AGENTS MODULANT LA REPONSE DE CELLULES NEURONALES A L'INHIBITION PAR LA MYELINE DU SYSTEME NERVEUX CENTRAL DES MAMMIFERES

AGENTS MODULATING THE RESPONSE OF NEURONAL CELLS TO INHIBITION BY MAMMALIAN CENTRAL NERVOUS SYSTEM MYELIN

Abrégé anglais

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.

Revendications

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THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
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

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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

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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.

Description

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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

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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

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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

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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

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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

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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

21 74025
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

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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

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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

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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

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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;

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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

- 13 - 2174025
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;

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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

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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.

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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

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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).

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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

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(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

21 74025
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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

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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

21 74025
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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

21 74025
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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

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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

21 74025
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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.

21 74025
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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
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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

21 74025
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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

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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

21 74025
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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,

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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.

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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

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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

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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
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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.

- 21 74025
- 36 -
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

21 74025
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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
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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

21 74025
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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

21 74025
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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

21 74025
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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

21 74025
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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

21 74025
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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

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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

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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

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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

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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

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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

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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

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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

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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.
EXAMPLE 1
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

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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

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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.
EXAMPLE 2

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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
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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
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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.
EXAMPLE 3
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.
EXAMPLE 4
CELLULAR PROTEINS RECOGNIZED BY THE 10D ANTIBODY
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

21 74025
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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.
EXAMPLE 5
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

21 74025
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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

21 74025
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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

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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

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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.
EXAMPLE 6
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.

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EXAMPLE 7
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.
EXAMPLE 8
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

21 74025
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- 63 -
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.
EXAMPLE 9
To study the relevance of the CNS myelin/neurite

`- 21 74025
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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.
EXAMPLE 10
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%.

21 74025
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-- 65 --
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

21 74025
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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.

- 2174025
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

21 74025
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 10..276
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GAATTCCGG GGT AAA TGT TAC ACA ACA AAG ACA GAC CGT CAC CTT AGA 48
Gly Lys Cys Tyr Thr Thr Lys Thr Asp Arg His Leu Arg
1 5 10
CAT TGC TCT GGA CAA AAC TAT GGG GCA CAG AAC ATG GGA CTA GTC AGA 96
His Cys Ser Gly Gln Asn Tyr Gly Ala Gln Asn Met Gly Leu Val Arg
15 20 25
ATG GGC TGG TTT CTG ATC TGG AAA CTG TCC AGT GAC AAT TTG GAA AGT 144
Met Gly Trp Phe Leu Ile Trp Lys Leu Ser Ser Asp Asn Leu Glu Ser
30 35 40 45
CCC GGT GGA GGG AAA TGG GAA AGA TGG GAG AAA TGT CAA AAA AAC AAA 192
Pro Gly Gly Gly Lys Trp Glu Arg Trp Glu Lys Cys Gln Lys Asn Lys
50 55 60
AAC AAA ACA AAA AAA AAA CCC AAA AAA ACT CTG CCT CAC CCC ACC ATC 240
Asn Lys Thr Lys Lys Lys Pro Lys Lys Thr Leu Pro His Pro Thr Ile
65 70 75
ACC ACA AAA GAA TAT AGA GAA ACG GAG GGG GCA GGA AATGGGGGCA 286
Thr Thr Lys Glu Tyr Arg Glu Thr Glu Gly Ala Gly
80 85
GGAAGGATCC CTGAAATAAC TGGAACACAC AATGAGATGA CTGCTCGTAC TTT 339
(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
1 5 10 15
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
65 70 75 80
Glu Tyr Arg Glu Thr Glu Gly Ala Gly
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:

21 74025
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- 69 -
(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
CCTGGCTACT GATGGACTCT GGGATGTCTT ATCAAATGAA GAAGTAGCGG AAGCAATCAC 180
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
GTCATGAAAG TGACCCAGGG GACTAGGAAG ACAGAAGAAG GGAAGAAAAC TGGGGTGCCT 420
CCAAGCAGGG CGGC 434
(2) INFORMATION FOR SEQ ID NO:4:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 449 base pairs
(B) TYPE: nucleic acid
(C) STRAN~N~SS: 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) 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:
TGCAGGTCGA CACTAGTGGA TCCCTGAAAT AACTGGAACA CACAATGAGA TGACTGCTCG 60
TA~ A GGTATGGTCC CCCAACTTTT CAATGTGGCT CTTCTCCTGG AGAGATGCCT 120

21 74025
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- 70 -
GGCAGCTTGA CAGGCTGAGA AGTCTTTTAC CAGTTACACC CGGAATGGCT GTCCCTGCTA 180
TTCTAGGTAA AATAAATAAA CGTGTGCCTG CGCCCCAAAT AGGCTGCTGC CGCCCTAGGA 240
GCCCTACTGT AGAAACCAAG AGAGATGTAA CCCTCAGTAG CAGGCTGTGC GTTTTGCCTC 300
CTTATTCAGT GTCCAGACCC TGTTTACCCA AAGAAGAAAC GGAAACCCAA GGGTCTCCCT 360
TGAAGCCAAA GCCCAGAGGC CCTTTTGGCC ACAAAATTCC CAACCTGGGC TGGGGGAAAA 420
ATTGTTCCGT ACCCTGCGAA TTCCACCCA 449
~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:
TGCAGGTCGA CACTAGTGGA TCCAAAGAAT TCACCCCTGA GACTGAGCGT CAGAGACTTC 60
AGTACCTGGC GTTCATGCAG CCTCACTTGC TGGGAAATGA GTTCACACAC TTGGAGTTTC 120
CAAGGAGAGT ACAGAGGAAA GAACTCGGGA AGAAGATGCT GTACCGGGAC TTTAACATGA 180
CAGGCTGGGC ATACAAAACC ATTGAGGATG ATGACTTGAA GTTTCCCCTT ATATATGGAG 240
AAGGCAAGAA GGCCCGGGTA ATGGCAACTA TTGGAGTGAC AAGGGGACTT GGGGACCATG 300
ACCTGAAGGT TCATGACTCC AACATCTACA TAAAACCATT CCTGTCTTCA GCTCCAGAGG 360
TCAGAGTCTA CGATCTCTCC AAATACGAGC ACGGAGCGGA TGACGTGCTG ATCCTGGCTA 420
CTGATGGACT CTGGGATGTC TTATCAAATG AAGAAGTAGC GGAAGCAATC ACTCAGTTT 479
(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

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- 71 -
(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:
GCCAAAAGCC AGCCCGGCCG CGCAGAAGAC CTCGAGAAGC TTTTTGAATT CTGAGTGTGT 60
TGGCCTTTGG CCTTCCCAGG AGTTCCTCCA AGGGCCAACT TGCTACACAG GTCCATCCTG 120
~ GAAG GGGTGACTTA GAGTCTCAGG GAACAACGAG GCCCACACAG AGTAAGCCCA 180
AAGCACAGAG CAATATCAGT GCTGGGCAGA TCTGCCATGG GCATTAACAA ATCACTTTCC 240
CCACTTTGGT TTATTCACTT CATTTGTAGT CTTCTCCCCT GAGCCCCACC CCTGCAACAC 300
AAACTAAGAT CTTTCCACAC AGCGGCGGCC TCACGGAGAG AACTCCTTTC CAACTAAAAC 360
CAGCAAGGAC TCCGGGTTTT AGGGTAATTT GAATTTGGGT TTTCGGGTTT GGTTTGGTTT 420
GGTTGACGGT ACATGAACTG GGGAGAATGT TGTCATGACA CCTACAGGAT ATTCACACTC 480
CAATTCG 487
(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:
GAATTCCGGG CCCGTGCAGC CTGATCATCA CATGACTCGA CGGCGGTAGC GTTGGGCAAG 60
CAAGGAGCGG CGGGTCCGCG GGCTCGCCGG GTGGGCTCAG CTCCGCGCAC GCAGAAACGG 120
GGCGCAGGGG GGCGGGGAAG AGACCTGCGG CAGCCGCCGC CCGCCAGCCG TTCCCGGGAC 180
TCTCGCTGTC CCTCCTCAGC CGCCCGCTCG CTCCATGTCG CCCGGTTGCG GAAGTGTCGC 240
TGGGAGAGGT GGCGGCGTCG TCGTCAGGGA GCACGGGAGG CCGGGGCTCG GCCCCTGCAG 300
CACTGAGCTC CCGGAGCCGC GCGTCCAGCG TCCCCACCGC CCGTGCGCCC CGCGCCCGCC 360
GCAGCCTGCA TGCCCCGCGC TGCACCTCGG CCGGCCGCCG CCTCCTGCTC GCTCAGCGGC 420
TGCCCGGCGC CGGAGTAATA TGCTCACTCG GGTGAAATCT GCCGTGGCCA ATTTCATGGG 480
CGGCATCATG GCTGGCAGCT CCGGCTCCGA GCACGGCGGC AGCGGCTGCG GAGGCTCGGA 540
CCTGCCCCTG CGCTTCCCGT ACGGGCGGCC AGAGTTCCTC GGGCTGTCTC AGGATGAGGT 600
GGAGTGCAGC GCAGACCACA TCGCCCGCCC CATCCTCATC CTCAAGGAGA CCCNNNNNNN 660

21 74025
-
- 72 -
~N~ NCGGCCTGCT GCAGCACCAA TCACACAACA GCTGCAGGAC ATCGTGGAGA 720
TCCTGAAGAA CTCCGCCATC CTTCCCCCGA CCTGCCTAGG GGAGGAGCCA GAGAGCACGC 780
CGGCCCATGG CAGGACCCTA ACCCGCGCAG CCTACTGCGT GGAGGGGTGG GTGCCCCCGG 840
CTCCCCCAGC ACACCACCCA CGCGCTTCTT CACGGAGAAG AAGATTCCTC ATGAGTGTTT 900
GGTCATCGGG GCCCTGGAGA GCGCCTTCAA GGAAATGGAC CTTCAAATTG AACGGGAGAG 960
GAGTGCATAT AATATATCCG GTGGCTGCAC AGCCCTCATC GTGGTTTGCC TTCTGGGGAA 1020
GCTCTACGTG GCAAATGCAG GTGACAGCAG GGCCATAATC ATCCGAAATG GAGAAATCAT 1080
CCCCATGTCT TCCGAATTCA CCCCTGAGAC TGAGCGTCAG AGACTTCAGT ACCTGGCGTT 1140
CATGCAGCCT CACTTGCTGG GAAATGAGTT CACACACTTG GAGTTTCCAA GGAGAGTACA 1200
GAGGAAAGAA CTCGGGAAGA AGATGCTGTA CCGGGACTTT AACATGACAG GCTGGGCATA 1260
CAAAACCATT GAGGATGATG ACTTGAAGTT TCCCCTTATA TATGGAGAAG GCAAGAAGGC 1320
CCGGGTAATG GCAACTATTG GAGTGACAAG GGGACTTGGG GACCATGACC TGAAGGTTCA 1380
TGACTCCAAC ATCTACATAA AACCATTCCT GTCTTCAGCT CCAGAGGTCA GAGTCTACGA 1440
TCTCTCCAAA TACGAGCACG GAGCGGATGA CGTGCTGATC CTGGCTACTG ATGGACTCTG 1500
GGATGTCTTA TCAAATGAAG AAGTAGCGGA AGCAATCACT CA~ C CTAACTGTGA 1560
TCCAGATGAC CCTCACAGGT ACACACTGGC AGCTCAGGAC CTGGTGATGC GTGCCCGAGG 1620
CGTCCTGAAG GACCGAGGAT GGCGGATATC AAATGACCGA CTGGGCTCAG GAGATGACAT 1680
TTCTGTATAC GTCATTCCTT TAATACACGG AAACAAACTG TCATGAAAGT GACCCAGGGG 1740
ACTAGGAAGA CAGAAGAAGG GAAGAAAACT GGGGTGCCTC CAAGCAGGGC GGGACTGGGG 1800
AGTAAGTACC TGGGCTGGAT TCCAGGTGAC GCACATTTTC CCCAGCCCAG GTGGGGAATT 1860
TGCTGCCAAA AGGCCTCCTG GCTTTGCTTC AAGGAGACCC TTGGGTTTCC ~'l"l"l'~'l"l'~'l"l' 1920
TGGTAACAGT CTGGACACTG AATAAGAGCA AAACGCACAG CCTGCTACTG AGGGTTACAT 1980
~ GT TTCTACAGTA GGGCTCCTAG GCGGCAGCAG CCTATTTGGG GCGCAGGCAC 2040
ACGTTTATTT ATTTTACCTA GAATAGCAGG GACAGCCATT CCGGGTGTAA CTGGTAAAAG 2100
ACTTCTCAGC CTGTCAAGCT GCCAGCATCT CTCCAGGAGA AGAGCCACAT TGAAAAGTTG 2160
GGGGACCATA CCTCAGAAAG TACGAGCAGT CATCTCATTG TGTGTTCCAG TTATTTCAGG 2220
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
TCTCCCATCT TTCCCATTTC CCTCCACCGG GACTTTCCAA ATTGTCACTG GACAGTTTCC 2400
AGATCAGAAA CCAGCCCATT CTGACTAGTC CCATGTTCTG TGCCCCATAG ~l"l"l~l~lCCAG 2460
AGCAATGTCT AAGGTGACGG TCTGTCTTTG TTGTGTAACA TTTACCCCGG GGTTCTGTTT 2520
TTCTCCCCAA ATAGATATGT TTGCTTCAAA ACATGGGTGT TTCATTGGAC CAGTGGTTCC 2580
TGGGGTTATC TTTAAGGCCC ~l~l~l~l~GT CTGGAGGCTC TGCCACGAGA GGCTGGGTTT 2640

- 2174025
- 73 -
GCGGTTCTGG ATTGGCGATA CTCCCCGCCT TCTGTGTCCT GGAGAGGCAT AGGAAGCAGC 2700
GTTCAGGACC ACGGTCTAAG CCAGGCTCTT GTTATCCAGC ACTTGACCAT GTTCGCGTTG 2760
AGGGAAGGGG GTGTGGGATT CAGGCTCCTT GGTCGCTGAC ~ lCCAG GGCACAGGAG 2820
GATCGAGTGT CACAGCTAGC TAAGCAGCAG CTCTTCCTGA CAC~lll~l~G CAAGGATAAC 2880
TAGGATGACA CTTGAATAAA AGTGAATTTG AATTGCAGTT GGTCATTGTG ATGCCCCCCT 2940
CCCCTTTCAC ATTGCTGAGA TCTCCTTCCT TTTATGCATC CACTGGTGTG TGTGCCTCAG 3000
TGGGCACACA CGGGCACATG AGCACCTGAG CACAGTATCT GTCCCCTGCT TCCTTGCTGG 3060
GAGGACCAGC AAAGTCCAGT TTAAAAATCA GCCGTCTCTT GGGCAGACTG CTGCTCTGCC 3120
CAGGGGCCTT CAGAGTAGCA TCCGGTTGCC TATTAGTCCT GTTCCTGTTG TCTTCCAGGA 3180
TATCAGCTTT CTGACAACTG GGTATAAATC AGACATTTCC AACCCAAGAA TGGATCCAAT 3240
GGTGTCATTT CCCTAAAATG CTTGAGGAGA AGGCAGTTCC AACCTCCCAG GGCAGCGGGG 3300
CATTCCCTCC CCGCCGGAAA GCCGTCCACA TTCCTAGAAT TGTAGATATT TTCTTAGGGG 3360
AAGGCCTGGT GCCATCCCAC TCAGGAACAA AGTCACCCCA CTGTGTAGAG CCAGGGCCCA 3420
GCCCGGCAGG TGACATACTG TGAGl~ G CCAACTCGCT GCCTGAGGAC TGAGCTGTGG 3480
CCATGCTGGG GGCACCTACG TGTGCCTCTT TTTCAGGATG CTTCCCACTC CTGACACCGA 3540
TGCTGGAAGT ~ GGC AACCATTGCT TBCCTGACAG AATACAATGC TGTGGGAAAC 3600
TGTTCAGGCA CGCTACAGCA GCGTAGTCCT CTTCCAGCCC GTGCCCCGTT CTCAAAGTCA 3660
CACACAAACG GGAAACTTGA GAAGGTCTTG AACTCTGCGG AAGACCTGAG CTGCCTTCCA 3720
TAGGGAGTAT ~ GG~ C CCCGTGCCTT TCATATTTTT G~ GA CCTCCCGAGT 3780
CTCACTTTGA CCTTCTTCAA TCACATTCAA GCCTCCTGTC GAATTGGAGT GTGAATATCC 3840
TGTAGGTGTC ATGACAACAT TCTCCCAGTT CATGTACCGT CAAACCAAAC CAAACCAAAC 3900
CCGAAAACCC AAATTCAAAT TACCCTAAAA CCCGGAGTCC TTGCTGGTTT TAGTTGGAAA 3960
GGA~ CCGTGAGGCC GCCGCTGTGT GGAAAGATCT TAGTTTGTGT TGCAGGGGGT 4020
GGGGCTCAGG GGAGAAGACT ACAAATGAAG TGAATAAATC AAAAGTGGGG AAAGTGATTT 4080
GTTAATGCCC ATGGCAGATC TGCCCAGCAC TGATATTGCT CTGTGCTTTG GGCTTACTCT 4140
GTGTGGGCCT C~ll~llCCC TGAGACTCTA AGTCACCCCT TCACAGAACA GGATGGACCT 4200
GTGTAGCAAG TTGGCCCTTG GAGGAACTCC TGGGAAGCCA AAGGCCAACA CACTCAGAAT 4260
TC 4262
(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

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( 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:
CGG CCT GCT GCA GCA CCA ATC ACA CAA CAG CTG CAG GAC ATC GTG GAG 48
Arg Pro Ala Ala Ala Pro Ile Thr Gln Gln Leu Gln Asp Ile Val Glu
5 10 15
ATC CTG AAG AAC TCC GCC ATC CTT CCC CCG ACC TGC CTA GGG GAG GAG 96
Ile Leu Lys Asn Ser Ala Ile Leu Pro Pro Thr Cys Leu Gly Glu Glu
20 25 30
CCA GAG AGC ACG CCG GCC CAT GGC AGG ACC CTA ACC CGC GCA GCC TAC 144
Pro Glu Ser Thr Pro Ala His Gly Arg Thr Leu Thr Arg Ala Ala Tyr
35 40 45
TGC GTG GAG GGG TGG GTG CCC CCG GCT CCC CCA GCA CAC CAC CCA CGC 192
Cys Val Glu Gly Trp Val Pro Pro Ala Pro Pro Ala His His Pro Arg
50 55 60
GCT TCT TCA CGG AGA AGA AGA TTC CTC ATG AGT GTT TGG TCA TCG GGG 240
Ala Ser Ser Arg Arg Arg Arg Phe Leu Met Ser Val Trp Ser Ser Gly
65 70 75 80
CCC TGG AGA GCG CCT TCA AGG AAA TGG ACC TTC AAA TTG AAC GGG AGA 288
Pro Trp Arg Ala Pro Ser Arg Lys Trp Thr Phe Lys Leu Asn Gly Arg
85 90 95
GGA GTG CAT ATA ATA TAT CCG GTG GCT GCA CAG CCC TCA TCG TGG TTT 336
Gly Val His Ile Ile Tyr Pro Val Ala Ala Gln Pro Ser Ser Trp Phe
100 105 110
GCC TTC TGG GGA AGC TCT ACG TGG CAA ATG CAG GTG ACA GCA GGG CCA 384
Ala Phe Trp Gly Ser Ser Thr Trp Gln Met Gln Val Thr Ala Gly Pro
115 120 125
TAA TCA TCC GAA ATG GAG AAA TCA TCC CCA TGT CTT CCG AAT TCA CCC 432
* Ser Ser Glu Met Glu Lys Ser Ser Pro Cys Leu Pro Asn Ser Pro
130 135 140
CTG AGA CTG AGC GTC AGA GAC TTC AGT ACC TGG CGT TCA TGC AGC CTC 480
Leu Arg Leu Ser Val Arg Asp Phe Ser Thr Trp Arg Ser Cys Ser Leu
145 150 155 160
ACT TGC TGG GAA ATG AGT TCA CAC ACT TGG AGT TTC CAA GGA GAG TAC 528
Thr Cys Trp Glu Met Ser Ser His Thr Trp Ser Phe Gln Gly Glu Tyr
165 170 175
AGA GGA AAG AAC TCG GGA AGA AGA TGC TGT ACC GGG ACT TTA ACA TGA 576
Arg Gly Lys Asn Ser Gly Arg Arg Cys Cys Thr Gly Thr Leu Thr *
180 185 190
CAG GCT GGG CAT ACA AAA CCA TTG AGG ATG ATG ACT TGA AGT TTC CCC 624
Gln Ala Gly His Thr Lys Pro Leu Arg Met Met Thr * Ser Phe Pro
195 200 205
TTA TAT ATG GAG AAG GCA AGA AGG CCC GGG TAA TGG CAA CTA TTG GAG 672
Leu Tyr Met Glu Lys Ala Arg Arg Pro Gly * Trp Gln Leu Leu Glu
210 215 220

21 74025
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TGA CAA GGG GAC TTG GGG ACC ATG ACC TGA AGG TTC ATG ACT CCA ACA 720
* Gln Gly Asp Leu Gly Thr Met Thr * Arg Phe Met Thr Pro Thr
225 230 235 240
TCT ACA TAA AAC CAT TCC TGT CTT CAG CTC CAG AGG TCA GAG TCT ACG 768
Ser Thr * Asn His Ser Cys Leu Gln Leu Gln Arg Ser Glu Ser Thr
245 250 255
ATC TCT CCA AAT ACG AGC ACG GAG CGG ATG ACG TGC TGA TCC TGG CTA 816
Ile Ser Pro Asn Thr Ser Thr Glu Arg Met Thr Cys * Ser Trp Leu
260 265 270
CTG ATG GAC TCT GGG ATG TCT TAT CAA ATG AAG AAG TAG CGG AAG CAA 864
Leu Met Asp Ser Gly Met Ser Tyr Gln Met Lys Lys * Arg Lys Gln
275 280 285
TCA CTC AGT TTC TTC CTA ACT GTG ATC CAG ATG ACC CTC ACA GGT ACA 912
Ser Leu Ser Phe Phe Leu Thr Val Ile Gln Met Thr Leu Thr Gly Thr
290 295 300
CAC TGG CAG CTC AGG ACC TGG TGA TGC GTG CCC GAG GCG TCC TGA AGG 960
His Trp Gln Leu Arg Thr Trp * Cys Val Pro Glu Ala Ser * Arg
305 310 315 320
ACC GAG GAT GGC GGA TAT CAA ATG ACC GAC TGG GCT CAG GAG ATG ACA 1008
Thr Glu Asp Gly Gly Tyr Gln Met Thr Asp Trp Ala Gln Glu Met Thr
325 330 335
TTT CTG TAT ACG TCA TTC CTT TAA TAC ACG GAA ACA AAC TGT CAT GAA 1056
Phe Leu Tyr Thr Ser Phe Leu * Tyr Thr Glu Thr Asn Cys His Glu
340 345 350
AGT GAC CCA GGG GAC TAG GAA GAC AGA AGA AGG GAA GAA AAC TGG GGT 1104
Ser Asp Pro Gly Asp * Glu Asp Arg Arg Arg Glu Glu Asn Trp Gly
355 360 365
GCC TCC AAG CAG GGC GGG ACT GGG GAG TAA GTA CCT GGG CTG GAT TCC 1152
Ala Ser Lys Gln Gly Gly Thr Gly Glu * Val Pro Gly Leu Asp Ser
370 375 380
AGG TGA CGC ACA TTT TCC CCA GCC CAG GTG GGG AAT TTG CTG CCA AAA 1200
Arg * Arg Thr Phe Ser Pro Ala Gln Val Gly Asn Leu Leu Pro Lys
385 390 395 400
GGC CTC CTG GCT TTG CTT CAA GGA GAC CCT TGG GTT TCC GTT TCT TCT 1248
Gly Leu Leu Ala Leu Leu Gln Gly Asp Pro Trp Val Ser Val Ser Ser
405 410 415
TTG GTA ACA GTC TGG ACA CTG AAT AAG AGC AAA ACG CAC AGC CTG CTA 1296
Leu Val Thr Val Trp Thr Leu Asn Lys Ser Lys Thr His Ser Leu Leu
420 425 430
CTG AGG GTT ACA TCT CTC TTG GTT TCT ACA GTA GGG CTC CTA GGC GGC 1344
Leu Arg Val Thr Ser Leu Leu Val Ser Thr Val Gly Leu Leu Gly Gly
435 440 445
AGC AGC CTA TTT GGG GCG CAG GCA CAC GTT TAT TTA TTT TAC CTA GAA 1392
Ser Ser Leu Phe Gly Ala Gln Ala His Val Tyr Leu Phe Tyr Leu Glu
450 455 460
TAG CAG GGA CAG CCA TTC CGG GTG TAA CTG GTA AAA GAC TTC TCA GCC 1440
* Gln Gly Gln Pro Phe Arg Val * Leu Val Lys Asp Phe Ser Ala
465 470 475 480
TGT CAA GCT GCC AGC ATC TCT CCA GGA GAA GAG CCA CAT TGA AAA GTT 1488
Cys Gln Ala Ala Ser Ile Ser Pro Gly Glu Glu Pro His * Lys Val

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485 490 495
GGG GGA CCA TAC CTC AGA AAG TAC GAG CAG TCA TCT CAT TGT GTG TTC 1536
Gly Gly Pro Tyr Leu Arg Lys Tyr Glu Gln Ser Ser His Cys Val Phe
500 505 510
CAG TTA TTT CAG GGA TCC TTC CTG CCC CCC ATT TCC TGC CCC CTC CGT 1584
Gln Leu Phe Gln Gly Ser Phe Leu Pro Pro Ile Ser Cys Pro Leu Arg
515 520 525
TTC TCT ATA TTC TTT TGT GGT GAT GGT GGG GTG AGG CAG AGT TTT TTT 1632
Phe Ser Ile Phe Phe Cys Gly Asp Gly Gly Val Arg Gln Ser Phe Phe
530 535 540
GGG TTT TTT TTT GTT TTG TTT TTG TTT TTT TGA CAT TTC TCC CAT CTT 1680
Gly Phe Phe Phe Val Leu Phe Leu Phe Phe * His Phe Ser His Leu
545 550 555 560
TCC CAT TTC CCT CCA CCG GGA CTT TCC AAA TTG TCA CTG GAC AGT TTC 1728
Ser His Phe Pro Pro Pro Gly Leu Ser Lys Leu Ser Leu Asp Ser Phe
565 570 575
CAG ATC AGA AAC CAG CCC ATT CTG ACT AGT CCC ATG TTC TGT GCC CCA 1776
Gln Ile Arg Asn Gln Pro Ile Leu Thr Ser Pro Met Phe Cys Ala Pro
580 585 590
TAG TTT TGT CCA GAG CAA TGT CTA AGG TGA CGG TCT GTC TTT GTT GTG 1824
* Phe Cys Pro Glu Gln Cys Leu Arg * Arg Ser Val Phe Val Val
595 600 605
TAA CAT TTA CCC CGG GGT TCT GTT TTT CTC CCC AAA TAG ATA TGT TTG 1872
* His Leu Pro Arg Gly Ser Val Phe Leu Pro Lys * Ile Cys Leu
610 615 620
CTT CAA AAC ATG GGT GTT TCA TTG GAC CAG TGG TTC CTG GGG TTA TCT 19 20
Leu Gln Asn Met Gly Val Ser Leu Asp Gln Trp Phe Leu Gly Leu Ser
625 630 635 640
TTA AGG CCC CTC TGT GTG TCT GGA GGC TCT GCC ACG AGA GGC TGG GTT 1968
Leu Arg Pro Leu Cys Val Ser Gly Gly Ser Ala Thr Arg Gly Trp Val
645 650 655
TGC GGT TCT GGA TTG GCG ATA CTC CCC GCC TTC TGT GTC CTG GAG AGG 2016
Cys Gly Ser Gly Leu Ala Ile Leu Pro Ala Phe Cys Val Leu Glu Arg
660 665 670
CAT AGG AAG CAG CGT TCA GGA CCA CGG TCT AAG CCA GGC TCT TGT TAT 2064
His Arg Lys Gln Arg Ser Gly Pro Arg Ser Lys Pro Gly Ser Cys Tyr
675 680 685
CCA GCA CTT GAC CAT GTT CGC GTT GAG GGA AGG GGG TGT GGG ATT CAG 2112
Pro Ala Leu Asp His Val Arg Val Glu Gly Arg Gly Cys Gly Ile Gln
690 695 700
GCT CCT TGG TCG CTG ACT GTT CTC CAG GGC ACA GGA GGA TCG AGT GTC 2160
Ala Pro Trp Ser Leu Thr Val Leu Gln Gly Thr Gly Gly Ser Ser Val
705 710 715 720
ACA GCT AGC TAA GCA GCA GCT CTT CCT GAC ACC TTT GTG CAA GGA TAA 2208
Thr Ala Ser * Ala Ala Ala Leu Pro Asp Thr Phe Val Gln Gly *
725 730 735
CTA GGA TGA CAC TTG AAT AAA AGT GAA TTT GAA TTG CAG TTG GTC ATT 2256
Leu Gly * His Leu Asn Lys Ser Glu Phe Glu Leu Gln Leu Val Ile
740 745 750

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.
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GTG ATG CCC CCC TCC CCT TTC ACA TTG CTG AGA TCT CCT TCC TTT TAT 2304
Val Met Pro Pro Ser Pro Phe Thr Leu Leu Arg Ser Pro Ser Phe Tyr
755 760 765
GCA TCC ACT GGT GTG TGT GCC TCA GTG GGC ACA CAC GGG CAC ATG AGC 2352
Ala Ser Thr Gly Val Cys Ala Ser Val Gly Thr His Gly His Met Ser
770 775 780
ACC TGA GCA CAG TAT CTG TCC CCT GCT TCC TTG CTG GGA GGA CCA GCA 2400
Thr * Ala Gln Tyr Leu Ser Pro Ala Ser Leu Leu Gly Gly Pro Ala
785 790 795 800
AAG TCC AGT TTA AAA ATC AGC CGT CTC TTG GGC AGA CTG CTG CTC TGC 2448
Lys Ser Ser Leu Lys Ile Ser Arg Leu Leu Gly Arg Leu Leu Leu Cys
805 810 815
CCA GGG GCC TTC AGA GTA GCA TCC GGT TGC CTA TTA GTC CTG TTC CTG 2496
Pro Gly Ala Phe Arg Val Ala Ser Gly Cys Leu Leu Val Leu Phe Leu
820 825 830
TTG TCT TCC AGG ATA TCA GCT TTC TGA CAA CTG GGT ATA AAT CAG ACA 2544
Leu Ser Ser Arg Ile Ser Ala Phe * Gln Leu Gly Ile Asn Gln Thr
835 840 845
TTT CCA ACC CAA GAA TGG ATC CAA TGG TGT CAT TTC CCT AAA ATG CTT 2592
Phe Pro Thr Gln Glu Trp Ile Gln Trp Cys His Phe Pro Lys Met Leu
850 855 860
GAG GAG AAG GCA GTT CCA ACC TCC CAG GGC AGC GGG GCA TTC CCT CCC 2640
Glu Glu Lys Ala Val Pro Thr Ser Gln Gly Ser Gly Ala Phe Pro Pro
865 870 875 880
CGC CGG AAA GCC GTC CAC ATT CCT AGA ATT GTA GAT ATT TTC TTA GGG 2688
Arg Arg Lys Ala Val His Ile Pro Arg Ile Val Asp Ile Phe Leu Gly
885 890 895
GAA GGC CTG GTG CCA TCC CAC TCA GGA ACA AAG TCA CCC CAC TGT GTA 2736
Glu Gly Leu Val Pro Ser His Ser Gly Thr Lys Ser Pro His Cys Val
900 905 910
GAG CCA GGG CCC AGC CCG GCA GGT GAC ATA CTG TGA GTG TGT GCC AAC 2784
Glu Pro Gly Pro Ser Pro Ala Gly Asp Ile Leu * Val Cys Ala Asn
915 920 925
TCG CTG CCT GAG GAC TGA GCT GTG GCC ATG CTG GGG GCA CCT ACG TGT 2832
Ser Leu Pro Glu Asp * Ala Val Ala Met Leu Gly Ala Pro Thr Cys
930 935 940
GCC TCT TTT TCA GGA TGC TTC CCA CTC CTG ACA CCG ATG CTG GAA GTG 2880
Ala Ser Phe Ser Gly Cys Phe Pro Leu Leu Thr Pro Met Leu Glu Val
945 950 955 960
TTC TGT GGC AAC CAT TGC TTC CTG ACA GAA TAC AAT GCT GTG GGA AAC 2928
Phe Cys Gly Asn His Cys Phe Leu Thr Glu Tyr Asn Ala Val Gly Asn
965 970 975
TGT TCA GGC ACG CTA CAG CAG CGT AGT CCT CTT CCA GCC CGT GCC CCG 2976
Cys Ser Gly Thr Leu Gln Gln Arg Ser Pro Leu Pro Ala Arg Ala Pro
980 985 990
TTC TCA AAG TCA CAC ACA AAC GGG AAA CTT GAG AAG GTC TTG AAC TCT 3024
Phe Ser Lys Ser His Thr Asn Gly Lys Leu Glu Lys Val Leu Asn Ser
995 1000 1005
GCG GAA GAC CTG AGC TGC CTT CCA TAG GGA GTA TTT CTG GGT TCC CCG 3072
Ala Glu Asp Leu Ser Cys Leu Pro * Gly Val Phe Leu Gly Ser Pro

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1010 1015 1020
TGC CTT TCA TAT TTT TGC TTT TCT GAC CTC CCG AGT CTC ACT TTG ACC 3120
Cys Leu Ser Tyr Phe Cys Phe Ser Asp Leu Pro Ser Leu Thr Leu Thr
1025 1030 1035 1040
TTC TTC AAT CAC ATT CAA GCC TCC TGT CGA ATT GGA GTG TGA ATA TCC 3168
Phe Phe Asn His Ile Gln Ala Ser Cys Arg Ile Gly Val * Ile Ser
1045 1050 1055
TGT AGG TGT CAT GAC AAC ATT CTC CCA GTT CAT GTA CCG TCA AAC CAA 3216
Cys Arg Cys His Asp Asn Ile Leu Pro Val His Val Pro Ser Asn Gln
1060 1065 1070
ACC AAA CCA AAC CCG AAA ACC CAA ATT CAA ATT ACC CTA AAA CCC GGA 3264
Thr Lys Pro Asn Pro Lys Thr Gln Ile Gln Ile Thr Leu Lys Pro Gly
1075 1080 1085
GTC CTT GCT GGT TTT AGT TGG AAA GGA GTT CTC TCC GTG AGG CCG CCG 3312
Val Leu Ala Gly Phe Ser Trp Lys Gly Val Leu Ser Val Arg Pro Pro
1090 1095 1100
CTG TGT GGA AAG ATC TTA GTT TGT GTT GCA GGG GGT GGG GCT CAG GGG 3360
Leu Cys Gly Lys Ile Leu Val Cys Val Ala Gly Gly Gly Ala Gln Gly
1105 1110 1115 1120
AGA AGA CTA CAA ATG AAG TGA ATA AAT CAA AAG TGG GGA AAG TGA TTT 3408
Arg Arg Leu Gln Met Lys * Ile Asn Gln Lys Trp Gly Lys * Phe
1125 1130 1135
GTT AAT GCC CAT GGC AGA TCT GCC CAG CAC TGA TAT TGC TCT GTG CTT 3456
Val Asn Ala His Gly Arg Ser Ala Gln His * Tyr Cys Ser Val Leu
1140 1145 1150
TGG GCT TAC TCT GTG TGG GCC TCG TTG TTC CCT GAG ACT CTA AGT CAC 3504
Trp Ala Tyr Ser Val Trp Ala Ser Leu Phe Pro Glu Thr Leu Ser His
1155 1160 1165
CCC TTC ACA GAA CAG GAT GGA CCT GTG TAG CAA GTT GGC CCT TGG AGG 3552
Pro Phe Thr Glu Gln Asp Gly Pro Val * Gln Val Gly Pro Trp Arg
1170 1175 1180
AAC TCC TGG GAA GCC AAA GGC CAA CAC ACT CAG AAT TC 3590
Asn Ser Trp Glu Ala Lys Gly Gln His Thr Gln Asn
1185 1190 1195
(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
1 5 10 15
Ile Leu Lys Asn Ser Ala Ile Leu Pro Pro Thr Cys Leu Gly Glu Glu
20 25 30
Pro Glu Ser Thr Pro Ala His Gly Arg Thr Leu Thr Arg Ala Ala Tyr

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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
100 105 110
Ala Phe Trp Gly Ser Ser Thr Trp Gln Met Gln Val Thr Ala Gly Pro
115 120 125
* Ser Ser Glu Met Glu Lys Ser Ser Pro Cys Leu Pro Asn Ser Pro
130 135 140
Leu Arg Leu Ser Val Arg Asp Phe Ser Thr Trp Arg Ser Cys Ser Leu
145 150 155 160
Thr Cys Trp Glu Met Ser Ser HiS Thr Trp Ser Phe Gln Gly Glu Tyr
165 170 175
Arg Gly Lys Asn Ser Gly Arg Arg Cys Cys Thr Gly Thr Leu Thr *
180 185 190
Gln Ala Gly His Thr Lys Pro Leu Arg Met Met Thr * Ser Phe Pro
195 200 205
Leu Tyr Met Glu Lys Ala Arg Arg Pro Gly * Trp Gln Leu Leu Glu
210 215 220
* Gln Gly Asp Leu Gly Thr Met Thr * Arg Phe Met Thr Pro Thr
225 230 235 240
Ser Thr * Asn His Ser Cys Leu Gln Leu Gln Arg Ser Glu Ser Thr
245 250 255
Ile Ser Pro Asn Thr Ser Thr Glu Arg Met Thr Cys * Ser Trp Leu
260 265 270
Leu Met Asp Ser Gly Met Ser Tyr Gln Met Lys Lys * Arg Lys Gln
275 280 285
Ser Leu Ser Phe Phe Leu Thr Val Ile Gln Met Thr Leu Thr Gly Thr
290 295 300
HiS Trp Gln Leu Arg Thr Trp * Cys Val Pro Glu Ala Ser * Arg
305 310 315 320
Thr Glu Asp Gly Gly Tyr Gln Met Thr Asp Trp Ala Gln Glu Met Thr
325 330 335
Phe Leu Tyr Thr Ser Phe Leu * Tyr Thr Glu Thr Asn Cys His Glu
340 345 350
Ser Asp Pro Gly Asp * Glu Asp Arg Arg Arg Glu Glu Asn Trp Gly
355 360 365
Ala Ser Lys Gln Gly Gly Thr Gly Glu * Val Pro Gly Leu Asp Ser
370 375 380
Arg * Arg Thr Phe Ser Pro Ala Gln Val Gly Asn Leu Leu Pro Lys
385 390 395 400

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Gly Leu Leu Ala Leu Leu Gln Gly Asp Pro Trp Val Ser Val Ser Ser
405 410 415
Leu Val Thr Val Trp Thr Leu Asn Lys Ser Lys Thr His Ser Leu Leu
420 425 430
Leu Arg Val Thr Ser Leu Leu Val Ser Thr Val Gly Leu Leu Gly Gly
435 440 445
Ser Ser Leu Phe Gly Ala Gln Ala His Val Tyr Leu Phe Tyr Leu Glu
450 455 460
* Gln Gly Gln Pro Phe Arg Val * Leu Val Lys Asp Phe Ser Ala
465 470 475 480
Cys Gln Ala Ala Ser Ile Ser Pro Gly Glu Glu Pro His * Lys Val
485 490 495
Gly Gly Pro Tyr Leu Arg Lys Tyr Glu Gln Ser Ser His Cys Val Phe
500 505 510
Gln Leu Phe Gln Gly Ser Phe Leu Pro Pro Ile Ser Cys Pro Leu Arg
515 520 525
Phe Ser Ile Phe Phe Cys Gly Asp Gly Gly Val Arg Gln Ser Phe Phe
530 535 540
Gly Phe Phe Phe Val Leu Phe Leu Phe Phe * His Phe Ser His Leu
545 550 555 560
Ser His Phe Pro Pro Pro Gly Leu Ser Lys Leu Ser Leu Asp Ser Phe
565 570 575
Gln Ile Arg Asn Gln Pro Ile Leu Thr Ser Pro Met Phe Cys Ala Pro
580 585 590
* Phe Cys Pro Glu Gln Cys Leu Arg * Arg Ser Val Phe Val Val
595 600 605
* His Leu Pro Arg Gly Ser Val Phe Leu Pro Lys * Ile Cys Leu
610 615 620
Leu Gln Asn Met Gly Val Ser Leu Asp Gln Trp Phe Leu Gly Leu Ser
625 630 635 640
Leu Arg Pro Leu Cys Val Ser Gly Gly Ser Ala Thr Arg Gly Trp Val
645 650 655
Cys Gly Ser Gly Leu Ala Ile Leu Pro Ala Phe Cys Val Leu Glu Arg
660 665 670
His Arg Lys Gln Arg Ser Gly Pro Arg Ser Lys Pro Gly Ser Cys Tyr
675 680 685
Pro Ala Leu Asp His Val Arg Val Glu Gly Arg Gly Cys Gly Ile Gln
690 695 700
Ala Pro Trp Ser Leu Thr Val Leu Gln Gly Thr Gly Gly Ser Ser Val
705 710 715 720
Thr Ala Ser * Ala Ala Ala Leu Pro Asp Thr Phe Val Gln Gly *
725 730 735
Leu Gly * His Leu Asn Lys Ser Glu Phe Glu Leu Gln Leu Val Ile
740 745 750

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Val Met Pro Pro Ser Pro Phe Thr Leu Leu Arg Ser Pro Ser Phe Tyr
755 760 765
Ala Ser Thr Gly Val Cys Ala Ser Val Gly Thr His Gly His Met Ser
770 775 780
Thr * Ala Gln Tyr Leu Ser Pro Ala Ser Leu Leu Gly Gly Pro Ala
785 790 795 800
Lys Ser Ser Leu Lys Ile Ser Arg Leu Leu Gly Arg Leu Leu Leu Cys
805 810 815
Pro Gly Ala Phe Arg Val Ala Ser Gly Cys Leu Leu Val Leu Phe Leu
820 825 830
Leu Ser Ser Arg Ile Ser Ala Phe * Gln Leu Gly Ile Asn Gln Thr
835 840 845
Phe Pro Thr Gln Glu Trp Ile Gln Trp Cys His Phe Pro Lys Met Leu
850 855 860
Glu Glu Lys Ala Val Pro Thr Ser Gln Gly Ser Gly Ala Phe Pro Pro
865 870 875 880
Arg Arg Lys Ala Val His Ile Pro Arg Ile Val Asp Ile Phe Leu Gly
885 890 895
Glu Gly Leu Val Pro Ser His Ser Gly Thr Lys Ser Pro His Cys Val
900 905 910
Glu Pro Gly Pro Ser Pro Ala Gly Asp Ile Leu * Val Cys Ala Asn
915 920 925
Ser Leu Pro Glu Asp * Ala Val Ala Met Leu Gly Ala Pro Thr Cys
930 935 940
Ala Ser Phe Ser Gly Cys Phe Pro Leu Leu Thr Pro Met Leu Glu Val
945 950 955 960
Phe Cys Gly Asn His Cys Phe Leu Thr Glu Tyr Asn Ala Val Gly Asn
965 970 975
Cys Ser Gly Thr Leu Gln Gln Arg Ser Pro Leu Pro Ala Arg Ala Pro
980 985 990
Phe Ser Lys Ser His Thr Asn Gly Lys Leu Glu Lys Val Leu Asn Ser
995 1000 1005
Ala Glu Asp Leu Ser Cys Leu Pro * Gly Val Phe Leu Gly Ser Pro
1010 1015 1020
Cys Leu Ser Tyr Phe Cys Phe Ser Asp Leu Pro Ser Leu Thr Leu Thr
1025 1030 1035 1040
Phe Phe Asn His Ile Gln Ala Ser Cys Arg Ile Gly Val * Ile Ser
1045 1050 1055
Cys Arg Cys His Asp Asn Ile Leu Pro Val His Val Pro Ser Asn Gln
1060 1065 1070
Thr Lys Pro Asn Pro Lys Thr Gln Ile Gln Ile Thr Leu Lys Pro Gly
1075 1080 1085
Val Leu Ala Gly Phe Ser Trp Lys Gly Val Leu Ser Val Arg Pro Pro
1090 1095 - 1100

21 74025
.
- 82 -
Leu Cys Gly Lys Ile Leu Val Cys Val Ala Gly Gly Gly Ala Gln Gly
1105 1110 1115 1120
Arg Arg Leu Gln Met Lys * Ile Asn Gln Lys Trp Gly Lys * Phe
1125 1130 1135
Val Asn Ala His Gly Arg Ser Ala Gln His * Tyr Cys Ser Val Leu
1140 1145 1150
Trp Ala Tyr Ser Val Trp Ala Ser Leu Phe Pro Glu Thr Leu Ser His
1155 1160 1165
Pro Phe Thr Glu Gln Asp Gly Pro Val * Gln Val Gly Pro Trp Arg
1170 1175 1180
Asn Ser Trp Glu Ala Lys Gly Gln His Thr Gln Asn
1185 1190 1195
(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
1 5 10 15
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
100 105 110
Val Trp Ser Pro Asn Phe Ser Met Trp Leu Phe Ser Trp Arg Asp Ala
115 120 125
Trp Gln Leu Asp Arg Leu Arg Ser Leu Leu Pro Val Thr Pro Gly Met
130 135 140
Ala Val Pro Ala Ile Leu Gly Lys Ile Asn Lys Arg Val Pro Ala Pro
145 150 155 160
Gln Ile Gly Cys Cys Arg Pro Arg Ser Pro Thr Val Glu Thr Lys Arg
165 170 175

21 74025
-
- 83 -
Asp Val Thr Leu Ser Ser Arg Leu Cys Val Leu Pro Pro Tyr Ser Val
180 185 190
Ser Arg Pro Cys Leu Pro Lys Glu Glu Thr Glu Thr Gln Gly Ser Pro
195 200 205
Leu Lys Pro Lys Pro Arg Gly Pro Phe Xaa Pro Xaa Lys Phe Pro Thr
210 215 220
Trp Ala Gly Gly Lys Ile Val Pro Tyr Pro Ala Asn Ser Xaa Pro
225 230 235