U.S. patent application number 11/013897 was filed with the patent office on 2005-07-21 for 68999, a human ubiquitin carboxyl-terminal hydrolase family member and uses therefor.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Kapeller-Libermann, Rosana.
Application Number | 20050158759 11/013897 |
Document ID | / |
Family ID | 26805052 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158759 |
Kind Code |
A1 |
Kapeller-Libermann, Rosana |
July 21, 2005 |
68999, a human ubiquitin carboxyl-terminal hydrolase family member
and uses therefor
Abstract
The invention provides isolated nucleic acids molecules,
designated 68999 nucleic acid molecules, which encode novel
ubiquitin carboxyl-terminal hydrolase family members. The invention
also provides antisense nucleic acid molecules, recombinant
expression vectors containing 68999 nucleic acid molecules, host
cells into which the expression vectors have been introduced, and
nonhuman transgenic animals in which a 68999 gene has been
introduced or disrupted. The invention still further provides
isolated 68999 proteins, fusion proteins, antigenic peptides and
anti-68999 antibodies. Diagnostic and therapeutic methods utilizing
compositions of the invention are also provided.
Inventors: |
Kapeller-Libermann, Rosana;
(Chestnut Hill, MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
40 Landsdowne Street
CAMBRIDGE
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
26805052 |
Appl. No.: |
11/013897 |
Filed: |
December 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11013897 |
Dec 16, 2004 |
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10107695 |
Mar 27, 2002 |
|
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6900044 |
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60279184 |
Mar 27, 2001 |
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Current U.S.
Class: |
435/6.14 ;
435/226; 435/320.1; 435/325; 435/69.1; 530/388.26; 536/23.2 |
Current CPC
Class: |
C12N 9/18 20130101; C12Y
301/02015 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/226; 435/320.1; 435/325; 530/388.26; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/10; C12N 009/64 |
Claims
1-29. (canceled)
30. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
b) a. polypeptide consisting of the amino acid sequence of SEQ ID
NO:2; and c) a polypeptide which is encoded by a nucleic acid
molecule comprising the nucleotide sequence of SEQ ID NO:1 or SEQ
ID NO:3
31. An isolated polypeptide selected from the group consisting of:
a) a polypeptide which is encoded by a nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3; and b) a
polypeptide comprising an amino acid sequence which is at least 95%
identical to the amino acid sequence of SEQ ID NO:2; wherein the
polypeptide has hydrolase activity.
32. The polypeptide of claim 30, further comprising a heterologous
amino acid sequence.
33. The polypeptide of claim 31, further comprising a heterologous
amino acid sequence.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/107,695, filed Mar. 27, 2002, which claims the benefit
of U.S. Provisional Application No. 60/279,184, filed Mar. 27,
2001, the contents of which are incorporated herein by this
reference.
BACKGROUND OF THE INVENTION
[0002] Living cells are capable of modulating the levels of
proteins that they express. A variety of different mechanisms exist
through which protein levels can be modulated. The ubiquitin
pathway is one example of a post-translational mechanism used to
regulate protein levels. Ubiquitin is a highly conserved
polypeptide expressed in all eukaryotic cells that marks proteins
for degradation. Ubiquitin is attached as a single molecule or as a
conjugated form to lysine residue(s) of proteins via formation of
an isopeptide bond at the C-terminal glycine residue. Most
ubiquitinated proteins are subsequently targeted to the 26S
proteasome, a multicatalytic protease, which cleaves the marked
protein into peptide fragments.
[0003] Only the protein conjugated to ubiquitin is degraded via the
proteasome; ubiquitin itself is recycled by ubiquitin
carboxy-terminal hydrolases (UCH; sometimes abbreviated UCTH),
which cleave the bond between ubiquitin and the protein targeted
for degradation. These enzymes constitute a family of thio
proteases, and homologues have been found in, for example, yeast
(Miller et al., (1989) BioTechnology 7: 698-704; Tobias and
Varshavsky, (1991) J. Biol. Chem. 266: 12021-12028; Baker et al.,
(1992) J. Biol. Chem. 267: 23364-23375), bovine (Papa and
Hochstrasser, (1993) Nature 366: 313-319), avian (Woo et al.,
(1995) J. Biol. Chem. 270: 18766-18773), Drosophila (Zhang et al.,
(1993) Dev. Biol. 17: 214) and human (Wilkinson et al., (1989)
Science 246: 670) cells.
[0004] Ubiquitination has been implicated in regulating numerous
cellular processes including, for example, proliferation,
differentiation, apoptosis (programmed cell death), transcription,
signal-transduction, cell-cycle progression, receptor-mediated
endocytosis, organelle biogenesis and others. The presence of
abnormal amounts of ubiquitinated proteins in neuropathological
conditions such as Alzheimer's and Pick's disease indicates that
ubiquitination plays a role in various physiological disorders.
Oncogenes (e.g., v-jun and v-fos) are often found to be resistant
to ubiquitination in comparison to their normal cell counterparts,
suggesting that a failure to degrade oncogene protein products
accounts for some of their cell transformation capability. Combined
with the observation that not all ubiquitinated proteins are
degraded by the proteosome, these findings indicate that the
process of ubiquitination and de-ubiquitination of particular
substrates have important functional roles apart from recycling
ubiquitin.
SUMMARY OF THE INVENTION
[0005] The present invention is based, in part, on the discovery of
a novel ubiquitin carboxyl-terminal hydrolase protein, referred to
herein as "68999". The nucleotide sequence of a cDNA encoding 68999
is shown in SEQ ID NO:1, and the amino acid sequence of a 68999
polypeptide is shown in SEQ ID NO:2. In addition, the nucleotide
sequence of the coding region is depicted in SEQ ID NO:3.
[0006] Accordingly, in one aspect, the invention features a nucleic
acid molecule which encodes a 68999 protein or polypeptide, e.g., a
biologically active portion of the 68999 protein. In a preferred
embodiment, the isolated nucleic acid molecule encodes a
polypeptide having the amino acid sequence of SEQ ID NO:2. In other
embodiments, the invention provides isolated 68999 nucleic acid
molecules having the nucleotide sequence shown in SEQ ID NO: 1, SEQ
ID NO:3 or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC Accession Number ______. In still other
embodiments, the invention provides nucleic acid molecules that are
sufficiently or substantially identical (e.g., naturally occurring
allelic variants) to the nucleotide sequence shown in SEQ ID NO: 1,
SEQ ID NO:3 or the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC Accession Number ______. In other
embodiments, the invention provides a nucleic acid molecule which
hybridizes under stringent hybridization conditions to a nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO:1,
SEQ ID NO:3 or the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC Accession Number ______, wherein the
nucleic acid encodes a full length 68999 protein or an active
fragment thereof.
[0007] In a related aspect, the invention further provides nucleic
acid constructs which include a 68999 nucleic acid molecule
described herein. In certain embodiments, the nucleic acid
molecules of the invention are operatively linked to native or
heterologous regulatory sequences. Also included are vectors and
host cells containing the 68999 nucleic acid molecules of the
invention e.g., vectors and host cells suitable for producing
polypeptides.
[0008] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection of 68999-encoding nucleic acids.
[0009] In still another related aspect, isolated nucleic acid
molecules that are antisense to a 68999 encoding nucleic acid
molecule are provided.
[0010] In another aspect, the invention features 68999
polypeptides, and biologically active or antigenic fragments
thereof that are useful, e.g., as reagents or targets in assays
applicable to treatment and diagnosis of ubiquitin
carboxyl-terminal hydrolase-associated or other 68999-associated
disorders. In another embodiment, the invention provides 68999
polypeptides having a 68999 activity. Preferred polypeptides are
68999 proteins including at least one ubiquitin carboxyl-terminal
hydrolase-1 domain, and at least one ubiquitin carboxyl-terminal
hydrolase-2 domain, and, preferably, having a 68999 activity, e.g.,
a 68999 activity as described herein.
[0011] In other embodiments, the invention provides 68999
polypeptides, e.g., a 68999 polypeptide having the amino acid
sequence shown in SEQ ID NO:2 or the amino acid sequence encoded by
the cDNA insert of the plasmid deposited with ATCC Accession Number
______; an amino acid sequence that is sufficiently or
substantially identical to the amino acid sequence shown in SEQ ID
NO:2 or the amino acid sequence encoded by the cDNA insert of the
plasmid deposited with ATCC Accession Number ______; or an amino
acid sequence encoded by a nucleic acid molecule having a
nucleotide sequence which hybridizes under stringent hybridization
conditions to a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO: 1 or SEQ ID NO:3 or the nucleotide sequence
of the insert of the plasmid deposited with ATCC Accession Number
______, wherein the nucleic acid encodes a full length 68999
protein or an active fragment thereof.
[0012] In a related aspect, the invention further provides nucleic
acid constructs which include a 68999 nucleic acid molecule
described herein.
[0013] In a related aspect, the invention provides 68999
polypeptides or fragments operatively linked to non-68999
polypeptides to form fusion proteins.
[0014] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically or selectively bind 68999 polypeptides.
[0015] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the 68999 polypeptides or nucleic acids.
[0016] In still another aspect, the invention provides a process
for modulating 68999 polypeptide or nucleic acid expression or
activity, e.g., using the compounds identified in the screens
described herein. In certain embodiments, the methods involve
treatment of conditions related to aberrant activity or expression
of the 68999 polypeptides or nucleic acids, such as conditions
involving aberrant or deficient ubiquitin carboxyl-terminal
hydrolase function. Examples of such disorders, e.g., ubiquitin
carboxyl-terminal hydrolase-associated or other 68999-associated
disorders, include but are not limited to, disorders associated
with aberrant or deficient activity of ubiquitin, or other stress
proteins that target the degradation of abnormal proteins
characteristic of human neurodegenerative disease, neurological
disorders, cellular proliferative and/or differentiative disorders,
disorders associated with bone metabolism, immune e.g.,
inflammatory, disorders, cardiovascular disorders, including
endothelial cell disorders, liver disorders, viral diseases, pain
or metabolic disorders.
[0017] The invention also provides assays for determining the
activity of or the presence or absence of 68999 polypeptides or
nucleic acid molecules in a biological sample, including for
disease diagnosis.
[0018] In a further aspect, the invention provides assays for
determining the presence or absence of a genetic alteration in a
68999 polypeptide or nucleic acid molecule, including for disease
diagnosis.
[0019] In another aspect, the invention features a two dimensional
array having a plurality of addresses, each address of the
plurality being positionally distinguishable from each other
address of the plurality, and each address of the plurality having
a unique capture probe, e.g., a nucleic acid or peptide sequence.
At least one address of the plurality has a capture probe that
recognizes a 68999 molecule. In one embodiment, the capture probe
is a nucleic acid, e.g., a probe complementary to a 68999 nucleic
acid sequence. In another embodiment, the capture probe is a
polypeptide, e.g., an antibody specific for 68999 polypeptides.
Also featured is a method of analyzing a sample by contacting the
sample to the aforementioned array and detecting binding of the
sample to the array.
[0020] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 depicts a hydropathy plot of human 68999. Relatively
hydrophobic residues are shown above the dashed horizontal line,
and relatively hydrophilic residues are below the dashed horizontal
line. The cysteinee residues (cys) are indicated by short vertical
lines just below the hydropathy trace. The numbers corresponding to
the amino acid sequence of human 68999 are indicated. Polypeptides
of the invention include fragments which include: all or part of a
hydrophobic sequence, e.g., a sequence above the dashed line, e.g.,
the sequence from about amino acid 272 to 280, from about 315 to
325, and from about 360 to 370 of SEQ ID NO:2; all or part of a
hydrophilic sequence, e.g., a sequence below the dashed line, e.g.,
the sequence from about amino acid 110 to 120, from about 180 to
190, and from about 420 to 430 of SEQ ID NO:2; a sequence which
includes a Cys, or a glycosylation site.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The human 68999 sequence, which is approximately 1763
nucleotides long including untranslated regions, contains a
predicted methionine-initiated coding sequence of about 1590
nucleotides, including the termination codon. The coding sequence
encodes a 530 amino acid protein (SEQ ID NO:2).
[0023] Human 68999 contains the following regions or other
structural features (for general information regarding PFAM
identifiers, PS prefix and PF prefix domain identification numbers,
refer to Sonnhammer et al. (1997) Protein 28: 405-420 and
http://www.psc.edu/general/software/packag- es/pfam/pfam.html):
[0024] a ubiquitin carboxyl-terminal hydrolase-1 domain (PFAM
Accession Number PF00442) located at about amino acid residues 80
to 111 of SEQ ID NO:2;
[0025] a ubiquitin carboxyl-terminal hydrolase-2 domain (PFAM
Accession Number PF00443) located at about amino acid residues 313
to 374 of SEQ ID NO:2;
[0026] three predicted N-glycosylation sites (Prosite Pattern
Number PS00001) at about amino acid residues 92-95, 444-447, and
488-491 of SEQ ID NO:2;
[0027] two cAMP and cGMP-dependent protein kinase phosphorylation
sites (Prosite Pattern Number PS00004) at about amino acid residues
281 to 284, and 401 to 404 of SEQ ID NO:2;
[0028] five protein kinase C phosphorylation sites (Prosite Pattern
Number PS00005) at about amino acids 22 to 24, 71 to 73, 272 to
274, 399 to 401, and 512 to 514 of SEQ ID NO:2;
[0029] seven casein kinase II phosphorylation sites (Prosite
Pattern Number PS00006) located at about amino acids 23 to 26, 36
to 39, 376 to 379, 404 to 407, 432 to 435, 446 to 449, and 495 to
498, of SEQ ID NO:2;
[0030] one tyrosine kinase phosphorylation site (Prosite Pattern
Number PS00007) located at about amino acid residues 340 to 347 of
SEQ ID NO:2;
[0031] five N-myristoylation sites (Prosite Pattern Number PS00008)
from about amino acids 122 to 127, 212 to 217, 288 to 293, 395 to
400, and 503 to 508, of SEQ ID NO:2;
[0032] one `helix-loop-helix` dimerization domain site signature
(Prosite Pattern Number PS00038) from about amino acids 264 to 279
of SEQ ID NO:2;
[0033] one cytochrome c family heme-binding site signature (Prosite
Pattern Number PS00190) from about amino acids 207 to 212 of SEQ ID
NO:2;
[0034] one ubiquitin carboxyl-terminal hydrolases family 2
signature 1 domain (Prosite Pattern Number PS00972) from about
amino acids 81 to 96 of SEQ ID NO:2;
[0035] one ubiquitin carboxyl-terminal hydrolases family 2
signature 2 domain (Prosite Pattern Number PS00973) from about
amino acids 317 to 335 of SEQ ID NO:2;
[0036] A plasmid containing the nucleotide sequence encoding human
68999, named Fbh68999F1, was deposited with American Type Culture
Collection (ATCC), 10801 University Boulevard, Manassas, Va.
20110-2209, on ______ and assigned Accession Number ______. This
deposit will be maintained under the terms of the Budapest Treaty
on the International Recognition of the Deposit of Microorganisms
for the Purposes of Patent Procedure. This deposit was made merely
as a convenience for those of skill in the art and is not an
admission that a deposit is required under 35 U.S.C. .sctn.
112.
[0037] The 68999 protein contains a significant number of
structural characteristics in common with members of the ubiquitin
carboxyl-terminal hydrolase family.
[0038] In one embodiment of the invention, a 68999 polypeptide
includes at least one ubiquitin carboxyl terminal hydrolase-1
domain.
[0039] In another embodiment of the invention, a 68999 polypeptide
includes at least one ubiquitin carboxyl terminal hydrolase-2
domain.
[0040] The term "family" when referring to the protein and nucleic
acid molecules of the invention means two or more proteins or
nucleic acid molecules having a common structural domain or motif
and having sufficient amino acid or nucleotide sequence homology as
defined herein. Such family members can be naturally or
non-naturally occurring and can be from either the same or
different species. For example, a family can contain a first
protein of human origin as well as other distinct proteins of human
origin, or alternatively, can contain homologs of non-human origin,
e.g., rat or mouse proteins. Members of a family also can have
common functional characteristics.
[0041] As used herein, the term "ubiquitin carboxyl-terminal
hydrolase" includes a protein or polypeptide which is capable of
participating in the removal of one or more ubiquitin molecules
from a protein that has one or more molecules of ubiquitin attached
to it. The definition also includes cleavage of conjugated forms of
ubiquitin (e.g., in a head to tail orientation linked via a peptide
bond) whether or not the ubiquitin conjugate is attached to a
protein. For example, a ubiquitin-ubiquitin conjugate (dimer) can
be cleaved into monomers, a tri-ubiquitin conjugate could be
cleaved into three monomers, or a dimer and a single monomer. In
either of these particular examples, the monomer or dimer can
remain attached to or be cleaved from the ubiquitinated protein.
Ubiquitin carboxyl-terminal hydrolase family of proteins share two
regions of similarity. The first region (described herein as
ubiquitin carboxyl-terminal hydrolase-1) contains a conserved
cysteine which is implicated in the catalytic mechanism. The second
region (described herein as ubiquitin carboxyl-terminal
hydrolase-2) contains two conserved histidines residues, one of
which is involved in the catalytic mechanism.
[0042] A 68999 polypeptide can include a "ubiquitin
carboxyl-terminal hydrolase-1 domain" or regions homologous with a
"ubiquitin carboxyl-terminal hydrolase domain-1". A 68999
polypeptide can further include a "ubiquitin carboxyl-terminal
hydrolase domain-2" or regions homologous with a "ubiquitin
carboxyl-terminal hydrolase domain-2.
[0043] As used herein, the term "ubiquitin carboxyl-terminal
hydrolase domain-1" includes an amino acid sequence of about 30 to
35 amino acid residues in length and having a bit score for the
alignment of the sequence to the ubiquitin carboxyl-terminal
hydrolase domain (HMM) of at least 54.6. Preferably, a ubiquitin
carboxyl-terminal hydrolase-1 domain mediates the catalytic
mechanism of the protein via a conserved cysteine. Preferably, a
ubiquitin carboxyl-terminal hydrolase domain includes at least
about 10 to 60 amino acids, more preferably about 20 to 50 amino
acid residues, or about 30 to 35 amino acids and has a bit score
for the alignment of the sequence to the ubiquitin
carboxyl-terminal hydrolase domain (HMM) (PFAM Accession Number
PF00442 (http://genome.wustl.edu/Pfam- /.html)) of at least 60, 70,
80, or greater.
[0044] The ubiquitin carboxyl-terminal hydrolase-1 domain (amino
acids 80 to 111 of SEQ ID NO:2) of human 68999 shares homology with
a consensus amino acid sequence (SEQ ID NO:4) derived from a hidden
Markov model (PFAM Accession Number PF00442
(http://genome.wustl.edu/Pfam/.html)).
[0045] Ubiquitin carboxyl-terminal hydrolase-1 domains typically
contain a conserved ubiquitin carboxyl terminal hydrolase-1 (UCH-1)
signature pattern which participates in the catalytic mechanism.
The conserved UCH-1 signature pattern is as follows:
G-[LIVMFY]-x(1,3)-[AGC]-[NASM]-x-C-
-[FYW]-[LIVMFC]-[NST]-[SACV]-x-[LIVMS]-Q (SEQ ID NO:6).
[0046] In the above conserved motif, and other motifs described
herein, the standard IUPAC one-letter code for the amino acids is
used. Each element in the pattern is separated by a dash (-);
square brackets ([ ]) indicate the particular residues that are
accepted at that position; x indicates that any residue is accepted
at that position; and numbers in parentheses (( )) indicate the
number of residues represented by the accompanying amino acid.
[0047] A 68999 protein contains a conserved UCH-1 pattern at about
amino acid residues 81 to 96 of SEQ ID NO:2.
[0048] In a preferred embodiment, a 68999 polypeptide or protein
has a "ubiquitin carboxyl-terminal hydrolase-1 domain" or a region
which includes at least about 10 to 60 more preferably about 20 to
40 or 30 to 35 amino acid residues and has at least about 60%, 70%
80% 90% 95%, 99%, or 100% homology with a "ubiquitin
carboxyl-terminal hydrolase-1 domain," e.g., the ubiquitin
carboxyl-terminal hydrolase-1 domain of human 68999 (e.g., residues
80 to 111 of SEQ ID NO:2).
[0049] To identify the presence of a "ubiquitin carboxyl-terminal
hydrolase-1" domain in a 68999 protein sequence, and make the
determination that a polypeptide or protein of interest has a
particular profile, the amino acid sequence of the protein can be
searched against the Pfam database of HMMs (e.g., the Pfam
database, release 2.1) using the default parameters
(http://www.sanger.ac.uk/Software/Pfam/HMM_search)- . For example,
the hmmsf program, which is available as part of the HMMER package
of search programs, is a family specific default program for
MILPAT0063 and a score of 15 is the default threshold score for
determining a hit. Alternatively, the threshold score for
determining a hit can be lowered (e.g., to 8 bits). A description
of the Pfam database can be found in Sonhammer et al. (1997)
Proteins 28: 405-420 and a detailed description of HMMs can be
found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:
146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:
4355-4358; Krogh et al. (1994) J. Mol. Biol. 235: 1501-1531; and
Stultz et al. (1993) Protein Sci. 2: 305-314, the contents of which
are incorporated herein by reference. A search was performed
against the HMM database resulting in the identification of a
"ubiquitin carboxyl-terminal hydrolase-1" domain in the amino acid
sequence of human 68999 at about residues 80 to 111 of SEQ ID
NO:2.
[0050] As used herein, the term "ubiquitin carboxyl-terminal
hydrolase-2 domain" includes an amino acid sequence of about 58 to
63 amino acid residues in length and having a bit score for the
alignment of the sequence to the ubiquitin carboxyl-terminal
hydrolase domain (HMM) of at least 80. Preferably a ubiquitin
carboxyl-terminal hydrolase-2 domain contains two conserved
histidine residues, one of which mediates the catalytic mechanism
of the ubiquitin carboxyl-terminal hydrolase. Preferably, a
ubiquitin carboxyl-terminal hydrolase-2 domain includes at least
about 20 to 100 amino acids, more preferably about 40 to 80 amino
acid residues, or about 55 to 65 amino acids and has a bit score
for the alignment of the sequence to the ubiquitin
carboxyl-terminal hydrolase-2 domain (HMM) of at least 90, 100 or
greater.
[0051] The ubiquitin carboxyl-terminal hydrolase-2 domain (HMM) has
been assigned the PFAM Accession Number PF000443
(http;//genome.wustl.edu/Pfam- /.html). The ubiquitin
carboxyl-terminal hydrolase-2 domain (amino acids 313 to 374 of SEQ
ID NO:2) of human 68999 shares homology with a consensus amino acid
sequence (SEQ ID NO:5) derived from a hidden Markov model.
[0052] In a preferred embodiment, a 68999 polypeptide or protein
has a "ubiquitin carboxyl-terminal hydrolase domain-2" or a region
which includes at least about 20 to 100 more preferably about 40 to
80 or 55 to 65 amino acid residues and has at least about 60%, 70%
80% 90% 95%, 99%, or 100% homology with a "ubiquitin
carboxyl-terminal hydrolase-2 domain," e.g., the ubiquitin
carboxyl-terminal hydrolase-2 domain of human 68999 (e.g., residues
313 to 374 of SEQ ID NO:2).
[0053] Ubiquitin carboxyl-terminal hydrolase-2 domains typically
contain a conserved ubiquitin carboxyl hydrolase-2 (UCH-2)
signature pattern which participates in the catalytic mechanism.
The conserved UCH-2 signature pattern is as follows:
Y-x-L-x-[SAG]-[LIVMFT]-x(2)-H-x-G-x(4,5)-G-H--Y (SEQ ID NO:7).
[0054] A 68999 protein contains a conserved UCH-2 signature
patttern at about amino acid residues 317 to 335 of SEQ ID
NO:2.
[0055] To identify the presence of a "ubiquitin carboxyl-terminal
hydrolase-2" domain in a 68999 protein sequence, and make the
determination that a polypeptide or protein of interest has a
particular profile, the amino acid sequence of the protein can be
searched against the Pfam database of HMMs (e.g., the Pfam
database, release 2.1) using the default parameters
(http://www.sanger.ac.uk/Software/Pfam/HMM_search)- . For example,
the hmmsf program, which is available as part of the HMMER package
of search programs, is a family specific default program for
MILPAT0063 and a score of 15 is the default threshold score for
determining a hit. Alternatively, the threshold score for
determining a hit can be lowered (e.g., to 8 bits). A description
of the Pfam database can be found in Sonhammer et al. (1997)
Proteins 28: 405-420 and a detailed description of HMMs can be
found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:
146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:
4355-4358; Krogh et al. (1994) J. Mol. Biol. 235: 1501-1531; and
Stultz et al. (1993) Protein Sci. 2: 305-314, the contents of which
are incorporated herein by reference. A search was performed
against the HMM database resulting in the identification of a
"ubiquitin carboxyl-terminal hydrolase-2" domain in the amino acid
sequence of human 68999 at about residues 313-374 of SEQ ID
NO:2.
[0056] A 68999 family member can include at least one ubiquitin
carboxyl-terminal hydrolase-1 domain and at least one ubiquitin
carboxyl-terminal hydrolase-1 domain. Furthermore, a 68999 family
member can include at least one, two, preferably three
N-glycosylation sites (PS00001), at least one, preferably two cAMP
and cGMP dependent protein kinase phosphorylation sites (PS00004),
at least one, two, three, four, preferably five protein kinase C
phosphorylation sites (PS00005); at least one, two, three, four,
five, six, and preferably seven casein kinase II phosphorylation
sites (PS00006); at least one tyrosine kinase phosphorylation site
(PS00007); and at least one, two, three, four, and preferably five
N-myristoylation sites (PS00008); at least one `helix-loop-helix`
dimerization domain site (PS00038), and at least one cytochrome c
family heme-binding site signature (PS000169).
[0057] As the 68999 polypeptides of the invention can modulate
68999-mediated activities, they can be useful for developing novel
diagnostic and therapeutic agents for ubiquitin carboxyl-terminal
hydrolase-associated or other 68999-associated disorders, as
described herein.
[0058] Localized expression of ubiquitin carboxyl-terminal
hydrolase is indicative of this molecules biological function. For
instance, the discovery of ubiquitin carboxyl-terminal hydrolase in
the brain suggests that the role of this protein can be studied in
relation to ubiquitinated cellular inclusions characteristic of
several chronic human degenerative diseases. Formalin-fixed,
paraffin-processed sections have been shown to contain
ubiquitin-protein conjugate immunoreactivity in cortical Lewy
bodies, neurofibrillary tangles, Rosenthal fibres, Pick bodies,
spinal inclusions in motor neuron disease, and Mallory's hyaline in
alcoholic liver disease, when immunostained to localize ubiquitin
carboxyl-terminal hydrolase. Lowe, et al., (1985) J Biol Chem 5:
260(13): 7903-10.
[0059] As used herein, a "ubiquitin carboxyl-terminal
hydrolase-mediated activity" includes an activity which involves
the processing of poly-ubiquitin precursors as well as that of
ubiquinated proteins. Ubiquitin carboxyl-terminal hydrolases have
been shown to hydrolyze thiol esters formed between the ubiquitin
carboxyl terminus and small thiols (e.g. glutathione), as well as
free ubiquitin adenylate (Rose, I. et.al (1983) Biochemistry 22,
4234-4237). These enzymes hydrolyze amide derivatives of the
ubiquitin carboxyl terminus, including those of lysine
(epsilon-amino), glycine methyl ester, and spermidine. They also
hydrolyze ubiquitin COOH-terminal hydroxamic acid. Thus, these
enzymes are general hydrolases that recognize the ubiquitin moiety,
but are highly selective for small ubiquitin derivatives. Such
enzymes function to regenerate ubiquitin from adventitiously formed
ubiquitin amides and thiol esters. They also have the correct
specificity to function in regenerating ubiquitin from small
ubiquitin peptides that are probable end products of
ubiquitin-dependent proteolysis. (Pickart C M, et.al. J (1985) Biol
Chem July 5; 260(13): 7903-10).
[0060] As used herein, a "68999 activity", "biological activity of
68999" or "functional activity of 68999", refers to an activity
exerted by a 68999 protein, polypeptide or nucleic acid molecule on
e.g., a 68999-responsive cell or on a 68999 substrate, e.g., a
protein substrate, as determined in vivo or in vitro. In one
embodiment, a 68999 activity is a direct activity, such as an
association with a 68999 target molecule. A "target molecule" or
"binding partner" is a molecule with which a 68999 protein binds or
interacts in nature. A 68999 activity can also be an indirect
activity, e.g., a cellular signaling activity mediated by
interaction of the 68999 protein with a 68999 receptor.
[0061] Based on the above-described sequence structures and
similarities to molecules of known function, the 68999 molecules of
the present invention have similar biological activities as
ubiquitin carboxyl-terminal hydrolase family members, e.g.,
biological activities of ubiquitin carboxyl-terminal hydrolases
described herein. For example, the 68999 proteins of the present
invention can modulate (directly or indirectly) any one or more of
the following activities: (1) de-ubiquitination of a substrate,
e.g., a ubiquitinated protein targeted for degradation; (2)
processing of poly-ubiquitin precursors; (3) cellular proliferation
and/or differentiation; (4) apoptosis; (5)transcription and/or
cell-cycle progression; (6) signal-transduction; (7) antigen
processing; (8) cell-cell adhesion; (9) receptor-mediated
endocytosis; (10) organelle biogenesis and development; (11)
neuropathological conditions; (12) oncogenesis, and (13) protein
levels, e.g., cellular protein levels.
[0062] Thus, the 68999 molecules can act as novel diagnostic
targets and therapeutic agents for controlling one or more
ubiquitin carboxyl-terminal hydrolase-associated or other
68999-associated disorders. Examples of such disorders (e.g.,
ubiquitin carboxyl-terminal hydrolase-associated disorders) include
but are not limited to, disorders involving neurons, and disorders
involving glia, such as astrocytes, oligodendrocytes, ependymal
cells, and microglia; cerebral edema, raised intracranial pressure
and herniation, and hydrocephalus; malformations and developmental
diseases, such as neural tube defects, forebrain anomalies,
posterior fossa anomalies, and syringomyelia and hydromyelia;
perinatal brain injury; cerebrovascular diseases, such as those
related to hypoxia, ischemia, and infarction, including
hypotension, hypoperfusion, and low-flow states, global cerebral
ischemia and focal cerebral ischemia--infarction from obstruction
of local blood supply, intracranial hemorrhage, including
intracerebral (intraparenchymal) hemorrhage, subarachnoid
hemorrhage and ruptured berry aneurysms, and vascular
malformations, hypertensive cerebrovascular disease, including
lacunar infarcts, slit hemorrhages, and hypertensive
encephalopathy; infections, such as acute meningitis, including
acute pyogenic (bacterial) meningitis and acute aseptic (viral)
meningitis, acute focal suppurative infections, including brain
abscess, subdural empyema, and extradural abscess, chronic
bacterial meningoencephalitis, including tuberculosis and
mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme
disease), viral meningoencephalitis, including arthropod-borne
(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes
simplex virus Type 2, Varicella-zoster virus (Herpes zoster),
cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency
virus 1, including HIV-1 meningoencephalitis (subacute
encephalitis), vacuolar myelopathy, AIDS-associated myopathy,
peripheral neuropathy, and AIDS in children, progressive multifocal
leukoencephalopathy, subacute sclerosing panencephalitis, fungal
meningoencephalitis, other infectious diseases of the nervous
system; transmissible spongiform encephalopathies (prion diseases);
demyelinating diseases, including multiple sclerosis, multiple
sclerosis variants, acute disseminated encephalomyelitis and acute
necrotizing hemorrhagic encephalomyelitis, and other diseases with
demyelination; degenerative diseases, such as degenerative diseases
affecting the cerebral cortex, including Alzheimer's disease and
Pick's disease, degenerative diseases of basal ganglia and brain
stem, including Parkinsonism, idiopathic Parkinson's disease
(paralysis agitans), progressive supranuclear palsy, corticobasal
degenration, multiple system atrophy, including striatonigral
degenration, Shy-Drager syndrome, and olivopontocerebellar atrophy,
and Huntington's disease; spinocerebellar degenerations, including
spinocerebellar ataxias, including Friedreich ataxia, and
ataxia-telanglectasia, degenerative diseases affecting motor
neurons, including amyotrophic lateral sclerosis (motor neuron
disease), bulbospinal atrophy (Kennedy syndrome), and spinal
muscular atrophy; inborn errors of metabolism, such as
leukodystrophies, including Krabbe disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease,
and Canavan disease, mitochondrial encephalomyopathies, including
Leigh disease and other mitochondrial encephalomyopathies; toxic
and acquired metabolic diseases, including vitamin deficiencies
such as thiamine (vitamin B.sub.1) deficiency and vitamin B.sub.12
deficiency, neurologic sequelae of metabolic disturbances,
including hypoglycemia, hyperglycemia, and hepatic encephatopathy,
toxic disorders, including carbon monoxide, methanol, ethanol, and
radiation, including combined methotrexate and radiation-induced
injury; tumors, such as gliomas, including astrocytoma, including
fibrillary (diffuse) astrocytoma and glioblastoma multiforme,
pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain
stem glioma, oligodendroglioma, and ependymoma and related
paraventricular mass lesions, neuronal tumors, poorly
differentiated neoplasms, including medulloblastoma, other
parenchymal tumors, including primary brain lymphoma, germ cell
tumors, and pineal parenchymal tumors, meningiomas, metastatic
tumors, paraneoplastic syndromes, peripheral nerve sheath tumors,
including schwannoma, neurofibroma, and malignant peripheral nerve
sheath tumor (malignant schwannoma), and neurocutaneous syndromes
(phakomatoses), including neurofibromotosis, including Type 1
neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2),
tuberous sclerosis, and Von Hippel-Lindau disease.
[0063] The 68999 molecules and modulators thereof can act as novel
therapeutic agents for controlling one or more of cellular
proliferative and/or differentiative disorders, neurological
disorders, and hormonal disorders as described herein.
[0064] Examples of cellular proliferative and/or differentiative
disorders include cancer, e.g., carcinoma, sarcoma, metastatic
disorders or hematopoietic neoplastic disorders, e.g., leukemias. A
metastatic tumor can arise from a multitude of primary tumor types,
including but not limited to those of prostate, colon, lung, breast
and liver origin.
[0065] As used herein, the term "cancer" (also used interchangeably
with the terms, "hyperproliferative" and "neoplastic") refers to
cells having the capacity for autonomous growth, i.e., an abnormal
state or condition characterized by rapidly proliferating cell
growth. Cancerous disease states may be categorized as pathologic,
i.e., characterizing or constituting a disease state, e.g.,
malignant tumor growth, or may be categorized as non-pathologic,
i.e., a deviation from normal but not associated with a disease
state, e.g., cell proliferation associated with wound repair. The
term is meant to include all types of cancerous growths or
oncogenic processes, metastatic tissues or malignantly transformed
cells, tissues, or organs, irrespective of histopathologic type or
stage of invasiveness. The term "cancer" includes malignancies of
the various organ systems, such as those affecting lung, breast,
thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as
well as adenocarcinomas which include malignancies such as most
colon cancers, renal-cell carcinoma, prostate cancer and/or
testicular tumors, non-small cell carcinoma of the lung, cancer of
the small intestine and cancer of the esophagus. The term
"carcinoma" is art recognized and refers to malignancies of
epithelial or endocrine tissues including respiratory system
carcinomas, gastrointestinal system carcinomas, genitourinary
system carcinomas, testicular carcinomas, breast carcinomas,
prostatic carcinomas, endocrine system carcinomas, and melanomas.
Exemplary carcinomas include those forming from tissue of the
cervix, lung, prostate, breast, head and neck, colon and ovary. The
term "carcinoma" also includes carcinosarcomas, e.g., which include
malignant tumors composed of carcinomatous and sarcomatous tissues.
An "adenocarcinoma" refers to a carcinoma derived from glandular
tissue or in which the tumor cells form recognizable glandular
structures. The term "sarcoma" is art recognized and refers to
malignant tumors of mesenchymal derivation.
[0066] The 68999 molecules of the invention can be used to monitor,
treat and/or diagnose a variety of proliferative disorders. Such
disorders include hematopoietic neoplastic disorders. As used
herein, the term "hematopoietic neoplastic disorders" includes
diseases involving hyperplastic/neoplastic cells of hematopoietic
origin, e.g., arising from myeloid, lymphoid or erythroid lineages,
or precursor cells thereof. Preferably, the diseases arise from
poorly differentiated acute leukemias, e.g., erythroblastic
leukemia and acute megakaryoblastic leukemia. Additional exemplary
myeloid disorders include, but are not limited to, acute promyeloid
leukemia (APML), acute myelogenous leukemia (AML) and chronic
myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit
Rev. in Oncol./Hemotol. 11: 267-97); lymphoid malignancies include,
but are not limited to acute lymphoblastic leukemia (ALL) which
includes B-lineage ALL and T-lineage ALL, chronic lymphocytic
leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia
(HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of
malignant lymphomas include, but are not limited to non-Hodgkin
lymphoma and variants thereof, peripheral T cell lymphomas, adult T
cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),
large granular lymphocytic leukemia (LGF), Hodgkin's disease and
Reed-Sternberg disease.
[0067] Neurological disorders include disorders of the central
nervous system (CNS) and the peripheral nervous system, e.g.,
cognitive and neurodegenerative disorders, Examples of neurological
disorders include, but are not limited to, autonomic function
disorders such as hypertension and sleep disorders, and
neuropsychiatric disorders, such as depression, schizophrenia,
schizoaffective disorder, Korsakoff's psychosis, alcoholism,
anxiety disorders, or phobic disorders; learning or memory
disorders, e.g., amnesia or age-related memory loss, attention
deficit disorder, dysthymic disorder, major depressive disorder,
mania, obsessive-compulsive disorder, psychoactive substance use
disorders, anxiety, phobias, panic disorder, as well as bipolar
affective disorder, e.g., severe bipolar affective (mood) disorder
(BP-1), and bipolar affective neurological disorders, e.g.,
migraine and obesity. Such neurological disorders include, for
example, disorders involving neurons, and disorders involving glia,
such as astrocytes, oligodendrocytes, ependymal cells, and
microglia; cerebral edema, raised intracranial pressure and
herniation, and hydrocephalus; malformations and developmental
diseases, such as neural tube defects, forebrain anomalies,
posterior fossa anomalies, and syringomyelia and hydromyelia;
perinatal brain injury; cerebrovascular diseases, such as those
related to hypoxia, ischemia, and infarction, including
hypotension, hypoperfusion, and low-flow states--global cerebral
ischemia and focal cerebral ischemia--infarction from obstruction
of local blood supply, intracranial hemorrhage, including
intracerebral (intraparenchymal) hemorrhage, subarachnoid
hemorrhage and ruptured berry aneurysms, and vascular
malformations, hypertensive cerebrovascular disease, including
lacunar infarcts, slit hemorrhages, and hypertensive
encephalopathy; infections, such as acute meningitis, including
acute pyogenic (bacterial) meningitis and acute aseptic (viral)
meningitis, acute focal suppurative infections, including brain
abscess, subdural empyema, and extradural abscess, chronic
bacterial meningoencephalitis, including tuberculosis and
mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme
disease), viral meningoencephalitis, including arthropod-borne
(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes
simplex virus Type 2, Varicella-zoster virus (Herpes zoster),
cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency
virus 1, including HIV-1 meningoencephalitis (subacute
encephalitis), vacuolar myelopathy, AIDS-associated myopathy,
peripheral neuropathy, and AIDS in children, progressive multifocal
leukoencephalopathy, subacute sclerosing panencephalitis, fungal
meningoencephalitis, other infectious diseases of the nervous
system; transmissible spongiform encephalopathies (prion diseases);
demyelinating diseases, including multiple sclerosis, multiple
sclerosis variants, acute disseminated encephalomyelitis and acute
necrotizing hemorrhagic encephalomyelitis, and other diseases with
demyelination; degenerative diseases, such as degenerative diseases
affecting the cerebral cortex, including Alzheimer's disease and
Pick's disease, degenerative diseases of basal ganglia and brain
stem, including Parkinsonism, idiopathic Parkinson's disease
(paralysis agitans) and other Lewy diffuse body diseases,
progressive supranuclear palsy, corticobasal degenration, multiple
system atrophy, including striatonigral degenration, Shy-Drager
syndrome, and olivopontocerebellar atrophy, and Huntington's
disease, senile dementia, Gilles de la Tourette's syndrome,
epilepsy, and Jakob-Creutzfieldt disease; spinocerebellar
degenerations, including spinocerebellar ataxias, including
Friedreich ataxia, and ataxiatelanglectasia, degenerative diseases
affecting motor neurons, including amyotrophic lateral sclerosis
(motor neuron disease), bulbospinal atrophy (Kennedy syndrome), and
spinal muscular atrophy; inborn errors of metabolism, such as
leukodystrophies, including Krabbe disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease,
and Canavan disease, mitochondrial encephalomyopathies, including
Leigh disease and other mitochondrial encephalomyopathies; toxic
and acquired metabolic diseases, including vitamin deficiencies
such as thiamine (vitamin B.sub.1) deficiency and vitamin B.sub.12
deficiency, neurologic sequelae of metabolic disturbances,
including hypoglycemia, hyperglycemia, and hepatic encephatopathy,
toxic disorders, including carbon monoxide, methanol, ethanol, and
radiation, including combined methotrexate and radiation-induced
injury; tumors, such as gliomas, including astrocytoma, including
fibrillary (diffuse) astrocytoma and glioblastoma multiforme,
pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain
stem glioma, oligodendroglioma, and ependymoma and related
paraventricular mass lesions, neuronal tumors, poorly
differentiated neoplasms, including medulloblastoma, other
parenchymal tumors, including primary brain lymphoma, germ cell
tumors, and pineal parenchymal tumors, meningiomas, metastatic
tumors, paraneoplastic syndromes, peripheral nerve sheath tumors,
including schwannoma, neurofibroma, and malignant peripheral nerve
sheath tumor (malignant schwannoma), and neurocutaneous syndromes
(phakomatoses), including neurofibromotosis, including Type 1
neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2),
tuberous sclerosis, and Von Hippel-Lindau disease. Further
CNS-related disorders include, for example, those listed in the
American Psychiatric Association's Diagnostic and Statistical
manual of Mental Disorders (DSM), the most current version of which
is incorporated herein by reference in its entirety.
[0068] Ubiquitin carboxyl terminal hydrolase, or other 68999
associated disorders also can include hormonal disorders, such as
conditions or diseases in which the production and/or regulation of
hormones in an organism is aberrant. Examples of such disorders and
diseases include type I and type II diabetes mellitus, pituitary
disorders (e.g., growth disorders), thyroid disorders (e.g.,
hypothyroidism or hyperthyroidism), and reproductive or fertility
disorders (e.g., disorders which affect the organs of the
reproductive system, e.g., the prostate gland, the uterus, or the
vagina; disorders which involve an imbalance in the levels of a
reproductive hormone in a subject; disorders affecting the ability
of a subject to reproduce; and disorders affecting secondary sex
characteristic development, e.g., adrenal hyperplasia).
[0069] The 68999 protein, fragments thereof, and derivatives and
other variants of the sequence in SEQ ID NO:2 thereof are
collectively referred to as "polypeptides or proteins of the
invention" or "68999 polypeptides or proteins". Nucleic acid
molecules encoding such polypeptides or proteins are collectively
referred to as "nucleic acids of the invention" or "68999 nucleic
acids."
[0070] As used herein, the term "nucleic acid molecule" includes
DNA molecules (e.g., a cDNA or genomic DNA) and RNA molecules
(e.g., an mRNA) and analogs of the DNA or RNA generated, e.g., by
the use of nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0071] The term "isolated or purified nucleic acid molecule"
includes nucleic acid molecules which are separated from other
nucleic acid molecules which are present in the natural source of
the nucleic acid. For example, with regards to genomic DNA, the
term "isolated" includes nucleic acid molecules which are separated
from the chromosome with which the genomic DNA is naturally
associated. Preferably, an "isolated" nucleic acid is free of
sequences which naturally flank the nucleic acid (i.e., sequences
located at the 5' and/or 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
For example, in various embodiments, the isolated nucleic acid
molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,
0.5 kb or 0.1 kb of 5' and/or 3' nucleotide sequences which
naturally flank the nucleic acid molecule in genomic DNA of the
cell from which the nucleic acid is derived. Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically
synthesized.
[0072] As used herein, the term "hybridizes under low stringency,
medium stringency, high stringency, or very high stringency
conditions" describes conditions for hybridization and washing.
Guidance for performing hybridization reactions can be found in
Current Protocols in Molecular Biology (1989) John Wiley &
Sons, N.Y., 6.3.1-6.3.6, which is incorporated by reference.
Aqueous and nonaqueous methods are described in that reference and
either can be used. Specific hybridization conditions referred to
herein are as follows: 1) low stringency hybridization conditions
in 6.times. sodium chloride/sodium citrate (SSC) at about
45.degree. C., followed by two washes in 0.2.times. SSC, 0.1% SDS
at least at 50.degree. C. (the temperature of the washes can be
increased to 55.degree. C. for low stringency conditions); 2)
medium stringency hybridization conditions in 6.times. SSC at about
45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 60.degree. C.; 3) high stringency hybridization
conditions in 6.times. SSC at about 45.degree. C., followed by one
or more washes in 0.2.times.SSC, 0.1% SDS at 65.degree. C.; and
preferably 4) very high stringency hybridization conditions are
0.5M sodium phosphate, 7% SDS at 65.degree. C., followed by one or
more washes at 0.2.times.SSC, 1% SDS at 65.degree. C. Very high
stringency conditions (4) are the preferred conditions and the ones
that should be used unless otherwise specified.
[0073] As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural
protein).
[0074] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules which include an open reading frame
encoding a 68999 protein, preferably a mammalian 68999 protein, and
can further include non-coding regulatory sequences, and
introns.
[0075] An "isolated" or "purified" polypeptide or protein is
substantially free of cellular material or other contaminating
proteins from the cell or tissue source from which the protein is
derived, or substantially free from chemical precursors or other
chemicals when chemically synthesized. In one embodiment, the
language "substantially free" means preparation of 68999 protein
having less than about 30%, 20%, 10% and more preferably 5% (by dry
weight), of non-68999 protein (also referred to herein as a
"contaminating protein"), or of chemical precursors or non-68999
chemicals. When the 68999 protein or biologically active portion
thereof is recombinantly produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, more preferably less than about
10%, and most preferably less than about 5% of the volume of the
protein preparation. The invention includes isolated or purified
preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry
weight.
[0076] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of 68999 (e.g., the sequence
of SEQ ID NO:1 or 3) without abolishing or more preferably, without
substantially altering a biological activity, whereas an
"essential" amino acid residue results in such a change. For
example, amino acid residues that are conserved among the
polypeptides of the present invention, e.g., those present in the
ubiquitin carboxyl-terminal hydrolase-1 domain, or the ubiquitin
carboxyl-terminal hydrolase 2 domain, are predicted to be
particularly unamenable to alteration.
[0077] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteinee), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a 68999 protein is
preferably replaced with another amino acid residue from the same
side chain family. Alternatively, in another embodiment, mutations
can be introduced randomly along all or part of a 68999 coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for 68999 biological activity to identify
mutants that retain activity. Following mutagenesis of SEQ ID NO:1
or SEQ ID NO: 3, the encoded protein can be expressed recombinantly
and the activity of the protein can be determined.
[0078] As used herein, a "biologically active portion" of a 68999
protein includes a fragment of a 68999 protein which participates
in an interaction between a 68999 molecule and a non-68999
molecule. Biologically active portions of a 68999 protein include
peptides comprising amino acid sequences sufficiently homologous to
or derived from the amino acid sequence of the 68999 protein, e.g.,
the amino acid sequence shown in SEQ ID NO:2, which include fewer
amino acids than the full length 68999 protein, and exhibit at
least one activity of a 68999 protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the 68999 protein, e.g., a domain or motif capable of
catalyzing an activity described herein, such as the ubiquitination
and de-ubiquitination of substrates. A biologically active portion
of a 68999 protein can be a polypeptide which is, for example, 10,
25, 50, 100, 200 or more amino acids in length. Biologically active
portions of a 68999 protein can be used as targets for developing
agents which modulate a 68999 mediated activity, e.g., a domain or
motif capable of catalyzing an activity described herein, such as
the ubiquitination and de-ubiquitination of substrates.
[0079] Calculations of homology or sequence identity (the terms
"homology" and "identity" are used interchangeably herein) between
sequences are performed as follows:
[0080] To determine the percent identity of two amino acid
sequences, or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, 90%, 100% of the length
of the reference sequence (e.g., when aligning a second sequence to
the 68999 amino acid sequence of SEQ ID NO:2 having 530 amino acid
residues, at least 159, preferably at least 212, more preferably at
least 265, even more preferably at least 318, and even more
preferably at least 371, 424, or 477 amino acid residues are
aligned). The amino acid residues or nucleotides at corresponding
amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same amino
acid residue or nucleotide as the corresponding position in the
second sequence, then the molecules are identical at that position
(as used herein amino acid or nucleic acid "identity" is equivalent
to amino acid or nucleic acid "homology"). The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which need to be introduced
for optimal alignment of the two sequences.
[0081] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch (1970) J. Mol. Biol. 48: 444-453 algorithm
which has been incorporated into the GAP program in the GCG
software package (available at http://www.gcg.com), using either a
Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In
yet another preferred embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (available at http://www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred
set of parameters (and the one that should be used if the
practitioner is uncertain about what parameters should be applied
to determine if a molecule is within a sequence identity or
homology limitation of the invention) are a Blossum 62 scoring
matrix with a gap penalty of 12, a gap extend penalty of 4, and a
frameshift gap penalty of 5.
[0082] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of E. Meyers and W.
Miller ((1989) CABIOS, 4: 11-17) which has been incorporated into
the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4.
[0083] The nucleic acid and protein sequences described herein can
be used as a "query sequence" to perform a search against public
databases to, for example, identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215: 403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to 68999 nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to 68999 protein molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al., (1997) Nucleic Acids Res.
25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the
default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
[0084] Particular 68999 polypeptides of the present invention have
an amino acid sequence substantially identical to the amino acid
sequence of SEQ ID NO:2. In the context of an amino acid sequence,
the term "substantially identical" is used herein to refer to a
first amino acid that contains a sufficient or minimum number of
amino acid residues that are i) identical to, or ii) conservative
substitutions of aligned amino acid residues in a second amino acid
sequence such that the first and second amino acid sequences can
have a common structural domain and/or common functional activity.
For example, amino acid sequences that contain a common structural
domain having at least about 60%, or 65% identity, likely 75%
identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:2 are termed substantially
identical.
[0085] In the context of nucleotide sequence, the term
"substantially identical" is used herein to refer to a first
nucleic acid sequence that contains a sufficient or minimum number
of nucleotides that are identical to aligned nucleotides in a
second nucleic acid sequence such that the first and second
nucleotide sequences encode a polypeptide having common functional
activity, or encode a common structural polypeptide domain or a
common functional polypeptide activity. For example, nucleotide
sequences having at least about 60%, or 65% identity, likely 75%
identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:1 or 3 are termed substantially
identical.
[0086] "Misexpression or aberrant expression", as used herein,
refers to a non-wild type pattern of gene expression, at the RNA or
protein level. It includes: expression at non-wild type levels,
i.e., over or under expression; a pattern of expression that
differs from wild type in terms of the time or stage at which the
gene is expressed, e.g., increased or decreased expression (as
compared with wild type) at a predetermined developmental period or
stage; a pattern of expression that differs from wild type in terms
of decreased expression (as compared with wild type) in a
predetermined cell type or tissue type; a pattern of expression
that differs from wild type in terms of the splicing size, amino
acid sequence, post-transitional modification, or biological
activity of the expressed polypeptide; a pattern of expression that
differs from wild type in terms of the effect of an environmental
stimulus or extracellular stimulus on expression of the gene, e.g.,
a pattern of increased or decreased expression (as compared with
wild type) in the presence of an increase or decrease in the
strength of the stimulus.
[0087] "Subject", as used herein, can refer to a mammal, e.g., a
human, or to an experimental or animal or disease model. The
subject can also be a non-human animal, e.g., a horse, cow, goat,
or other domestic animal.
[0088] A "purified preparation of cells", as used herein, refers
to, in the case of plant or animal cells, an in vitro preparation
of cells and not an entire intact plant or animal. In the case of
cultured cells or microbial cells, it consists of a preparation of
at least 10% and more preferably 50% of the subject cells.
[0089] Various aspects of the invention are described in further
detail below.
[0090] Isolated Nucleic Acid Molecules
[0091] In one aspect, the invention provides, an isolated or
purified, nucleic acid molecule that encodes a 68999 polypeptide
described herein, e.g., a full length 68999 protein or a fragment
thereof, e.g., a biologically active portion of 68999 protein. Also
included is a nucleic acid fragment suitable for use as a
hybridization probe, which can be used, e.g., to identify a nucleic
acid molecule encoding a polypeptide of the invention, 68999 mRNA,
and fragments suitable for use as primers, e.g., PCR primers for
the amplification or mutation of nucleic acid molecules.
[0092] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO:1, or
a portion of any of this nucleotide sequence. In one embodiment,
the nucleic acid molecule includes sequences encoding the human
68999 protein (i.e., "the coding region" of SEQ ID NO:1, as shown
in SEQ ID NO:3), as well as 5' untranslated sequences (nucleotides
1 to 170 of SEQ ID NO:1). Alternatively, the nucleic acid molecule
can include only the coding region of SEQ ID NO:1 (e.g., SEQ ID
NO:3) and, e.g., no flanking sequences which normally accompany the
subject sequence. In another embodiment, the nucleic acid molecule
encodes a sequence corresponding to a fragment of the protein from
about amino acid 80 to 111 of SEQ ID NO:2, or from amino 313 to 374
of SEQ ID NO:2.
[0093] In another embodiment, an isolated nucleic acid molecule o
the invention includes a nucleic acid molecule which is a
complement of the nucleotide sequence shown in SEQ ID NO:1 or SEQ
ID NO:3, or a portion of any of these nucleotide sequences. In
other embodiments, the nucleic acid molecule of the invention is
sufficiently complementary to the nucleotide sequence shown in SEQ
ID NO:1 or SEQ ID NO:3 such that it can hybridize to the nucleotide
sequence shown in SEQ ID NO:1 or 3, thereby forming a stable
duplex.
[0094] In one embodiment, an isolated nucleic acid molecule of the
present invention includes a nucleotide sequence which is at least
about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more homologous to the entire length of the
nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3, or a
portion, preferably of the same length, of any of these nucleotide
sequences.
[0095] 68999 Nucleic Acid Fragments
[0096] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO:1 or 3. For
example, such a nucleic acid molecule can include a fragment which
can be used as a probe or primer or a fragment encoding a portion
of a 68999 protein, e.g., an immunogenic or biologically active
portion of a 68999 protein. A fragment can comprise those
nucleotides of SEQ ID NO:1 which encode a ubiquitin
carboxyl-terminal hydrolase-1 domain, or a ubiquitin
carboxyl-terminal hydrolase-2 domain of human 68999. The nucleotide
sequence determined from the cloning of the 68999 gene allows for
the generation of probes and primers designed for use in
identifying and/or cloning other 68999 family members, or fragments
thereof, as well as 68999 homologs, or fragments thereof, from
other species.
[0097] In another embodiment, a nucleic acid includes a nucleotide
sequence that includes part, or all, of the coding region and
extends into either (or both) the 5' or 3' noncoding region. Other
embodiments include a fragment which includes a nucleotide sequence
encoding an amino acid fragment described herein. Nucleic acid
fragments can encode a specific domain or site described herein or
fragments thereof, particularly fragments thereof which are at
least 100 amino acids in length. Fragments also include nucleic
acid sequences corresponding to specific amino acid sequences
described above or fragments thereof. Nucleic acid fragments should
not to be construed as encompassing those fragments that may have
been disclosed prior to the invention.
[0098] A nucleic acid fragment can include a sequence corresponding
to a domain, region, or functional site described herein. A nucleic
acid fragment can also include one or more domain, region, or
functional site described herein. Thus, for example, a 68999
nucleic acid fragment can include a sequence corresponding to a
ubiquitin carboxyl-terminal hydrolase domain-1, as described
herein.
[0099] 68999 probes and primers are provided. Typically a
probe/primer is an isolated or purified oligonucleotide. The
oligonucleotide typically includes a region of nucleotide sequence
that hybridizes under stringent conditions to at least about 7, 12
or 15, preferably about 20 or 25, more preferably about 30, 35, 40,
45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or
antisense sequence of SEQ ID NO:1 or SEQ ID NO:3, or of a naturally
occurring allelic variant or mutant of SEQ ID NO:1 or SEQ ID
NO:3.
[0100] In a preferred embodiment the nucleic acid is a probe which
is at least 5 or 10, and less than 200, more preferably less than
100, or less than 50, base pairs in length. It should be identical,
or differ by 1, or less than in 5 or 10 bases, from a sequence
disclosed herein. If alignment is needed for this comparison the
sequences should be aligned for maximum homology. "Looped" out
sequences from deletions or insertions, or mismatches, are
considered differences.
[0101] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid which encodes:
[0102] a ubiquitin carboxyl-terminal hydrolase-1 domain at about
amino acid residues 80-111 of SEQ ID NO:2;
[0103] a ubiquitin carboxyl terminal hydrolase-2 domain at about
amino acid residues 313-374 of SEQ NO:2.
[0104] In another embodiment a set of primers is provided, e.g.,
primers suitable for use in a PCR, which can be used to amplify a
selected region of a 68999 sequence, e.g., a domain, region, site
or other sequence described herein. The primers should be at least
5, 10, or 50 base pairs in length and less than 100, or less than
200, base pairs in length. The primers should be identical, or
differ by one base from a sequence disclosed herein or from a
naturally occurring variant. For example, primers suitable for
amplifying all or a portion of any of the following regions are
provided: a ubiquitin carboxyl-terminal hydrolase-1 domain from
about amino acid 80 to 111 of SEQ ID NO:2, and a ubiquitin
carboxyl-terminal hydrolase-2 domain from about amino acid residues
313-374 of SEQ ID NO:2.
[0105] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0106] A nucleic acid fragment encoding a "biologically active
portion of a 68999 polypeptide" can be prepared by isolating a
portion of the nucleotide sequence of SEQ ID NO:1 or 3, which
encodes a polypeptide having a 68999 biological activity (e.g., the
biological activities of the 68999 proteins are described herein),
expressing the encoded portion of the 68999 protein (e.g., by
recombinant expression in vitro) and assessing the activity of the
encoded portion of the 68999 protein. For example, a nucleic acid
fragment encoding a biologically active portion of 68999 includes a
ubiquitin carboxyl-terminal hydrolase-1 domain, e.g., amino acid
residues about 80 to 111 of SEQ ID NO:2, or a ubiquitin
carboxyl-terminal hydrolase-2 domain, e.g., amino acid residues
about 313 to 374 of SEQ ID NO:2. A nucleic acid fragment encoding a
biologically active portion of a 68999 polypeptide, can comprise a
nucleotide sequence which is greater than 550 or more nucleotides
in length.
[0107] In preferred embodiments, a nucleic acid includes a
nucleotide sequence which is about 300, 400, 500, 510, 520, 530,
540, 550, 560, 570, 580, 590, 600, 700, 800, 900, 1000, 1100, 1200,
1300, 1400, 1500, 1600 or more nucleotides in length and hybridizes
under stringent hybridization conditions to a nucleic acid molecule
of SEQ ID NO:1 or SEQ ID NO:3.
[0108] 68999 Nucleic Acid Variants
[0109] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence shown in SEQ ID NO:1 or
SEQ ID NO:3. Such differences can be due to degeneracy of the
genetic code (and result in a nucleic acid which encodes the same
68999 proteins as those encoded by the nucleotide sequence
disclosed herein. In another embodiment, an isolated nucleic acid
molecule of the invention has a nucleotide sequence encoding a
protein having an amino acid sequence which differs, by at least 1,
but less than 5, 10, 20, 50, or 100 amino acid residues that shown
in SEQ ID NO:2. If alignment is needed for this comparison the
sequences should be aligned for maximum homology. "Looped" out
sequences from deletions or insertions, or mismatches, are
considered differences.
[0110] Nucleic acids of the inventor can be chosen for having
codons, which are preferred, or non-preferred, for a particular
expression system. E.g., the nucleic acid can be one in which at
least one codon, at preferably at least 10%, or 20% of the codons
has been altered such that the sequence is optimized for expression
in E. coli, yeast, human, insect, or CHO cells.
[0111] Nucleic acid variants can be naturally occurring, such as
allelic variants (same locus), homologs (different locus), and
orthologs (different organism) or can be non naturally occurring.
Non-naturally occurring variants can be made by mutagenesis
techniques, including those applied to polynucleotides, cells, or
organisms. The variants can contain nucleotide substitutions,
deletions, inversions and insertions. Variation can occur in either
or both the coding and non-coding regions. The variations can
produce both conservative and non-conservative amino acid
substitutions (as compared in the encoded product).
[0112] In a preferred embodiment, the nucleic acid differs from
that of SEQ ID NO:1 or 3, e.g., as follows: by at least one but
less than 10, 20, 30, or 40 nucleotides; at least one but less than
1%, 5%, 10% or 20% of the nucleotides in the subject nucleic acid.
If necessary for this analysis the sequences should be aligned for
maximum homology. "Looped" out sequences from deletions or
insertions, or mismatches, are considered differences.
[0113] Orthologs, homologs, and allelic variants can be identified
using methods known in the art. These variants comprise a
nucleotide sequence encoding a polypeptide that is 50%, at least
about 55%, typically at least about 70-75%, more typically at least
about 80-85%, and most typically at least about 90-95% or more
identical to the nucleotide sequence shown in SEQ ID NO:2 or a
fragment of this sequence. Such nucleic acid molecules can readily
be identified as being able to hybridize under stringent
conditions, to the nucleotide sequence shown in SEQ ID NO 2 or a
fragment of the sequence. Nucleic acid molecules corresponding to
orthologs, homologs, and allelic variants of the 68999 cDNAs of the
invention can further be isolated by mapping to the same chromosome
or locus as the 68999 gene.
[0114] Preferred variants include those that are correlated with a
domain or motif capable of catalyzing an activity described herein,
such as the ubiquitination and de-ubiquitination of substrates.
Allelic variants of 68999, e.g., human 68999, include both
functional and non-functional proteins. Functional allelic variants
are naturally occurring amino acid sequence variants of the 68999
protein within a population that maintain the ability of catalyzing
an activity described herein, such as the ubiquitination and
de-ubiquitination of substrates. Functional allelic variants will
typically contain only conservative substitution of one or more
amino acids of SEQ ID NO:2, or substitution, deletion or insertion
of non-critical residues in non-critical regions of the protein.
Non-functional allelic variants are naturally-occurring amino acid
sequence variants of the 68999, e.g., human 68999, protein within a
population that do not have the ability to catalyze an activity
described herein, such as the ubiquitination and de-ubiquitination
of substrates. Non-functional allelic variants will typically
contain a non-conservative substitution, a deletion, or insertion,
or premature truncation of the amino acid sequence of SEQ ID NO:2,
or a substitution, insertion, or deletion in critical residues or
critical regions of the protein.
[0115] Moreover, nucleic acid molecules encoding other 68999 family
members and, thus, which have a nucleotide sequence which differs
from the 68999 sequences of SEQ ID NO:1 or SEQ ID NO:3 are intended
to be within the scope of the invention.
[0116] Antisense Nucleic Acid Molecules, Ribozymes and Modified
68999 Nucleic Acid Molecules
[0117] In another aspect, the invention features, an isolated
nucleic acid molecule which is antisense to 68999. An "antisense"
nucleic acid can include a nucleotide sequence which is
complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. The antisense
nucleic acid can be complementary to an entire 68999 coding strand,
or to only a portion thereof (e.g., the coding region of human
68999 corresponding to SEQ ID NO:3). In another embodiment, the
antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding
68999 (e.g., the 5' and 3' untranslated regions).
[0118] An antisense nucleic acid can be designed such that it is
complementary to the entire coding region of 68999 mRNA, but more
preferably is an oligonucleotide which is antisense to only a
portion of the coding or noncoding region of 68999 mRNA. For
example, the antisense oligonucleotide can be complementary to the
region surrounding the translation start site of 68999 mRNA, e.g.,
between the -10 and +10 regions of the target gene nucleotide
sequence of interest. An antisense oligonucleotide can be, for
example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, or more nucleotides in length.
[0119] An antisense nucleic acid of the invention can be
constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used. The antisense nucleic acid also can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0120] The antisense nucleic acid molecules of the invention are
typically administered to a subject (e.g., by direct injection at a
tissue site), or generated in situ such that they hybridize with or
bind to cellular mRNA and/or genomic DNA encoding a 68999 protein
to thereby inhibit expression of the protein, e.g., by inhibiting
transcription and/or translation. Alternatively, antisense nucleic
acid molecules can be modified to target selected cells and then
administered systemically. For systemic administration, antisense
molecules can be modified such that they specifically or
selectively bind to receptors or antigens expressed on a selected
cell surface, e.g., by linking the antisense nucleic acid molecules
to peptides or antibodies which bind to cell surface receptors or
antigens. The antisense nucleic acid molecules can also be
delivered to cells using the vectors described herein. To achieve
sufficient intracellular concentrations of the antisense molecules,
vector constructs in which the antisense nucleic acid molecule is
placed under the control of a strong pol II or pol III promoter are
preferred.
[0121] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15: 6625-6641). The
antisense nucleic acid molecule can also comprise a 2'--O--
methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:
6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS
Lett. 215: 327-330).
[0122] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. A ribozyme having specificity for a
68999-encoding nucleic acid can include one or more sequences
complementary to the nucleotide sequence of a 68999 cDNA disclosed
herein (i.e., SEQ ID NO:1 or SEQ ID NO:3), and a sequence having
known catalytic sequence responsible for mRNA cleavage (see U.S.
Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:
585-591). For example, a derivative of a Tetrahymena L-19 IVS RNA
can be constructed in which the nucleotide sequence of the active
site is complementary to the nucleotide sequence to be cleaved in a
68999-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No.
4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,
68999 mRNA can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules. See, e.g.,
Bartel, D. and Szostak, J. W. (1993) Science 261: 1411-1418.
[0123] 68999 gene expression can be inhibited by targeting
nucleotide sequences complementary to the regulatory region of the
68999 (e.g., the 68999 promoter and/or enhancers) to form triple
helical structures that prevent transcription of the 68999 gene in
target cells. See generally, Helene, C. (1991) Anticancer Drug Des.
6: 569-84; Helene, C. (1992) Ann. N.Y. Acad. Sci. 660: 27-36; and
Maher, L. J. (1992) Bioassays 14: 807-15. The potential sequences
that can be targeted for triple helix formation can be increased by
creating a so-called "switchback" nucleic acid molecule. Switchback
molecules are synthesized in an alternating 5'-3',3'-5' manner,
such that they base pair with first one strand of a duplex and then
the other, eliminating the necessity for a sizeable stretch of
either purines or pyrimidines to be present on one strand of a
duplex.
[0124] The invention also provides detectably labeled
oligonucleotide primer and probe molecules. Typically, such labels
are chemiluminescent, fluorescent, radioactive, or
colorimetric.
[0125] A 68999 nucleic acid molecule can be modified at the base
moiety, sugar moiety or phosphate backbone to improve, e.g., the
stability, hybridization, or solubility of the molecule. For
example, the deoxyribose phosphate backbone of the nucleic acid
molecules can be modified to generate peptide nucleic acids (see
Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4:
5-23). As used herein, the terms "peptide nucleic acid" or "PNA"
refers to a nucleic acid mimic, e.g., a DNA mimic, in which the
deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of a PNA can allow for specific hybridization to
DNA and RNA under conditions of low ionic strength. The synthesis
of PNA oligomers can be performed using standard solid phase
peptide synthesis protocols as described in Hyrup B. et al. (1996)
supra; Perry-O'Keefe et a.l (1996) Proc. Natl. Acad. Sci. 93:
14670-675.
[0126] PNAs of 68999 nucleic acid molecules can be used in
therapeutic and diagnostic applications. For example, PNAs can be
used as antisense or antigene agents for sequence-specific
modulation of gene expression by, for example, inducing
transcription or translation arrest or inhibiting replication. PNAs
of 68999 nucleic acid molecules can also be used in the analysis of
single base pair mutations in a gene, (e.g., by PNA-directed PCR
clamping); as `artificial restriction enzymes` when used in
combination with other enzymes, (e.g., S1 nucleases (Hyrup B. et
al. (1996) supra)); or as probes or primers for DNA sequencing or
hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe
supra).
[0127] In other embodiments, the oligonucleotide can include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad.
Sci. USA 86: 6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.
Sci. USA 84: 648-652; PCT Publication No. WO88/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In
addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (see, e.g., Krol et al.
(1988) Bio-Techniques 6: 958-976) or intercalating agents. (see,
e.g., Zon (1988) Pharm. Res. 5: 539-549). To this end, the
oligonucleotide can be conjugated to another molecule, (e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, or hybridization-triggered cleavage agent).
[0128] The invention also includes molecular beacon oligonucleotide
primer and probe molecules having at least one region which is
complementary to a 68999 nucleic acid of the invention, two
complementary regions one having a fluorophore and one a quencher
such that the molecular beacon is useful for quantitating the
presence of the 68999 nucleic acid of the invention in a sample.
Molecular beacon nucleic acids are described, for example, in
Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al., U.S.
Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.
[0129] Isolated 68999 Polypeptides
[0130] In another aspect, the invention features, an isolated 68999
protein, or fragment, e.g., a biologically active portion, for use
as immunogens or antigens to raise or test (or more generally to
bind) anti-68999 antibodies. 68999 protein can be isolated from
cells or tissue sources using standard protein purification
techniques. 68999 protein or fragments thereof can be produced by
recombinant DNA techniques or synthesized chemically.
[0131] Polypeptides of the invention include those which arise as a
result of the existence of multiple genes, alternative
transcription events, alternative RNA splicing events, and
alternative translational and post-translational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same post-translational
modifications present when expressed the polypeptide is expressed
in a native cell, or in systems which result in the alteration or
omission of post-translational modifications, e.g., glycosylation
or cleavage, present in a native cell.
[0132] In a preferred embodiment, a 68999 polypeptide has one or
more of the following characteristics:
[0133] For example, the 68999 proteins of the present invention can
have one or more of the following activities:
[0134] the ability to modulate de-ubiquitination of a substrate,
e.g., a ubiquitinated protein targeted for degradation;
[0135] the ability to modulate the processing of poly-ubiquitin
precursors;
[0136] the ability to modulate cellular proliferation and/or
differentiation;
[0137] the ability to modulate apoptosis;
[0138] the ability to modulate transcription and/or cell-cycle
progression;
[0139] the ability to modulate signal-transduction;
[0140] the ability to modulate antigen processing;
[0141] the ability to modulate cell-cell adhesion;
[0142] the ability to modulate receptor-mediated endocytosis;
[0143] the ability to modulate neuropathological conditions;
[0144] the ability to modulate oncogenesis, and;
[0145] the ability to modulate protein levels, e.g., cellular
protein levels.
[0146] it has a molecular weight, e.g., a deduced molecular weight,
preferably ignoring any contribution of post translational
modifications, amino acid composition or other physical
characteristic of a 68999 polypeptide, e.g., a polypeptide of SEQ
ID NO:2;
[0147] it has an overall sequence similarity of at least 60%,
preferably at least 70%, more preferably at least 80%, 90%, or 95%,
with a polypeptide of SEQ ID NO:2;
[0148] it has a ubiquitin carboxyl-terminal hydrolase-1 domain
which is preferably about 70%, 80%, 90% or 95% identical to about
amino acid residues 80 to 111 of SEQ ID NO:2;
[0149] it has a ubiquitin carboxyl-terminal hydrolase-2 domain
which is preferably about 70%, 80%, 90% or 95% identical to about
amino acid residues 313 to 374 of SEQ ID NO:2;
[0150] it has a conserved cysteine in the ubiquitin carboxyl
hydrolase-1 domain, and;
[0151] it has two conserved histidines in the ubiquitin carboxyl
hydrolase-2 domain.
[0152] In a preferred embodiment the 68999 protein, or fragment
thereof, differs from the corresponding sequence in SEQ ID NO:2. In
one embodiment it differs by at least one but by less than 15, 10
or 5 amino acid residues. In another it differs from the
corresponding sequence in SEQ ID NO:2 by at least one residue but
less than 20%, 15%, 10% or 5% of the residues in it differ from the
corresponding sequence in SEQ ID NO:2. (If this comparison requires
alignment the sequences should be aligned for maximum homology.
"Looped" out sequences from deletions or insertions, or mismatches,
are considered differences.) The differences are, preferably,
differences or changes at a non-essential residue or a conservative
substitution. In a preferred embodiment the differences are not in
the ubiquitin carboxyl-terminal hydrolase-1 domain, or the
ubiquitin carboxyl-terminal hydrolase-2 domain. In another
embodiment one or more differences are in the ubiquitin
carboxyl-terminal hydrolase-1, or the ubiquitin carboxyl-terminal
hydrolase-2 domain.
[0153] Other embodiments include a protein that contains one or
more changes in amino acid sequence, e.g., a change in an amino
acid residue which is not essential for activity. Such 68999
proteins differ in amino acid sequence from SEQ ID NO:2, yet retain
biological activity.
[0154] In one embodiment, the protein includes an amino acid
sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%
or more homologous to SEQ ID NO:2.
[0155] In one embodiment, a 68999 protein or fragment is provided
which varies from the sequence of SEQ ID NO:2 in regions defined by
amino acids about 160 to 280 by at least one but by less than 15,
10 or 5 amino acid residues in the protein or fragment but which
does not differ from SEQ ID NO:2 in regions defined by amino acids
about 80 to 111 or 313 to 374. (If this comparison requires
alignment the sequences should be aligned for maximum homology.
"Looped" out sequences from deletions or insertions, or mismatches,
are considered differences.) In some embodiments the difference is
at a non-essential residue or is a conservative substitution, while
in others the difference is at an essential residue or is a
non-conservative substitution.
[0156] In one embodiment, a biologically active portion of a 68999
protein includes a ubiquitin carboxyl-terminal hydrolase-1 domain,
and/or a ubiquitin carboxyl-terminal hydrolase-2 domain. Moreover,
other biologically active portions, in which other regions of the
protein are deleted, can be prepared by recombinant techniques and
evaluated for one or more of the functional activities of a native
68999 protein.
[0157] In a preferred embodiment, the 68999 protein has an amino
acid sequence shown in SEQ ID NO:2. In other embodiments, the 68999
protein is sufficiently or substantially identical to SEQ ID NO:2.
In yet another embodiment, the 68999 protein is sufficiently or
substantially identical to SEQ ID NO:2 and retains the functional
activity of the protein of SEQ ID NO:2, as described in detail in
the subsections above.
[0158] 68999 Chimeric or Fusion Proteins
[0159] In another aspect, the invention provides 68999 chimeric or
fusion proteins. As used herein, a 68999 "chimeric protein" or
"fusion protein" includes a 68999 polypeptide linked to a non-68999
polypeptide. A "non-68999 polypeptide" refers to a polypeptide
having an amino acid sequence corresponding to a protein which is
not substantially homologous to the 68999 protein, e.g., a protein
which is different from the 68999 protein and which is derived from
the same or a different organism. The 68999 polypeptide of the
fusion protein can correspond to all or a portion e.g., a fragment
described herein of a 68999 amino acid sequence. In a preferred
embodiment, a 68999 fusion protein includes at least one (or two)
biologically active portion of a 68999 protein. The non-68999
polypeptide can be fused to the N-terminus or C-terminus of the
68999 polypeptide.
[0160] The fusion protein can include a moiety which has a high
affinity for a ligand. For example, the fusion protein can be a
GST-68999 fusion protein in which the 68999 sequences are fused to
the C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant 68999. Alternatively,
the fusion protein can be a 68999 protein containing a heterologous
signal sequence at its N-terminus. In certain host cells (e.g.,
mammalian host cells), expression and/or secretion of 68999 can be
increased through use of a heterologous signal sequence.
[0161] Fusion proteins can include all or a part of a serum
protein, e.g., a portion of an immunoglobulin (e.g., IgG, IgA, or
IgE), e.g., an Fc region and/or the hinge C1 and C2 sequences of an
immunoglobulin or human serum albumin.
[0162] The 68999 fusion proteins of the invention can be
incorporated into pharmaceutical compositions and administered to a
subject in vivo. The 68999 fusion proteins can be used to affect
the bioavailability of a 68999 substrate. 68999 fusion proteins can
be useful therapeutically for the treatment of disorders caused by,
for example, (i) aberrant modification or mutation of a gene
encoding a 68999 protein; (ii) mis-regulation of the 68999 gene;
and (iii) aberrant post-translational modification of a 68999
protein.
[0163] Moreover, the 68999-fusion proteins of the invention can be
used as immunogens to produce anti-68999 antibodies in a subject,
to purify 68999 ligands and in screening assays to identify
molecules which inhibit the interaction of 68999 with a 68999
substrate.
[0164] Expression vectors are commercially available that already
encode a fusion moiety (e.g., a GST polypeptide). A 68999-encoding
nucleic acid can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the 68999 protein.
[0165] Variants of 68999 Proteins
[0166] In another aspect, the invention also features a variant of
a 68999 polypeptide, e.g., which functions as an agonist (mimetics)
or as an antagonist. Variants of the 68999 proteins can be
generated by mutagenesis, e.g., discrete point mutation, the
insertion or deletion of sequences or the truncation of a 68999
protein. An agonist of the 68999 proteins can retain substantially
the same, or a subset, of the biological activities of the
naturally occurring form of a 68999 protein. An antagonist of a
68999 protein can inhibit one or more of the activities of the
naturally occurring form of the 68999 protein by, for example,
competitively modulating a 68999-mediated activity of a 68999
protein. Thus, specific biological effects can be elicited by
treatment with a variant of limited function. Preferably, treatment
of a subject with a variant having a subset of the biological
activities of the naturally occurring form of the protein has fewer
side effects in a subject relative to treatment with the naturally
occurring form of the 68999 protein.
[0167] Variants of a 68999 protein can be identified by screening
combinatorial libraries of mutants, e.g., truncation mutants, of a
68999 protein for agonist or antagonist activity.
[0168] Libraries of fragments e.g., N terminal, C terminal, or
internal fragments, of a 68999 protein coding sequence can be used
to generate a variegated population of fragments for screening and
subsequent selection of variants of a 68999 protein.
[0169] Variants in which a cysteinee residues is added or deleted
or in which a residue which is glycosylated is added or deleted are
particularly preferred.
[0170] Methods for screening gene products of combinatorial
libraries made by point mutations or truncation, and for screening
cDNA libraries for gene products having a selected property.
Recursive ensemble mutagenesis (REM), a new technique which
enhances the frequency of functional mutants in the libraries, can
be used in combination with the screening assays to identify 68999
variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:
7811-7815; Delgrave et al. (1993) Protein Engineering 6:
327-331).
[0171] Cell based assays can be exploited to analyze a variegated
68999 library. For example, a library of expression vectors can be
transfected into a cell line, e.g., a cell line, which ordinarily
responds to 68999 in a substrate-dependent manner. The transfected
cells are then contacted with 68999 and the effect of the
expression of the mutant on signaling by the 68999 substrate can be
detected, e.g., measuring an activity, such as the ubiquitination
and de-ubiquitination of substrates. Plasmid DNA can then be
recovered from the cells which score for inhibition, or
alternatively, potentiation of signaling by the 68999 substrate,
and the individual clones further characterized.
[0172] In another aspect, the invention features a method of making
a 68999 polypeptide, e.g., a peptide having a non-wild type
activity, e.g., an antagonist, agonist, or super agonist of a
naturally occurring 68999 polypeptide, e.g., a naturally occurring
68999 polypeptide. The method includes altering the sequence of a
68999 polypeptide, e.g., altering the sequence, e.g., by
substitution or deletion of one or more residues of a non-conserved
region, a domain or residue disclosed herein, and testing the
altered polypeptide for the desired activity.
[0173] In another aspect, the invention features a method of making
a fragment or analog of a 68999 polypeptide a biological activity
of a naturally occurring 68999 polypeptide. The method includes
altering the sequence, e.g., by substitution or deletion of one or
more residues, of a 68999 polypeptide, e.g., altering the sequence
of a non-conserved region, or a domain or residue described herein,
and testing the altered polypeptide for the desired activity.
[0174] Anti-68999 Antibodies
[0175] In another aspect, the invention provides an anti-68999
antibody. The term "antibody" as used herein refers to an
immunoglobulin molecule or immunologically active portion thereof,
i.e., an antigen-binding portion. Examples of immunologically
active portions of immunoglobulin molecules include scFV and dcFV
fragments, Fab and F(ab').sub.2 fragments which can be generated by
treating the antibody with an enzyme such as papain or pepsin,
respectively.
[0176] The antibody can be a polyclonal, monoclonal, recombinant,
e.g., a chimeric or humanized, fully human, non-human, e.g.,
murine, or single chain antibody. In a preferred embodiment it has
effector function and can fix complement. The antibody can be
coupled to a toxin or imaging agent.
[0177] A full-length 68999 protein or, antigenic peptide fragment
of 68999 can be used as an immunogen or can be used to identify
anti-68999 antibodies made with other immunogens, e.g., cells,
membrane preparations, and the like. The antigenic peptide of 68999
should include at least 8 amino acid residues of the amino acid
sequence shown in SEQ ID NO:2 and encompasses an epitope of 68999.
Preferably, the antigenic peptide includes at least 10 amino acid
residues, more preferably at least 15 amino acid residues, even
more preferably at least 20 amino acid residues, and most
preferably at least 30 amino acid residues.
[0178] Fragments of 68999 which include residues about 114 to 122,
about 374 to 381, or about 430 to 436 of SEQ ID NO:2 can be used to
make, e.g., used as immunogens or used to characterize the
specificity of an antibody, antibodies against hydrophilic regions
of the 68999 protein. Similarly, fragments of 68999 which include
residues about 273 to 280, about 315 to 324, or about 352 to 359 of
SEQ ID NO:2 can be used to make an antibody against a hydrophobic
region of the 68999 protein; a fragment of 68999 which include
residues about 80 to 90, about 91 to 100 or about 101 to 111 of SEQ
ID NO:2 can be used to make an antibody against the ubiquitin
carboxyl-terminal hydrolase-1 domain of the 68999 protein; a
fragment of 68999 which include residues about 313 to 333, about
334 to 354 or about 355 to 374 of SEQ ID NO:2 can be used to make
an antibody against the ubiquitin carboxyl-terminal hydrolase-2
domain of the 68999 protein.
[0179] Antibodies reactive with, or specific or selective for, any
of these regions, or other regions or domains described herein are
provided.
[0180] Preferred epitopes encompassed by the antigenic peptide are
regions of 68999 are located on the surface of the protein, e.g.,
hydrophilic regions, as well as regions with high antigenicity. For
example, an Emini surface probability analysis of the human 68999
protein sequence can be used to indicate the regions that have a
particularly high probability of being localized to the surface of
the 68999 protein and are thus likely to constitute surface
residues useful for targeting antibody production.
[0181] In a preferred embodiment the antibody binds an epitope on
any domain or region on 68999 proteins described herein.
[0182] Additionally, chimeric, humanized, and completely human
antibodies are also within the scope of the invention. Chimeric,
humanized, but most preferably, completely human antibodies are
desirable for applications which include repeated administration,
e.g., therapeutic treatment of human patients, and some diagnostic
applications.
[0183] Chimeric and humanized monoclonal antibodies, comprising
both human and non-human portions, can be made using standard
recombinant DNA techniques. Such chimeric and humanized monoclonal
antibodies can be produced by recombinant DNA techniques known in
the art, for example using methods described in Robinson et al.
International Application No. PCT/US86/02269; Akira, et al.
European Patent Application 184,187; Taniguchi, M., European Patent
Application 171,496; Morrison et al. European Patent Application
173,494; Neuberger et al. PCT International Publication No. WO
86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.
European Patent Application 125,023; Better et al. (1988) Science
240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:
3439-3443; Liu et al. (1987) J. Immunol. 139: 3521-3526; Sun et al.
(1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al.
(1987) Canc. Res. 47: 999-1005; Wood et al. (1985) Nature 314:
446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:
1553-1559); Morrison, S. L. (1985) Science 229: 1202-1207; Oi et
al. (1986) BioTechniques 4: 214; Winter U.S. Pat. No. 5,225,539;
Jones et al. (1986) Nature 321: 552-525; Verhoeyan et al. (1988)
Science 239: 1534; and Beidler et al. (1988) J. Immunol. 141:
4053-4060.
[0184] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Such antibodies can be
produced using transgenic mice that are incapable of expressing
endogenous immunoglobulin heavy and light chains genes, but which
can express human heavy and light chain genes. See, for example,
Lonberg and Huszar (1995) Int. Rev. Immunol. 13: 65-93); and U.S.
Pat. Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and
5,545,806. In addition, companies such as Abgenix, Inc. (Fremont,
Calif.) and Medarex, Inc. (Princeton, N.J.), can be engaged to
provide human antibodies directed against a selected antigen using
technology similar to that described above.
[0185] Completely human antibodies that recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a murine antibody, is used to guide the selection
of a completely human antibody recognizing the same epitope. This
technology is described by Jespers et al. (1994) Bio/Technology 12:
899-903).
[0186] The anti-68999 antibody can be a single chain antibody. A
single-chain antibody (scFV) can be engineered as described, for
example, in Colcher, D. et al. (1999) Ann. N Y Acad. Sci. 880:
263-80; and Reiter, Y. (1996) Clin. Cancer Res. 2: 245-52. The
single chain antibody can be dimerized or multimerized to generate
multivalent antibodies having specificities for different epitopes
of the same target 68999 protein.
[0187] In a preferred embodiment, the antibody has reduced or no
ability to bind an Fc receptor. For example, it is an isotype or
subtype, fragment or other mutant, which does not support binding
to an Fc receptor, e.g., it has a mutagenized or deleted Fc
receptor binding region.
[0188] An antibody (or fragment thereof) may be conjugated to a
therapeutic moiety such as a cytotoxin, a therapeutic agent or a
radioactive ion. A cytotoxin or cytotoxic agent includes any agent
that is detrimental to cells. Examples include taxol, cytochalasin
B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g.,
maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat.
Nos. 5,475,092, 5,585,499, 5,846,545) and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065,
melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine,
taxol and maytansinoids). Radioactive ions include, but are not
limited to iodine, yttrium and praseodymium. The conjugates of the
invention can be used for modifying a given biological response,
the therapeutic moiety is not to be construed as limited to
classical chemical therapeutic agents. For example, the therapeutic
moiety may be a protein or polypeptide possessing a desired
biological activity. Such proteins may include, for example, a
toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria
toxin; a protein such as tumor necrosis factor, .alpha.-interferon,
.beta.-interferon, nerve growth factor, platelet derived growth
factor, tissue plasminogen activator; or, biological response
modifiers such as, for example, lymphokines, interleukin-1
("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"),
granulocyte macrophase colony stimulating factor ("GM-CSF"),
granulocyte colony stimulating factor ("G-CSF"), or other growth
factors.
[0189] The conjugates of the invention can be used for modifying a
given biological response, the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety may be a protein or polypeptide possessing a
desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor,
.alpha.-interferon, .beta.-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophase colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[0190] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980.
[0191] An anti-68999 antibody (e.g., monoclonal antibody) can be
used to isolate 68999 by standard techniques, such as affinity
chromatography or immunoprecipitation. Moreover, an anti-68999
antibody can be used to detect 68999 protein (e.g., in a cellular
lysate or cell supernatant) in order to evaluate the abundance and
pattern of expression of the protein. Anti-68999 antibodies can be
used diagnostically to monitor protein levels in tissue as part of
a clinical testing procedure, e.g., to determine the efficacy of a
given treatment regimen. Detection can be facilitated by coupling
(i.e., physically linking) the antibody to a detectable substance
(i.e., antibody labelling). Examples of detectable substances
include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S, .sup.3H.
[0192] In preferred embodiments, an antibody can be made by
immunizing with a purified 68999 antigen, or a fragment thereof,
e.g., a fragment described herein, a membrane associated antigen,
tissues, e.g., crude tissue preparations, whole cells, preferably
living cells, lysed cells, or cell fractions.
[0193] Antibodies which bind only a native 68999 protein, only
denatured or otherwise non-native 68999 protein, or which bind
both, are within the invention. Antibodies with linear or
conformational epitopes are within the invention. Conformational
epitopes sometimes can be identified by identifying antibodies
which bind to native but not denatured 68999 protein.
[0194] Recombinant Expression Vectors, Host Cells and Genetically
Engineered Cells
[0195] In another aspect, the invention includes, vectors,
preferably expression vectors, containing a nucleic acid encoding a
polypeptide described herein. As used herein, the term "vector"
refers to a nucleic acid molecule capable of transporting another
nucleic acid to which it has been linked and can include a plasmid,
cosmid or viral vector. The vector can be capable of autonomous
replication or it can integrate into a host DNA. Viral vectors
include, e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses.
[0196] A vector can include a 68999 nucleic acid in a form suitable
for expression of the nucleic acid in a host cell. Preferably the
recombinant expression vector includes one or more regulatory
sequences operatively linked to the nucleic acid sequence to be
expressed. The term "regulatory sequence" includes promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence, as well as
tissue-specific regulatory and/or inducible sequences. The design
of the expression vector can depend on such factors as the choice
of the host cell to be transformed, the level of expression of
protein desired, and the like. The expression vectors of the
invention can be introduced into host cells to thereby produce
proteins or polypeptides, including fusion proteins or
polypeptides, encoded by nucleic acids as described herein (e.g.,
68999 proteins, mutant forms of 68999 proteins, fusion proteins,
and the like).
[0197] The recombinant expression vectors of the invention can be
designed for expression of 68999 proteins in prokaryotic or
eukaryotic cells. For example, polypeptides of the invention can be
expressed in E. coli, insect cells (e.g., using baculovirus
expression vectors), yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel, (1990) Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego,
Calif. Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0198] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
protein to enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion protein.
Such enzymes, and their cognate recognition sequences, include
Factor Xa, thrombin and enterokinase. Typical fusion expression
vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and
Johnson, K. S. (1988) Gene 67: 31-40), pMAL (New England Biolabs,
Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant protein.
[0199] Purified fusion proteins can be used in 68999 activity
assays, (e.g., direct assays or competitive assays described in
detail below), or to generate antibodies specific or selective for
68999 proteins. In a preferred embodiment, a fusion protein
expressed in a retroviral expression vector of the present
invention can be used to infect bone marrow cells which are
subsequently transplanted into irradiated recipients. The pathology
of the subject recipient is then examined after sufficient time has
passed (e.g., six weeks).
[0200] To maximize recombinant protein expression in E. coli is to
express the protein in a host bacteria with an impaired capacity to
proteolytically cleave the recombinant protein (Gottesman, S.,
(1990) Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. 119-128). Another strategy is to
alter the nucleic acid sequence of the nucleic acid to be inserted
into an expression vector so that the individual codons for each
amino acid are those preferentially utilized in E. coli (Wada et
al., (1992) Nucleic Acids Res. 20: 2111-2118). Such alteration of
nucleic acid sequences of the invention can be carried out by
standard DNA synthesis techniques.
[0201] The 68999 expression vector can be a yeast expression
vector, a vector for expression in insect cells, e.g., a
baculovirus expression vector or a vector suitable for expression
in mammalian cells.
[0202] When used in mammalian cells, the expression vector's
control functions are often provided by viral regulatory elements.
For example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40.
[0203] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43: 235-275), in particular promoters of T
cell receptors (Winoto and Baltimore (1989) EMBO J. 8: 729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33: 729-740; Queen and
Baltimore (1983) Cell 33: 741-748), neuron-specific promoters
(e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc.
Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters
(Edlund et al. (1985) Science 230: 912-916), and mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example, the murine hox promoters (Kessel and Gruss (1990) Science
249: 374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3: 537-546).
[0204] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. Regulatory sequences
(e.g., viral promoters and/or enhancers) operatively linked to a
nucleic acid cloned in the antisense orientation can be chosen
which direct the constitutive, tissue specific or cell type
specific expression of antisense RNA in a variety of cell types.
The antisense expression vector can be in the form of a recombinant
plasmid, phagemid or attenuated virus. For a discussion of the
regulation of gene expression using antisense genes see Weintraub,
H. et al., (1986) Reviews--Trends in Genetics 1: 1.
[0205] Another aspect the invention provides a host cell which
includes a nucleic acid molecule described herein, e.g., a 68999
nucleic acid molecule within a recombinant expression vector or a
68999 nucleic acid molecule containing sequences which allow it to
homologously recombine into a specific site of the host cell's
genome. The terms "host cell" and "recombinant host cell" are used
interchangeably herein. Such terms refer not only to the particular
subject cell but to the progeny or potential progeny of such a
cell. Because certain modifications can occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term as used herein.
[0206] A host cell can be any prokaryotic or eukaryotic cell. For
example, a 68999 protein can be expressed in bacterial cells such
as E. coli, insect cells, yeast or mammalian cells (such as Chinese
hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to those skilled in the art.
[0207] Vector DNA can be introduced into host cells via
conventional transformation or transfection techniques. As used
herein, the terms "transformation" and "transfection" are intended
to refer to a variety of art-recognized techniques for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation.
[0208] A host cell of the invention can be used to produce (i.e.,
express) a 68999 protein. Accordingly, the invention further
provides methods for producing a 68999 protein using the host cells
of the invention. In one embodiment, the method includes culturing
the host cell of the invention (into which a recombinant expression
vector encoding a 68999 protein has been introduced) in a suitable
medium such that a 68999 protein is produced. In another
embodiment, the method further includes isolating a 68999 protein
from the medium or the host cell.
[0209] In another aspect, the invention features, a cell or
purified preparation of cells which include a 68999 transgene, or
which otherwise misexpress 68999. The cell preparation can consist
of human or non-human cells, e.g., rodent cells, e.g., mouse or rat
cells, rabbit cells, or pig cells. In preferred embodiments, the
cell or cells include a 68999 transgene, e.g., a heterologous form
of a 68999, e.g., a gene derived from humans (in the case of a
non-human cell). The 68999 transgene can be misexpressed, e.g.,
overexpressed or underexpressed. In other preferred embodiments,
the cell or cells include a gene which misexpresses an endogenous
68999, e.g., a gene the expression of which is disrupted, e.g., a
knockout. Such cells can serve as a model for studying disorders
which are related to mutated or misexpressed 68999 alleles or for
use in drug screening.
[0210] In another aspect, the invention features, a human cell,
e.g., a hematopoietic stem cell, transformed with nucleic acid
which encodes a subject 68999 polypeptide.
[0211] Also provided are cells, preferably human cells, e.g., human
hematopoietic or fibroblast cells, in which an endogenous 68999 is
under the control of a regulatory sequence that does not normally
control the expression of the endogenous 68999 gene. The expression
characteristics of an endogenous gene within a cell, e.g., a cell
line or microorganism, can be modified by inserting a heterologous
DNA regulatory element into the genome of the cell such that the
inserted regulatory element is operably linked to the endogenous
68999 gene. For example, an endogenous 68999 gene which is
"transcriptionally silent," e.g., not normally expressed, or
expressed only at very low levels, can be activated by inserting a
regulatory element which is capable of promoting the expression of
a normally expressed gene product in that cell. Techniques such as
targeted homologous recombinations, can be used to insert the
heterologous DNA as described in, e.g., Chappel, U.S. Pat. No.
5,272,071; WO 91/06667, published in May 16, 1991.
[0212] Transgenic Animals
[0213] The invention provides non-human transgenic animals. Such
animals are useful for studying the function and/or activity of a
68999 protein and for identifying and/or evaluating modulators of
68999 activity. As used herein, a "transgenic animal" is a
non-human animal, preferably a mammal, more preferably a rodent
such as a rat or mouse, in which one or more of the cells of the
animal includes a transgene. Other examples of transgenic animals
include non-human primates, sheep, dogs, cows, goats, chickens,
amphibians, and the like. A transgene is exogenous DNA or a
rearrangement, e.g., a deletion of endogenous chromosomal DNA,
which preferably is integrated into or occurs in the genome of the
cells of a transgenic animal. A transgene can direct the expression
of an encoded gene product in one or more cell types or tissues of
the transgenic animal, other transgenes, e.g., a knockout, reduce
expression. Thus, a transgenic animal can be one in which an
endogenous 68999 gene has been altered by, e.g., by homologous
recombination between the endogenous gene and an exogenous DNA
molecule introduced into a cell of the animal, e.g., an embryonic
cell of the animal, prior to development of the animal.
[0214] Intronic sequences and polyadenylation signals can also be
included in the transgene to increase the efficiency of expression
of the transgene. A tissue-specific regulatory sequence(s) can be
operably linked to a transgene of the invention to direct
expression of a 68999 protein to particular cells. A transgenic
founder animal can be identified based upon the presence of a 68999
transgene in its genome and/or expression of 68999 mRNA in tissues
or cells of the animals. A transgenic founder animal can then be
used to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene encoding a 68999 protein
can further be bred to other transgenic animals carrying other
transgenes.
[0215] 68999 proteins or polypeptides can be expressed in
transgenic animals or plants, e.g., a nucleic acid encoding the
protein or polypeptide can be introduced into the genome of an
animal. In preferred embodiments the nucleic acid is placed under
the control of a tissue specific promoter, e.g., a milk or egg
specific promoter, and recovered from the milk or eggs produced by
the animal. Suitable animals are mice, pigs, cows, goats, and
sheep.
[0216] The invention also includes a population of cells from a
transgenic animal, as discussed, e.g., below.
[0217] Uses
[0218] The nucleic acid molecules, proteins, protein homologs, and
antibodies described herein can be used in one or more of the
following methods: a) screening assays; b) predictive medicine
(e.g., diagnostic assays, prognostic assays, monitoring clinical
trials, and pharmacogenetics); and c) methods of treatment (e.g.,
therapeutic and prophylactic).
[0219] The isolated nucleic acid molecules of the invention can be
used, for example, to express a 68999 protein (e.g., via a
recombinant expression vector in a host cell in gene therapy
applications), to detect a 68999 mRNA (e.g., in a biological
sample) or a genetic alteration in a 68999 gene, and to modulate
68999 activity, as described further below. The 68999 proteins can
be used to treat disorders characterized by insufficient or
excessive production of a 68999 substrate or production of 68999
inhibitors. In addition, the 68999 proteins can be used to screen
for naturally occurring 68999 substrates, to screen for drugs or
compounds which modulate 68999 activity, as well as to treat
disorders characterized by insufficient or excessive production of
68999 protein or production of 68999 protein forms which have
decreased, aberrant or unwanted activity compared to 68999 wild
type protein (e.g., the presence of abnormal amounts of
ubiquitinated proteins in neuropathological conditions such as
Alzheimer's and Pick's disease indicates that ubiquitination and
de-ubiquitination plays a role in various physiological disorders).
Moreover, the anti-68999 antibodies of the invention can be used to
detect and isolate 68999 proteins, regulate the bioavailability of
68999 proteins, and modulate 68999 activity.
[0220] A method of evaluating a compound for the ability to
interact with, e.g., bind, a subject 68999 polypeptide is provided.
The method includes: contacting the compound with the subject 68999
polypeptide; and evaluating ability of the compound to interact
with, e.g., to bind or form a complex with the subject 68999
polypeptide. This method can be performed in vitro, e.g., in a cell
free system, or in vivo, e.g., in a two-hybrid interaction trap
assay. This method can be used to identify naturally occurring
molecules which interact with subject 68999 polypeptide. It can
also be used to find natural or synthetic inhibitors of subject
68999 polypeptide. Screening methods are discussed in more detail
below.
[0221] Screening Assays:
[0222] The invention provides methods (also referred to herein as
"screening assays") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., proteins, peptides,
peptidomimetics, peptoids, small molecules or other drugs) which
bind to 68999 proteins, have a stimulatory or inhibitory effect on,
for example, 68999 expression or 68999 activity, or have a
stimulatory or inhibitory effect on, for example, the expression or
activity of a 68999 substrate. Compounds thus identified can be
used to modulate the activity of target gene products (e.g., 68999
genes) in a therapeutic protocol, to elaborate the biological
function of the target gene product, or to identify compounds that
disrupt normal target gene interactions.
[0223] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a
68999 protein or polypeptide or a biologically active portion
thereof. In another embodiment, the invention provides assays for
screening candidate or test compounds which bind to or modulate the
activity of a 68999 protein or polypeptide or a biologically active
portion thereof.
[0224] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries; peptoid
libraries (libraries of molecules having the functionalities of
peptides, but with a novel, non-peptide backbone which are
resistant to enzymatic degradation but which nevertheless remain
bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem.
37: 2678-85); spatially addressable parallel solid phase or
solution phase libraries; synthetic library methods requiring
deconvolution; the `one-bead one-compound` library method; and
synthetic library methods using affinity chromatography selection.
The biological library and peptoid library approaches are limited
to peptide libraries, while the other four approaches are
applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:
145).
[0225] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90: 6909-13; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91: 11422-426; Zuckermann et al. (1994). J. Med.
Chem. 37: 2678-85; Cho et al. (1993) Science 261: 1303; Carrell et
al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al.
(1994) Angew. Chem. Int. Ed. Engl. 33: 2061; and in Gallop et al.
(1994) J. Med. Chem. 37: 1233-51.
[0226] Libraries of compounds can be presented in solution (e.g.,
Houghten (1992) Biotechniques 13: 412-421), or on beads (Lam (1991)
Nature 354: 82-84), chips (Fodor (1993) Nature 364: 555-556),
bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner USP
'409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:
1865-1869) or on phage (Scott and Smith (1990) Science 249:
386-390; Devlin (1990) Science 249: 404-406; Cwirla et al. (1990)
Proc. Natl. Acad. Sci. 87: 6378-6382; Felici (1991) J. Mol. Biol.
222: 301-310; Ladner supra.).
[0227] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a 68999 protein or biologically active portion
thereof is contacted with a test compound, and the ability of the
test compound to modulate 68999 activity is determined. Determining
the ability of the test compound to modulate 68999 activity can be
accomplished by monitoring, for example, formalin-fixed,
paraffin-processed sections known to contain ubiquitin-protein
conjugate immunoreactivity can be immunostained to localize
ubiquitin carboxyl terminal hydrolase activity (i.e. in cortical
Lewy bodies, neurofibrillary tangles, Rosenthal fibres, Pick
bodies, spinal inclusions in motor neuron disease, and Mallory's
hyaline in alcoholic liver disease). The cell, for example, can be
of mammalian origin, e.g., human.
[0228] The ability of the test compound to modulate 68999 binding
to a compound, e.g., a 68999 substrate, or to bind to 68999 can
also be evaluated. This can be accomplished, for example, by
coupling the compound, e.g., the substrate, with a radioisotope or
enzymatic label such that binding of the compound, e.g., the
substrate, to 68999 can be determined by detecting the labeled
compound, e.g., substrate, in a complex. Alternatively, 68999 could
be coupled with a radioisotope or enzymatic label to monitor the
ability of a test compound to modulate 68999 binding to a 68999
substrate in a complex. For example, compounds (e.g., 68999
substrates) can be labeled with .sup.125I, .sup.35S, .sup.14C, or
.sup.3H, either directly or indirectly, and the radioisotope
detected by direct counting of radioemmission or by scintillation
counting. Alternatively, compounds can be enzymatically labeled
with, for example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product.
[0229] The ability of a compound (e.g., a 68999 substrate) to
interact with 68999 with or without the labeling of any of the
interactants can be evaluated. For example, a microphysiometer can
be used to detect the interaction of a compound with 68999 without
the labeling of either the compound or the 68999. McConnell, H. M.
et al. (1992) Science 257: 1906-1912. As used herein, a
"microphysiometer" (e.g., Cytosensor) is an analytical instrument
that measures the rate at which a cell acidifies its environment
using a light-addressable potentiometric sensor (LAPS). Changes in
this acidification rate can be used as an indicator of the
interaction between a compound and 68999.
[0230] In yet another embodiment, a cell-free assay is provided in
which a 68999 protein or biologically active portion thereof is
contacted with a test compound and the ability of the test compound
to bind to the 68999 protein or biologically active portion thereof
is evaluated. Preferred biologically active portions of the 68999
proteins to be used in assays of the present invention include
fragments which participate in interactions with non-68999
molecules, e.g., fragments with high surface probability
scores.
[0231] Soluble and/or membrane-bound forms of isolated proteins
(e.g., 68999 proteins or biologically active portions thereof) can
be used in the cell-free assays of the invention. When
membrane-bound forms of the protein are used, it may be desirable
to utilize a solubilizing agent. Examples of such solubilizing
agents include non-ionic detergents such as n-octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton.RTM. X-100, Triton.RTM. X-114,
Thesit.RTM., Isotridecypoly(ethylene glycol ether).sub.n,
3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),
3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane
sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane
sulfonate.
[0232] Cell-free assays involve preparing a reaction mixture of the
target gene protein and the test compound under conditions and for
a time sufficient to allow the two components to interact and bind,
thus forming a complex that can be removed and/or detected.
[0233] The interaction between two molecules can also be detected,
e.g., using fluorescence energy transfer (FET) (see, for example,
Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al.,
U.S. Pat. No. 4,868,103). A fluorophore label on the first, `donor`
molecule is selected such that its emitted fluorescent energy will
be absorbed by a fluorescent label on a second, `acceptor`
molecule, which in turn is able to fluoresce due to the absorbed
energy. Alternately, the `donor` protein molecule can simply
utilize the natural fluorescent energy of tryptophan residues.
Labels are chosen that emit different wavelengths of light, such
that the `acceptor` molecule label can be differentiated from that
of the `donor`. Since the efficiency of energy transfer between the
labels is related to the distance separating the molecules, the
spatial relationship between the molecules can be assessed. In a
situation in which binding occurs between the molecules, the
fluorescent emission of the `acceptor` molecule label in the assay
should be maximal. An FET binding event can be conveniently
measured through standard fluorometric detection means well known
in the art (e.g., using a fluorimeter).
[0234] In another embodiment, determining the ability of the 68999
protein to bind to a target molecule can be accomplished using
real-time Biomolecular Interaction Analysis (BIA) (see, e.g.,
Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63: 2338-2345
and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5: 699-705).
"Surface plasmon resonance" or "BIA" detects biospecific
interactions in real time, without labeling any of the interactants
(e.g., BIAcore). Changes in the mass at the binding surface
(indicative of a binding event) result in alterations of the
refractive index of light near the surface (the optical phenomenon
of surface plasmon resonance (SPR)), resulting in a detectable
signal which can be used as an indication of real-time reactions
between biological molecules.
[0235] In one embodiment, the target gene product or the test
substance is anchored onto a solid phase. The target gene
product/test compound complexes anchored on the solid phase can be
detected at the end of the reaction. Preferably, the target gene
product can be anchored onto a solid surface, and the test
compound, (which is not anchored), can be labeled, either directly
or indirectly, with detectable labels discussed herein.
[0236] It may be desirable to immobilize either 68999, an
anti-68999 antibody or its target molecule to facilitate separation
of complexed from uncomplexed forms of one or both of the proteins,
as well as to accommodate automation of the assay. Binding of a
test compound to a 68999 protein, or interaction of a 68999 protein
with a target molecule in the presence and absence of a candidate
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtiter plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/68999 fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtiter plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or 68999 protein, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtiter plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described above. Alternatively, the complexes can be dissociated
from the matrix, and the level of 68999 binding or activity
determined using standard techniques.
[0237] Other techniques for immobilizing either a 68999 protein or
a target molecule on matrices include using conjugation of biotin
and streptavidin. Biotinylated 68999 protein or target molecules
can be prepared from biotin-NHS(N-hydroxy-succinimide) using
techniques known in the art (e.g., biotinylation kit, Pierce
Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
[0238] In order to conduct the assay, the non-immobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously non-immobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
non-immobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific or selective for the immobilized
component (the antibody, in turn, can be directly labeled or
indirectly labeled with, e.g., a labeled anti-Ig antibody).
[0239] In one embodiment, this assay is performed utilizing
antibodies reactive with 68999 protein or target molecules but
which do not interfere with binding of the 68999 protein to its
target molecule. Such antibodies can be derivatized to the wells of
the plate, and unbound target or 68999 protein trapped in the wells
by antibody conjugation. Methods for detecting such complexes, in
addition to those described above for the GST-immobilized
complexes, include immunodetection of complexes using antibodies
reactive with the 68999 protein or target molecule, as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the 68999 protein or target molecule.
[0240] Alternatively, cell free assays can be conducted in a liquid
phase. In such an assay, the reaction products are separated from
unreacted components, by any of a number of standard techniques,
including but not limited to: differential centrifugation (see, for
example, Rivas, G., and Minton, A. P., (1993) Trends Biochem Sci
18: 284-7); chromatography (gel filtration chromatography,
ion-exchange chromatography); electrophoresis (see, e.g., Ausubel,
F. et al., eds. (1999) Current Protocols in Molecular Biology, J.
Wiley, New York.); and immunoprecipitation (see, for example,
Ausubel, F. et al., eds. (1999) Current Protocols in Molecular
Biology, J. Wiley, New York). Such resins and chromatographic
techniques are known to one skilled in the art (see, e.g.,
Heegaard, N. H., (1998) J Mol Recognit 11: 141-8; Hage, D. S., and
Tweed, S. A. (1997) J Chromatogr B Biomed Sci Appl. 699: 499-525).
Further, fluorescence energy transfer can also be conveniently
utilized, as described herein, to detect binding without further
purification of the complex from solution.
[0241] In a preferred embodiment, the assay includes contacting the
68999 protein or biologically active portion thereof with a known
compound which binds 68999 to form an assay mixture, contacting the
assay mixture with a test compound, and determining the ability of
the test compound to interact with a 68999 protein, wherein
determining the ability of the test compound to interact with a
68999 protein includes determining the ability of the test compound
to preferentially bind to 68999 or biologically active portion
thereof, or to modulate the activity of a target molecule, as
compared to the known compound.
[0242] The target gene products of the invention can, in vivo,
interact with one or more cellular or extracellular macromolecules,
such as proteins. For the purposes of this discussion, such
cellular and extracellular macromolecules are referred to herein as
"binding partners." Compounds that disrupt such interactions can be
useful in regulating the activity of the target gene product. Such
compounds can include, but are not limited to molecules such as
antibodies, peptides, and small molecules. The preferred target
genes/products for use in this embodiment are the 68999 genes
herein identified. In an alternative embodiment, the invention
provides methods for determining the ability of the test compound
to modulate the activity of a 68999 protein through modulation of
the activity of a downstream effector of a 68999 target molecule.
For example, the activity of the effector molecule on an
appropriate target can be determined, or the binding of the
effector to an appropriate target can be determined, as previously
described.
[0243] To identify compounds that interfere with the interaction
between the target gene product and its cellular or extracellular
binding partner(s), a reaction mixture containing the target gene
product and the binding partner is prepared, under conditions and
for a time sufficient, to allow the two products to form complex.
In order to test an inhibitory agent, the reaction mixture is
provided in the presence and absence of the test compound. The test
compound can be initially included in the reaction mixture, or can
be added at a time subsequent to the addition of the target gene
and its cellular or extracellular binding partner. Control reaction
mixtures are incubated without the test compound or with a placebo.
The formation of any complexes between the target gene product and
the cellular or extracellular binding partner is then detected. The
formation of a complex in the control reaction, but not in the
reaction mixture containing the test compound, indicates that the
compound interferes with the interaction of the target gene product
and the interactive binding partner. Additionally, complex
formation within reaction mixtures containing the test compound and
normal target gene product can also be compared to complex
formation within reaction mixtures containing the test compound and
mutant target gene product. This comparison can be important in
those cases wherein it is desirable to identify compounds that
disrupt interactions of mutant but not normal target gene
products.
[0244] These assays can be conducted in a heterogeneous or
homogeneous format. Heterogeneous assays involve anchoring either
the target gene product or the binding partner onto a solid phase,
and detecting complexes anchored on the solid phase at the end of
the reaction. In homogeneous assays, the entire reaction is carried
out in a liquid phase. In either approach, the order of addition of
reactants can be varied to obtain different information about the
compounds being tested. For example, test compounds that interfere
with the interaction between the target gene products and the
binding partners, e.g., by competition, can be identified by
conducting the reaction in the presence of the test substance.
Alternatively, test compounds that disrupt preformed complexes,
e.g., compounds with higher binding constants that displace one of
the components from the complex, can be tested by adding the test
compound to the reaction mixture after complexes have been formed.
The various formats are briefly described below.
[0245] In a heterogeneous assay system, either the target gene
product or the interactive cellular or extracellular binding
partner, is anchored onto a solid surface (e.g., a microtiter
plate), while the non-anchored species is labeled, either directly
or indirectly. The anchored species can be immobilized by
non-covalent or covalent attachments. Alternatively, an immobilized
antibody specific or selective for the species to be anchored can
be used to anchor the species to the solid surface.
[0246] In order to conduct the assay, the partner of the
immobilized species is exposed to the coated surface with or
without the test compound. After the reaction is complete,
unreacted components are removed (e.g., by washing) and any
complexes formed will remain immobilized on the solid surface.
Where the non-immobilized species is pre-labeled, the detection of
label immobilized on the surface indicates that complexes were
formed. Where the non-immobilized species is not pre-labeled, an
indirect label can be used to detect complexes anchored on the
surface; e.g., using a labeled antibody specific or selective for
the initially non-immobilized species (the antibody, in turn, can
be directly labeled or indirectly labeled with, e.g., a labeled
anti-Ig antibody). Depending upon the order of addition of reaction
components, test compounds that inhibit complex formation or that
disrupt preformed complexes can be detected.
[0247] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from unreacted components, and complexes
detected; e.g., using an immobilized antibody specific or selective
for one of the binding components to anchor any complexes formed in
solution, and a labeled antibody specific or selective for the
other partner to detect anchored complexes. Again, depending upon
the order of addition of reactants to the liquid phase, test
compounds that inhibit complex or that disrupt preformed complexes
can be identified.
[0248] In an alternate embodiment of the invention, a homogeneous
assay can be used. For example, a preformed complex of the target
gene product and the interactive cellular or extracellular binding
partner product is prepared in that either the target gene products
or their binding partners are labeled, but the signal generated by
the label is quenched due to complex formation (see, e.g., U.S.
Pat. No. 4,109,496 that utilizes this approach for immunoassays).
The addition of a test substance that competes with and displaces
one of the species from the preformed complex will result in the
generation of a signal above background. In this way, test
substances that disrupt target gene product-binding partner
interaction can be identified.
[0249] In yet another aspect, the 68999 proteins can be used as
"bait proteins" in a two-hybrid assay or three-hybrid assay (see,
e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:
223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046-12054;
Bartel et al. (1993) Biotechniques 14: 920-924; Iwabuchi et al.
(1993) Oncogene 8: 1693-1696; and Brent WO94/10300), to identify
other proteins, which bind to or interact with 68999
("68999-binding proteins" or "68999-bp") and are involved in 68999
activity. Such 68999-bps can be activators or inhibitors of signals
by the 68999 proteins or 68999 targets as, for example, downstream
elements of a 68999-mediated signaling pathway.
[0250] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a 68999
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
(Alternatively the: 68999 protein can be the fused to the activator
domain.) If the "bait" and the "prey" proteins are able to
interact, in vivo, forming a 68999-dependent complex, the
DNA-binding and activation domains of the transcription factor are
brought into close proximity. This proximity allows transcription
of a reporter gene (e.g., lacZ) which is operably linked to a
transcriptional regulatory site responsive to the transcription
factor. Expression of the reporter gene can be detected and cell
colonies containing the functional transcription factor can be
isolated and used to obtain the cloned gene which encodes the
protein which interacts with the 68999 protein.
[0251] In another embodiment, modulators of 68999 expression are
identified. For example, a cell or cell free mixture is contacted
with a candidate compound and the expression of 68999 mRNA or
protein evaluated relative to the level of expression of 68999 mRNA
or protein in the absence of the candidate compound. When
expression of 68999 mRNA or protein is greater in the presence of
the candidate compound than in its absence, the candidate compound
is identified as a stimulator of 68999 mRNA or protein expression.
Alternatively, when expression of 68999 mRNA or protein is less
(statistically significantly less) in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of 68999 mRNA or protein expression. The level of
68999 mRNA or protein expression can be determined by methods
described herein for detecting 68999 mRNA or protein.
[0252] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a cell
free assay, and the ability of the agent to modulate the activity
of a 68999 protein can be confirmed in vivo, e.g., in an animal
such as an animal model for aberrant or deficient neurological
function.
[0253] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein (e.g., a 68999 modulating agent, an antisense
68999 nucleic acid molecule, a 68999-specific antibody, or a
68999-binding partner) in an appropriate animal model to determine
the efficacy, toxicity, side effects, or mechanism of action, of
treatment with such an agent. Furthermore, novel agents identified
by the above-described screening assays can be used for treatments
as described herein.
[0254] Detection Assays
[0255] Portions or fragments of the nucleic acid sequences
identified herein can be used as polynucleotide reagents. For
example, these sequences can be used to: (i) map their respective
genes on a chromosome e.g., to locate gene regions associated with
genetic disease or to associate 68999 with a disease; (ii) identify
an individual from a minute biological sample (tissue typing); and
(iii) aid in forensic identification of a biological sample. These
applications are described in the subsections below.
[0256] Chromosome Mapping
[0257] The 68999 nucleotide sequences or portions thereof can be
used to map the location of the 68999 genes on a chromosome. This
process is called chromosome mapping. Chromosome mapping is useful
in correlating the 68999 sequences with genes associated with
disease.
[0258] Briefly, 68999 genes can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp in length) from the
68999 nucleotide sequences. These primers can then be used for PCR
screening of somatic cell hybrids containing individual human
chromosomes. Only those hybrids containing the human gene
corresponding to the 68999 sequences will yield an amplified
fragment.
[0259] A panel of somatic cell hybrids in which each cell line
contains either a single human chromosome or a small number of
human chromosomes, and a full set of mouse chromosomes, can allow
easy mapping of individual genes to specific human chromosomes.
(D'Eustachio P. et al. (1983) Science 220: 919-924).
[0260] Other mapping strategies e.g., in situ hybridization
(described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:
6223-27), pre-screening with labeled flow-sorted chromosomes, and
pre-selection by hybridization to chromosome specific cDNA
libraries can be used to map 68999 to a chromosomal location.
[0261] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. The FISH technique can be
used with a DNA sequence as short as 500 or 600 bases. However,
clones larger than 1,000 bases have a higher likelihood of binding
to a unique chromosomal location with sufficient signal intensity
for simple detection. Preferably 1,000 bases, and more preferably
2,000 bases will suffice to get good results at a reasonable amount
of time. For a review of this technique, see Verma et al. (1988)
Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,
New York).
[0262] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0263] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between a gene and a disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland, J. et al. (1987) Nature, 325: 783-787.
[0264] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the 68999 gene, can be determined. If a mutation is observed in
some or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
[0265] Tissue Typing
[0266] 68999 sequences can be used to identify individuals from
biological samples using, e.g., restriction fragment length
polymorphism (RFLP). In this technique, an individual's genomic DNA
is digested with one or more restriction enzymes, the fragments
separated, e.g., in a Southern blot, and probed to yield bands for
identification. The sequences of the present invention are useful
as additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
[0267] Furthermore, the sequences of the present invention can also
be used to determine the actual base-by-base DNA sequence of
selected portions of an individual's genome. Thus, the 68999
nucleotide sequences described herein can be used to prepare two
PCR primers from the 5' and 3' ends of the sequences. These primers
can then be used to amplify an individual's DNA and subsequently
sequence it. Panels of corresponding DNA sequences from
individuals, prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences.
[0268] Allelic variation occurs to some degree in the coding
regions of these sequences, and to a greater degree in the
noncoding regions. Each of the sequences described herein can, to
some degree, be used as a standard against which DNA from an
individual can be compared for identification purposes. Because
greater numbers of polymorphisms occur in the noncoding regions,
fewer sequences are necessary to differentiate individuals. The
noncoding sequences of SEQ ID NO:1 can provide positive individual
identification with a panel of perhaps 10 to 1,000 primers which
each yield a noncoding amplified sequence of 100 bases. If
predicted coding sequences, such as those in SEQ ID NO:3 are used,
a more appropriate number of primers for positive individual
identification would be 500-2,000.
[0269] If a panel of reagents from 68999 nucleotide sequences
described herein is used to generate a unique identification
database for an individual, those same reagents can later be used
to identify tissue from that individual. Using the unique
identification database, positive identification of the individual,
living or dead, can be made from extremely small tissue
samples.
[0270] Use of Partial 68999 Sequences in Forensic Biology
[0271] DNA-based identification techniques can also be used in
forensic biology. To make such an identification, PCR technology
can be used to amplify DNA sequences taken from very small
biological samples such as tissues, e.g., hair or skin, or body
fluids, e.g., blood, saliva, or semen found at a crime scene. The
amplified sequence can then be compared to a standard, thereby
allowing identification of the origin of the biological sample.
[0272] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As mentioned
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to
noncoding regions of SEQ ID NO:1 (e.g., fragments derived from the
noncoding regions of SEQ ID NO:1 having a length of at least 20
bases, preferably at least 30 bases) are particularly appropriate
for this use.
[0273] The 68999 nucleotide sequences described herein can further
be used to provide polynucleotide reagents, e.g., labeled or
labelable probes which can be used in, for example, an in situ
hybridization technique, to identify a specific tissue. This can be
very useful in cases where a forensic pathologist is presented with
a tissue of unknown origin. Panels of such 68999 probes can be used
to identify tissue by species and/or by organ type.
[0274] In a similar fashion, these reagents, e.g., 68999 primers or
probes can be used to screen tissue culture for contamination (i.e.
screen for the presence of a mixture of different types of cells in
a culture).
[0275] Predictive Medicine
[0276] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual.
[0277] Generally, the invention provides, a method of determining
if a subject is at risk for a disorder related to a lesion in or
the misexpression of a gene which encodes 68999.
[0278] Such disorders include, e.g., a disorder associated with the
misexpression of 68999 gene; a disorder of the necrological
system.
[0279] The method includes one or more of the following:
[0280] detecting, in a tissue of the subject, the presence or
absence of a mutation which affects the expression of the 68999
gene, or detecting the presence or absence of a mutation in a
region which controls the expression of the gene, e.g., a mutation
in the 5' control region;
[0281] detecting, in a tissue of the subject, the presence or
absence of a mutation which alters the structure of the 68999
gene;
[0282] detecting, in a tissue of the subject, the misexpression of
the 68999 gene, at the mRNA level, e.g., detecting a non-wild type
level of a mRNA;
[0283] detecting, in a tissue of the subject, the misexpression of
the gene, at the protein level, e.g., detecting a non-wild type
level of a 68999 polypeptide.
[0284] In preferred embodiments the method includes: ascertaining
the existence of at least one of: a deletion of one or more
nucleotides from the 68999 gene; an insertion of one or more
nucleotides into the gene, a point mutation, e.g., a substitution
of one or more nucleotides of the gene, a gross chromosomal
rearrangement of the gene, e.g., a translocation, inversion, or
deletion.
[0285] For example, detecting the genetic lesion can include: (i)
providing a probe/primer including an oligonucleotide containing a
region of nucleotide sequence which hybridizes to a sense or
antisense sequence from SEQ ID NO:1, or naturally occurring mutants
thereof or 5' or 3' flanking sequences naturally associated with
the 68999 gene; (ii) exposing the probe/primer to nucleic acid of
the tissue; and detecting, by hybridization, e.g., in situ
hybridization, of the probe/primer to the nucleic acid, the
presence or absence of the genetic lesion.
[0286] In preferred embodiments detecting the misexpression
includes ascertaining the existence of at least one of: an
alteration in the level of a messenger RNA transcript of the 68999
gene; the presence of a non-wild type splicing pattern of a
messenger RNA transcript of the gene; or a non-wild type level of
68999.
[0287] Methods of the invention can be used prenatally or to
determine if a subject's offspring will be at risk for a
disorder.
[0288] In preferred embodiments the method includes determining the
structure of a 68999 gene, an abnormal structure being indicative
of risk for the disorder.
[0289] In preferred embodiments the method includes contacting a
sample from the subject with an antibody to the 68999 protein or a
nucleic acid, which hybridizes specifically with the gene. These
and other embodiments are discussed below.
[0290] Diagnostic and Prognostic Assays
[0291] The presence, level, or absence of 68999 protein or nucleic
acid in a biological sample can be evaluated by obtaining a
biological sample from a test subject and contacting the biological
sample with a compound or an agent capable of detecting 68999
protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes
68999 protein such that the presence of 68999 protein or nucleic
acid is detected in the biological sample. The term "biological
sample" includes tissues, cells and biological fluids isolated from
a subject, as well as tissues, cells and fluids present within a
subject. A preferred biological sample is serum. The level of
expression of the 68999 gene can be measured in a number of ways,
including, but not limited to: measuring the mRNA encoded by the
68999 genes; measuring the amount of protein encoded by the 68999
genes; or measuring the activity of the protein encoded by the
68999 genes.
[0292] The level of mRNA corresponding to the 68999 gene in a cell
can be determined both by in situ and by in vitro formats.
[0293] The isolated mRNA can be used in hybridization or
amplification assays that include, but are not limited to, Southern
or Northern analyses, polymerase chain reaction analyses and probe
arrays. One preferred diagnostic method for the detection of mRNA
levels involves contacting the isolated mRNA with a nucleic acid
molecule (probe) that can hybridize to the mRNA encoded by the gene
being detected. The nucleic acid probe can be, for example, a
full-length 68999 nucleic acid, such as the nucleic acid of SEQ ID
NO:1, or a portion thereof, such as an oligonucleotide of at least
7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient
to specifically hybridize under stringent conditions to 68999 mRNA
or genomic DNA. Other suitable probes for use in the diagnostic
assays are described herein.
[0294] In one format, mRNA (or cDNA) is immobilized on a surface
and contacted with the probes, for example by running the isolated
mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as nitrocellulose. In an alternative format, the
probes are immobilized on a surface and the mRNA (or cDNA) is
contacted with the probes, for example, in a two-dimensional gene
chip array. A skilled artisan can adapt known mRNA detection
methods for use in detecting the level of mRNA encoded by the 68999
genes.
[0295] The level of mRNA in a sample that is encoded by one of
68999 can be evaluated with nucleic acid amplification, e.g., by
rtPCR (Mullis (1987) U.S. Pat. No. 4,683,202), ligase chain
reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189-193),
self sustained sequence replication (Guatelli et al., (1990) Proc.
Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification
system (Kwoh et al., (1989), Proc. Natl. Acad. Sci. USA 86:
1173-1177), Q-Beta Replicase (Lizardi et al., (1988) Bio/Technology
6: 1197), rolling circle replication (Lizardi et al., U.S. Pat. No.
5,854,033) or any other nucleic acid amplification method, followed
by the detection of the amplified molecules using techniques known
in the art. As used herein, amplification primers are defined as
being a pair of nucleic acid molecules that can anneal to 5' or 3'
regions of a gene (plus and minus strands, respectively, or
vice-versa) and contain a short region in between. In general,
amplification primers are from about 10 to 30 nucleotides in length
and flank a region from about 50 to 200 nucleotides in length.
Under appropriate conditions and with appropriate reagents, such
primers permit the amplification of a nucleic acid molecule
comprising the nucleotide sequence flanked by the primers.
[0296] For in situ methods, a cell or tissue sample can be
prepared/processed and immobilized on a support, typically a glass
slide, and then contacted with a probe that can hybridize to mRNA
that encodes the 68999 gene being analyzed.
[0297] In another embodiment, the methods further contacting a
control sample with a compound or agent capable of detecting 68999
mRNA, or genomic DNA, and comparing the presence of 68999 mRNA or
genomic DNA in the control sample with the presence of 68999 mRNA
or genomic DNA in the test sample.
[0298] A variety of methods can be used to determine the level of
protein encoded by 68999. In general, these methods include
contacting an agent that selectively binds to the protein, such as
an antibody with a sample, to evaluate the level of protein in the
sample. In a preferred embodiment, the antibody bears a detectable
label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or
F(ab').sub.2) can be used. The term "labeled", with regard to the
probe or antibody, is intended to encompass direct labeling of the
probe or antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as indirect
labeling of the probe or antibody by reactivity with a detectable
substance. Examples of detectable substances are provided
herein.
[0299] The detection methods can be used to detect 68999 protein in
a biological sample in vitro as well as in vivo. In vitro
techniques for detection of 68999 protein include enzyme linked
immunosorbent assays (ELISAs), immunoprecipitations,
immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay
(RIA), and Western blot analysis. In vivo techniques for detection
of 68999 protein include introducing into a subject a labeled
anti-68999 antibody. For example, the antibody can be labeled with
a radioactive marker whose presence and location in a subject can
be detected by standard imaging techniques.
[0300] In another embodiment, the methods further include
contacting the control sample with a compound or agent capable of
detecting 68999 protein, and comparing the presence of 68999
protein in the control sample with the presence of 68999 protein in
the test sample.
[0301] The invention also includes kits for detecting the presence
of 68999 in a biological sample. For example, the kit can include a
compound or agent capable of detecting 68999 protein or mRNA in a
biological sample; and a standard. The compound or agent can be
packaged in a suitable container. The kit can further comprise
instructions for using the kit to detect 68999 protein or nucleic
acid.
[0302] For antibody-based kits, the kit can include: (1) a first
antibody (e.g., attached to a solid support) which binds to a
polypeptide corresponding to a marker of the invention; and,
optionally, (2) a second, different antibody which binds to either
the polypeptide or the first antibody and is conjugated to a
detectable agent.
[0303] For oligonucleotide-based kits, the kit can include: (1) an
oligonucleotide, e.g., a detectably labeled oligonucleotide, which
hybridizes to a nucleic acid sequence encoding a polypeptide
corresponding to a marker of the invention or (2) a pair of primers
useful for amplifying a nucleic acid molecule corresponding to a
marker of the invention. The kit can also includes a buffering
agent, a preservative, or a protein stabilizing agent. The kit can
also includes components necessary for detecting the detectable
agent (e.g., an enzyme or a substrate). The kit can also contain a
control sample or a series of control samples which can be assayed
and compared to the test sample contained. Each component of the
kit can be enclosed within an individual container and all of the
various containers can be within a single package, along with
instructions for interpreting the results of the assays performed
using the kit.
[0304] The diagnostic methods described herein can identify
subjects having, or at risk of developing, a disease or disorder
associated with misexpressed or aberrant or unwanted 68999
expression or activity. As used herein, the term "unwanted"
includes an unwanted phenomenon involved in a biological response
such as pain or deregulated cell proliferation.
[0305] In one embodiment, a disease or disorder associated with
aberrant or unwanted 68999 expression or activity is identified. A
test sample is obtained from a subject and 68999 protein or nucleic
acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level,
e.g., the presence or absence, of 68999 protein or nucleic acid is
diagnostic for a subject having or at risk of developing a disease
or disorder associated with aberrant or unwanted 68999 expression
or activity. As used herein, a "test sample" refers to a biological
sample obtained from a subject of interest, including a biological
fluid (e.g., serum), cell sample, or tissue.
[0306] The prognostic assays described herein can be used to
determine whether a subject can be administered an agent (e.g., an
agonist, antagonist, peptidomimetic, protein, peptide, nucleic
acid, small molecule, or other drug candidate) to treat a disease
or disorder associated with aberrant or unwanted 68999 expression
or activity. For example, such methods can be used to determine
whether a subject can be effectively treated with an agent for a
cell filamentous inclusion found in neurons in the major human
neurodegenerative diseases disorder.
[0307] The methods of the invention can also be used to detect
genetic alterations in a 68999 gene, thereby determining if a
subject with the altered gene is at risk for a disorder
characterized by misregulation in 68999 protein activity or nucleic
acid expression, such as a neurodegenerative disease. In preferred
embodiments, the methods include detecting, in a sample from the
subject, the presence or absence of a genetic alteration
characterized by at least one of an alteration affecting the
integrity of a gene encoding a 68999-protein, or the mis-expression
of the 68999 gene. For example, such genetic alterations can be
detected by ascertaining the existence of at least one of 1) a
deletion of one or more nucleotides from a 68999 gene; 2) an
addition of one or more nucleotides to a 68999 gene; 3) a
substitution of one or more nucleotides of a 68999 gene, 4) a
chromosomal rearrangement of a 68999 gene; 5) an alteration in the
level of a messenger RNA transcript of a 68999 gene, 6) aberrant
modification of a 68999 gene, such as of the methylation pattern of
the genomic DNA, 7) the presence of a non-wild type splicing
pattern of a messenger RNA transcript of a 68999 gene, 8) a
non-wild type level of a 68999-protein, 9) allelic loss of a 68999
gene, and 10) inappropriate post-translational modification of a
68999-protein.
[0308] An alteration can be detected without a probe/primer in a
polymerase chain reaction, such as anchor PCR or RACE PCR, or,
alternatively, in a ligation chain reaction (LCR), the latter of
which can be particularly useful for detecting point mutations in
the 68999-gene. This method can include the steps of collecting a
sample of cells from a subject, isolating nucleic acid (e.g.,
genomic, mRNA or both) from the sample, contacting the nucleic acid
sample with one or more primers which specifically hybridize to a
68999 gene under conditions such that hybridization and
amplification of the 68999 gene (if present) occurs, and detecting
the presence or absence of an amplification product, or detecting
the size of the amplification product and comparing the length to a
control sample. It is anticipated that PCR and/or LCR may be
desirable to use as a preliminary amplification step in conjunction
with any of the techniques used for detecting mutations described
herein. Alternatively, other amplification methods described herein
or known in the art can be used.
[0309] In another embodiment, mutations in a 68999 gene from a
sample cell can be identified by detecting alterations in
restriction enzyme cleavage patterns. For example, sample and
control DNA is isolated, amplified (optionally), digested with one
or more restriction endonucleases, and fragment length sizes are
determined, e.g., by gel electrophoresis and compared. Differences
in fragment length sizes between sample and control DNA indicates
mutations in the sample DNA. Moreover, the use of sequence specific
ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used
to score for the presence of specific mutations by development or
loss of a ribozyme cleavage site.
[0310] In other embodiments, genetic mutations in 68999 can be
identified by hybridizing a sample and control nucleic acids, e.g.,
DNA or RNA, two dimensional arrays, e.g., chip based arrays. Such
arrays include a plurality of addresses, each of which is
positionally distinguishable from the other. A different probe is
located at each address of the plurality. The arrays can have a
high density of addresses, e.g., can contain hundreds or thousands
of oligonucleotides probes (Cronin, M. T. et al. (1996) Human
Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2:
753-759). For example, genetic mutations in 68999 can be identified
in two dimensional arrays containing light-generated DNA probes as
described in Cronin, M. T. et al. supra. Briefly, a first
hybridization array of probes can be used to scan through long
stretches of DNA in a sample and control to identify base changes
between the sequences by making linear arrays of sequential
overlapping probes. This step allows the identification of point
mutations. This step is followed by a second hybridization array
that allows the characterization of specific mutations by using
smaller, specialized probe arrays complementary to all variants or
mutations detected. Each mutation array is composed of parallel
probe sets, one complementary to the wild-type gene and the other
complementary to the mutant gene.
[0311] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
68999 gene and detect mutations by comparing the sequence of the
sample 68999 with the corresponding wild-type (control) sequence.
Automated sequencing procedures can be utilized when performing the
diagnostic assays (Naeve C. W. et al. (1995) Biotechniques 19:
448-53), including sequencing by mass spectrometry.
[0312] Other methods for detecting mutations in the 68999 gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al. (1985) Science 230: 1242; Cotton et al. (1988) Proc. Natl.
Acad Sci USA 85: 4397; Saleeba et al. (1992) Methods Enzymol. 217:
286-295).
[0313] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in 68999
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.
(1994) Carcinogenesis 15: 1657-1662; U.S. Pat. No. 5,459,039).
[0314] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in 68999 genes. For
example, single strand conformation polymorphism (SSCP) can be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad.
Sci USA: 86: 2766, see also Cotton (1993) Mutat. Res. 285: 125-144;
and Hayashi (1992) Genet. Anal. Tech. Appl. 9: 73-79).
Single-stranded DNA fragments of sample and control 68999 nucleic
acids will be denatured and allowed to renature. The secondary
structure of single-stranded nucleic acids varies according to
sequence, the resulting alteration in electrophoretic mobility
enables the detection of even a single base change. The DNA
fragments can be labeled or detected with labeled probes. The
sensitivity of the assay can be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In a preferred embodiment, the subject method
utilizes heteroduplex analysis to separate double stranded
heteroduplex molecules on the basis of changes in electrophoretic
mobility (Keen et al. (1991) Trends Genet 7: 5).
[0315] In yet another embodiment, the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313: 495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem
265: 12753).
[0316] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension (Saiki et al. (1986) Nature 324: 163); Saiki et al.
(1989) Proc. Natl. Acad. Sci USA 86: 6230).
[0317] Alternatively, allele specific amplification technology
which depends on selective PCR amplification can be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification can carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res. 17: 2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent, or
reduce polymerase extension (Prossner (1993) Tibtech 11: 238). In
addition it may be desirable to introduce a novel restriction site
in the region of the mutation to create cleavage-based detection
(Gasparini et al. (1992) Mol. Cell Probes 6: 1). It is anticipated
that in certain embodiments amplification can also be performed
using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad.
Sci USA 88: 189-93). In such cases, ligation will occur only if
there is a perfect match at the 3' end of the 5' sequence making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[0318] The methods described herein can be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which can
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving a 68999 gene.
[0319] Use of 68999 Molecules as Surrogate Markers
[0320] The 68999 molecules of the invention are also useful as
markers of disorders or disease states, as markers for precursors
of disease states, as markers for predisposition of disease states,
as markers of drug activity, or as markers of the pharmacogenomic
profile of a subject. Using the methods described herein, the
presence, absence and/or quantity of the 68999 molecules of the
invention can be detected, and can be correlated with one or more
biological states in vivo. For example, the 68999 molecules of the
invention can serve as surrogate markers for one or more disorders
or disease states or for conditions leading up to disease states.
As used herein, a "surrogate marker" is an objective biochemical
marker which correlates with the absence or presence of a disease
or disorder, or with the progression of a disease or disorder
(e.g., with the presence or absence of a tumor). The presence or
quantity of such markers is independent of the disease. Therefore,
these markers can serve to indicate whether a particular course of
treatment is effective in lessening a disease state or disorder.
Surrogate markers are of particular use when the presence or extent
of a disease state or disorder is difficult to assess through
standard methodologies (e.g., early stage tumors), or when an
assessment of disease progression is desired before a potentially
dangerous clinical endpoint is reached (e.g., an assessment of
cardiovascular disease can be made using cholesterol levels as a
surrogate marker, and an analysis of HIV infection can be made
using HIV RNA levels as a surrogate marker, well in advance of the
undesirable clinical outcomes of myocardial infarction or
fully-developed AIDS). Examples of the use of surrogate markers in
the art include: Koomen et al. (2000) J. Mass. Spectrom. 35:
258-264; and James (1994) AIDS Treatment News Archive 209.
[0321] The 68999 molecules of the invention are also useful as
pharmacodynamic markers. As used herein, a "pharmacodynamic marker"
is an objective biochemical marker which correlates specifically
with drug effects. The presence or quantity of a pharmacodynamic
marker is not related to the disease state or disorder for which
the drug is being administered; therefore, the presence or quantity
of the marker is indicative of the presence or activity of the drug
in a subject. For example, a pharmacodynamic marker can be
indicative of the concentration of the drug in a biological tissue,
in that the marker is either expressed or transcribed or not
expressed or transcribed in that tissue in relationship to the
level of the drug. In this fashion, the distribution or uptake of
the drug can be monitored by the pharmacodynamic marker. Similarly,
the presence or quantity of the pharmacodynamic marker can be
related to the presence or quantity of the metabolic product of a
drug, such that the presence or quantity of the marker is
indicative of the relative breakdown rate of the drug in vivo.
Pharmacodynamic markers are of particular use in increasing the
sensitivity of detection of drug effects, particularly when the
drug is administered in low doses. Since even a small amount of a
drug can be sufficient to activate multiple rounds of marker (e.g.,
a 68999 marker) transcription or expression, the amplified marker
can be in a quantity which is more readily detectable than the drug
itself. Also, the marker can be more easily detected due to the
nature of the marker itself; for example, using the methods
described herein, anti-68999 antibodies can be employed in an
immune-based detection system for a 68999 protein marker, or
68999-specific radiolabeled probes can be used to detect a 68999
mRNA marker. Furthermore, the use of a pharmacodynamic marker can
offer mechanism-based prediction of risk due to drug treatment
beyond the range of possible direct observations. Examples of the
use of pharmacodynamic markers in the art include: Matsuda et al.
U.S. Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect.
90: 229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl.
3: S21-S24; and Nicolau (1999) Am. J. Health-Syst. Pharm. 56 Suppl.
3: S16-S20.
[0322] The 68999 molecules of the invention are also useful as
pharmacogenomic markers. As used herein, a "pharmacogenomic marker"
is an objective biochemical marker which correlates with a specific
clinical drug response or susceptibility in a subject (see, e.g.,
McLeod et al. (1999) Eur. J. Cancer 35: 1650-1652). The presence or
quantity of the pharmacogenomic marker is related to the predicted
response of the subject to a specific drug or class of drugs prior
to administration of the drug. By assessing the presence or
quantity of one or more pharmacogenomic markers in a subject, a
drug therapy which is most appropriate for the subject, or which is
predicted to have a greater degree of success, can be selected. For
example, based on the presence or quantity of RNA, or protein
(e.g., 68999 protein or RNA) for specific tumor markers in a
subject, a drug or course of treatment can be selected that is
optimized for the treatment of the specific tumor likely to be
present in the subject. Similarly, the presence or absence of a
specific sequence mutation in 68999 DNA can correlate with a 68999
drug response. The use of pharmacogenomic markers therefore permits
the application of the most appropriate treatment for each subject
without having to administer the therapy.
[0323] Pharmaceutical Compositions
[0324] The nucleic acid and polypeptides, fragments thereof, as
well as anti-68999 antibodies (also referred to herein as "active
compounds") of the invention can be incorporated into
pharmaceutical compositions. Such compositions typically include
the nucleic acid molecule, protein, or antibody and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" includes solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. Supplementary active compounds can
also be incorporated into the compositions.
[0325] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral (e.g., inhalation), transdermal (topical),
transmucosal, and rectal administration. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0326] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It should be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0327] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0328] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules, e.g., gelatin capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash. Pharmaceutically compatible binding agents,
and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0329] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0330] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0331] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0332] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0333] It is advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
[0334] 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 LD50 (the dose
lethal to 50% of the population) and the ED50 (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 LD50/ED50. Compounds which exhibit
high therapeutic indices are preferred. While compounds that
exhibit toxic side effects can be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0335] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage can vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose can be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma can be measured, for example, by
high performance liquid chromatography.
[0336] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The
protein or polypeptide can be administered one time per week for
between about 1 to 10 weeks, preferably between 2 to 8 weeks, more
preferably between about 3 to 7 weeks, and even more preferably for
about 4, 5, or 6 weeks. The skilled artisan will appreciate that
certain factors can influence the dosage and timing required to
effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of a protein, polypeptide, or antibody can include
a single treatment or, preferably, can include a series of
treatments.
[0337] For antibodies, the preferred dosage is 0.1 mg/kg of body
weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act
in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually
appropriate. Generally, partially human antibodies and fully human
antibodies have a longer half-life within the human body than other
antibodies. Accordingly, lower dosages and less frequent
administration is often possible. Modifications such as lipidation
can be used to stabilize antibodies and to enhance uptake and
tissue penetration (e.g., into the brain). A method for lipidation
of antibodies is described by Cruikshank et al. ((1997) J. Acquired
Immune Deficiency Syndromes and Human Retrovirology 14: 193).
[0338] The present invention encompasses agents which modulate
expression or activity. An agent can, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics (e.g., peptoids), amino
acids, amino acid analogs, polynucleotides, polynucleotide analogs,
nucleotides, nucleotide analogs, organic or inorganic compounds
(i.e.,. including heteroorganic and organometallic compounds)
having a molecular weight less than about 10,000 grams per mole,
organic or inorganic compounds having a molecular weight less than
about 5,000 grams per mole, organic or inorganic compounds having a
molecular weight less than about 1,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 500
grams per mole, and salts, esters, and other pharmaceutically
acceptable forms of such compounds.
[0339] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram. It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. When one or more of these small molecules is to be
administered to an animal (e.g., a human) in order to modulate
expression or activity of a polypeptide or nucleic acid of the
invention, a physician, veterinarian, or researcher can, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0340] An antibody (or fragment thereof) can be conjugated to a
therapeutic moiety such as a cytotoxin, a therapeutic agent or a
radioactive metal ion. A cytotoxin or cytotoxic agent includes any
agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[0341] The conjugates of the invention can be used for modifying a
given biological response, the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety can be a protein or polypeptide possessing a
desired biological activity. Such proteins can include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor,
.alpha.-interferon, .alpha.-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophase colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[0342] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980.
[0343] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl.
Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of
the gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells which produce the gene
delivery system.
[0344] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0345] Methods of Treatment:
[0346] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant or unwanted 68999 expression or activity. As used herein,
the term "treatment" is defined as the application or
administration of a therapeutic agent to a patient, or application
or administration of a therapeutic agent to an isolated tissue or
cell line from a patient, who has a disease, a symptom of disease
or a predisposition toward a disease, with the purpose to cure,
heal, alleviate, relieve, alter, remedy, ameliorate, improve or
affect the disease, the symptoms of disease or the predisposition
toward disease. A therapeutic agent includes, but is not limited
to, small molecules, peptides, antibodies, ribozymes and antisense
oligonucleotides.
[0347] With regards to both prophylactic and therapeutic methods of
treatment, such treatments can be specifically tailored or
modified, based on knowledge obtained from the field of
pharmacogenomics. "Pharmacogenomics", as used herein, refers to the
application of genomics technologies such as gene sequencing,
statistical genetics, and gene expression analysis to drugs in
clinical development and on the market. More specifically, the term
refers the study of how a patient's genes determine his or her
response to a drug (e.g., a patient's "drug response phenotype", or
"drug response genotype".) Thus, another aspect of the invention
provides methods for tailoring an individual's prophylactic or
therapeutic treatment with either the 68999 molecules of the
present invention or 68999 modulators according to that
individual's drug response genotype. Pharmacogenomics allows a
clinician or physician to target prophylactic or therapeutic
treatments to patients who will most benefit from the treatment and
to avoid treatment of patients who will experience toxic
drug-related side effects.
[0348] In one aspect, the invention provides a method for
preventing in a subject, a disease or condition associated with an
aberrant or unwanted 68999 expression or activity, by administering
to the subject a 68999 or an agent which modulates 68999 expression
or at least one 68999 activity. Subjects at risk for a disease
which is caused or contributed to by aberrant or unwanted 68999
expression or activity can be identified by, for example, any or a
combination of diagnostic or prognostic assays as described herein.
Administration of a prophylactic agent can occur prior to the
manifestation of symptoms characteristic of the 68999 aberrance,
such that a disease or disorder is prevented or, alternatively,
delayed in its progression. Depending on the type of 68999
aberrance, for example, a 68999, 68999 agonist or 68999 antagonist
agent can be used for treating the subject. The appropriate agent
can be determined based on screening assays described herein.
[0349] It is possible that some 68999 disorders can be caused, at
least in part, by an abnormal level of gene product, or by the
presence of a gene product exhibiting abnormal activity. As such,
the reduction in the level and/or activity of such gene products
would bring about the amelioration of disorder symptoms.
[0350] The 68999 molecules and modulators thereof can act as novel
diagnostic and therapeutic agents for controlling one or more of
cellular proliferative and/or differentiative disorders, hormonal
disorders, and neurological disorders described above, as well as
the skeletal or bone metabolism disorders, metabolism or pain
disorders, immune and inflammatory disorders, cardiovascular
disorders, blood vessel disorders, endothelial disorders, liver
disorders, viral diseases, and neurological disorders as described
herein.
[0351] Aberrant expression and/or activity of 68999 molecules can
mediate disorders associated with bone metabolism. "Bone
metabolism" refers to direct or indirect effects in the formation
or degeneration of bone structures, e.g., bone formation, bone
resorption, etc., which can ultimately affect the concentrations in
serum of calcium and phosphate. This term also includes activities
mediated by 68999 molecules effects in bone cells, e.g. osteoclasts
and osteoblasts, that can in turn result in bone formation and
degeneration. For example, 68999 molecules can support different
activities of bone resorbing osteoclasts such as the stimulation of
differentiation of monocytes and mononuclear phagocytes into
osteoclasts. Accordingly, 68999 molecules that modulate the
production of bone cells can influence bone formation and
degeneration, and thus can be used to treat bone disorders.
Examples of such disorders include, but are not limited to,
osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis
fibrosa cystica, renal osteodystrophy, osteosclerosis,
anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta
ossium, secondary hyperparathyrodism, hypoparathyroidism,
hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced
metabolism, medullary carcinoma, chronic renal disease, rickets,
sarcoidosis, glucocorticoid antagonism, malabsorption syndrome,
steatorrhea, tropical sprue, idiopathic hypercalcemia and milk
fever.
[0352] Additionally, 68999 can play an important role in the
regulation of metabolism or pain disorders. Diseases of metabolic
imbalance include, but are not limited to, obesity, anorexia
nervosa, cachexia, lipid disorders, and diabetes. Examples of pain
disorders include, but are not limited to, pain response elicited
during various forms of tissue injury, e.g., inflammation,
infection, and ischemia, usually referred to as hyperalgesia
(described in, for example, Fields, H. L. (1987) Pain, New
York:McGraw-Hill); pain associated with musculoskeletal disorders,
e.g., joint pain; tooth pain; headaches; pain associated with
surgery; pain related to irritable bowel syndrome; or chest
pain.
[0353] The 68999 nucleic acid and protein of the invention can be
used to treat and/or diagnose a variety of immune, e.g.,
inflammatory (e.g. respiratory inflammatory) disorders. Examples
immune and inflammatory disorders or diseases include, but are not
limited to, autoimmune diseases (including, for example, diabetes
mellitus, arthritis (including rheumatoid arthritis, juvenile
rheumatoid arthritis, osteoarthritis, psoriatic arthritis),
multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic
lupus erythematosis, autoimmune thyroiditis, dermatitis (including
atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's
Syndrome, inflammatory bowel disease, e.g. Crohn's disease and
ulcerative colitis, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, asthma, allergic asthma, chronic obstructive
pulmonary disease, cutaneous lupus erythematosus, scleroderma,
vaginitis, proctitis, drug eruptions, leprosy reversal reactions,
erythema nodosum leprosum, autoimmune uveitis, allergic
encephalomyelitis, acute necrotizing hemorrhagic encephalopathy,
idiopathic bilateral progressive sensorineural hearing loss,
aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia,
polychondritis, Wegener's granulomatosis, chronic active hepatitis,
Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves'
disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior,
and interstitial lung fibrosis), graft-versus-host disease, cases
of transplantation, and allergy such as, atopic allergy.
[0354] As used herein, disorders involving the heart, or
"cardiovascular disease" or a "cardiovascular disorder" includes a
disease or disorder which affects the cardiovascular system, e.g.,
the heart, the blood vessels, and/or the blood. A cardiovascular
disorder can be caused by an imbalance in arterial pressure, a
malfunction of the heart, or an occlusion of a blood vessel, e.g.,
by a thrombus. A cardiovascular disorder includes, but is not
limited to disorders such as arteriosclerosis, atherosclerosis,
cardiac hypertrophy, ischemia reperfusion injury, restenosis,
arterial inflammation, vascular wall remodeling, ventricular
remodeling, rapid ventricular pacing, coronary microembolism,
tachycardia, bradycardia, pressure overload, aortic bending,
coronary artery ligation, vascular heart disease, valvular disease,
including but not limited to, valvular degeneration caused by
calcification, rheumatic heart disease, endocarditis, or
complications of artificial valves; atrial fibrillation, long-QT
syndrome, congestive heart failure, sinus node dysfunction, angina,
heart failure, hypertension, atrial fibrillation, atrial flutter,
pericardial disease, including but not limited to, pericardial
effusion and pericarditis; cardiomyopathies, e.g., dilated
cardiomyopathy or idiopathic cardiomyopathy, myocardial infarction,
coronary artery disease, coronary artery spasm, ischemic disease,
arrhythmia, sudden cardiac death, and cardiovascular developmental
disorders (e.g., arteriovenous malformations, arteriovenous
fistulae, raynaud's syndrome, neurogenic thoracic outlet syndrome,
causalgia/reflex sympathetic dystrophy, hemangioma, aneurysm,
cavernous angioma, aortic valve stenosis, atrial septal defects,
atrioventricular canal, coarctation of the aorta, ebsteins anomaly,
hypoplastic left heart syndrome, interruption of the aortic arch,
mitral valve prolapse, ductus arteriosus, patent foramen ovale,
partial anomalous pulmonary venous return, pulmonary atresia with
ventricular septal defect, pulmonary atresia without ventricular
septal defect, persistance of the fetal circulation, pulmonary
valve stenosis, single ventricle, total anomalous pulmonary venous
return, transposition of the great vessels, tricuspid atresia,
truncus arteriosus, ventricular septal defects). A cardiovasular
disease or disorder also includes an endothelial cell disorder.
[0355] As used herein, "blood vessel disorders" include disorders
involving blood vessels include, but are not limited to, responses
of vascular cell walls to injury, such as endothelial dysfunction
and endothelial activation and intimal thickening; vascular
diseases including, but not limited to, congenital anomalies, such
as arteriovenous fistula, atherosclerosis, and hypertensive
vascular disease, such as hypertension; inflammatory disease--the
vasculitides, such as giant cell (temporal) arteritis, Takayasu
arteritis, polyarteritis nodosa (classic), Kawasaki syndrome
(mucocutaneous lymph node syndrome), microscopic polyanglitis
(microscopic polyarteritis, hypersensitivity or leukocytoclastic
anglitis), Wegener granulomatosis, thromboanglitis obliterans
(Buerger disease), vasculitis associated with other disorders, and
infectious arteritis; Raynaud disease; aneurysms and dissection,
such as abdominal aortic aneurysms, syphilitic (luetic) aneurysms,
and aortic dissection (dissecting hematoma); disorders of veins and
lymphatics, such as varicose veins, thrombophlebitis and
phlebothrombosis, obstruction of superior vena cava (superior vena
cava syndrome), obstruction of inferior vena cava (inferior vena
cava syndrome), and lymphangitis and lymphedema; tumors, including
benign tumors and tumor-like conditions, such as hemangioma,
lymphangioma, glomus tumor (glomangioma), vascular ectasias, and
bacillary angiomatosis, and intermediate-grade (borderline
low-grade malignant) tumors, such as Kaposi sarcoma and
hemangloendothelioma, and malignant tumors, such as angiosarcoma
and hemangiopericytoma; and pathology of therapeutic interventions
in vascular disease, such as balloon angioplasty and related
techniques and vascular replacement, such as coronary artery bypass
graft surgery.
[0356] As used herein, an "endothelial cell disorder" includes a
disorder characterized by aberrant, unregulated, or unwanted
endothelial cell activity, e.g., proliferation, migration,
angiogenesis, or vascularization; or aberrant expression of cell
surface adhesion molecules or genes associated with angiogenesis,
e.g., TIE-2, FLT and FLK. Endothelial cell disorders include
tumorigenesis, tumor metastasis, psoriasis, diabetic retinopathy,
endometriosis, Grave's disease, ischemic disease (e.g.,
atherosclerosis), and chronic inflammatory diseases (e.g.,
rheumatoid arthritis).
[0357] Disorders which can be treated or diagnosed by methods
described herein include, but are not limited to, disorders
associated with an accumulation in the liver of fibrous tissue,
such as that resulting from an imbalance between production and
degradation of the extracellular matrix accompanied by the collapse
and condensation of preexisting fibers. The methods described
herein can be used to diagnose or treat hepatocellular necrosis or
injury induced by a wide variety of agents including processes
which disturb homeostasis, such as an inflammatory process, tissue
damage resulting from toxic injury or altered hepatic blood flow,
and infections (e.g., bacterial, viral and parasitic). For example,
the methods can be used for the early detection of hepatic injury,
such as portal hypertension or hepatic fibrosis. In addition, the
methods can be employed to detect liver fibrosis attributed to
inborn errors of metabolism, for example, fibrosis resulting from a
storage disorder such as Gaucher's disease (lipid abnormalities) or
a glycogen storage disease, A1-antitrypsin deficiency; a disorder
mediating the accumulation (e.g., storage) of an exogenous
substance, for example, hemochromatosis (iron-overload syndrome)
and copper storage diseases (Wilson's disease), disorders resulting
in the accumulation of a toxic metabolite (e.g., tyrosinemia,
fructosemia and galactosemia) and peroxisomal disorders (e.g.,
Zellweger syndrome). Additionally, the methods described herein can
be useful for the early detection and treatment of liver injury
associated with the administration of various chemicals or drugs,
such as for example, methotrexate, isonizaid, oxyphenisatin,
methyldopa, chlorpromazine, tolbutamide or alcohol, or which
represents a hepatic manifestation of a vascular disorder such as
obstruction of either the intrahepatic or extrahepatic bile flow or
an alteration in hepatic circulation resulting, for example, from
chronic heart failure, veno-occlusive disease, portal vein
thrombosis or Budd-Chiari syndrome.
[0358] Additionally, 68999 molecules can play an important role in
the etiology of certain viral diseases, including but not limited
to Hepatitis B, Hepatitis C and Herpes Simplex Virus (HSV).
Modulators of 68999 activity could be used to control viral
diseases. The modulators can be used in the treatment and/or
diagnosis of viral infected tissue or virus-associated tissue
fibrosis, especially liver and liver fibrosis. Also, 68999
modulators can be used in the treatment and/or diagnosis of
virus-associated carcinoma, especially hepatocellular cancer.
[0359] As discussed, successful treatment of 68999 disorders can be
brought about by techniques that serve to inhibit the expression or
activity of target gene products. For example, compounds, e.g., an
agent identified using an assays described above, that proves to
exhibit negative modulatory activity, can be used in accordance
with the invention to prevent and/or ameliorate symptoms of 68999
disorders. Such molecules can include, but are not limited to
peptides, phosphopeptides, small organic or inorganic molecules, or
antibodies (including, for example, polyclonal, monoclonal,
humanized, human, anti-idiotypic, chimeric or single chain
antibodies, and Fab, F(ab).sub.2 and Fab expression library
fragments, scFV molecules, and epitope-binding fragments
thereof).
[0360] Further, antisense and ribozyme molecules that inhibit
expression of the target gene can also be used in accordance with
the invention to reduce the level of target gene expression, thus
effectively reducing the level of target gene activity. Still
further, triple helix molecules can be utilized in reducing the
level of target gene activity. Antisense, ribozyme and triple helix
molecules are discussed above.
[0361] It is possible that the use of antisense, ribozyme, and/or
triple helix molecules to reduce or inhibit mutant gene expression
can also reduce or inhibit the transcription (triple helix) and/or
translation (antisense, ribozyme) of mRNA produced by normal target
gene alleles, such that the concentration of normal target gene
product present can be lower than is necessary for a normal
phenotype. In such cases, nucleic acid molecules that encode and
express target gene polypeptides exhibiting normal target gene
activity can be introduced into cells via gene therapy method.
Alternatively, in instances in that the target gene encodes an
extracellular protein, it can be preferable to co-administer normal
target gene protein into the cell or tissue in order to maintain
the requisite level of cellular or tissue target gene activity.
[0362] Another method by which nucleic acid molecules can be
utilized in treating or preventing a disease characterized by 68999
expression is through the use of aptamer molecules specific for
68999 protein. Aptamers are nucleic acid molecules having a
tertiary structure which permits them to specifically or
selectively bind to protein ligands (see, e.g., Osborne, et al.
(1997) Curr. Opin. Chem Biol. 1: 5-9; and Patel, D. J. (1997) Curr
Opin Chem Biol 1: 32-46). Since nucleic acid molecules can in many
cases be more conveniently introduced into target cells than
therapeutic protein molecules can be, aptamers offer a method by
which 68999 protein activity can be specifically decreased without
the introduction of drugs or other molecules which can have
pluripotent effects.
[0363] Antibodies can be generated that are both specific for
target gene product and that reduce target gene product activity.
Such antibodies can, therefore, by administered in instances
whereby negative modulatory techniques are appropriate for the
treatment of 68999 disorders. For a description of antibodies, see
the Antibody section above.
[0364] In circumstances wherein injection of an animal or a human
subject with a 68999 protein or epitope for stimulating antibody
production is harmful to the subject, it is possible to generate an
immune response against 68999 through the use of anti-idiotypic
antibodies (see, for example, Herlyn, D. (1999) Ann Med 31: 66-78;
and Bhattacharya-Chattetjee- , M., and Foon, K. A. (1998) Cancer
Treat Res. 94: 51-68). If an anti-idiotypic antibody is introduced
into a mammal or human subject, it should stimulate the production
of anti-anti-idiotypic antibodies, which should be specific to the
68999 protein. Vaccines directed to a disease characterized by
68999 expression can also be generated in this fashion.
[0365] In instances where the target antigen is intracellular and
whole antibodies are used, internalizing antibodies can be
preferred. Lipofectin or liposomes can be used to deliver the
antibody or a fragment of the Fab region that binds to the target
antigen into cells. Where fragments of the antibody are used, the
smallest inhibitory fragment that binds to the target antigen is
preferred. For example, peptides having an amino acid sequence
corresponding to the Fv region of the antibody can be used.
Alternatively, single chain neutralizing antibodies that bind to
intracellular target antigens can also be administered. Such single
chain antibodies can be administered, for example, by expressing
nucleotide sequences encoding single-chain antibodies within the
target cell population (see e.g., Marasco et al. (1993) Proc. Natl.
Acad. Sci. USA 90: 7889-7893).
[0366] The identified compounds that inhibit target gene
expression, synthesis and/or activity can be administered to a
patient at therapeutically effective doses to prevent, treat or
ameliorate 68999 disorders. A therapeutically effective dose refers
to that amount of the compound sufficient to result in amelioration
of symptoms of the disorders. Toxicity and therapeutic efficacy of
such compounds can be determined by standard pharmaceutical
procedures as described above.
[0367] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. 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 can vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose can be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound that achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma can
be measured, for example, by high performance liquid
chromatography.
[0368] Another example of determination of effective dose for an
individual is the ability to directly assay levels of "free" and
"bound" compound in the serum of the test subject. Such assays can
utilize antibody mimics and/or "biosensors" that have been created
through molecular imprinting techniques. The compound which is able
to modulate 68999 activity is used as a template, or "imprinting
molecule", to spatially organize polymerizable monomers prior to
their polymerization with catalytic reagents. The subsequent
removal of the imprinted molecule leaves a polymer matrix which
contains a repeated "negative image" of the compound and is able to
selectively rebind the molecule under biological assay conditions.
A detailed review of this technique can be seen in Ansell, R. J. et
al (1996) Current Opinion in Biotechnology 7: 89-94 and in Shea, K.
J. (1994) Trends in Polymer Science 2: 166-173. Such "imprinted"
affinity matrixes are amenable to ligand-binding assays, whereby
the immobilized monoclonal antibody component is replaced by an
appropriately imprinted matrix. An example of the use of such
matrixes in this way can be seen in Vlatakis, G. et al (1993)
Nature 361: 645-647. Through the use of isotope-labeling, the
"free" concentration of compound which modulates the expression or
activity of 68999 can be readily monitored and used in calculations
of IC.sub.50.
[0369] Such "imprinted" affinity matrixes can also be designed to
include fluorescent groups whose photon-emitting properties
measurably change upon local and selective binding of target
compound. These changes can be readily assayed in real time using
appropriate fiberoptic devices, in turn allowing the dose in a test
subject to be quickly optimized based on its individual IC.sub.50.
An rudimentary example of such a "biosensor" is discussed in Kriz,
D. et al (1995) Analytical Chemistry 67: 2142-2144.
[0370] Another aspect of the invention pertains to methods of
modulating 68999 expression or activity for therapeutic purposes.
Accordingly, in an exemplary embodiment, the modulatory method of
the invention involves contacting a cell with a 68999 or agent that
modulates one or more of the activities of 68999 protein activity
associated with the cell. An agent that modulates 68999 protein
activity can be an agent as described herein, such as a nucleic
acid or a protein, a naturally-occurring target molecule of a 68999
protein (e.g., a 68999 substrate or receptor), a 68999 antibody, a
68999 agonist or antagonist, a peptidomimetic of a 68999 agonist or
antagonist, or other small molecule.
[0371] In one embodiment, the agent stimulates one or 68999
activities. Examples of such stimulatory agents include active
68999 protein and a nucleic acid molecule encoding 68999. In
another embodiment, the agent inhibits one or more 68999
activities. Examples of such inhibitory agents include antisense
68999 nucleic acid molecules, anti-68999 antibodies, and 68999
inhibitors. These modulatory methods can be performed in vitro
(e.g., by culturing the cell with the agent) or, alternatively, in
vivo (e.g., by administering the agent to a subject). As such, the
present invention provides methods of treating an individual
afflicted with a disease or disorder characterized by aberrant or
unwanted expression or activity of a 68999 protein or nucleic acid
molecule. In one embodiment, the method involves administering an
agent (e.g., an agent identified by a screening assay described
herein), or combination of agents that modulates (e.g., up
regulates or down regulates) 68999 expression or activity. In
another embodiment, the method involves administering a 68999
protein or nucleic acid molecule as therapy to compensate for
reduced, aberrant, or unwanted 68999 expression or activity.
[0372] Stimulation of 68999 activity is desirable in situations in
which 68999 is abnormally downregulated and/or in which increased
68999 activity is likely to have a beneficial effect. For example,
stimulation of 68999 activity is desirable in situations in which a
68999 is downregulated and/or in which increased 68999 activity is
likely to have a beneficial effect. Likewise, inhibition of 68999
activity is desirable in situations in which 68999 is abnormally
upregulated and/or in which decreased 68999 activity is likely to
have a beneficial effect.
[0373] Pharmacogenomics
[0374] The 68999 molecules of the present invention, as well as
agents, or modulators which have a stimulatory or inhibitory effect
on 68999 activity (e.g., 68999 gene expression) as identified by a
screening assay described herein can be administered to individuals
to treat (prophylactically or therapeutically) 68999-associated
disorders (e.g., aberrant or deficient ubiquitin activity the major
human neurodegenerative diseases) associated with aberrant or
unwanted 68999 activity. In conjunction with such treatment,
pharmacogenomics (i.e., the study of the relationship between an
individual's genotype and that individual's response to a foreign
compound or drug) can be considered. Differences in metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by
altering the relation between dose and blood concentration of the
pharmacologically active drug. Thus, a physician or clinician can
consider applying knowledge obtained in relevant pharmacogenomics
studies in determining whether to administer a 68999 molecule or
68999 modulator as well as tailoring the dosage and/or therapeutic
regimen of treatment with a 68999 molecule or 68999 modulator.
[0375] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol.
Physiol. 23: 983-985 and Linder, M. W. et al. (1997) Clin. Chem.
43: 254-266. In general, two types of pharmacogenetic conditions
can be differentiated. Genetic conditions transmitted as a single
factor altering the way drugs act on the body (altered drug action)
or genetic conditions transmitted as single factors altering the
way the body acts on drugs (altered drug metabolism). These
pharmacogenetic conditions can occur either as rare genetic defects
or as naturally-occurring polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0376] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP can occur once per every 1000
bases of DNA. A SNP can be involved in a disease process, however,
the vast majority can not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that can be common among
such genetically similar individuals.
[0377] Alternatively, a method termed the "candidate gene
approach", can be utilized to identify genes that predict drug
response. According to this method, if a gene that encodes a drug's
target is known (e.g., a 68999 protein of the present invention),
all common variants of that gene can be fairly easily identified in
the population and it can be determined if having one version of
the gene versus another is associated with a particular drug
response.
[0378] Alternatively, a method termed the "gene expression
profiling", can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a 68999 molecule or 68999 modulator of the present
invention) can give an indication whether gene pathways related to
toxicity have been turned on.
[0379] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment of an individual. This knowledge, when applied to dosing
or drug selection, can avoid adverse reactions or therapeutic
failure and thus enhance therapeutic or prophylactic efficiency
when treating a subject with a 68999 molecule or 68999 modulator,
such as a modulator identified by one of the exemplary screening
assays described herein.
[0380] The present invention further provides methods for
identifying new agents, or combinations, that are based on
identifying agents that modulate the activity of one or more of the
gene products encoded by one or more of the 68999 genes of the
present invention, wherein these products can be associated with
resistance of the cells to a therapeutic agent. Specifically, the
activity of the proteins encoded by the 68999 genes of the present
invention can be used as a basis for identifying agents for
overcoming agent resistance. By blocking the activity of one or
more of the resistance proteins, target cells, e.g., human cells,
will become sensitive to treatment with an agent that the
unmodified target cells were resistant to.
[0381] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a 68999 protein can be applied in
clinical trials. For example, the effectiveness of an agent
determined by a screening assay as described herein to increase
68999 gene expression, protein levels, or upregulate 68999
activity, can be monitored in clinical trials of subjects
exhibiting decreased 68999 gene expression, protein levels, or
downregulated 68999 activity. Alternatively, the effectiveness of
an agent determined by a screening assay to decrease 68999 gene
expression, protein levels, or downregulate 68999 activity, can be
monitored in clinical trials of subjects exhibiting increased 68999
gene expression, protein levels, or upregulated 68999 activity. In
such clinical trials, the expression or activity of a 68999 gene,
and preferably, other genes that have been implicated in, for
example, a ubiquitin carboxyl-terminal hydrolase-associated or
another 68999-associated disorder can be used as a "read out" or
markers of the phenotype of a particular cell.
Other Embodiments
[0382] In another aspect, the invention features a method of
analyzing a plurality of capture probes. The method is useful,
e.g., to analyze gene expression. The method includes: providing a
two dimensional array having a plurality of addresses, each address
of the plurality being positionally distinguishable from each other
address of the plurality, and each address of the plurality having
a unique capture probe, e.g., a nucleic acid or peptide sequence,
wherein the capture probes are from a cell or subject which
expresses 68999 or from a cell or subject in which a 68999 mediated
response has been elicited; contacting the array with a 68999
nucleic acid (preferably purified), a 68999 polypeptide (preferably
purified), or an anti-68999 antibody, and thereby evaluating the
plurality of capture probes. Binding, e.g., in the case of a
nucleic acid, hybridization with a capture probe at an address of
the plurality, is detected, e.g., by a signal generated from a
label attached to the 68999 nucleic acid, polypeptide, or
antibody.
[0383] The capture probes can be a set of nucleic acids from a
selected sample, e.g., a sample of nucleic acids derived from a
control or non-stimulated tissue or cell.
[0384] The method can include contacting the 68999 nucleic acid,
polypeptide, or antibody with a first array having a plurality of
capture probes and a second array having a different plurality of
capture probes. The results of each hybridization can be compared,
e.g., to analyze differences in expression between a first and
second sample. The first plurality of capture probes can be from a
control sample, e.g., a wild type, normal, or non-diseased,
non-stimulated, sample, e.g., a biological fluid, tissue, or cell
sample. The second plurality of capture probes can be from an
experimental sample, e.g., a mutant type, at risk, disease-state or
disorder-state, or stimulated, sample, e.g., a biological fluid,
tissue, or cell sample.
[0385] The plurality of capture probes can be a plurality of
nucleic acid probes each of which specifically hybridizes, with an
allele of 68999. Such methods can be used to diagnose a subject,
e.g., to evaluate risk for a disease or disorder, to evaluate
suitability of a selected treatment for a subject, to evaluate
whether a subject has a disease or disorder.
[0386] The method can be used to detect SNPs, as described
above.
[0387] In another aspect, the invention features, a method of
analyzing 68999, e.g., analyzing structure, function, or
relatedness to other nucleic acid or amino acid sequences. The
method includes: providing a 68999 nucleic acid or amino acid
sequence; comparing the 68999 sequence with one or more preferably
a plurality of sequences from a collection of sequences, e.g., a
nucleic acid or protein sequence database; to thereby analyze
68999.
[0388] The method can include evaluating the sequence identity
between a 68999 sequence and a database sequence. The method can be
performed by accessing the database at a second site, e.g., over
the internet. Preferred databases include GenBank.TM. and
SwissProt.
[0389] In another aspect, the invention features, a set of
oligonucleotides, useful, e.g., for identifying SNP's, or
identifying specific alleles of 68999. The set includes a plurality
of oligonucleotides, each of which has a different nucleotide at an
interrogation position, e.g., an SNP or the site of a mutation. In
a preferred embodiment, the oligonucleotides of the plurality
identical in sequence with one another (except for differences in
length). The oligonucleotides can be provided with differential
labels, such that an oligonucleotide which hybridizes to one allele
provides a signal that is distinguishable from an oligonucleotides
which hybridizes to a second allele.
[0390] The sequences of 68999 molecules are provided in a variety
of mediums to facilitate use thereof. A sequence can be provided as
a manufacture, other than an isolated nucleic acid or amino acid
molecule, which contains a 68999 molecule. Such a manufacture can
provide a nucleotide or amino acid sequence, e.g., an open reading
frame, in a form which allows examination of the manufacture using
means not directly applicable to examining the nucleotide or amino
acid sequences, or a subset thereof, as they exist in nature or in
purified form.
[0391] A 68999 nucleotide or amino acid sequence can be recorded on
computer readable media. As used herein, "computer readable media"
refers to any medium that 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 compact disc and
CD-ROM; electrical storage media such as RAM, ROM, EPROM, EEPROM,
and the like; and general hard disks and hybrids of these
categories such as magnetic/optical storage media. The medium is
adapted or configured for having thereon 68999 sequence information
of the present invention.
[0392] As used herein, the term "electronic apparatus" is intended
to include any suitable computing or processing apparatus of other
device configured or adapted for storing data or information.
Examples of electronic apparatus suitable for use with the present
invention include stand-alone computing apparatus; networks,
including a local area network (LAN), a wide area network (WAN)
Internet, Intranet, and Extranet; electronic appliances such as
personal digital assistants (PDAs), cellular phones, pagers, and
the like; and local and distributed processing systems.
[0393] As used herein, "recorded" refers to a process for storing
or encoding information on the electronic apparatus readable
medium. Those skilled in the art can readily adopt any of the
presently known methods for recording information on known media to
generate manufactures comprising the 68999 sequence
information.
[0394] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a 68999 nucleotide or amino acid 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. The 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.
[0395] By providing the 68999 nucleotide or amino acid sequences of
the invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. A search is used to identify fragments or regions of the
sequences of the invention which match a particular target sequence
or target motif.
[0396] The present invention therefore provides a medium for
holding instructions for performing a method for determining
whether a subject has a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder or a pre-disposition to a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder, wherein the method comprises the steps of determining
68999 sequence information associated with the subject and based on
the 68999 sequence information, determining whether the subject has
a ubiquitin carboxyl-terminal hydrolase-associated or another
68999-associated disease or disorder and/or recommending a
particular treatment for the disease, disorder, or pre-disease
condition.
[0397] The present invention further provides in an electronic
system and/or in a network, a method for determining whether a
subject has a ubiquitin carboxyl-terminal hydrolase-associated or
another 68999-associated disease or disorder or a pre-disposition
to a disease associated with 68999, wherein the method comprises
the steps of determining 68999 sequence information associated with
the subject, and based on the 68999 sequence information,
determining whether the subject has a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder or a pre-disposition to a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder, and/or recommending a particular treatment for the
disease, disorder, or pre-disease condition. The method may further
comprise the step of receiving phenotypic information associated
with the subject and/or acquiring from a network phenotypic
information associated with the subject.
[0398] The present invention also provides in a network, a method
for determining whether a subject has a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder or a pre-disposition to a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder, said method comprising the steps of receiving 68999
sequence information from the subject and/or information related
thereto, receiving phenotypic information associated with the
subject, acquiring information from the network corresponding to
68999 and/or corresponding to a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder, and based on one or more of the phenotypic information,
the 68999 information (e.g., sequence information and/or
information related thereto), and the acquired information,
determining whether the subject has a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder or a pre-disposition to a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder. The method may further comprise the step of recommending
a particular treatment for the disease, disorder, or pre-disease
condition.
[0399] The present invention also provides a business method for
determining whether a subject has a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder or a pre-disposition to a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder, said method comprising the steps of receiving information
related to 68999 (e.g., sequence information and/or information
related thereto), receiving phenotypic information associated with
the subject, acquiring information from the network related to
68999 and/or related to a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder, and based on one or more of the phenotypic information,
the 68999 information, and the acquired information, determining
whether the subject has a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder or a pre-disposition to a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder. The method may further comprise the step of recommending
a particular treatment for the disease, disorder, or pre-disease
condition.
[0400] The invention also includes an array comprising a 68999
sequence of the present invention. The array can be used to assay
expression of one or more genes in the array. In one embodiment,
the array can be used to assay gene expression in a tissue to
ascertain tissue specificity of genes in the array. In this manner,
up to about 7600 genes can be simultaneously assayed for
expression, one of which can be 68999. This allows a profile to be
developed showing a battery of genes specifically expressed in one
or more tissues.
[0401] In addition to such qualitative information, the invention
allows the quantitation of gene expression. Thus, not only tissue
specificity, but also the level of expression of a battery of genes
in the tissue if ascertainable. Thus, genes can be grouped on the
basis of their tissue expression per se and level of expression in
that tissue. This is useful, for example, in ascertaining the
relationship of gene expression in that tissue. Thus, one tissue
can be perturbed and the effect on gene expression in a second
tissue can be determined. In this context, the effect of one cell
type on another cell type in response to a biological stimulus can
be determined. In this context, the effect of one cell type on
another cell type in response to a biological stimulus can be
determined. Such a determination is useful, for example, to know
the effect of cell-cell interaction at the level of gene
expression. If an agent is administered therapeutically to treat
one cell type but has an undesirable effect on another cell type,
the invention provides an assay to determine the molecular basis of
the undesirable effect and thus provides the opportunity to
co-administer a counteracting agent or otherwise treat the
undesired effect. Similarly, even within a single cell type,
undesirable biological effects can be determined at the molecular
level. Thus, the effects of an agent on expression of other than
the target gene can be ascertained and counteracted.
[0402] In another embodiment, the array can be used to monitor the
time course of expression of one or more genes in the array. This
can occur in various biological contexts, as disclosed herein, for
example development of a ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder, progression of ubiquitin carboxyl-terminal
hydrolase-associated or another 68999-associated disease or
disorder, and processes, such a cellular transformation associated
with the ubiquitin carboxyl-terminal hydrolase-associated or
another 68999-associated disease or disorder.
[0403] The array is also useful for ascertaining the effect of the
expression of a gene on the expression of other genes in the same
cell or in different cells (e.g., acertaining the effect of 68999
expression on the expression of other genes). This provides, for
example, for a selection of alternate molecular targets for
therapeutic intervention if the ultimate or downstream target
cannot be regulated.
[0404] The array is also useful for ascertaining differential
expression patterns of one or more genes in normal and abnormal
cells. This provides a battery of genes (e.g., including 68999)
that could serve as a molecular target for diagnosis or therapeutic
intervention.
[0405] As used herein, a "target sequence" can be any DNA 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. Typical sequence
lengths of a target sequence are from about 10 to 100 amino acids
or from about 30 to 300 nucleotide residues. However, it is well
recognized that commercially important fragments, such as sequence
fragments involved in gene expression and protein processing, may
be of shorter length.
[0406] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. 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 include, but are not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBI).
[0407] Thus, the invention features a method of making a computer
readable record of a sequence of a 68999 sequence which includes
recording the sequence on a computer readable matrix. In a
preferred embodiment the record includes one or more of the
following: identification of an ORF; identification of a domain,
region, or site; identification of the start of transcription;
identification of the transcription terminator; the full length
amino acid sequence of the protein, or a mature form thereof; the
5' end of the translated region.
[0408] In another aspect, the invention features a method of
analyzing a sequence. The method includes: providing a 68999
sequence, or record, in computer readable form; comparing a second
sequence to the 68999 sequence; thereby analyzing a sequence.
Comparison can include comparing to sequences for sequence identity
or determining if one sequence is included within the other, e.g.,
determining if the 68999 sequence includes a sequence being
compared. In a preferred embodiment the 68999 or second sequence is
stored on a first computer, e.g., at a first site and the
comparison is performed, read, or recorded on a second computer,
e.g., at a second site. E.g., the 68999 or second sequence can be
stored in a public or proprietary database in one computer, and the
results of the comparison performed, read, or recorded on a second
computer. In a preferred embodiment the record includes one or more
of the following: identification of an ORF; identification of a
domain, region, or site; identification of the start of
transcription; identification of the transcription terminator; the
full length amino acid sequence of the protein, or a mature form
thereof; the 5' end of the translated region.
[0409] The contents of all references, patents and published patent
applications cited throughout this application are incorporated
herein by reference.
[0410] Equivalents
[0411] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein.
Sequence CWU 1
1
7 1 1763 DNA Homo sapiens CDS (171)...(1763) 1 tatcgcgcac
ctgatgagtg ggtggggtgt tcgcggttgg tgggggtgac ttacagaagg 60
gctgatgcgg ccagagagct cgtcatttga agactctctc ggaagggata gcgtctttct
120 gcaacctgcg gtcccagcag acaaaccttg tgatcctcgt tccagtcgac atg gag
176 Met Glu 1 gac gac tca ctc tac ttg gga ggt gag tgg cag ttc aac
cac ttt tca 224 Asp Asp Ser Leu Tyr Leu Gly Gly Glu Trp Gln Phe Asn
His Phe Ser 5 10 15 aaa ctc aca tct tct cgg ccc gat gca gct ttt gct
gaa atc cag cgg 272 Lys Leu Thr Ser Ser Arg Pro Asp Ala Ala Phe Ala
Glu Ile Gln Arg 20 25 30 act tct ctc cct gag aag tca cca ctc tca
tgt gag gcc cgt gtc gac 320 Thr Ser Leu Pro Glu Lys Ser Pro Leu Ser
Cys Glu Ala Arg Val Asp 35 40 45 50 ctc tgt gat gat ttg gct cct gtg
gca aga cag ctt gct ccc agg gag 368 Leu Cys Asp Asp Leu Ala Pro Val
Ala Arg Gln Leu Ala Pro Arg Glu 55 60 65 aag ctt cct ctg agt agc
agg aga cct gct gcg gtg ggg gct ggg ctc 416 Lys Leu Pro Leu Ser Ser
Arg Arg Pro Ala Ala Val Gly Ala Gly Leu 70 75 80 cag aat atg gga
aat acc tgc tac gtg aac gct tcc ttg cag tgc ctg 464 Gln Asn Met Gly
Asn Thr Cys Tyr Val Asn Ala Ser Leu Gln Cys Leu 85 90 95 aca tac
aca ccg ccc ctt gcc aac tac atg ctg tcc cgg gag cac tct 512 Thr Tyr
Thr Pro Pro Leu Ala Asn Tyr Met Leu Ser Arg Glu His Ser 100 105 110
caa acg tgt cat cgt cac aag ggc tgc atg ctc tgt act atg caa gct 560
Gln Thr Cys His Arg His Lys Gly Cys Met Leu Cys Thr Met Gln Ala 115
120 125 130 cac atc aca cgg gcc ctc cac aat cct ggc cac gtc atc cag
ccc tca 608 His Ile Thr Arg Ala Leu His Asn Pro Gly His Val Ile Gln
Pro Ser 135 140 145 cag gca ttg gct gct ggc ttc cat aga ggc aag cag
gaa gat gcc cat 656 Gln Ala Leu Ala Ala Gly Phe His Arg Gly Lys Gln
Glu Asp Ala His 150 155 160 gaa ttt ctc atg ttc act gtg gat gcc atg
aaa aag gca tgc ctt ccc 704 Glu Phe Leu Met Phe Thr Val Asp Ala Met
Lys Lys Ala Cys Leu Pro 165 170 175 ggg cac aag cag gta gat cat cac
tct aag gac acc acc ctc atc cac 752 Gly His Lys Gln Val Asp His His
Ser Lys Asp Thr Thr Leu Ile His 180 185 190 caa ata ttt gga ggc tac
tgg aga tct caa atc aag tgt ctc cac tgc 800 Gln Ile Phe Gly Gly Tyr
Trp Arg Ser Gln Ile Lys Cys Leu His Cys 195 200 205 210 cac ggc att
tca gac act ttt gac cct tac ctg gac atc gcc ctg gat 848 His Gly Ile
Ser Asp Thr Phe Asp Pro Tyr Leu Asp Ile Ala Leu Asp 215 220 225 atc
cag gca gct cag agt gtc cag caa gct ttg gaa cag ttg gtg aag 896 Ile
Gln Ala Ala Gln Ser Val Gln Gln Ala Leu Glu Gln Leu Val Lys 230 235
240 ccc gaa gaa ctc aat gga gag aat gcc tat cat tgt ggt gtt tgt ctc
944 Pro Glu Glu Leu Asn Gly Glu Asn Ala Tyr His Cys Gly Val Cys Leu
245 250 255 cag agg gcg ccg gcc tcc aag acg tta act tta cac acc tct
gcc aag 992 Gln Arg Ala Pro Ala Ser Lys Thr Leu Thr Leu His Thr Ser
Ala Lys 260 265 270 gtc ctc atc ctt gta ttg aag aga ttc tcc gat gtc
aca ggc aac aag 1040 Val Leu Ile Leu Val Leu Lys Arg Phe Ser Asp
Val Thr Gly Asn Lys 275 280 285 290 att gcc aag aat gtg caa tat cct
gag tgc ctt gac atg cag cca tac 1088 Ile Ala Lys Asn Val Gln Tyr
Pro Glu Cys Leu Asp Met Gln Pro Tyr 295 300 305 atg tct cag cag aac
aca gga cct ctt gtc tat gtc ctc tat gct gtg 1136 Met Ser Gln Gln
Asn Thr Gly Pro Leu Val Tyr Val Leu Tyr Ala Val 310 315 320 ctg gtc
cac gct ggg tgg agt tgt cac aac gga cat tac ttc tct tat 1184 Leu
Val His Ala Gly Trp Ser Cys His Asn Gly His Tyr Phe Ser Tyr 325 330
335 gtc aaa gct caa gaa ggc cag tgg tat aaa atg gat gat gcc gag gtc
1232 Val Lys Ala Gln Glu Gly Gln Trp Tyr Lys Met Asp Asp Ala Glu
Val 340 345 350 acc gcc tct agc atc act tct gtc ctg agt caa cag gcc
tac gtc ctc 1280 Thr Ala Ser Ser Ile Thr Ser Val Leu Ser Gln Gln
Ala Tyr Val Leu 355 360 365 370 ttt tac atc cag aag agt gaa tgg gaa
aga cac agt gag agt gtg tca 1328 Phe Tyr Ile Gln Lys Ser Glu Trp
Glu Arg His Ser Glu Ser Val Ser 375 380 385 aga ggc agg gaa cca aga
gcc ctt ggc gca gaa gac aca gac agg cga 1376 Arg Gly Arg Glu Pro
Arg Ala Leu Gly Ala Glu Asp Thr Asp Arg Arg 390 395 400 gca acg caa
gga gag ctc aag aga gac cac ccc tgc ctc cag gcc ccc 1424 Ala Thr
Gln Gly Glu Leu Lys Arg Asp His Pro Cys Leu Gln Ala Pro 405 410 415
gag ttg gac gag cac ttg gtg gaa aga gcc act cag gaa agc acc tta
1472 Glu Leu Asp Glu His Leu Val Glu Arg Ala Thr Gln Glu Ser Thr
Leu 420 425 430 gac cac tgg aaa ttc ctt caa gag caa aac aaa acg aag
cct gag ttc 1520 Asp His Trp Lys Phe Leu Gln Glu Gln Asn Lys Thr
Lys Pro Glu Phe 435 440 445 450 aac gtc aga aaa gtc gaa ggt acc ctg
cct ccc gac gta ctt gtg att 1568 Asn Val Arg Lys Val Glu Gly Thr
Leu Pro Pro Asp Val Leu Val Ile 455 460 465 cat caa tca aaa tac aag
tgt ggg atg aaa aac cat cat cct gaa cag 1616 His Gln Ser Lys Tyr
Lys Cys Gly Met Lys Asn His His Pro Glu Gln 470 475 480 caa agc tcc
ctg cta aac ctc tct tcg acg aac ccg aca gat cag gag 1664 Gln Ser
Ser Leu Leu Asn Leu Ser Ser Thr Asn Pro Thr Asp Gln Glu 485 490 495
tcc atg aac act ggc aca ctc gct tct ctg caa ggg agg acc agg aga
1712 Ser Met Asn Thr Gly Thr Leu Ala Ser Leu Gln Gly Arg Thr Arg
Arg 500 505 510 gcc aaa ggg aag aac aaa cac tgc aag aga tct ctg ctt
gtg tgc cag 1760 Ala Lys Gly Lys Asn Lys His Cys Lys Arg Ser Leu
Leu Val Cys Gln 515 520 525 530 tga 1763 * 2 530 PRT Homo sapiens 2
Met Glu Asp Asp Ser Leu Tyr Leu Gly Gly Glu Trp Gln Phe Asn His 1 5
10 15 Phe Ser Lys Leu Thr Ser Ser Arg Pro Asp Ala Ala Phe Ala Glu
Ile 20 25 30 Gln Arg Thr Ser Leu Pro Glu Lys Ser Pro Leu Ser Cys
Glu Ala Arg 35 40 45 Val Asp Leu Cys Asp Asp Leu Ala Pro Val Ala
Arg Gln Leu Ala Pro 50 55 60 Arg Glu Lys Leu Pro Leu Ser Ser Arg
Arg Pro Ala Ala Val Gly Ala 65 70 75 80 Gly Leu Gln Asn Met Gly Asn
Thr Cys Tyr Val Asn Ala Ser Leu Gln 85 90 95 Cys Leu Thr Tyr Thr
Pro Pro Leu Ala Asn Tyr Met Leu Ser Arg Glu 100 105 110 His Ser Gln
Thr Cys His Arg His Lys Gly Cys Met Leu Cys Thr Met 115 120 125 Gln
Ala His Ile Thr Arg Ala Leu His Asn Pro Gly His Val Ile Gln 130 135
140 Pro Ser Gln Ala Leu Ala Ala Gly Phe His Arg Gly Lys Gln Glu Asp
145 150 155 160 Ala His Glu Phe Leu Met Phe Thr Val Asp Ala Met Lys
Lys Ala Cys 165 170 175 Leu Pro Gly His Lys Gln Val Asp His His Ser
Lys Asp Thr Thr Leu 180 185 190 Ile His Gln Ile Phe Gly Gly Tyr Trp
Arg Ser Gln Ile Lys Cys Leu 195 200 205 His Cys His Gly Ile Ser Asp
Thr Phe Asp Pro Tyr Leu Asp Ile Ala 210 215 220 Leu Asp Ile Gln Ala
Ala Gln Ser Val Gln Gln Ala Leu Glu Gln Leu 225 230 235 240 Val Lys
Pro Glu Glu Leu Asn Gly Glu Asn Ala Tyr His Cys Gly Val 245 250 255
Cys Leu Gln Arg Ala Pro Ala Ser Lys Thr Leu Thr Leu His Thr Ser 260
265 270 Ala Lys Val Leu Ile Leu Val Leu Lys Arg Phe Ser Asp Val Thr
Gly 275 280 285 Asn Lys Ile Ala Lys Asn Val Gln Tyr Pro Glu Cys Leu
Asp Met Gln 290 295 300 Pro Tyr Met Ser Gln Gln Asn Thr Gly Pro Leu
Val Tyr Val Leu Tyr 305 310 315 320 Ala Val Leu Val His Ala Gly Trp
Ser Cys His Asn Gly His Tyr Phe 325 330 335 Ser Tyr Val Lys Ala Gln
Glu Gly Gln Trp Tyr Lys Met Asp Asp Ala 340 345 350 Glu Val Thr Ala
Ser Ser Ile Thr Ser Val Leu Ser Gln Gln Ala Tyr 355 360 365 Val Leu
Phe Tyr Ile Gln Lys Ser Glu Trp Glu Arg His Ser Glu Ser 370 375 380
Val Ser Arg Gly Arg Glu Pro Arg Ala Leu Gly Ala Glu Asp Thr Asp 385
390 395 400 Arg Arg Ala Thr Gln Gly Glu Leu Lys Arg Asp His Pro Cys
Leu Gln 405 410 415 Ala Pro Glu Leu Asp Glu His Leu Val Glu Arg Ala
Thr Gln Glu Ser 420 425 430 Thr Leu Asp His Trp Lys Phe Leu Gln Glu
Gln Asn Lys Thr Lys Pro 435 440 445 Glu Phe Asn Val Arg Lys Val Glu
Gly Thr Leu Pro Pro Asp Val Leu 450 455 460 Val Ile His Gln Ser Lys
Tyr Lys Cys Gly Met Lys Asn His His Pro 465 470 475 480 Glu Gln Gln
Ser Ser Leu Leu Asn Leu Ser Ser Thr Asn Pro Thr Asp 485 490 495 Gln
Glu Ser Met Asn Thr Gly Thr Leu Ala Ser Leu Gln Gly Arg Thr 500 505
510 Arg Arg Ala Lys Gly Lys Asn Lys His Cys Lys Arg Ser Leu Leu Val
515 520 525 Cys Gln 530 3 1590 DNA Homo sapiens CDS (1)...(1590) 3
atg gag gac gac tca ctc tac ttg gga ggt gag tgg cag ttc aac cac 48
Met Glu Asp Asp Ser Leu Tyr Leu Gly Gly Glu Trp Gln Phe Asn His 1 5
10 15 ttt tca aaa ctc aca tct tct cgg ccc gat gca gct ttt gct gaa
atc 96 Phe Ser Lys Leu Thr Ser Ser Arg Pro Asp Ala Ala Phe Ala Glu
Ile 20 25 30 cag cgg act tct ctc cct gag aag tca cca ctc tca tgt
gag gcc cgt 144 Gln Arg Thr Ser Leu Pro Glu Lys Ser Pro Leu Ser Cys
Glu Ala Arg 35 40 45 gtc gac ctc tgt gat gat ttg gct cct gtg gca
aga cag ctt gct ccc 192 Val Asp Leu Cys Asp Asp Leu Ala Pro Val Ala
Arg Gln Leu Ala Pro 50 55 60 agg gag aag ctt cct ctg agt agc agg
aga cct gct gcg gtg ggg gct 240 Arg Glu Lys Leu Pro Leu Ser Ser Arg
Arg Pro Ala Ala Val Gly Ala 65 70 75 80 ggg ctc cag aat atg gga aat
acc tgc tac gtg aac gct tcc ttg cag 288 Gly Leu Gln Asn Met Gly Asn
Thr Cys Tyr Val Asn Ala Ser Leu Gln 85 90 95 tgc ctg aca tac aca
ccg ccc ctt gcc aac tac atg ctg tcc cgg gag 336 Cys Leu Thr Tyr Thr
Pro Pro Leu Ala Asn Tyr Met Leu Ser Arg Glu 100 105 110 cac tct caa
acg tgt cat cgt cac aag ggc tgc atg ctc tgt act atg 384 His Ser Gln
Thr Cys His Arg His Lys Gly Cys Met Leu Cys Thr Met 115 120 125 caa
gct cac atc aca cgg gcc ctc cac aat cct ggc cac gtc atc cag 432 Gln
Ala His Ile Thr Arg Ala Leu His Asn Pro Gly His Val Ile Gln 130 135
140 ccc tca cag gca ttg gct gct ggc ttc cat aga ggc aag cag gaa gat
480 Pro Ser Gln Ala Leu Ala Ala Gly Phe His Arg Gly Lys Gln Glu Asp
145 150 155 160 gcc cat gaa ttt ctc atg ttc act gtg gat gcc atg aaa
aag gca tgc 528 Ala His Glu Phe Leu Met Phe Thr Val Asp Ala Met Lys
Lys Ala Cys 165 170 175 ctt ccc ggg cac aag cag gta gat cat cac tct
aag gac acc acc ctc 576 Leu Pro Gly His Lys Gln Val Asp His His Ser
Lys Asp Thr Thr Leu 180 185 190 atc cac caa ata ttt gga ggc tac tgg
aga tct caa atc aag tgt ctc 624 Ile His Gln Ile Phe Gly Gly Tyr Trp
Arg Ser Gln Ile Lys Cys Leu 195 200 205 cac tgc cac ggc att tca gac
act ttt gac cct tac ctg gac atc gcc 672 His Cys His Gly Ile Ser Asp
Thr Phe Asp Pro Tyr Leu Asp Ile Ala 210 215 220 ctg gat atc cag gca
gct cag agt gtc cag caa gct ttg gaa cag ttg 720 Leu Asp Ile Gln Ala
Ala Gln Ser Val Gln Gln Ala Leu Glu Gln Leu 225 230 235 240 gtg aag
ccc gaa gaa ctc aat gga gag aat gcc tat cat tgt ggt gtt 768 Val Lys
Pro Glu Glu Leu Asn Gly Glu Asn Ala Tyr His Cys Gly Val 245 250 255
tgt ctc cag agg gcg ccg gcc tcc aag acg tta act tta cac acc tct 816
Cys Leu Gln Arg Ala Pro Ala Ser Lys Thr Leu Thr Leu His Thr Ser 260
265 270 gcc aag gtc ctc atc ctt gta ttg aag aga ttc tcc gat gtc aca
ggc 864 Ala Lys Val Leu Ile Leu Val Leu Lys Arg Phe Ser Asp Val Thr
Gly 275 280 285 aac aag att gcc aag aat gtg caa tat cct gag tgc ctt
gac atg cag 912 Asn Lys Ile Ala Lys Asn Val Gln Tyr Pro Glu Cys Leu
Asp Met Gln 290 295 300 cca tac atg tct cag cag aac aca gga cct ctt
gtc tat gtc ctc tat 960 Pro Tyr Met Ser Gln Gln Asn Thr Gly Pro Leu
Val Tyr Val Leu Tyr 305 310 315 320 gct gtg ctg gtc cac gct ggg tgg
agt tgt cac aac gga cat tac ttc 1008 Ala Val Leu Val His Ala Gly
Trp Ser Cys His Asn Gly His Tyr Phe 325 330 335 tct tat gtc aaa gct
caa gaa ggc cag tgg tat aaa atg gat gat gcc 1056 Ser Tyr Val Lys
Ala Gln Glu Gly Gln Trp Tyr Lys Met Asp Asp Ala 340 345 350 gag gtc
acc gcc tct agc atc act tct gtc ctg agt caa cag gcc tac 1104 Glu
Val Thr Ala Ser Ser Ile Thr Ser Val Leu Ser Gln Gln Ala Tyr 355 360
365 gtc ctc ttt tac atc cag aag agt gaa tgg gaa aga cac agt gag agt
1152 Val Leu Phe Tyr Ile Gln Lys Ser Glu Trp Glu Arg His Ser Glu
Ser 370 375 380 gtg tca aga ggc agg gaa cca aga gcc ctt ggc gca gaa
gac aca gac 1200 Val Ser Arg Gly Arg Glu Pro Arg Ala Leu Gly Ala
Glu Asp Thr Asp 385 390 395 400 agg cga gca acg caa gga gag ctc aag
aga gac cac ccc tgc ctc cag 1248 Arg Arg Ala Thr Gln Gly Glu Leu
Lys Arg Asp His Pro Cys Leu Gln 405 410 415 gcc ccc gag ttg gac gag
cac ttg gtg gaa aga gcc act cag gaa agc 1296 Ala Pro Glu Leu Asp
Glu His Leu Val Glu Arg Ala Thr Gln Glu Ser 420 425 430 acc tta gac
cac tgg aaa ttc ctt caa gag caa aac aaa acg aag cct 1344 Thr Leu
Asp His Trp Lys Phe Leu Gln Glu Gln Asn Lys Thr Lys Pro 435 440 445
gag ttc aac gtc aga aaa gtc gaa ggt acc ctg cct ccc gac gta ctt
1392 Glu Phe Asn Val Arg Lys Val Glu Gly Thr Leu Pro Pro Asp Val
Leu 450 455 460 gtg att cat caa tca aaa tac aag tgt ggg atg aaa aac
cat cat cct 1440 Val Ile His Gln Ser Lys Tyr Lys Cys Gly Met Lys
Asn His His Pro 465 470 475 480 gaa cag caa agc tcc ctg cta aac ctc
tct tcg acg aac ccg aca gat 1488 Glu Gln Gln Ser Ser Leu Leu Asn
Leu Ser Ser Thr Asn Pro Thr Asp 485 490 495 cag gag tcc atg aac act
ggc aca ctc gct tct ctg caa ggg agg acc 1536 Gln Glu Ser Met Asn
Thr Gly Thr Leu Ala Ser Leu Gln Gly Arg Thr 500 505 510 agg aga gcc
aaa ggg aag aac aaa cac tgc aag aga tct ctg ctt gtg 1584 Arg Arg
Ala Lys Gly Lys Asn Lys His Cys Lys Arg Ser Leu Leu Val 515 520 525
tgc cag 1590 Cys Gln 530 4 32 PRT Artificial Sequence Consensus 4
Thr Gly Leu Ile Asn Leu Gly Asn Thr Cys Tyr Met Asn Ser Val Leu 1 5
10 15 Gln Cys Leu Phe Ser Ile Pro Pro Leu Arg Asp Tyr Leu Leu Asp
Ile 20 25 30 5 69 PRT Artificial Sequence Consensus 5 Gly Pro Gly
Lys Tyr Glu Leu Tyr Ala Val Val Val His Ser Gly Ser 1 5 10 15 Ser
Leu Ser Gly Gly His Tyr Thr Ala Tyr Val Lys Lys Glu Asn Trp 20 25
30 Tyr Lys Phe Asp Asp Asp Lys Val Ser Arg Val Thr Glu Glu Glu Val
35 40 45 Leu Lys Glu Ser Gly Gly Glu Ser Gly Asp Thr Ser Ser Ala
Tyr Ile 50 55 60 Leu Phe Tyr Glu Arg 65 6 16 PRT Artificial
Sequence consensus 6 Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa Gln 1 5 10 15 7 19 PRT Artificial Sequence
consensus 7 Tyr Xaa Leu Xaa Xaa Xaa Xaa Xaa His Xaa Gly Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 Gly His Tyr
* * * * *
References