U.S. patent application number 10/461791 was filed with the patent office on 2004-11-04 for methods and materials relating to semaphorin-like polynucleotides.
Invention is credited to Arterburn, Matthew C., Boyle, Bryan J., Drmanac, Radoje T., Liu, Chenghua, Mize, Nancy K., Tang, Y. Tom, Yeung, George.
Application Number | 20040220383 10/461791 |
Document ID | / |
Family ID | 23952072 |
Filed Date | 2004-11-04 |
United States Patent
Application |
20040220383 |
Kind Code |
A1 |
Boyle, Bryan J. ; et
al. |
November 4, 2004 |
METHODS AND MATERIALS RELATING TO SEMAPHORIN-LIKE
POLYNUCLEOTIDES
Abstract
The invention provides novel polynucleotides and polypeptides
encoded by such polynucleotides and mutants or variants thereof
that correspond to a novel human secreted semaphorin-like
polypeptide. These polynucleotides comprise nucleic acid sequences
isolated from cDNA library from fetal liver-spleen (Hyseq clone
identification numbers 5688868 (SEQ ID NO. 1)). Other aspects of
the invention include vectors containing processes for producing
novel human secreted semaphorin-like polypeptides, and antibodies
specific for such polypeptides.
Inventors: |
Boyle, Bryan J.; (San
Francisco, CA) ; Yeung, George; (Mountain View,
CA) ; Arterburn, Matthew C.; (Los Gatos, CA) ;
Mize, Nancy K.; (Mountain View, CA) ; Tang, Y.
Tom; (San Jose, CA) ; Liu, Chenghua; (San
Jose, CA) ; Drmanac, Radoje T.; (Palo Alto,
CA) |
Correspondence
Address: |
Renee S. Polizotto, Ph.D.
NUVELO, INC.
675 Almanor Avenue
Sunnyvale
CA
94085
US
|
Family ID: |
23952072 |
Appl. No.: |
10/461791 |
Filed: |
June 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10461791 |
Jun 12, 2003 |
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09653274 |
Aug 31, 2000 |
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6635742 |
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09653274 |
Aug 31, 2000 |
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09491404 |
Jan 25, 2000 |
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Current U.S.
Class: |
530/350 ;
435/320.1; 435/325; 435/6.16; 435/69.1; 530/388.1; 536/23.5 |
Current CPC
Class: |
C07K 14/715 20130101;
C07K 14/70503 20130101; C12N 9/6432 20130101; C12N 9/16 20130101;
C12Y 304/21006 20130101; C07K 14/4703 20130101; C07K 16/00
20130101; A61K 38/00 20130101 |
Class at
Publication: |
530/350 ;
435/006; 435/069.1; 435/320.1; 435/325; 530/388.1; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/47 |
Claims
1: An isolated polynucleotide comprising a nucleotide sequence of
SEQ ID NO: 3, the translated protein coding portion thereof, the
mature protein coding portion thereof, the extracellular portion
thereof, or the active domain thereof.
2: An isolated polynucleotide encoding a polypeptide with
biological activity, which polynucleotide hybridizes to the
complement of a polynucleotide of claim 1 under stringent
hybridization conditions, wherein said conditions are hybridization
to a filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate
(SDS), 1 mM EDTA at 65.degree. C., and washing in 0.133 SSC/0.1%
SDS at 68.degree. C.
3: An isolated polynucleotide encoding a polypeptide with
biological activity, said polynucleotide having greater than about
90% sequence identity with the polynucleotide of claim 1.
4: The polynucleotide of claim 1 which is a DNA sequence.
5: An isolated polynucleotide which comprises the complement of the
polynucleotide of claim 1
6: A vector comprising the polynucleotide of claim 1.
7: An expression vector comprising the polynucleotide of claim
1.
8: A host cell genetically engineered to express the polynucleotide
of claim 1.
9: The host cell of claim 8 wherein the polynucleotide is in
operative association with a regulatory sequence that controls
expression of the polynucleotide in the host cell.
10-17. (Canceled).
18: A method for detecting the polynucleotide of claim 1 in a
sample, comprising: a) contacting the sample with a compound that
binds to and forms a complex with the polynucleotide of claim 1 for
a period sufficient to form the complex; and b) detecting the
complex, so that if a complex is detected, the polynucleotide of
claim 1 is detected.
19: A method for detecting the polynucleotide of claim 1 in a
sample, comprising: a) contacting the sample under stringent
hybridization conditions with nucleic acid primers that anneal to
the polynucleotide of claim 1 under such conditions; b) amplifying
a product comprising at least a portion of the polynucleotide of
claim 1; and c) detecting said product and thereby the
polynucleotide of claim 1 in the sample.
20: The method of claim 19, wherein the polynucleotide comprises an
RNA molecule and the method further comprises reverse transcribing
an annealed RNA molecule into a cDNA polynucleotide.
21-25. (Canceled).
26: A nucleic acid array comprising the polynucleotide of claim 1
or a unique segment of the polynucleotide of claim 1 attached to a
surface.
27: The array of claim 26, wherein the array detects full-matches
to the polynucleotide or a unique segment of the polynucleotide of
claim 1.
28: The array of claim 26, wherein the array detects mismatches to
the polynucleotide or a unique segment of the polynucleotide of
claim 1.
29-30 (Canceled)
Description
1. CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
U.S. application Ser. No. 09/491,404 filed Jan. 10, 2000, entitled
"Novel Contigs Obtained from Various Libraries", Attorney Docket
No. 784, which is incorporated herein by reference in its
entirety.
2. BACKGROUND
[0002] 2.1 Technical Field
[0003] The present invention provides novel polynucleotides and
proteins encoded by such polynucleotides, along with uses for these
polynucleotides and proteins, for example in therapeutic,
diagnostic and research methods. In particular, the invention
relates to a novel semaphorin-like polypeptide.
[0004] 2.2 Background Art
[0005] Identified polynucleotide and polypeptide sequences have
numerous applications in, for example, diagnostics, forensics, gene
mapping; identification of mutations responsible for genetic
disorders or other traits, to assess biodiversity, and to produce
many other types of data and products dependent on DNA and amino
acid sequences. Proteins are known to have biological activity, for
example, by virtue of their secreted nature in the case of leader
sequence cloning, by virtue of their cell or tissue source in the
case of PCR-based techniques, or by virtue of structural similarity
to other genes of known biological activity. It is to these
polypeptides and the polynucleotides encoding them that the present
invention is directed. In particular, this invention is directed to
novel soluble semaphorin-like polypeptides and polynucleotides.
[0006] In the developing nervous system, growing axons are targeted
to their correct targets by different mechanisms. Among these
mechanisms are soluble and contact-mediated chemoattraction and
chemrepulsion (Goodman (1996) Annu. Rev. Neurosci. 19, 341-377).
Semaphorins, netrins, and ephrins family members have been
identified as molecular cues for axonal guidance during development
(Kikuchi et al (1999) Mol. Cell. Neurosci. 13, 9-23). Semaphorins
are secreted and transmembrane proteins containing a characteristic
domain of about 500 amino acids in their extracellular domain (sema
domain). In contrast to the extracellular region, no common domain
structures have been mapped to the cytoplasmic domains of
semaphorins. However, many proline residues are found in the
cytoplasmic domain and they may serve as a binding module for src
homology 3 (SH3) containing proteins (Kikuchi et al (1999) Mol.
Cell. Neurosci. 13, 9-23). Semaphorins are found in all eukaryotes
from worm to mammals and some viruses, and are grouped into six
classes based on the sema domain homology.
[0007] Sema III has been shown to be chemorepellent to sympathetic,
sensory and spinal motor axons. Sema I has also been shown to be a
contact-mediated chemorepellent in Drosophila for motor axons, and
as a chemoattractant for sensory axons in grasshopper.
[0008] Semaphorin class VI family members are comprised of sema Y,
rat sema Y and sema Z, mouse sema VIa and sema VIb. Sema Y mRNA is
expressed in rat embryos but the levels decrease after birth. In
the embryo, sema Y expression was initially seen in the dorsal
spinal cord and the dermamyotome. By embryonic day 13 (E13) sema Y
expression is highest in the ventral horn, dorsal root ganglia
(DRG), dermatome, myotome, notochord, motor nuclei of cranial
nerves, and throughout the brain surface including the marginal
zone of the developing neocortex, thalamus, cerebellum and retina.
Sema Y is also expressed in the granule cell progenitors, immature
muscle and dermis. In adult tissues, sema Y is expressed in
skeletal muscle, brain, and all areas of central nervous system
examined. Thus, semaphorins may play a crucial role in development
of nervous system.
[0009] Recently, plexins, which contain a distantly related sema
domain, and neuropilins have been characterized as semaphorin
receptors.
[0010] Although regulating aspects of neural development,
semaphorins are also expressed in the immune system. In fact, while
many semaphorins including sema Y have been shown to exhibit
inhibitory growth cone collapsing activity and sema3A have been
shown to attract cortical apical dendrites, sema4D (CD100) has been
shown to modulate T and B lymphocyte function. Also, the virally
encoded semaphorins are secreted by the infected cells and
postulated to interfere with the host immune response.
[0011] Semaphorins and semaphorin receptors are crucial for the
normal development and regulation of the nervous system. These are
the molecular cues that guide the axons and neurons. The
semaphorins and their receptors could be very useful in modulating
neuronal growth regenerative capacity (such as in the case of
spinal cord damage), treating neurodegenerative diseases,
diagnosing and mapping genetic neuronal defects. They may be
helpful in treating immunological disorders arising from T and B
lymphocyte dysfunction, or treating viral infections and
cancers.
3. SUMMARY OF THE INVENTION
[0012] This invention is based on the discovery of novel
semaphorin-like polypeptides, novel isolated polynucleotides
encoding such polypeptides, including recombinant DNA molecules,
cloned genes or degenerate variants thereof, especially naturally
occurring variants such as allelic variants, antisense
polynucleotide molecules, and antibodies that specifically
recognize one or more epitopes present on such polypeptides, as
well as hybridomas producing such antibodies. Specifically, the
polynucleotides of the present invention are based on a
semaphorin-like polynucleotide isolated from a cDNA library
prepared from fetal liver-spleen (Hyseq clone identification
numbers 5688868 (SEQ ID NO: 1).
[0013] The compositions of the present invention additionally
include vectors such as expression vectors containing the
polynucleotides of the invention, cells genetically engineered to
contain such polynucleotides and cells genetically engineered to
express such polynucleotides.
[0014] The compositions of the invention provide isolated
polynucleotides that include, but are not limited to, a
polynucleotide comprising the nucleotide sequence set forth in the
SEQ ID NO: 1-3, 5 or 12; or a fragment of SEQ ID NO: 1-3, 5 or 12;
a polynucleotide comprising the full length protein coding sequence
of the SEQ ID NO: 1-3, 5 or 12 (for example, SEQ ID NO: 4); and a
polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of any of SEQ ID NO: 1-3, 5 or 12. The
polynucleotides of the present invention also include, but are not
limited to, a polynucleotide that hybridizes under stringent
hybridization conditions to (a) the complement of any of the
nucleotide sequences set forth in SEQ ID NO: 1-3, 5 or 12; (b) a
nucleotide sequence encoding any of SEQ ID NO: 4, 6-8, 11 or 13; a
polynucleotide which is an allelic variant of any polynucleotides
recited above having at least 70% polynucleotide sequence identity
to the polynucleotides; a polynucleotide which encodes a species
homolog (e.g. orthologs) of any of the peptides recited above; or a
polynucleotide that encodes a polypeptide comprising a specific
domain or truncation of the polypeptide comprising SEQ ID NO:
4.
[0015] A collection as used in this application can be a collection
of only one polynucleotide. The collection of sequence information
or unique identifying information of each sequence can be provided
on a nucleic acid array. In one embodiment, segments of sequence
information are provided on a nucleic acid array to detect the
polynucleotide that contains the segment. The array can be designed
to detect full-match or mismatch to the polynucleotide that
contains the segment. The collection can also be provided in a
computer-readable format.
[0016] This invention further provides cloning or expression
vectors comprising at least a fragment of the polynucleotides set
forth above and host cells or organisms transformed with these
expression vectors. Useful vectors include plasmids, cosmids,
lambda phage derivatives, phagemids, and the like, that are well
known in the art. Accordingly, the invention also provides a vector
including a polynucleotide of the invention and a host cell
containing the polynucleotide. In general, the vector contains an
origin of replication functional in at least one organism,
convenient restriction endonuclease sites, and a selectable marker
for the host cell. Vectors according to the invention include
expression vectors, replication vectors, probe generation vectors,
and sequencing vectors. A host cell according to the invention can
be a prokaryotic or eukaryotic cell and can be a unicellular
organism or part of a multicellular organism.
[0017] The compositions of the present invention include
polypeptides comprising, but not limited to, an isolated
polypeptide selected from the group comprising the amino acid
sequence of SEQ ID NO: 4, 6-8, 11 or 13; or the corresponding full
length or mature protein. Polypeptides of the invention also
include polypeptides with biological activity that are encoded by
(a) any of the polynucleotides having a nucleotide sequence set
forth in the SEQ ID NO: 1-3, 5 or 12; or (b) polynucleotides that
hybridize to the complement of the polynucleotides of (a) under
stringent hybridization conditions. Biologically or immunologically
active variants of any of the protein sequences listed as SEQ ID
NO: 4, 6-8, 11 or 13 and substantial equivalents thereof that
retain biological or immunological activity are also contemplated.
The polypeptides of the invention may be wholly or partially
chemically synthesized but are preferably produced by recombinant
means using the genetically engineered cells (e.g. host cells) of
the invention.
[0018] The invention also provides compositions comprising a
polypeptide of the invention. Pharmaceutical compositions of the
invention may comprise a polypeptide of the invention and an
acceptable carrier, such as a hydrophilic, e.g., pharmaceutically
acceptable, carrier.
[0019] The invention also relates to methods for producing a
polypeptide of the invention comprising culturing host cells
comprising an expression vector containing at least a fragment of a
polynucleotide encoding the polypeptide of the invention in a
suitable culture medium under conditions permitting expression of
the desired polypeptide, and purifying the protein or peptide from
the culture or from the host cells. Preferred embodiments include
those in which the protein produced by such a process is a mature
form of the protein.
[0020] Polynucleotides according to the invention have numerous
applications in a variety of techniques known to those skilled in
the art of molecular biology. These techniques include use as
hybridization probes, use as oligomers, or primers, for PCR, use in
an array, use in computer-readable media, use for chromosome and
gene mapping, use in the recombinant production of protein, and use
in generation of antisense DNA or RNA, their chemical analogs and
the like. For example, when the expression of an mRNA is largely
restricted to a particular cell or tissue type, polynucleotides of
the invention can be used as hybridization probes to detect the
presence of the particular cell or tissue mRNA in a sample using,
e.g., in situ hybridization.
[0021] In other exemplary embodiments, the polynucleotides are used
in diagnostics as expressed sequence tags for identifying expressed
genes or, as well known in the art and exemplified by Vollrath et
al., Science 258:52-59 (1992), as expressed sequence tags for
physical mapping of the human genome.
[0022] The polypeptides according to the invention can be used in a
variety of conventional procedures and methods that are currently
applied to other proteins. For example, a polypeptide of the
invention can be used to generate an antibody that specifically
binds the polypeptide. Such antibodies, particularly monoclonal
antibodies, are useful for detecting or quantitating the
polypeptide in tissue. The polypeptides of the invention can also
be used as molecular weight markers, and as a food supplement.
[0023] Methods are also provided for preventing, treating, or
ameliorating a medical condition which comprises the step of
administering to a mammalian subject a therapeutically effective
amount of a composition comprising a peptide of the present
invention and a pharmaceutically acceptable carrier.
[0024] In particular, the polypeptides and polynucleotides of the
invention can be utilized, for example, in modulating neuronal
growth regenerative capacity, treating neurodegenerative diseases,
diagnosing and mapping genetic neuronal defects. They may also be
helpful in treating immunological disorders arising from T and B
lymphocyte dysfunction, or treating viral infections and
cancers.
[0025] The methods of the invention also provides methods for the
treatment of disorders as recited herein which comprise the
administration of a therapeutically effective amount of a
composition comprising a polynucleotide or polypeptide of the
invention and a pharmaceutically acceptable carrier to a mammalian
subject exhibiting symptoms or tendencies related to disorders as
recited herein. In addition, the invention encompasses methods for
treating diseases or disorders as recited herein comprising the
step of administering a composition comprising compounds and other
substances that modulate the overall activity of the target gene
products and a pharmaceutically acceptable carrier. Compounds and
other substances can effect such modulation either on the level of
target gene/protein expression or target protein activity. The
modulators maybe agonists or antagonists of the semaphorin-like
polypeptide. Specifically, methods are provided for preventing,
treating or ameliorating a medical condition, including viral
diseases, which comprises administering to a mammalian subject,
including but not limited to humans, a therapeutically effective
amount of a composition comprising a polypeptide of the invention
or a therapeutically effective amount of a composition comprising a
binding partner of (e.g., antibody specifically reactive for)
semaphorin-like polypeptides of the invention. The mechanics of the
particular condition or pathology will dictate whether the
polypeptides of the invention or binding partners (or inhibitors)
of these would be beneficial to the individual in need of
treatment.
[0026] According to this method, polypeptides of the invention can
be administered to produce an in vitro or in vivo inhibition of
cellular function. A polypeptide of the invention can be
administered in vivo alone or as an adjunct to other therapies.
Conversely, protein or other active ingredients of the present
invention may be included in formulations of a particular agent to
minimize side effects of such an agent.
[0027] The invention further provides methods for manufacturing
medicaments useful in the above-described methods.
[0028] The present invention further relates to methods for
detecting the presence of the polynucleotides or polypeptides of
the invention in a sample (e.g., tissue or sample). Such methods
can, for example, be utilized as part of prognostic and diagnostic
evaluation of disorders as recited herein and for the
identification of subjects exhibiting a predisposition to such
conditions.
[0029] The invention provides a method for detecting a polypeptide
of the invention in a sample comprising contacting the sample with
a compound that binds to and forms a complex with the polypeptide
under conditions and for a period sufficient to form the complex
and detecting formation of the complex, so that if a complex is
formed, the polypeptide is detected.
[0030] The invention also provides kits comprising polynucleotide
probes and/or monoclonal antibodies, and optionally quantitative
standards, for carrying out methods of the invention. Furthermore,
the invention provides methods for evaluating the efficacy of
drugs, and monitoring the progress of patients, involved in
clinical trials for the treatment of disorders as recited
above.
[0031] The invention also provides methods for the identification
of compounds that modulate (i.e., increase or decrease) the
expression or activity of the polynucleotides and/or polypeptides
of the invention. Such methods can be utilized, for example, for
the identification of compounds that can ameliorate symptoms of
disorders as recited herein. Such methods can include, but are not
limited to, assays for identifying compounds and other substances
that interact with (e.g. bind to) the polypeptides of the
invention.
[0032] The invention provides a method for identifying a compound
that binds to the polypeptide of the present invention comprising
contacting the compound with the polypeptide under conditions and
for a time sufficient to form a polypeptide/compound complex and
detecting the complex, so that if the polypeptide/compound complex
is detected, a compound that binds to the polypeptide is
identified.
[0033] Also provided is a method for identifying a compound that
binds to the polypeptide comprising contacting the compound with
the polypeptide in a cell for a time sufficient to form a
polypeptide/compound complex wherein the complex drives expression
of a reporter gene sequence in the cell and detecting the complex
by detecting reporter gene sequence expression so that if the
polypeptide/compound complex is detected a compound that binds to
the polypeptide is identified.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 shows the BLASTX amino acid sequence alignment
between the protein encoded by SEQ ID NO: 3 (i.e. SEQ ID NO: 4)
semaphorin-like polypeptide and human KIAA1479 protein (Nagase et
al., (2000) DNA Res 7(2): 143-50) [SEQ ID NO: 9], indicating that
the two sequences share 100% similarity over 429 amino acid
residues of SEQ ID NO: 4 and 100% identity over the same 429 amino
acid residues of SEQ ID NO: 4, wherein A=Alanine, C=Cysteine,
D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,
H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,
N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine,
T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented
as dashes.
[0035] FIG. 2 shows the BLASTX amino acid sequence alignment
between the protein encoded by SEQ ID NO: 3 (i.e. SEQ ID NO: 4)
semaphorin-like polypeptide and human Semaphorin Y protein
(International Patent Application No. WO98/11216) [SEQ ID NO: 10],
indicating that the two sequences share 71% similarity over 540
amino acid residues of SEQ ID NO: 4 and 52% identity over the same
540 amino acid residues of SEQ ID NO: 4, wherein A=Alanine,
C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,
G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine,
M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine,
S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are
presented as dashes.
5. DETAILED DESCRIPTION OF THE INVENTION
[0036] The semaphorin-like polypeptide of SEQ ID NO: 4 is an
approximately 1086-amino acid secreted, transmembrane protein with
a predicted molecular mass of approximately 121 kDa unglycosylated.
Protein database searches with the BLASTX algorithm (Altschul S. F.
et al., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al.,
J. Mol. Biol. 21:403-10 (1990), herein incorporated by reference)
indicate that SEQ ID NO: 4 is homologous to human KIAA1479 and
human Semaphorin Y proteins.
[0037] FIG. 1 shows the BLASTX amino acid sequence alignment
between the protein encoded by SEQ ID NO: 3 (i.e. SEQ ID NO: 4) and
the human KIAA1479 protein (Nagase et al., (2000) DNA Res 7(2):
143-50) (SEQ ID NO: 9) indicating that the two sequences share 100%
similarity over 429 amino acid residues of SEQ ID NO: 4 and 100%
identity over the same 429 amino acid residues of SEQ ID NO: 4.
FIG. 2 shows the BLASTX amino acid sequence alignment between the
protein encoded by SEQ ID NO: 3 (i.e. SEQ ID NO: 4) and the human
semaphorin Y protein (International Patent Application No.
WO98/11216) [SEQ ID NO: 10] indicating that the two sequences share
71% similarity over 540 amino acid residues of SEQ ID NO: 4 and 52%
identity over the same 540 amino acid residues of SEQ ID NO: 4. The
sequences of the present invention are expected to have secreted,
transmembrane semaphorin-like activity.
[0038] A predicted approximately sixteen-residue signal peptide is
encoded from approximately residue 1 through residue 16 inclusive
of SEQ ID NO: 4 (SEQ ID NO: 6). The extracellular portion is useful
on its own. This can be confirmed by expression in mammalian cells
and sequencing of the cleaved product. The signal peptide region
was predicted using the Kyte-Doolittle hydrophobicity prediction
algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein
by reference).
[0039] A predicted approximately twenty-nine-residue transmembrane
peptide is encoded from approximately residue 671 to residue 699
inclusive of SEQ ID NO: 4 (SEQ ID NO: 7). The transmembrane peptide
region was predicted using the Kyte-Doolittle hydrophobicity
prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982),
incorporated herein by reference). One of skill in the art will
recognize that the actual cleavage site may be different than that
predicted by the computer program.
[0040] The polypeptides and polynucleotides of the invention and
molecules capable of modulating the polypeptides and
polynucloetides of the invention can be utilized, for example, in
modulating neuronal growth regenerative capacity, treating
neurodegenerative diseases, diagnosing and mapping genetic neuronal
defects. They may also be helpful in treating immunological
disorders arising from T and B lymphocyte dysfunction, or treating
viral infections and cancers.
[0041] 5.1 Definitions
[0042] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an" and "the" include plural
references unless the context clearly dictates otherwise.
[0043] The term "active" refers to those forms of the polypeptide
that retain the biologic and/or immunologic activities of any
naturally occurring polypeptide. According to the invention, the
terms "biologically active" or "biological activity" refer to a
protein or peptide having structural, regulatory or biochemical
functions of a naturally occurring molecule. Likewise "biologically
active" or "biological activity" refers to the capability of the
natural, recombinant or synthetic semaphorin-like peptide, or any
peptide thereof, to induce a specific biological response in
appropriate animals or cells and to bind with specific antibodies.
The term "semaphorin-like biological activity" refers to biological
activity that is similar to the biological activity of a semaphorin
protein.
[0044] The term "activated cells" as used in this application are
those cells which are engaged in extracellular or intracellular
membrane trafficking, including the export of secretory or
enzymatic molecules as part of a normal or disease process.
[0045] The terms "complementary" or "complementarity" refer to the
natural binding of polynucleotides by base pairing. For example,
the sequence 5'-AGT-3' binds to the complementary sequence
3'-TCA-5'. Complementarity between two single-stranded molecules
may be "partial" such that only some of the nucleic acids bind or
it may be "complete" such that total complementarity exists between
the single stranded molecules. The degree of complementarity
between the nucleic acid strands has significant effects on the
efficiency and strength of the hybridization between the nucleic
acid strands.
[0046] The term "embryonic stem cells (ES)" refers to a cell that
can give rise to many differentiated cell types in an embryo or an
adult, including the germ cells. The term "germ line stem cells
(GSCs)" refers to stem cells derived from primordial stem cells
that provide a steady and continuous source of germ cells for the
production of gametes. The term "primordial germ cells (PGCs)"
refers to a small population of cells set aside from other cell
lineages particularly from the yolk sac, mesenteries, or gonadal
ridges during embryogenesis that have the potential to
differentiate into germ cells and other cells. PGCs are the source
from which GSCs and ES cells are derived The PGCs, the GSCs and the
ES cells are capable of self-renewal. Thus these cells not only
populate the germ line and give rise to a plurality of terminally
differentiated cells that comprise the adult specialized organs,
but are able to regenerate themselves.
[0047] The term "expression modulating fragment," EMF, means a
series of nucleotides that modulates the expression of an operably
linked ORF or another EMF.
[0048] As used herein, a sequence is said to "modulate the
expression of an operably linked sequence" when the expression of
the sequence is altered by the presence of the EMF. EMFs include,
but are not limited to, promoters, and promoter modulating
sequences (inducible elements). One class of EMFs is nucleic acid
fragments which induce the expression of an operably linked ORF in
response to a specific regulatory factor or physiological
event.
[0049] The terms "nucleotide sequence" or "nucleic acid" or
"polynucleotide" or "oligonculeotide" are used interchangeably and
refer to a heteropolymer of nucleotides or the sequence of these
nucleotides. These phrases also refer to DNA or RNA of genomic or
synthetic origin which may be single-stranded or double-stranded
and may represent the sense or the antisense strand, to peptide
nucleic acid (PNA) or to any DNA-like or RNA-like material. It its
understood that where the nucleic acid is RNA, the T's in the
nucleic acid sequences provided herein will be substituted with
U's. Generally, nucleic acid segments provided by this invention
may be assembled from fragments of the genome and short
oligonucleotide linkers, or from a series of oligonucleotides, or
from individual nucleotides, to provide a synthetic nucleic acid
which is capable of being expressed in a recombinant
transcriptional unit comprising regulatory elements derived from a
microbial or viral operon, or a eukaryotic gene.
[0050] The terms "oligonucleotide fragment" or a "polynucleotide
fragment", "portion," or "segment" or "probe" or "primer" are used
interchangeably and refer to a sequence of nucleotide residues
which are at least about 5 nucleotides, more preferably at least
about 7 nucleotides, more preferably at least about 9 nucleotides,
more preferably at least about 11 nucleotides and most preferably
at least about 17 nucleotides. The fragment is preferably less than
about 500 nucleotides, preferably less than about 200 nucleotides,
more preferably less than about 100 nucleotides, more preferably
less than about 50 nucleotides and most preferably less than 30
nucleotides. Preferably the probe is from about 6 nucleotides to
about 200 nucleotides, preferably from about 15 to about 50
nucleotides, more preferably from about 17 to 30 nucleotides and
most preferably from about 20 to 25 nucleotides. Preferably the
fragments can be used in polymerase chain reaction (PCR), various
hybridization procedures or microarray procedures to identify or
amplify identical or related parts of mRNA or DNA molecules. A
fragment or segment may uniquely identify each polynucleotide
sequence of the present invention. Preferably the fragment
comprises a sequence substantially similar to a portion of SEQ ID
NO: 1-3, 5 or 12.
[0051] Probes may, for example, be used to determine whether
specific mRNA molecules are present in a cell or tissue or to
isolate similar nucleic acid sequences from chromosomal DNA as
described by Walsh et al. (Walsh, P. S. et al., 1992, PCR Methods
Appl 1:241-250). They may be labeled by nick translation, Klenow
fill-in reaction, PCR, or other methods well known in the art.
Probes of the present invention, their preparation and/or labeling
are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel,
F. M. et al., 1989, Current Protocols in Molecular Biology, John
Wiley & Sons, New York N.Y., both of which are incorporated
herein by reference in their entirety.
[0052] The nucleic acid sequences of the present invention also
include the sequence information from any of the nucleic acid
sequences of SEQ ID NO: 1-3, 5 or 12. The sequence information can
be a segment of SEQ ID NO: 1-3, 5 or 12 that uniquely identifies or
represents the sequence information of SEQ ID NO: 1-3, 5 or 12. One
such segment can be a twenty-mer nucleic acid sequence because the
probability that a twenty-mer is fully matched in the human genome
is 1 in 300. In the human genome, there are three billion base
pairs in one set of chromosomes. Because 4.sup.20 possible
twenty-mers exist, there are 300 times more twenty-mers than there
are base pairs in a set of human chromosome. Using the same
analysis, the probability for a seventeen-mer to be fully matched
in the human genome is approximately 1 in 5. When these segments
are used in arrays for expression studies, fifteen-mer segments can
be used. The probability that the fifteen-mer is fully matched in
the expressed sequences is also approximately one in five because
expressed sequences comprise less than approximately 5% of the
entire genome sequence.
[0053] Similarly, when using sequence information for detecting a
single mismatch, a segment can be a twenty-five mer. The
probability that the twenty-five mer would appear in a human genome
with a single mismatch is calculated by multiplying the probability
for a full match (1.div.4.sup.25) times the increased probability
for mismatch at each nucleotide position (3.times.25). The
probability that an eighteen mer with a single mismatch can be
detected in an array for expression studies is approximately one in
five. The probability that a twenty-mer with a single mismatch can
be detected in a human genome is approximately one in five.
[0054] The term "open reading frame," ORF, means a series of
nucleotide triplets coding for amino acids without any termination
codons and is a sequence translatable into protein.
[0055] The terms "operably linked" or "operably associated" refer
to functionally related nucleic acid sequences. For example, a
promoter is operably associated or operably linked with a coding
sequence if the promoter controls the transcription of the coding
sequence. While operably linked nucleic acid sequences can be
contiguous and in the same reading frame, certain genetic elements
e.g. repressor genes are not contiguously linked to the coding
sequence but still control transcription/translation of the coding
sequence.
[0056] The term "pluripotent" refers to the capability of a cell to
differentiate into a number of differentiated cell types that are
present in an adult organism. A pluripotent cell is restricted in
its differentiation capability in comparison to a totipotent
cell.
[0057] The terms "polypeptide" or "peptide" or "amino acid
sequence" refer to an oligopeptide, peptide, polypeptide or protein
sequence or fragment thereof and to naturally occurring or
synthetic molecules. A polypeptide "fragment," "portion," or
"segment" is a stretch of amino acid residues of at least about 5
amino acids, preferably at least about 7 amino acids, more
preferably at least about 9 amino acids and most preferably at
least about 17 or more amino acids. The peptide preferably is not
greater than about 200 amino acids, more preferably less than 150
amino acids and most preferably less than 100 amino acids.
Preferably the peptide is from about 5 to about 200 amino acids. To
be active, any polypeptide must have sufficient length to display
biological and/or immunological activity.
[0058] The term "naturally occurring polypeptide" refers to
polypeptides produced by cells that have not been genetically
engineered and specifically contemplates various polypeptides
arising from post-translational modifications of the polypeptide
including, but not limited to, acetylation, carboxylation,
glycosylation, phosphorylation, lipidation and acylation.
[0059] The term "translated protein coding portion" means a
sequence which encodes for the full length protein which may
include any leader sequence or a processing sequence.
[0060] The term "mature protein coding sequence" refers to a
sequence which encodes a peptide or protein without any
leader/signal sequence. The peptide may have the leader sequences
removed during processing in the cell or the protein may have been
produced synthetically or using a polynucleotide only encoding for
the mature protein coding sequence.
[0061] The term "derivative" refers to polypeptides chemically
modified by such techniques as ubiquitination, labeling (e.g., with
radionuclides or various enzymes), covalent polymer attachment such
as pegylation (derivatization with polyethylene glycol) and
insertion or substitution by chemical synthesis of amino acids such
as ornithine, which do hot normally occur in human proteins.
[0062] The term "variant" (or "analog") refers to any polypeptide
differing from naturally occurring polypeptides by amino acid
insertions, deletions, and substitutions, created using, e g.,
recombinant DNA techniques. Guidance in determining which amino
acid residues may be replaced, added or deleted without abolishing
activities of interest, may be found by comparing the sequence of
the particular polypeptide with that of homologous peptides and
minimizing the number of amino acid sequence changes made in
regions of high homology (conserved regions) or by replacing amino
acids with consensus sequence.
[0063] Alternatively, recombinant variants encoding these same or
similar polypeptides may be synthesized or selected by making use
of the "redundancy" in the genetic code. Various codon
substitutions, such as the silent changes which produce various
restriction sites, may be introduced to optimize cloning into a
plasmid or viral vector or expression in a particular prokaryotic
or eukaryotic system. Mutations in the polynucleotide sequence may
be reflected in the polypeptide or domains of other peptides added
to the polypeptide to modify the properties of any part of the
polypeptide, to change characteristics such as ligand-binding
affinities, interchain affinities, or degradation/turnover
rate.
[0064] Preferably, amino acid "substitutions" are the result of
replacing one amino acid with another amino acid having similar
structural and/or chemical properties, i.e., conservative amino
acid replacements. "Conservative" amino acid substitutions may be
made on the basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues involved. For example, nonpolar (hydrophobic) amino
acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan, and methionine; polar neutral amino
acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine, and glutamine; positively charged (basic) amino acids
include arginine, lysine, and histidine; and negatively charged
(acidic) amino acids include aspartic acid and glutamic acid.
"Insertions" or "deletions" are preferably in the range of about 1
to 20 amino acids, more preferably 1 to 10 amino acids. The
variation allowed may be experimentally determined by
systematically making insertions, deletions, or substitutions of
amino acids in a polypeptide molecule using recombinant DNA
techniques and assaying the resulting recombinant variants for
activity.
[0065] Alternatively, where alteration of function is desired,
insertions, deletions or non-conservative alterations can be
engineered to produce altered polypeptides. Such alterations can,
for example, alter one or more of the biological functions or
biochemical characteristics of the polypeptides of the invention.
For example, such alterations may change polypeptide
characteristics such as ligand-binding affinities, interchain
affinities, or degradation/turnover rate. Further, such alterations
can be selected so as to generate polypeptides that are better
suited for expression, scale up and the like in the host cells
chosen for expression. For example, cysteine residues can be
deleted or substituted with another amino acid residue in order to
eliminate disulfide bridges.
[0066] The terms "purified" or "substantially purified" as used
herein denotes that the indicated nucleic acid or polypeptide is
present in the substantial absence of other biological
macromolecules, e.g., polynucleotides, proteins, and the like. In
one embodiment, the polynucleotide or polypeptide is purified such
that it constitutes at least 95% by weight, more preferably at
least 99% by weight, of the indicated biological macromolecules
present (but water, buffers, and other small molecules, especially
molecules having a molecular weight of less than 1000 daltons, can
be present).
[0067] The term "isolated" as used herein refers to a nucleic acid
or polypeptide separated from at least one other component (e.g.,
nucleic acid or polypeptide) present with the nucleic acid or
polypeptide in its natural source. In one embodiment, the nucleic
acid or polypeptide is found in the presence of (if anything) only
a solvent, buffer, ion, or other components normally present in a
solution of the same. The terms "isolated" and "purified" do not
encompass nucleic acids or polypeptides present in their natural
source.
[0068] The term "recombinant," when used herein to refer to a
polypeptide or protein, means that a polypeptide or protein is
derived from recombinant (e.g., microbial, insect, or mammalian)
expression systems. "Microbial" refers to recombinant polypeptides
or proteins made in bacterial or fungal (e.g., yeast) expression
systems. As a product, "recombinant microbial" defines a
polypeptide or protein essentially free of native endogenous
substances and unaccompanied by associated native glycosylation.
Polypeptides or proteins expressed in most bacterial cultures,
e.g., E. coli, will be free of glycosylation modifications;
polypeptides or proteins expressed in yeast will have a
glycosylation pattern in general different from those expressed in
mammalian cells.
[0069] The term "recombinant expression vehicle or vector" refers
to a plasmid or phage or virus or vector, for expressing a
polypeptide from a DNA (RNA) sequence. An expression vehicle can
comprise a transcriptional unit comprising an assembly of (1) a
genetic element or elements having a regulatory role in gene
expression, for example, promoters or enhancers, (2) a structural
or coding sequence which is transcribed into mRNA and translated
into protein, and (3) appropriate transcription initiation and
termination sequences. Structural units intended for use in yeast
or eukaryotic expression systems preferably include a leader
sequence enabling extracellular secretion of translated protein by
a host cell. Alternatively, where recombinant protein is expressed
without a leader or transport sequence, it may include an amino
terminal methionine residue. This residue may or may not be
subsequently cleaved from the expressed recombinant protein to
provide a final product.
[0070] The term "recombinant expression system" means host cells
which have stably integrated a recombinant transcriptional unit
into chromosomal DNA or carry the recombinant transcriptional unit
extrachromosomally. Recombinant expression systems as defined
herein will express heterologous polypeptides or proteins upon
induction of the regulatory elements linked to the DNA segment or
synthetic gene to be expressed. This term also means host cells
which have stably integrated a recombinant genetic element or
elements having a regulatory role in gene expression, for example,
promoters or enhancers. Recombinant expression systems as defined
herein will express polypeptides or proteins endogenous to the cell
upon induction of the regulatory elements linked to the endogenous
DNA segment or gene to be expressed. The cells can be prokaryotic
or eukaryotic.
[0071] The term "secreted" includes a protein that is transported
across or through a membrane, including transport as a result of
signal sequences in its amino acid sequence when it is expressed in
a suitable host cell. "Secreted" proteins include without
limitation proteins secreted wholly (e.g., soluble proteins) or
partially (e.g., receptors) from the cell in which they are
expressed. "Secreted" proteins also include without limitation
proteins that are transported across the membrane of the
endoplasmic reticulum. "Secreted" proteins are also intended to
include proteins containing non-typical signal sequences (e.g.
Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992)
Cytokine 4(2):134-143) and factors released from damanged cells
(e.g. Interleukin-1 Receptor Antagonist, see Arend, W. P. et al.
(1998) Annu. Rev. Immunol. 16:27-55)
[0072] Where desired, an expression vector may be designed to
contain a "signal or leader sequence" which will direct the
polypeptide through the membrane of a cell. Such a sequence may be
naturally present on the polypeptides of the present invention or
provided from heterologous protein sources by recombinant DNA
techniques.
[0073] The term "stringent" is used to refer to conditions that are
commonly understood in the art as stringent. Stringent conditions
can include highly stringent conditions (i.e., hybridization to
filter-bound DNA in 0.5 M NaHPO.sub.4, 7% sodium dodecyl sulfate
(SDS), 1 mM EDTA at 65.degree. C., and washing in
0.1.times.SSC/0.1% SDS at 68.degree. C.), and moderately stringent
conditions (i.e., washing in 0.2.times.SSC/0.1% SDS at 42.degree.
C.). Other exemplary hybridization conditions are described herein
in the examples.
[0074] In instances of hybridization of deoxyoligonucleotides,
additional exemplary stringent hybridization conditions include
washing in 6.times.SSC/0.05% sodium pyrophosphate at 37.degree. C.
(for 14-base oligonucleotides), 48.degree. C. (for 17-base
oligonucleotides), 55.degree. C. (for 20-base oligonucleotides),
and 60.degree. C. (for 23-base oligonucleotides).
[0075] As used herein, "substantially equivalent" can refer both to
nucleotide and amino acid sequences, for example a mutant
-sequence, that varies from a reference sequence by one or more
substitutions, deletions, or additions, the net effect of which
does not result in an adverse functional dissimilarity between the
reference and subject sequences. Typically, such a substantially
equivalent sequence varies from one of those listed herein by no
more than about 35% (i.e., the number of individual residue
substitutions, additions, and/or deletions in a substantially
equivalent sequence, as compared to the corresponding reference
sequence, divided by the total number of residues in the
substantially equivalent sequence is about 0.35 or less). Such a
sequence is said to have 65% sequence identity to the listed
sequence. In one embodiment, a substantially equivalent, e.g.,
mutant, sequence of the invention varies from a listed sequence by
no more than 30% (70% sequence identity); in a variation of this
embodiment, by no more than 25% (75% sequence identity); and in a
further variation of this embodiment, by no more than 20% (80%
sequence identity) and in a further variation of this embodiment,
by no more than 10% (90% sequence identity) and in a further
variation of this embodiment, by no more that 5% (95% sequence
identity). Substantially equivalent, e.g., mutant, amino acid
sequences according to the invention preferably have at least 80%
sequence identity with a listed amino acid sequence, more
preferably at least 90% sequence identity. Substantially equivalent
nucleotide sequence of the invention can have lower percent
sequence identities, taking into account, for example, the
redundancy or degeneracy of the genetic code. Preferably,
nucleotide sequence has at least about 65% identity, more
preferably at least about 75% identity, and most preferably at
least about 95% identity. For the purposes of the present
invention, sequences having substantially equivalent biological
activity and substantially equivalent expression characteristics
are considered substantially equivalent. For the purposes of
determining equivalence, truncation of the mature sequence (e.g.,
via a mutation which creates a spurious stop codon) should be
disregarded. Sequence identity may be determined, e.g., using the
Jotun Hein method (Hein, J. (1990) Methods Enzymol. 183:626-645).
Identity between sequences can also be determined by other methods
known in the art, e.g. by varying hybridization conditions.
[0076] The term "totipotent" refers to the capability of a cell to
differentiate into all of the cell types of an adult organism.
[0077] The term "transformation" means introducing DNA into a
suitable host cell so that the DNA is replicable, either as an
extrachromosomal element, or by chromosomal integration. The term
"transfection" refers to the taking up of an expression vector by a
suitable host cell, whether or not any coding sequences are in fact
expressed. The term "infection" refers to the introduction of
nucleic acids into a suitable host cell by use of a virus or viral
vector.
[0078] As used herein, an "uptake modulating fragment," UMF, means
a series of nucleotides which mediate the uptake of a linked DNA
fragment into a cell. UMFs can be readily identified using known
UMFs as a target sequence or target motif with the computer-based
systems described below. The presence and activity of a UMF can be
confirmed by attaching the suspected UMF to a marker sequence. The
resulting nucleic acid molecule is then incubated with an
appropriate host under appropriate conditions and the uptake of the
marker sequence is determined. As described above, a UMF will
increase the frequency of uptake of a linked marker sequence.
[0079] Each of the above terms is meant to encompass all that is
described for each, unless the context dictates otherwise.
[0080] 5.2 Nucleic Acids of the Invention
[0081] The invention is based on the discovery of a novel secreted
semaphorin-like polypeptide, the polynucleotides encoding the
semaphorin-like polypeptide and the use of these compositions for
the diagnosis, treatment or prevention of neurological conditions
and disorders.
[0082] The isolated polynucleotides of the invention include, but
are not limited to a polynucleotide comprising any of the
nucleotide sequences of SEQ ID NO: 1-3, 5 or 12; a fragment of SEQ
ID NO: 1-3, 5 or 12; a polynucleotide comprising the full length
protein coding sequence of SEQ ID NO: 1-3, 5 or 12 (for example SEQ
ID NO: 4); and a polynucleotide comprising the nucleotide sequence
encoding the mature protein coding sequence of the polynucleotides
of any one of SEQ ID NO: 1-3, 5 or 12. The polynucleotides of the
present invention also include, but are not limited to, a
polynucleotide that hybridizes under stringent conditions to (a)
the complement of any of the nucleotides sequences of the SEQ ID
NO: 1-3, 5 or 12; (b) a polynucleotide encoding any one of the
polypeptides of SEQ ID NO: 4, 6-8, 11 or 13; (c) a polynucleotide
which is an allelic variant of any polynucleotides recited above;
(d) a polynucleotide which encodes a species homolog of any of the
proteins recited above; or (e) a polynucleotide that encodes a
polypeptide comprising a specific domain or truncation of the
polypeptides of SEQ ID NO: 4, 6-8, 11 or 13. Domains of interest
may depend on the nature of the encoded polypeptide; e.g., domains
in receptor-like polypeptides include ligand-binding,
extracellular, transmembrane, or cytoplasmic domains, or
combinations thereof; domains in immunoglobulin-like proteins
include the variable immunoglobulin-like domains; domains in
enzyme-like polypeptides include catalytic and substrate binding
domains; and domains in ligand polypeptides include
receptor-binding domains.
[0083] The polynucleotides of the invention include naturally
occurring or wholly or partially synthetic DNA, e.g., cDNA and
genomic DNA, and RNA, e.g., mRNA. The polynucleotides may include
all of the coding region of the cDNA or may represent a portion of
the coding region of the cDNA.
[0084] The present invention also provides genes corresponding to
the cDNA sequences disclosed herein. The corresponding genes can be
isolated in accordance with known methods using the sequence
information disclosed herein. Such methods include the preparation
of probes or primers from the disclosed sequence information for
identification and/or amplification of genes in appropriate genomic
libraries or other sources of genomic materials. Further 5' and 3'
sequence can be obtained using methods known in the art. For
example, full length cDNA or genomic DNA that corresponds to any of
the polynucleotides of the SEQ ID NO: 1-3, 5 or 12 can be obtained
by screening appropriate cDNA or genomic DNA libraries under
suitable hybridization conditions using any of the polynucleotides
of the SEQ ID NO: 1-3, 5 or 12 or a portion thereof as a probe.
Alternatively, the polynucleotides of the SEQ ID NO: 1-3, 5 or 12
may be used as the basis for suitable primer(s) that allow
identification and/or amplification of genes in appropriate genomic
DNA or cDNA libraries.
[0085] The nucleic acid sequences of the invention can be assembled
from ESTs and sequences (including cDNA and genomic sequences)
obtained from one or more public databases, such as dbEST, gbpri,
and UniGene. The EST sequences can provide identifying sequence
information, representative fragment or segment information, or
novel segment information for the full-length gene.
[0086] The polynucleotides of the invention also provide
polynucleotides including nucleotide sequences that are
substantially equivalent to the polynucleotides recited above.
Polynucleotides according to the invention can have, e.g., at least
about 65%, at least about 70%, at least about 75%, at least about
80%, more typically at least about 90%, and even more typically at
least about 95%, sequence identity to a polynucleotide recited
above.
[0087] Included within the scope of the nucleic acid sequences of
the invention are nucleic acid sequence fragments that hybridize
under stringent conditions to any of the nucleotide sequences of
the SEQ ID NO: 1-3, 5 or 12, or complements thereof, which fragment
is greater than about 5 nucleotides, preferably 7 nucleotides, more
preferably greater than 9 nucleotides and most preferably greater
than 17 nucleotides. Fragments of, e.g. 15, 17, or 20 nucleotides
or more that are selective for (i.e. specifically hybridize to any
one of the polynucleotides of the invention) are contemplated.
Probes capable of specifically hybridizing to a polynucleotide can
differentiate polynucleotide sequences of the invention from other
polynucleotide sequences in the same family of genes or can
differentiate human genes from genes of other species, and are
preferably based on unique nucleotide sequences.
[0088] The sequences falling within the scope of the present
invention are not limited to these specific sequences, but also
include allelic and species variations thereof. Allelic and species
variations can be routinely determined by comparing the sequence
provided in SEQ ID NO: 1-3, 5 or 12, a representative fragment
thereof, or a nucleotide sequence at least 90% identical,
preferably 95% identical, to SEQ ID NO: 1-3, 5 or 12 with a
sequence from another isolate of the same species. Furthermore, to
accommodate codon variability, the invention includes nucleic acid
molecules coding for the same amino acid sequences as do the
specific ORFs disclosed herein. In other words, in the coding
region of an ORF, substitution of one codon for another codon that
encodes the same amino acid is expressly contemplated.
[0089] The nearest neighbor result for the nucleic acids of the
present invention, including SEQ ID NO: 1-3, 5 or 12, can be
obtained by searching a database using an algorithm or a program.
Preferably, a BLAST which stands for Basic Local alignment Search
Tool is used to search for local sequence alignments (Altshul, S.
F. J Mol. Evol. 36 290-300 (1993) and Altschul S. F. et al. J. Mol.
Biol. 21:403-410 (1990))
[0090] Species homologs (or orthologs) of the disclosed
polynucleotides and proteins are also provided by the present
invention. Species homologs may be isolated and identified by
making suitable probes or primers from the sequences provided
herein and screening a suitable nucleic acid source from the
desired species.
[0091] The invention also encompasses allelic variants of the
disclosed polynucleotides or proteins; that is, naturally-occurring
alternative forms of the isolated polynucleotide which also encode
proteins which are identical, homologous or related to that encoded
by the polynucleotides.
[0092] The nucleic acid sequences of the invention are further
directed to sequences which encode variants of the described
nucleic acids. These amino acid sequence variants may be prepared
by methods known in the art by introducing appropriate nucleotide
changes into a native or variant polynucleotide. There are two
variables in the construction of amino acid sequence variants: the
location of the mutation and the nature of the mutation. Nucleic
acids encoding the amino acid sequence variants are preferably
constructed by mutating the polynucleotide to encode an amino acid
sequence that does not occur in nature. These nucleic acid
alterations can be made at sites that differ in the nucleic acids
from different species (variable positions) or in highly conserved
regions (constant regions). Sites at such locations will typically
be modified in series, e.g., by substituting first with
conservative choices (e.g., hydrophobic amino acid to a different
hydrophobic amino acid) and then with more distant choices (e.g.,
hydrophobic amino acid to a charged amino acid), and then deletions
or insertions may be made at the target site. Amino acid sequence
deletions generally range from about 1 to 30 residues, preferably
about 1 to 10 residues, and are typically contiguous. Amino acid
insertions include amino- and/or carboxyl-terminal fusions ranging
in length from one to one hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Intrasequence insertions may range generally from about 1 to 10
amino residues, preferably from 1 to 5 residues. Examples of
terminal insertions include the heterologous signal sequences
necessary for secretion or for intracellular targeting in different
host cells and sequences such as FLAG or poly-histidine sequences
useful for purifying the expressed protein.
[0093] In a preferred method, polynucleotides encoding the novel
amino acid sequences are changed via site-directed mutagenesis.
This method uses oligonucleotide sequences to alter a
polynucleotide to encode the desired amino acid variant, as well as
sufficient adjacent nucleotides on both sides of the changed amino
acid to form a stable duplex on either side of the site being
changed. In general, the techniques of site-directed mutagenesis
are well known to those of skill in the art and this technique is
exemplified by publications such as, Edelman et al., DNA 2:183
(1983). A versatile and efficient method for producing
site-specific changes in a polynucleotide sequence was published by
Zoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may
also be used to create amino acid sequence variants of the novel
nucleic acids. When small amounts of template DNA are used as
starting material, primer(s) that differs slightly in sequence from
the corresponding region in the template DNA can generate the
desired amino acid variant. PCR amplification results in a
population of product DNA fragments that differ from the
polynucleotide template encoding the polypeptide at the position
specified by the primer. The product DNA fragments replace the
corresponding region in the plasmid and this gives a polynucleotide
encoding the desired amino acid variant.
[0094] A further technique for generating amino acid variants is
the cassette mutagenesis technique described in Wells et al., Gene
34:315 (1985); and other mutagenesis techniques well known in the
art, such as, for example, the techniques in Sambrook et al.,
supra, and Current Protocols in Molecular Biology, Ausubel et al.
Due to the inherent degeneracy of the genetic code, other DNA
sequences which encode substantially the same or a functionally
equivalent amino acid sequence may be used in the practice of the
invention for the cloning and expression of these novel nucleic
acids. Such DNA sequences include those which are capable of
hybridizing to the appropriate novel nucleic acid sequence under
stringent conditions.
[0095] Polynucleotides encoding preferred polypeptide truncations
of the invention can be used to generate polynucleotides encoding
chimeric or fusion proteins comprising one or more domains of the
invention and heterologous protein sequences.
[0096] The polynucleotides of the invention additionally include
the complement of any of the polynucleotides recited above. The
polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic)
or RNA. Methods and algorithms for obtaining such polynucleotides
are well known to those of skill in the art and can include, for
example, methods for determining hybridization conditions that can
routinely isolate polynucleotides of the desired sequence
identities.
[0097] In accordance with the invention, polynucleotide sequences
comprising the mature protein coding sequences corresponding to any
one of SEQ ID NO: 4, 6-8, 11 or 13 or functional equivalents
thereof, may be used to generate recombinant DNA molecules that
direct the expression of that nucleic acid, or a functional
equivalent thereof, in appropriate host cells. Also included are
the cDNA inserts of any of the clones identified herein.
[0098] A polynucleotide according to the invention can be joined to
any of a variety of other nucleotide sequences by well-established
recombinant DNA techniques (see Sambrook J et al. (1989) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).
Useful nucleotide sequences for joining to polynucleotides include
an assortment of vectors, e.g., plasmids, cosmids, lambda phage
derivatives, phagemids, and the like, that are well known in the
art. Accordingly, the invention also provides a vector including a
polynucleotide of the invention and a host cell containing the
polynucleotide. In general, the vector contains an origin of
replication functional in at least one organism, convenient
restriction endonuclease sites, and a selectable marker for the
host cell. Vectors according to the invention include expression
vectors, replication vectors, probe generation vectors, and
sequencing vectors. A host cell according to the invention can be a
prokaryotic or eukaryotic cell and can be a unicellular organism or
part of a multicellular organism.
[0099] The present invention further provides recombinant
constructs comprising a nucleic acid having any of the nucleotide
sequences of the SEQ ID NO: 1-3, 5 or 12 or a fragment thereof or
any other polynucleotides of the invention. In one embodiment, the
recombinant constructs of the present invention comprise a vector,
such as a plasmid or viral vector, into which a nucleic acid having
any of the nucleotide sequences of the SEQ ID NO: 1-3, 5 or 12 or a
fragment thereof is inserted, in a forward or reverse orientation.
In the case of a vector comprising one of the ORFs of the present
invention, the vector may further comprise regulatory sequences,
including for example, a promoter, operably linked to the ORF.
Large numbers of suitable vectors and promoters are known to those
of skill in the art and are commercially available for generating
the recombinant constructs of the present invention. The following
vectors are provided by way of example. Bacterial: pBs,
phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a,
pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540,
pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
[0100] The isolated polynucleotide of the invention may be operably
linked to an expression control sequence such as the pMT2 or pED
expression vectors disclosed in Kaufman et al., Nucleic Acids Res.
19, 4485-4490 (1991), in order to produce the protein
recombinantly. Many suitable expression control sequences are known
in the art. General methods of expressing recombinant proteins are
also known and are exemplified in R. Kaufman, Methods in Enzymology
185, 537-566 (1990). As defined herein "operably linked" means that
the isolated polynucleotide of the invention and an expression
control sequence are situated within a vector or cell in such a way
that the protein is expressed by a host cell which has been
transformed (transfected) with the ligated
polynucleotide/expression control sequence.
[0101] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lacI, lacZ, T3, T7,
gpt, lambda PR, and trc. Eukaryotic promoters include CMV immediate
early, HSV thymidine kinase, early and late SV40, LTRs from
retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art. Generally, recombinant expression
vectors will include origins of replication and selectable markers
permitting transformation of the host cell, e.g., the ampicillin
resistance gene of E. coli and S. cerevisiae TRP1 gene, and a
promoter derived from a highly expressed gene to direct
transcription of a downstream structural sequence. Such promoters
can be derived from operons encoding glycolytic enzymes such as
3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or
heat shock proteins, among others. The heterologous structural
sequence is assembled in appropriate phase with translation
initiation and termination sequences, and preferably, a leader
sequence capable of directing secretion of translated protein into
the periplasmic space or extracellular medium. Optionally, the
heterologous sequence can encode a fusion protein including an
amino terminal identification peptide imparting desired
characteristics, e.g., stabilization or simplified purification of
expressed recombinant product. Useful expression vectors for
bacterial use are constructed by inserting a structural DNA
sequence encoding a desired protein together with suitable
translation initiation and termination signals in operable reading
phase with a functional promoter. The vector will comprise one or
more phenotypic selectable markers and an origin of replication to
ensure maintenance of the vector and to, if desirable, provide
amplification within the host. Suitable prokaryotic hosts for
transformation include E. coli, Bacillus subtilis, Salmonella
typhimurium and various species within the genera Pseudomonas,
Streptomyces, and Staphylococcus, although others may also be
employed as a matter of choice.
[0102] As a representative but non-limiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed. Following
transformation of a suitable host strain and growth of the host
strain to an appropriate cell density, the selected promoter is
induced or derepressed by appropriate means (e.g., temperature
shift or chemical induction) and cells are cultured for an
additional period. Cells are typically harvested by centrifugation,
disrupted by physical or chemical means, and the resulting crude
extract retained for further purification.
[0103] Polynucleotides of the invention can also be used to induce
immune responses. For example, as described in Fan et al., Nat.
Biotech. 17:870-872 (1999), incorporated herein by reference,
nucleic acid sequences encoding a polypeptide may be used to
generate antibodies against the encoded polypeptide following
topical administration of naked plasmid DNA or following injection,
and preferably intramuscular injection of the DNA. The nucleic acid
sequences are preferably inserted in a recombinant expression
vector and may be in the form of naked DNA.
[0104] 5.3 Hosts
[0105] The present invention further provides host cells
genetically engineered to contain the polynucleotides of the
invention. For example, such host cells may contain nucleic acids
of the invention introduced into the host cell using known
transformation, transfection or infection methods. The present
invention still further provides host cells genetically engineered
to express the polynucleotides of the invention, wherein such
polynucleotides are in operative association with a regulatory
sequence heterologous to the host cell which drives expression of
the polynucleotides in the cell.
[0106] Knowledge of semaphorin-like DNA sequences allows for
modification of cells to permit, or increase, expression of
semaphorin-like polypeptide. Cells can be modified (e.g., by
homologous recombination) to provide increased semaphorin-like
polypeptide expression by replacing, in whole or in part, the
naturally occurring semaphorin-like promoter with all or part of a
heterologous promoter so that the cells semaphorin-like polypeptide
is expressed at higher levels. The heterologous promoter is
inserted in such a manner that it is operatively linked to
semaphorin-like encoding sequences. See, for example, PCT
International Publication No. WO94/12650, PCT International
Publication No. WO92/20808, and PCT International Publication No.
WO91/09955. It is also contemplated that, in addition to
heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr,
and the multifunctional CAD gene which encodes carbamyl phosphate
synthase, aspartate transcarbamylase, and dihydroorotase) and/or
intron DNA may be inserted along with the heterologous promoter
DNA. If linked to the semaphorin-like coding sequence,
amplification of the marker DNA by standard selection methods
results in co-amplification of the semaphorin-like coding sequences
in the cells.
[0107] The host cell can be a higher eukaryotic host cell, such as
a mammalian cell, a lower eukaryotic host cell, such as a yeast
cell, or the host cell can be a prokaryotic cell, such as a
bacterial cell. Introduction of the recombinant construct into the
host cell can be effected by calcium phosphate transfection, DEAE,
dextran-mediated transfection, or electroporation (Davis, L. et
al., Basic Methods in Molecular Biology (1986)). The host cells
containing one of the polynucleotides of the invention, can be used
in conventional manners to produce the gene product encoded by the
isolated fragment (in the case of an ORF) or can be used to produce
a heterologous protein under the control of the EMF.
[0108] Any host/vector system can be used to express one or more of
the ORFs of the present invention. These include, but are not
limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS
cells, 293 cells, and Sf9 cells, as well as prokaryotic host such
as E. coli and B. subtilis. The most preferred cells are those
which do not normally express the particular polypeptide or protein
or which expresses the polypeptide or protein at low natural level.
Mature proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which
is hereby incorporated by reference.
[0109] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell 23:175 (1981). Other cell lines capable
of expressing a compatible vector are, for example, the C127,
monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney
293 cells, human epidermal A431 cells, human Colo205 cells, 3T3
cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell strains derived from in vitro culture of
primary tissue, primary explants, HeLa cells, mouse L cells, BHK,
HL-60, U937, HaK or Jurkat cells. Mammalian expression vectors will
comprise an origin of replication, a suitable promoter and also any
necessary ribosome binding sites, polyadenylation site, splice
donor and acceptor sites, transcriptional termination sequences,
and 5' flanking nontranscribed sequences. DNA sequences derived
from the SV40 viral genome, for example, SV40 origin, early
promoter, enhancer, splice, and polyadenylation sites may be used
to provide the required nontranscribed genetic elements.
Recombinant polypeptides and proteins produced in bacterial culture
are usually isolated by initial extraction from cell pellets,
followed by one or more salting-out, aqueous ion exchange or size
exclusion chromatography steps. Protein refolding steps can be
used, as necessary, in completing configuration of the mature
protein. Finally, high performance liquid chromatography (HPLC) can
be employed for final purification steps. Microbial cells employed
in expression of proteins can be disrupted by any convenient
method, including freeze-thaw cycling, sonication, mechanical
disruption, or use of cell lysing agents.
[0110] Alternatively, it may be possible to produce the protein in
lower eukaryotes such as yeast or insects or in prokaryotes such as
bacteria. Potentially suitable yeast strains include Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any yeast strain capable of expressing heterologous
proteins. Potentially suitable bacterial strains include
Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any
bacterial strain capable of expressing heterologous proteins. If
the protein is made in yeast or bacteria, it may be necessary to
modify the protein produced therein, for example by phosphorylation
or glycosylation of the appropriate sites, in order to obtain the
functional protein. Such covalent attachments may be accomplished
using known chemical or enzymatic methods.
[0111] In another embodiment of the present invention, cells and
tissues may be engineered to express an endogenous gene comprising
the polynucleotides of the invention under the control of inducible
regulatory elements, in which case the regulatory sequences of the
endogenous gene may be replaced by homologous recombination. As
described herein, gene targeting can be used to replace a gene's
existing regulatory region with a regulatory sequence isolated from
a different gene or a novel regulatory sequence synthesized by
genetic engineering methods. Such regulatory sequences may be
comprised of promoters, enhancers, scaffold-attachment regions,
negative regulatory elements, transcriptional initiation sites,
regulatory protein binding sites or combinations of said sequences.
Alternatively, sequences which affect the structure or stability of
the RNA or protein produced may be replaced, removed, added, or
otherwise modified by targeting. These sequence include
polyadenylation signals, mRNA stability elements, splice sites,
leader sequences for enhancing or modifying transport or secretion
properties of the protein, or other sequences which alter or
improve the function or stability of protein or RNA molecules.
[0112] The targeting event may be a simple insertion of the
regulatory sequence, placing the gene under the control of the new
regulatory sequence, e.g., inserting a new promoter or enhancer or
both upstream of a gene. Alternatively, the targeting event may be
a simple deletion of a regulatory element, such as the deletion of
a tissue-specific negative regulatory element. Alternatively, the
targeting event may replace an existing element; for example, a
tissue-specific enhancer can be replaced by an enhancer that has
broader or different cell-type specificity than the naturally
occurring elements. Here, the naturally occurring sequences are
deleted and new sequences are added. In all cases, the
identification of the targeting event may be facilitated by the use
of one or more selectable marker genes that are contiguous with the
targeting DNA, allowing for the selection of cells in which the
exogenous DNA has integrated into the host cell genome. The
identification of the targeting event may also be facilitated by
the use of one or more marker genes exhibiting the property of
negative selection, such that the negatively selectable marker is
linked to the exogenous DNA, but configured such that the
negatively selectable marker flanks the targeting sequence, and
such that a correct homologous recombination event with sequences
in the host cell genome does not result in the stable integration
of the negatively selectable marker. Markers useful for this
purpose include the Herpes Simplex Virus thymidine kinase (TK) gene
or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt)
gene.
[0113] The gene targeting or gene activation techniques which can
be used in accordance with this aspect of the invention are more
particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S.
Pat. No. 5,578,461 to Sherwin et al.; International Application No.
PCT/US92/09627 (WO93/09222) by Selden et al.; and International
Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al.,
each of which is incorporated by reference herein in its
entirety.
[0114] 5.4 Polypeptides of the Invention
[0115] The isolated polypeptides of the invention include, but are
not limited to, a polypeptide comprising: the amino acid sequence
set forth as any one of SEQ ID NO: 4, 6-8, 11 or 13 or an amino
acid sequence encoded by any one of the nucleotide sequences SEQ ID
NO: 1-3, 5 or 12 or the corresponding full length or mature
protein. Polypeptides of the invention also include polypeptides
preferably with biological or immunological activity that are
encoded by: (a) a polynucleotide having any one of the nucleotide
sequences set forth in the SEQ ID NO: 1-3, 5 or 12 or (b)
polynucleotides encoding any one of the amino acid sequences set
forth as SEQ ID NO: 4, 6-8, 11 or 13 or (c) polynucleotides that
hybridize to the complement of the polynucleotides of either (a) or
(b) under stringent hybridization conditions. The invention also
provides biologically active or immunologically active variants of
any of the amino acid sequences set forth as SEQ ID NO: 4, 6-8, 11
or 13 or the corresponding full length or mature protein; and
"substantial equivalents" thereof (e.g., with at least about 65%,
at least about 70%, at least about 75%, at least about 80%, at
least about 85%, at least about 90%, typically at least about 95%,
more typically at least about 98%, or most typically at least about
99% amino acid identity) that retain biological activity.
Polypeptides encoded by allelic variants may have a similar,
increased, or decreased activity compared to polypeptides
comprising SEQ ID NO: 4, 6-8, 11 or 13.
[0116] Fragments of the proteins of the present invention which are
capable of exhibiting biological activity are also encompassed by
the present invention. Fragments of the protein may be in linear
form or they may be cyclized using known methods, for example, as
described in H. U. Saragovi, et al., Bio/Technology 10, 773-778
(1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114,
9245-9253 (1992), both of which are incorporated herein by
reference. Such fragments may be fused to carrier molecules such as
immunoglobulins for many purposes, including increasing the valency
of protein binding sites.
[0117] The present invention also provides both full-length and
mature forms (for example, without a signal sequence or precursor
sequence) of the disclosed proteins. The protein coding sequence is
identified in the sequence listing by translation of the disclosed
nucleotide sequences. The mature form of such protein may be
obtained by expression of a full-length polynucleotide in a
suitable mammalian cell or other host cell. The sequence of the
mature form of the protein is also determinable from the amino acid
sequence of the full-length form. Where proteins of the present
invention are membrane bound, soluble forms of the proteins are
also provided. In such forms, part or all of the regions causing
the proteins to be membrane bound are deleted so that the proteins
are fully secreted from the cell in which it is expressed.
[0118] Protein compositions of the present invention may further
comprise an acceptable carrier, such as a hydrophilic, e.g.,
pharmaceutically acceptable, carrier.
[0119] The present invention further provides isolated polypeptides
encoded by the nucleic acid fragments of the present invention or
by degenerate variants of the nucleic acid fragments of the present
invention. By "degenerate variant" is intended nucleotide fragments
which differ from a nucleic acid fragment of the present invention
(e.g., an ORF) by nucleotide sequence but, due to the degeneracy of
the genetic code, encode an identical polypeptide sequence.
Preferred nucleic acid fragments of the present invention are the
ORFs that encode proteins.
[0120] A variety of methodologies known in the art can be utilized
to obtain any one of the isolated polypeptides or proteins of the
present invention. At the simplest level, the amino acid sequence
can be synthesized using commercially available peptide
synthesizers. The synthetically-constructed protein sequences, by
virtue of sharing primary, secondary or tertiary structural and/or
conformational characteristics with proteins may possess biological
properties in common therewith, including protein activity. This
technique is particularly useful in producing small peptides and
fragments of larger polypeptides. Fragments are useful, for
example, in generating antibodies against the native polypeptide.
Thus, they may be employed as biologically active or immunological
substitutes for natural, purified proteins in screening of
therapeutic compounds and in immunological processes for the
development of antibodies.
[0121] The polypeptides and proteins of the present invention can
alternatively be purified from cells which have been altered to
express the desired polypeptide or protein. As used herein, a cell
is said to be altered to express a desired polypeptide or protein
when the cell, through genetic manipulation, is made to produce a
polypeptide or protein which it normally does not produce or which
the cell normally produces at a lower level. One skilled in the art
can readily adapt procedures for introducing and expressing either
recombinant or synthetic sequences into eukaryotic or prokaryotic
cells in order to generate a cell which produces one of the
polypeptides or proteins of the present invention.
[0122] The invention also relates to methods for producing a
polypeptide comprising growing a culture of host cells of the
invention in a suitable culture medium, and purifying the protein
from the cells or the culture in which the cells are grown. For
example, the methods of the invention include a process for
producing a polypeptide in which a host cell containing a suitable
expression vector that includes a polynucleotide of the invention
is cultured under conditions that allow expression of the encoded
polypeptide. The polypeptide can be recovered from the culture,
conveniently from the culture medium, or from a lysate prepared
from the host cells and further purified. Preferred embodiments
include those in which the protein produced by such process is a
full length or mature form of the protein.
[0123] In an alternative method, the polypeptide or protein is
purified from bacterial cells which naturally produce the
polypeptide or protein. One skilled in the art can readily follow
known methods for isolating polypeptides and proteins in order to
obtain one of the isolated polypeptides or proteins of the present
invention. These include, but are not limited to,
immunochromatography, HPLC, size-exclusion chromatography,
ion-exchange chromatography, and immuno-affinity chromatography.
See, e.g., Scopes, Protein Purification: Principles and Practice,
Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A
Laboratory Manual; Ausubel et al., Current Protocols in Molecular
Biology. Polypeptide fragments that retain biological/immunological
activity include fragments comprising greater than about 100 amino
acids, or greater than about 200 amino acids, and fragments that
encode specific protein domains.
[0124] The purified polypeptides can be used in in vitro binding
assays which are well known in the art to identify molecules which
bind to the polypeptides. These molecules include but are not
limited to, for e.g., small molecules, molecules from combinatorial
libraries, antibodies or other proteins. The molecules identified
in the binding assay are then tested for antagonist or agonist
activity in in vivo tissue culture or animal models that are well
known in the art. In brief, the molecules are titrated into a
plurality of cell cultures or animals and then tested for either
cell/animal death or prolonged survival of the animal/cells.
[0125] In addition, the peptides of the invention or molecules
capable of binding to the peptides may be complexed with toxins,
e.g., ricin or cholera, or with other compounds that are toxic to
cells. The toxin-binding molecule complex is then targeted to a
tumor or other cell by the specificity of the binding molecule for
SEQ ID NO: 4, 6-8, 11 or 13.
[0126] The protein of the invention may also be expressed as a
product of transgenic animals, e.g., as a component of the milk of
transgenic cows, goats, pigs, or sheep which are characterized by
somatic or germ cells containing a nucleotide sequence encoding the
protein.
[0127] The proteins provided herein also include proteins
characterized by amino acid sequences similar to those of purified
proteins but into which modification are naturally provided or
deliberately engineered. For example, modifications, in the peptide
or DNA sequence, can be made by those skilled in the art using
known techniques. Modifications of interest in the protein
sequences may include the alteration, substitution, replacement,
insertion or deletion of a selected amino acid, residue in the
coding sequence. For example, one or more of the cysteine residues
may be deleted or replaced with another amino acid to alter the
conformation of the molecule. Techniques for such alteration,
substitution, replacement, insertion or deletion are well known to
those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584).
Preferably, such alteration, substitution, replacement, insertion
or deletion retains the desired activity of the protein. Regions of
the protein that are important for the protein function can be
determined by various methods known in the art including the
alanine-scanning method which involved systematic substitution of
single or strings of amino acids with alanine, followed by testing
the resulting alanine-containing variant for biological activity.
This type of analysis determines the importance of the substituted
amino acid(s) in biological activity. Regions of the protein that
are important for protein function may be determined by the eMATRIX
program.
[0128] Other fragments and derivatives of the sequences of proteins
which would be expected to retain protein activity in whole or in
part and are useful for screening or other immunological
methodologies may also be easily made by those skilled in the art
given the disclosures herein. Such modifications are encompassed by
the present invention.
[0129] The protein may also be produced by operably linking the
isolated polynucleotide of the invention to suitable control
sequences in one or more insect expression vectors, and employing
an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially
available in kit form from, e.g., Invitrogen, San Diego, Calif.,
U.S.A. (the MaxBat.TM. kit), and such methods are well known in the
art, as described in Summers and Smith, Texas Agricultural
Experiment Station Bulletin No. 1555 (1987), incorporated herein by
reference. As used herein, an insect cell capable of expressing a
polynucleotide of the present invention is "transformed."
[0130] The protein of the invention may be prepared by culturing
transformed host cells under culture conditions suitable to express
the recombinant protein. The resulting expressed protein may then
be purified from such culture (i.e., from culture medium or cell
extracts) using known purification processes, such as gel
filtration and ion exchange chromatography. The purification of the
protein may also include an affinity column containing agents which
will bind to the protein; one or more column steps over such
affinity resins as concanavalin A-agarose, heparin-toyopear.TM. or
Cibacrom blue 3GA Sepharose.TM.; one or more steps involving
hydrophobic interaction chromatography using such resins as phenyl
ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
[0131] Alternatively, the protein of the invention may also be
expressed in a form which will facilitate purification. For
example, it may be expressed as a fusion protein, such as those of
maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin (TRX), or as a His tag. Kits for expression and
purification of such fusion proteins are commercially available
from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway,
N.J.) and Invitrogen, respectively. The protein can also be tagged
with an epitope and subsequently purified by using a specific
antibody directed to such epitope. One such epitope ("FLAG.RTM.")
is commercially available from Kodak (New Haven, Conn.).
[0132] Finally, one or more reverse-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify the protein. Some or all of the
foregoing purification steps, in various combinations, can also be
employed to provide a substantially homogeneous isolated
recombinant protein. The protein thus purified is substantially
free of other mammalian proteins and is defined in accordance with
the present invention as an "isolated protein."
[0133] The polypeptides of the invention include analogs
(variants). The polypeptides of the invention include
semaphorin-like analogs. This embraces fragments of semaphorin-like
polypeptide of the invention, as well semaphorin-like polypeptides
which comprise one or more amino acids deleted, inserted, or
substituted. Also, analogs of the semaphorin-like polypeptide of
the invention embrace fusions of the semaphorin-like polypeptides
or modifications of the semaphorin-like polypeptides, wherein the
semaphorin-like polypeptide or analog is fused to another moiety or
moieties, e.g., targeting moiety or another therapeutic agent. Such
analogs may exhibit improved properties such as activity and/or
stability. Examples of moieties which may be fused to the
semaphorin-like polypeptide or an analog include, for example,
targeting moieties which provide for the delivery of polypeptide to
neurons, e.g., antibodies to central nervous system, or antibodies
to receptor and ligands expressed on neuronal cells. Other moieties
which may be fused to semaphorin-like polypeptide include
therapeutic agents which are used for treatment, for example
anti-depressant drugs or other medications for neurological
disorders. Also, semaphorin-like polypeptides may be fused to
neuron growth modulators, and other chemokines for targeted
delivery.
[0134] 5.4.1 Determining Polypeptide and Polynucleotide Identity
and Similarity
[0135] Preferred identity and/or similarity are designed to give
the largest match between the sequences tested. Methods to
determine identity and similarity are codified in computer programs
including, but are not limited to, the GCG program package,
including GAP (Devereux, J., et al., Nucleic Acids Research
12(1):387 (1984); Genetics Computer Group, University of Wisconsin,
Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et
al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST (Altschul S. F.
et al., Nucleic Acids Res. vol. 25, pp. 3389-3402, herein
incorporated by reference), eMatrix software (Wu et al., J. Comp.
Biol., vol. 6, pp. 219-235 (1999), herein incorporated by
reference), eMotif software (Nevill-Manning et al, ISMB-97, vol 4,
pp. 202-209, herein incorporated by reference) and the
Kyte-Doolittle hydrophobicity prediction algorithm (J. Mol Biol,
157, pp. 105-31 (1982), incorporated herein by reference). The
BLAST programs are publicly available from the National Center for
Biotechnology Information (NCBI) and other sources (BLAST Manual,
Altschul, S., et al. NCB NLM NIH Bethesda, Md. 20894; Altschul, S.,
et al., J. Mol. Biol. 215:403-410 (1990).
[0136] 5.5 Gene Therapy
[0137] Mutations in the polynucleotides of the invention gene may
result in loss of normal function of the encoded protein. The
invention thus provides gene therapy to restore normal activity of
the polypeptides of the invention; or to treat disease states
involving polypeptides of the invention. Delivery of a functional
gene encoding polypeptides of the invention to appropriate cells is
effected ex vivo, in situ, or in vivo by use of vectors, and more
particularly viral vectors (e.g., adenovirus, adeno-associated
virus, or a retrovirus), or ex vivo by use of physical DNA transfer
methods (e.g., liposomes or chemical treatments). See, for example,
Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20
(1998). For additional reviews of gene therapy technology see
Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific
American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).
Introduction of any one of the nucleotides of the present invention
or a gene encoding the polypeptides of the present invention can
also be accomplished with extrachromosomal substrates (transient
expression) or artificial chromosomes (stable expression). Cells
may also be cultured ex vivo in the presence of proteins of the
present invention in order to proliferate or to produce a desired
effect on or activity in such cells. Treated cells can then be
introduced in vivo for therapeutic purposes. Alternatively, it is
contemplated that in other human disease states, preventing the
expression of or inhibiting the activity of polypeptides of the
invention will be useful in treating the disease states. It is
contemplated that antisense therapy or gene therapy could be
applied to negatively regulate the expression of polypeptides of
the invention.
[0138] Other methods inhibiting expression of a protein include the
introduction of antisense molecules to the nucleic acids of the
present invention, their complements, or their translated RNA
sequences, by methods known in the art. Further, the polypeptides
of the present invention can be inhibited by using targeted
deletion methods, or the insertion of a negative regulatory element
such as a silencer, which is tissue specific.
[0139] The present invention still further provides cells
genetically engineered in vivo to express the polynucleotides of
the invention, wherein such polynucleotides are in operative
association with a regulatory sequence heterologous to the host
cell which drives expression of the polynucleotides in the cell.
These methods can be used to increase or decrease the expression of
the polynucleotides of the present invention.
[0140] Knowledge of DNA sequences provided by the invention allows
for modification of cells to permit, increase, or decrease,
expression of endogenous polypeptide. Cells can be modified (e.g.,
by homologous recombination) to provide increased polypeptide
expression by replacing, in whole or in part, the naturally
occurring promoter with all or part of a heterologous promoter so
that the cells express the protein at higher levels. The
heterologous promoter is inserted in such a manner that it is
operatively linked to the desired protein encoding sequences. See,
for example, PCT International Publication No. WO 94/12650, PCT
International Publication No. WO 92/20808, and PCT International
Publication No. WO 91/09955. It is also contemplated that, in
addition to heterologous promoter DNA, amplifiable marker DNA
(e.g., ada, dhfr, and the multifunctional CAD gene which encodes
carbamyl phosphate synthase, aspartate transcarbamylase, and
dihydroorotase) and/or intron DNA may be inserted along with the
heterologous promoter DNA. If linked to the desired protein coding
sequence, amplification of the marker DNA by standard selection
methods results in co-amplification of the desired protein coding
sequences in the cells.
[0141] In another embodiment of the present invention, cells and
tissues may be engineered to express an endogenous gene comprising
the polynucleotides of the invention under the control of inducible
regulatory elements, in which case the regulatory sequences of the
endogenous gene may be replaced by homologous recombination. As
described herein, gene targeting can be used to replace a gene's
existing regulatory region with a regulatory sequence isolated from
a different gene or a novel regulatory sequence synthesized by
genetic engineering methods. Such regulatory sequences may be
comprised of promoters, enhancers, scaffold-attachment regions,
negative regulatory elements, transcriptional initiation sites,
regulatory protein binding sites or combinations of said sequences.
Alternatively, sequences which affect the structure or stability of
the RNA or protein produced may be replaced, removed, added, or
otherwise modified by targeting. These sequences include
polyadenylation signals, mRNA stability elements, splice sites,
leader sequences for enhancing or modifying transport or secretion
properties of the protein, or other sequences which alter or
improve the function or stability of protein or RNA molecules.
[0142] The targeting event may be a simple insertion of the
regulatory sequence, placing the gene under the control of the new
regulatory sequence, e.g., inserting a new promoter or enhancer or
both upstream of a gene. Alternatively, the targeting event may be
a simple deletion of a regulatory element, such as the deletion of
a tissue-specific negative regulatory element. Alternatively, the
targeting event may replace an existing element; for example, a
tissue-specific enhancer can be replaced by an enhancer that has
broader or different cell-type specificity than the naturally
occurring elements. Here, the naturally occurring sequences are
deleted and new sequences are added. In all cases, the
identification of the targeting event may be facilitated by the use
of one or more selectable marker genes that are contiguous with the
targeting DNA, allowing for the selection of cells in which the
exogenous DNA has integrated into the cell genome. The
identification of the targeting event may also be facilitated by
the use of one or more marker genes exhibiting the property of
negative selection, such that the negatively selectable marker is
linked to the exogenous DNA, but configured such that the
negatively selectable marker flanks the targeting sequence, and
such that a correct homologous recombination event with sequences
in the host cell genome does not result in the stable integration
of the negatively selectable marker. Markers useful for this
purpose include the Herpes Simplex Virus thymidine kinase (TK) gene
or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt)
gene.
[0143] The gene targeting or gene activation techniques which can
be used in accordance with this aspect of the invention are more
particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S.
Pat. No. 5,578,461 to Sherwin et al.; International Application No.
PCT/US92/09627 (WO93/09222) by Selden et al.; and International
Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al.,
each of which is incorporated by reference herein in its
entirety.
[0144] 5.6 Transgenic Animals
[0145] In preferred methods to determine biological functions of
the polypeptides of the invention in vivo, one or more genes
provided by the invention are either over expressed or inactivated
in the germ line of animals using homologous recombination
[Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene
is over expressed, under the regulatory control of exogenous or
endogenous promoter elements, are known as transgenic animals.
Animals in which an endogenous gene has been inactivated by
homologous recombination are referred to as "knockout" animals.
Knockout animals, preferably non-human mammals, can be prepared as
described in U.S. Pat. No. 5,557,032, incorporated herein by
reference. Transgenic animals are useful to determine the roles
polypeptides of the invention play in biological processes, and
preferably in disease states. Transgenic animals are useful as
model systems to identify compounds that modulate lipid metabolism.
Transgenic animals, preferably non-human mammals, are produced
using methods as described in U.S. Pat. No. 5,489,743 and PCT
Publication No. WO94/28122, incorporated herein by reference.
[0146] Transgenic animals can be prepared wherein all or part of a
promoter of the polynucleotides of the invention is either
activated or inactivated to alter the level of expression of the
polypeptides of the invention. Inactivation can be carried out
using homologous recombination methods described above. Activation
can be achieved by supplementing or even replacing the homologous
promoter to provide for increased protein expression. The
homologous promoter can be supplemented by insertion of one or more
heterologous enhancer elements known to confer promoter activation
in a particular tissue.
[0147] The polynucleotides of the present invention also make
possible the development, through, e.g., homologous recombination
or knock out strategies, of animals that fail to express functional
semaphorin-like polypeptide or that express a variant of
semaphorin-like polypeptide. Such animals are useful as models for
studying the in vivo activities of semaphorin-like polypeptide as
well as for studying modulators of the semaphorin-like
polypeptide.
[0148] In preferred methods to determine biological functions of
the polypeptides of the invention in vivo, one or more genes
provided by the invention are either over expressed or inactivated
in the germ line of animals using homologous recombination
[Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene
is over expressed, under the regulatory control of exogenous or
endogenous promoter elements, are known as transgenic animals.
Animals in which an endogenous gene has been inactivated by
homologous recombination are referred to as "knockout" animals.
Knockout animals, preferably non-human mammals, can be prepared as
described in U.S. Pat. No. 5,557,032, incorporated herein by
reference. Transgenic animals are useful to determine the roles
polypeptides of the invention play in biological processes, and
preferably in disease states. Transgenic animals are useful as
model systems to identify compounds that modulate lipid metabolism.
Transgenic animals, preferably non-human mammals, are produced
using methods as described in U.S. Pat. No. 5,489,743 and PCT
Publication No. WO94/28122, incorporated herein by reference.
[0149] Transgenic animals can be prepared wherein all or part of
the polynucleotides of the invention promoter is either activated
or inactivated to alter the level of expression of the polypeptides
of the invention. Inactivation can be carried out using homologous
recombination methods described above. Activation can be achieved
by supplementing or even replacing the homologous promoter to
provide for increased protein expression. The homologous promoter
can be supplemented by insertion of one or more heterologous
enhancer elements known to confer promoter activation in a
particular tissue.
[0150] 5.7 Uses and Biological Activity of Human Semaphorin-Like
Polypeptide
[0151] The polynucleotides and proteins of the present invention
are expected to exhibit one or more of the uses or biological
activities (including those associated with assays cited herein)
identified herein. Uses or activities described for proteins of the
present invention may be provided by administration or use of such
proteins or of polynucleotides encoding such proteins (such as, for
example, in gene therapies or vectors suitable for introduction of
DNA). The mechanism underlying the particular condition or
pathology will dictate whether the polypeptides of the invention,
the polynucleotides of the invention or modulators (activators or
inhibitors) thereof would be beneficial to the subject in need of
treatment. Thus, "therapeutic compositions of the invention"
include compositions comprising isolated polynucleotides (including
recombinant DNA molecules, cloned genes and degenerate variants
thereof) or polypeptides of the invention (including full length
protein, mature protein and truncations or domains thereof), or
compounds and other substances that modulate the overall activity
of the target gene products, either at the level of target
gene/protein expression or target protein activity. Such modulators
include polypeptides, analogs, (variants), including fragments and
fusion proteins, antibodies and other binding proteins; chemical
compounds that directly or indirectly activate or inhibit the
polypeptides of the invention (identified, e.g., via drug screening
assays as described herein); antisense polynucleotides and
polynucleotides suitable for triple helix formation; and in
particular antibodies or other binding partners that specifically
recognize one or more epitopes of the polypeptides of the
invention.
[0152] The polypeptides of the present invention may likewise be
involved in cellular activation or in one of the other
physiological pathways described herein.
[0153] 5.7.1 Research Uses and Utilities
[0154] The polynucleotides provided by the present invention can be
used by the research community for various purposes. The
polynucleotides can be used to express recombinant protein for
analysis, characterization or therapeutic use; as markers for
tissues in which the corresponding protein is preferentially
expressed (either constitutively or at a particular stage of tissue
differentiation or development or in disease states); as molecular
weight markers on gels; as chromosome markers or tags (when
labeled) to identify chromosomes or to map related gene positions;
to compare with endogenous DNA sequences in patients to identify
potential genetic disorders; as probes to hybridize and thus
discover novel, related DNA sequences; as a source of information
to derive PCR primers for genetic fingerprinting; as a probe to
"subtract-out" known sequences in the process of discovering other
novel polynucleotides; for selecting and making oligomers for
attachment to a "gene chip" or other support, including for
examination of expression patterns; to raise anti-protein
antibodies using DNA immunization techniques; and as an antigen to
raise anti-DNA antibodies or elicit another immune response. Where
the polynucleotide encodes a protein which binds or potentially
binds to another protein (such as, for example, in a
receptor-ligand interaction), the polynucleotide can also be used
in interaction trap assays (such as, for example, that described in
Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides
encoding the other protein with which binding occurs or to identify
inhibitors of the binding interaction.
[0155] The polypeptides provided by the present invention can
similarly be used in assays to determine biological activity,
including in a panel of multiple proteins for high-throughput
screening; to raise antibodies or to elicit another immune
response; as a reagent (including the labeled reagent) in assays
designed to quantitatively determine levels of the protein (or its
receptor) in biological fluids; as markers for tissues in which the
corresponding polypeptide is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation
or development or in a disease state); and, of course, to isolate
correlative receptors or ligands. Proteins involved in these
binding interactions can also be used to screen for peptide or
small molecule inhibitors or agonists of the binding
interaction.
[0156] The polypeptides of the invention are also useful for making
antibody substances that are specifically immunoreactive with
semaphorin-like proteins. Antibodies and portions thereof (e.g.,
Fab fragments) which bind to the polypeptides of the invention can
be used to identify the presence of such polypeptides in a sample.
Such determinations are carried out using any suitable immunoassay
format, and any polypeptide of the invention that is specifically
bound by the antibody can be employed as a positive control.
[0157] Any or all of these research utilities are capable of being
developed into reagent grade or kit format for commercialization as
research products.
[0158] Methods for performing the uses listed above are well known
to those skilled in the art. References disclosing such methods
include without limitation "Molecular Cloning: A Laboratory
Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J.,
E. F. Fritsch and T. Maniatis eds., 1989, and "Methods in
Enzymology: Guide to Molecular Cloning Techniques", Academic Press,
Berger, S. L. and A. R. Kimmel eds., 1987.
[0159] 5.7.2 Nutritional Uses
[0160] Polynucleotides and polypeptides of the present invention
can also be used as nutritional sources or supplements. Such uses
include without limitation use as a protein or amino acid
supplement, use as a carbon source, use as a nitrogen source and
use as a source of carbohydrate. In such cases the polypeptide or
polynucleotide of the invention can be added to the feed of a
particular organism or can be administered as a separate solid or
liquid preparation, such as in the form of powder, pills,
solutions, suspensions or capsules. In the case of microorganisms,
the polypeptide or polynucleotide of the invention can be added to
the medium in or on which the microorganism is cultured.
[0161] Additionally, the polypeptides of the invention can be used
as molecular weight markers, and as a food supplement. A
polypeptide consisting of SEQ ID NO: 4, for example, has a
molecular mass of approximately 121 kDa in its unprocessed and
unglycosylated state. Protein food supplements are well known and
the formulation of suitable food supplements including polypeptides
of the invention is within the level of skill in the food
preparation art.
[0162] 5.7.3 Cytokine and Cell Proliferation/Differentiation
Activity
[0163] A polypeptide of the present invention may exhibit activity
relating to cytokine, cell proliferation (either inducing or
inhibiting) or cell differentiation (either inducing or inhibiting)
activity or may induce production of other cytokines in certain
cell populations. A polynucleotide of the invention can encode a
polypeptide exhibiting such attributes. Many protein factors
discovered to date, including all known cytokines, have exhibited
activity in one or more factor-dependent cell proliferation assays,
and hence the assays serve as a convenient confirmation of cytokine
activity. The activity of therapeutic compositions of the present
invention is evidenced by any one of a number of routine factor
dependent cell proliferation assays for cell lines including,
without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G,
M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e,
CMK, HUVEC, and Caco. Therapeutic compositions of the invention can
be used in the following:
[0164] Assays for T-cell or thymocyte proliferation include without
limitation those described in: Current Protocols in Immunology, Ed
by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach,
W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai
et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J.
Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular
Immunology 133:327-341, 1991; Bertagnolli, et al., I. Immunol.
149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761,
1994.
[0165] Assays for cytokine production and/or proliferation of
spleen cells, lymph node cells or thymocytes include, without
limitation, those described in: Polyclonal T cell stimulation,
Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in
Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John
Wiley and Sons, Toronto. 1994; and Measurement of mouse and human
interleukin- , Schreiber, R. D. In Current Protocols in Immunology.
J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons,
Toronto. 1994.
[0166] Assays for proliferation and differentiation of
hematopoietic and lymphopoietic cells include, without limitation,
those described in: Measurement of Human and Murine Interleukin 2
and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In
Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp.
6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; devries et al.,
J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature
336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci.
U.S.A. 80:2931-2938, 1983; Measurement of mouse and human
interleukin 6--Nordan, R. In Current Protocols in Immunology. J. E.
Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto.
1991; Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861,
1986; Measurement of human Interleukin 11--Bennett, F., Giannotti,
J., Clark, S. C. and Turner, K. J. In Current Protocols in
Immunology. J. E. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and
Sons, Toronto. 1991; Measurement of mouse and human Interleukin
9--Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J. In
Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp.
6.13.1, John Wiley and Sons, Toronto. 1991.
[0167] Assays for T-cell clone responses to antigens (which will
identify, among others, proteins that affect APC-T cell
interactions as well as direct T-cell effects by measuring
proliferation and cytokine production) include, without limitation,
those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W
Strober, Pub. Greene Publishing Associates and Wiley-Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter
6, Cytokines and their cellular receptors; Chapter 7, Immunologic
studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA
77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411,
1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol. 140:508-512, 1988.
[0168] 5.7.4 Stem Cell Growth Factor Activity
[0169] A polypeptide of the present invention may exhibit stem cell
growth factor activity and be involved in the proliferation,
differentiation and survival of pluripotent and totipotent stem
cells including primordial germ cells, embryonic stem cells,
hematopoietic stem cells, neuronal stem cells and/or germ line stem
cells. Administration of the polypeptide of the invention to stem
cells in vivo or ex vivo may maintain and expand cell populations
in a totipotential or pluripotential state which would be useful
for re-engineering damaged or diseased tissues, transplantation,
manufacture of bio-pharmaceuticals and the development of
bio-sensors. The ability to produce large quantities of human cells
has important working applications for the production of human
proteins which currently must be obtained from non-human sources or
donors, implantation of cells to treat diseases such as
Parkinson's, Alzheimer's and other neurodegenerative diseases;
tissues for grafting such as bone marrow, skin, cartilage, tendons,
bone, muscle (including cardiac muscle), blood vessels, cornea,
neural cells, gastrointestinal cells and others; and organs for
transplantation such as kidney, liver, pancreas (including islet
cells), heart and lung.
[0170] It is contemplated that the polypeptide of the present
invention may inhibit the growth of neural stem cells. Accordingly,
administration of a polypeptide antagonist to stem cells may result
in the proliferation, differentiation and survival of the stem
cells.
[0171] It is also contemplated that multiple different exogenous
growth factors and/or cytokines may be administered in combination
with the polypeptide of the invention to achieve the desired
effect, including any of the growth factors listed herein, other
stem cell maintenance factors, and specifically including stem cell
factor (SCF), leukemia inhibitory factor (LIF), Flt-3 ligand
(Flt-3L), any of the interleukins, recombinant soluble IL-6
receptor fused to IL-6, macrophage inflammatory protein 1-alpha
(MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor
4 (PF-4), platelet-derived growth factor (PDGF), neural growth
factors and basic fibroblast growth factor (bFGF).
[0172] Since totipotent stem cells can give rise to virtually any
mature cell type, expansion of these cells in culture will
facilitate the production of large quantities of mature cells.
Techniques for culturing stem cells are known in the art and
administration of polypeptides of the invention, optionally with
other growth factors and/or cytokines, is expected to enhance the
survival and proliferation of the stem cell populations. This can
be accomplished by direct administration of the polypeptide of the
invention to the culture medium. Alternatively, stroma cells
transfected with a polynucleotide that encodes for the polypeptide
of the invention can be used as a feeder layer for the stem cell
populations in culture or in vivo. Stromal support cells for feeder
layers may include embryonic bone marrow fibroblasts, bone marrow
stromal cells, fetal liver cells, or cultured embryonic fibroblasts
(see U.S. Pat. No. 5,690,926).
[0173] Stem cells themselves can be transfected with a
polynucleotide of the invention to induce autocrine expression of
the polypeptide of the invention. This will allow for generation of
undifferentiated totipotential/pluripotential stem cell lines that
are useful as is or that can then be differentiated into the
desired mature cell types. These stable cell lines can also serve
as a source of undifferentiated totipotential/pluripotential mRNA
to create cDNA libraries and templates for polymerase chain
reaction experiments. These studies would allow for the isolation
and identification of differentially expressed genes in stem cell
populations that regulate stem cell proliferation and/or
maintenance.
[0174] Expansion and maintenance of totipotent stem cell
populations will be useful in the treatment of many pathological
conditions. For example, polypeptides of the present invention may
be used to manipulate stem cells in culture to give rise to
neuroepithelial cells that can be used to augment or replace cells
damaged by illness, autoimmune disease, accidental damage or
genetic disorders. The polypeptide of the invention may be useful
for inducing the proliferation of neural cells and for the
regeneration of nerve and brain tissue, i.e. for the treatment of
central and peripheral nervous system diseases and neuropathies, as
well as mechanical and traumatic disorders which involve
degeneration, death or trauma to neural cells or nerve tissue. In
addition, the expanded stem cell populations can also be
genetically altered for gene therapy purposes and to decrease host
rejection of replacement tissues after grafting or
implantation.
[0175] Expression of the polypeptide of the invention and its
effect on stem cells can also be manipulated to achieve controlled
differentiation of the stem cells into more differentiated cell
types. A broadly applicable method of obtaining pure populations of
a specific differentiated cell type from undifferentiated stem cell
populations involves the use of a cell-type specific promoter
driving a selectable marker. The selectable marker allows only
cells of the desired type to survive. For example, stem cells can
be induced to differentiate into cardiomyocytes (Wobus et al.,
Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.
Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder,
L. W. In: Principles of Tissue Engineering eds. Lanza et al.,
Academic Press (1997)). Alternatively, directed differentiation of
stem cells can be accomplished by culturing the stem cells in the
presence of a differentiation factor such as retinoic acid and an
antagonist of the polypeptide of the invention which would inhibit
the effects of endogenous stem cell factor activity and allow
differentiation to proceed.
[0176] In vitro cultures of stem cells can be used to determine if
the polypeptide of the invention exhibits stem cell growth factor
activity. Stem cells are isolated from any one of various cell
sources (including hematopoietic stem cells and embryonic stem
cells) and cultured on a feeder layer, as described by Thompson et
al. Proc. Natl. Acad. Sci, U.S.A., 92: 7844-7848 (1995), in the
presence of the polypeptide of the invention alone or in
combination with other growth factors or cytokines. The ability of
the polypeptide of the invention to induce stem cells proliferation
is determined by colony formation on semi-solid support e.g. as
described by Bernstein et al., Blood, 77: 2316-2321 (1991).
[0177] 5.7.5 Hematopoiesis Regulating Activity
[0178] A polypeptide of the present invention may be involved in
regulation of hematopoiesis and, consequently, in the treatment of
myeloid or lymphoid cell disorders. Even marginal biological
activity in support of colony forming cells or of factor-dependent
cell lines indicates involvement in regulating hematopoiesis, e.g.
in supporting the growth and proliferation of erythroid progenitor
cells alone or in combination with other cytokines, thereby
indicating utility, for example, in treating various anemias or for
use in conjunction with irradiation/chemotherapy to stimulate the
production of erythroid precursors and/or erythroid cells; in
supporting the growth and proliferation of myeloid cells such as
granulocytes and monocytes/macrophages (i.e., traditional CSF
activity) useful, for example, in conjunction with chemotherapy to
prevent or treat consequent myelo-suppression; in supporting the
growth and proliferation of megakaryocytes and consequently of
platelets thereby allowing prevention or treatment of various
platelet disorders such as thrombocytopenia, and generally for use
in place of or complimentary to platelet transfusions; and/or in
supporting the growth and proliferation of hematopoietic stem cells
which are capable of maturing to any and all of the above-mentioned
hematopoietic cells and therefore find therapeutic utility in
various stem cell disorders (such as those usually treated with
transplantation, including, without limitation, aplastic anemia and
paroxysmal nocturnal hemoglobinuria), as well as in repopulating
the stem cell compartment post irradiation/chemotherapy, either
in-vivo or ex-vivo (i.e., in conjunction with bone marrow
transplantation or with peripheral progenitor cell transplantation
(homologous or heterologous)) as normal cells or genetically
manipulated for gene therapy.
[0179] Therapeutic compositions of the invention can be used in the
following:
[0180] Suitable assays for proliferation and differentiation of
various hematopoietic lines are cited above.
[0181] Assays for embryonic stem cell differentiation (which will
identify, among others, proteins that influence embryonic
differentiation hematopoiesis) include, without limitation, those
described in: Johansson et al. Cellular Biology 15:141-151, 1995;
Keller et al., Molecular and Cellular Biology 13:473-486, 1993;
McClanahan et al., Blood 81:2903-2915, 1993.
[0182] Assays for stem cell survival and differentiation (which
will identify, among others, proteins that regulate
lympho-hematopoiesis) include, without limitation, those described
in: Methylcellulose colony forming assays, Freshney, M. G. In
Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.
265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al.,
Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive
hematopoietic colony forming cells with high proliferative
potential, McNiece, I. K. and Briddell, R. A. In Culture of
Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,
Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental
Hematology 22:353-359, 1994; Cobblestone area forming cell assay,
Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I.
Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York,
N.Y. 1994; Long term bone marrow cultures in the presence of
stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of
Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 163-179,
Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating
cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R.
I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New
York, N.Y. 1994.
[0183] 5.7.6 Tissue Growth Activity
[0184] A polypeptide of the present invention also may be involved
in bone, cartilage, tendon, ligament and/or nerve tissue growth or
regeneration, as well as in wound healing and tissue repair and
replacement, and in healing of burns, incisions and ulcers.
[0185] A polypeptide of the present invention which induces
cartilage and/or bone growth in circumstances where bone is not
normally formed, has application in the healing of bone fractures
and cartilage damage or defects in humans and other animals.
Compositions of a polypeptide, antibody, binding partner, or other
modulator of the invention may have prophylactic use in closed as
well as open fracture reduction and also in the improved fixation
of artificial joints. De novo bone formation induced by an
osteogenic agent contributes to the repair of congenital, trauma
induced, or oncologic resection induced craniofacial defects, and
also is useful in cosmetic plastic surgery.
[0186] A polypeptide of this invention may also be involved in
attracting bone-forming cells, stimulating growth of bone-forming
cells, or inducing differentiation of progenitors of bone-forming
cells. Treatment of osteoporosis, osteoarthritis, bone degenerative
disorders, or periodontal disease, such as through stimulation of
bone and/or cartilage repair or by blocking inflammation or
processes of tissue destruction (collagenase activity, osteoclast
activity, etc.) mediated by inflammatory processes may also be
possible using the composition of the invention.
[0187] Another category of tissue regeneration activity that may
involve the polypeptide of the present invention is tendon/ligament
formation. Induction of tendon/ligament-like tissue or other tissue
formation in circumstances where such tissue is not normally
formed, has application in the healing of tendon or ligament tears,
deformities and other tendon or ligament defects in humans and
other animals. Such a preparation employing a tendon/ligament-like
tissue inducing protein may have prophylactic use in preventing
damage to tendon or ligament tissue, as well as use in the improved
fixation of tendon or ligament to bone or other tissues, and in
repairing defects to tendon or ligament tissue. De novo
tendon/ligament-like tissue formation induced by a composition of
the present invention contributes to the repair of congenital,
trauma induced, or other tendon or ligament defects of other
origin, and is also useful in cosmetic plastic surgery for
attachment or repair of tendons or ligaments. The compositions of
the present invention may provide environment to attract tendon- or
ligament-forming cells, stimulate growth of tendon- or
ligament-forming cells, induce differentiation of progenitors of
tendon- or ligament-forming cells, or induce growth of
tendon/ligament cells or progenitors ex vivo for return in vivo to
effect tissue repair. The compositions of the invention may also be
useful in the treatment of tendinitis, carpal tunnel syndrome and
other tendon or ligament defects. The compositions may also include
an appropriate matrix and/or sequestering agent as a carrier as is
well known in the art.
[0188] The compositions of the present invention may also be useful
for proliferation of neural stem cells and for regeneration of
nerve and brain tissue, i.e. for the treatment of central and
peripheral nervous system diseases and neuropathies, as well as
mechanical and traumatic disorders, which involve degeneration,
death or trauma to neural cells or nerve tissue. More specifically,
a composition may be used in the treatment of peripheral and
central nerve injuries and of diseases of the peripheral nervous
system, such as peripheral neuropathy and localized neuropathies,
and central nervous system diseases, such as Alzheimer's,
Parkinson's disease, Huntington's disease, amyotrophic lateral
sclerosis, and Shy-Drager syndrome. Further conditions which may be
treated in accordance with the present invention include mechanical
and traumatic disorders, such as spinal cord disorders, head trauma
and cerebrovascular diseases such as stroke. Peripheral
neuropathies resulting from chemotherapy or other medical therapies
may also be treatable using a composition of the invention.
[0189] Compositions of the invention may also be useful to promote
better or faster closure of non-healing wounds, including without
limitation pressure ulcers, ulcers associated with vascular
insufficiency, surgical and traumatic wounds, and the like.
[0190] Compositions of the present invention may also be involved
in the generation or regeneration of other tissues, such as organs
(including, for example, pancreas, liver, intestine, kidney, skin,
endothelium), muscle (smooth, skeletal or cardiac) and vascular
(including vascular endothelium) tissue, or for promoting the
growth of cells comprising such tissues. Part of the desired
effects may be by inhibition or modulation of fibrotic scarring may
allow normal tissue to regenerate. A polypeptide of the present
invention may also exhibit angiogenic activity.
[0191] A composition of the present invention may also be useful
for gut protection or regeneration and treatment of lung or liver
fibrosis, reperfusion injury in various tissues, and conditions
resulting from systemic cytokine damage.
[0192] A composition of the present invention may also be useful
for promoting or inhibiting differentiation of tissues described
above from precursor tissues or cells; or for inhibiting the growth
of tissues described above.
[0193] Therapeutic compositions of the invention can be used in the
following:
[0194] Assays for tissue generation activity include, without
limitation, those described in: International Patent Publication
No. WO95/16035 (bone, cartilage, tendon); International Patent
Publication No. WO95/05846 (nerve, neuronal); International Patent
Publication No. WO91/07491 (skin, endothelium).
[0195] Assays for wound healing activity include, without
limitation, those described in: Winter, Epidermal Wound Healing,
pp. 71-112 (Maibach, H. I. and Rovee, D. T., eds.), Year Book
Medical Publishers, Inc., Chicago, as modified by Eaglstein and
Mertz, J. Invest. Dermatol 71:382-84 (1978).
[0196] 5.7.7 Immune Function Stimulating or Suppressing
Activity
[0197] A polypeptide of the present invention may also exhibit
immune stimulating or immune suppressing activity, including
without limitation the activities for which assays are described
herein. A polynucleotide of the invention can encode a polypeptide
exhibiting such activities. A protein may be useful in the
treatment of various immune deficiencies and disorders (including
severe combined immunodeficiency (SCID)), e.g., in regulating (up
or down) growth and proliferation of T and/or B lymphocytes, as
well as effecting the cytolytic activity of NK cells and other cell
populations. These immune deficiencies may be genetic or be caused
by viral (e.g., HIV) as well as bacterial or fungal infections, or
may result from autoimmune disorders. More specifically, infectious
diseases causes by viral, bacterial, fungal or other infection may
be treatable using a protein of the present invention, including
infections by HIV, hepatitis viruses, herpes viruses, mycobacteria,
Leishmania spp., malaria spp. and various fungal infections such as
candidiasis. Of course, in this regard, proteins of the present
invention may also be useful where a boost to the immune system
generally may be desirable, i.e., in the treatment of cancer.
[0198] Autoimmune disorders which may be treated using a protein of
the present invention include, for example, connective tissue
disease, multiple sclerosis, systemic lupus erythematosus,
rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent
diabetes mellitis, myasthenia gravis, graft-versus-host disease and
autoimmune inflammatory eye disease. Such a protein (or antagonists
thereof, including antibodies) of the present invention may also to
be useful in the treatment of allergic reactions and conditions
(e.g., anaphylaxis, serum sickness, drug reactions, food allergies,
insect venom allergies, mastocytosis, allergic rhinitis,
hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic
dermatitis, allergic contact dermatitis, erythema multiforme,
Stevens-Johnson syndrome, allergic conjunctivitis, atopic
keratoconjunctivitis, venereal keratoconjunctivitis, giant
papillary conjunctivitis and contact allergies), such as asthma
(particularly allergic asthma) or other respiratory problems. Other
conditions, in which immune suppression is desired (including, for
example, organ transplantation), may also be treatable using a
protein (or antagonists thereof) of the present invention. The
therapeutic effects of the polypeptides or antagonists thereof on
allergic reactions can be evaluated by in vivo animals models such
as the cumulative contact enhancement test (Lastbom et al.,
Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al.,
Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr
et al., Arch. Toxocol. 73: 501-9), and murine local lymph node
assay (Kimber et al., J. Toxicol. Environ. Health 53: 563-79).
[0199] Using the proteins of the invention it may also be possible
to modulate immune responses, in a number of ways. Down regulation
may be in the form of inhibiting or blocking an immune response
already in progress or may involve preventing the induction of an
immune response. The functions of activated T cells may be
inhibited by suppressing T cell responses or by inducing specific
tolerance in T cells, or both. Immunosuppression of T cell
responses is generally an active, non-antigen-specific, process
which requires continuous exposure of the T cells to the
suppressive agent. Tolerance, which involves inducing
non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and
persists after exposure to the tolerizing agent has ceased.
Operationally, tolerance can be demonstrated by the lack of a T
cell response upon reexposure to specific antigen in the absence of
the tolerizing agent.
[0200] Down regulating or preventing one or more antigen functions
(including without limitation B lymphocyte antigen functions (such
as, for example, B7)), e.g., preventing high level lymphokine
synthesis by activated T cells, will be useful in situations of
tissue, skin and organ transplantation and in graft-versus-host
disease (GVHD). For example, blockage of T cell function should
result in reduced tissue destruction in tissue transplantation.
Typically, in tissue transplants, rejection of the transplant is
initiated through its recognition as foreign by T cells, followed
by an immune reaction that destroys the transplant. The
administration of a therapeutic composition of the invention may
prevent cytokine synthesis by immune cells, such as T cells, and
thus acts as an immunosuppressant. Moreover, a lack of
costimulation may also be sufficient to anergize the T cells,
thereby inducing tolerance in a subject. Induction of long-term
tolerance by B lymphocyte antigen-blocking reagents may avoid the
necessity of repeated administration of these blocking reagents. To
achieve sufficient immunosuppression or tolerance in a subject, it
may also be necessary to block the function of a combination of B
lymphocyte antigens.
[0201] The efficacy of particular therapeutic compositions in
preventing organ transplant rejection or GVHD can be assessed using
animal models that are predictive of efficacy in humans. Examples
of appropriate systems which can be used include allogeneic cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice,
both of which have been used to examine the immunosuppressive
effects of CTLA4Ig fusion proteins in vivo as described in Lenschow
et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl.
Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of
GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York,
1989, pp. 846-847) can be used to determine the effect of
therapeutic compositions of the invention on the development of
that disease.
[0202] Blocking antigen function may also be therapeutically useful
for treating autoimmune diseases. Many autoimmune disorders are the
result of inappropriate activation of T cells that are reactive
against self tissue and which promote the production of cytokines
and autoantibodies involved in the pathology of the diseases.
Preventing the activation of autoreactive T cells may reduce or
eliminate disease symptoms. Administration of reagents which block
stimulation of T cells can be used to inhibit T cell activation and
prevent production of autoantibodies or T cell-derived cytokines
which may be involved in the disease process. Additionally,
blocking reagents may induce antigen-specific tolerance of
autoreactive T cells which could lead to long-term relief from the
disease. The efficacy of blocking reagents in preventing or
alleviating autoimmune disorders can be determined using a number
of well-characterized animal models of human autoimmune diseases.
Examples include murine experimental autoimmune encephalitis,
systemic lupus erythematosus in MRL/lpr/lpr mice or NZB hybrid
mice, murine autoimmune collagen arthritis, diabetes mellitus in
NOD mice and BB rats, and murine experimental myasthenia gravis
(see Paul ed., Fundamental Immunology, Raven Press, New York, 1989,
pp. 840-856).
[0203] Upregulation of an antigen function (e.g., a B lymphocyte
antigen function), as a means of up regulating immune responses,
may also be useful in therapy. Upregulation of immune responses may
be in the form of enhancing an existing immune response or
eliciting an initial immune response. For example, enhancing an
immune response may be useful in cases of viral infection,
including systemic viral diseases such as influenza, the common
cold, and encephalitis.
[0204] Alternatively, anti-viral immune responses may be enhanced
in an infected patient by removing T cells from the patient,
costimulating the T cells in vitro with viral antigen-pulsed APCs
either expressing a peptide of the present invention or together
with a stimulatory form of a soluble peptide of the present
invention and reintroducing the in vitro activated T cells into the
patient. Another method of enhancing anti-viral immune responses
would be to isolate infected cells from a patient, transfect them
with a nucleic acid encoding a protein of the present invention as
described herein such that the cells express all or a portion of
the protein on their surface, and reintroduce the transfected cells
into the patient. The infected cells would now be capable of
delivering a costimulatory signal to, and thereby activate, T cells
in vivo.
[0205] A polypeptide of the present invention may provide the
necessary stimulation signal to T cells to induce a T cell mediated
immune response against the transfected tumor cells. In addition,
tumor cells which lack MHC class I or MHC class II molecules, or
which fail to reexpress sufficient mounts of MHC class I or MHC
class II molecules, can be transfected with nucleic acid encoding
all or a portion of (e.g., a cytoplasmic-domain truncated portion)
of an MHC class I alpha chain protein and .beta..sub.2
microglobulin protein or an MHC class II alpha chain protein and an
MHC class II beta chain protein to thereby express MHC class I or
MHC class II proteins on the cell surface. Expression of the
appropriate class I or class II MHC in conjunction with a peptide
having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2,
B7-3) induces a T cell mediated immune response against the
transfected tumor cell. Optionally, a gene encoding an antisense
construct which blocks expression of an MHC class II associated
protein, such as the invariant chain, can also be cotransfected
with a DNA encoding a peptide having the activity of a B lymphocyte
antigen to promote presentation of tumor associated antigens and
induce tumor specific immunity. Thus, the induction of a T cell
mediated immune response in a human subject may be sufficient to
overcome tumor-specific tolerance in the subject.
[0206] The activity of a protein of the invention may, among other
means, be measured by the following methods:
[0207] Suitable assays for thymocyte or splenocyte cytotoxicity
include, without limitation, those described in: Current Protocols
in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.
Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing
Associates and Wiley-Interscience (Chapter 3, In Vitro assays for
Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies
in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974,
1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al.,
I. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.
140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998;
Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et
al., J. Immunol. 153:3079-3092, 1994.
[0208] Assays for T-cell-dependent immunoglobulin responses and
isotype switching (which will identify, among others, proteins that
modulate T-cell dependent antibody responses and that affect
Th1/Th2 profiles) include, without limitation, those described in:
Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell
function: In vitro antibody production, Mond, J. J. and Brunswick,
M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol
1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
[0209] Mixed lymphocyte reaction (MLR) assays (which will identify,
among others, proteins that generate predominantly Th1 and CTL
responses) include, without limitation, those described in: Current
Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D.
H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing
Associates and Wiley-Interscience (Chapter 3, In Vitro assays for
Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies
in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et
al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783, 1992.
[0210] Dendritic cell-dependent assays (which will identify, among
others, proteins expressed by dendritic cells that activate naive
T-cells) include, without limitation, those described in: Guery et
al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal
of Immunology 154:5071-5079, 1995; Porgador et al., Journal of
Experimental Medicine 182:255-260, 1995; Nair et al., Journal of
Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine
169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical
Investigation 94:797-807, 1994; and Inaba et al., Journal of
Experimental Medicine 172:631-640, 1990.
[0211] Assays for lymphocyte survival/apoptosis (which will
identify, among others, proteins that prevent apoptosis after
superantigen induction and proteins that regulate lymphocyte
homeostasis) include, without limitation, those described in:
Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al.,
Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research
53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry
14:891-897, 1993; Gorczyca et al., International Journal of
Oncology 1:639-648, 1992.
[0212] Assays for proteins that influence early steps of T-cell
commitment and development include, without limitation, those
described in: Antica et al., Blood 84:111-117, 1994; Fine et al.,
Cellular Immunology 155:111-122, 1994; Galy et al., Blood
85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA
88:7548-7551, 1991.
[0213] 5.7.8 Activin/Inhibin Activity
[0214] A polypeptide of the present invention may also exhibit
activin- or inhibin-related activities. A polynucleotide of the
invention may encode a polypeptide exhibiting such characteristics.
Inhibins are characterized by their ability to inhibit the release
of follicle stimulating hormone (FSH), while activins and are
characterized by their ability to stimulate the release of follicle
stimulating hormone (FSH). Thus, a polypeptide of the present
invention, alone or in heterodimers with a member of the inhibin
family, may be useful as a contraceptive based on the ability of
inhibins to decrease fertility in female mammals and decrease
spermatogenesis in male mammals. Administration of sufficient
amounts of other inhibins can induce infertility in these mammals.
Alternatively, the polypeptide of the invention, as a homodimer or
as a heterodimer with other protein subunits of the inhibin group,
may be useful as a fertility inducing therapeutic, based upon the
ability of activin molecules in stimulating FSH release from cells
of the anterior pituitary. See, for example, U.S. Pat. No.
4,798,885. A polypeptide of the invention may also be useful for
advancement of the onset of fertility in sexually immature mammals,
so as to increase the lifetime reproductive performance of domestic
animals such as, but not limited to, cows, sheep and pigs.
[0215] The activity of a polypeptide of the invention may, among
other means, be measured by the following methods.
[0216] Assays for activin/inhibin activity include, without
limitation, those described in: Vale et al., Endocrinology
91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et
al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663,
1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095,
1986.
[0217] 5.7.9 Chemotactic/Chemokinetic Activity
[0218] A polypeptide of the present invention may be involved in
chemotactic or chemokinetic activity for mammalian cells,
including, for example, monocytes, fibroblasts, neutrophils,
T-cells, mast cells, eosinophils, epithelial and/or endothelial
cells. A polynucleotide of the invention can encode a polypeptide
exhibiting such attributes. Chemotactic and chemokinetic receptor
activation can be used to mobilize or attract a desired cell
population to a desired site of action. Chemotactic or chemokinetic
compositions (e.g. proteins, antibodies, binding partners, or
modulators of the invention) provide particular advantages in
treatment of wounds and other trauma to tissues, as well as in
treatment of localized infections. For example, attraction of
lymphocytes, monocytes or neutrophils to tumors or sites of
infection may result in improved immune responses against the tumor
or infecting agent.
[0219] A protein or peptide has chemotactic activity for a
particular cell population if it can stimulate, directly or
indirectly, the directed orientation or movement of such cell
population. Preferably, the protein or peptide has the ability to
directly stimulate directed movement of cells. Whether a particular
protein has chemotactic activity for a population of cells can be
readily determined by employing such protein or peptide in any
known assay for cell chemotaxis.
[0220] Therapeutic compositions of the invention can be used in the
following:
[0221] Assays for chemotactic activity (which will identify
proteins that induce or prevent chemotaxis) consist of assays that
measure the ability of a protein to induce the migration of cells
across a membrane as well as the ability of a protein to induce the
adhesion of one cell population to another cell population.
Suitable assays for movement and adhesion include, without
limitation, those described in: Current Protocols in Immunology, Ed
by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach,
W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta
Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest.
95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et
al Eur. J. Immunol. 25:1744-1748; Gruber et al. J. of Immunol.
152:5860-5867, 1994; Johnston et al. J. of Immunol. 153:1762-1768,
1994.
[0222] 5.7.10 Hemostatic and Thrombolytic Activity
[0223] A polypeptide of the invention may also be involved in
hemostatis or thrombolysis or thrombosis. A polynucleotide of the
invention can encode a polypeptide exhibiting such attributes.
Compositions may be useful in treatment of various coagulation
disorders (including hereditary disorders, such as hemophilias) or
to enhance coagulation and other hemostatic events in treating
wounds resulting from trauma, surgery or other causes. A
composition of the invention may also be useful for dissolving or
inhibiting formation of thromboses and for treatment and prevention
of conditions resulting therefrom (such as, for example, infarction
of cardiac and central nervous system vessels (e.g., stroke).
[0224] Therapeutic compositions of the invention can be used in the
following:
[0225] Assay for hemostatic and thrombolytic activity include,
without limitation, those described in: Linet et al., J. Clin.
Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res.
45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991);
Schaub, Prostaglandins 35:467-474, 1988.
[0226] 5.7.11 Cancer Diagnosis and Therapy
[0227] Polypeptides of the invention may be involved in cancer cell
generation, proliferation or metastasis. Detection of the presence
or amount of polynucleotides or polypeptides of the invention may
be useful for the diagnosis and/or prognosis of one or more types
of cancer. For example, the presence or increased expression of a
polynucleotide/polypeptide of the invention may indicate a
hereditary risk of cancer, a precancerous condition, or an ongoing
malignancy. Conversely, a defect in the gene or absence of the
polypeptide may be associated with a cancer condition.
Identification of single nucleotide polymorphisms associated with
cancer or a predisposition to cancer may also be useful for
diagnosis or prognosis.
[0228] Cancer treatments promote tumor regression by inhibiting
tumor cell proliferation, inhibiting angiogenesis (growth of new
blood vessels that is necessary to support tumor growth) and/or
prohibiting metastasis by reducing tumor cell motility or
invasiveness. Therapeutic compositions of the invention may be
effective in adult and pediatric oncology including in solid phase
tumors/malignancies, locally advanced tumors, human soft tissue
sarcomas, metastatic cancer, including lymphatic metastases, blood
cell malignancies including multiple myeloma, acute and chronic
leukemias, and lymphomas, head and neck cancers including mouth
cancer, larynx cancer and thyroid cancer, lung cancers including
small cell carcinoma and non-small cell cancers, breast cancers
including small cell carcinoma and ductal carcinoma,
gastrointestinal cancers including esophageal cancer, stomach
cancer, colon cancer, colorectal cancer and polyps associated with
colorectal neoplasia, pancreatic cancers, liver cancer, urologic
cancers including bladder cancer and prostate cancer, malignancies
of the female genital tract including ovarian carcinoma, uterine
(including endometrial) cancers, and solid tumor in the ovarian
follicle, kidney cancers including renal cell carcinoma, brain
cancers including intrinsic brain tumors, neuroblastoma, astrocytic
brain tumors, gliomas, metastatic tumor cell invasion in the
central nervous system, bone cancers including osteomas, skin
cancers including malignant melanoma, tumor progression of human
skin keratinocytes, squamous cell carcinoma, basal cell carcinoma,
hemangiopericytoma and Karposi's sarcoma.
[0229] Polypeptides, polynucleotides, or modulators of polypeptides
of the invention (including inhibitors and stimulators of the
biological activity of the polypeptide of the invention) may be
administered to treat cancer. Therapeutic compositions can be
administered in therapeutically effective dosages alone or in
combination with adjuvant cancer therapy such as surgery,
chemotherapy, radiotherapy, thermotherapy, and laser therapy, and
may provide a beneficial effect, e.g. reducing tumor size, slowing
rate of tumor growth, inhibiting metastasis, or otherwise improving
overall clinical condition, without necessarily eradicating the
cancer.
[0230] The composition can also be administered in therapeutically
effective amounts as a portion of an anti-cancer cocktail. An
anti-cancer cocktail is a mixture of the polypeptide or modulator
of the invention with one or more anti-cancer drugs in addition to
a pharmaceutically acceptable carrier for delivery. The use of
anti-cancer cocktails as a cancer treatment is routine. Anti-cancer
drugs that are well known in the art and can be used as a treatment
in combination with the polypeptide or modulator of the invention
include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin,
Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin
(cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside),
Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl,
Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine,
5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide),
Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide
acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine
HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna,
Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide,
Plicamycin, Procarbazine HCl, Streptozocin,, Tamoxifen citrate,
Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate,
Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2,
Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine
sulfate.
[0231] In addition, therapeutic compositions of the invention may
be used for prophylactic treatment of cancer. There are hereditary
conditions and/or environmental situations (e.g. exposure to
carcinogens) known in the art that predispose an individual to
developing cancers. Under these circumstances, it may be beneficial
to treat these individuals with therapeutically effective doses of
the polypeptide of the invention to reduce the risk of developing
cancers.
[0232] In vitro models can be used to determine the effective doses
of the polypeptide of the invention as a potential cancer
treatment. These in vitro models include proliferation assays of
cultured tumor cells, growth of cultured tumor cells in soft agar
(see Freshney, (1987) Culture of Animal Cells: A Manual of Basic
Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor
systems in nude mice as described in Giovanella et al., J. Natl.
Can. Inst., 52: 921-30 (1974), mobility and invasive potential of
tumor cells in Boyden Chamber assays as described in Pilkington et
al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays
such as induction of vascularization of the chick chorioallantoic
membrane or induction of vascular endothelial cell migration as
described in Ribatta et al., Intl. J. Dev. Biol., 40: 1189-97
(1999) and Li et al., Clin. Exp. Metastasis, 17:423-9 (1999),
respectively. Suitable tumor cells lines are available, e.g. from
American Type Tissue Culture Collection catalogs.
[0233] 5.7.12 Receptor/Ligand Activity
[0234] A polypeptide of the present invention may also demonstrate
activity as receptor, receptor ligand or inhibitor or agonist of
receptor/ligand interactions. A polynucleotide of the invention can
encode a polypeptide exhibiting such characteristics. Examples of
such receptors and ligands include, without limitation, cytokine
receptors and their ligands, receptor kinases and their ligands,
receptor phosphatases and their ligands, receptors involved in
cell-cell interactions and their ligands (including without
limitation, cellular adhesion molecules (such as selectins,
integrins and their ligands) and receptor/ligand pairs involved in
antigen presentation, antigen recognition and development of
cellular and humoral immune responses. Receptors and ligands are
also useful for screening of potential peptide or small molecule
inhibitors of the relevant receptor/ligand interaction. A protein
of the present invention (including, without limitation, fragments
of receptors and ligands) may themselves be useful as inhibitors of
receptor/ligand interactions.
[0235] The activity of a polypeptide of the invention may, among
other means, be measured by the following methods:
[0236] Suitable assays for receptor-ligand activity include without
limitation those described in: Current Protocols in Immunology, Ed
by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach,
W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion
under static conditions 7.28.1- 7.28.22), Takai et al., Proc. Natl.
Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med.
168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160
1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994;
Stitt et al., Cell 80:661-670, 1995.
[0237] By way of example, the polypeptides of the invention may be
used as a receptor for a ligand(s) thereby transmitting the
biological activity of that ligand(s). Ligands may be identified
through binding assays, affinity chromatography, dihybrid screening
assays, BIAcore assays, gel overlay assays, or other methods known
in the art.
[0238] Studies characterizing drugs or proteins as agonist or
antagonist or partial agonists or a partial antagonist require the
use of other proteins as competing ligands. The polypeptides of the
present invention or ligand(s) thereof may be labeled by being
coupled to radioisotopes, calorimetric molecules or a toxin
molecules by conventional methods. ("Guide to Protein Purification"
Murray P. Deutscher (ed) Methods in Enzymology Vol. 182 (1990)
Academic Press, Inc. San Diego). Examples of radioisotopes include,
but are not limited to, tritium and carbon-14. Examples of
calorimetric molecules include, but are not limited to, fluorescent
molecules such as fluorescamine, or rhodamine or other calorimetric
molecules. Examples of toxins include, but are not limited, to
ricin.
[0239] 5.7.13 Drug Screening
[0240] This invention is particularly useful for screening chemical
compounds by using the novel polypeptides or binding fragments
thereof in any of a variety of drug screening techniques. The
polypeptides or fragments employed in such a test may either be
free in solution, affixed to a solid support, borne on a cell
surface or located intracellularly. One method of drug screening
utilizes eukaryotic or prokaryotic host cells which are stably
transformed with recombinant nucleic acids expressing the
polypeptide or a fragment thereof. Drugs are screened against such
transformed cells in competitive binding assays. Such cells, either
in viable or fixed form, can be used for standard binding assays.
One may measure, for example, the formation of complexes between
polypeptides of the invention or fragments and the agent being
tested or examine the diminution in complex formation between the
novel polypeptides and an appropriate cell line, which are well
known in the art.
[0241] Sources for test compounds that may be screened for ability
to bind to or modulate (i.e., increase or decrease) the activity of
polypeptides of the invention include (1) inorganic and organic
chemical libraries, (2) natural product libraries, and (3)
combinatorial libraries comprised of either random or mimetic
peptides, oligonucleotides or organic molecules.
[0242] Chemical libraries may be readily synthesized or purchased
from a number of commercial sources, and may include structural
analogs of known compounds or compounds that are identified as
"hits" or "leads" via natural product screening.
[0243] The sources of natural product libraries are microorganisms
(including bacteria and fungi), animals, plants or other
vegetation, or marine organisms, and libraries of mixtures for
screening may be created by: (1) fermentation and extraction of
broths from soil, plant or marine microorganisms or (2) extraction
of the organisms themselves. Natural product libraries include
polyketides, non-ribosomal peptides, and (non-naturally occurring)
variants thereof. For a review, see Science 282:63-68 (1998).
[0244] Combinatorial libraries are composed of large numbers of
peptides, oligonucleotides or organic compounds and can be readily
prepared by traditional automated synthesis methods, PCR, cloning
or proprietary synthetic methods. Of particular interest are
peptide and oligonucleotide combinatorial libraries. Still other
libraries of interest include peptide, protein, peptidomimetic,
multiparallel synthetic collection, recombinatorial, and
polypeptide libraries. For a review of combinatorial chemistry and
libraries created therefrom, see Myers, Curr. Opin. Biotechnol.
8:701-707 (1997). For reviews and examples of peptidomimetic
libraries, see Al-Obeidi et al., Mol. Biotechnol, 9(3):205-23
(1998); Hruby et al., Curr Opin Chem Biol, 1(1):114-19 (1997);
Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated
dipeptides).
[0245] Identification of modulators through use of the various
libraries described herein permits modification of the candidate
"hit" (or "lead") to optimize the capacity of the "hit" to bind a
polypeptide of the invention. The molecules identified in the
binding assay are then tested for antagonist or agonist activity in
in vivo tissue culture or animal models that are well known in the
art. In brief, the molecules are titrated into a plurality of cell
cultures or animals and then tested for either cell/animal death or
prolonged survival of the animal/cells.
[0246] The binding molecules thus identified may be complexed with
toxins, e.g., ricin or cholera, or with other compounds that are
toxic to cells such as radioisotopes. The toxin-binding molecule
complex is then targeted to a tumor or other cell by the
specificity of the binding molecule for a polypeptide of the
invention. Alternatively, the binding molecules may be complexed
with imaging agents for targeting and imaging purposes.
[0247] 5.7.14 Assay for Receptor Activity
[0248] The invention also provides methods to detect specific
binding of a polypeptide e.g. a ligand or a receptor. The art
provides numerous assays particularly useful for identifying
previously unknown binding partners for receptor polypeptides of
the invention. For example, expression cloning using mammalian or
bacterial cells, or dihybrid screening assays can be used to
identify polynucleotides encoding binding partners. As another
example, affinity chromatography with the appropriate immobilized
polypeptide of the invention can be used to isolate polypeptides
that recognize and bind polypeptides of the invention. There are a
number of different libraries used for the identification of
compounds, and-in particular small molecules, that modulate (i. e.,
increase or decrease). biological activity of a polypeptide of the
invention. Ligands for receptor polypeptides of the invention can
also be identified by adding exogenous ligands, or cocktails of
ligands to two cells populations that are genetically identical
except for the expression of the receptor of the invention: one
cell population expresses the receptor of the invention whereas the
other does not. The response of the two cell populations to the
addition of ligands,(s) are then compared. Alternatively, an
expression library can be co-expressed with the polypeptide of the
invention in cells and assayed for an autocrine response to
identify potential ligand(s). As still another example, BIAcore
assays, gel overlay assays, or other methods known in the art can
be used to identify binding partner polypeptides, including, (1)
organic and inorganic chemical libraries, (2) natural product
libraries, and (3) combinatorial libraries comprised of random
peptides, oligonucleotides or organic molecules.
[0249] The role of downstream intracellular signaling molecules in
the signaling cascade of the polypeptide of the invention can be
determined. For example, a chimeric protein in which the
cytoplasmic domain of the polypeptide of the invention is fused to
the extracellular portion of a protein, whose ligand has been
identified, is produced in a host cell. The cell is then incubated
with the ligand specific for the extracellular portion of the
chimeric protein, thereby activating the chimeric receptor. Known
downstream proteins involved in intracellular signaling can then be
assayed for expected modifications i.e. phosphorylation. Other
methods known to those in the art can also be used to identify
signaling molecules involved in receptor activity.
[0250] 5.7.15 Anti-Inflammatory Activity
[0251] Compositions of the present invention may also exhibit
anti-inflammatory activity. The anti-inflammatory activity may be
achieved by providing a stimulus to cells involved in the
inflammatory response, by inhibiting or promoting cell-cell
interactions (such as, for example, cell adhesion), by inhibiting
or promoting chemotaxis of cells involved in the inflammatory
process, inhibiting or promoting cell extravasation, or by
stimulating or suppressing production of other factors which more
directly inhibit or promote an inflammatory response. Compositions
with such activities can be used to treat inflammatory conditions
including chronic or acute conditions, including without limitation
intimation associated with infection (such as septic shock, sepsis
or systemic inflammatory response syndrome (SIRS)),
ischemia-reperfusion injury, endotoxin lethality, arthritis,
complement-mediated hyperacute rejection, nephritis, cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's
disease or resulting from over production of cytokines such as TNF
or IL-1. Compositions of the invention may also be useful to treat
anaphylaxis and, hypersensitivity to an antigenic substance or
material. Compositions of this invention may be utilized to prevent
or treat conditions such as, but not limited to, sepsis, acute
pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid
arthritis, chronic inflammatory arthritis, pancreatic cell damage
from diabetes mellitus type 1, graft versus host disease,
inflammatory bowel disease, inflamation associated with pulmonary
disease, other autoimmune disease or inflammatory disease, an
antiproliferative agent such as for acute or chronic mylegenous
leukemia or in the prevention of premature labor secondary to
intrauterine infections.
[0252] 5.7.16 Leukemias
[0253] Leukemias and related disorders may be treated or prevented
by administration of a therapeutic that promotes or inhibits
function of the polynucleotides and/or polypeptides of the
invention. Such leukemias and related disorders include but are not
limited to acute leukemia, acute lymphocytic leukemia, acute
myelocytic leukemia, myeloblastic, promyclocytic, myelomonocytic,
monocytic, erythroleukemia, chronic leukemia, chronic myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia (for a
review of such disorders, see Fishman et al., 1985, Medicine, 2d
Ed., J. B. Lippincott Co., Philadelphia).
[0254] 5.7.17 Nervous System Disorders
[0255] Nervous system disorders, involving cell types which can be
tested for efficacy of intervention with compounds that modulate
the activity of the polynucleotides and/or polypeptides of the
invention, and which can be treated upon thus observing an
indication of therapeutic utility, include but are not limited to
nervous system injuries, and diseases or disorders which result in
either a disconnection of axons, a diminution or degeneration of
neurons, or demyelination. Nervous system lesions which may be
treated in a patient (including human and non-human mammalian
patients) according to the invention include but are not limited to
the following lesions of either the central (including spinal cord,
brain) or peripheral nervous systems:
[0256] (i) traumatic lesions, including lesions caused by physical
injury or associated with surgery, for example, lesions which sever
a portion of the nervous system, or compression injuries;
[0257] (ii) ischemic lesions, in which a lack of oxygen in a
portion of the nervous system results in neuronal injury or death,
including cerebral infarction or ischemia, or spinal cord
infarction or ischemia;
[0258] (iii) infectious lesions, in which a portion of the nervous
system is destroyed or injured as a result of infection, for
example, by an abscess or associated with infection by human
immunodeficiency virus, herpes zoster, or herpes simplex virus or
with Lyme disease, tuberculosis, syphilis;
[0259] (iv) degenerative lesions, in which a portion of the nervous
system is destroyed or injured as a result of a degenerative
process including but not limited to degeneration associated with
Parkinson's disease, Alzheimer's disease, Huntington's chorea, or
amyotrophic lateral sclerosis;
[0260] (v) lesions associated with nutritional diseases or
disorders, in which a portion of the nervous system is destroyed or
injured by a nutritional disorder or disorder of metabolism
including but not limited to, vitamin B12 deficiency, folic acid
deficiency, Wernicke disease, tobacco-alcohol amblyopia,
Marchiafava-Bignami disease (primary degeneration of the corpus
callosum), and alcoholic cerebellar degeneration;
[0261] (vi) neurological lesions associated with systemic diseases
including but not limited to diabetes (diabetic neuropathy, Bell's
palsy), systemic lupus erythematosus, carcinoma, or
sarcoidosis;
[0262] (vii) lesions caused by toxic substances including alcohol,
lead, or particular neurotoxins; and
[0263] (viii) demyelinated lesions in which a portion of the
nervous system is destroyed or injured by a demyelinating disease
including but not limited to multiple sclerosis, human
immunodeficiency virus-associated myelopathy, transverse myelopathy
or various etiologies, progressive multifocal leukoencephalopathy,
and central pontine myelinolysis.
[0264] Therapeutics which are useful according to the invention for
treatment of a nervous system disorder may be selected by testing
for biological activity in promoting the survival or
differentiation of neurons. For example, and not by way of
limitation, therapeutics which elicit any of the following effects
may be useful according to the invention:
[0265] (i) increased survival time of neurons in culture;
[0266] (ii) increased sprouting of neurons in culture or in
vivo;
[0267] (iii) increased production of a neuron-associated molecule
in culture or in vivo, e.g., choline acetyltransferase or
acetylcholinesterase with respect to motor neurons;
[0268] (iv) decreased symptoms of neuron dysfunction in vivo, such
as observed during chronic pain; or
[0269] (v) proliferation and differentiation of neural precursors
for the treatment of neural degenerative diseases, e.g.,
Parkinson's, Alzheimer's.
[0270] Such effects may be measured by any method known in the art.
In preferred, non-limiting embodiments, increased survival of
neurons may be measured by the method set forth in Arakawa et
al.,(1990, J. Neurosci. 10:3507-3515); increased sprouting of
neurons may be-detected by methods set forth in Pestronk et al.
(1980, Exp. Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev.
Neurosci. 4:17-42); increased production of neuron-associated
molecules may be measured by bioassay, enzymatic assay, antibody
binding, Northern blot assay, etc., depending on the molecule to be
measured; and motor neuron dysfunction may be measured by assessing
the physical manifestation of motor neuron disorder, e.g.,
weakness, motor neuron conduction velocity, or functional
disability.
[0271] In specific embodiments, motor neuron disorders that may be
treated according to the invention include but are not limited to
disorders such as infarction, infection, exposure to toxin, trauma,
surgical damage, degenerative disease or malignancy that may affect
motor neurons as well as other components of the nervous system, as
well as disorders that selectively affect neurons such as
amyotrophic lateral sclerosis, and including but not limited to
progressive spinal muscular atrophy, progressive bulbar palsy,
primary lateral sclerosis, infantile and juvenile muscular atrophy,
progressive bulbar paralysis of childhood (Fazio-Londe syndrome),
poliomyelitis and the post polio syndrome, and Hereditary
Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
[0272] 5.7.18 Other Activities
[0273] A polypeptide of the invention may also exhibit one or more
of the following additional activities or effects: inhibiting the
growth, infection or function of, or killing, infectious agents,
including, without limitation, bacteria, viruses, fungi and other
parasites; effecting (suppressing or enhancing) bodily
characteristics, including, without limitation, height, weight,
hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ or body part size or shape (such as, for
example, breast augmentation or diminution, change in bone form or
shape); effecting biorhythms or circadian cycles or rhythms;
effecting the fertility of male or female subjects; effecting the
metabolism, catabolism, anabolism, processing, utilization, storage
or elimination of dietary fat, lipid, protein, carbohydrate,
vitamins, minerals, co-factors or other nutritional factors or
component(s); effecting behavioral characteristics, including,
without limitation, appetite, libido, stress, cognition (including
cognitive disorders), depression (including depressive disorders)
and violent behaviors; providing analgesic effects or other pain
reducing effects; promoting differentiation and growth of embryonic
stem cells in lineages other than hematopoietic lineages; hormonal
or endocrine activity; in the case of enzymes, correcting
deficiencies of the enzyme and treating deficiency-related
diseases; treatment of hyperproliferative disorders (such as, for
example, psoriasis); immunoglobulin-like activity (such as, for
example, the ability to bind antigens or complement); and the
ability to act as an antigen in a vaccine composition to raise an
immune response against such protein or another material or entity
which is cross-reactive with such protein.
[0274] 5.7.19 Identification of Polymorphisms
[0275] The demonstration of polymorphisms makes possible the
identification of such polymorphisms in human subjects and the
pharmacogenetic use of this information for diagnosis and
treatment. Such polymorphisms may be associated with, e.g.,
differential predisposition or susceptibility to various disease
states (such as disorders involving inflammation or immune
response) or a differential response to drug administration, and
this genetic information can be used to tailor preventive or
therapeutic treatment appropriately. For example, the existence of
a polymorphism associated with a predisposition to inflammation or
autoimmune disease makes possible the diagnosis of this condition
in humans by identifying the presence of the polymorphism.
[0276] Polymorphisms can be identified in a variety of ways known
in the art which all generally involve obtaining a sample from a
patient, analyzing DNA from the sample, optionally involving
isolation or amplification of the DNA, and identifying the presence
of the polymorphism in the DNA. For example, PCR may be used to
amplify an appropriate fragment of genomic DNA which may then be
sequenced. Alternatively, the DNA may be subjected to
allele-specific oligonucleotide hybridization (in which appropriate
oligonucleotides are hybridized to the DNA under conditions
permitting detection of a single base mismatch) or to a single
nucleotide extension assay (in which an oligonucleotide that
hybridizes immediately adjacent to the position of the polymorphism
is extended with one or more labeled nucleotides). In addition,
traditional restriction fragment length polymorphism analysis
(using restriction enzymes that provide differential digestion of
the genomic DNA depending on the presence or absence of the
polymorphism) may be performed. Arrays with nucleotide sequences of
the present invention can be used to detect polymorphisms. The
array can comprise modified nucleotide sequences of the present
invention in order to detect the nucleotide sequences of the
present invention. In the alternative, any one of the nucleotide
sequences of the present invention can be placed on the array to
detect changes from those sequences.
[0277] Alternatively a polymorphism resulting in a change in the
amino acid sequence could also be detected by detecting a
corresponding change in amino acid sequence of the protein, e.g.,
by an antibody specific to the variant sequence.
[0278] 5.7.20 Arthritis and Inflammation
[0279] The immunosuppressive effects of the compositions of the
invention against rheumatoid arthritis is determined in an
experimental animal model system. The experimental model system is
adjuvant induced arthritis in rats, and the protocol is described
by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et
al., 1963, Int. Arch. Allergy Appl, Immunol., 23:129. Induction of
the disease can be caused by a single injection, generally
intradermally, of a suspension of killed Mycobacterium tuberculosis
in complete Freund's adjuvant (CFA). The route of injection can
vary, but rats may be injected at the base of the tail with an
adjuvant mixture. The polypeptide is administered in phosphate
buffered solution (PBS) at a dose of about 1-5 mg/kg. The control
consists of administering PBS only.
[0280] The procedure for testing the effects of the test compound
would consist of intradermally injecting killed Mycobacterium
tuberculosis in CFA followed by immediately administering the test
compound and subsequent treatment every other day until day 24. At
14, 15; 18, 20, 22, and 24 days after injection of Mycobacterium
CFA, an overall arthritis score may be obtained as described by J.
Holoskitz above. An analysis of the data would reveal that the test
compound would have a dramatic affect on the swelling of the joints
as measured by a decrease of the arthritis score.
[0281] 5.8 Therapeutic Methods
[0282] The compositions (including polypeptide fragments, analogs,
variants and antibodies or other binding partners or modulators
including antisense polynucleotides) of the invention have numerous
applications in a variety of therapeutic methods. Examples of
therapeutic applications include, but are not limited to, those
exemplified herein.
[0283] 5.8.1 Example
[0284] One embodiment of the invention is the administration of an
effective amount of the semaphorin-like polypeptides or other
composition of the invention to individuals affected by a disease
or disorder that can be modulated by regulating the peptides of the
invention. While the mode of administration is not particularly
important, parenteral administration is preferred. An exemplary
mode of administration is to deliver an intravenous bolus. The
dosage of semaphorin-like polypeptides or other composition of the
invention will normally be determined by the prescribing physician.
It is to be expected that the dosage will vary according to the
age, weight, condition and response of the individual patient.
Typically, the amount of polypeptide administered per dose will be
in the range of about 0.01 .mu.g/kg to 100 mg/kg of body weight,
with the preferred dose being about 0.1 .mu.g/kg to 10 mg/kg of
patient body weight. For parenteral administration, semaphorin-like
polypeptides of the invention will be formulated in an injectable
form combined with a pharmaceutically acceptable parenteral
vehicle. Such vehicles are well known in the art and examples
include water, saline, Ringer's solution, dextrose solution, and
solutions consisting of small amounts of the human serum albumin.
The vehicle may contain minor amounts of additives that maintain
the isotonicity and stability of the polypeptide or other active
ingredient. The preparation of such solutions is within the skill
of the art.
[0285] 5.9 Pharmaceutical Formulations and Routes of
Administration
[0286] A protein or other composition of the present invention
(from whatever source derived, including without limitation from
recombinant and non-recombinant sources and including antibodies
and other binding partners of the polypeptides of the invention)
may be administered to a patient in need, by itself, or in
pharmaceutical compositions where it is mixed with suitable
carriers or excipient(s) at doses to treat or ameliorate a variety
of disorders. Such a composition may optionally contain (in
addition to protein or other active ingredient and a carrier)
diluents, fillers, salts, buffers, stabilizers, solubilizers, and
other materials well known in the art. The term "pharmaceutically
acceptable" means a non-toxic material that does not interfere with
the effectiveness of the biological activity of the active
ingredient(s). The characteristics of the carrier will depend on
the route of administration. The pharmaceutical composition of the
invention may also contain cytokines, lymphokines, or other
hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,
IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem cell factor, and erythropoietin. In further
compositions, proteins of the invention may be combined with other
agents beneficial to the treatment of the disease or disorder in
question. These agents include various growth factors such as
epidermal growth factor (EGF), platelet-derived growth factor
(PDGF), transforming growth factors (TGF-.alpha. and TGF-.beta.),
insulin-like growth factor (IGF), as well as cytokines described
herein.
[0287] The pharmaceutical composition may further contain other
agents which either enhance the activity of the protein or other
active ingredient or complement its activity or use in treatment.
Such additional factors and/or agents may be included in the
pharmaceutical composition to produce a synergistic effect with
protein or other active ingredient of the invention, or to minimize
side effects. Conversely, protein or other active ingredient of the
present invention may be included in formulations of the particular
clotting factor, cytokine, lymphokine, other hematopoietic factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent
to minimize side effects of the clotting factor, cytokine,
lymphokine, other hematopoietic factor, thrombolytic or
anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra,
IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive
agents). A protein of the present invention may be active in
multimers (e.g., heterodimers or homodimers) or complexes with
itself or other proteins. As a result, pharmaceutical compositions
of the invention may comprise a protein of the invention in such
multimeric or complexed form.
[0288] As an alternative to being included in a pharmaceutical
composition of the invention including a first protein, a second
protein or a therapeutic agent may be concurrently administered
with the first protein (e.g., at the same time, or at differing
times provided that therapeutic concentrations of the combination
of agents is achieved at the treatment site). Techniques for
formulation and administration of the compounds of the instant
application may be found in "Remington's Pharmaceutical Sciences,"
Mack Publishing Co., Easton, Pa., latest edition. A therapeutically
effective dose further refers to that amount of the compound
sufficient to result in amelioration of symptoms, e.g., treatment,
healing, prevention or amelioration of the relevant medical
condition, or an increase in rate of treatment, healing, prevention
or amelioration of such conditions. When applied to an individual
active ingredient, administered alone, a therapeutically effective
dose refers to that ingredient alone. When applied to a,
combination, a therapeutically effective dose refers to combined
amounts of the active ingredients that result in the therapeutic
effect, whether administered in combination, serially or
simultaneously.
[0289] In practicing the method of treatment or use of the present
invention, a therapeutically effective amount of protein or other
active ingredient of the present invention is administered to a
mammal having a condition to be treated. Protein or other active
ingredient of the present invention may be administered in
accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing
cytokines, lymphokines or other hematopoietic factors. When
co-administered with one or more cytokines, lymphokines or other
hematopoietic factors, protein or other active ingredient of the
present invention may be administered either simultaneously with
the cytokine(s), lymphokine(s), other hematopoietic factor(s),
thrombolytic or anti-thrombotic factors, or sequentially. If
administered sequentially, the attending physician will decide on
the appropriate sequence of administering protein or other active
ingredient of the present invention in combination with
cytokine(s), lymphokine(s), other hematopoietic factor(s),
thrombolytic or anti-thrombotic factors.
[0290] 5.9.1 Routes of Administration
[0291] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections. Administration of protein or other active
ingredient of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can
be carried out in a variety of conventional ways, such as oral
ingestion, inhalation, topical application or cutaneous,
subcutaneous, intraperitoneal, parenteral or intravenous injection.
Intravenous administration to the patient is preferred.
[0292] Alternately, one may administer the compound in a local
rather than systemic manner, for example, via injection of the
compound directly into a arthritic joints or in fibrotic tissue,
often in a depot or sustained release formulation. In order to
prevent the scarring process frequently occurring as complication
of glaucoma surgery, the compounds may be administered topically,
for example, as eye drops. Furthermore, one may administer the drug
in a targeted drug delivery system, for example, in a liposome
coated with a specific antibody, targeting, for example, arthritic
or fibrotic tissue. The liposomes will be targeted to and taken up
selectively by the afflicted tissue.
[0293] The polypeptides of the invention are administered by any
route that delivers an effective dosage to the desired site of
action. The determination of a suitable route of administration and
an effective dosage for a particular indication is within the level
of skill in the art. Preferably for wound treatment, one
administers the therapeutic compound directly to the site. Suitable
dosage ranges for the polypeptides of the invention can be
extrapolated from these dosages or from similar studies in
appropriate animal models. Dosages can then be adjusted as
necessary by the clinician to provide maximal therapeutic
benefit.
[0294] 5.9.2 Compositions/Formulations
[0295] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in a conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the
active compounds into preparations which can be used
pharmaceutically. These pharmaceutical compositions may be
manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes. Proper formulation is dependent upon the route of
administration chosen. When a therapeutically effective amount of
protein or other active ingredient of the present invention is
administered orally, protein or other active ingredient of the
present invention will be in the form of a tablet, capsule, powder,
solution or elixir. When administered in tablet form, the
pharmaceutical composition of the invention may additionally
contain a solid carrier such as a gelatin or an adjuvant. The
tablet, capsule, and powder contain from about 5 to 95% protein or
other active ingredient of the present invention, and-preferably
from about 25 to 90% protein or other active ingredient of the
present invention. When administered in liquid form, a liquid
carrier such as water, petroleum, oils of animal or plant origin
such as peanut oil, mineral oil, soybean oil, or sesame oil, or
synthetic oils may be added. The liquid form of the pharmaceutical
composition may further contain physiological saline solution,
dextrose or other saccharide solution, or glycols such as ethylene
glycol, propylene glycol or polyethylene glycol. When administered
in liquid form, the pharmaceutical composition contains from about
0.5 to 90% by weight of protein or other active ingredient of the
present invention, and preferably from about 1 to 50% protein or
other active ingredient of the present invention.
[0296] When a therapeutically effective amount of protein or other
active ingredient of the present invention is administered by
intravenous, cutaneous or subcutaneous injection, protein or other
active ingredient of the present invention will be in the form of a
pyrogen-free, parenterally acceptable aqueous solution. The
preparation of such parenterally acceptable protein or other active
ingredient solutions, having due regard to pH, isotonicity,
stability, and the like, is within the skill in the art. A
preferred pharmaceutical composition for intravenous, cutaneous, or
subcutaneous injection should contain, in addition to protein or
other active ingredient of the present invention, an isotonic
vehicle such as Sodium Chloride Injection, Ringer's Injection,
Dextrose Injection, Dextrose and Sodium Chloride Injection,
Lactated Ringer's Injection, or other vehicle as known in the art.
The pharmaceutical composition of the present invention may also
contain stabilizers, preservatives, buffers, antioxidants, or other
additives known to those of skill in the art. For injection, the
agents of the invention may be formulated in aqueous solutions,
preferably in physiologically compatible buffers such as Hanks's
solution, Ringer's solution, or physiological saline buffer. For
transmucosal administration, penetrants appropriate to the barrier
to be permeated are used in the formulation. Such penetrants are
generally known in the art.
[0297] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a patient to be treated.
Pharmaceutical preparations for oral use can be obtained solid
excipient, optionally grinding a resulting mixture, and processing
the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. Suitable excipients
are, in particular, fillers such as sugars, including lactose,
sucrose, mannitol, or sorbitol; cellulose preparations such as, for
example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate. Dragee cores are provided with suitable coatings. For
this purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0298] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration. For buccal
administration, the compositions may take the form of tablets or
lozenges formulated in conventional manner.
[0299] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g., gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch. The compounds may
be formulated for parenteral administration by injection, e.g., by
bolus injection or continuous infusion. Formulations for injection
may be presented in unit dosage form, e.g., in ampules or in
multi-dose containers, with an added preservative. The compositions
may take such forms as suspensions, solutions or emulsions in oily
or aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents.
[0300] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions. Alternatively,
the active ingredient may be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0301] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides. In addition to the formulations described previously,
the compounds may also be formulated as a depot preparation. Such
long acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0302] A pharmaceutical carrier for the hydrophobic compounds of
the invention is a co-solvent system comprising benzyl alcohol, a
nonpolar surfactant, a water-miscible organic polymer, and an
aqueous phase. The co-solvent system may be the VPD co-solvent
system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the
nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol
300, made up to volume in absolute ethanol. The VPD co-solvent
system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in
water solution. This co-solvent system dissolves hydrophobic
compounds well, and itself produces low toxicity upon systemic
administration. Naturally, the proportions of a co-solvent system
may be varied considerably without destroying its solubility and
toxicity characteristics. Furthermore, the identity of the
co-solvent components may be varied: for example, other
low-toxicity nonpolar surfactants may be used instead of
polysorbate 80; the fraction size of polyethylene glycol may be
varied; other biocompatible polymers may replace polyethylene
glycol, e.g. polyvinyl pyrrolidone; and other sugars or
polysaccharides may substitute for dextrose. Alternatively, other
delivery systems for hydrophobic pharmaceutical compounds may be
employed. Liposomes and emulsions are well known examples of
delivery vehicles or carriers for hydrophobic drugs. Certain
organic solvents such as dimethylsulfoxide also may be employed,
although usually at the cost of greater toxicity. Additionally, the
compounds may be delivered using a sustained-release system, such
as semipermeable matrices of solid hydrophobic polymers containing
the therapeutic agent. Various types of sustained-release materials
have been established and are well known by those skilled in the
art. Sustained-release capsules may, depending on their chemical
nature, release the compounds for a few weeks up to over 100 days.
Depending on the chemical nature and the biological stability of
the therapeutic reagent, additional strategies for protein or other
active ingredient stabilization may be employed.
[0303] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene glycols.
Many of the active ingredients of the invention may be provided as
salts with pharmaceutically compatible counter ions. Such
pharmaceutically acceptable base addition salts are those salts
which retain the biological effectiveness and properties of the
free acids and which are obtained by reaction with inorganic or
organic bases such as sodium hydroxide, magnesium hydroxide,
ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino
acids, sodium acetate, potassium benzoate, triethanol amine and the
like.
[0304] The pharmaceutical composition of the invention may be in
the form of a complex of the protein(s) or other active ingredient
of present invention along with protein or peptide antigens. The
protein and/or peptide antigen will deliver a stimulatory signal to
both B and T lymphocytes. B lymphocytes will respond to antigen
through their surface immunoglobulin receptor. T lymphocytes will
respond to antigen through the T cell receptor (TCR) following
presentation of the antigen by MHC proteins. MHC and structurally
related proteins including those encoded by class I and class II
MHC genes on host cells will serve to present the peptide
antigen(s) to T lymphocytes. The antigen components could also be
supplied as purified MHC-peptide complexes alone or with
co-stimulatory molecules that can directly signal T cells.
Alternatively antibodies able to bind surface immunoglobulin and
other molecules on B cells as well as antibodies able to bind the
TCR and other molecules on T cells can be combined with the
pharmaceutical composition of the invention.
[0305] The pharmaceutical composition of the invention may be in
the form of a liposome in which protein of the present invention is
combined, in addition to other pharmaceutically acceptable
carriers, with amphipathic agents such as lipids which exist in
aggregated form as micelles, insoluble monolayers, liquid crystals,
or lamellar layers in aqueous solution. Suitable lipids for
liposomal formulation include, without limitation, monoglycerides,
diglycerides, sulfatides, lysolecithins, phospholipids, saponin,
bile acids, and the like. Preparation of such liposomal
formulations is within the level of skill in the art, as disclosed,
for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and
4,737,323, all of which are incorporated herein by reference.
[0306] The amount of protein or other active ingredient of the
present invention in the pharmaceutical composition of the present
invention will depend upon the nature and severity of the condition
being treated, and on the nature of prior treatments which the
patient has undergone. Ultimately, the attending physician will
decide the amount of protein or other active ingredient of the
present invention with which to treat each individual patient.
Initially, the attending physician will administer low doses of
protein or other active ingredient of the present invention and
observe the patient's response. Larger doses of protein or other
active ingredient of the present invention may be administered
until the optimal therapeutic effect is obtained for the patient,
and at that point the dosage is not increased further. It is
contemplated that the various pharmaceutical compositions used to
practice the method of the present invention should contain about
0.01 .mu.g to about 100 mg (preferably about 0.1 .mu.g to about 10
mg, more preferably about 0.1 .mu.g to about 1 mg) of protein or
other active ingredient of the present invention per kg body
weight. For compositions of the present invention which are useful
for bone, cartilage, tendon or ligament regeneration, the
therapeutic method includes administering the composition
topically, systematically, or locally as an implant or device. When
administered, the therapeutic composition for use in this invention
is, of course, in a pyrogen-free, physiologically acceptable form.
Further, the composition may desirably be encapsulated or injected
in a viscous form for delivery to the site of bone, cartilage or
tissue damage. Topical administration may be suitable for wound
healing and tissue repair. Therapeutically useful agents other than
a protein or other active ingredient of the invention which may
also optionally be included in the composition as described above,
may alternatively or additionally, be administered simultaneously
or sequentially with the composition in the methods of the
invention. Preferably for bone and/or cartilage formation, the
composition would include a matrix capable of delivering the
protein-containing or other active ingredient-containing
composition to the site of bone and/or cartilage damage, providing
a structure for the developing bone and cartilage and optimally
capable of being resorbed into the body. Such matrices may be
formed of materials presently in use for other implanted medical
applications.
[0307] The choice of matrix material is based on biocompatibility,
biodegradability, mechanical properties, cosmetic appearance and
interface properties. The particular application of the
compositions will define the appropriate formulation. Potential
matrices for the compositions may be biodegradable and chemically
defined calcium sulfate, tricalcium phosphate, hydroxyapatite,
polylactic acid, polyglycolic acid and polyanhydrides. Other
potential materials are biodegradable and biologically
well-defined, such as bone or dermal collagen. Further matrices are
comprised of pure proteins or extracellular matrix components.
Other potential matrices are nonbiodegradable and chemically
defined, such as sintered hydroxyapatite, bioglass, aluminates, or
other ceramics. Matrices may be comprised of combinations of any of
the above mentioned types of material, such as polylactic acid and
hydroxyapatite or collagen and tricalcium phosphate. The
bioceramics may be altered in composition, such as in
calcium-aluminate-phosphate and processing to alter pore size,
particle size, particle shape, and biodegradability. Presently
preferred is a 50:50 (mole weight) copolymer of lactic acid and
glycolic acid in the form of porous particles having diameters
ranging from 150 to 800 microns. In some applications, it will be
useful to utilize a sequestering agent, such as carboxymethyl
cellulose or autologous blood clot, to prevent the protein
compositions from disassociating from the matrix.
[0308] A preferred family of sequestering agents is cellulosic
materials such as alkylcelluloses (including
hydroxyalkylcelluloses), including methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most
preferred being cationic salts of carboxymethylcellulose (CMC).
Other preferred sequestering agents include hyaluronic acid, sodium
alginate, poly(ethylene glycol), polyoxyethylene oxide,
carboxyvinyl polymer and poly(vinyl alcohol). The amount of
sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt
% based on total formulation weight, which represents the amount
necessary to prevent desorption of the protein from the polymer
matrix and to provide appropriate handling of the composition, yet
not so much that the progenitor cells are prevented from
infiltrating the matrix, thereby providing the protein the
opportunity to assist the osteogenic activity of the progenitor
cells. In further compositions, proteins or other active ingredient
of the invention may be combined with other agents beneficial to,
the treatment of the bone and/or cartilage defect, wound, or tissue
in question. These agents include various growth factors such as
epidermal growth factor (EGF), platelet derived growth factor
(PDGF), transforming growth factors (TGF-.alpha. and TGF-.beta.),
and insulin-like growth factor (IGF).
[0309] The therapeutic compositions are also presently valuable for
veterinary applications. Particularly domestic animals and
thoroughbred horses, in addition to humans, are desired patients
for such treatment with proteins or other active ingredient of the
present invention. The dosage regimen of a protein-containing
pharmaceutical composition to be used in tissue regeneration will
be determined by the attending physician considering various
factors which modify the action of the proteins, e.g., amount of
tissue weight desired to be formed, the site of damage, the
condition of the damaged tissue, the size of a wound, type of
damaged tissue (e.g., bone), the patient's age, sex, and diet, the
severity of any infection, time of administration and other
clinical factors. The dosage may vary with the type of matrix used
in the reconstitution and with inclusion of other proteins in the
pharmaceutical composition. For example, the addition of other
known growth factors, such as IGF I (insulin like growth factor I),
to the final composition, may also effect the dosage. Progress can
be monitored by periodic assessment of tissue/bone growth and/or
repair, for example, X-rays, histomorphometric determinations and
tetracycline labeling.
[0310] Polynucleotides of the present invention can also be used
for gene therapy. Such polynucleotides can be introduced either in
vivo or ex vivo into cells for expression in a mammalian subject.
Polynucleotides of the invention may also be administered by other
known methods for introduction of nucleic acid into a cell or
organism (including, without limitation, in the form of viral
vectors or naked DNA). Cells may also be cultured ex vivo in the
presence of proteins of the present invention in order to
proliferate or to produce a desired effect on or activity in such
cells. Treated cells can then be introduced in vivo for therapeutic
purposes.
[0311] 5.9.3 Effective Dosage
[0312] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
More specifically, a therapeutically effective amount means an
amount effective to prevent development of or to alleviate the
existing symptoms of the subject being treated. Determination of
the effective amount is well within the capability of those skilled
in the art, especially in light of the detailed disclosure provided
herein. For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from
appropriate in vitro assays. For example, a dose can be formulated
in animal models to achieve a circulating concentration range that
can be used to more accurately determine useful doses in humans.
For example, a dose can be formulated in animal models to achieve a
circulating concentration range that includes the IC.sub.50 as
determined in cell culture (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of the protein's
biological activity). Such information can be used to more
accurately determine useful doses in humans.
[0313] A therapeutically effective dose refers to that amount of
the compound that results in amelioration of symptoms or a
prolongation of survival in a patient. Toxicity and therapeutic
efficacy of such compounds can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio between LD.sub.50 and ED.sub.50. Compounds
which exhibit high therapeutic indices are preferred. The data
obtained from these cell culture assays and animal studies can be
used in formulating a range of dosage for use in human. The dosage
of such compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. The
exact formulation, route of administration and dosage can be chosen
by the individual physician in view of the patient's condition.
See, e.g., Fingl et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1. Dosage amount and interval may be
adjusted individually to provide plasma levels of the active moiety
which are sufficient to maintain the desired effects, or minimal
effective concentration (MEC). The MEC will vary for each compound
but can be estimated from in vitro data. Dosages necessary to
achieve the MEC will depend on individual characteristics and route
of administration. However, HPLC assays or bioassays can be used to
determine plasma concentrations.
[0314] Dosage intervals can also be determined using MEC value.
Compounds should be administered using a regimen which maintains
plasma levels above the MEC for 10-90% of the time, preferably
between 30-90% and most preferably between 50-90%. In cases of
local administration or selective uptake, the effective local
concentration of the drug may not be related to plasma
concentration.
[0315] An exemplary dosage regimen for polypeptides or other
compositions of the invention will be in the range of about 0.01
.mu.g/kg to 100 mg/kg of body weight daily, with the preferred dose
being about 0.1 .mu.g/kg to 25 mg/kg of patient body weight daily,
varying in adults and children. Dosing may be once daily, or
equivalent doses may be delivered at longer or shorter
intervals.
[0316] The amount of composition administered will, of course, be
dependent on the subject being treated, on the subject's age and
weight, the severity of the affliction, the manner of
administration and the judgment of the prescribing physician.
[0317] 5.9.4 Packaging
[0318] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may, for example,
comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. Compositions comprising a compound of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition.
[0319] 5.10 Antibodies
[0320] Another aspect of the invention is an antibody that
specifically binds the polypeptide of the invention. Such
antibodies include monoclonal and polyclonal antibodies, single
chain antibodies, chimeric antibodies, bifunctional/bispecific
antibodies, humanized antibodies, human antibodies, and
complementary determining region (CDR)-grafted antibodies,
including compounds which include CDR and/or antigen-binding
sequences, which specifically recognize a polypeptide of the
invention. Preferred antibodies of the invention are human
antibodies which are produced and identified according to methods
described in WO93/11236, published Jun. 20, 1993, which is
incorporated herein by reference in its entirety. Antibody
fragments, including Fab, Fab', F(ab').sub.2, and F.sub.v, are also
provided by the invention. The term "specific for" indicates that
the variable regions of the antibodies of the invention recognize
and bind polypeptides of the invention exclusively (i.e., able to
distinguish the polypeptide of the invention from other similar
polypeptides despite sequence identity, homology, or similarity
found in the family of polypeptides), but may also interact with
other proteins (for example, S. aureus protein A or other
antibodies in ELISA techniques) through interactions with sequences
outside the variable region of the antibodies, and in particular,
in the constant region of the molecule. Screening assays to
determine binding specificity of an antibody of the invention are
well known and routinely practiced in the art. For a comprehensive
discussion of such assays, see Harlow et al. (Eds), Antibodies A
Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring
Harbor, N.Y. (1988), Chapter 6. Antibodies that recognize and bind
fragments of the polypeptides of the invention are also
contemplated, provided that the antibodies are first and foremost
specific for, as defined above, full length polypeptides of the
invention. As with antibodies that are specific for full length
polypeptides of the invention, antibodies of the invention that
recognize fragments are those which can distinguish polypeptides
from the same family of polypeptides despite inherent sequence
identity, homology, or similarity found in the family of proteins.
Antibodies of the invention can be produced using any method well
known and routinely practiced in the art.
[0321] Non-human antibodies may be humanized by any methods known
in the art. In one method, the non-human CDRs are inserted into a
human antibody or consensus antibody framework sequence. Further
changes can then be introduced into the antibody framework to
modulate affinity or immunogenicity.
[0322] Antibodies of the invention are useful for, for example,
therapeutic purposes (by modulating activity of a polypeptide of
the invention), diagnostic purposes to detect or quantitate a
polypeptide of the invention, as well as purification of a
polypeptide of the invention. Kits comprising an antibody of the
invention for any of the purposes described herein are also
comprehended. In general, a kit of the invention also includes a
control antigen for which the antibody is immunospecific. The
invention further provides a hybridoma that produces an antibody
according to the invention. Antibodies of the invention are useful
for detection and/or purification of the polypeptides of the
invention.
[0323] Polypeptides of the invention may also be used to immunize
animals to obtain polyclonal and monoclonal antibodies which
specifically react with the protein. Such antibodies may be
obtained using either the entire protein or fragments thereof as an
immunogen. The peptide immunogens additionally may contain a
cysteine residue at the carboxyl terminus, and are conjugated, to a
hapten such as keyhole limpet hemocyanin (KLH). Methods for
synthesizing such peptides are known in the art, for example, as in
R. P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963); J. L.
Krstenansky, et al., FEBS Lett. 211, 10 (1987).
[0324] Monoclonal antibodies binding to the protein of the
invention may be useful diagnostic agents for the immunodetection
of the protein. Neutralizing monoclonal antibodies binding to the
protein may also be useful therapeutics for both conditions
associated with the protein and also in the treatment of some forms
of cancer where abnormal expression of the protein is involved. In
the case of cancerous cells or leukemic cells, neutralizing
monoclonal antibodies against the protein may be useful in
detecting and preventing the metastatic spread of the cancerous
cells, which may be mediated by the protein. In general, techniques
for preparing polyclonal and monoclonal antibodies as well as
hybridomas capable of producing the desired antibody are well known
in the art (Campbell, A. M., Monoclonal Antibodies Technology:
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1984); St.
Groth et al., J. Immunol. 35:1-21 (1990); Kohler and Milstein,
Nature 256:495-497 (1975)), the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., Immunology Today 4:72 (1983);
Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc. (1985), pp. 77-96).
[0325] Any animal (mouse, rabbit, etc.) which is known to produce
antibodies can be immunized with a peptide or polypeptide of the
invention. Methods for immunization are well known in the art. Such
methods include subcutaneous or intraperitoneal injection of the
polypeptide. One skilled in the art will recognize that the amount
of the protein encoded by the ORF of the present invention used for
immunization will vary based on the animal which is immunized, the
antigenicity of the peptide and the site of injection. The protein
that is used as an immunogen may be modified or administered in an
adjuvant in order to increase the protein's antigenicity. Methods
of increasing the antigenicity of a protein are well known in the
art and include, but are not limited to, coupling the antigen with
a heterologous protein (such as globulin or .beta.-galactosidase)
or through the inclusion of an adjuvant during immunization.
[0326] For monoclonal antibodies, spleen cells from the immunized
animals are removed, fused with myeloma cells, such as SP2/0-Ag14
myeloma cells, and allowed to become monoclonal antibody producing
hybridoma cells. Any one of a number of methods well known in the
art can be used to identify the hybridoma cell which produces an
antibody with the desired characteristics. These include screening
the hybridomas with an ELISA assay, Western blot analysis, or
radioimmunoassay (Lutz et al., Exp. Cell Research. 175:109-124
(1988)). Hybridomas secreting the desired antibodies are cloned and
the class and subclass is determined using procedures known in the
art (Campbell, A.M., Monoclonal Antibody Technology: Laboratory
Techniques in Biochemistry and Molecular Biology, Elsevier Science
Publishers, Amsterdam, The Netherlands (1984)). Techniques
described for the production of single chain antibodies (U.S. Pat.
No. 4,946,778) can be adapted to produce single chain antibodies to
proteins of the present invention.
[0327] For polyclonal antibodies, antibody-containing antiserum is
isolated from the immunized animal and is screened for the presence
of antibodies with the desired specificity using one of the
above-described procedures. The present invention further provides
the above-described antibodies in detectably labeled form.
Antibodies can be detectably labeled through the use of
radioisotopes, affinity labels (such as biotin, avidin, etc.),
enzymatic labels (such as horseradish peroxidase, alkaline
phosphatase, etc.) fluorescent labels (such as FITC or rhodamine,
etc.), paramagnetic atoms, etc. Procedures for accomplishing such
labeling are well known in the art; for example, see (Sternberger,
L. A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A.
et al., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol.
109:129 (1972); Goding, J. W. J. Immunol. Meth. 13:215 (1976)).
[0328] The labeled antibodies of the present invention can be used
for in vitro, in vivo, and in situ assays to identify cells or
tissues in which a fragment of the polypeptide of interest is
expressed. The antibodies may also be used directly in therapies or
other diagnostics. The present invention further provides the
above-described antibodies immobilized on a solid support. Examples
of such solid supports include plastics such as polycarbonate,
complex carbohydrates such as agarose and Sepharose.RTM., acrylic
resins and such as polyacrylamide and latex beads. Techniques for
coupling antibodies to such solid supports are well known in the
art (Weir, D. M. et al., "Handbook of Experimental Immunology" 4th
Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10
(1986); Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press, N.Y.
(1974)). The immobilized antibodies of the present invention can be
used for in vitro, in vivo, and in situ assays as well as for
immuno-affinity purification of the proteins of the present
invention.
[0329] 5.11 Computer Readable Sequences
[0330] In one application of this embodiment, a nucleotide sequence
of the present invention can be recorded on computer readable
media. As used herein, "computer readable media" refers to any
medium which can be read and accessed directly by a computer. Such
media include, but are not limited to: magnetic storage media, such
as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. A skilled artisan can readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide sequence of the present
invention. As used herein, "recorded" refers to a process for
storing information on computer readable medium. A skilled artisan
can readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide sequence information of the present
invention.
[0331] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide sequence of the present invention.
The choice of the data storage structure will generally be based on
the means chosen to access the stored information. In addition, a
variety of data processor programs and formats can be used to store
the nucleotide sequence information of the present invention on
computer readable medium. The sequence information can be
represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. A
skilled artisan can readily adapt any number of data processor
structuring formats (e.g. text file or database) in order to obtain
computer readable medium having recorded thereon the nucleotide
sequence information of the present invention.
[0332] By providing any of the nucleotide sequences SEQ ID NO: 1-3,
5 or 12 or a representative fragment thereof; or a nucleotide
sequence at least 95% identical to any of the nucleotide sequences
of the SEQ ID NO: 1-3, 5 or 12 in computer readable form, a skilled
artisan can routinely access the sequence information for a variety
of purposes. Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium. The examples which follow demonstrate how
software which implements the BLAST (Altschul et al., J. Mol. Biol.
215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem.
17:203-207 (1993)) search algorithms on a Sybase system is used to
identify open reading frames (ORFs) within a nucleic acid sequence.
Such ORFs may be protein encoding fragments and may be useful in
producing commercially important proteins such as enzymes used in
fermentation reactions and in the production of commercially useful
metabolites.
[0333] As used herein, "a computer-based system" refers to the
hardware means, software means, and data storage means used to
analyze the nucleotide sequence information of the present
invention. The minimum hardware means of the computer-based systems
of the present invention comprises a central processing unit (CPU),
input means, output means, and data storage means. A skilled
artisan can readily appreciate that any one of the currently
available computer-based systems are suitable for use in the
present invention. As stated above, the computer-based systems of
the present invention comprise a data storage means having stored
therein a nucleotide sequence of the present invention and the
necessary hardware means and software means for supporting and
implementing a search means. As used herein, "data storage means"
refers to memory which can store nucleotide sequence information of
the present invention, or a memory access means which can access
manufactures having recorded thereon the nucleotide sequence
information of the present invention.
[0334] As used herein, "search means" refers to one or more
programs which are implemented on the computer-based system to
compare a target sequence or target structural motif with the
sequence information stored within the data storage means. Search
means are used to identify fragments or regions of a known sequence
which match a particular target sequence or target motif. A variety
of known algorithms are disclosed publicly and a variety of
commercially available software for conducting search means are and
can be used in the computer-based systems of the present invention.
Examples of such software includes, but is not limited to,
Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA
(NPOLYPEPTIDEIA). A skilled artisan can readily recognize that any
one of the available algorithms or implementing software packages
for conducting homology searches can be adapted for use in the
present computer-based systems. As used herein, a "target sequence"
can be any nucleic acid or amino acid sequence of six or more
nucleotides or two or more amino acids. A skilled artisan can
readily recognize that the longer a target sequence is, the less
likely a target sequence will be present as a random occurrence in
the database. The most preferred sequence length of a target
sequence is from about 10 to 100 amino acids, or from about 30 to
300 nucleotide residues. However, it is well recognized that
searches for commercially important fragments, such as sequence
fragments involved in gene expression and protein processing, may
be of shorter length.
[0335] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzyme
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
[0336] 5.12 Triple Helix Formation
[0337] In addition, the fragments of the present invention, as
broadly described, can be used to control gene expression through
triple helix formation or antisense DNA or RNA, both of which
methods are based on the binding of a polynucleotide sequence to
DNA or RNA. Polynucleotides suitable for use in these methods are
usually 20 to 40 bases in length and are designed to be
complementary to a region of the gene involved in transcription
(triple helix--see Lee et al., Nucl. Acids Res. 6:3073 (1979);
Cooney et al., Science 15241:456 (1988); and Dervan et al., Science
251:1360 (1991)) or to the mRNA itself (antisense--Olmno, J.
Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).
Triple helix-formation optimally results in a shut-off of RNA
transcription from DNA, while antisense RNA hybridization blocks
translation of an mRNA molecule into polypeptide. Both techniques
have been demonstrated to be effective in model systems.
Information contained in the sequences of the present invention is
necessary for the design of an antisense or triple helix
oligonucleotide.
[0338] 5.13 Diagnostic Assays and Kits
[0339] The present invention further provides methods to identify
the presence or expression of one of the ORFs of the present
invention, or homolog thereof, in a test sample, using a nucleic
acid probe or antibodies of the present invention, optionally
conjugated or otherwise associated with a suitable label.
[0340] In general, methods for detecting a polynucleotide of the
invention can comprise contacting a sample with a compound that
binds to and forms a complex with the polynucleotide for a period
sufficient to form the complex, and detecting the complex, so that
if a complex is detected, a polynucleotide of the invention is
detected in the sample. Such methods can also comprise contacting a
sample under stringent hybridization conditions with nucleic acid
primers that anneal to a polynucleotide of the invention under such
conditions, and amplifying annealed polynucleotides, so that if a
polynucleotide is amplified, a polynucleotide of the invention is
detected in the sample.
[0341] In general, methods for detecting a polypeptide of the
invention can comprise contacting a sample with a compound that
binds to and forms a complex with the polypeptide for a period
sufficient to form the complex, and detecting the complex, so that
if a complex is detected, a polypeptide of the invention is
detected in the sample.
[0342] In detail, such methods comprise incubating a test sample
with one or more of the antibodies or one or more of the nucleic
acid probes of the present invention and assaying for binding of
the nucleic acid probes or antibodies to components within the test
sample.
[0343] Conditions for incubating a nucleic acid probe or antibody
with a test sample vary. Incubation conditions depend on the format
employed in the assay, the detection methods employed, and the type
and nature of the nucleic acid probe or antibody used in the assay.
One skilled in the art will recognize that any one of the commonly
available hybridization, amplification or immunological assay
formats can readily be adapted to employ the nucleic acid probes or
antibodies of the present invention. Examples of such assays can be
found in Chard, T., An Introduction to Radioimmunoassay and Related
Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands
(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,
Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3
(1985); Tijssen, P., Practice and Theory of immunoassays:
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The
test samples of the present invention include cells, protein or
membrane extracts of cells, or biological fluids such as sputum,
blood, serum, plasma, or urine. The test sample used in the
above-described method will vary based on the assay format, nature
of the detection method and the tissues, cells or extracts used as
the sample to be assayed. Methods for preparing protein extracts or
membrane extracts of cells are well known in the art and can be
readily be adapted in order to obtain a sample which is compatible
with the system utilized.
[0344] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention. Specifically, the invention
provides a compartment kit to receive, in close confinement, one or
more containers which comprises: (a) a first container comprising
one of the probes or antibodies of the present invention; and (b)
one or more other containers comprising one or more of the
following: wash reagents, reagents capable of detecting presence of
a bound probe or antibody.
[0345] In detail, a compartment kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers or strips of
plastic or paper. Such containers allows one to efficiently
transfer reagents from one compartment to another compartment such
that the samples and reagents are not cross-contaminated, and the
agents or solutions of each container can be added in a
quantitative fashion from one compartment to another. Such
containers will include a container which will accept the test
sample, a container which contains the antibodies used in the
assay, containers which contain wash reagents (such as phosphate
buffered saline, Tris-buffers, etc.), and containers which contain
the reagents used to detect the bound antibody or probe. Types of
detection reagents include labeled nucleic acid probes, labeled
secondary antibodies, or in the alternative, if the primary
antibody is labeled, the enzymatic, or antibody binding reagents
which are capable of reacting with the labeled antibody. One
skilled in the art will readily recognize that the disclosed probes
and antibodies of the present invention can be readily incorporated
into one of the established kit formats which are well known in the
art.
[0346] 5.14 Medical Imaging
[0347] The novel polypeptides and binding partners of the invention
are useful in medical imaging of sites expressing the molecules of
the invention (e.g., where the polypeptide of the invention is
involved in the immune response, for imaging sites of inflammation
or infection). See, e.g., Kunkel et al., U.S. Pat. No. 5,413,778.
Such methods involve chemical attachment of a labeling or imaging
agent, administration of the labeled polypeptide to a subject in a
pharmaceutically acceptable carrier, and imaging the labeled
polypeptide in vivo at the target site.
[0348] 5.15 Screening Assays
[0349] Using the isolated proteins and polynucleotides of the
invention, the present invention further provides methods of
obtaining and identifying agents which bind to a polypeptide
encoded by an ORF corresponding to any of the nucleotide sequences
set forth in the SEQ ID NO: 1-3, 5 or 12, or bind to a specific
domain of the polypeptide encoded by the nucleic acid. In detail,
said method comprises the steps of:
[0350] (a) contacting an agent with an isolated protein encoded by
an ORF of the present invention, or nucleic acid of the invention;
and
[0351] (b) determining whether the agent binds to said protein or
said nucleic acid.
[0352] In general, therefore, such methods for identifying
compounds that bind to a polynucleotide of the invention can
comprise contacting a compound with a polynucleotide of the
invention for a time sufficient to form a polynucleotide/compound
complex, and detecting the complex, so that if a
polynucleotide/compound complex is detected, a compound that binds
to a polynucleotide of the invention is identified.
[0353] Likewise, in general, therefore, such methods for
identifying compounds that bind to a polypeptide of the invention
can comprise contacting a compound with a polypeptide of the
invention for a time sufficient to form a polypeptide/compound
complex, and detecting the complex, so that if a
polypeptide/compound complex is detected, a compound that binds to
a polynucleotide of the invention is identified.
[0354] Methods for identifying compounds that bind to a polypeptide
of the invention can also comprise contacting a compound with a
polypeptide of the invention in a cell for a time sufficient to
form a polypeptide/compound complex, wherein the complex drives
expression of a receptor gene sequence in the cell, and detecting
the complex by detecting reporter gene sequence expression, so that
if a polypeptide/compound complex is detected, a compound that
binds a polypeptide of the invention is identified.
[0355] Compounds identified via such methods can include compounds
which modulate the activity of a polypeptide of the invention (that
is, increase or decrease its activity, relative to activity
observed in the absence of the compound). Alternatively, compounds
identified via such methods can include compounds which modulate
the expression of a polynucleotide of the invention (that is,
increase or decrease expression relative to expression levels
observed in the absence of the compound). Compounds, such as
compounds identified via the methods of the invention, can be
tested using standard assays well known to those of skill in the
art for their ability to modulate activity/expression.
[0356] The agents screened in the above assay can be, but are not
limited to, peptides, carbohydrates, vitamin derivatives, or other
pharmaceutical agents. The agents can be selected and screened at
random or rationally selected or designed using protein modeling
techniques.
[0357] For random screening, agents such as peptides,
carbohydrates, pharmaceutical agents and the like are selected at
random and are assayed for their ability to bind to the protein
encoded by the ORF of the present invention. Alternatively, agents
may be rationally selected or designed. As used herein, an agent is
said to be "rationally selected or designed" when the agent is
chosen based on the configuration of the particular protein. For
example, one skilled in the art can readily adapt currently
available procedures to generate peptides, pharmaceutical agents
and the like, capable of binding to a specific peptide sequence, in
order to generate rationally designed antipeptide peptides, for
example see Hurby et al., Application of Synthetic Peptides:
Antisense Peptides," In Synthetic Peptides, A User's Guide, W. H.
Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry
28:9230-8 (1989), or pharmaceutical agents, or the like.
[0358] In addition to the foregoing, one class of agents of the
present invention, as broadly described, can be used to control
gene expression through binding to one of the ORFs or EMFs of the
present invention. As described above, such agents can be randomly
screened or rationally designed/selected. Targeting the ORF or EMF
allows a skilled artisan to design sequence specific or element
specific agents, modulating the expression of either a single ORF
or multiple ORFs which rely on the same EMF for expression control.
One class of DNA binding agents are agents which contain base
residues which hybridize or form a triple helix formation by
binding to DNA or RNA. Such agents can be based on the classic
phosphodiester, ribonucleic acid backbone, or can be a variety of
sulfhydryl or polymeric derivatives which have base attachment
capacity.
[0359] Agents suitable for use in these methods usually contain 20
to 40 bases and are designed to be complementary to a region of the
gene involved in transcription (triple helix--see Lee et al., Nucl.
Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988);
and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself
(antisense--Okano, J. Neurochem. 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation
optimally results in a shut-off of RNA transcription from DNA,
while antisense RNA hybridization blocks translation of an mRNA
molecule into polypeptide. Both techniques have been demonstrated
to be effective in model systems. Information contained in the
sequences of the present invention is necessary for the design of
an antisense or triple helix oligonucleotide and other DNA binding
agents.
[0360] Agents which bind to a protein encoded by one of the ORFs of
the present invention can be used as a diagnostic agent. Agents
which bind to a protein encoded by one of the ORFs of the present
invention can be formulated using known techniques to generate a
pharmaceutical composition.
[0361] 5.16 Use of Nucleic Acids as Probes
[0362] Another aspect of the subject invention is to provide for
polypeptide-specific nucleic acid hybridization probes capable of
hybridizing with naturally occurring nucleotide sequences. The
hybridization probes of the subject invention may be derived from
any of the nucleotide sequences SEQ ID NO: 1-3, 5 or 12. Because
the corresponding gene is only expressed in a limited number of
tissues, a hybridization probe derived from of any of the
nucleotide sequences SEQ ID NO: 1-3, 5 or 12 can be used as an
indicator of the presence of RNA of cell type of such a tissue in a
sample.
[0363] Any suitable hybridization technique can be employed, such
as, for example, in situ hybridization. PCR as described in U.S.
Pat. Nos. 4,683,195 and 4,965,188 provides additional uses for
oligonucleotides based upon the nucleotide sequences. Such probes
used in PCR may be of recombinant origin, may be chemically
synthesized, or a mixture of both. The probe will comprise a
discrete nucleotide sequence for the detection of identical
sequences or a degenerate pool of possible sequences for
identification of closely related genomic sequences.
[0364] Other means for producing specific hybridization probes for
nucleic acids include the cloning of nucleic acid sequences into
vectors for the production of mRNA probes. Such vectors are known
in the art and are commercially available and may be used to
synthesize RNA probes in vitro by means of the addition of the
appropriate RNA polymerase as T7 or SP6 RNA polymerase and the
appropriate radioactively labeled nucleotides. The nucleotide
sequences may be used to construct hybridization probes for mapping
their respective genomic sequences. The nucleotide sequence
provided herein may be mapped to a chromosome or specific regions
of a chromosome using well known genetic and/or chromosomal mapping
techniques. These techniques include in situ hybridization, linkage
analysis against known chromosomal markers, hybridization screening
with libraries or flow-sorted chromosomal preparations specific to
known chromosomes, and the like. The technique of fluorescent in
situ hybridization of chromosome spreads has been described, among
other places, in Verma et al (1988) Human Chromosomes: A Manual of
Basic Techniques, Pergamon Press, New York N.Y.
[0365] Fluorescent in situ hybridization of chromosomal
preparations and other physical chromosome mapping techniques may
be correlated with additional genetic map data. Examples of genetic
map data can be found in the 1994 Genome Issue of Science
(265:1981f). Correlation between the location of a nucleic acid on
a physical chromosomal map and a specific disease (or
predisposition to a specific disease) may help delimit the region
of DNA associated with that genetic disease. The nucleotide
sequences of the subject invention may be used to detect
differences in gene sequences between normal, carrier or affected
individuals.
[0366] 5.17 Preparation of Support Bound Oligonucleotides
[0367] Oligonucleotides, i.e., small nucleic acid segments, may be
readily prepared by, for example, directly synthesizing the
oligonucleotide by chemical means, as is commonly practiced using
an automated oligonucleotide synthesizer.
[0368] Support bound oligonucleotides may be prepared by any of the
methods known to those of skill in the art using any suitable
support such as glass, polystyrene or Teflon. One strategy is to
precisely spot oligonucleotides synthesized by standard
synthesizers. Immobilization can be achieved using passive
adsorption (Inouye & Hondo, 1990 J. Clin Microbiol 28(6)
1462-72); using UV light (Nagata et al., 1985; Dahlen et al., 1987;
Morrissey & Collins, Mol. Cell Probes 1989 3(2) 189-207) or by
covalent binding of base modified DNA (Keller et al., 1988; 1989);
all references being specifically incorporated herein.
[0369] Another strategy that may be employed is the use of the
strong biotin-streptavidin interaction as a linker. For example,
Broude et al. (1994) Proc. Natl. Acad. Sci USA 91(8) 3072-6
describe the use of biotinylated probes, although these are duplex
probes, that are immobilized on streptavidin-coated magnetic beads.
Streptavidin-coated beads may be purchased from Dynal, Oslo. Of
course, this same linking chemistry is applicable to coating any
surface with streptavidin. Biotinylated probes may be purchased
from various sources, such as, e.g., Operon Technologies (Alameda,
Calif.).
[0370] Nunc Laboratories (Naperville, Ill.) is also selling
suitable material that could be used. Nunc Laboratories have
developed a method by which DNA can be covalently bound to the
microwell surface termed Covalink NH. CovaLink NH is a polystyrene
surface grafted with secondary amino groups (>NH) that serve as
bridge-heads for further covalent coupling. CovaLink Modules may be
purchased from Nunc Laboratories. DNA molecules may be bound to
CovaLink exclusively at the 5'-end by a phosphoramidate bond,
allowing immobilization of more than 1 pmol of DNA (Rasmussen et
al., (1991) Anal Biochem 198(1) 138-42.
[0371] The use of CovaLink NH strips for covalent binding of DNA
molecules at the 5'-end has been described (Rasmussen et al.,
1991). In this technology, a phosphoramidate bond is employed (Chu
et al., 1983 Nucleic Acids 11(18) 6513-29). This is beneficial as
immobilization using only a single covalent bond is preferred. The
phosphoramidate bond joins the DNA to the CovaLink NH secondary
amino groups that are positioned at the end of spacer arms
covalently grafted onto the polystyrene surface through a 2 nm long
spacer arm. To link an oligonucleotide to CovaLink NH via an
phosphoramidate bond, the oligonucleotide terminus must have a
5'-end phosphate group. It is, perhaps, even possible for biotin to
be covalently bound to CovaLink and then streptavidin used to bind
the probes.
[0372] More specifically, the linkage method includes dissolving
DNA in water (7.5 ng/ul) and denaturing for 10 min. at 95.degree.
C. and cooling on ice for 10 min. Ice-cold 0.1 M 1-methylimidazole,
pH 7.0 (1-MeIm.sub.7), is then added to a final concentration of 10
mM 1-MeIm.sub.7. A ss DNA solution is then dispensed into CovaLink
NH strips (75 ul/well) standing on ice.
[0373] Carbodiimide 0.2 M
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in
10 mM 1-MeIm.sub.7, is made fresh and 25 ul added per well. The
strips are incubated for 5 hours at 50.degree. C. After incubation
the strips are washed using, e.g., Nunc-Immuno Wash; first the
wells are washed 3 times, then they are soaked with washing
solution for 5 min., and finally they are washed 3 times (where in
the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50.degree.
C.).
[0374] It is contemplated that a further suitable method for use
with the present invention is that described in PCT Patent
Application WO 90/03382 (Southern & Maskos), incorporated
herein by reference. This method of preparing an oligonucleotide
bound to a support involves attaching a nucleoside 3'-reagent
through the phosphate group by a covalent phosphodiester link to
aliphatic hydroxyl groups carried by the support. The
oligonucleotide is then synthesized on the supported nucleoside and
protecting groups removed from the synthetic oligonucleotide chain
under standard conditions that do not cleave the oligonucleotide
from the support. Suitable reagents include nucleoside
phosphoramidite and nucleoside hydrogen phosphorate.
[0375] An on-chip strategy for the preparation of DNA probe for the
preparation of DNA probe arrays may be employed. For example,
addressable laser-activated photodeprotection may be employed in
the chemical synthesis of oligonucleotides directly on a glass
surface, as described by Fodor et al. (1991) Science 251(4995)
767-73, incorporated herein by reference. Probes may also be
immobilized on nylon supports as described by Van Ness et al.
(1991) Nucleic Acids Res. 19(12) 3345-50; or linked to Teflon using
the method of Duncan & Cavalier (1988) Anal Biochem 169(1)
104-8; all references being specifically incorporated herein.
[0376] To link an oligonucleotide to a nylon support, as described
by Van Ness et al. (1991), requires activation of the nylon surface
via alkylation and selective activation of the 5'-amine of
oligonucleotides with cyanuric chloride.
[0377] One particular way to prepare support bound oligonucleotides
is to utilize the light-generated synthesis described by Pease et
al., (1994) Proc. Natl. Acad. Sci USA 91(11) 5022-6. These authors
used current photolithographic techniques to generate arrays of
immobilized oligonucleotide probes (DNA chips). These methods, in
which light is used to direct the synthesis of oligonucleotide
probes in high-density, miniaturized arrays, utilize photolabile
5'-protected N-acyl-deoxynucleoside phosphoramidites, surface
linker chemistry and versatile combinatorial synthesis strategies.
A matrix of 256 spatially defined oligonucleotide probes may be
generated in this manner.
[0378] 5.18 Preparation of Nucleic Acid Fragments
[0379] The nucleic acids may be obtained from any appropriate
source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected
chromosome bands, cosmid or YAC inserts, and RNA, including mRNA
without any amplification steps. For example, Sambrook et al.
(1989) describes three protocols for the isolation of high
molecular weight DNA from mammalian cells (p. 9.14-9.23).
[0380] DNA fragments may be prepared as clones in M13, plasmid or
lambda vectors and/or prepared directly from genomic DNA or cDNA by
PCR or other amplification methods. Samples may be prepared or
dispensed in multiwell plates. About 100-1000 ng of DNA samples may
be prepared in 2-500 ml of final volume.
[0381] The nucleic acids would then be fragmented by any of the
methods known to those of skill in the art including, for example,
using restriction enzymes as described at 9.24-9.28 of Sambrook et
al. (1989), shearing by ultrasound and NaOH treatment.
[0382] Low pressure shearing is also appropriate, as described by
Schriefer et al. (1990) Nucleic Acids Res. 18(24) 7455-6. In this
method, DNA samples are passed through a small French pressure cell
at a variety of low to intermediate pressures. A lever device
allows controlled application of low to intermediate pressures to
the cell. The results of these studies indicate that low-pressure
shearing is a useful alternative to sonic and enzymatic DNA
fragmentation methods.
[0383] One particularly suitable way for fragmenting DNA is
contemplated to be that using the two base recognition
endonuclease, CviJI, described, by Fitzgerald et al. (1992) Nucleic
Acids Res. 20(14) 3753-62. These authors described an approach for
the rapid fragmentation and fractionation of DNA into particular
sizes that they contemplated to be suitable for shotgun cloning and
sequencing.
[0384] The restriction endonuclease CviJI normally cleaves the
recognition sequence PuGCPy between the G and C to leave blunt
ends. Atypical reaction conditions, which alter the specificity of
this enzyme (CviJI**), yield a quasi-random distribution of DNA
fragments form the small molecule pUC19 (2688 base pairs).
Fitzgerald et al. (1992) quantitatively evaluated the randomness of
this fragmentation strategy, using a CviJI** digest of pUC19 that
was size fractionated by a rapid gel filtration method and directly
ligated, without end repair, to a lac Z minus M13 cloning vector.
Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy
and PuGCPu, in addition to PuGCPy sites, and that new sequence data
is accumulated at a rate consistent with random fragmentation.
[0385] As reported in the literature, advantages of this approach
compared to sonication and agarose gel fractionation include:
smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug);
and fewer steps are involved (no preligation, end repair, chemical
extraction, or agarose gel electrophoresis and elution are
needed).
[0386] Irrespective of the manner in which the nucleic acid
fragments are obtained or prepared, it is important to denature the
DNA to give single stranded pieces available for hybridization.
This is achieved by incubating the DNA solution for 2-5 minutes at
80-90.degree. C. The solution is then cooled quickly to 2.degree.
C. to prevent renaturation of the DNA fragments before they are
contacted with the chip. Phosphate groups must also be removed from
genomic DNA by methods known in the art.
[0387] 5.19 Preparation of DNA Arrays
[0388] Arrays may be prepared by spotting DNA samples on a support
such as a nylon membrane. Spotting may be performed by using arrays
of metal pins (the positions of which correspond to an array of
wells in a microtiter plate) to repeated by transfer of about 20 nl
of a DNA solution to a nylon membrane. By offset printing, a
density of dots higher than the density of the wells is achieved.
One to 25 dots may be accommodated in 1 mm.sup.2, depending on the
type of label used. By avoiding spotting in some preselected number
of rows and columns, separate subsets (subarrays) may be formed.
Samples in one subarray may be the same genomic segment of DNA (or
the same gene) from different individuals, or may be different,
overlapped genomic clones. Each of the subarrays may represent
replica spotting of the same samples. In one example, a selected
gene segment may be amplified from 64 patients. For each patient,
the amplified gene segment may be in one 96-well plate (all 96
wells containing the same sample). A plate for each of the 64
patients is prepared. By using a 96-pin device, all samples may be
spotted on one 8.times.12 cm membrane. Subarrays may contain 64
samples, one from each patient. Where the 96 subarrays are
identical, the dot span may be 1 mm.sup.2 and there may be a 1 mm
space between subarrays.
[0389] Another approach is to use membranes or plates (available
from NUNC, Naperville, Ill.) which may be partitioned by physical
spacers e.g. a plastic grid molded over the membrane, the grid
being similar to the sort of membrane applied to the bottom of
multiwell plates, or hydrophobic strips. A fixed physical spacer is
not preferred for imaging by exposure to flat phosphor-storage
screens or x-ray films.
[0390] The present invention is illustrated in the following
examples. Upon consideration of the present disclosure, one of
skill in the art will appreciate that many other embodiments and
variations may be made in the scope of the present invention.
Accordingly, it is intended that the broader aspects of the present
invention not be limited to the disclosure of the following
examples. The present invention is not to be limited in scope by
the exemplified embodiments which are intended as illustrations of
single aspects of the invention, and compositions and methods which
are functionally equivalent are within the scope of the invention.
Indeed, numerous modifications and variations in the practice of
the invention are expected to occur to those skilled in the art
upon consideration of the present preferred embodiments.
Consequently, the only limitations which should be placed upon the
scope of the invention are those which appear in the appended
claims.
[0391] All references cited within the body of the instant
specification are hereby incorporated by reference in their
entirety.
6.0 EXAMPLES
Example 1
Isolation of SEQ ID NO: 1 from a cDNA Library of Fetal
Liver-Spleen
[0392] A plurality of novel nucleic acids were obtained from a cDNA
library prepared from fetal liver-spleen (Hyseq clone
identification numbers 5688868 (SEQ ID NO: 1)) using standard PCR,
sequencing by hybridization sequence signature analysis, and Sanger
sequencing techniques. The inserts of the library were amplified
with PCR using primers specific for vector sequences flanking the
inserts. These samples were spotted onto nylon membranes and
interrogated with oligonucleotide probes to give sequence
signatures. The clones were clustered into groups of similar or
identical sequences, and single representative clones were selected
from each group for gel sequencing. The 5' sequence of the
amplified inserts was then deduced using the reverse M13 sequencing
primer in a typical Sanger sequencing protocol. PCR products were
purified and subjected to fluorescent dye terminator cycle
sequencing. Single-pass gel sequencing was done using a 377 Applied
Biosystems (ABI) sequencer. The insert was identified as a novel
sequence not previously obtained from this library and not
previously reported in public databases. The sequence was
designated as SEQ ID NO: 1.
Example 2
Assemblage of SEQ ID NO: 2
[0393] The nucleic acid of the present invention, designated as SEQ
ID NO: 2 was assembled using SEQ ID NO:1 as a seed. Then a
recursive algorithm was used to extend the seed into an extended
assemblage, by pulling additional sequences from different
databases (i.e., Hyseq's database containing EST sequences, dbEST
version 114, gb pri 114, and UniGene version 101) that belong to
this assemblage. The algorithm terminated when there was no
additional sequences from the above databases that would extend the
assemblage. Inclusion of component sequences into the assemblage
was based on a BLASTN hit to the extending assemblage with BLAST
score greater than 300 and percent identity greater than 95%.
[0394] The nearest neighbor result for the assembled contigs were
obtained by a FASTA version 3 search against Genpept release 114,
using FASTXY algorithm. FASTXY is an improved version of FASTA
alignment which allows in-codon frame shifts. The nearest neighbor
result showed the closest homologue for each assemblage from
Genpept (and contains the translated amino acid sequences for which
the assemblage encodes). The nearest neighbor results is set forth
below:
1 SEQ ID Accession Smith- NO: No. Description Waterman Score %
Identity 2 AF030430 Mus musculus 170 30.769 Semaphorin VIa
[0395] A polypeptide was predicted to be encoded by SEQ ID NO: 2 as
set forth below. The polypeptide was predicted using a software
program called FASTY (available from
http://fasta.bioch.virginia.edu) which selects a polypeptide based
on a comparison of translated novel polynucleotide to known
polypeptides (W. R. Pearson, Methods in Enzymology, 183: 63-98
(1990), herein incorporated by reference).
2 Predicted Predicted AMINO ACID ENCODED BY SEQ ID NO: 2 beginning
end (A = Alanine, C = Cysteine, D = Aspartic Acid, E = nucleotide
nucleotide Glutamic Acid, F = Phenylalanine, G = Glycine, location
location H = Histidine, I = Isoleucine, K = Lysine, L = Leucine,
correspond- correspond- M = Methionine, N = Asparagine, P =
Proline, ing to first ing to last Q = Glutamine, R = Arginine, S =
Serine, T = Threonine, amino acid amino acid V = Valine, W =
Tryptophan, Y = Tyrosine, residue of residue of X = Unknown, * =
Stop Codon, / = possible nucleotide amino acid amino acid deletion,
.backslash. = possible nucleotide insertion) segment segment 2 868
IAGVAVFFYRDMFVRKDRKIHKDAESAQSCTDSSG
SFAKLNGLFDSPVKEYQQNIDSPKLIVT/SLTSRKEL
PPNGDTKSMVMDHRGQPPELAALPTPESTPVLHQK
TLQAMKSHSEKAHGHGASRKETPQFFPSSPPPHSPL
SHGHIPSAIVLPNATHDYNTSFSNSNAHKAEKKLQN
IDHPLTKSSSKRDHRRSVDSRNTLNDLLKHLNDPNS
NPKAIMGDIQMAHQNLMLDPMGSMSEVPPKVPNR
EASLYSPPSTLPRNSPTKRVDVPTTPGVPMTSLERQ RGYHK
Example 3
Assemblage of SEQ ID NOs: 3 and 4
[0396] Assembly of novel nucleotide sequence of SEQ ID NO: 3 was
accomplished by using an EST sequence SEQ ID NO: 1 as a seed. The
seed was extended by gel sequencing (377 Applied Biosystems (ABI)
sequencer) using primers to extend the 3' end (primer extension).
The 5' end was extended using RACE, as disclosed in
Marathon-Ready.TM. cDNA User Manual (PT1156-1) (Clontech) herein
incorporated by reference.
[0397] A polypeptide (SEQ ID NO:4) was predicted to be encoded by
SEQ ID NO:3 as set forth below. The polypeptide was predicted using
a software program called BLASTX which selects a polypeptide based
on a comparison of translated novel polynucleotides to known
polypeptides. The initial methionine starts at position 434 of SEQ
ID NO:3 and the putative stop codon, TAG, begins at position 3692
of the nucleotide sequence.
[0398] FIG. 1 shows the BLASTX amino acid sequence alignment
between the protein encoded by SEQ ID NO: 3 (i.e. SEQ ID NO: 4) and
the human KIAA1479 protein (SEQ ID NO: 9) indicating that the two
sequences share 100% similarity over 429 amino acid residues of SEQ
ID NO: 4 and 100% identity over the same 429 amino acid residues of
SEQ ID NO: 4.
[0399] FIG. 2 shows the BLASTX amino acid sequence alignment
between the protein encoded by SEQ ID NO: 3 (i.e. SEQ ID NO: 4) and
the human semaphorin Y protein (SEQ ID NO: 10) indicating that the
two sequences share 71% similarity over 540 amino acid residues of
SEQ ID NO: 4 and 52% identity over the same 540 amino acid residues
of SEQ ID NO: 4.
[0400] A predicted approximately sixteen-residue signal peptide is
encoded from approximately residue 1 to residue 16 inclusive of SEQ
ID NO: 4 (SEQ ID NO: 6). The signal peptide region was predicted
using the Kyte-Doolittle hydrophobicity prediction algorithm (J.
Mol Biol, 157, pp. 105-31 (1982), incorporated herein by
reference).
[0401] A predicted approximately twenty-nine-residue transmembrane
peptide is encoded from approximately residue 671 to residue 699
inclusive of SEQ ID NO: 4 (SEQ ID NO: 7). The transmembrane peptide
region was predicted using the Kyte-Doolittle hydrophobicity
prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982),
incorporated herein by reference).
Example 4
A. Expression of SEQ ID NO: 4 in Cells
[0402] Chinese Hamster Ovary (CHO) cells or other suitable cell
types are grown in DMEM (ATCC) and 10% fetal bovine serum (FBS)
(Gibco) to 70% confluence. Prior to transfection the media is
changed to DMEM and 0.5% FCS. Cells are transfected with cDNAs for
SEQ ID NO: 1-3, 5 or 12 or with pBGal vector by the FuGENE-6
transfection reagent (Boehringer). In summary, 4 .mu.l of FuGENE-6
is diluted in 100 .mu.l of DMEM and incubated for 5 minutes. Then,
this is added to 1 .mu.g of DNA and incubated for 15 minutes before
adding it to a 35 mm dish of CHO cells. The CHO cells are incubated
at 37.degree. C. with 5% CO.sub.2. After 24 hours, media and cell
lysates are collected, centrifuged and dialyzed against assay
buffer (15 mM Tris pH 7.6, 134 mM NaCl, 5 mM glucose, 3 mM
CaCl.sub.2 and MgCl.sub.2.
B. Expression Study Using SEQ ID NO: 1-3, 5 or 12
[0403] The expression of SEQ ID NO: 1-3, 5 or 12 in various tissues
is analyzed using a semi-quantitative polymerase chain
reaction-based technique. Human cDNA libraries are used as sources
of expressed genes from tissues of interest (adult bladder, adult
brain, adult heart, adult kidney, adult lymph node, adult liver,
adult lung, adult ovary, adult placenta, adult rectum, adult
spleen, adult testis, bone marrow, thymus, thyroid gland, fetal
kidney, fetal liver, fetal liver-spleen, fetal skin, fetal brain,
fetal leukocyte and macrophage). Gene-specific primers are used to
amplify portions of the SEQ ID NO: 1-3, 5 or 12 sequence from the
samples. Amplified products are separated on an agarose gel,
transferred and chemically linked to a nylon filter. The filter is
then hybridized with a radioactively labeled (.sup.33P-dCTP)
double-stranded probe generated from SEQ ID NO: 1-3, 5 or 12 using
a Klenow polymerase, random-prime method. The filters are washed
(high stringency) and used to expose a phosphorimaging screen for
several hours. Bands indicate the presence of cDNA including SEQ ID
NO: 1-3, 5 or 12 sequences in a specific library, and thus mRNA
expression in the corresponding cell type or tissue.
Sequence CWU 1
1
13 1 348 DNA Homo sapiens 1 ggcacgagca aaacattgat caccctctca
caaagtcatc cagtaagaga gatcaccggc 60 gttctgttga ttccagaaat
accctcaatg atctcctgaa gcatctgaat gacccaaata 120 gtaaccccaa
agccatcatg ggagacatcc agatggcaca ccagaactta atgctggatc 180
ccatgggatc gatgtctgag gtcccaccta aagtccctaa ccgggaggca tcgctatact
240 cccctccttc aactctcccc agaaatagcc caaccaagcg agtggatgtc
cccaccactc 300 ctggagtccc aatgacttct ctggaaagac aaagaggtta tcacaaaa
348 2 868 DNA Homo sapiens 2 cattgcaggt gtggcagtat tcttctatcg
agacatgttt gttcggaaag acagaaagat 60 ccataaagat gcagagtccg
cccagtcatg cacagactcc agtggaagtt ttgccaaact 120 gaatggtctc
tttgacagcc ctgtcaagga ataccaacag aatattgatt ctcctaaact 180
tatagtaacc tgctaaccag tcggaaagag ctaccaccca atggagatac taaatccatg
240 gtaatggacc atcgagggca acctccagag ttggctgctc ttcccactcc
tgagtctaca 300 cccgtgcttc accagaagac cctgcaggcc atgaagagcc
actcagaaaa ggcccatggc 360 catggagctt caaggaaaga aacccctcag
ttttttccgt ctagtccgcc acctcattcc 420 ccattaagtc atgggcatat
ccccagtgcc attgttcttc caaatgctac ccatgactac 480 aacacgtctt
tctcaaactc caatgctcac aaagctgaaa agaagcttca aaacattgat 540
caccctctca caaagtcatc cagtaagaga gatcaccggc gttctgttga ttccagaaat
600 accctcaatg atctcctgaa gcatctgaat gacccaaata gtaaccccaa
agccatcatg 660 ggagacatcc agatggcaca ccagaactta atgctggatc
ccatgggatc gatgtctgag 720 gtcccaccta aagtccctaa ccgggaggca
tcgctatact cccctccttc aactctcccc 780 agaaatagcc caaccaagcg
agtggatgtc cccaccactc ctggagtccc aatgacttct 840 ctggaaagac
aaagaggtta tcacaaaa 868 3 3694 DNA Homo sapiens CDS (434)..(3694) 3
cgggcgttcc caccgtccct ctccccttac tggcagagct gcgggcggat tcccgggccc
60 ggagcagccc accggccacc ccaccgccca cccggctccc ggtgtctcct
cccggccgct 120 ctacccagca actttccgtg ctttgttccc cgactggaaa
tgctttacgg aagcgtcttg 180 gacagggtct ccgccaggcg acaagagctc
ggtgctgaga tgtgttacgt tctcatctcc 240 ccatcaatta tggatggaaa
caaataagga agagtcaatt ctgctgagcc ccttctccgg 300 caacgagagg
cgttctgcag ccgggaggga gccgccgctc gcgccggcag ccgctggcag 360
gggcatggtg aggaggaagg tagctcagtg gcatttctga gcaggggcca ccctgacttc
420 accttggccc acc atg agg gtc ttc ctg ctt tgt gcc tac ata ctg ctg
469 Met Arg Val Phe Leu Leu Cys Ala Tyr Ile Leu Leu 1 5 10 ctg atg
gtt tcc cag ttg agg gca gtc agc ttt cct gaa gat gat gaa 517 Leu Met
Val Ser Gln Leu Arg Ala Val Ser Phe Pro Glu Asp Asp Glu 15 20 25
ccc ctt aat act gtc gac tat cac tat tca agg caa tat ccg gtt ttt 565
Pro Leu Asn Thr Val Asp Tyr His Tyr Ser Arg Gln Tyr Pro Val Phe 30
35 40 aga gga cgc cct tca ggc aat gaa tcg cag cac agg ctg gac ttt
cag 613 Arg Gly Arg Pro Ser Gly Asn Glu Ser Gln His Arg Leu Asp Phe
Gln 45 50 55 60 ctg atg ttg aaa att cga gac aca ctt tat att gct ggc
agg gat caa 661 Leu Met Leu Lys Ile Arg Asp Thr Leu Tyr Ile Ala Gly
Arg Asp Gln 65 70 75 gtt tat aca gta aac tta aat gaa atg ccc aaa
aca gaa gta ata ccc 709 Val Tyr Thr Val Asn Leu Asn Glu Met Pro Lys
Thr Glu Val Ile Pro 80 85 90 aac aag aaa ctg aca tgg cga tca aga
caa cag gat cga gaa aac tgt 757 Asn Lys Lys Leu Thr Trp Arg Ser Arg
Gln Gln Asp Arg Glu Asn Cys 95 100 105 gct atg aaa ggc aag cat aaa
gat gaa tgc cac aac ttt atc aaa gta 805 Ala Met Lys Gly Lys His Lys
Asp Glu Cys His Asn Phe Ile Lys Val 110 115 120 ttt gtt cca aga aac
gat gag atg gtt ttt gtt tgt ggt acc aat gca 853 Phe Val Pro Arg Asn
Asp Glu Met Val Phe Val Cys Gly Thr Asn Ala 125 130 135 140 ttc aat
ccc atg tgt aga tac tac agg ttg agt acc tta gaa tat gat 901 Phe Asn
Pro Met Cys Arg Tyr Tyr Arg Leu Ser Thr Leu Glu Tyr Asp 145 150 155
ggg gaa gaa att agt ggc ctg gca aga tgc cca ttt gat gcc aga caa 949
Gly Glu Glu Ile Ser Gly Leu Ala Arg Cys Pro Phe Asp Ala Arg Gln 160
165 170 acc aat gtt gcc ctc ttt gct gat ggg aag ctg tat tct gcc aca
gtg 997 Thr Asn Val Ala Leu Phe Ala Asp Gly Lys Leu Tyr Ser Ala Thr
Val 175 180 185 gct gac ttc ttg gcc agc gat gcc gtt att tat cga agc
atg ggt gat 1045 Ala Asp Phe Leu Ala Ser Asp Ala Val Ile Tyr Arg
Ser Met Gly Asp 190 195 200 gga tct gcc ctt cgc aca ata aaa tat gat
tcc aaa tgg ata aaa gag 1093 Gly Ser Ala Leu Arg Thr Ile Lys Tyr
Asp Ser Lys Trp Ile Lys Glu 205 210 215 220 cca cac ttt ctt cat gcc
ata gaa tat gga aac tat gtc tat ttc ttc 1141 Pro His Phe Leu His
Ala Ile Glu Tyr Gly Asn Tyr Val Tyr Phe Phe 225 230 235 ttt cga gaa
atc gct gtc gaa cat aat aat tta ggc aag gct gtg tat 1189 Phe Arg
Glu Ile Ala Val Glu His Asn Asn Leu Gly Lys Ala Val Tyr 240 245 250
tcc cgc gtg gcc cgc ata tgt aaa aac gac atg ggt ggt tcc cag cgg
1237 Ser Arg Val Ala Arg Ile Cys Lys Asn Asp Met Gly Gly Ser Gln
Arg 255 260 265 gtc ctg gag aaa cac tgg act tca ttt cta aag gct cgg
ctg aac tgt 1285 Val Leu Glu Lys His Trp Thr Ser Phe Leu Lys Ala
Arg Leu Asn Cys 270 275 280 tct gtc cct gga gat ccg ttt ttc tac ttt
gat gtt ctg cag tct att 1333 Ser Val Pro Gly Asp Pro Phe Phe Tyr
Phe Asp Val Leu Gln Ser Ile 285 290 295 300 aca gac ata ata caa atc
aat ggc atc ccc act gtg gtc ggg gtg ttt 1381 Thr Asp Ile Ile Gln
Ile Asn Gly Ile Pro Thr Val Val Gly Val Phe 305 310 315 acc acg cag
ctc aat agc atc cct ggt tct gct gtc tgt gca ttt agc 1429 Thr Thr
Gln Leu Asn Ser Ile Pro Gly Ser Ala Val Cys Ala Phe Ser 320 325 330
atg gat gac att gaa aaa gta ttc aaa gga cgg ttt aag gaa cag aaa
1477 Met Asp Asp Ile Glu Lys Val Phe Lys Gly Arg Phe Lys Glu Gln
Lys 335 340 345 act cca gat tct gtt tgg aca gca gtt ccc gaa gac aaa
gtg cca aag 1525 Thr Pro Asp Ser Val Trp Thr Ala Val Pro Glu Asp
Lys Val Pro Lys 350 355 360 cca agg cct ggc tgt tgt gca aaa cac ggc
ctt gcc gaa gct tat aaa 1573 Pro Arg Pro Gly Cys Cys Ala Lys His
Gly Leu Ala Glu Ala Tyr Lys 365 370 375 380 acc tcc atc gat ttc ccg
gat gaa act ctg tca ttc atc aaa tct cat 1621 Thr Ser Ile Asp Phe
Pro Asp Glu Thr Leu Ser Phe Ile Lys Ser His 385 390 395 ccc ctg atg
gac tct gcc gtt cca ccc att gcc gat gag ccc tgg ttc 1669 Pro Leu
Met Asp Ser Ala Val Pro Pro Ile Ala Asp Glu Pro Trp Phe 400 405 410
aca aag act cgg gtc agg tac aga ctg acg gcc atc tca gtg gac cat
1717 Thr Lys Thr Arg Val Arg Tyr Arg Leu Thr Ala Ile Ser Val Asp
His 415 420 425 tca gcc gga ccc tac cag aac tac aca gtc atc ttt gtt
ggc tct gaa 1765 Ser Ala Gly Pro Tyr Gln Asn Tyr Thr Val Ile Phe
Val Gly Ser Glu 430 435 440 gct ggc atg gta ctt aaa gtt ctg gca aag
acc agt cct ttc tct ttg 1813 Ala Gly Met Val Leu Lys Val Leu Ala
Lys Thr Ser Pro Phe Ser Leu 445 450 455 460 aac gac agc gta tta ctg
gaa gag att gaa gcc tac aac cat gca aag 1861 Asn Asp Ser Val Leu
Leu Glu Glu Ile Glu Ala Tyr Asn His Ala Lys 465 470 475 tgc agt gct
gag aat gag gaa gac aaa aag gtc atc tca tta cag ttg 1909 Cys Ser
Ala Glu Asn Glu Glu Asp Lys Lys Val Ile Ser Leu Gln Leu 480 485 490
gat aaa gat cac cac gct tta tat gtg gcg ttc tct agc tgc att atc
1957 Asp Lys Asp His His Ala Leu Tyr Val Ala Phe Ser Ser Cys Ile
Ile 495 500 505 cgc atc ccc ctc agt cgc tgt gag cgt tat gga tca tgt
aaa aag tct 2005 Arg Ile Pro Leu Ser Arg Cys Glu Arg Tyr Gly Ser
Cys Lys Lys Ser 510 515 520 tgt att gca tct cgt gac ccg tat tgt ggc
tgg tta agc cag gga tcc 2053 Cys Ile Ala Ser Arg Asp Pro Tyr Cys
Gly Trp Leu Ser Gln Gly Ser 525 530 535 540 tgt ggt aga gtg acc cca
ggg atg ctg ctg tta acc gaa gac ttc ttt 2101 Cys Gly Arg Val Thr
Pro Gly Met Leu Leu Leu Thr Glu Asp Phe Phe 545 550 555 gct ttc cat
aac cac agt gct gaa gga tat gaa caa gac aca gaa ttc 2149 Ala Phe
His Asn His Ser Ala Glu Gly Tyr Glu Gln Asp Thr Glu Phe 560 565 570
ggc aac aca gct cat cta ggg gac tgc cat gaa att ttg cct act tca
2197 Gly Asn Thr Ala His Leu Gly Asp Cys His Glu Ile Leu Pro Thr
Ser 575 580 585 act aca cca gat tac aaa ata ttt ggc ggt cca aca tct
gac atg gag 2245 Thr Thr Pro Asp Tyr Lys Ile Phe Gly Gly Pro Thr
Ser Asp Met Glu 590 595 600 gta tct tca tct tct gtt acc aca atg gca
agt atc cca gaa atc aca 2293 Val Ser Ser Ser Ser Val Thr Thr Met
Ala Ser Ile Pro Glu Ile Thr 605 610 615 620 cct aaa gtg att gat acc
tgg aga cct aaa ctg aca agc tct cgg aaa 2341 Pro Lys Val Ile Asp
Thr Trp Arg Pro Lys Leu Thr Ser Ser Arg Lys 625 630 635 ttt gta gtt
caa gat gat cca aac act tct gat ttt act gat cct tta 2389 Phe Val
Val Gln Asp Asp Pro Asn Thr Ser Asp Phe Thr Asp Pro Leu 640 645 650
tcg ggt atc cca aag ggt gta cga tgg gaa gtc cag tct gga gag tcc
2437 Ser Gly Ile Pro Lys Gly Val Arg Trp Glu Val Gln Ser Gly Glu
Ser 655 660 665 aac cag atg gtc cac atg aat gtc ctc atc acc tgt gtc
ttt gct gct 2485 Asn Gln Met Val His Met Asn Val Leu Ile Thr Cys
Val Phe Ala Ala 670 675 680 ttt gtc ttg ggg gca ttc att gca ggt gtg
gca gta tac tgc tat cga 2533 Phe Val Leu Gly Ala Phe Ile Ala Gly
Val Ala Val Tyr Cys Tyr Arg 685 690 695 700 gac atg ttt gtt cgg aaa
aac aga aag atc cat aaa gat gca gag tcc 2581 Asp Met Phe Val Arg
Lys Asn Arg Lys Ile His Lys Asp Ala Glu Ser 705 710 715 gcc cag tca
tgc aca gac tcc agt gga agt ttt gcc aaa ctg aat ggt 2629 Ala Gln
Ser Cys Thr Asp Ser Ser Gly Ser Phe Ala Lys Leu Asn Gly 720 725 730
ctc ttt gac agc cct gtc aag gaa tac caa cag aat att gat tct cct
2677 Leu Phe Asp Ser Pro Val Lys Glu Tyr Gln Gln Asn Ile Asp Ser
Pro 735 740 745 aaa ctg tat agt aac ctg cta acc agt cgg aaa gag cta
cca ccc aat 2725 Lys Leu Tyr Ser Asn Leu Leu Thr Ser Arg Lys Glu
Leu Pro Pro Asn 750 755 760 gga gat act aaa tcc atg gta atg gac cat
cga ggg caa cct cca gag 2773 Gly Asp Thr Lys Ser Met Val Met Asp
His Arg Gly Gln Pro Pro Glu 765 770 775 780 ttg gct gct ctt ccc act
cct gag tct aca ccc gtg ctt cac cag aag 2821 Leu Ala Ala Leu Pro
Thr Pro Glu Ser Thr Pro Val Leu His Gln Lys 785 790 795 acc ctg cag
gcc atg aag agc cac tca gaa aag gcc cat ggc cat gga 2869 Thr Leu
Gln Ala Met Lys Ser His Ser Glu Lys Ala His Gly His Gly 800 805 810
gct tca agg aaa gaa acc cct cag ttt ttt ccg tct agt ccg cca cct
2917 Ala Ser Arg Lys Glu Thr Pro Gln Phe Phe Pro Ser Ser Pro Pro
Pro 815 820 825 cat tcc cca tta agt cat ggg cat atc ccc agt gcc att
gtt ctt cca 2965 His Ser Pro Leu Ser His Gly His Ile Pro Ser Ala
Ile Val Leu Pro 830 835 840 aat gct acc cat gac tac aac acg tct ttc
tca aac tcc aat gct cac 3013 Asn Ala Thr His Asp Tyr Asn Thr Ser
Phe Ser Asn Ser Asn Ala His 845 850 855 860 aaa gct gaa aag aag ctt
caa aac att gat cac cct ctc aca aag tca 3061 Lys Ala Glu Lys Lys
Leu Gln Asn Ile Asp His Pro Leu Thr Lys Ser 865 870 875 tcc agt aag
aga gat cac cgg cgt tct gtt gat tcc aga aat acc ctc 3109 Ser Ser
Lys Arg Asp His Arg Arg Ser Val Asp Ser Arg Asn Thr Leu 880 885 890
aat gat ctc ctg aag cat ctg aat gac cca aat agt aac ccc aaa gcc
3157 Asn Asp Leu Leu Lys His Leu Asn Asp Pro Asn Ser Asn Pro Lys
Ala 895 900 905 atc atg gga gac atc cag atg gca cac cag aac tta atg
ctg gat ccc 3205 Ile Met Gly Asp Ile Gln Met Ala His Gln Asn Leu
Met Leu Asp Pro 910 915 920 atg gga tcg atg tct gag gtc cca cct aaa
gtc cct aac cgg gag gca 3253 Met Gly Ser Met Ser Glu Val Pro Pro
Lys Val Pro Asn Arg Glu Ala 925 930 935 940 tcg cta tac tcc cct cct
tca act ctc ccc aga aat agc cca acc aag 3301 Ser Leu Tyr Ser Pro
Pro Ser Thr Leu Pro Arg Asn Ser Pro Thr Lys 945 950 955 cga gtg gat
gtc ccc acc act cct gga gtc cca atg act tct ctg gaa 3349 Arg Val
Asp Val Pro Thr Thr Pro Gly Val Pro Met Thr Ser Leu Glu 960 965 970
aga caa aga ggt tat cac aaa aat tcc tcc cag agg cac tct ata tct
3397 Arg Gln Arg Gly Tyr His Lys Asn Ser Ser Gln Arg His Ser Ile
Ser 975 980 985 gct atg cct aaa aac tta aac tca cca aat ggt gtt ttg
tta tcc aga 3445 Ala Met Pro Lys Asn Leu Asn Ser Pro Asn Gly Val
Leu Leu Ser Arg 990 995 1000 cag cct agt atg aac cgt gga gga tat
atg ccc acc ccc act ggg gcg 3493 Gln Pro Ser Met Asn Arg Gly Gly
Tyr Met Pro Thr Pro Thr Gly Ala 1005 1010 1015 1020 aag gtg gac tat
att cag gga aca cca gtg agt gtt cat ctg cag cct 3541 Lys Val Asp
Tyr Ile Gln Gly Thr Pro Val Ser Val His Leu Gln Pro 1025 1030 1035
tcc ctc tcc aga cag agc agc tac acc agt aat ggc act ctt cct agg
3589 Ser Leu Ser Arg Gln Ser Ser Tyr Thr Ser Asn Gly Thr Leu Pro
Arg 1040 1045 1050 acg gga cta aag agg acg ccg tcc tta aaa cct gac
gtg cca cca aag 3637 Thr Gly Leu Lys Arg Thr Pro Ser Leu Lys Pro
Asp Val Pro Pro Lys 1055 1060 1065 cct tcc ttt gtt cct caa acc cca
tct gtc aga cca ctg aac aaa tac 3685 Pro Ser Phe Val Pro Gln Thr
Pro Ser Val Arg Pro Leu Asn Lys Tyr 1070 1075 1080 aca tac tag 3694
Thr Tyr 1085 4 1086 PRT Homo sapiens 4 Met Arg Val Phe Leu Leu Cys
Ala Tyr Ile Leu Leu Leu Met Val Ser 1 5 10 15 Gln Leu Arg Ala Val
Ser Phe Pro Glu Asp Asp Glu Pro Leu Asn Thr 20 25 30 Val Asp Tyr
His Tyr Ser Arg Gln Tyr Pro Val Phe Arg Gly Arg Pro 35 40 45 Ser
Gly Asn Glu Ser Gln His Arg Leu Asp Phe Gln Leu Met Leu Lys 50 55
60 Ile Arg Asp Thr Leu Tyr Ile Ala Gly Arg Asp Gln Val Tyr Thr Val
65 70 75 80 Asn Leu Asn Glu Met Pro Lys Thr Glu Val Ile Pro Asn Lys
Lys Leu 85 90 95 Thr Trp Arg Ser Arg Gln Gln Asp Arg Glu Asn Cys
Ala Met Lys Gly 100 105 110 Lys His Lys Asp Glu Cys His Asn Phe Ile
Lys Val Phe Val Pro Arg 115 120 125 Asn Asp Glu Met Val Phe Val Cys
Gly Thr Asn Ala Phe Asn Pro Met 130 135 140 Cys Arg Tyr Tyr Arg Leu
Ser Thr Leu Glu Tyr Asp Gly Glu Glu Ile 145 150 155 160 Ser Gly Leu
Ala Arg Cys Pro Phe Asp Ala Arg Gln Thr Asn Val Ala 165 170 175 Leu
Phe Ala Asp Gly Lys Leu Tyr Ser Ala Thr Val Ala Asp Phe Leu 180 185
190 Ala Ser Asp Ala Val Ile Tyr Arg Ser Met Gly Asp Gly Ser Ala Leu
195 200 205 Arg Thr Ile Lys Tyr Asp Ser Lys Trp Ile Lys Glu Pro His
Phe Leu 210 215 220 His Ala Ile Glu Tyr Gly Asn Tyr Val Tyr Phe Phe
Phe Arg Glu Ile 225 230 235 240 Ala Val Glu His Asn Asn Leu Gly Lys
Ala Val Tyr Ser Arg Val Ala 245 250 255 Arg Ile Cys Lys Asn Asp Met
Gly Gly Ser Gln Arg Val Leu Glu Lys 260 265 270 His Trp Thr Ser Phe
Leu Lys Ala Arg Leu Asn Cys Ser Val Pro Gly 275 280 285 Asp Pro Phe
Phe Tyr Phe Asp Val Leu Gln Ser Ile Thr Asp Ile Ile 290 295 300 Gln
Ile Asn Gly Ile Pro Thr Val Val Gly Val Phe Thr Thr Gln Leu 305 310
315 320 Asn Ser Ile Pro Gly Ser Ala Val Cys Ala Phe Ser Met Asp Asp
Ile 325 330 335 Glu Lys Val Phe Lys Gly Arg Phe Lys Glu Gln Lys Thr
Pro Asp Ser 340 345 350 Val Trp Thr Ala Val Pro Glu Asp Lys Val Pro
Lys Pro Arg Pro Gly 355 360 365 Cys Cys Ala Lys His Gly Leu Ala Glu
Ala Tyr Lys Thr Ser Ile Asp 370 375 380 Phe Pro Asp Glu Thr Leu Ser
Phe Ile Lys Ser His Pro Leu Met Asp 385
390 395 400 Ser Ala Val Pro Pro Ile Ala Asp Glu Pro Trp Phe Thr Lys
Thr Arg 405 410 415 Val Arg Tyr Arg Leu Thr Ala Ile Ser Val Asp His
Ser Ala Gly Pro 420 425 430 Tyr Gln Asn Tyr Thr Val Ile Phe Val Gly
Ser Glu Ala Gly Met Val 435 440 445 Leu Lys Val Leu Ala Lys Thr Ser
Pro Phe Ser Leu Asn Asp Ser Val 450 455 460 Leu Leu Glu Glu Ile Glu
Ala Tyr Asn His Ala Lys Cys Ser Ala Glu 465 470 475 480 Asn Glu Glu
Asp Lys Lys Val Ile Ser Leu Gln Leu Asp Lys Asp His 485 490 495 His
Ala Leu Tyr Val Ala Phe Ser Ser Cys Ile Ile Arg Ile Pro Leu 500 505
510 Ser Arg Cys Glu Arg Tyr Gly Ser Cys Lys Lys Ser Cys Ile Ala Ser
515 520 525 Arg Asp Pro Tyr Cys Gly Trp Leu Ser Gln Gly Ser Cys Gly
Arg Val 530 535 540 Thr Pro Gly Met Leu Leu Leu Thr Glu Asp Phe Phe
Ala Phe His Asn 545 550 555 560 His Ser Ala Glu Gly Tyr Glu Gln Asp
Thr Glu Phe Gly Asn Thr Ala 565 570 575 His Leu Gly Asp Cys His Glu
Ile Leu Pro Thr Ser Thr Thr Pro Asp 580 585 590 Tyr Lys Ile Phe Gly
Gly Pro Thr Ser Asp Met Glu Val Ser Ser Ser 595 600 605 Ser Val Thr
Thr Met Ala Ser Ile Pro Glu Ile Thr Pro Lys Val Ile 610 615 620 Asp
Thr Trp Arg Pro Lys Leu Thr Ser Ser Arg Lys Phe Val Val Gln 625 630
635 640 Asp Asp Pro Asn Thr Ser Asp Phe Thr Asp Pro Leu Ser Gly Ile
Pro 645 650 655 Lys Gly Val Arg Trp Glu Val Gln Ser Gly Glu Ser Asn
Gln Met Val 660 665 670 His Met Asn Val Leu Ile Thr Cys Val Phe Ala
Ala Phe Val Leu Gly 675 680 685 Ala Phe Ile Ala Gly Val Ala Val Tyr
Cys Tyr Arg Asp Met Phe Val 690 695 700 Arg Lys Asn Arg Lys Ile His
Lys Asp Ala Glu Ser Ala Gln Ser Cys 705 710 715 720 Thr Asp Ser Ser
Gly Ser Phe Ala Lys Leu Asn Gly Leu Phe Asp Ser 725 730 735 Pro Val
Lys Glu Tyr Gln Gln Asn Ile Asp Ser Pro Lys Leu Tyr Ser 740 745 750
Asn Leu Leu Thr Ser Arg Lys Glu Leu Pro Pro Asn Gly Asp Thr Lys 755
760 765 Ser Met Val Met Asp His Arg Gly Gln Pro Pro Glu Leu Ala Ala
Leu 770 775 780 Pro Thr Pro Glu Ser Thr Pro Val Leu His Gln Lys Thr
Leu Gln Ala 785 790 795 800 Met Lys Ser His Ser Glu Lys Ala His Gly
His Gly Ala Ser Arg Lys 805 810 815 Glu Thr Pro Gln Phe Phe Pro Ser
Ser Pro Pro Pro His Ser Pro Leu 820 825 830 Ser His Gly His Ile Pro
Ser Ala Ile Val Leu Pro Asn Ala Thr His 835 840 845 Asp Tyr Asn Thr
Ser Phe Ser Asn Ser Asn Ala His Lys Ala Glu Lys 850 855 860 Lys Leu
Gln Asn Ile Asp His Pro Leu Thr Lys Ser Ser Ser Lys Arg 865 870 875
880 Asp His Arg Arg Ser Val Asp Ser Arg Asn Thr Leu Asn Asp Leu Leu
885 890 895 Lys His Leu Asn Asp Pro Asn Ser Asn Pro Lys Ala Ile Met
Gly Asp 900 905 910 Ile Gln Met Ala His Gln Asn Leu Met Leu Asp Pro
Met Gly Ser Met 915 920 925 Ser Glu Val Pro Pro Lys Val Pro Asn Arg
Glu Ala Ser Leu Tyr Ser 930 935 940 Pro Pro Ser Thr Leu Pro Arg Asn
Ser Pro Thr Lys Arg Val Asp Val 945 950 955 960 Pro Thr Thr Pro Gly
Val Pro Met Thr Ser Leu Glu Arg Gln Arg Gly 965 970 975 Tyr His Lys
Asn Ser Ser Gln Arg His Ser Ile Ser Ala Met Pro Lys 980 985 990 Asn
Leu Asn Ser Pro Asn Gly Val Leu Leu Ser Arg Gln Pro Ser Met 995
1000 1005 Asn Arg Gly Gly Tyr Met Pro Thr Pro Thr Gly Ala Lys Val
Asp Tyr 1010 1015 1020 Ile Gln Gly Thr Pro Val Ser Val His Leu Gln
Pro Ser Leu Ser Arg 1025 1030 1035 1040 Gln Ser Ser Tyr Thr Ser Asn
Gly Thr Leu Pro Arg Thr Gly Leu Lys 1045 1050 1055 Arg Thr Pro Ser
Leu Lys Pro Asp Val Pro Pro Lys Pro Ser Phe Val 1060 1065 1070 Pro
Gln Thr Pro Ser Val Arg Pro Leu Asn Lys Tyr Thr Tyr 1075 1080 1085
5 3261 DNA Homo sapiens 5 atgagggtct tcctgctttg tgcctacata
ctgctgctga tggtttccca gttgagggca 60 gtcagctttc ctgaagatga
tgaacccctt aatactgtcg actatcacta ttcaaggcaa 120 tatccggttt
ttagaggacg cccttcaggc aatgaatcgc agcacaggct ggactttcag 180
ctgatgttga aaattcgaga cacactttat attgctggca gggatcaagt ttatacagta
240 aacttaaatg aaatgcccaa aacagaagta atacccaaca agaaactgac
atggcgatca 300 agacaacagg atcgagaaaa ctgtgctatg aaaggcaagc
ataaagatga atgccacaac 360 tttatcaaag tatttgttcc aagaaacgat
gagatggttt ttgtttgtgg taccaatgca 420 ttcaatccca tgtgtagata
ctacaggttg agtaccttag aatatgatgg ggaagaaatt 480 agtggcctgg
caagatgccc atttgatgcc agacaaacca atgttgccct ctttgctgat 540
gggaagctgt attctgccac agtggctgac ttcttggcca gcgatgccgt tatttatcga
600 agcatgggtg atggatctgc ccttcgcaca ataaaatatg attccaaatg
gataaaagag 660 ccacactttc ttcatgccat agaatatgga aactatgtct
atttcttctt tcgagaaatc 720 gctgtcgaac ataataattt aggcaaggct
gtgtattccc gcgtggcccg catatgtaaa 780 aacgacatgg gtggttccca
gcgggtcctg gagaaacact ggacttcatt tctaaaggct 840 cggctgaact
gttctgtccc tggagatccg tttttctact ttgatgttct gcagtctatt 900
acagacataa tacaaatcaa tggcatcccc actgtggtcg gggtgtttac cacgcagctc
960 aatagcatcc ctggttctgc tgtctgtgca tttagcatgg atgacattga
aaaagtattc 1020 aaaggacggt ttaaggaaca gaaaactcca gattctgttt
ggacagcagt tcccgaagac 1080 aaagtgccaa agccaaggcc tggctgttgt
gcaaaacacg gccttgccga agcttataaa 1140 acctccatcg atttcccgga
tgaaactctg tcattcatca aatctcatcc cctgatggac 1200 tctgccgttc
cacccattgc cgatgagccc tggttcacaa agactcgggt caggtacaga 1260
ctgacggcca tctcagtgga ccattcagcc ggaccctacc agaactacac agtcatcttt
1320 gttggctctg aagctggcat ggtacttaaa gttctggcaa agaccagtcc
tttctctttg 1380 aacgacagcg tattactgga agagattgaa gcctacaacc
atgcaaagtg cagtgctgag 1440 aatgaggaag acaaaaaggt catctcatta
cagttggata aagatcacca cgctttatat 1500 gtggcgttct ctagctgcat
tatccgcatc cccctcagtc gctgtgagcg ttatggatca 1560 tgtaaaaagt
cttgtattgc atctcgtgac ccgtattgtg gctggttaag ccagggatcc 1620
tgtggtagag tgaccccagg gatgctgctg ttaaccgaag acttctttgc tttccataac
1680 cacagtgctg aaggatatga acaagacaca gaattcggca acacagctca
tctaggggac 1740 tgccatgaaa ttttgcctac ttcaactaca ccagattaca
aaatatttgg cggtccaaca 1800 tctgacatgg aggtatcttc atcttctgtt
accacaatgg caagtatccc agaaatcaca 1860 cctaaagtga ttgatacctg
gagacctaaa ctgacaagct ctcggaaatt tgtagttcaa 1920 gatgatccaa
acacttctga ttttactgat cctttatcgg gtatcccaaa gggtgtacga 1980
tgggaagtcc agtctggaga gtccaaccag atggtccaca tgaatgtcct catcacctgt
2040 gtctttgctg cttttgtctt gggggcattc attgcaggtg tggcagtata
ctgctatcga 2100 gacatgtttg ttcggaaaaa cagaaagatc cataaagatg
cagagtccgc ccagtcatgc 2160 acagactcca gtggaagttt tgccaaactg
aatggtctct ttgacagccc tgtcaaggaa 2220 taccaacaga atattgattc
tcctaaactg tatagtaacc tgctaaccag tcggaaagag 2280 ctaccaccca
atggagatac taaatccatg gtaatggacc atcgagggca acctccagag 2340
ttggctgctc ttcccactcc tgagtctaca cccgtgcttc accagaagac cctgcaggcc
2400 atgaagagcc actcagaaaa ggcccatggc catggagctt caaggaaaga
aacccctcag 2460 ttttttccgt ctagtccgcc acctcattcc ccattaagtc
atgggcatat ccccagtgcc 2520 attgttcttc caaatgctac ccatgactac
aacacgtctt tctcaaactc caatgctcac 2580 aaagctgaaa agaagcttca
aaacattgat caccctctca caaagtcatc cagtaagaga 2640 gatcaccggc
gttctgttga ttccagaaat accctcaatg atctcctgaa gcatctgaat 2700
gacccaaata gtaaccccaa agccatcatg ggagacatcc agatggcaca ccagaactta
2760 atgctggatc ccatgggatc gatgtctgag gtcccaccta aagtccctaa
ccgggaggca 2820 tcgctatact cccctccttc aactctcccc agaaatagcc
caaccaagcg agtggatgtc 2880 cccaccactc ctggagtccc aatgacttct
ctggaaagac aaagaggtta tcacaaaaat 2940 tcctcccaga ggcactctat
atctgctatg cctaaaaact taaactcacc aaatggtgtt 3000 ttgttatcca
gacagcctag tatgaaccgt ggaggatata tgcccacccc cactggggcg 3060
aaggtggact atattcaggg aacaccagtg agtgttcatc tgcagccttc cctctccaga
3120 cagagcagct acaccagtaa tggcactctt cctaggacgg gactaaagag
gacgccgtcc 3180 ttaaaacctg acgtgccacc aaagccttcc tttgttcctc
aaaccccatc tgtcagacca 3240 ctgaacaaat acacatacta g 3261 6 16 PRT
Homo sapiens 6 Met Arg Val Phe Leu Leu Cys Ala Tyr Ile Leu Leu Leu
Met Val Ser 1 5 10 15 7 29 PRT Homo sapiens 7 Met Val His Met Asn
Val Leu Ile Thr Cys Val Phe Ala Ala Phe Val 1 5 10 15 Leu Gly Ala
Phe Ile Ala Gly Val Ala Val Tyr Cys Tyr 20 25 8 1070 PRT Homo
sapiens 8 Gln Leu Arg Ala Val Ser Phe Pro Glu Asp Asp Glu Pro Leu
Asn Thr 1 5 10 15 Val Asp Tyr His Tyr Ser Arg Gln Tyr Pro Val Phe
Arg Gly Arg Pro 20 25 30 Ser Gly Asn Glu Ser Gln His Arg Leu Asp
Phe Gln Leu Met Leu Lys 35 40 45 Ile Arg Asp Thr Leu Tyr Ile Ala
Gly Arg Asp Gln Val Tyr Thr Val 50 55 60 Asn Leu Asn Glu Met Pro
Lys Thr Glu Val Ile Pro Asn Lys Lys Leu 65 70 75 80 Thr Trp Arg Ser
Arg Gln Gln Asp Arg Glu Asn Cys Ala Met Lys Gly 85 90 95 Lys His
Lys Asp Glu Cys His Asn Phe Ile Lys Val Phe Val Pro Arg 100 105 110
Asn Asp Glu Met Val Phe Val Cys Gly Thr Asn Ala Phe Asn Pro Met 115
120 125 Cys Arg Tyr Tyr Arg Leu Ser Thr Leu Glu Tyr Asp Gly Glu Glu
Ile 130 135 140 Ser Gly Leu Ala Arg Cys Pro Phe Asp Ala Arg Gln Thr
Asn Val Ala 145 150 155 160 Leu Phe Ala Asp Gly Lys Leu Tyr Ser Ala
Thr Val Ala Asp Phe Leu 165 170 175 Ala Ser Asp Ala Val Ile Tyr Arg
Ser Met Gly Asp Gly Ser Ala Leu 180 185 190 Arg Thr Ile Lys Tyr Asp
Ser Lys Trp Ile Lys Glu Pro His Phe Leu 195 200 205 His Ala Ile Glu
Tyr Gly Asn Tyr Val Tyr Phe Phe Phe Arg Glu Ile 210 215 220 Ala Val
Glu His Asn Asn Leu Gly Lys Ala Val Tyr Ser Arg Val Ala 225 230 235
240 Arg Ile Cys Lys Asn Asp Met Gly Gly Ser Gln Arg Val Leu Glu Lys
245 250 255 His Trp Thr Ser Phe Leu Lys Ala Arg Leu Asn Cys Ser Val
Pro Gly 260 265 270 Asp Pro Phe Phe Tyr Phe Asp Val Leu Gln Ser Ile
Thr Asp Ile Ile 275 280 285 Gln Ile Asn Gly Ile Pro Thr Val Val Gly
Val Phe Thr Thr Gln Leu 290 295 300 Asn Ser Ile Pro Gly Ser Ala Val
Cys Ala Phe Ser Met Asp Asp Ile 305 310 315 320 Glu Lys Val Phe Lys
Gly Arg Phe Lys Glu Gln Lys Thr Pro Asp Ser 325 330 335 Val Trp Thr
Ala Val Pro Glu Asp Lys Val Pro Lys Pro Arg Pro Gly 340 345 350 Cys
Cys Ala Lys His Gly Leu Ala Glu Ala Tyr Lys Thr Ser Ile Asp 355 360
365 Phe Pro Asp Glu Thr Leu Ser Phe Ile Lys Ser His Pro Leu Met Asp
370 375 380 Ser Ala Val Pro Pro Ile Ala Asp Glu Pro Trp Phe Thr Lys
Thr Arg 385 390 395 400 Val Arg Tyr Arg Leu Thr Ala Ile Ser Val Asp
His Ser Ala Gly Pro 405 410 415 Tyr Gln Asn Tyr Thr Val Ile Phe Val
Gly Ser Glu Ala Gly Met Val 420 425 430 Leu Lys Val Leu Ala Lys Thr
Ser Pro Phe Ser Leu Asn Asp Ser Val 435 440 445 Leu Leu Glu Glu Ile
Glu Ala Tyr Asn His Ala Lys Cys Ser Ala Glu 450 455 460 Asn Glu Glu
Asp Lys Lys Val Ile Ser Leu Gln Leu Asp Lys Asp His 465 470 475 480
His Ala Leu Tyr Val Ala Phe Ser Ser Cys Ile Ile Arg Ile Pro Leu 485
490 495 Ser Arg Cys Glu Arg Tyr Gly Ser Cys Lys Lys Ser Cys Ile Ala
Ser 500 505 510 Arg Asp Pro Tyr Cys Gly Trp Leu Ser Gln Gly Ser Cys
Gly Arg Val 515 520 525 Thr Pro Gly Met Leu Leu Leu Thr Glu Asp Phe
Phe Ala Phe His Asn 530 535 540 His Ser Ala Glu Gly Tyr Glu Gln Asp
Thr Glu Phe Gly Asn Thr Ala 545 550 555 560 His Leu Gly Asp Cys His
Glu Ile Leu Pro Thr Ser Thr Thr Pro Asp 565 570 575 Tyr Lys Ile Phe
Gly Gly Pro Thr Ser Asp Met Glu Val Ser Ser Ser 580 585 590 Ser Val
Thr Thr Met Ala Ser Ile Pro Glu Ile Thr Pro Lys Val Ile 595 600 605
Asp Thr Trp Arg Pro Lys Leu Thr Ser Ser Arg Lys Phe Val Val Gln 610
615 620 Asp Asp Pro Asn Thr Ser Asp Phe Thr Asp Pro Leu Ser Gly Ile
Pro 625 630 635 640 Lys Gly Val Arg Trp Glu Val Gln Ser Gly Glu Ser
Asn Gln Met Val 645 650 655 His Met Asn Val Leu Ile Thr Cys Val Phe
Ala Ala Phe Val Leu Gly 660 665 670 Ala Phe Ile Ala Gly Val Ala Val
Tyr Cys Tyr Arg Asp Met Phe Val 675 680 685 Arg Lys Asn Arg Lys Ile
His Lys Asp Ala Glu Ser Ala Gln Ser Cys 690 695 700 Thr Asp Ser Ser
Gly Ser Phe Ala Lys Leu Asn Gly Leu Phe Asp Ser 705 710 715 720 Pro
Val Lys Glu Tyr Gln Gln Asn Ile Asp Ser Pro Lys Leu Tyr Ser 725 730
735 Asn Leu Leu Thr Ser Arg Lys Glu Leu Pro Pro Asn Gly Asp Thr Lys
740 745 750 Ser Met Val Met Asp His Arg Gly Gln Pro Pro Glu Leu Ala
Ala Leu 755 760 765 Pro Thr Pro Glu Ser Thr Pro Val Leu His Gln Lys
Thr Leu Gln Ala 770 775 780 Met Lys Ser His Ser Glu Lys Ala His Gly
His Gly Ala Ser Arg Lys 785 790 795 800 Glu Thr Pro Gln Phe Phe Pro
Ser Ser Pro Pro Pro His Ser Pro Leu 805 810 815 Ser His Gly His Ile
Pro Ser Ala Ile Val Leu Pro Asn Ala Thr His 820 825 830 Asp Tyr Asn
Thr Ser Phe Ser Asn Ser Asn Ala His Lys Ala Glu Lys 835 840 845 Lys
Leu Gln Asn Ile Asp His Pro Leu Thr Lys Ser Ser Ser Lys Arg 850 855
860 Asp His Arg Arg Ser Val Asp Ser Arg Asn Thr Leu Asn Asp Leu Leu
865 870 875 880 Lys His Leu Asn Asp Pro Asn Ser Asn Pro Lys Ala Ile
Met Gly Asp 885 890 895 Ile Gln Met Ala His Gln Asn Leu Met Leu Asp
Pro Met Gly Ser Met 900 905 910 Ser Glu Val Pro Pro Lys Val Pro Asn
Arg Glu Ala Ser Leu Tyr Ser 915 920 925 Pro Pro Ser Thr Leu Pro Arg
Asn Ser Pro Thr Lys Arg Val Asp Val 930 935 940 Pro Thr Thr Pro Gly
Val Pro Met Thr Ser Leu Glu Arg Gln Arg Gly 945 950 955 960 Tyr His
Lys Asn Ser Ser Gln Arg His Ser Ile Ser Ala Met Pro Lys 965 970 975
Asn Leu Asn Ser Pro Asn Gly Val Leu Leu Ser Arg Gln Pro Ser Met 980
985 990 Asn Arg Gly Gly Tyr Met Pro Thr Pro Thr Gly Ala Lys Val Asp
Tyr 995 1000 1005 Ile Gln Gly Thr Pro Val Ser Val His Leu Gln Pro
Ser Leu Ser Arg 1010 1015 1020 Gln Ser Ser Tyr Thr Ser Asn Gly Thr
Leu Pro Arg Thr Gly Leu Lys 1025 1030 1035 1040 Arg Thr Pro Ser Leu
Lys Pro Asp Val Pro Pro Lys Pro Ser Phe Val 1045 1050 1055 Pro Gln
Thr Pro Ser Val Arg Pro Leu Asn Lys Tyr Thr Tyr 1060 1065 1070 9
429 PRT Homo sapiens 9 Gly Val Arg Trp Glu Val Gln Ser Gly Glu Ser
Asn Gln Met Val His 1 5 10 15 Met Asn Val Leu Ile Thr Cys Val Phe
Ala Ala Phe Val Leu Gly Ala 20 25 30 Phe Ile Ala Gly Val Ala Val
Tyr Cys Tyr Arg Asp Met Phe Val Arg 35 40 45 Lys Asn Arg Lys Ile
His Lys Asp Ala Glu Ser Ala Gln Ser Cys Thr 50 55 60 Asp Ser Ser
Gly Ser Phe Ala Lys Leu Asn Gly Leu Phe Asp Ser Pro 65 70 75 80 Val
Lys Glu Tyr Gln
Gln Asn Ile Asp Ser Pro Lys Leu Tyr Ser Asn 85 90 95 Leu Leu Thr
Ser Arg Lys Glu Leu Pro Pro Asn Gly Asp Thr Lys Ser 100 105 110 Met
Val Met Asp His Arg Gly Gln Pro Pro Glu Leu Ala Ala Leu Pro 115 120
125 Thr Pro Glu Ser Thr Pro Val Leu His Gln Lys Thr Leu Gln Ala Met
130 135 140 Lys Ser His Ser Glu Lys Ala His Gly His Gly Ala Ser Arg
Lys Glu 145 150 155 160 Thr Pro Gln Phe Phe Pro Ser Ser Pro Pro Pro
His Ser Pro Leu Ser 165 170 175 His Gly His Ile Pro Ser Ala Ile Val
Leu Pro Asn Ala Thr His Asp 180 185 190 Tyr Asn Thr Ser Phe Ser Asn
Ser Asn Ala His Lys Ala Glu Lys Lys 195 200 205 Leu Gln Asn Ile Asp
His Pro Leu Thr Lys Ser Ser Ser Lys Arg Asp 210 215 220 His Arg Arg
Ser Val Asp Ser Arg Asn Thr Leu Asn Asp Leu Leu Lys 225 230 235 240
His Leu Asn Asp Pro Asn Ser Asn Pro Lys Ala Ile Met Gly Asp Ile 245
250 255 Gln Met Ala His Gln Asn Leu Met Leu Asp Pro Met Gly Ser Met
Ser 260 265 270 Glu Val Pro Pro Lys Val Pro Asn Arg Glu Ala Ser Leu
Tyr Ser Pro 275 280 285 Pro Ser Thr Leu Pro Arg Asn Ser Pro Thr Lys
Arg Val Asp Val Pro 290 295 300 Thr Thr Pro Gly Val Pro Met Thr Ser
Leu Glu Arg Gln Arg Gly Tyr 305 310 315 320 His Lys Asn Ser Ser Gln
Arg His Ser Ile Ser Ala Met Pro Lys Asn 325 330 335 Leu Asn Ser Pro
Asn Gly Val Leu Leu Ser Arg Gln Pro Ser Met Asn 340 345 350 Arg Gly
Gly Tyr Met Pro Thr Pro Thr Gly Ala Lys Val Asp Tyr Ile 355 360 365
Gln Gly Thr Pro Val Ser Val His Leu Gln Pro Ser Leu Ser Arg Gln 370
375 380 Ser Ser Tyr Thr Ser Asn Gly Thr Leu Pro Arg Thr Gly Leu Lys
Arg 385 390 395 400 Thr Pro Ser Leu Lys Pro Asp Val Pro Pro Lys Pro
Ser Phe Val Pro 405 410 415 Gln Thr Pro Ser Val Arg Pro Leu Asn Lys
Tyr Thr Tyr 420 425 10 536 PRT Homo sapiens 10 Leu Leu Leu Leu Leu
Leu Leu Leu Ser Leu Pro His Thr Gln Ala Ala 1 5 10 15 Phe Pro Gln
Asp Pro Leu Pro Leu Leu Ile Ser Asp Leu Gln Gly Thr 20 25 30 Ser
Pro Leu Ser Trp Phe Arg Gly Leu Glu Asp Asp Ala Val Ala Ala 35 40
45 Glu Leu Gly Leu Asp Phe Gln Arg Phe Leu Thr Leu Asn Arg Thr Leu
50 55 60 Leu Val Ala Ala Arg Asp His Val Phe Ser Phe Asp Leu Gln
Ala Glu 65 70 75 80 Glu Glu Gly Glu Gly Leu Val Pro Asn Lys Tyr Leu
Thr Trp Arg Ser 85 90 95 Gln Asp Val Glu Asn Cys Ala Val Arg Gly
Lys Leu Thr Asp Glu Cys 100 105 110 Tyr Asn Tyr Ile Arg Val Leu Val
Pro Trp Asp Ser Gln Thr Leu Leu 115 120 125 Ala Cys Gly Thr Asn Ser
Phe Ser Pro Val Cys Arg Ser Tyr Gly Ile 130 135 140 Thr Ser Leu Gln
Gln Glu Gly Glu Glu Leu Ser Gly Gln Ala Arg Cys 145 150 155 160 Pro
Phe Asp Ala Thr Gln Ser Asn Val Ala Ile Phe Ala Glu Gly Ser 165 170
175 Leu Tyr Ser Ala Thr Ala Ala Asp Phe Gln Ala Ser Asp Ala Val Val
180 185 190 Tyr Arg Ser Leu Gly Pro Gln Pro Pro Leu Arg Ser Ala Lys
Tyr Asp 195 200 205 Ser Lys Trp Leu Arg Glu Pro His Phe Val Gln Ala
Leu Glu His Gly 210 215 220 Asp His Val Tyr Phe Phe Phe Arg Glu Val
Ser Val Glu Asp Ala Arg 225 230 235 240 Leu Gly Lys Val Gln Phe Ser
Arg Val Ala Arg Val Cys Lys Arg Asp 245 250 255 Met Gly Gly Ser Pro
Arg Ala Leu Asp Arg His Trp Thr Ser Phe Leu 260 265 270 Lys Leu Arg
Leu Asn Cys Ser Val Pro Gly Asp Ser Thr Phe Tyr Phe 275 280 285 Asp
Val Leu Gln Ala Leu Thr Gly Pro Val Asn Leu His Gly Arg Ser 290 295
300 Ala Leu Phe Gly Val Phe Thr Thr Gln Thr Asn Ser Ile Pro Gly Ser
305 310 315 320 Ala Val Cys Ala Phe Tyr Leu Asp Glu Ile Glu Arg Gly
Phe Glu Gly 325 330 335 Lys Phe Lys Glu Gln Arg Ser Leu Asp Gly Ala
Trp Thr Pro Val Ser 340 345 350 Glu Asp Arg Val Pro Ser Pro Arg Pro
Gly Ser Cys Ala Gly Val Gly 355 360 365 Gly Ala Ala Leu Phe Ser Ser
Ser Arg Asp Leu Pro Asp Asp Val Leu 370 375 380 Thr Phe Ile Lys Ala
His Pro Leu Leu Asp Pro Ala Val Pro Pro Val 385 390 395 400 Thr His
Gln Pro Leu Leu Thr Leu Thr Ser Arg Ala Leu Leu Thr Gln 405 410 415
Val Ala Val Asp Gly Met Ala Gly Pro His Ser Asn Ile Thr Val Met 420
425 430 Phe Leu Gly Ser Asn Asp Gly Thr Val Leu Lys Val Leu Thr Pro
Gly 435 440 445 Gly Arg Ser Gly Gly Pro Glu Pro Ile Leu Leu Glu Glu
Ile Asp Ala 450 455 460 Tyr Ser Pro Ala Arg Cys Ser Gly Lys Arg Thr
Ala Gln Thr Ala Arg 465 470 475 480 Arg Ile Ile Gly Leu Glu Leu Asp
Thr Glu Gly His Arg Leu Phe Val 485 490 495 Ala Phe Ser Gly Cys Ile
Val Tyr Leu Pro Leu Ser Arg Cys Ala Arg 500 505 510 His Gly Ala Cys
Gln Arg Ser Cys Leu Ala Ser Gln Asp Pro Tyr Cys 515 520 525 Gly Trp
His Ser Ser Arg Gly Cys 530 535 11 289 PRT Homo sapiens 11 Ile Ala
Gly Val Ala Val Phe Phe Tyr Arg Asp Met Phe Val Arg Lys 1 5 10 15
Asp Arg Lys Ile His Lys Asp Ala Glu Ser Ala Gln Ser Cys Thr Asp 20
25 30 Ser Ser Gly Ser Phe Ala Lys Leu Asn Gly Leu Phe Asp Ser Pro
Val 35 40 45 Lys Glu Tyr Gln Gln Asn Ile Asp Ser Pro Lys Leu Ile
Val Thr Ser 50 55 60 Leu Thr Ser Arg Lys Glu Leu Pro Pro Asn Gly
Asp Thr Lys Ser Met 65 70 75 80 Val Met Asp His Arg Gly Gln Pro Pro
Glu Leu Ala Ala Leu Pro Thr 85 90 95 Pro Glu Ser Thr Pro Val Leu
His Gln Lys Thr Leu Gln Ala Met Lys 100 105 110 Ser His Ser Glu Lys
Ala His Gly His Gly Ala Ser Arg Lys Glu Thr 115 120 125 Pro Gln Phe
Phe Pro Ser Ser Pro Pro Pro His Ser Pro Leu Ser His 130 135 140 Gly
His Ile Pro Ser Ala Ile Val Leu Pro Asn Ala Thr His Asp Tyr 145 150
155 160 Asn Thr Ser Phe Ser Asn Ser Asn Ala His Lys Ala Glu Lys Lys
Leu 165 170 175 Gln Asn Ile Asp His Pro Leu Thr Lys Ser Ser Ser Lys
Arg Asp His 180 185 190 Arg Arg Ser Val Asp Ser Arg Asn Thr Leu Asn
Asp Leu Leu Lys His 195 200 205 Leu Asn Asp Pro Asn Ser Asn Pro Lys
Ala Ile Met Gly Asp Ile Gln 210 215 220 Met Ala His Gln Asn Leu Met
Leu Asp Pro Met Gly Ser Met Ser Glu 225 230 235 240 Val Pro Pro Lys
Val Pro Asn Arg Glu Ala Ser Leu Tyr Ser Pro Pro 245 250 255 Ser Thr
Leu Pro Arg Asn Ser Pro Thr Lys Arg Val Asp Val Pro Thr 260 265 270
Thr Pro Gly Val Pro Met Thr Ser Leu Glu Arg Gln Arg Gly Tyr His 275
280 285 Lys 12 1923 DNA Homo sapiens CDS (1)..(1923) 12 cag ttg agg
gca gtc agc ttt cct gaa gat gat gaa ccc ctt aat act 48 Gln Leu Arg
Ala Val Ser Phe Pro Glu Asp Asp Glu Pro Leu Asn Thr 1 5 10 15 gtc
gac tat cac tat tca agg caa tat ccg gtt ttt aga gga cgc cct 96 Val
Asp Tyr His Tyr Ser Arg Gln Tyr Pro Val Phe Arg Gly Arg Pro 20 25
30 tca ggc aat gaa tcg cag cac agg ctg gac ttt cag ctg atg ttg aaa
144 Ser Gly Asn Glu Ser Gln His Arg Leu Asp Phe Gln Leu Met Leu Lys
35 40 45 att cga gac aca ctt tat att gct ggc agg gat caa gtt tat
aca gta 192 Ile Arg Asp Thr Leu Tyr Ile Ala Gly Arg Asp Gln Val Tyr
Thr Val 50 55 60 aac tta aat gaa atg ccc aaa aca gaa gta ata ccc
aac aag aaa ctg 240 Asn Leu Asn Glu Met Pro Lys Thr Glu Val Ile Pro
Asn Lys Lys Leu 65 70 75 80 aca tgg cga tca aga caa cag gat cga gaa
aac tgt gct atg aaa ggc 288 Thr Trp Arg Ser Arg Gln Gln Asp Arg Glu
Asn Cys Ala Met Lys Gly 85 90 95 aag cat aaa gat gaa tgc cac aac
ttt atc aaa gta ttt gtt cca aga 336 Lys His Lys Asp Glu Cys His Asn
Phe Ile Lys Val Phe Val Pro Arg 100 105 110 aac gat gag atg gtt ttt
gtt tgt ggt acc aat gca ttc aat ccc atg 384 Asn Asp Glu Met Val Phe
Val Cys Gly Thr Asn Ala Phe Asn Pro Met 115 120 125 tgt aga tac tac
agg ttg agt acc tta gaa tat gat ggg gaa gaa att 432 Cys Arg Tyr Tyr
Arg Leu Ser Thr Leu Glu Tyr Asp Gly Glu Glu Ile 130 135 140 agt ggc
ctg gca aga tgc cca ttt gat gcc aga caa acc aat gtt gcc 480 Ser Gly
Leu Ala Arg Cys Pro Phe Asp Ala Arg Gln Thr Asn Val Ala 145 150 155
160 ctc ttt gct gat ggg aag ctg tat tct gcc aca gtg gct gac ttc ttg
528 Leu Phe Ala Asp Gly Lys Leu Tyr Ser Ala Thr Val Ala Asp Phe Leu
165 170 175 gcc agc gat gcc gtt att tat cga agc atg ggt gat gga tct
gcc ctt 576 Ala Ser Asp Ala Val Ile Tyr Arg Ser Met Gly Asp Gly Ser
Ala Leu 180 185 190 cgc aca ata aaa tat gat tcc aaa tgg ata aaa gag
cca cac ttt ctt 624 Arg Thr Ile Lys Tyr Asp Ser Lys Trp Ile Lys Glu
Pro His Phe Leu 195 200 205 cat gcc ata gaa tat gga aac tat gtc tat
ttc ttc ttt cga gaa atc 672 His Ala Ile Glu Tyr Gly Asn Tyr Val Tyr
Phe Phe Phe Arg Glu Ile 210 215 220 gct gtc gaa cat aat aat tta ggc
aag gct gtg tat tcc cgc gtg gcc 720 Ala Val Glu His Asn Asn Leu Gly
Lys Ala Val Tyr Ser Arg Val Ala 225 230 235 240 cgc ata tgt aaa aac
gac atg ggt ggt tcc cag cgg gtc ctg gag aaa 768 Arg Ile Cys Lys Asn
Asp Met Gly Gly Ser Gln Arg Val Leu Glu Lys 245 250 255 cac tgg act
tca ttt cta aag gct cgg ctg aac tgt tct gtc cct gga 816 His Trp Thr
Ser Phe Leu Lys Ala Arg Leu Asn Cys Ser Val Pro Gly 260 265 270 gat
ccg ttt ttc tac ttt gat gtt ctg cag tct att aca gac ata ata 864 Asp
Pro Phe Phe Tyr Phe Asp Val Leu Gln Ser Ile Thr Asp Ile Ile 275 280
285 caa atc aat ggc atc ccc act gtg gtc ggg gtg ttt acc acg cag ctc
912 Gln Ile Asn Gly Ile Pro Thr Val Val Gly Val Phe Thr Thr Gln Leu
290 295 300 aat agc atc cct ggt tct gct gtc tgt gca ttt agc atg gat
gac att 960 Asn Ser Ile Pro Gly Ser Ala Val Cys Ala Phe Ser Met Asp
Asp Ile 305 310 315 320 gaa aaa gta ttc aaa gga cgg ttt aag gaa cag
aaa act cca gat tct 1008 Glu Lys Val Phe Lys Gly Arg Phe Lys Glu
Gln Lys Thr Pro Asp Ser 325 330 335 gtt tgg aca gca gtt ccc gaa gac
aaa gtg cca aag cca agg cct ggc 1056 Val Trp Thr Ala Val Pro Glu
Asp Lys Val Pro Lys Pro Arg Pro Gly 340 345 350 tgt tgt gca aaa cac
ggc ctt gcc gaa gct tat aaa acc tcc atc gat 1104 Cys Cys Ala Lys
His Gly Leu Ala Glu Ala Tyr Lys Thr Ser Ile Asp 355 360 365 ttc ccg
gat gaa act ctg tca ttc atc aaa tct cat ccc ctg atg gac 1152 Phe
Pro Asp Glu Thr Leu Ser Phe Ile Lys Ser His Pro Leu Met Asp 370 375
380 tct gcc gtt cca ccc att gcc gat gag ccc tgg ttc aca aag act cgg
1200 Ser Ala Val Pro Pro Ile Ala Asp Glu Pro Trp Phe Thr Lys Thr
Arg 385 390 395 400 gtc agg tac aga ctg acg gcc atc tca gtg gac cat
tca gcc gga ccc 1248 Val Arg Tyr Arg Leu Thr Ala Ile Ser Val Asp
His Ser Ala Gly Pro 405 410 415 tac cag aac tac aca gtc atc ttt gtt
ggc tct gaa gct ggc atg gta 1296 Tyr Gln Asn Tyr Thr Val Ile Phe
Val Gly Ser Glu Ala Gly Met Val 420 425 430 ctt aaa gtt ctg gca aag
acc agt cct ttc tct ttg aac gac agc gta 1344 Leu Lys Val Leu Ala
Lys Thr Ser Pro Phe Ser Leu Asn Asp Ser Val 435 440 445 tta ctg gaa
gag att gaa gcc tac aac cat gca aag tgc agt gct gag 1392 Leu Leu
Glu Glu Ile Glu Ala Tyr Asn His Ala Lys Cys Ser Ala Glu 450 455 460
aat gag gaa gac aaa aag gtc atc tca tta cag ttg gat aaa gat cac
1440 Asn Glu Glu Asp Lys Lys Val Ile Ser Leu Gln Leu Asp Lys Asp
His 465 470 475 480 cac gct tta tat gtg gcg ttc tct agc tgc att atc
cgc atc ccc ctc 1488 His Ala Leu Tyr Val Ala Phe Ser Ser Cys Ile
Ile Arg Ile Pro Leu 485 490 495 agt cgc tgt gag cgt tat gga tca tgt
aaa aag tct tgt att gca tct 1536 Ser Arg Cys Glu Arg Tyr Gly Ser
Cys Lys Lys Ser Cys Ile Ala Ser 500 505 510 cgt gac ccg tat tgt ggc
tgg tta agc cag gga tcc tgt ggt aga gtg 1584 Arg Asp Pro Tyr Cys
Gly Trp Leu Ser Gln Gly Ser Cys Gly Arg Val 515 520 525 acc cca ggg
atg ctg ctg tta acc gaa gac ttc ttt gct ttc cat aac 1632 Thr Pro
Gly Met Leu Leu Leu Thr Glu Asp Phe Phe Ala Phe His Asn 530 535 540
cac agt gct gaa gga tat gaa caa gac aca gaa ttc ggc aac aca gct
1680 His Ser Ala Glu Gly Tyr Glu Gln Asp Thr Glu Phe Gly Asn Thr
Ala 545 550 555 560 cat cta ggg gac tgc cat gaa att ttg cct act tca
act aca cca gat 1728 His Leu Gly Asp Cys His Glu Ile Leu Pro Thr
Ser Thr Thr Pro Asp 565 570 575 tac aaa ata ttt ggc ggt cca aca tct
gac atg gag gta tct tca tct 1776 Tyr Lys Ile Phe Gly Gly Pro Thr
Ser Asp Met Glu Val Ser Ser Ser 580 585 590 tct gtt acc aca atg gca
agt atc cca gaa atc aca cct aaa gtg att 1824 Ser Val Thr Thr Met
Ala Ser Ile Pro Glu Ile Thr Pro Lys Val Ile 595 600 605 gat acc tgg
aga cct aaa ctg aca agc tct cgg aaa ttt gta gtt caa 1872 Asp Thr
Trp Arg Pro Lys Leu Thr Ser Ser Arg Lys Phe Val Val Gln 610 615 620
gat gat cca aac act tct gat ttt act gat cct tta tcg ggt atc cca
1920 Asp Asp Pro Asn Thr Ser Asp Phe Thr Asp Pro Leu Ser Gly Ile
Pro 625 630 635 640 aag 1923 Lys 13 641 PRT Homo sapiens 13 Gln Leu
Arg Ala Val Ser Phe Pro Glu Asp Asp Glu Pro Leu Asn Thr 1 5 10 15
Val Asp Tyr His Tyr Ser Arg Gln Tyr Pro Val Phe Arg Gly Arg Pro 20
25 30 Ser Gly Asn Glu Ser Gln His Arg Leu Asp Phe Gln Leu Met Leu
Lys 35 40 45 Ile Arg Asp Thr Leu Tyr Ile Ala Gly Arg Asp Gln Val
Tyr Thr Val 50 55 60 Asn Leu Asn Glu Met Pro Lys Thr Glu Val Ile
Pro Asn Lys Lys Leu 65 70 75 80 Thr Trp Arg Ser Arg Gln Gln Asp Arg
Glu Asn Cys Ala Met Lys Gly 85 90 95 Lys His Lys Asp Glu Cys His
Asn Phe Ile Lys Val Phe Val Pro Arg 100 105 110 Asn Asp Glu Met Val
Phe Val Cys Gly Thr Asn Ala Phe Asn Pro Met 115 120 125 Cys Arg Tyr
Tyr Arg Leu Ser Thr Leu Glu Tyr Asp Gly Glu Glu Ile 130 135 140 Ser
Gly Leu Ala Arg Cys Pro Phe Asp Ala Arg Gln Thr Asn Val Ala 145 150
155 160 Leu Phe Ala Asp Gly Lys Leu Tyr Ser Ala Thr Val Ala Asp Phe
Leu 165 170 175 Ala Ser Asp Ala Val Ile Tyr Arg Ser Met Gly Asp Gly
Ser Ala Leu 180 185 190 Arg Thr Ile Lys Tyr Asp Ser Lys Trp Ile Lys
Glu Pro His Phe Leu 195 200 205 His Ala Ile Glu Tyr Gly Asn Tyr Val
Tyr Phe Phe Phe Arg Glu Ile 210 215
220 Ala Val Glu His Asn Asn Leu Gly Lys Ala Val Tyr Ser Arg Val Ala
225 230 235 240 Arg Ile Cys Lys Asn Asp Met Gly Gly Ser Gln Arg Val
Leu Glu Lys 245 250 255 His Trp Thr Ser Phe Leu Lys Ala Arg Leu Asn
Cys Ser Val Pro Gly 260 265 270 Asp Pro Phe Phe Tyr Phe Asp Val Leu
Gln Ser Ile Thr Asp Ile Ile 275 280 285 Gln Ile Asn Gly Ile Pro Thr
Val Val Gly Val Phe Thr Thr Gln Leu 290 295 300 Asn Ser Ile Pro Gly
Ser Ala Val Cys Ala Phe Ser Met Asp Asp Ile 305 310 315 320 Glu Lys
Val Phe Lys Gly Arg Phe Lys Glu Gln Lys Thr Pro Asp Ser 325 330 335
Val Trp Thr Ala Val Pro Glu Asp Lys Val Pro Lys Pro Arg Pro Gly 340
345 350 Cys Cys Ala Lys His Gly Leu Ala Glu Ala Tyr Lys Thr Ser Ile
Asp 355 360 365 Phe Pro Asp Glu Thr Leu Ser Phe Ile Lys Ser His Pro
Leu Met Asp 370 375 380 Ser Ala Val Pro Pro Ile Ala Asp Glu Pro Trp
Phe Thr Lys Thr Arg 385 390 395 400 Val Arg Tyr Arg Leu Thr Ala Ile
Ser Val Asp His Ser Ala Gly Pro 405 410 415 Tyr Gln Asn Tyr Thr Val
Ile Phe Val Gly Ser Glu Ala Gly Met Val 420 425 430 Leu Lys Val Leu
Ala Lys Thr Ser Pro Phe Ser Leu Asn Asp Ser Val 435 440 445 Leu Leu
Glu Glu Ile Glu Ala Tyr Asn His Ala Lys Cys Ser Ala Glu 450 455 460
Asn Glu Glu Asp Lys Lys Val Ile Ser Leu Gln Leu Asp Lys Asp His 465
470 475 480 His Ala Leu Tyr Val Ala Phe Ser Ser Cys Ile Ile Arg Ile
Pro Leu 485 490 495 Ser Arg Cys Glu Arg Tyr Gly Ser Cys Lys Lys Ser
Cys Ile Ala Ser 500 505 510 Arg Asp Pro Tyr Cys Gly Trp Leu Ser Gln
Gly Ser Cys Gly Arg Val 515 520 525 Thr Pro Gly Met Leu Leu Leu Thr
Glu Asp Phe Phe Ala Phe His Asn 530 535 540 His Ser Ala Glu Gly Tyr
Glu Gln Asp Thr Glu Phe Gly Asn Thr Ala 545 550 555 560 His Leu Gly
Asp Cys His Glu Ile Leu Pro Thr Ser Thr Thr Pro Asp 565 570 575 Tyr
Lys Ile Phe Gly Gly Pro Thr Ser Asp Met Glu Val Ser Ser Ser 580 585
590 Ser Val Thr Thr Met Ala Ser Ile Pro Glu Ile Thr Pro Lys Val Ile
595 600 605 Asp Thr Trp Arg Pro Lys Leu Thr Ser Ser Arg Lys Phe Val
Val Gln 610 615 620 Asp Asp Pro Asn Thr Ser Asp Phe Thr Asp Pro Leu
Ser Gly Ile Pro 625 630 635 640 Lys
* * * * *
References