neurite growth inhibition. The improved outgrowth with 10D antibody on this inhibitory substrate is unlikely to be due to non-specific blocking of myelin components 30 in the substrate since myelin-specific antibodies recognizing galactose cerebroside (GalC), myelin basic protein (MBP) and myelin associated `- 2174025 glycoprotein (MAG) did not promote neurite outgrowth. Similarly, immunoglobulin binding to neuronal cells is not sufficient to overcome myelin inhibition of neurite outgrowth since antibodies to tyrosine hydroxylase (TH), neural cell adhesion molecule (NCAM) and Thy-1 did not 5 block the growth inhibitory properties of the CNS myelin. The interaction between these control antibodies and neural cells or myelin was confirmed using immunocytochemistry and western blots.

10D RECOGNIZES dbCAMPNG108-15 AND PC12 CELLS
To confirm the interaction between antibody 10D and dbCAMPNG108 cells, these cells were processed for immunocytochemistry. In particular, dbcAMPNG108-15cells were grown on poly-L-lysine coated glass slides for 48 hours, fixed and processed for immunocytochemistry with 10D
ascites (b) or control ascites (a) each diluted 1:1000. A secondary antibody 15 linked to a biotin-avidin and diaminobenzidine ~y~Lem was used. As shown in Figure 4, the antibody reacted with the cellular soma, processes and growth cones. In certain cells, staining is heaviest at the cell surface but there was often a more diffuse staining throughout the cell body (Figure 4). Using peroxidase conjugated fluorescent labelled secondary antibodies gave similar 2 o results.

The 10D antibody blocks the effect on neurons in culture of a CNS myelin inhibitor that has been reported to affect the interactions of both 25 neurons and fibroblasts with substrates. To determine the molecular species recognized by the 10D antibody, proteins from tissues and cell lines were separated on denaturing SDS-polyacrylamide gels, transferred to nitrocellulose and reacted with serum-free hybridoma supernatant. In particular, two identical denaturing 13% poyacrylamide-SDS gels were each 30 loaded with marker proteins and 10 ',lg of protein from liver, cerebrum (both from 2 day old rats), dbCAMPNG108 cells and adult CNS myelin (same -preparation as was used as an inhibitory growth substrate), and run simultaneously. One gel was stained for total proteins with Coomassie Brilliant Blue (left) and the other transferred to nitrocellulose and reacted with the 10D monoclonal antibody. Also shown in Figure 5 is a lane from a 5 gel run and blotted separately, loaded with 10 ,ug of protein from NIH3T3 cells.
Enhanced chemiluminescence detection revealed several immunoreactive species (Figure 5). Prominent in adult rat cerebrum, rat liver, dbCAMPNG108 cells and mouse NIH3T3 fibroblast samples was a band of 10 Mr 35,000. This band did not correspond to any prominent Coomassie Blue stained species. All four samples also contained a slightly faster migrating band (Mr 33,000), reduced in intensity relative to the Mr 35,000 band to a similar degree in each case. Less intense immunoreactivity against some higher Mr species (Mr 60,000 to 100,000) was seen less consistently in some 15 samples. It was not determined whether these bands represent aggregates of the faster migrating species, or immunologically cross-reactive but molecularly distinct proteins. Two bands at Mr 14,000 and 18,000 were seen in many samples and correlated consistently in several independent experiments with the presence of two prominent Coomassie Blue bands.
2 0 These may be the result of a lower-affinity interaction with a pair of abundant, widely-expressed proteins. Reaction of 10D with myelin proteins resulted in only a weak signal co-localizing with the highly abundant myelin basic proleins.

25 D1: A cDNA CLONE CAPABLE OF MODIFYING NEURITE GROWTH
INHIBITION ON CNS MYELIN SUBSTRATES.
In order to clone a gene encoding a neuronally-expressed protein required for sensitivity to CNS myelin inhibition, the 10D MAb was used to screen a rat brain cDNA (adult) library in the vector ~gtll. Of 106 30 plaques, 11 rescreened positive and partial sequence data was obtained to permit preliminary identification. In addition, each insert was used to probe -blots of RNA from NG108 cells and brains of postnatal day 1 and adult rats.
Six of the eleven represented known brain-expressed sequences, while five were previously unreported. The following set of criteria were used to determine which of these eleven to pursue with further studies.
5 1) the cDNA must be expressed in NG108 cells, since these cells are inhibited by CNS myelin and this inhibition is modulated by MAb lOD.
2) the cDNA must be expressed in the CNS.
3) if previously described, the gene product must be one that can accommodate a role in the regulation of growth on inhibitory substrates.
Figure 6 is a schematic representing the strategy used to characterize clones selected with the lOD monoclonal antibody.
Clones Dl, D5, Dll and D12 met these criteria most clearly.
To determine which if any of these might represent a gene regulating neurite growth on CNS inhibitory substrates, a functional strategy was pursued. This 15 was to down-regulate, by cellular expression of antisense RNA, the gene products corresponding to specific cDNAs and then assay neurite growth characteristics on CNS myelin and non-inhibitory substrates (see Figure 7).
The cDNA inserts were subcloned from three novel clones, in antisense orientation, into the vector pBK-CMV, in which the strong 20 HCMV promoter can drive transcription in mammalian cells. These constructs were electroporated into NG108 cells, stable transfectants were selected with G418, and assayed for neurite growth on a permissive (PLL) and an inhibitory CNS myelin substrate. Ten individual lines derived from antisense D5 and D12 constructs all showed normal growth on PLL
25 (approximately 60-90% cells with identifiable processes) and normal inhibition on CNS myelin (1-4% cells with processes). In contrast, 15 out of 19 lines transfected with the antisense Dl construct showed significant enhancement of growth on myelin, with normal growth on several permissive substrates. The extent of neurite growth by antisense transformed 30 lines varied. For the line presented in Figure 7 (Dl/A3), 56% of cells grew processes on myelin, and addition of lOD antibody had little effect. Three -lines transformed with the D1 antisense cDNA construct showed no significant effects on growth on a variety of permissive substrates (data not shown).
To test whether the enhanced ability of some D1 antisense transformants to extend neurites on myelin was due to antisense inhibition of D1 gene expression, the selected clones were analyzed in greater detail.
The presence of stable integrated copies of the pBK-CMV-D1 construct was determined by probing gel blots of cellular DNA with the D1 cDNA insert.
Intact copies would be expected to include a 1.0 kb EcoRI fragment representing the cDNA sequence in the construct, that would not be present in the genomic DNA of the parental NG108 cells. Figure 8 shows the Southern blot of EcoRI digested genomic DNA from NG108 cells (parental cells) and the transformed cell line A3 probed with the lkb D1 cDNA insert.
The control lane contains 10pg of D1 insert (EcoRI-fragment). Figure 8 shows that clone A3 has, in addition to numerous bands also present in NG108 cells and presumed to arise from the endogenous D1 genes, the expected 1 kb hybridizing fragment. Thus, it was shown that some clones of NG108 cells transfected with the D1 antisense construct, but not other cDNA antisense constructs, have acquired the ability to grow neurites on an inhibitory CNS
myelin substrate.
Figures 9 and 11 (SEQ. ID. NOS. 1 and 3) show the sequence of two fragments from each end of the cDNA clone D1. The first nucleotides of each fragment is the EcoRI site added in the linker used in the library construction. There is an unsequenced gap of apporximately 200 bp separating the two sequences. Computerized database searching of the portion sequenced (Figures 9 and 11 and SEQ. ID. NOS. 1 and 3) indicated no previous reports of substantially similar sequences from any species (Genbank release 84.0; EMBL
release 39.0). Sequences of other fragments of the cDNA clone are shown in Figures 12 to 14 (SEQ. ID. NOS.4 to 6).
Additional cDNA sequence data was obtained (SEQ. ID.
NOS.7-9, Figure 21). The salient features of the data are, 1) that it extends the open reading frame significantly in the 5' direction, and 2) that there is a gapof about 100-300 bp near the 5' end. It is likely that the open reading frame continues on the 5' side of this gap (see ORFs in this region).SEQ ID. NOS. 8 and 9 show the nucleotide and amino acid sequence of the downstream 5 portion of the gene. A new diagram of the cloned regions of D1 is shown in Figure 22.
The partial cDNA sequence of D1 will be extended by rescreening to isolate overlapping cDNAs. Both commercially obtained libraries, as well as a ~gtlO library rigorously selected for long inserts, will be 10 screened. The inhibition-reversing properties of D1 will be tested independently by transfecting NG 108 cells with antisense constructs of non-overlapping fragments derived from the D1 gene. Additional sequence data will be determined to predict the primary structure of the encoded protein.
Additional independently-isolated clonal D1 transfectants (both sense and antisense) are being grown up for functional and molecular characterization. Cells will be grown on both inhibitory CNS and permissive substrates, and neurite growth quanititated by the standard procedures described herein. Lines with different levels of neurite growth will be identified for correlation with molecular data. It is expected that antisense (AS) RNA derived from other regions of the D1 gene will be effective;
therefore, AS constructs will be made with newly-isolated portions of the D1 cDNA as they are obtained. PC12 cells will also be transfected with the antisense DI construct. The resulting lines will provide an independent test of the inhibition-blocking activity of D1 antisense, and will also be used to probe the intracellular pathways mediating inhibition and its reversal.
Since the level of expression in NG108 cells is low an RNase protection assay may be used which is capable of measuring individually sense and antisense D1 transcripts (Melton, D.A., 1984, Nucleic Acids Res.
12:7035-7056). This assay will be used to correlate steady-state mRNA levels with myelin growth characteristics. If the mechanism of reversal of -inhibition is interference with mRNA processing or promotion of degradation due to duplex formation, mRNA levels will be reduced.
Alternatively mRNA levels could remain constant if the mechanism involves a specific inhibition of translation.

REGENERATION OF OPTIC AXONS
Adult female Sprague-Dawley rats are anaesthetized using ketamine (40-80mg/kg) and Xylazine (5-lOmg/kg) IP or IM. In anaesthetized rats the left optic nerve is exposed with the aid of a microscope and crushed with a liquid nitrogen cooled jewellers forceps 3mm behind the globe. The supraorbital incision is closed and the animal is allowed to recover for 2-4 weeks. Animals are then reanaesthetized and 5 ,ul of anterograde tracer (3%
Rhodamine Isothiocyanate or Horseradish Peroxidase) is injected intraoccularly with the Hamilton Syringe. After two days animals are given an anaestheitc overdose and perfused transcardiacly with 4%
paraformaldehyde in phosphate buffered saline (PBS). The optic nerve is removed and sectioned with a cryostat. The sections are processed for neurofilament immuncytochemistry and to detect anterograde tracers to determine the position of the distal end of the optic axons. In experimental animals 108 hybridoma cells in 100 ~l secreting 10D antibody or an irrelevant sister control antibody are deposited at the site of optic nerve crush at the time of surgery. At the time of sacrifice, a sample of cerebrospinal fluid will be obtained for the detection of secreted mouse Ig to prove the production and delivery of 10D antibody within the spinal canal. In certain experiments the application of 10D secreting hybridoma cells will be combined with the intraoccular administration of 300-500 llg of NGF, BDBF, FGF or saline using a Hamilton syringe. Because these growth factors have been shown to enhance RGC survival after injury, it is expected that in combination with 10D antibody, they will lead to a greater enhancement of axonal growth in the 3 o injured CNS over the use of 10D antibody alone.

-REGROWTH OF CENTRAL PROCESSES OF DORSAL ROOT GANGLION
NEURONS
Adult female Sprague-Dawley rats are anaesthetized using ketamine (40-80mg/kg) and Xylazine (5-lOmg/kg) IP or IM. Using the microscope animals have a laminectomy to expose the lumbar spinal cord.
The L5 dorsal root is crushed proximal to the dorsal root ganglia 106 antibody secreting hybridoma cells are deposited at the surgical site. 10D secreting cells and sister clones producing irrelevant control antibodies will be used. Axons are allowed to regenerate towards the spinal cord for 2-4 weeks. Animals are re-anaesthetized, the L5 dorsal root ganglion is re-exposed and injected with an anterograde tracer as described above. After 48 hours anmials are sacrificed by anaesthetic overdose and they are perfused with 4% paraformaldehyde in PBS. The spinal cord is collected and processed for GAP-43 immunocytochemistry to visualize axons in a phase of growth and to visualize the anterograde tracers. At the time of sacrifice, a sample of cerebrospinal fluid will be obtained for the detection of secreted mouse immunoglobulin by Western blotting to demonstrate the production and delivery of 10D antibody within the spinal canal. These techniques will determine the extent of axonal regrowth within the spinal cord.

The D1 cDNA can be used to identify, study and isolate the corresponding human gene. This will make possible the study of the suspected role of the D1 gene and its ~rolei~l product in development of the nervous ~yslen- and in regeneration in the adult, as well as other more general roles in cell-substrate interactions, as suggested by in vitro data. It will allow the cloning of the human gene and its expression for use in drug discovery applications to find potential therapeutic agents that enhance regrowth of injured nerve fibres in the CNS.
The D1 cDNA sequences from position bp230 to bpl,095 (as shown in the Sequence Listing as SEQ. ID. NO. 7 and in Figure 21) were '_ shown to specifically recognize human genomic DNA fragments similar in number to those recognized in rat and mouse DNA (Figures 17 and 18).
Further, it has been shown using a panel of human-rodent hybrid cell lines (NIGMS Human Genetic Mutant Cell Repository, Coriell Inst. for Medical 5 Res., Camden, N.J. 08103, USA) that all the D1 gene sequences detected in the human genome reside on chromosome 12 (Figures 17 and 18). The success of this mapping indicates that it will be possible to use the D1 probes described herein to determine whether human disorders are genetically linked to the D1 gene.
In particular, Figures 17 and 18 shows that the D1 cDNA
recognises corresponding human sequences and localizes them to chromosome 12. 10~1g of DNA was digested with EcoRI, electrophoresed, transferred to nylon membrane, and hybridized with the D1 (bp230 to bpl,O95) probe. Samples were hamster, human or mouse genomic DNA (as marked), or DNA from hybrid cell lines containing mostly mouse or hamster chromosomes and the human chromosome marked. The human specific bands appear only in the hybrid DNA from the "Hamster/Chl2" line.
It has also been demonstrated that the rat D1 cDNA described herein can be used to detect human D1 mRNA and study its expression in normal tissue and in disease. Figure 19 shows that the rat D1 seqences bp230 to bpl,O95 can detect the corresponding human RNA, in this case isolated from a surgically removed lung metastatic tumour. The transcripts detected are of the same gel migration and similar abundance to those detected in RNA from rat brain tissue and cell lines.
In particular, Figure 19 shows that the D1 cDNA recognizes human RNA transcripts. 12,ug of total RNA from a human metastatic tumour of lung origin, and adult rat brain, was denatured, electrophoresed, transferred to nylon and hybridized with D1 cDNA (bp230 to bplO95) probe.

To study the relevance of the CNS myelin/neurite `- 21 74025 outgrowth assay of the invention to the human, a bioassay was developed which uses human brain-derived myelin as a substrate. The results indicate that powerful neurite outgrowth inhibitory activity is present in human CNS myelin. Human CNS myelin strongly inhibits neuritic outgrowth from 5 newborn rat dorsal root ganglion neurons and NG-108-15 cells. The inhibition increases with increasing myelin concentration (Figure 20). It is dependent on the direct contact between neurites and the myelin substrate.
The inhibitory activity in human CNS myelin closely resembles the myelin inhibition of neurite growth that is observed with adult rodent CNS myelin.
10 The inhibition of neurite outgrowth by human CNS myelin can be used as a model to develop strategies to enhance neural recovery and repair in the injured Human CNS.

Sequence Analysis Based on overlapping partial cDNA clones (isolated from a ratbrain cDNA expression library) 4515bp were sequenced which contain an open reading frame (ORF) of 1941bp encoding 647aa. Three methionine codons are located at positions 436, 475 and 486, the third of which is precededby a Kozak consensus sequence. Based on this finding, this ATG is the most likely site for translational initiation and the predicted molecular weight for the encoded protein would be approximately 60kD. The protein, designated as Petrin, contains two putative tyrosine phosphorylation sites at the C-terminus. No transmembrane domain or other known motifs could be found in the sequence. The most related known protein to date is protein phosphatase 2C (PP2C), a ser/thr phoshatase isolated from a number of species and different tissues ( Mann, D.J. Et al., Biochim Biophys Acta 1130:100-4, 1992; Hou, E.W. Et al., Biochem Mol Biol Int 32:773-780, 1994;
Terasawa, T. Et al., Arch Biochem Biophys 307:342-9, 1993; Kato, S., et al., Arch Biochem Biophys 318:387-393, 1995; andT. Kuromori & Yamamoto, Nucleic Acids Res 22: 5296-5301, 1994). Distinct regions of petrin exhibit amino acid identities/similarities of up to 63%, the over all homology is below 20%.

-DNA and RNA hybridization experiments confirmed the presence of highly related genes in mouse, hamster and human. The human petrin homologue could be localized on chromosome 12.
ession: Northern blot and in situ Analyses Expression of petrin appears to be brain specific. Petrin mRNA is not detectable in liver, spleen, muscle, or fibroblasts. In rat brain, its expression is developmentally regulated. It is first detectable after embryonic day E13, increases steadily with age and is highest in the adult rat.
In situ hybridizations using a DIG-UTP-labeled RNA probe (3') revealed that petrin is specifically expressed in neurons. Staining is found all over the brain to different degrees, with some regions containing intensely stained neurons (e.g.: cerebellum: Purkinje cells; cortex: cells in the 3rd and 4th layer; hippocampus).
Functional analysis Polyclonal antibodies were generated in rabbits against a GST-fusion protein containing the C-terminal 210aa of Petrin. One of two antisera (#11) specifically precipitates a 60-64kD protein (from 35S-labelled NG108 cell lysates). The antibody (Ab) is not functional in western blots, nor does it block neurite growth inhibition on myelin substrate.
The immunoprecipitates from rat brain and NG108 cells exhibit Mg2+ dependent phosphatase (Pase) activity. This activity is ca. 5-fold higher when performed with a ser/thr phospho-peptide than with a tyr phospho-peptide. When transfecting COS cells with an expression construct containing the complete ORF, specific Pase activity can be precipitated using Ab #11. The majority of the activity is found in the cytosolic fraction of ccll lysatc after crude fractionation into membranes and soluble protein.
Peptide sequences containing an HA (hemagglutinine) epitope were regenerated and cloned into the N'- and C'-terminal regions of the protein (into the BsiWI and the EcoRV sites, respectively). When introducing expression constructs containing the HA-tagged derivatives of Petrin into COS cells and using an anti-HA monoclonal antibody for -immunoprecipitation of COS cell lysates, Pase activity could be detected with the 3'HA-tagged petrin, and to a lesser extent (1/3) with the 5'HA-tagged petrin (control negative). In Western blots the anti-HA Ab specifically detects a band at appr. 60kD in lysates from ORF-HA transfected COS cells.
Application of antisense oligonucleotides on NG108 cells and growth on myelin sul,slrale An oligonucleotide (GCT GCC AGC CAT GAT ) overlapping the two distal ATGs, i.e. the assumed translational start site, was applied for 4 days to cAMP-treated NG108 cells (final concentration l,umol, for 3 days every 24th and on the fourth day every 12h), and those cells where subsequently plated on myelin. 45% of the antisense treated cells extended neurites on myelin, whereas 7% of cells treated with a scrambled version and 2% of the untreated cells showed neurite growth. On permissive substrate poly-L-lysine 80-90% of the cells extended neurites.
Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those skilled in the art that the invention can be modified in arrangement and detail without departure from such principles. We claim all modifications coming within the scope of the following claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
The following sequence listings form part of the application.

SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: Roach, Arthur Lozano, Andres Labes, Monika Roder, John (ii) TITLE OF INVENTION: Novel Agents Modulating the Response of Neuronal Cells to Inhibition by Mammalian Central Nervous System Myelin (iii) NUMBER OF SEQUENCES: 10 (iv) CORRESPO~N~ ADDRESS:
(A) ADDRESSEE: BERESKIN & PARR
(B) STREET: 40 King Street West (C) CITY: Toronto (D) STATE: Ontario (E) COUNTRY: Canada (F) ZIP: M5H 3Y2 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Kurdydyk, Linda M.
(B) REGISTRATION NUMBER: 34,971 (C) REFERENCE/DOCKET NUMBER: 3153-167 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (416) 364-7311 (B) TELEFAX: (416) 361-1398 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 339 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Rattus (F) TISSUE TYPE: Central Nervous System (G) CELL TYPE: Neuroblastoma (H) CELL LINE: NG108 (vii) IMMEDIATE SOURCE:
(A) LIBRARY: Lambda gt 11 Adult Rat Brain cDNA Expression Library (B) CLONE: Dl T7 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 10..276 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Gly Lys Cys Tyr Thr Thr Lys Thr Asp Arg His Leu Arg His Cys Ser Gly Gln Asn Tyr Gly Ala Gln Asn Met Gly Leu Val Arg Met Gly Trp Phe Leu Ile Trp Lys Leu Ser Ser Asp Asn Leu Glu Ser Pro Gly Gly Gly Lys Trp Glu Arg Trp Glu Lys Cys Gln Lys Asn Lys Asn Lys Thr Lys Lys Lys Pro Lys Lys Thr Leu Pro His Pro Thr Ile Thr Thr Lys Glu Tyr Arg Glu Thr Glu Gly Ala Gly (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 89 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) ~Q~ DESCRIPTION: SEQ ID NO:2:
Gly Lys Cys Tyr Thr Thr Lys Thr Asp Arg His Leu Arg His Cys Ser ly Gln Asn Tyr Gly Ala Gln Asn Met Gly Leu Val Arg Met Gly Trp Phe Leu Ile Trp Lys Leu Ser Ser Asp Asn Leu Glu Ser Pro Gly Gly Gly Lys Trp Glu Arg Trp Glu Lys Cys Gln Lys Asn Lys Asn Lys Thr Lys Lys Lys Pro Lys Lys Thr Leu Pro His Pro Thr Ile Thr Thr Lys Glu Tyr Arg Glu Thr Glu Gly Ala Gly (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:

-(A) LENGTH: 434 base pairs (B) TYPE: nucleic acid (C) STRAN~ S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) ORGANISN: Rattus (F) TISSUE TYPE: Central Nervous System (G) CELL TYPE: Neuroblastoma (H) CELL LINE: NG108 (vii) INNEDIATE SOURCE:
(A) LIBRARY: Lambda gt 11 Adult Rat Brain cDNA Expression Library (B) CLONE: Dl T3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GAATTCCGGC CTGAAGGTTC ATGACTCCAA CATCTACATA AAACCATTCC ~ CAGC 60 TCCAGAGGTC AGAGTCTACG Al~l~lCCAA ATACGAGCAC GGAGCGGATG ACGTGCTGAT 120 TCA~ll~ CCTAACTGTG ATCCAGATGA CCCTCACAGG TACACACTGG CAGCTCA6GA 240 C~l~G~lGATG CGTGCCCGAG GCGTCCTGAA GGACCGAGGA TGGCGGATAT CAAATGACCG 300 ACTGGGCTCA GGAGATGACA lll~l~lATA CGTCATTCCT TTAATACACG GAAACAAACT 360 (2) INFORMATION FOR SEQ ID NO:4:
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(vi) ORIGINAL SOURCE:
(A) ORGANISM: Rattus (F) TISSUE TYPE: Central Nervous System (G) CELL TYPE: Neuroblastoma (H) CELL LINE: NG108 (vii) INMEDIATE SOURCE:
(A) LIBRARY: Lambda gt 11 Adult Rat Brain cDNA Expression Library (B) CLONE: Dl-l T3 (xi) ~Q~N~ DESCRIPTION: SEQ ID NO:4:

TA~ A GGTATGGTCC CCCAACTTTT CAATGTGGCT CTTCTCCTGG AGAGATGCCT 120 -~2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 479 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Rattus (F) TISSUE TYPE: Central Nervous System (G) CELL TYPE: Neuroblastoma (H) CELL LINE: NG108 (vii) IMMEDIATE SOURCE:
(A) LIBRARY: Lambda gt 11 Adult Rat Brain cDNA Expression (B) CLONE: D1 4T3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

(2) INFORMATION FOR SEQ ID NO:6:

(i) ~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 487 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Rattus (F) TISSUE TYPE: Central Nervous System (G) CELL TYPE: Neuroblastoma (H) CELL LINE: NG108 tvii) IMMEDIATE SOURCE:
(A) LIBRARY: Lambda gt 11 Adult Rat Brain cDNA Expression Library (B) CLONE: D1 5T7 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

~ GAAG GGGTGACTTA GAGTCTCAGG GAACAACGAG GCCCACACAG AGTAAGCCCA 180 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4262 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) ~Q~ DESCRIPTION: SEQ ID NO:7:

-~N~ NCGGCCTGCT GCAGCACCAA TCACACAACA GCTGCAGGAC ATCGTGGAGA 720 GGATGTCTTA TCAAATGAAG AAGTAGCGGA AGCAATCACT CA~ C CTAACTGTGA 1560 TGCTGCCAAA AGGCCTCCTG GCTTTGCTTC AAGGAGACCC TTGGGTTTCC ~'l"l"l'~'l"l'~'l"l' 1920 ~ GT TTCTACAGTA GGGCTCCTAG GCGGCAGCAG CCTATTTGGG GCGCAGGCAC 2040 GATCCTTCCT GCCCCCCATT TCCTGCCCCC TCC~'l"l"l'~'lC TATATTCTTT TGTGGTGATG 2280 GTGGGGTGAG GCAGAGTTTT TTTGGGTTTT 'l"l"l"l"l'~'l"l"l"l' GTTTTTGTTT TTTTGACATT 2340 AGATCAGAAA CCAGCCCATT CTGACTAGTC CCATGTTCTG TGCCCCATAG ~l"l"l~l~lCCAG 2460 TGGGGTTATC TTTAAGGCCC ~l~l~l~l~GT CTGGAGGCTC TGCCACGAGA GGCTGGGTTT 2640 AGGGAAGGGG GTGTGGGATT CAGGCTCCTT GGTCGCTGAC ~ lCCAG GGCACAGGAG 2820 GATCGAGTGT CACAGCTAGC TAAGCAGCAG CTCTTCCTGA CAC~lll~l~G CAAGGATAAC 2880 GCCCGGCAGG TGACATACTG TGAGl~ G CCAACTCGCT GCCTGAGGAC TGAGCTGTGG 3480 TGCTGGAAGT ~ GGC AACCATTGCT TBCCTGACAG AATACAATGC TGTGGGAAAC 3600 TAGGGAGTAT ~ GG~ C CCCGTGCCTT TCATATTTTT G~ GA CCTCCCGAGT 3780 GGA~ CCGTGAGGCC GCCGCTGTGT GGAAAGATCT TAGTTTGTGT TGCAGGGGGT 4020 GTGTGGGCCT C~ll~llCCC TGAGACTCTA AGTCACCCCT TCACAGAACA GGATGGACCT 4200 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3590 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

-( ix ) FEATURE:
(A ) NAME / KEY: CDS
(B) LOCATION: 1. .3590 ( ix ) FEATURE:
(A) NAME/KEY: mat_peptide (B) LOCATION: 1. .3590 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Arg Pro Ala Ala Ala Pro Ile Thr Gln Gln Leu Gln Asp Ile Val Glu Ile Leu Lys Asn Ser Ala Ile Leu Pro Pro Thr Cys Leu Gly Glu Glu Pro Glu Ser Thr Pro Ala His Gly Arg Thr Leu Thr Arg Ala Ala Tyr Cys Val Glu Gly Trp Val Pro Pro Ala Pro Pro Ala His His Pro Arg Ala Ser Ser Arg Arg Arg Arg Phe Leu Met Ser Val Trp Ser Ser Gly Pro Trp Arg Ala Pro Ser Arg Lys Trp Thr Phe Lys Leu Asn Gly Arg Gly Val His Ile Ile Tyr Pro Val Ala Ala Gln Pro Ser Ser Trp Phe Ala Phe Trp Gly Ser Ser Thr Trp Gln Met Gln Val Thr Ala Gly Pro * Ser Ser Glu Met Glu Lys Ser Ser Pro Cys Leu Pro Asn Ser Pro Leu Arg Leu Ser Val Arg Asp Phe Ser Thr Trp Arg Ser Cys Ser Leu Thr Cys Trp Glu Met Ser Ser His Thr Trp Ser Phe Gln Gly Glu Tyr Arg Gly Lys Asn Ser Gly Arg Arg Cys Cys Thr Gly Thr Leu Thr *

Gln Ala Gly His Thr Lys Pro Leu Arg Met Met Thr * Ser Phe Pro Leu Tyr Met Glu Lys Ala Arg Arg Pro Gly * Trp Gln Leu Leu Glu -* Gln Gly Asp Leu Gly Thr Met Thr * Arg Phe Met Thr Pro Thr Ser Thr * Asn His Ser Cys Leu Gln Leu Gln Arg Ser Glu Ser Thr Ile Ser Pro Asn Thr Ser Thr Glu Arg Met Thr Cys * Ser Trp Leu Leu Met Asp Ser Gly Met Ser Tyr Gln Met Lys Lys * Arg Lys Gln Ser Leu Ser Phe Phe Leu Thr Val Ile Gln Met Thr Leu Thr Gly Thr His Trp Gln Leu Arg Thr Trp * Cys Val Pro Glu Ala Ser * Arg Thr Glu Asp Gly Gly Tyr Gln Met Thr Asp Trp Ala Gln Glu Met Thr Phe Leu Tyr Thr Ser Phe Leu * Tyr Thr Glu Thr Asn Cys His Glu Ser Asp Pro Gly Asp * Glu Asp Arg Arg Arg Glu Glu Asn Trp Gly Ala Ser Lys Gln Gly Gly Thr Gly Glu * Val Pro Gly Leu Asp Ser Arg * Arg Thr Phe Ser Pro Ala Gln Val Gly Asn Leu Leu Pro Lys Gly Leu Leu Ala Leu Leu Gln Gly Asp Pro Trp Val Ser Val Ser Ser Leu Val Thr Val Trp Thr Leu Asn Lys Ser Lys Thr His Ser Leu Leu Leu Arg Val Thr Ser Leu Leu Val Ser Thr Val Gly Leu Leu Gly Gly Ser Ser Leu Phe Gly Ala Gln Ala His Val Tyr Leu Phe Tyr Leu Glu * Gln Gly Gln Pro Phe Arg Val * Leu Val Lys Asp Phe Ser Ala Cys Gln Ala Ala Ser Ile Ser Pro Gly Glu Glu Pro His * Lys Val .

Gly Gly Pro Tyr Leu Arg Lys Tyr Glu Gln Ser Ser His Cys Val Phe Gln Leu Phe Gln Gly Ser Phe Leu Pro Pro Ile Ser Cys Pro Leu Arg Phe Ser Ile Phe Phe Cys Gly Asp Gly Gly Val Arg Gln Ser Phe Phe Gly Phe Phe Phe Val Leu Phe Leu Phe Phe * His Phe Ser His Leu Ser His Phe Pro Pro Pro Gly Leu Ser Lys Leu Ser Leu Asp Ser Phe Gln Ile Arg Asn Gln Pro Ile Leu Thr Ser Pro Met Phe Cys Ala Pro * Phe Cys Pro Glu Gln Cys Leu Arg * Arg Ser Val Phe Val Val * His Leu Pro Arg Gly Ser Val Phe Leu Pro Lys * Ile Cys Leu Leu Gln Asn Met Gly Val Ser Leu Asp Gln Trp Phe Leu Gly Leu Ser Leu Arg Pro Leu Cys Val Ser Gly Gly Ser Ala Thr Arg Gly Trp Val Cys Gly Ser Gly Leu Ala Ile Leu Pro Ala Phe Cys Val Leu Glu Arg His Arg Lys Gln Arg Ser Gly Pro Arg Ser Lys Pro Gly Ser Cys Tyr Pro Ala Leu Asp His Val Arg Val Glu Gly Arg Gly Cys Gly Ile Gln Ala Pro Trp Ser Leu Thr Val Leu Gln Gly Thr Gly Gly Ser Ser Val Thr Ala Ser * Ala Ala Ala Leu Pro Asp Thr Phe Val Gln Gly *

Leu Gly * His Leu Asn Lys Ser Glu Phe Glu Leu Gln Leu Val Ile 2l 74025 .

Val Met Pro Pro Ser Pro Phe Thr Leu Leu Arg Ser Pro Ser Phe Tyr Ala Ser Thr Gly Val Cys Ala Ser Val Gly Thr His Gly His Met Ser Thr * Ala Gln Tyr Leu Ser Pro Ala Ser Leu Leu Gly Gly Pro Ala Lys Ser Ser Leu Lys Ile Ser Arg Leu Leu Gly Arg Leu Leu Leu Cys Pro Gly Ala Phe Arg Val Ala Ser Gly Cys Leu Leu Val Leu Phe Leu Leu Ser Ser Arg Ile Ser Ala Phe * Gln Leu Gly Ile Asn Gln Thr Phe Pro Thr Gln Glu Trp Ile Gln Trp Cys His Phe Pro Lys Met Leu Glu Glu Lys Ala Val Pro Thr Ser Gln Gly Ser Gly Ala Phe Pro Pro Arg Arg Lys Ala Val His Ile Pro Arg Ile Val Asp Ile Phe Leu Gly Glu Gly Leu Val Pro Ser His Ser Gly Thr Lys Ser Pro His Cys Val Glu Pro Gly Pro Ser Pro Ala Gly Asp Ile Leu * Val Cys Ala Asn Ser Leu Pro Glu Asp * Ala Val Ala Met Leu Gly Ala Pro Thr Cys Ala Ser Phe Ser Gly Cys Phe Pro Leu Leu Thr Pro Met Leu Glu Val Phe Cys Gly Asn His Cys Phe Leu Thr Glu Tyr Asn Ala Val Gly Asn Cys Ser Gly Thr Leu Gln Gln Arg Ser Pro Leu Pro Ala Arg Ala Pro Phe Ser Lys Ser His Thr Asn Gly Lys Leu Glu Lys Val Leu Asn Ser Ala Glu Asp Leu Ser Cys Leu Pro * Gly Val Phe Leu Gly Ser Pro -Cys Leu Ser Tyr Phe Cys Phe Ser Asp Leu Pro Ser Leu Thr Leu Thr Phe Phe Asn His Ile Gln Ala Ser Cys Arg Ile Gly Val * Ile Ser Cys Arg Cys His Asp Asn Ile Leu Pro Val His Val Pro Ser Asn Gln Thr Lys Pro Asn Pro Lys Thr Gln Ile Gln Ile Thr Leu Lys Pro Gly Val Leu Ala Gly Phe Ser Trp Lys Gly Val Leu Ser Val Arg Pro Pro Leu Cys Gly Lys Ile Leu Val Cys Val Ala Gly Gly Gly Ala Gln Gly Arg Arg Leu Gln Met Lys * Ile Asn Gln Lys Trp Gly Lys * Phe Val Asn Ala His Gly Arg Ser Ala Gln His * Tyr Cys Ser Val Leu Trp Ala Tyr Ser Val Trp Ala Ser Leu Phe Pro Glu Thr Leu Ser His Pro Phe Thr Glu Gln Asp Gly Pro Val * Gln Val Gly Pro Trp Arg Asn Ser Trp Glu Ala Lys Gly Gln His Thr Gln Asn (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1196 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Arg Pro Ala Ala Ala Pro Ile Thr Gln Gln Leu Gln Asp Ile Val Glu Ile Leu Lys Asn Ser Ala Ile Leu Pro Pro Thr Cys Leu Gly Glu Glu Pro Glu Ser Thr Pro Ala His Gly Arg Thr Leu Thr Arg Ala Ala Tyr -Cys Val Glu Gly Trp Val Pro Pro Ala Pro Pro Ala His HiS Pro Arg Ala Ser Ser Arg Arg Arg Arg Phe Leu Met Ser Val Trp Ser Ser Gly Pro Trp Arg Ala Pro Ser Arg Lys Trp Thr Phe Lys Leu Asn Gly Arg Gly Val His Ile Ile Tyr Pro Val Ala Ala Gln Pro Ser Ser Trp Phe Ala Phe Trp Gly Ser Ser Thr Trp Gln Met Gln Val Thr Ala Gly Pro * Ser Ser Glu Met Glu Lys Ser Ser Pro Cys Leu Pro Asn Ser Pro Leu Arg Leu Ser Val Arg Asp Phe Ser Thr Trp Arg Ser Cys Ser Leu Thr Cys Trp Glu Met Ser Ser HiS Thr Trp Ser Phe Gln Gly Glu Tyr Arg Gly Lys Asn Ser Gly Arg Arg Cys Cys Thr Gly Thr Leu Thr *

Gln Ala Gly His Thr Lys Pro Leu Arg Met Met Thr * Ser Phe Pro Leu Tyr Met Glu Lys Ala Arg Arg Pro Gly * Trp Gln Leu Leu Glu * Gln Gly Asp Leu Gly Thr Met Thr * Arg Phe Met Thr Pro Thr Ser Thr * Asn His Ser Cys Leu Gln Leu Gln Arg Ser Glu Ser Thr Ile Ser Pro Asn Thr Ser Thr Glu Arg Met Thr Cys * Ser Trp Leu Leu Met Asp Ser Gly Met Ser Tyr Gln Met Lys Lys * Arg Lys Gln Ser Leu Ser Phe Phe Leu Thr Val Ile Gln Met Thr Leu Thr Gly Thr HiS Trp Gln Leu Arg Thr Trp * Cys Val Pro Glu Ala Ser * Arg Thr Glu Asp Gly Gly Tyr Gln Met Thr Asp Trp Ala Gln Glu Met Thr Phe Leu Tyr Thr Ser Phe Leu * Tyr Thr Glu Thr Asn Cys His Glu Ser Asp Pro Gly Asp * Glu Asp Arg Arg Arg Glu Glu Asn Trp Gly Ala Ser Lys Gln Gly Gly Thr Gly Glu * Val Pro Gly Leu Asp Ser Arg * Arg Thr Phe Ser Pro Ala Gln Val Gly Asn Leu Leu Pro Lys -Gly Leu Leu Ala Leu Leu Gln Gly Asp Pro Trp Val Ser Val Ser Ser Leu Val Thr Val Trp Thr Leu Asn Lys Ser Lys Thr His Ser Leu Leu Leu Arg Val Thr Ser Leu Leu Val Ser Thr Val Gly Leu Leu Gly Gly Ser Ser Leu Phe Gly Ala Gln Ala His Val Tyr Leu Phe Tyr Leu Glu * Gln Gly Gln Pro Phe Arg Val * Leu Val Lys Asp Phe Ser Ala Cys Gln Ala Ala Ser Ile Ser Pro Gly Glu Glu Pro His * Lys Val Gly Gly Pro Tyr Leu Arg Lys Tyr Glu Gln Ser Ser His Cys Val Phe Gln Leu Phe Gln Gly Ser Phe Leu Pro Pro Ile Ser Cys Pro Leu Arg Phe Ser Ile Phe Phe Cys Gly Asp Gly Gly Val Arg Gln Ser Phe Phe Gly Phe Phe Phe Val Leu Phe Leu Phe Phe * His Phe Ser His Leu Ser His Phe Pro Pro Pro Gly Leu Ser Lys Leu Ser Leu Asp Ser Phe Gln Ile Arg Asn Gln Pro Ile Leu Thr Ser Pro Met Phe Cys Ala Pro * Phe Cys Pro Glu Gln Cys Leu Arg * Arg Ser Val Phe Val Val * His Leu Pro Arg Gly Ser Val Phe Leu Pro Lys * Ile Cys Leu Leu Gln Asn Met Gly Val Ser Leu Asp Gln Trp Phe Leu Gly Leu Ser Leu Arg Pro Leu Cys Val Ser Gly Gly Ser Ala Thr Arg Gly Trp Val Cys Gly Ser Gly Leu Ala Ile Leu Pro Ala Phe Cys Val Leu Glu Arg His Arg Lys Gln Arg Ser Gly Pro Arg Ser Lys Pro Gly Ser Cys Tyr Pro Ala Leu Asp His Val Arg Val Glu Gly Arg Gly Cys Gly Ile Gln Ala Pro Trp Ser Leu Thr Val Leu Gln Gly Thr Gly Gly Ser Ser Val Thr Ala Ser * Ala Ala Ala Leu Pro Asp Thr Phe Val Gln Gly *

Leu Gly * His Leu Asn Lys Ser Glu Phe Glu Leu Gln Leu Val Ile -Val Met Pro Pro Ser Pro Phe Thr Leu Leu Arg Ser Pro Ser Phe Tyr Ala Ser Thr Gly Val Cys Ala Ser Val Gly Thr His Gly His Met Ser Thr * Ala Gln Tyr Leu Ser Pro Ala Ser Leu Leu Gly Gly Pro Ala Lys Ser Ser Leu Lys Ile Ser Arg Leu Leu Gly Arg Leu Leu Leu Cys Pro Gly Ala Phe Arg Val Ala Ser Gly Cys Leu Leu Val Leu Phe Leu Leu Ser Ser Arg Ile Ser Ala Phe * Gln Leu Gly Ile Asn Gln Thr Phe Pro Thr Gln Glu Trp Ile Gln Trp Cys His Phe Pro Lys Met Leu Glu Glu Lys Ala Val Pro Thr Ser Gln Gly Ser Gly Ala Phe Pro Pro Arg Arg Lys Ala Val His Ile Pro Arg Ile Val Asp Ile Phe Leu Gly Glu Gly Leu Val Pro Ser His Ser Gly Thr Lys Ser Pro His Cys Val Glu Pro Gly Pro Ser Pro Ala Gly Asp Ile Leu * Val Cys Ala Asn Ser Leu Pro Glu Asp * Ala Val Ala Met Leu Gly Ala Pro Thr Cys Ala Ser Phe Ser Gly Cys Phe Pro Leu Leu Thr Pro Met Leu Glu Val Phe Cys Gly Asn His Cys Phe Leu Thr Glu Tyr Asn Ala Val Gly Asn Cys Ser Gly Thr Leu Gln Gln Arg Ser Pro Leu Pro Ala Arg Ala Pro Phe Ser Lys Ser His Thr Asn Gly Lys Leu Glu Lys Val Leu Asn Ser Ala Glu Asp Leu Ser Cys Leu Pro * Gly Val Phe Leu Gly Ser Pro Cys Leu Ser Tyr Phe Cys Phe Ser Asp Leu Pro Ser Leu Thr Leu Thr Phe Phe Asn His Ile Gln Ala Ser Cys Arg Ile Gly Val * Ile Ser Cys Arg Cys His Asp Asn Ile Leu Pro Val His Val Pro Ser Asn Gln Thr Lys Pro Asn Pro Lys Thr Gln Ile Gln Ile Thr Leu Lys Pro Gly Val Leu Ala Gly Phe Ser Trp Lys Gly Val Leu Ser Val Arg Pro Pro .

Leu Cys Gly Lys Ile Leu Val Cys Val Ala Gly Gly Gly Ala Gln Gly Arg Arg Leu Gln Met Lys * Ile Asn Gln Lys Trp Gly Lys * Phe Val Asn Ala His Gly Arg Ser Ala Gln His * Tyr Cys Ser Val Leu Trp Ala Tyr Ser Val Trp Ala Ser Leu Phe Pro Glu Thr Leu Ser His Pro Phe Thr Glu Gln Asp Gly Pro Val * Gln Val Gly Pro Trp Arg Asn Ser Trp Glu Ala Lys Gly Gln His Thr Gln Asn (2) INFORMATION FOR SEQ ID NO:10:
(i) ~Q~N~ CHARACTERISTICS:
(A) LENGTH: 239 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Gly Lys Cys Tyr Thr Thr Lys Thr Asp Arg His Leu Arg His Cys Ser Gly Gln Asn Tyr Gly Ala Gln Asn Met Gly Leu Val Arg Met Gly Trp Phe Leu Ile Trp Lys Leu Ser Ser Asp Asn Leu Glu Ser Pro Gly Gly Gly Lys Trp Glu Arg Trp Glu Lys Cys Gln Lys Asn Lys Asn Lys Thr Lys Lys Lys Pro Lys Lys Thr Leu Pro His Pro Thr Ile Thr Thr Lys Glu Tyr Arg Glu Thr Glu Gly Ala Gly Asn Xaa Gly Gln Glu Gly Ser Leu Lys Xaa Leu Glu His Thr Met Arg Xaa Leu Leu Val Leu Ser Glu Val Trp Ser Pro Asn Phe Ser Met Trp Leu Phe Ser Trp Arg Asp Ala Trp Gln Leu Asp Arg Leu Arg Ser Leu Leu Pro Val Thr Pro Gly Met Ala Val Pro Ala Ile Leu Gly Lys Ile Asn Lys Arg Val Pro Ala Pro Gln Ile Gly Cys Cys Arg Pro Arg Ser Pro Thr Val Glu Thr Lys Arg -Asp Val Thr Leu Ser Ser Arg Leu Cys Val Leu Pro Pro Tyr Ser Val Ser Arg Pro Cys Leu Pro Lys Glu Glu Thr Glu Thr Gln Gly Ser Pro Leu Lys Pro Lys Pro Arg Gly Pro Phe Xaa Pro Xaa Lys Phe Pro Thr Trp Ala Gly Gly Lys Ile Val Pro Tyr Pro Ala Asn Ser Xaa Pro

Claims (15)

1. A method of assaying for a substance which modulates the response of neuronal cells to inhibition by adult central nervous system myelin comprising growing neuronal cells which have a propensity for neurite growth, on mammalian central nervous system (CNS) myelin in the presence of a test substance which is suspected of affecting neurite outgrowth, and assaying for neurite outgrowth.

2. An isolated nucleic acid molecule which is present in neuronal cells; its expression is required for neurite growth inhibition by mammalian central nervous system myelin; and it comprises the nucleic acid sequences shown in the Sequence Listing as SEQ. ID. No. 1, SEQ. ID. No.3, SEQ. ID. NO.4, SEQ. ID. NO. 5, SEQ ID. NO. 6, SEQ ID. NO. 7. and/or SEQ ID.
NO. 8.

3. The isolated nucleic acid molecule as claimed in claim 2 which comprises (a) a nucleic acid sequence as shown in SEQ. ID NO:1, SEQ.
ID. NO:3, SEQ. ID. NO:4, SEQ. ID. NO. 5, SEQ. ID. NO.6, SEQ. ID. NO.7 and/or SEQ. ID. NO.8, or in Figures 9, 11 to 14, and 21 wherein T can also be U; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences having at least 80-90% identity, preferably 90% identity with SEQ. ID NO:1, SEQ.ID.NO:3, SEQ. ID. NO:4, SEQ. ID. NO.5, SEQ. ID. NO.6, SEQ. ID. NO.7 and SEQ.ID. NO.8; (d) a fragment of (a) to (c) that is at least 15 bases and that will hybridize to (a) or (b) under stringent hybridization conditions, or (e) a nucleic acid molecule differing from any of the nucleic acids of (a) to (d) in codon sequences due to the degeneracy of the genetic code.

4. An isolated and purified nucleic acid molecule comprising (a) a nucleic acid sequence encoding a protein having the amino acid sequence as shown in Figure 24 (or SEQ. ID. NO. 12); (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which are at least 80%, preferably 90% identical to (a); or, (d) a fragment of (a) or (b) that is at least 15 bases and which will hybridize to (a) or (b) under stringent hybridization conditions.

5. A nucleic acid sequence as claimed in claim 4 in an antisense orientation.

6. A recombinant molecule adapted for transformation of a host cell comprising a nucleic acid molecule as claimed in claim 4.

7. A transformed host cell containing a recombinant molecule as claimed in claim 6.

8. A method for preparing a protein comprising (a) transferring a recombinant expression vector as claimed in claim 6; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected transformed host cell under conditions which allow expression of the protein; and (d) isolating the protein.

9. A isolated and purified protein encoded by the nucleic acid molecule as claimed in claim 3 including the amino acid sequence as shown in the Sequence Listing as SEQ. ID. NO. 2 or SEQ ID. NO. 9 and sequences having at least 80-90% identity thereto, and which is expressed in brain, NG108, PC12, and fibroblast cells.

10. An isolated and purified protein comprising an amino acid sequence as shown in Figure 24 or SEQ ID NO:12, and truncations, analogs, homologs, and isoforms of the protein and truncations thereof.

11. A method for assaying for the presence of an activator or inhibitor of the protein as claimed in claim 10, comprising growing neuronal cells which have a propensity for neurite growth on mammalian central nervous system (CNS) myelin and which express the protein in the presence of a suspected activator or inhibitor substance, and assaying for neurite outgrowth.

12. A method for identifying a substance which is capable of binding to a protein as claimed in claim 10, comprising reacting the protein with at least one substance which potentially can bind with the protein, or part of the protein, under conditions which permit the formation of substance-protein complexes, and assaying for substance-protein complexes, for free substance, and/or for non-complexed protein.

13. A method for assaying for a substance that affects the phosphatase activity of a protein as claimed in claim 10 comprising reacting the protein with a substrate which is capable of being dephosphorylated by the protein to produce a dephosphorylated product, in the presence of a substance which is suspected of affecting the phosphatase activity of the protein, and under conditions which permit dephosphorylation of the substrate; assaying for dephosphorylated product; and, comparing to product obtained in the absence of the substance to determine the affect of the substance on the phosphatase activity of the protein.

14. Antibodies having specificity against an epitope of a protein as claimed in claim 10.

15. Monoclonal antibodies which (a) immunoreact with neuronal membrane proteins; (b) neutralize the inhibition of neurite growth by mammalian central nervous system myelin; and, (c) recognize bands of Mr 35,000 and Mr 33,000 expressed in neuronal and fibroblast cell lines and in rat cerebrum and rat liver.