U.S. patent application number 10/606366 was filed with the patent office on 2004-01-22 for isolated human enzyme, nucleic acid molecules encoding human enzyme, and uses thereof.
This patent application is currently assigned to APPLERA CORPORATION. Invention is credited to Beasley, Ellen M., DiFrancesco, Valentina, Wei, Ming-Hui, Yan, Chunhua.
Application Number | 20040014666 10/606366 |
Document ID | / |
Family ID | 27124026 |
Filed Date | 2004-01-22 |
United States Patent
Application |
20040014666 |
Kind Code |
A1 |
Wei, Ming-Hui ; et
al. |
January 22, 2004 |
Isolated human enzyme, nucleic acid molecules encoding human
enzyme, and uses thereof
Abstract
The present invention provides amino acid sequences of peptides
that are encoded by genes within the human genome, the enzyme
peptides of the present invention. The present invention
specifically provides isolated peptide and nucleic acid molecules,
methods of identifying orthologs and paralogs of the enzyme
peptides, and methods of identifying modulators of the enzyme
peptides.
Inventors: |
Wei, Ming-Hui; (Germantown,
MD) ; Yan, Chunhua; (Boyds, MD) ; DiFrancesco,
Valentina; (Rockville, MD) ; Beasley, Ellen M.;
(Darnestown, MD) |
Correspondence
Address: |
CELERA GENOMICS CORP.
ATTN: WAYNE MONTGOMERY, VICE PRES, INTEL PROPERTY
45 WEST GUDE DRIVE
C2-4#20
ROCKVILLE
MD
20850
US
|
Assignee: |
APPLERA CORPORATION
Global Headquarters, 301 Merritt 7 P.O. Box 5435
Norwalk
CT
06856-5435
|
Family ID: |
27124026 |
Appl. No.: |
10/606366 |
Filed: |
June 26, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10606366 |
Jun 26, 2003 |
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09956993 |
Sep 21, 2001 |
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6613554 |
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09956993 |
Sep 21, 2001 |
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09816088 |
Mar 26, 2001 |
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6326180 |
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Current U.S.
Class: |
435/183 ;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12Q 1/6876 20130101;
C12N 9/0006 20130101 |
Class at
Publication: |
514/12 ;
435/69.1; 435/183; 435/320.1; 435/325; 536/23.2 |
International
Class: |
A61K 038/43; C07H
021/04; C12N 009/00; C12P 021/02; C12N 005/06 |
Claims
That which is claimed is:
1. An isolated peptide consisting of an amino acid sequence
selected from the group consisting of: (a) an amino acid sequence
shown in SEQ ID NO: 2; (b) an amino acid sequence of an allelic
variant of an amino acid sequence shown in SEQ ID NO: 2, wherein
said allelic variant is encoded by a nucleic acid molecule that
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (c) an amino
acid sequence of an ortholog of an amino acid sequence shown in SEQ
ID NO: 2, wherein said ortholog is encoded by a nucleic acid
molecule that hybridizes under stringent conditions to the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; and
(d) a fragment of an amino acid sequence shown in SEQ ID NO: 2,
wherein said fragment comprises at least 10 contiguous amino
acids.
2. An isolated peptide comprising an amino acid sequence selected
from the group consisting of: (a) an amino acid sequence shown in
SEQ ID NO: 2; (b) an amino acid sequence of an allelic variant of
an amino acid sequence shown in SEQ ID NO: 2, wherein said allelic
variant is encoded by a nucleic acid molecule that hybridizes under
stringent conditions to the opposite strand of a nucleic acid
molecule shown in SEQ ID NOS: 1 or 3; (c) an amino acid sequence of
an ortholog of an amino acid sequence shown in SEQ ID NO: 2,
wherein said ortholog is encoded by a nucleic acid molecule that
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS: 1or 3; and (d) a
fragment of an amino acid sequence shown in SEQ ID NO: 2, wherein
said fragment comprises at least 10 contiguous amino acids.
3. An isolated antibody that selectively binds to a peptide of
claim 2.
4. An isolated nucleic acid molecule consisting of a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence that encodes an amino acid sequence shown in SEQ ID NO: 2;
(b) a nucleotide sequence that encodes of an allelic variant of an
amino acid sequence shown in SEQ ID NO: 2, wherein said nucleotide
sequence hybridizes under stringent conditions to: the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (c)
a nucleotide sequence that encodes an ortholog of an amino acid
sequence shown in SEQ ID NO: 2, wherein said nucleotide sequence
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotide
sequence that encodes a fragment of an amino acid sequence shown in
SEQ ID NO: 2, wherein said fragment comprises at least 10
contiguous amino acids; and (e) a nucleotide sequence that is the
complement of a nucleotide sequence of (a)-(d).
5. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence that encodes an amino acid sequence shown in SEQ ID NO: 2;
(b) a nucleotide sequence that encodes of an allelic variant of an
amino acid sequence shown in SEQ ID NO: 2, wherein said nucleotide
sequence hybridizes under stringent conditions to the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (c)
a nucleotide sequence that encodes an ortholog of an amino acid
sequence shown in SEQ ID NO: 2, wherein said nucleotide sequence
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotide
sequence that encodes a fragment of an amino acid sequence shown in
SEQ ID NO: 2, wherein said fragment comprises at least 10
contiguous amino acids; and (e) a nucleotide sequence that is the
complement of a nucleotide sequence of (a)-(d).
6. A gene chip comprising a nucleic acid molecule of claim 5.
7. A transgenic non-human animal comprising a nucleic acid molecule
of claim 5.
8. A nucleic acid vector comprising a nucleic acid molecule of
claim 5.
9. A host cell containing the vector of claim 8.
10. A method for producing any of the peptides of claim 1
comprising introducing a nucleotide sequence encoding any of the
amino acid sequences in (a)-(d) into a host cell, and culturing the
host cell under conditions in which the peptides are expressed from
the nucleotide, sequence.
11. A method for producing any of the peptides of claim 2
comprising introducing a nucleotide sequence encoding any of the
amino acid sequences in (a)-(d) into a host cell, and culturing the
host cell under conditions in which the peptides are expressed from
the nucleotide sequence.
12. A method for detecting the presence of any of the peptides of
claim 2 in a sample, said method comprising contacting said sample
with a detection agent that specifically allows detection of the
presence of the peptide in the sample and then detecting the
presence of the peptide.
13. A method for detecting the presence of a nucleic acid molecule
of claim 5 in a sample, said method comprising Contacting the
sample with an oligonucleotide that hybridizes to said nucleic acid
molecule under stringent conditions and determining whether the
oligonucleotide binds to said nucleic acid molecule in the
sample.
14. A method for identifying a modulator of a peptide of claim 2,
said method comprising contacting said peptide with an agent and
determining if said agent has modulated the function or activity of
said peptide.
15. The method of claim 14, wherein said agent is administered to a
host cell comprising an expression vector that expresses said
peptide.
16. A method for identifying an agent that binds to any of the
peptides of claim 2, said method comprising contacting the peptide
with an agent and assaying the contacted mixture to determine
whether a complex is formed with the agent bound to the
peptide.
17. A pharmaceutical composition comprising an agent identified by
the method of claim 16 and a pharmaceutically acceptable carrier
therefor.
18. A method for treating a disease or condition mediated by a
human enzyme protein, said method comprising administering to a
patient a pharmaceutically effective amount of an agent identified
by the method of claim 16.
19. A method for identifying a modulator of the expression of a
peptide of claim 2, said method comprising contacting a cell
expressing said peptide with an agent, and determining if said
agent has modulated the expression of said peptide.
20. An isolated human enzyme peptide having an amino acid sequence
that shares at least 70% homology with an amino acid sequence shown
in SEQ ID NO: 2.
21. A peptide according to claim 20 that shares at least 90 percent
homology with an amino acid sequence shown in SEQ ID NO: 2.
22. An isolated nucleic; acid molecule encoding a human enzyme
peptide, said nucleic acid molecule sharing at least 80 percent
homology with a nucleic acid molecule shown in SEQ ID NOS: 1 or
3.
23. A nucleic acid molecule according to claim 22 that shares at
least 90 percent homology with a nucleic acid molecule shown in SEQ
ID NOS: 1 or 3.
Description
FIELD OF THE INVENTION
[0001] The present invention is in the field of enzyme proteins
that are related to the steroid oxidoreductase subfamily,
recombinant DNA molecules, and protein production. The present
invention specifically provides novel peptides and proteins that
effect protein phosphorylation and nucleic acid molecules encoding
such peptide and protein molecules, all of which are useful in the
development of human therapeutics and diagnostic compositions and
methods.
BACKGROUND OF THE INVENTION
[0002] Many human enzymes serve as targets for the action of
pharmaceutically active compounds. Several classes of human enzymes
that serve as such targets include helicase, steroid esterase and
sulfatase, convertase, synthase, dehydrogenase, monoxygenase,
transferase, kinase, glutanase, decarboxylase, isomerase and
reductase. It is therefore important in developing new
pharmaceutical compounds to identify target enzyme proteins that
can be put into high-throughput screening formats. The present
invention advances the state of the art by providing novel human
drug target enzymes related to the steroid oxidoreductase
subfamily.
[0003] The present invention has substantial similarity to
3.beta.-hydroxy-5-C27-steroid oxidoreductase (C.sub.27
3.beta.-HSD). 3.beta.-hydroxy-5-C27-steroid oxidoreductase involves
in the synthesis of bile acids.
[0004] Bile acids are important components of normal physiology
with essential functions in the liver and small intestine. Their
synthesis in the liver provides a metabolic pathway for the
catabolism of cholesterol and their detergent properties promote
the solubilization of essential nutrients and vitamins in the small
intestine. The synthesis of bile acids was thought to involve as
many as three separate pathways. Each pathway differs in the
initial steps and in the involvement of distinct sterol
7-hydroxylase enzymes that add an essential hydroxyl group to
carbon seven of the sterol ring. After the addition of this group,
the next step in each pathway is catalyzed by a
3.beta.-hydroxy-5-C27-steroid oxidoreductase, which isomerizes the
5 bond to the 4 position and oxidizes the 3.beta.-hydroxyl group to
a 3-oxo moiety on intermediates with 27 carbon atoms.
[0005] The 3.beta.-HSD enzyme family consists of a large number of
proteins that ate present in both prokaryotes and eukaryotes. They
are proposed to play a wide variety of anabolic and catabolic roles
in intermediary metabolism, and, consistent with this broad
function, they are expressed in abundance in organisms ranging from
viruses to humans.
[0006] C.sub.27 3.beta.-HSD enzyme deficiency is marked by
accumulation of C.sub.27 sterol intermediates of bile acid
synthesis, progressive intrahepatic cholestasis, and no endocrine
abnormalities. Inherited conditions that prevent the synthesis of
bile acids can cause the accumulation of cholesterol and liver
dysfunction (cholestasis), underscoring the essential role of bile
acids in metabolism. Progressive neonatal intrahepatic cholestasis
is marked by jaundice, fat-soluble vitamin deficiency, and lipid
malabsorption and is a rare condition of diverse etiologies. Among
the causes of this disorder are inborn errors of metabolism that
affect the production and secretion of bile. In the absence of a
normal bile flow and bile acid pool size, the end products of heme
metabolism are not secreted, and their accumulation causes the
characteristic jaundice. Dietary fat-soluble vitamins are not
effectively taken up in the intestine leading to deficiencies in
hemiostasis, and hydrophobic lipids such as long-chain fatty acids
and cholesterol are poorly absorbed causing fatty stools. It has
been reported that a deficiency of this enzyme can be treated by
oral administration of bile acids. For a review relate to the
oxidoreductase, see Schwarz et al., J Clin Invest November 2000;
106(9):1175-84.
[0007] Enzyme proteins, particularly members of the steroid
oxidoreductase subfamily, are a major target for drug action and
development. Accordingly, it is valuable to the field of
pharmaceutical development to identify and characterize previously
unknown members of this subfamily of enzyme proteins. The present
invention advances the state of the art by providing previously
unidentified human enzyme proteins, and the polynucleotides
encoding them, that have homology to members of the steroid
oxidoreductase subfamily. These novel compositions are useful in
the diagnosis, prevention and treatment of biological processes
associated with human diseases.
SUMMARY OF THE INVENTION
[0008] The present invention is based in part on the identification
of amino acid sequences of human enzyme peptides and proteins that
are related to the steroid oxidoreductase subfamily, as well as
allelic variants and other mammalian orthologs thereof. These
unique peptide sequences, and nucleic acid sequences that encode
these peptides, can be used as models for the development of human
therapeutic targets, aid in the identification of therapeutic
proteins, and serve as targets for the development of human
therapeutic agents that modulate enzyme activity in cells and
tissues that express the enzyme. Experimental data as provided in
FIG. 1 indicates expression in humans in the brain, kidney, colon
and uterus.
DESCRIPTION OF THE FIGURE SHEETS
[0009] FIG. 1 provides the nucleotide sequence of a cDNA molecule
sequence that encodes the enzyme protein of the present invention.
(SEQ ID NO: 1) In addition, structure and functional information is
provided, such as ATG start, stop and tissue distribution, where
available, that allows one to readily determine specific uses of
inventions based on this molecular sequence. Experimental data as
provided in FIG. 1 indicates expression in humans in the brain,
kidney, colon and uterus.
[0010] FIG. 2 provides the predicted amino acid sequence of the
enzyme of the present invention. (SEQ ID NO: 2) In addition
structure and functional information such as protein family,
function, and modification sites is provided where available,
allowing one to readily determine specific uses of inventions based
on this molecular sequence.
[0011] FIG. 3 provides genomic sequences that span the gene
encoding the enzyme protein of the present invention. (SEQ ID NO:
3) In addition structure and functional information, such as
intron/exon structure, promoter location, etc., is provided where
available, allowing one to readily determine specific uses of
inventions based on this molecular sequence. As illustrated in FIG.
3, SNPs, were identified at 4 different nucleotide positions.
DETAILED DESCRIPTION OF THE INVENTION
[0012] General Description
[0013] The present invention is based on the sequencing of the
human genome. During the sequencing and assembly of the human
genome, analysis of the sequence information revealed previously
unidentified fragments of the human genome that encode peptides
that share structural and/or sequence homology to
protein/peptide/domains identified and characterized within the art
as being a enzyme protein or part of a enzyme protein and are
related to the steroid oxidoreductase subfamily. Utilizing these
sequences, additional genomic sequences were assembled and
transcript and/or cDNA sequences were isolated and characterized.
Based on this analysis, the present invention provides amino acid
sequences of human enzyme peptides and proteins that are related to
the steroid oxidoreductase subfamily, nucleic acid sequences in the
form of transcript sequences, cDNA sequences and/or genomic
sequences that encode these enzyme peptides and proteins, nucleic
acid variation (allelic information), tissue distribution of
expression, and information about the closest art known
protein/peptide/domain that has structural or sequence homology to
the enzyme of the present invention.
[0014] In addition to being previously unknown, the peptides that
are provided in the present invention are selected based on their
ability to be used for the development of commercially important
products and services. Specifically, the present peptides are
selected based on homology and/or structural relatedness to known
enzyme proteins of the steroid oxidoreductase subfamily and the
expression pattern observed. Experimental data as provided in FIG.
1 indicates expression in humans in the brain, kidney, colon and
uterus. The art has clearly established the commercial importance
of members of this family of proteins and proteins that have
expression patterns similar to that of the present gene. Some of
the more specific features of the peptides of the present
invention, and the uses thereof, are described herein, particularly
in the Background of the Invention and in the annotation provided
in the Figures, and/or are known within the art for each of the
known steroid oxidoreductase family or subfamily of enzyme
proteins.
[0015] Specific Embodiments
[0016] Peptide Molecules
[0017] The present invention provides nucleic acid sequences that
encode protein molecules that have been identified as being members
of the enzyme family of proteins and are related to the steroid
oxidoreductase subfamily (protein sequences are provided in FIG. 2,
transcript/cDNA sequences are provided in FIG. 1 and genomic
sequences are provided in FIG. 3). The peptide sequences provided
in FIG. 2, as well as the obvious variants described herein,
particularly allelic variants as identified herein and using the
information in FIG. 3, will be referred herein as the enzyme
peptides of the present invention, enzyme peptides, or
peptides/proteins of the present invention.
[0018] The present invention provides isolated peptide and protein
molecules that consist of, consist essentially of, or comprise the
amino acid sequences of the enzyme peptides disclosed in the FIG.
2, (encoded by the nucleic acid molecule shown in FIG. 1,
transcript/cDNA or FIG. 3, genomic sequence), as well as all
obvious variants of these peptides that are within the art to make
and use. Some of these variants are described in detail below.
[0019] As used herein, a peptide is said to be "isolated" or
"purified" when it is substantially free of cellular material or
free of chemical precursors or other chemicals. The peptides of the
present invention can be purified to homogeneity or other degrees
of purity. The level of purification will be based on the intended
use. The critical feature is that the preparation allows for the
desired function of the peptide, even if in the presence of
considerable amounts of other components (the features of an
isolated nucleic acid molecule is discussed below).
[0020] In some uses, "substantially free of cellular material"
includes preparations of the peptide having less than about 30% (by
dry weight) other proteins (i.e., contaminating protein), less than
about 20% other proteins, less than about 10% other proteins, or
less than about 5% other proteins. When the peptide is
recombinantly produced, it can also be substantially free of
culture medium, i.e., culture medium represents less than about 20%
of the volume of the protein preparation.
[0021] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the peptide in which it
is separated from chemical precursors or other chemicals that are
involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the enzyme peptide having less than about
30% (by dry weight) chemical precursors or other chemicals, less
than about 20% chemical precursors or other chemicals, less than
about 10% chemical precursors or other chemicals, or less than
about 5% chemical precursors or other chemicals.
[0022] The isolated enzyme peptide can be purified from cells that
naturally express it, purified from cells that have been altered to
express it (recombinant), or synthesized using known protein
synthesis methods. Experimental data as provided in FIG. 1
indicates expression in humans in the brain, kidney, colon and
uterus. For example, a nucleic acid molecule encoding the enzyme
peptide is cloned into an expression vector, the expression vector
introduced into a host cell and the protein expressed in the host
cell. The protein can then be isolated from the cells by an
appropriate purification scheme using standard protein purification
techniques. Many of these techniques are described in detail
below.
[0023] Accordingly, the present invention provides proteins that
consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO:
2), for example, proteins encoded by the transcript/cDNA nucleic
acid sequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic
sequences provided in FIG. 3 (SEQ ID NO: 3). The amino acid
sequence of such a protein is provided in FIG. 2. A protein
consists of an amino acid sequence when the amino acid sequence is
the final amino acid sequence of the protein.
[0024] The present invention further provides proteins that consist
essentially of the amino acid sequences provided in FIG. 2 (SEQ ID
NO: 2), for example, proteins encoded by the transcript/cDNA
nucleic acid sequences shown in FIG. 1 (SEQ ID NO: 1) and the
genomic sequences provided in FIG. 3 (SEQ ID NO: 3). A protein
consists essentially of an amino acid sequence when such an amino
acid sequence is present with only a few additional amino acid
residues, for example from about 1 to about 100 or so additional
residues, typically from 1 to about 20 additional residues in the
final protein.
[0025] The present invention further provides proteins that
comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:
2), for example, proteins encoded by the transcript/cDNA nucleic
acid sequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic
sequences provided in FIG. 3 (SEQ ID NO: 3). A protein comprises an
amino acid sequence when the amino acid sequence is at least part
of the final amino acid sequence of the protein. In such a fashion,
the protein can be only the peptide or have additional amino acid
molecules, such as amino acid residues (contiguous encoded
sequence) that are naturally associated with it or heterologous
amino acid residues/peptide sequences. Such a protein can have a
few additional amino acid residues or can comprise several hundred
or more additional amino acids. The preferred classes of proteins
that are comprised of the enzyme peptides of the present invention
are the naturally occurring mature proteins. A brief description of
how various types of these proteins can be made/isolated is
provided below.
[0026] The enzyme peptides of the present invention can be attached
to heterologous sequences to form chimeric or fusion proteins. Such
chimeric and fusion proteins comprise a enzyme peptide operatively
linked to a heterologous protein having an amino acid sequence not
substantially homologous to the enzyme peptide. "Operatively
linked" indicates that the enzyme peptide and the heterologous
protein are fused in-frame. The heterologous protein can be fused
to the N-terminus or C-terminus of the enzyme peptide.
[0027] In some uses, the fusion protein does not affect the
activity of the enzyme peptide per se. For example, the fusion
protein can include, but is not limited to, enzymatic fusion
proteins, for example beta-galactosidase fusions, yeast two-hybrid
GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig
fusions. Such fusion proteins, particularly poly-His fusions, can
facilitate the purification of recombinant enzyme peptide. In
certain host cells (e.g., mammalian host cells), expression and/or
secretion of a protein can be increased by using a heterologous
signal sequence.
[0028] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. In another embodiment, the
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (see Ausubel et al., Current
Protocols in Molecular Biology, 1992). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). A enzyme peptide-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the enzyme peptide.
[0029] As mentioned above, the present invention also provides and
enables obvious variants of the amino acid sequence of the proteins
of the present invention, such as naturally occurring mature forms
of the peptide, allelic/sequence variants of the peptides,
non-naturally occurring recombinantly derived variants of the
peptides, and orthologs and paralogs of the peptides. Such variants
can readily be generated using art-known techniques in the fields
of recombinant nucleic acid technology and protein biochemistry. It
is understood, however, that variants exclude any amino acid
sequences disclosed prior to the invention.
[0030] Such variants can readily be identified/made using molecular
techniques and the sequence information disclosed herein. Further,
such variants can readily be distinguished from other peptides
based on sequence and/or structural homology to the enzyme peptides
of the present invention. The degree of homology/identity present
will be based primarily on whether the peptide is a functional
variant or non-functional variant, the amount of divergence present
in the paralog family and the evolutionary distance between the
orthologs.
[0031] To determine the percent identity of two amino acid
sequences or 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, at
least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of
a reference sequence is aligned for companson purposes. 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.
[0032] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics, and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a
preferred embodiment, the percent identity between two amino acid
sequences is determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossom 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 (Devereux, J., et
al., Nucleic Acids Res. 12(1):387 (1984)) (available at
http://www.gcg.eorn), 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. In another embodiment, the percent identity between two amino
acid or nucleotide sequences is determined using the algorithm of
E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) 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.
[0033] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against sequence 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. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches
can be performed with the NBLAST program, score=100, wordlength=12
to obtain nucleotide sequences homologous to the 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 the proteins of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (Nucleic Acids Res.
25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used.
[0034] Full-length pre-processed forms, as well as mature processed
forms, of proteins that comprise one of the peptides of the present
invention can readily be identified as having complete sequence
identity to one of the enzyme peptides of the present invention as
well as being encoded by the same genetic locus as the enzyme
peptide provided herein. As indicated by the data presented in FIG.
3, the map position was determined to be on chromosome 12 by
ePCR.
[0035] Allelic variants of a enzyme peptide can readily be
identified as being a human protein having a high degree
(significant) of sequence homology/identity to at least a portion
of the enzyme peptide as well as being encoded by the same genetic
locus as the enzyme peptide provided herein. Genetic locus can
readily be determined based on the genomic information provided in
FIG. 3, such as the genomic sequence mapped to the reference human.
As indicated by the data presented in FIG. 3, the map position was
determined to be on chromosome 12 by ePCR. As used herein, two
proteins (or a region of the proteins) have, significant homology
when the amino acid sequences are typically at least about 70-80%,
80-90%, and more typically at least about 90-95% or more
homologous. A significantly homologous amino acid sequence,
according to the present invention, will be encoded by a nucleic
acid sequence that will hybridize to a enzyme peptide encoding
nucleic acid molecule under stringent conditions as more fully
described below.
[0036] FIG. 3 provides information on SNPs that have been found in
the gene encoding the enzyme of the present invention. SNPs were
identified at 4different nucleotide positions in exon, introns and
regions 5' and 3' of the ORF. Such SNPs in introns and outside the
ORF may affect control/regulatory elements. Changes in the amino
acid sequence caused by these SNPs is indicated in FIG. 3 and can
readily be determined, using the universal genetic code and the
protein sequence provided in FIG. 2 as a reference.
[0037] Paralogs of a enzyme peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the enzyme peptide, as being encoded by a gene
from humans, and as having similar activity or function. Two
proteins will typically be considered paralogs when the amino acid
sequences are typically at least about 60% or greater, and more
typically at least about 70% or greater homology through a given
region or domain. Such paralogs will be encoded by a nucleic acid
sequence that will hybridize toga enzyme peptide encoding nucleic
acid molecule under moderate to stringent conditions as more fully
described below.
[0038] Orthologs of a enzyme peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the enzyme peptide as well as being encoded by a
gene from another organism. Preferred orthologs will be isolated
from mammals, preferably primates, for the development of human
therapeutic targets and agents. Such orthologs will be encoded by a
nucleic acid sequence that will hybridize to a enzyme peptide
encoding nucleic acid molecule under moderate to stringent
conditions, as more fully described below, depending on the degree
of relatedness of the two organisms yielding the proteins.
[0039] Non-naturally occurring variants of the enzyme peptides of
the present invention can readily be generated using recombinant
techniques. Such variants include, but are not limited to
deletions, additions and substitutions in the amino acid sequence
of the enzyme peptide. For example, one class of substitutions are
conserved amino acid substitution. Such substitutions are those
that substitute a given amino acid in a enzyme peptide by another
amino acid of like characteristics. Typically seen as conservative
substitutions are the replacements, one for another, among the
aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the
hydroxyl residues Ser and Thr; exchange of the acidic residues Asp
and Glu; substitution between the amide residues Asn and Gln;
exchange of the basic residues Lys and Arg; and replacements among
the aromatic residues Phe and Tyr. Guidance concerning which amino
acid changes are likely to be phenotypically silent are found in
Bowie et al. Science 247:1306-1310 (1990).
[0040] Variant enzyme peptides can be fully functional or can lack
function in one or more activities, e.g. ability to bind substrate,
ability to phosphorylate substrate, ability to mediate signaling,
etc. Fully functional variants typically contain only conservative
variation or variation in non-critical residues or in non-critical
regions. FIG. 2 provides the result of protein analysis and can be
used to identify critical domains/regions. Functional variants can
also contain substitution of similar amino acids that result in no
change or an insignificant change in function. Alternatively, such
substitutions may positively or negatively affect function to some
degree.
[0041] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0042] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science 244:1081-1085 (1989)), particularly using the results
provided in FIG. 2. The latter procedure introduces single alanine
mutations at every residue in the molecule. The resulting mutant
molecules are then tested for biological activity such as enzyme
activity or in assays such as an in vitro proliferative activity.
Sites that are critical for binding partner/substrate binding can
also be determined by structural analysis such as crystallization,
nuclear magnetic resonance or photoaffinity labeling (Smith et al.,
J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312
(1992)).
[0043] The present invention further provides fragments of the
enzyme peptides, in addition to proteins and peptides that comprise
and consist of such fragments, particularly those comprising the
residues identified in FIG. 2. The fragments to which the invention
pertains, however, are not to be construed as encompassing
fragments that may be disclosed publicly prior to the present
invention.
[0044] As used herein, a fragment comprises at least 8, 10, 12, 14,
16, or more contiguous amino acid residues from a enzyme peptide.
Such fragments can be chosen based on the ability to retain one or
more of the biological activities of the enzyme peptide or could be
chosen for the ability to perform a function, e.g. bind a substrate
or act as an immunogen. Particularly important fragments are
biologically active fragments, peptides that are, for example,
about 8 or more amino acids in length. Such fragments will
typically comprise a domain or motif of the enzyme peptide, e.g.,
active site, a transmembrane domain or a substrate-binding domain.
Further, possible fragments include, but are not limited to, domain
or motif containing fragments, soluble peptide fragments, and
fragments containing immunogenic structures. Predicted domains and
functional sites are readily identifiable by computer programs well
known and readily available to those of skill in the art (e.g.,
PROSITE analysis). The results of one such analysis are provided in
FIG. 2.
[0045] Polypeptides often contain amino acids other than the 20
amino acids commonly referred to as the 20 naturally occurring
amino acids. Further, many amino acids, including the terminal
amino acids, may be modified by natural processes, such as
processing and other post-translational modifications, or by
chemical modification techniques well known in the art. Common
modifications that occur naturally in enzyme peptides are described
in basic texts, detailed monographs, and the research literature,
and they are well known to those of skill in the art (some of these
features are identified in FIG. 2).
[0046] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosyation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0047] Such modifications are well known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993). Many
detailed reviews are available on this subject, such as by Wold,
F., Posttranslational Covalent Modification of Proteins, B. C.
Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al.
(Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y.
Acad. Sci. 663:48-62 (1992)).
[0048] Accordingly, the enzyme peptides of the present invention
also encompass derivatives or analogs in which a substituted amino
acid residue is not one encoded by the genetic code, in which a
substituent group is included, in which the mature enzyme peptide
is fused with another compound, such as a compound to increase the
half-life of the enzyme peptide (for example, polyethylene glycol),
or in which the additional amino acids are fused to the mature
enzyme peptide, such as a leader or secretory sequence or a
sequence for purification of the mature enzyme peptide o a
pro-protein sequence.
[0049] Protein/Peptide Uses
[0050] The proteins of the present invention can be used in
substantial and specific assays related to the functional
information provided in the Figures; to raise antibodies or to
elicit another immune response; as a reagent (including the labeled
reagent) in assays designed to quantitatively determine levels of
the protein. (or its binding partner or ligand)in biological
fluids; and as markers for tissues in which the corresponding
protein is preferentially expressed (either constitutively or; at a
particular stage of tissue differentiation or development or in a
disease state). Where the protein binds or potentially binds to
another protein or ligand (such as, for example, in a
enzyme-effector protein interaction or enzyme-ligand interaction),
the protein can be used to identify the binding partner/ligand so
as to develop a system to identify inhibitors of the binding
interaction. Any or all of these uses are capable of being
developed into reagent grade or kit format for commercialization as
commercial products.
[0051] Methods for performing the uses listed above are well known
to those skilled in the art. References disclosing such methods
include "Molecular Cloning: A Laboratory Manual", 2d ed., Cold
Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T.
Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular
Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel
eds., 1987.
[0052] The potential uses of the peptides of the present invention
are based primarily on the source of the protein as well as the
class/action of the protein. For example, enzymes isolated from
humans and their human/mammalian orthologs serve as targets for
identifying agents for use in mammalian therapeutic applications,
e.g. a human drug, particularly in modulating a biological or
pathological response in a cell or tissue that expresses the
enzyme. Experimental data as provided in FIG. 1 indicates that the
enzymes of the present invention are expressed in humans in the
brain, kidney, colon, uterus detected by a virtual northern blot. A
large percentage of pharmaceutical agents are being developed that
modulate the activity of enzyme proteins, particularly members of
the steroid oxidoreductase subfamily (see Background of the
Invention). The structural and functional information provided in
the Background and Figures provide specific and substantial uses
for the molecules of the present invention, particularly in
combination with the expression information provided in FIG. 1.
Experimental data as provided in FIG. 1 indicates expression in
humans in the brain, kidney, colon and uterus. Such uses can
readily be determined using the information provided herein, that
which is known in the art, and routine experimentation.
[0053] The proteins of the present invention (including variants
and fragments that may have been disclosed prior to the present
invention) are useful for biological assays related to enzymes that
are related to members of the steroid oxidoreductase subfamily.
Such assays involve any of the known enzyme functions or activities
or properties useful for diagnosis and treatment of enzyme-related
conditions that are specific for the subfamily of enzymes that the
one of the present invention belongs to, particularly in cells and
tissues that express the enzyme. Experimental data as provided in
FIG. 1 indicates that the enzymes of the present invention are
expressed in humans in the brain, kidney, colon, uterus detected by
a virtual northern blot.
[0054] The proteins of the present invention are also useful in
drug screening assays, in cell-based or cell-free systems.
Cell-based systems can be native, i.e., cells that normally express
the enzyme, as a biopsy or expanded in cell culture. Experimental
data as provided in FIG. 1 indicates expression in humans in the
brain, kidney, colon and uterus. In an alternate embodiment,
cell-based assays involve recombinant host cells expressing the
enzyme protein.
[0055] The polypeptides can be used to identify compounds that
modulate enzyme activity of the protein in its natural state or an
altered form that causes a specific disease or pathology associated
with the enzyme. Both the enzymes of the present invention and
appropriate variants and fragments can be used in high-throughput
screens to assay candidate compounds for the ability to bind to the
enzyme. These compounds can be further screened against a
functional enzyme to determine the effect of the compound on the
enzyme activity. Further, these compounds can be tested in animal
or invertebrate systems to determine activity/effectiveness.
Compounds can be identified that activate (agonist) or inactivate
(antagonist) the enzyme to a desired degree.
[0056] Further, the proteins of the present invention can be used
to screen a compound for the ability to stimulate or inhibit
interaction between the enzyme protein and a molecule that normally
interacts with the enzyme protein, e.g. a substrate or a component
of the signal pathway that the enzyme protein normally interacts
(for example, another enzyme). Such assays typically include the
steps of combining the enzyme protein with a candidate compound
under conditions that allow the enzyme protein, or fragment, to
interact with the target molecule, and to detect the formation of a
complex between the protein and the target or to detect the
biochemical consequence of the interaction with the enzyme protein
and the target, such as any of the associated effects of signal
transduction such as protein phosphorylation, cAMP turnover, and
adenylate cyclase activation, etc.
[0057] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al., Nature
354:82-84 (1991), Houghten et al., Nature 354:84-86 (1991)) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L- configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0058] One candidate compound is a soluble fragment of the receptor
that competes for substrate binding. Other candidate compounds
include mutant enzymes or appropriate fragments containing
mutations that affect enzyme function and thus compete for
substrate. Accordingly, a fragment that competes for substrate, for
example with a higher affinity, or a fragment that binds substrate
but does not allow release, is encompassed by the invention.
[0059] The invention further includes other end point assays to
identify compounds that modulate (stimulate or inhibit) enzyme
activity. The assays typically involve an assay of events in the
signal transduction pathway that indicate enzyme activity. Thus,
the phosphorylation of a substrate, activation of a protein, a
change in the expression of genes that are up- or down-regulated in
response to the enzyme protein dependent signal cascade can be
assayed.
[0060] Any of the biological or biochemical functions mediated by
the enzyme can be used as an endpoint assay. These include all of
the biochemical or biochemical/biological events described herein,
in the references cited herein, incorporated by reference for these
endpoint assay targets, and other functions known to those of
ordinary skill in the art or that can be readily identified using
the information provided in the Figures, particularly FIG. 2.
Specifically, a biological function of a cell or tissues that
expresses the enzyme can be assayed. Experimental data as provided
in FIG. 1 indicates: that the enzymes of the present invention are
expressed in humans in the brain, kidney, colon, uterus detected by
a virtual northern blot.
[0061] Binding and/or activating compounds can also be screened by
using chimeric enzyme proteins in which the amino terminal
extracellular domain, or parts thereof, the entire transmembrane
domain or subregions, such as any of the seven transmembrane
segments or any of the intracellular or extracellular loops and the
carboxy terminal intracellular domain, or parts thereof, can be
replaced by heterologous domains or subregions. For example, a
substrate-binding region can be used that interacts with a
different substrate then that which is recognized by the native
enzyme. Accordingly, a different set of signal transduction
components is available as an end-point assay for activation. This
allows for assays to be performed in other than the specific host
cell from which the enzyme is derived.
[0062] The proteins of the present invention are also useful in
competition binding assays in methods designed to discover
compounds that interact with the enzyme (e.g. binding partners
and/or ligands). Thus, a compound is exposed to a enzyme
polypeptide under conditions that allow the compound to bind or to
otherwise interact with the polypeptide. Soluble enzyme polypeptide
is also added to the mixture. If the test compound interacts with
the soluble enzyme polypeptide, it decreases the amount of complex
formed or activity from the enzyme target. This type of assay is
particularly useful in cases in which compounds are sought that
interact with specific regions of the enzyme. Thus, the soluble
polypeptide that competes with the target enzyme region is designed
to contain peptide sequences corresponding to the region of
interest.
[0063] To perform cell free drug screening assays, it is sometimes
desirable to immobilize either the enzyme protein, or fragment, or
its target molecule to facilitate separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay.
[0064] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound, to a matrix. For example, glutathione-S-transferase fusion
proteins can be adsorbed onto glutathione sepharose beads (Sigma
Chemical, St. Louis, Mo.) or glutathione derivatized microtitre
plates, which are then combined with the cell lysates (e.g.,
.sup.35S-labeled) and the candidate compound, and the mixture
incubated under conditions conducive to complex formation (e.g. at
physiological conditions for salt and pH). Following incubation,
the beads are washed to remove any unbound label, and the matrix
immobilized and radiolabel determined directly, or in the
supematant after the complexes are dissociated. Alternatively, the
complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of enzyme-binding protein found in the bead
fraction quantitated from the gel using standard electrophoretic
techniques. For example, either the polypeptide or its target
molecule can be immobilized utilizing conjugation of biotin and
streptavidin using techniques well known in the art. Alternatively,
antibodies reactive with the protein but which do not interfere
with binding of the protein to its target molecule can be
derivatized to the wells of the plate, and the protein trapped in
the wells by antibody conjugation. Preparations of a enzyme-binding
protein and a candidate compound are incubated in the enzyme
protein-presenting wells and the amount of complex trapped in the
well can be quantitated. 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 enzyme protein target molecule, or which are
reactive with enzyme protein and compete with the target molecule,
as well as enzyme-linked assays which rely on detecting an
enzymatic activity associated with the target molecule.
[0065] Agents that modulate one of the enzymes of the present
invention can be identified using one or more of the above assays,
alone or in combination. It is generally preferable to use a
cell-based or cell free system first and then confirm activity in
an animal or other model system. Such model systems are well known
in the art and can readily be employed in this context.
[0066] Modulators of enzyme protein activity identified according
to these drug screening assays can be used to treat a subject with
a disorder mediated by the enzyme pathway, by treating cells or
tissues that express the enzyme. Experimental data as provided in
FIG. 1 indicates expression in humans in the brain, kidney, colon
and uterus. These methods of treatment include the steps of
administering a modulator of enzyme activity in a pharmaceutical
composition to a subject in need of such treatment, the modulator
being identified as described herein.
[0067] In yet another aspect of the invention, the enzyme 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 the
enzyme and are involved in enzyme activity. Such enzyme binding
proteins are also likely to be involved in the propagation of
signals by the enzyme proteins or enzyme targets as, for example,
downstream elements of a enzyme-mediated signaling pathway.
Alternatively, such enzyme-binding proteins are likely to be enzyme
inhibitors.
[0068] 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 enzyme
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.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming a enzyme-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 enzyme protein.
[0069] 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 in an appropriate animal model. For example, an
agent identified as described herein (e.g., a enzyme-modulating
agent, an antisense enzyme nucleic acid molecule, a enzyme-specific
antibody, or a enzyme-binding partner) can be used in an animal or
other model to determine the efficacy, toxicity, or side effects of
treatment with such an a gent. Alternatively, an agent identified
as described herein can be used in an animal or other model to
determine the mechanism of action of such an agent. Furthermore,
this invention pertains to uses of novel agents identified by the
above-described screening assays for treatments as described
herein.
[0070] The enzyme proteins of the present invention are also useful
to provide a target for diagnosing a disease or predisposition to
disease mediated by the peptide. Accordingly, the invention
provides methods for detecting the presence, or levels of, the
protein (or encoding mRNA) in a cell, tissue, or organism.
Experimental data as provided in FIG. 1 indicates expression in
humans in the brain, kidney, colon and uterus. The method involves
contacting A biological sample with a compound capable of
interacting with the enzyme protein such that the interaction can
be detected. Such an assay can be provided in a single detection
format or a multi-detection format such as an antibody chip
array.
[0071] One agent for detecting a protein in a sample is an antibody
capable of selectively binding to protein. A biological sample
includes tissues, cells and biological fluids isolated from a
subject, as well as tissues, cells and fluids present within a
subject.
[0072] The peptides of the present invention also provide targets
for diagnosing active protein activity, disease, or predisposition
to disease, in a patient having a variant peptide, particularly
activities and conditions that are known for other members of the
family of proteins to which the present one belongs. Thus, the
peptide can be isolated from a biological sample and assayed for
the presence of a genetic mutation that results in aberrant
peptide. This includes amino acid substitution, deletion,
insertion, rearrangement, (as the result of aberrant splicing
events), and inappropriate post-translational modification.
Analytic methods include altered electrophoretic mobility, altered
tryptic peptide digest, altered enzyme activity in cell-based or
cell-free assay, alteration in substrate or antibody-binding
pattern, altered isoelectric point, direct amino acid sequencing,
and any other of the known assay techniques useful for detecting
mutations in a protein. Such an assay can be provided in a single
detection format or a multi-detection format such as an antibody
chip array.
[0073] In vitro techniques for detection of peptide include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence using a detection
reagent, such as an antibody or protein binding agent.
Alternatively, the peptide can be detected in vivo in a subject by
introducing into the subject a labeled anti-peptide antibody or
other types of detection agent. 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.
Particularly useful are methods that detect the allelic variant of
a peptide expressed in a subject and methods which detect fragments
of a peptide in a sample.
[0074] The peptides are also useful in pharmacogenomic analysis.
Pharmacogenomics deal with clinically significant hereditary
variations in the response to drugs due to altered drug disposition
and abnormal action in affected persons. See, e.g., Eichelbaum, M.
(Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and
Linder, M. W. (Clin Chem. 43(2):254-266 (1997)). The clinical
outcomes of these variations result in severe toxicity of
therapeutic drugs in certain individuals or therapeutic failure of
drugs in certain individuals as a result of individual variation in
metabolism. Thus, the genotype of the individual can determine the
way a therapeutic compound acts on the body or the way the body
metabolizes the compound. Further, the activity of drug
metabolizing enzymes effects both the intensity and duration of
drug action. Thus, the pharmacogenomics of the individual permit
the selection of effective compounds and effective dosages of such
compounds for prophylactic or therapeutic treatment based on the
individual's genotype. The discovery of genetic polymorphisms in
some drug metabolizing enzymes has explained why some patients do
not obtain the expected drug effects, show an exaggerated drug
effect, or experience serious toxicity from standard drug dosages.
Polymorphisms can be expressed in the phenotype of the extensive
metabolizer and the phenotype of the poor metabolizer. Accordingly,
genetic polymorphism may lead to allelic protein variants of the
enzyme protein in which one or more of the enzyme functions in one
population is different from those in another population. The
peptides thus allow a target to ascertain a genetic predisposition
that can affect treatment modality. Thus, in a ligand-based
treatment, polymorphism may give rise to amino terminal
extracellular domains and/or other substrate-binding regions that
are more or less active in substrate binding, and enzyme
activation. Accordingly, substrate dosage would necessarily be
modified to maximize the therapeutic effect within a given
population containing a polymorphism. As an alternative to
genotyping, specific polymorphic peptides could be identified.
[0075] The peptides are also useful for treating a disorder
characterized by an absence of, inappropriate, or unwanted
expression of the protein. Experimental data as provided in FIG. 1
indicates expression in humans in the brain, kidney, colon and
uterus. Accordingly, methods for treatment include the use of the
enzyme protein or fragments.
[0076] Antibodies
[0077] The invention also provides antibodies that selectively bind
to one of the peptides of the present invention, a protein
comprising such a peptide, as well as variants and fragments
thereof. As used herein, an antibody selectively binds a target
peptide when it binds the target peptide and does not significantly
bind to unrelated proteins. An antibody is still considered to
selectively bind a peptide even if it also binds to other proteins
that are not substantially homologous with the target peptide so
long as such proteins share homology with a fragment or domain of
the peptide target of the antibody. In this case, it would be
understood that antibody binding to the peptide is still selective
despite some degree of cross-reactivity.
[0078] As used herein, an antibody is defined in terms consistent
with that recognized within the art: they are multi-subunit
proteins produced by a mammalian organism in response to an antigen
challenge. The antibodies of the present invention include
polyclonal antibodies and monoclonal antibodies, as well as
fragments of such antibodies, including, but not limited to, Fab or
F(ab').sub.2, and Fv fragments.
[0079] Many methods are known for generating and/or identifying
antibodies to a given target peptide. Several such methods are
described by Harlow, Antibodies, Cold Spring Harbor Press,
(1989).
[0080] In general, to generate antibodies, an isolated peptide is
used as an immunogen and is administered to a mammalian organism,
such as a rat, rabbit or mouse. The full-length protein, an
antigenic peptide fragment or a fusion protein can be used.
Particularly important fragments are those covering functional
domains, such as the domains identified in FIG. 2, and domain of
sequence homology or divergence amongst the family, such as those
that can readily be identified using protein alignment methods and
as presented in the Figures.
[0081] Antibodies are preferably prepared from regions or discrete
fragments of the enzyme proteins. Antibodies can be prepared from
any region of the peptide as described herein. However, preferred
regions will include those involved in function/activity and/or
enzyme/binding partner interaction. FIG. 2 can be used to identify
particularly important regions while sequence alignment can be used
to identify conserved and unique sequence, fragments.
[0082] An antigenic fragment will typically comprise at least 8
contiguous amino acid residues. The antigenic peptide can comprise,
however, at least 10, 12, 14, 16 or more amino acid residues. Such
fragments can be selected on a physical property, such as fragments
correspond to regions that are located on the surface of the
protein, e.g., hydrophilic regions or can be selected based on
sequence uniqueness (see FIG. 2).
[0083] Detection on an antibody of the present invention can be
facilitated by coupling (i.e., physically linking) the antibody to
a detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, biolummescent 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 or .sup.3H.
[0084] Antibody Uses
[0085] The antibodies can be used to isolate one of the proteins of
the present invention by standard techniques, such as affinity
chromatography or immunoprecipitation. The antibodies can
facilitate the purification of the natural protein from cells and
recombinantly produced protein expressed in host cells. In
addition, such antibodies are useful to detect the presence of one
of the proteins of the present invention in cells or tissues to
determine the pattern of expression of the protein among various
tissues in an organism and over the course of normal development.
Experimental data as provided in FIG. 1 indicates that the enzymes
of the present invention are expressed in humans in the brain,
kidney, colon, uterus detected by a virtual northern blot. Further,
such antibodies can be used to detect protein in situ, in vitro, or
in a cell lysate or supernatant in order to evaluate the abundance
and pattern of expression. Also, such antibodies can be used to
assess abnormal tissue distribution or abnormal expression during
development or progression of a biological condition. Antibody
detection of circulating fragments of the full length protein can
be used to identify turnover.
[0086] Further, the antibodies can be used to assess expression in
disease states such as in active stages of the disease or in an
individual with a predisposition toward disease related to the
protein's function. When a disorder is caused by an inappropriate
tissue distribution, developmental expression, level of expression
of the protein, or expressed/processed form, the antibody can be
prepared against the normal protein. Experimental data as provided
in FIG. 1 indicates expression in humans in the brain, kidney,
colon and uterus. If a disorder is characterized by a specific
mutation in the protein, antibodies specific for this mutant
protein can be used to assay for the presence of the specific
mutant protein.
[0087] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Experimental data as provided in FIG. 1 indicates
expression in humans in the brain, kidney, colon and uterus. The
diagnostic uses can be applied, not only in genetic testing, but
also in monitoring a treatment modality. Accordingly, where
treatment is ultimately aimed at correcting expression-level or the
presence of aberrant sequence and aberrant tissue distribution or
developmental expression, antibodies directed against the protein
or relevant fragments can be used to monitor therapeutic
efficacy.
[0088] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic proteins
can be used to identify individuals that require modified treatment
modalities. The antibodies are also useful as diagnostic tools as
an immunological marker for aberrant protein analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[0089] The antibodies are also useful for tissue typing.
Experimental data as provided in FIG. 1 indicates expression in
humans in the brain kidney, colon and uterus. Thus, where a
specific protein has been correlated with expression in a specific
tissue, antibodies that are specific for this protein can be used
to identify a tissue type.
[0090] The antibodies are also useful for inhibiting protein
function, for example, blocking the binding of the enzyme peptide
to a binding partner such as a substrate. These uses can also be
applied in a therapeutic context in which treatment involves
inhibiting the protein's function. An antibody can be used, for
example, to block binding, thus modulating (agonizing or
antagonizing) the peptides activity. Antibodies can be prepared
against specific fragments containing sites required for function
or against intact protein that is associated with a cell or cell
membrane: See FIG. 2 for structural information relating to the
proteins of the present invention.
[0091] The invention also encompasses kits for using antibodies to
detect the presence of a protein in a biological sample. The kit
can comprise antibodies such as a labeled or labelable antibody and
a compound or agent for detecting protein in a biological sample;
means for determining the amount of protein in the sample; means
for comparing the amount of protein in the sample with a: standard;
and instructions for use. Such a kit can be supplied to detect a
single protein or epitope or can be configured to detect one of a
multitude of epitopes, such as in an antibody detection array.
Arrays are described in detail below for nucleic acid arrays and
similar methods have been developed for antibody arrays.
[0092] Nucleic Acid Molecules
[0093] The present invention further provides isolated nucleic acid
molecules that encode a enzyme peptide or protein of the present
invention (cDNA, transcript and genomic sequence). Such nucleic
acid molecules will consist of, consist essentially of, or comprise
a nucleotide sequence that encodes one of the enzyme peptides of
the present invention, an allelic variant thereof, or an ortholog
or paralog thereof.
[0094] As used herein, an "isolated" nucleic acid molecule is one
that is separated from other nucleic acid present in the natural
source of the nucleic acid. Preferably, an "isolated" nucleic acid
is free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 340 ends of the nucleic acid) in
the genomic DNA of the organism from which the nucleic acid is
derived. However, there can be some flanking nucleotide sequences,
for example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less,
particularly contiguous peptide encoding sequences and peptide
encoding sequences within the same gene but separated by introns in
the genomic sequence. The important point is that the nucleic acid
is isolated from remote and unimportant flanking sequences such
that it can be subjected to the specific manipulations described
herein such as recombinant expression, preparation of probes and
primers, and other uses specific to the nucleic acid sequences.
[0095] Moreover, an "isolated" nucleic acid molecule, such as a
transcript/cDNA molecule, can be substantially free of other
cellular material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0096] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0097] Accordingly, the present invention provides nucleic acid
molecules that consist of the nucleotide sequence shown in FIGS. 1
or 3 (SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NO: 2. A nucleic acid molecule consists
of a nucleotide sequence when the nucleotide sequence is the
complete nucleotide sequence of the nucleic acid molecule.
[0098] The present invention further provides nucleic acid
molecules that consist essentially of the nucleotide sequence shown
in FIGS. 1 or 3 (SEQ ID NO: 1, transcript sequence and SEQ ID NO:
3, genomic sequence), or any nucleic acid molecule that encodes the
protein provided in FIG. 2, SEQ ID NO: 2. A nucleic acid molecule
consists essentially of a nucleotide sequence when such a
nucleotide sequence is present with only a few additional nucleic
acid residues in the final nucleic acid molecule.
[0099] The present invention further provides nucleic acid
molecules that comprise the nucleotide sequences shown in FIGS. 1
or 3. (SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NO: 2. A nucleic acid molecule comprises
a nucleotide sequence when the nucleotide sequence is at least part
of the final nucleotide sequence of the nucleic acid molecule. In
such a fashion, the nucleic acid molecule can be only the
nucleotide sequence or have additional nucleic acid residues, such
as nucleic acid residues that are naturally associated with it or
heterologous nucleotide sequences. Such a nucleic acid molecule can
have a few additional nucleotides or can comprises several hundred
or more additional nucleotides. A brief description of how various
types of these nucleic acid molecules can be readily made/isolated
is provided below.
[0100] In FIGS. 1 and 3, both coding and non-coding sequences are
provided. Because of the source of the present invention, humans
genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1),
the nucleic acid molecules in the Figures will contain genomic
intronic sequences, 5' and 3' non-coding sequences, gene regulatory
regions and non-coding intergenic sequences. In general such
sequence features are either noted in FIGS. 1 and 3 or can readily
be identified using computational tools known in the art. As
discussed below, some of the non-coding regions, particularly
generegulatory elements such as promoters, are useful for a variety
of purposes, e.g. control of heterologous gene expression, target
for identifying gene activity modulating compounds, and are
particularly claimed as fragments of the genomic sequence provided
herein.
[0101] The isolated nucleic acid molecules can encode the mature
protein plus additional amino or carboxyl-terminal amino acids, or
amino acids interior to the mature peptide (when the mature form
has more thanone peptide chain, for instance). Such sequences may
play a role in processing of a protein from precursor to a mature
form, facilitate protein trafficking, prolong or shorten protein
half-life or facilitate manipulation of a protein for assay or
production, among other things. As generally is the case in situ,
the additional amino acids may be processed away from the mature
protein by cellular enzymes.
[0102] As mentioned above, the isolated nucleic acid molecules
include, but are not limited to, the sequence encoding the enzyme
peptide alone, the sequence encoding the mature peptide and
additional coding sequences, such as a leader or secretory sequence
(e.g., a pre-pro or pro-protein sequence), the sequence encoding
the mature peptide,with or without the additional coding sequences,
plus additional non-coding sequences, for example introns and
non-coding 5' and 3' sequences such as transcribed but
non-translated sequences that play a role in transcription, mRNA
processing (including splicing and polyadenylation signals),
ribosome binding and stability of mRNA. In addition, the nucleic
acid molecule may be fused to a marker sequence encoding, for
example, a peptide that facilitates purification.
[0103] Isolated nucleic acid molecules can be in the form of RNA,
such as mRNA, or in the form DNA, including cDNA and genomic DNA
obtained by cloning or produced by chemical synthetic techniques or
by a combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[0104] The invention further provides nucleic acid molecules that
encode fragments of the peptides of the present invention as well
as nucleic acid molecules that encode obvious variants of the
enzyme proteins of the present invention that are described above.
Such nucleic acid molecules may be naturally occurring, such as
allelic variants (same locus), para logs (different locus), and
orthologs (different organism), or may be constructed by
recombinant DNA methods or by chemical synthesis. Such
non-naturally occurring variants may be made by mutagenesis
techniques, including those applied to nucleic acid molecules,
cells, or organisms. Accordingly, as discussed above, the variants
can contain nucleotide substitutions, deletions, versions and
insertions. Variation can occur in either or both the coding and
noncoding regions. The variations can produce both conservative and
non-conservative amino acid substitutions.
[0105] The present invention further provides non-coding fragments
of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred
non-coding fragments include, but are not limited to, promoter
sequences, enhancer sequences, gene modulating sequences and gene
termination sequences. Such fragments are useful in controlling
heterologous gene expression and in developing screens to identify
gene-modulating agents. A promoter can readily be identified as
being 5' to the ATG start site in the genomic sequence provided in
FIG. 3.
[0106] A fragment comprises a contiguous nucleotide sequence
greater than 12 or more nucleotides. Further, a fragment could at a
least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length
of the fragment will be based on its intended use. For example, the
fragment can encode epitope bearing regions of the peptide, or can
be useful as DNA probes and primers. Such fragments can be isolated
using the known nucleotide sequence to synthesize an
oligonucleotide probe. A labeled probe can then be used to screen a
cDNA library, genomic DNA library, or mRNA to isolate nucleic acid
corresponding to the coding region. Further, primers can be used in
PCR reactions to clone specific regions of gene.
[0107] A probe/primer typically comprises substantially a purified
oligonucleotide or oligonucleotide pair. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, 20, 25, 40, 50 or
more consecutive nucleotides.
[0108] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. As described in the Peptide
Section, these variants comprise a nucleotide sequence encoding a
peptide that is typically 60-70%, 70-80%, 80-90%, and more
typically at least about 90-95% or more homologous to the
nucleotide sequence shown in the Figure sheets or a fragment of
this sequence. Such nucleic acid molecules can readily be
identified as being able to hybridize under moderate to stringent
conditions, to the nucleotide sequence shown in the Figure sheets
or a fragment of the sequence. Allelic variants can readily be
determined by genetic locus of the encoding gene. As indicated by
the data presented in FIG. 3, the map position was determined to be
on chromosome 12 by ePCR.
[0109] FIG. 3 provides information on SNPs that have been found in
the gene encoding the enzyme of the present invention. SNPs were
identified at 4 different nucleotide positions in exon, introns and
regions 5' and 3' of the ORF. Such SNPs in introns and outside the
ORF may affect control/regulatory elements. Changes in the amino
acid sequence caused by these SNPs is indicated in FIG. 3 and can
readily be determined using the universal genetic code and the
protein sequence provided in FIG. 2 as a reference.
[0110] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a peptide at
least 60-70% homologous to each other typically remain hybridizedto
each other. The conditions can be such that sequences at least
about 60%, at least about 70%, or at least about 80% or more
homologous to each other typically remain hybridized to each other.
Such stringent conditions ame known to those skilled in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent
hybridization conditions are hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45 C., followed by one or
more washes in 0.2.times.SSC, 0.1% SDS at 50-65 C. Examples of
moderate to low stringency hybridization conditions are well known
in the art.
[0111] Nucleic Acid Molecule Uses
[0112] The nucleic acid molecules of the present invention are
useful for probes, primers, chemical intermediates, and in
biological assays. The nucleic acid molecules are useful as a
hybridization probe for messenger RNA, transcript/cDNA and genomic
DNA to isolate full-length cDNA and genomic clones encoding the
peptide described in FIG. 2 and to isolate cDNA and genomic clones
that correspond to variants (alleles, orthologs, etc.) producing
the same or related peptides shown in FIG. 2. As illustrated in
FIG. 3, SNPs, were identified at 4 different nucleotide
positions.
[0113] The probe can correspond to any sequence along the entire
length of the nucleic acid molecules provided in the Figures.
Accordingly, it could be derived from 5' noncoding regions, the
coding region, and 3' noncoding regions. However, as discussed,
fragments are not to be construed as encompassing fragments
disclosed prior to the present invention.
[0114] The nucleic acid molecules are also useful as primers for
PCR to amplify any given region of a nucleic acid molecule and are
useful to synthesize antisense molecules of desired length and
sequence.
[0115] The nucleic acid molecules are also useful for constructing
recombinant vectors. Such vectors include expression vectors that
express a portion of, or all of, the peptide sequences. Vectors
also include insertion vectors, used to integrate into another
nucleic acid molecule sequence, such as into the cellular genome,
to alter in situ expression of a gene and/or gene product. For
example, an endogenous coding sequence can be replaced via
homologous recombination with all or part of the coding region
containing one or more specifically introduced mutations.
[0116] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0117] The nucleic acid molecules are also useful as probes for
determining the chromosomal positions of the nucleic acid molecules
by means of in situ hybridization methods. As indicated by the data
presented in FIG. 3, the map position was determined to be on
chromosome 12 by ePCR.
[0118] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0119] The nucleic acid molecules are also useful for designing
ribozymes corresponding to all, or a part, of the mRNA produced
from the nucleic acid molecules described herein.
[0120] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0121] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0122] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0123] The nucleic acid molecules are also useful as hybridization
probes for determining the presence, level, form and distribution
of nucleic acid expression. Experimental data as provided in FIG.
1. indicates that the enzymes of the present invention are
expressed in humans in the brain, kidney, colon, uterus detected by
a virtual northern blot. Accordingly, the probes can be used to
detect the presence of, or to determine levels of, a specific
nucleic acid molecule in cells, tissues, and in organisms. The
nucleic acid whose level is determined can be DNA or RNA.
Accordingly, probes corresponding to the peptides described herein
can be used to assess expression and/or gene copy number in a given
cell, tissue, or organism. These uses are relevant for diagnosis of
disorders involving an increase or decrease in enzyme protein
expression relative to normal results.
[0124] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA includes Southern hybridizations and in situ
hybridization.
[0125] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express a enzyme protein, such as
by measuring a level of a enzyme-encoding nucleic acid in a sample
of cells from a subject e.g., mRNA or genomic DNA, or determining
if a enzyme gene has been mutated. Experimental data as provided in
FIG. 1 indicates that the enzymes of the present invention are
expressed in humans in the brain, kidney, colon, uterus detected by
a virtual northern blot.
[0126] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate enzyme nucleic acid
expression.
[0127] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of the enzyme gene, particularly biological
and pathological processes that are mediated by the enzyme in cells
and tissues that express it. Experimental data as provided in FIG.
1 indicates expression in humans in the brain, kidney, colon and
uterus. The method typically includes assaying the ability of the
compound to modulate the expression of the enzyme nucleic acid and
thus identifying a compound that can be used to treat a disorder
characterized by undesired enzyme nucleic acid expression. The
assays can be performed in cell-based and cell-free systems.
Cell-based assays include cells naturally expressing the enzyme
nucleic acid or recombinant cells genetically engineered to express
specific nucleic acid sequences.
[0128] The assay for enzyme nucleic acid expression can involve
direct assay of nucleic acid levels, such as mRNA levels, or on
collateral compounds involved in the signal pathway. Further, the
expression of genes that are up- or down-regulated in response to
the enzyme protein signal pathway can also be assayed. In this
embodiment the regulatory regions of these genes can be operably
linked to a reporter gene such as luciferase.
[0129] Thus, modulators of enzyme gene expression can be identified
in a method wherein a cell is contacted with a candidate compound
and the expression of mRNA determined. The level of expression of
enzyme mRNA in the presence of the candidate compound is compared
to the level of expression of enzyme mRNA in the absence of the
candidate compound. The candidate compound can then be identified
as a modulator of nucleic acid expression based on this comparison
and be used, for example to treat a disorder characterized by
aberrant nucleic acid expression. When expression of mRNA is
statistically significantly greater in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of nucleic acid expression. When nucleic
acid expression is statistically significantly less in the presence
of the candidate compound than in its absence, the candidate
compound is identified as an inhibitor of nucleic acid
expression.
[0130] The invention further provides methods of treatment, with
the nucleic acid as a target, using a compound identified through
drug screening as a gene modulator to modulate enzyme nucleic acid
expression in cells and tissues that express the enzyme.
Experimental data as provided in FIG. 1 indicates that the enzymes
of the present invention are expressed in humans in the brain,
kidney, colon, uterus detected by a virtual northern blot.
Modulation includes both up-regulation (i.e. activation or
agonization) or down-regulation (suppression or antagonization) or
nucleic acid expression.
[0131] Alternatively, a modulator for enzyme nucleic acid
expression can be a small molecule or drug identified using the
screening assays described herein as long as the drug or small
molecule inhibits the enzyme nucleic acid expression in the cells
and tissues that express the protein. Experimental data as provided
in FIG. 1 indicates expression in humans in the brain, kidney,
colon and uterus.
[0132] The nucleic acid molecules are also useful for monitoring
the effectiveness of modulating compounds on the expression or
activity of the enzyme gene in clinical trials or in a treatment
regimen. Thus, the gene expression pattern can serve as a barometer
for the continuing effectiveness of treatment with the compound,
particularly with compounds to which a patient can develop
resistance. The gene expression pattern can also serve as a marker
indicative of a physiological response of the affected cells to the
compound. Accordingly, such monitoring would allow either increased
administration of the compound or the administration of alternative
compounds to which the patient has not become resistant. Similarly,
if the level of nucleic acid expression falls below a desirable
level, administration of the compound could be commensurately
decreased.
[0133] The nucleic acid molecules are also useful in diagnostic
assays for qualitative changes in enzyme nucleic acid expression,
and particularly in qualitative changes that lead to pathology. The
nucleic acid molecules can be used to detect mutations in enzyme
genes and gene expression products such as mRNA. The nucleic acid
molecules can be used as hybridization probes to detect naturally
occurring genetic mutations in the enzyme gene and thereby to
determine whether a subject with the mutation is at risk for a
disorder caused by the mutation. Mutations include deletion,
addition, or substitution of one or more nucleotides in the gene,
chromosomal rearrangement, such as inversion or transposition,
modification of genomic DNA, such as aberrant methylation patterns
or changes in gene copy number, such as amplification. Detection of
a mutated form of the enzyme gene associated with a dysfunction
provides, a diagnostic tool for an active disease or susceptibility
to disease when the disease results from overexpression,
underexpression, or altered expression of a enzyme protein.
[0134] Individuals carrying mutations in the enzyme gene can be
detected at the nucleic acid level by a variety of techniques. FIG.
3 provides information on SNPs that have been found in the gene
encoding the enzyme of the present invention. SNPs were identified
at 4 different nucleotide positions in exon, introns and regions 5'
and 3' of the ORF. Such SNPs in introns and outside the ORF may
affect control/regulatory elements. Changes in the amino acid
sequence caused by these SNPs is indicated in FIG. 3 and can
readily be determined using the universal genetic code and the
protein sequence provided in FIG. 2 as a reference. As indicated by
the data presented in FIG. 3, the map position was determined to be
on chromosome 12by ePCR. Genomic DNA can be analyzed directly or
can be amplified by using PCR prior to analysis. RNA or cDNA can be
used in the same way. In some uses, detection of the mutation
involves the use of a probe/primer in a polymerase chain reaction
(PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as
anchor PCR or RACE PCR, or, alternatively, in a ligation, chain
reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080
(1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of
which can be particularly useful for detecting point mutations in
the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682
(1995)). This method can include the steps of collecting a sample
of cells from a patient, isolating nucleic acid (e.g., genomic,
mRNA or both) from the cells of the sample, contacting the nucleic
acid sample with one or more primers which specifically hybridize
to a gene under conditions such that hybridization and
amplification of the 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. Deletions and insertions can be detected by a
change in size of the amplified product compared to the normal
genotype. Point mutations can be identified by hybridizing
amplified DNA to normal RNA or antisense DNA sequences.
[0135] Alternatively, mutations in a enzyme gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0136] Further, sequence-specific nbozymes (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.
Perfectly matched sequences can be distinguished from mismatched
sequences by nuclease cleavage digestion assays or by differences
in melting temperature.
[0137] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method. Furthermore, sequence differences
between a mutant enzyme gene and a wild-type gene can be determined
by direct DNA sequencing. A variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
(Naeve, C. W., (1995) Biotechniques 19:448), including sequencing
by mass spectrometry (see, e.g., PCT International Publication No.
WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and
Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).
[0138] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.,
Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988);
Saleeba et al., Meth. Enzymol. 17:286-295 (1992)), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144
(1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79
(1992)), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al., Nature
313:495 (1985)). Examples of other techniques for detecting point
mutations include selective oligonucleotide hybridization,
selective amplification, and selective primer extension.
[0139] The nucleic acid molecules are also useful for testified an
individual for a genotype that while not necessarily causing the
disease, nevertheless affects the treatment modality. Thus, the
nucleic acid molecules can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship).
Accordingly, the nucleic acid molecules described herein can be
used to assess the mutation content of the enzyme gene in an
individual in order to select an appropriate compound or dosage
regimen for treatment. FIG. 3 provides information on SNPs that
have been found in the gene encoding the enzyme of the present
invention. SNPs were identified at 4 different nucleotide positions
in exon, introns and regions 5' and 3' of the ORF. Such SNPs in
introns and outside the ORF may affect control/regulatory elements.
Changes in the amino acid sequence caused by these SNPs is
indicated in FIG. 3 and can readily be determined using the
universal genetic code and the protein sequence provided in FIG. 2
as a reference.
[0140] Thus nucleic acid molecules displaying genetic variations
that affect treatment provide a diagnostic target that can be used
to tailor treatment in an individual. Accordingly, the production
of recombinant cells and animals containing these polymorphisms
allow effective clinical design of treatment compounds and dosage
regimens.
[0141] The nucleic acid molecules are thus useful as antisense
constructs to control enzyme gene expression in cells, tissues, and
organisms. A DNA antisense nucleic acid molecule is designed to be
complementary to a region of the gene involved in transcription,
preventing transcription and hence production of enzyme protein. An
antisense RNA or DNA nucleic acid molecule would hybridize to the
mRNA and thus block translation of mRNA into enzyme protein.
[0142] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of enzyme nucleic
acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired enzyme nucleic acid
expression. This technique involves cleavage by means of ribozymes
containing nucleotide sequences complementary to one or more
regions in the mRNA that attenuate the ability of the mRNA to be
translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the enzyme protein, such as
substrate binding.
[0143] The nucleic acid molecules also provide vectors for gene
therapy in patients containing cells that are aberrant in enzyme
gene expression. Thus, recombinant cells, which include the
patient's cells that have been engineered ex vivo and returned to
the patient, are introduced into an individual where the cells
produce the desired enzyme protein to treat the individual.
[0144] The invention also encompasses kits for detecting the
presence of a enzyme nucleic acid in a biological sample.
Experimental data as provided in FIG. 1 indicates that the enzymes
of the present invention are expressed in humans in the brain,
kidney, colon, uterus detected by a virtual northern blot. For
example, the kit can comprise reagents such as a labeled or
labelable nucleic acid or agent capable of detecting enzyme nucleic
acid in a biological sample; means for determining the amount of
enzyme nucleic acid in the sample; and means for comparing the
amount of enzyme nucleic acid in the sample with 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
enzyme protein mRNA or DNA.
[0145] Nucleic Acid Arrays
[0146] The present invention further provides nucleic acid
detection kits, such as arrays or microarrays of nucleic acid
molecules that are based on the sequence information provided in
FIGS. 1 and 3 (SEQ ID NOS: 1 and 3).
[0147] As used herein "Arrays" or "Microarrays" refers to an array
of distinct polynucleotides or oligonucleotides synthesized on a
substrate, such as paper, nylon or other type of membrane, filter,
chip, glass slide, or any other suitable solid support. In one
embodiment, the microarray is prepared and used according to the
methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT
application W095/11995 (Chee et al.), Lockhart, D. J. et al. (1996;
Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc.
Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated
herein in their entirety by reference. In other embodiments, such
arrays are produced by the methods described by, Brown et al., U.S.
Pat. No. 5,807,522.
[0148] The microarray or detection kit is preferably composed of a
large number of unique, single-stranded nucleic acid sequences,
usually either synthetic antisense oligonucleotides or fragments of
cDNAs, fixed to a solid support. The oligonucleotides are
preferably about 6-60 nucleotides in length, more preferably 15-30
nucleotides in length, and most preferably about 20-25 nucleotides
in length. For a certain type of microarray or detection kit, it
may be preferable to use oligonucleotides that are only 7-20
nucleotides in length. The microarray or detection kit may contain
oligonucleotides that cover the known 5', or 3', sequence,
sequential oligonucleotides which cover the full length sequence;
or unique oligonucleotides selected from particular areas along the
length of the sequence. Polynucleotides used in the microarray or
detection kit may be oligonucleotides that are specific to a gene
or genes of interest.
[0149] In order to produce oligonucleotides to a known sequence for
a microarray or detection kit, the gene(s) of interest (or an ORF
identified from the contigs of the present invention) is typically
examined using a computer algorithm which starts at the 5' or at
the 3' end of the nucleotide sequence. Typical algorithms will then
identify oligomers of defined length that are unique: to the gene,
have a GC content within a range suitable for hybridization, and
lack predicted secondary structure that may interfere with
hybridization. In certain situation it may be appropriate to use
pairs of oligonucleotides on a microarray or detection kit. The
"pairs" will be identical, except for one nucleotide that
preferably is located in the center of the sequence. The second
oligonucleotide in the pair (mismatched by one) serves as a
control. The number of oligonucleotide pairs may range from two to
one million. The oligomers are synthesized at designated areas on a
substrate using a light-directed chemical process. The substrate
may be paper, nylon or other type of membrane, filter, chip, glass
slide or any other suitable solid support.
[0150] In another aspect, an oligonucleotide may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, as described in PCT
application W095/251116 (Baldeschweiler et al.) which is
incorporated herein in its entirety by reference. In another
aspect, a "gridded" array analogous to a dot (or slot) blot may be
used to arrange and link cDNA fragments or oligonucleotides to the
surface of a substrate using a vacuum system, thermal, UV,
mechanical or chemical bonding procedures. An array, such as those
described above, may be produced by hand or by using available
devices (slot blot or dot blot apparatus), materials (any suitable
solid support), and machines (including robotic instruments), and
may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or
any other number between two and one million which lends itself to
the efficient use of commercially available instrumentation.
[0151] In order to conduct sample analysis using a microarray or
detection kit, the RNA or DNA from a biological sample is made into
hybridization probes. The mRNA is isolated, and cDNA is produced
and used as a template to make antisense RNA (aRNA). The aRNA is
amplified in the presence of fluorescent nucleotides, and labeled
probes are incubated with the microarray or detection kit so that
the probe sequences hybridize to complementary oligonucleotides of
the microarray or detection kit. Incubation conditions are adjusted
so that hybridization occurs with precise complementary matches or
with various degrees of less complementarity. After removal of
nonhybridized probes, a scanner is used to determine the levels and
patterns of fluorescence. The scanned images are examined to
determine degree of complementarity and the relative abundance of
each oligonucleotide sequence on the microarray or detection kit.
The biological samples may be obtained from any bodily fluids (such
as blood; urine, saliva, phlegm, gastric juices, etc.), cultured
cells, biopsies, or other tissue preparations. A detection system
may be used to measure the absence, presence, and amount of
hybridization for all of the distinct sequences simultaneously.
This data may be used for large-scale correlation studies on the
sequences, expression patterns, mutations, variants, or
polymorphisms among samples.
[0152] Using such arrays, the present invention provides methods to
identify the expression of the enzyme proteins/peptides of the
present invention. In detail, such methods comprise incubating a
test sample with one or more nucleic acid molecules and assaying
for binding of the nucleic acid molecule with components within the
test sample. Such assays will typically involve arrays comprising
many genes, at least one of which is a gene of the present
invention and or alleles of the enzyme gene of the present
invention. FIG. 3 provides information on SNPs that have been found
in the gene encoding the enzyme of the present invention. SNPs were
identified at 4 different nucleotide positions in exon, introns and
regions 5' and 3' of the ORF. Such SNPs in introns and outside the
ORF may affect control/regulatory elements. Changes in the amino
acid sequence caused by these SNPs is indicated in FIG. 3 and can
readily be determined using the universal genetic code and the
protein sequence provided in FIG. 2 as a reference.
[0153] Conditions for incubating a nucleic acid molecule with a
test sample vary. Incubation conditions depend on the format
employed in the assay, the detection methods employed, and the type
and nature of the nucleic acid molecule used in the assay. One
skilled in the art will recognize that any one of the commonly
available hybridization, amplification or array assay formats can
readily be adapted to employ the novel fragments of the Human
genome disclosed herein. Examples of such assays can be found in
Chard, T, An Introduction to Radioimmunoassay and Related
Techniques, Elsevier Science Publishers Amsterdam, The Netherlands
(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,
Academic Press, Orlando, Fla. Vol. 1 (1 982), Vol. 2 (1983), Vol. 3
(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
[0154] The test samples of the present invention include cells,
protein or membrane extracts of cells. The test sample used in the
above-described method will vary based on the assay format, nature
of the detection method and the tissues, cells or extracts used as
the sample to be assayed. Methods for preparing nucleic acid
extracts or of cells are well known in the art and can be readily
be adapted in order to obtain a sample that is compatible with the
system utilized.
[0155] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0156] Specifically, the invention provides a compartmentalized kit
to receive, in close confinement, one or more containers which
comprises: (a) a first container comprising one of the nucleic acid
molecules that can bind to a fragment of the Human genome disclosed
herein; and (b) one or more other containers comprising one or more
of the following: wash reagents, reagents capable of detecting
presence of a bound nucleic acid.
[0157] In detail, a compartmentalized kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers, strips of
plastic, glass or paper, or arraying material such as silica. Such
containers allows one to efficiently transfer reagents from one
compartment to another compartment such that the samples and
reagents are not cross-contaminated, and the agents or solutions of
each container can be added in a quantitative fashion from one
compartment to another. Such containers will include a container
which will accept the test sample, a container which contains the
nucleic acid probe, containers which contain wash reagents (such as
phosphate buffered saline, Tris-buffers, etc.), and containers
which contain the reagents used to detect the bound probe. One
skilled in the art will readily recognize that the previously
unidentified enzyme gene of the present invention can be routinely
identified using the sequence information disclosed herein can be
readily incorporated into one of the established kit formats which
are well known in the art, particularly expression arrays.
[0158] Vectors/Host Cells
[0159] The invention also provides vectors containing the nucleic
acid molecules described herein. The term "vector" refers to a
vehicle, preferably a nucleic acid molecule, which can transport
the nucleic acid molecules. When the vector is a nucleic acid
molecule, the nucleic acid molecules are covalently linked to the
vector nucleic acid. With this aspect of the invention, the vector
includes a plasmid, single or double stranded phage, a single or
double stranded RNA or DNA viral vector, or artificial chromosome,
such as a BAC, PAC, YAC, OR MAC.
[0160] A vector can be maintained in the host cell as an extra
chromosomal element where it replicates and produces additional
copies of the nucleic acid molecules. Alternatively, the vector may
integrate into the host cell genome and produce additional copies
of the nucleic acid molecules when the host cell replicates.
[0161] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
nucleic acid molecules. The vectors can function in prokaryotic or
eukaryotic cells or in both (shuttle vectors).
[0162] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the nucleic acid
molecules such that transcription of the nucleic acid molecules is
allowed in a host cell. The nucleic acid molecules can be
introduced into the host cell with a separate nucleic acid molecule
capable of affecting transcription. Thus, the second nucleic acid
molecule may provide a transacting factor interacting with the
cis-regulatory control region to allow transcription of the nucleic
acid molecules from the vector. Alternatively, a transacting factor
may be supplied by the host cell. Finally, a trans-acting factor
can be produced from the vector itself. It is understood, however,
that in some embodiments, transcription and/or translation of the
nucleic acid molecules can occur in a cell-free system.
[0163] The regulatory sequence to which the nucleic acid molecules
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lambda., the lac, TRP,
and TAC promoters from E. coli, the early and late promoters from
SV40, the CMV immediate early promoter, the adenovirus early and
late promoters, and retrovirus long-terminal repeats.
[0164] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0165] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al., Molecular
Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1989).
[0166] A variety of expression vectors can be used to express a
nucleic acid molecule. Such vectors include chromosomal, episomal,
and virus-derived vectors, for example vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, including yeast artificial chromosomes,
from viruses such as baculoviruses, papovaviruses such as SV40,
Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses,
and retroviruses. Vectors may also be derived from combinations of
these sources such as those derived from plasmid and bactenrophage
genetic elements, e.g. cosmids and phagemids. Appropriate cloning
and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual. 2nd. ed., Cold spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1989).
[0167] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e. tissue specific) or may provide for
inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[0168] The nucleic acid molecules can be inserted into the vector
nucleic acid by well-known methodology. Generally, the DNA sequence
that will ultimately be expressed is joined to an expression vector
by cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known to those of ordinary skill in the art.
[0169] The vector containing the appropriate nucleic acid molecule
can be introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[0170] As described herein, it may be desirable to express the
peptide as a fusion protein. Accordingly, the invention provides
fusion vectors that allow for the production of the peptides.
Fusion vectors can increase the expression of a recombinant
protein, increase the solubility of the recombinant protein, and
aid in the purification of the protein by acting for example as a
ligand for affinity purification. A proteolytic cleavage site may
be introduced at the junction of the fusion moiety so that the
desired peptide can ultimately be separated from the fusion moiety.
Proteolytic enzymes include, but are not limited to, factor Xa,
thrombin, and enteroenzryme. Typical fusion expression vectors
include pGEX (Smith et al., Gene 67:31-40 (1988)), 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. Examples of suitable inducible non-fusion E. coli
expression vectors include pTrc (Amann et al., Gene 69:301-315
(1988)) and pET 11d (Studier et al., Gene Expression Technology:
Methods in, Enzymology 185:60-89 (1990)).
[0171] Recombinant protein expression can be maximized in host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein. (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128).
Alternatively, the sequence of the nucleic acid molecule of
interest can be altered to provide preferential codon usage for a
specific host cell, for example E. coli. (Wada et al., Nucleic
Acids Res. 20:211 1-2118 (1992)).
[0172] The nucleic acid molecules can also be expressed by
expression vectors that are operative in yeast. Examples of vectors
for expression in yeast e.g., S. cerevisiae include pYepSec1
(Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kuian et al.,
Cell 30:933-943(1982)), pJRY88: (Schultz et al., Gene 54:1.13-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0173] The nucleic acid molecules can also be expressed in insect
cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf9 cells) include the pAc series
(Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL
series (Lucklow et al., Virology 170:3 1-39 (1989)).
[0174] In certain embodiments of the invention, the nucleic acid
molecules described herein are expressed in mammalian cells using
mammalian expression vectors. Examples of mammalian expression
vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC
(Kaufinan et al., EMBO J. 6:187-195 (1987)).
[0175] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the
nucleic acid molecules. The person of ordinary skill in the art
would be aware of other vectors suitable for maintenance
propagation or expression of the nucleic acid molecules described
herein. These are found for example in Sambrook, J., Fritsh, E. F.,
and Maniatis, T. Molecular Cloning A Laboratory Manual. 2nd, ed.,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989.
[0176] The invention also encompasses vectors in which the nucleic
acid sequence's described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the nucleic
acid molecule sequences described herein, including both coding and
non-coding regions. Expression of this antisense RNA is subject to
each of the parameters described above in relation to expression of
the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0177] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[0178] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook,
et al. (Molecular. Cloning: A Laboratory Manual. 2nd, ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989).
[0179] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the nucleic acid molecules can be introduced
either alone or with other nucleic acid molecules that are not
related to the nucleic acid molecules such as those providing
transacting factors for expression vectors. When more than one
vector is introduced into a cell, the vectors can be introduced
independently, co-introduced or joined to the nucleic acid molecule
vector.
[0180] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[0181] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the nucleic acid molecules described
herein or may be on a separate vector. Markers include tetracycline
or ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0182] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[0183] Where secretion of the peptide is desired, which is
difficult to achieve with multi-transmembrane domain containing
proteins such as enzymes, appropriate secretion signals are
incorporated into the vector. The signal sequence can be endogenous
to the peptides or heterologous to these peptides.
[0184] Where the peptide is not secreted into the medium, which is
typically the case with enzymes, the protein can be isolated from
the host cell by standard disruption procedures, including; freeze
thaw, sonication, mechanical disruption, use of lysing agents and
the like. The peptide can then be recovered and purified by
well-known purification methods including ammonium sulfate
precipitation, acid extraction, anion or cationic exchange
chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0185] It is also understood that depending upon the host cell in
recombinant production of the peptides described herein, the
peptides can have various glycosylation patterns, depending upon
the cell, or maybe non-glycosylated as when produced in bacteria.
In addition, the peptides may include an initial modified
methionine in some cases as a result of a host-mediated
process.
[0186] Uses of Vectors and Host Cells
[0187] The recombinant host cells expressing the peptides described
herein have a variety of uses. First, the cells are useful for
producing a enzyme protein or peptide that can be further purified
to produce desired amounts of enzyme protein or fragments. Thus,
host cells containing expression vectors are useful for peptide
production.
[0188] Host cells are also useful for conducting cell-based assays
involving the enzyme protein or enzyme protein fragments, such as
those described above as well as other formats known in the art.
Thus, a recombinant host cell expressing a native enzyme protein is
useful for assaying compounds that stimulate or inhibit enzyme
protein function.
[0189] Host cells are also useful for identifying enzyme protein
mutants in which these functions are affected. If the mutants
naturally occur and give rise to a pathology, host cells containing
the mutations ate useful to assay compounds that have a desired
effect on the mutant enzyme protein (for example, stimulating or
inhibiting function) which may not be indicated by their effect on
the native enzyme protein.
[0190] Genetically engineered host cells can be further used to
produce non-human transgenic animals. A transgenic animal is
preferably a mammal, for example a rodent, such as a rat or mouse,
in which one or more of the cells of the animal include a
transgene. A transgene is exogenous DNA which is integrated into
the genome of a cell from which a transgenic animal develops and
which remains in the genome of the mature animal in one or more
cell types or tissues of the transgenic animal. These animals are
useful for studying the function of a enzyme protein and
identifying and evaluating modulators of enzyme protein activity.
Other examples of transgenic animals include non-human primates,
sheep, dogs, cows, goats, chickens, and amphibians.
[0191] A transgenic animal can be produced by introducing nucleic
acid into the male pronuclei of a fertilized oocyte, e.g., by
microinjection, retroviral infection, and allowing the oocyte to
develop in a pseudopregnant female foster animal. Any of the enzyme
protein nucleotide sequences can be introduced as a transgene into
the genome of a non-human animal, such as a mouse.
[0192] Any of the, regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the enzyme
protein to particular cells.
[0193] Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice,
have become conventional in the art and are described, for example,
in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al.,
U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press,.Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of the transgene
in its genome and/or expression of transgenic mRNA in tissues or
cells of the animals. A transgenic founder animal canthen be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[0194] It, another embodiment, transgenic non-human animals can be
produced which contain selected systems that allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS
89:6232-6236 (1992). Another example of a recombinase system is the
FLP recombinase system of S. cerevisiae (O'Gorman et al. Science
251:1351-1355 (1991). If a cre/loxP recombinase system is used to
regulate expression of the transgene, animals containing transgenes
encoding both the Cre recombinase and a selected protein is
required. Such animals can be provided through the construction of
"double" transgenic animals, e.g., by mating two transgenic
animals, one containing a transgene encoding a selected protein and
the other containing a transgene encoding a recombinase.
[0195] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. Nature 385:810-813 (1997) and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter G.sub.o phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyst and then transferred to pseudopregnant female
foster animal. The offspring born of this female foster animal will
be a clone of the animal from which the cell, e.g., the somatic
cell, is isolated.
[0196] Transgenic animals containing recombinant cells that express
the peptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
effect substrate binding, enzyme protein activation, and signal
transduction, may not be evident from in vitro cell-free or
cell-based assays. Accordingly, it is useful to provide non-human
transgenic animals to assay in vivo enzyme protein function,
including substrate interaction, the effect of specific mutant
enzyme proteins on enzyme protein function and substrate
interaction, and the effect of chimeric enzyme proteins. It is also
possible to assess the effect of null mutations, that is, mutations
that substantially or completely eliminate one or more enzyme
protein functions.
[0197] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the above-described modes for carrying out
the invention which are obvious to those skilled in the field of
molecular biology or related fields are intended to be within the
scope of the following claims.
Sequence CWU 1
1
4 1 2071 DNA Homo sapiens 1 tggaggagcc agcggaagga cggtgtgcgg
gccggccagc cctggacgaa agaagagggc 60 ccctccaggc cagtctgggc
accctgggat agcggctgca gccaggcatg gccgactctg 120 cacaggccca
gaagctggtg tacctggtca cagggggctg tggcttcctg ggagagcacg 180
tggtgcgaat gctgctgcag cgggagcccc ggctcgggga gctgcgggtc tttgaccaac
240 acctgggtcc ctggctggag gagctgaaga caggtacccg gaacgtgatc
gaggcttgtg 300 tgcagaccgg aacacggttc ctggtctaca ccagcagcat
ggaagttgtg gggcctaaca 360 ccaaaggtca ccccttctac aggggcaacg
aagacacccc atacgaagca gtgcacaggc 420 acccctatcc ttgcagcaag
gccctggccg agtggctggt cctggaggcc aacgggagga 480 aggtccgtgg
ggggctgccc ctggtgacgt gtgcccttcg tcccacgggc atctacggtg 540
aaggccacca gatcatgagg gacttctacc gccagggcct gcgcctggga ggttggctct
600 tccgggccat cccggcctct gtggagcatg gccgggtcta tgtgggcaat
gttgcctgga 660 tgcacgtgct ggcagcccgg gagctggagc agcgggcagc
cctgatgggc ggccaggtat 720 acttctgcta cgatggatca ccctacagga
gctacgagga tttcaacatg gagttcctgg 780 gcccctgcgg actgcggctg
gtgggcgccc gcccattgct gccctactgg ctgctggtgt 840 tcctggctgc
cctcaatgcc ctgctgcagt ggctgctgcg gccactggtg ctctacgcac 900
ccctgctgaa cccctacacg ctggccgtgg ccaacaccac cttcaccgtc agcaccgaca
960 aggctcagcg ccatttcggc tatgagcccc tgttctcgtg ggaggatagc
cggacccgca 1020 ccattctctg ggtacaggcc gctacgggtt cagcccagtg
acggtggggc tggggcctgg 1080 aggcccagat acagcacatc cacccaggtc
ccgagccctc acaccctgga cgggaaggga 1140 cagctgcatt ccagagcagg
aggcagggct ctggggccag aatggctgtc cttgtcgtag 1200 agccctccac
attttctttt tcttttttga gacagggtct tgctctgtca cccagactgg 1260
agtgcagtgg tgtgatcata gctcactgca ccctcaacct cctgggttca agcaatcctc
1320 ctgcctcagc ctcctgaaca gctgggacca caggtgcacg ccaccatacc
tggctttttt 1380 ttgttgcttt tagagacagg gtctcactat attgctcaag
gctggacttg aactcctggg 1440 ctcaagtgat cttcccacgt gggcctccca
aaacgctgga actacaagtg tgagccaccg 1500 cgcctggccc accgcctctc
cacattttca atccaggagc cttgagtctg tggctgtgtc 1560 ctgacacctc
cagagttctg agggccgtca ggacacggga gggtttgggg acagagtgtc 1620
cttcctctgt cctatcatca ccagtcctga tggccgcttg gtgagtgtct ggtgccctgg
1680 tggcttgccc cagctctctt gtggctttct gagcaggaag cgagcactag
gctccacagg 1740 cttacgctgt gtctcctgcc agccacacag cgacccatcg
gtgcagagtg cagacgcggg 1800 tgtggttcct ccagcccacc tcagtccctc
tttgggaggt gatgttccca ttgtttttca 1860 aaggcctcac cttcaactgt
tctgttttag aattcccctc tggagggcta tggcctccct 1920 atggtttcac
ttcccaccta cttctaccta agttccttcc cagcacatcg ccagccctgg 1980
gcctggggat gtccccaatg ctgtacctgg ctgaccccgg attaaaagcc tcatccacga
2040 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 2071 2 317 PRT Homo sapiens
2 Met Ala Asp Ser Ala Gln Ala Gln Lys Leu Val Tyr Leu Val Thr Gly 1
5 10 15 Gly Cys Gly Phe Leu Gly Glu His Val Val Arg Met Leu Leu Gln
Arg 20 25 30 Glu Pro Arg Leu Gly Glu Leu Arg Val Phe Asp Gln His
Leu Gly Pro 35 40 45 Trp Leu Glu Glu Leu Lys Thr Gly Thr Arg Asn
Val Ile Glu Ala Cys 50 55 60 Val Gln Thr Gly Thr Arg Phe Leu Val
Tyr Thr Ser Ser Met Glu Val 65 70 75 80 Val Gly Pro Asn Thr Lys Gly
His Pro Phe Tyr Arg Gly Asn Glu Asp 85 90 95 Thr Pro Tyr Glu Ala
Val His Arg His Pro Tyr Pro Cys Ser Lys Ala 100 105 110 Leu Ala Glu
Trp Leu Val Leu Glu Ala Asn Gly Arg Lys Val Arg Gly 115 120 125 Gly
Leu Pro Leu Val Thr Cys Ala Leu Arg Pro Thr Gly Ile Tyr Gly 130 135
140 Glu Gly His Gln Ile Met Arg Asp Phe Tyr Arg Gln Gly Leu Arg Leu
145 150 155 160 Gly Gly Trp Leu Phe Arg Ala Ile Pro Ala Ser Val Glu
His Gly Arg 165 170 175 Val Tyr Val Gly Asn Val Ala Trp Met His Val
Leu Ala Ala Arg Glu 180 185 190 Leu Glu Gln Arg Ala Ala Leu Met Gly
Gly Gln Val Tyr Phe Cys Tyr 195 200 205 Asp Gly Ser Pro Tyr Arg Ser
Tyr Glu Asp Phe Asn Met Glu Phe Leu 210 215 220 Gly Pro Cys Gly Leu
Arg Leu Val Gly Ala Arg Pro Leu Leu Pro Tyr 225 230 235 240 Trp Leu
Leu Val Phe Leu Ala Ala Leu Asn Ala Leu Leu Gln Trp Leu 245 250 255
Leu Arg Pro Leu Val Leu Tyr Ala Pro Leu Leu Asn Pro Tyr Thr Leu 260
265 270 Ala Val Ala Asn Thr Thr Phe Thr Val Ser Thr Asp Lys Ala Gln
Arg 275 280 285 His Phe Gly Tyr Glu Pro Leu Phe Ser Trp Glu Asp Ser
Arg Thr Arg 290 295 300 Thr Ile Leu Trp Val Gln Ala Ala Thr Gly Ser
Ala Gln 305 310 315 3 7301 DNA Homo sapiens 3 atttgcatta gccggtggca
gccaacaggt gcctgttttg gagagaggtc cagggaggag 60 agatgagcag
ggtgccgttg gtgacatggc cagtcatttc aggagctgcc ccaaccccag 120
acttgcccca gcagtccggg accccactgt gaccaggcag atgctcgaag gagtcagtgg
180 ctctcttacc cagtgcagat ttccctggag ttccctgcgg gtgacttaga
atggccacca 240 gaggcttagg atgctgcccc aaagagggag ggctcctgga
agcagagtcg agagagtcag 300 tgccgggtta gcgggagctg gaggcagagc
tgcagctcca ggcctggtgg gcgtggacct 360 ggggtgctgg ctggcaggcg
tgctcagggg caggaagtgg gggactcttc cctgaccatc 420 gcatctcacc
ctggcagatg gtggccgaca tgcgggagaa gcgctacgtg caggagggca 480
ttggcagcag ctacctgttc cgggtggacc acgacaccat catcgatgcc accaagtgtg
540 gcaacctggc cagattcatc aaccactgct gcacggtgcg ccaggggcca
gccggggcag 600 gagttggggg tcggtggggg tggccacggc tcacacgccc
ttccatccgc agcctaactg 660 ctacgccaag gtcatcacca tcgagtccca
gaagaagatc gtgatctact ccaagcagcc 720 cattggcgtg gacgaggaga
tcacctacga ctacaagttc ccactggaag acaacaagat 780 cccgtgtctg
tgtggcacag agagctgccg gggctcccta aactgaggtg gggcaggatg 840
ggtgcccaca cccctattta ttccccctgg tgccctgagc tcccagcacc cccccagcct
900 tagtgggctc agcagggccc acatgccccc atctccaagc gtggggttgg
gggccccaag 960 cccagcgagg gagcctcagt ccctggaggc agcttctgcc
tctcctgtcg cccctgccca 1020 ccaccccctg attgtttttc tttgcggaga
agaagctgta aatgttttgt agcagccagc 1080 agctgtttcc tgtggaaacc
tggggtgccg gcctgtacag attctgtcct ggggggctac 1140 acagtcctct
cgctttgtgt taatggggac ttccccttac gccctgcgtg tacccctccc 1200
cagtttaggg gtctctgggg cagtggccat gttctccccc tgggggggct ctgcaccccc
1260 agtcctgggg actccgtgcc tggaaccctg cctcatctgt tcctgccaga
ccctgagggt 1320 cacccttcca ccctggtgtc actccccggc tcagccaggc
caggatggcg gggtgggtcc 1380 cttttgctgg gctggactgt acatatgtta
atagcgcaaa cccgacgcca catttttata 1440 attgtgatta aactttattg
tacaaaagtg tttggtcggt gtatttgggc aggagcgagg 1500 ggttgggggt
agagggcacg gagggttgtg caagttgaag agagggaaaa gtgggtacct 1560
gaagtgtggg gcaggtaaag gggccttcag gcaagagccc agacctgcag agacagtccg
1620 agactgtctc ggaccccctg acaggctgca gcagccgcac ccgcaccagg
aataccccac 1680 cagtgcccgc cagggtggtg ccaaggtcag gcctcccctt
cctacaatca cagctgcagc 1740 tggacctccg gcctcctggg aagcccagca
ggagggaagg cctgaggtca cactgtggga 1800 tgaggtcacc gctggctcca
cccacagccc cagacccctt cagcccactc tgcaagttcg 1860 agcttcatcc
ccaccaagtt ctccgctgga cccagatgcc agtggagcac agagcggccg 1920
ccagggggcg ccttggggca agagtggtgg gggttgtggc tgggcgggtc tctgttcctg
1980 gaatggggca ggagggagaa ggaggagcca gcggaaggac ggtgtgcggg
ccggccagcc 2040 ctggacgaaa gaagagggcc cctccaggcc agtctgggca
ccctgggata gcggctgcag 2100 gtaggcagag gcgctgccag tgcccaggtg
gcctttccct ccatccggcc cttcccacct 2160 tcctataacc ttccctccac
ctccctcaac tcctggcctc cccacccttt tactgccttc 2220 aaatctctct
ccctaaaccc tgaccccttc ctgcacccca agcccgcccc tctctccgta 2280
actcagccat cagcaggggc agacggcagg tggcctggtt gctgcagctc ccaggatcag
2340 ctctgccctc ccgccaaacg ccagcctcgt caccgctcca gggcacctcc
agcagtaaca 2400 ggtggttgca gcaggtggca gccagcccct ggatgagcca
aggtctcttc cccagccagg 2460 catggccgac tctgcacagg cccagaagct
ggtgtacctg gtcacagggg gctgtggctt 2520 cctgggagag cacgtggtgc
gaatgctgct gcagcgggag ccccggctcg gggagctgcg 2580 ggtctttgac
caacacctgg gtccctggct ggaggagctg aagacaggtt cttgttgggg 2640
gagcttgtgg tggagagggt gtggacgctt ccccaaccct tcccaagctg ggatccccac
2700 ccctgcagtg gaacagatga tgctggtttc tgtccacatg gatgggtcga
gtgagtcaca 2760 ttgggaacgt gactccaggg tggaagatga acccagcctc
tggcctctgg ccccagctct 2820 gacatggcct gtgtcctcca accccggcca
gggcctgtga gggtgactgc catccagggg 2880 gacgtgaccc aggcccatga
ggtggcagca gctgtggccg gagcccatgt ggtcatccac 2940 acggctgggc
tggtagacgt gtttggcagg gccagtccca agaccatcca tgaggtcaac 3000
gtgcagggtg aggagctctg gacactcctg gccatcttgc ctgtttgttc cccactctgt
3060 ctttggcctt gacctccggt gactcccctg ggacaagttg tcctattgac
agccctgccc 3120 ccgcctcccc tgacctgtca tggttttccc tggacctggg
atggggagga ggaagatgca 3180 gagagggaag aagctgcagc ttggatacgc
ctcctcctct gccaggtacc cggaacgtga 3240 tcgaggcttg tgtgcagacc
ggaacacggt tcctggtcta caccagcagc atggaagttg 3300 tggggcctaa
caccaaaggt caccccttct acaggtgagt ggcaggccct cttgtcctct 3360
aagagcccat ttccctcagc attgagtctt ccttctcctc ccaccagggg caacgaagac
3420 accccatacg aagcagtgca caggcacccc tatccttgca gcaaggccct
ggccgagtgg 3480 ctggtcctgg aggccaacgg gaggaaggtg agcccagaaa
aaggaggcgc agagatgggg 3540 ctcctgccct gcacaccccc ttaccctgcc
agcccaagga ggccggggcc gagagcaagc 3600 tgtcgggtcc caggtctcag
cagtacctgc ctttgccacc aggtccgtgg ggggctgccc 3660 ctggtgacgt
gtgcccttcg tcccacgggc atctacggtg aaggccacca gatcatgagg 3720
gacttctacc gccagggcct gcgcctggga ggttggctct tccgggccat cccggcctct
3780 gtggagcatg gccgggtcta tgtgggtgag gactgggcta ggcaggggga
ggctgagaat 3840 atggcaggag gacttgctct agaagggggc aggacccaca
tggccctggg agagaagtgt 3900 ggactctggc tagaaaaata tggtctatac
atgggccaag gtagactgtg attatgtctc 3960 cacagcctgc agagaataca
ggatccatgc aagttgggac attaaaaagt gtatcatagg 4020 ctacagagaa
gattgcagct atgggagcag ccattcccca ggagaggaga ggagagggac 4080
agtgtgtaca cagcactaaa agggctgggt tcagtggctc gcatctataa tcccagcact
4140 ttaggaggct gaggcgggag gatggcctga gcccaggagt tggaggctgc
agtgagctat 4200 gaccgcacca ctgcactcca gcctggatga cagagacaga
ccctgtctct aaaacttttt 4260 ttaaaggaag tagcatctac acagggaata
aggtcacctg ccactccatc ctgcagtccc 4320 caagcctctc agggcccacc
acgcaggtcc tggtttctct atcctctccc caggttcttt 4380 gcagatgcag
gctggcccag gagagcaagt gactaccagg gcgagggaga aggcagcctt 4440
tcccaggctg ctgtggggat gtgggcggca actacctggg cccaaagagg gggtggccca
4500 ggagagcagc ctcgatgtgg tgttgcaagg gcactcaggg gtgtgtccgc
ctctcttccg 4560 ccaccggcag gcaatgttgc ctggatgcac gtgctggcag
cccgggagct ggagcagcgg 4620 gcagccctga tgggcggcca ggtatacttc
tgctacgatg gatcacccta caggagctac 4680 gaggatttca acatggagtt
cctgggcccc tgcggactgc ggctggtggg cgcccgccca 4740 ttgctgccct
actggctgct ggtgttcctg gctgccctca atgccctgct gcagtggctg 4800
ctgcggccac tggtgctcta cgcacccctg ctgaacccct acacgctggc cgtggccaac
4860 accaccttca ccgtcagcac cgacaaggct cagcgccatt tcggctatga
gcccctgttc 4920 tcgtgggagg atagccggac ccgcaccatt ctctgggtac
aggccgctac gggttcagcc 4980 cagtgacggt ggggctgggg cctggaggcc
cagatacagc acatccaccc aggtcccgag 5040 ccctcacacc ctggacggga
agggacagct gcattccaga gcaggaggca gggcttctgg 5100 ggccagaatg
gctgtccttg tcgtagagcc ctccacattt tctttttctt ttttgagaca 5160
gggtcttgct ctgtcaccca gactggagtg cagtggtgtg atcatagctc actgcaccct
5220 caacctcctg ggttcaagca atcctcctgc ctcagcctcc ttgaacagct
gggaccacag 5280 gtgcacgcca ccacacctgg cttttttttg ttgtttttag
agacagggtc tcactatatt 5340 gctcaggctg gtcttgaact cctgggctca
agtgatcttc ccacgtgggc ctcccaaaac 5400 gctggaacta caagtgtgag
ccaccgcgcc tggcccaagc cctccacatt ttcaatccag 5460 gagccttgag
tctgtgttgt gtcctgacac ctccaagttc tagggccgtc aggacacggg 5520
agggtttggg gacagagtgt ccttcctctg tcctctcatc ccagtcctga tggccgcttg
5580 gtgagtgtct ggtgccctgg tggcctgccc cagctctctt ctggctttct
gagcaggaag 5640 cgagcagagg ctccacaggc ttacgctgct ctcctgacag
ccacacgcga ccctcggtgc 5700 agagtgcaga ggcggctctg gttcctccag
ccacctcagt ccctctttgg gaggtgatgt 5760 tcccattgtt tttcaaaggc
ctcaccttca actgtctgtc ttagaattcc cctctggagg 5820 gctatggcct
ccctatgctt tcacttccca cctctctacc taagttcctt cccagcacat 5880
cgccagccct gggcctgggg atgtccccaa tgctgtacct ggctgacccc ggattaaaag
5940 cctcatccac gaccgtgtcc atctgtctgt ccagctctcc ctcccatccc
cccaccccat 6000 gtccgcctcc ccacggcgcc catcccacgt ggggacagaa
ggaagtgagc acacggcaca 6060 cccgctgttg gattggttgc tatttctccc
gtcccacagg gcctgacctg gcccagggtg 6120 gggtgggggg ctctggggac
aggacatgca gggaggaagg ggggggcagg attttcctgt 6180 gttttatcca
tttgcaagtt ggtcaccaat agaaatggga ctctgagggc taacagaaat 6240
gggactctga gggctaacag gagagggcgg cctggctctg ggccccagcc aggccccagg
6300 agtcctgtcc cctctgagaa ggggagggag agagctctag aaaccaacgg
agaaacagag 6360 aagggggcag gggctcatgt cagcaaacac ggctacatca
cgtgacacgc cagtgacaca 6420 gaaacacacg ccaacgcaca cggctgcaca
gcgggcaggg gcggttaggg gaaagggagc 6480 cggggccacc catcttgtcc
tctgcagggc gggctggggg gcagggtgaa tgcatagaac 6540 acatcatgtg
tacacgctca gggcgtggca agagcgtgcg tcgacccacg ggtacatggg 6600
atggacacgc agtgtgcttc atgaggggtg ggaacaggga ggagggggaa gaggaagcac
6660 tgagccctgg ccaggcccgg gaccacccgc agggcacacg tggggcacat
gtgggctcaa 6720 tggttgcagg cgcctgggca ggtagcacac atttgtccaa
gaacatgcaa aagacaccag 6780 cctccagaca acatgccagg acgcacacag
acagcagcca acaagcaggc acatcatagg 6840 atgtggagga cgcatagaaa
gggcacagca gacccttaga gatcccctgg tccacctgag 6900 gcccagagat
gggcagctgt gggcccaatg ccactccagg tggggggagt ggtgccccag 6960
ccacgcttca acccttctcc tgtggcccca aggccgtggg acttccggaa acacctgggc
7020 tgaatggggg tcctgtccag gcggccggaa gaggggactg ggggctgggg
cctgctctga 7080 tgtctcccaa gcagcccgag atgggagcag gagggccgtg
gccagacttg gggcagactt 7140 cctgtcctgc agaggggcgt tctgggaagg
gacaggcagg cccccagctc aggacagccc 7200 acctggggtt acgcacgtgg
ccacactgac acacacacag gacaagggag agctcggctg 7260 tctgagctcg
ggtagaggtg gaggggtact gtgttctggg a 7301 4 369 PRT Homo sapiens 4
Met Ala Asp Ser Ala Gln Ala Gln Lys Leu Val Tyr Leu Val Thr Gly 1 5
10 15 Gly Cys Gly Phe Leu Gly Glu His Val Val Arg Met Leu Leu Gln
Arg 20 25 30 Glu Pro Arg Leu Gly Glu Leu Arg Val Phe Asp Gln His
Leu Gly Pro 35 40 45 Trp Leu Glu Glu Leu Lys Thr Gly Pro Val Arg
Val Thr Ala Ile Gln 50 55 60 Gly Asp Val Thr Gln Ala His Glu Val
Ala Ala Ala Val Ala Gly Ala 65 70 75 80 His Val Val Ile His Thr Ala
Gly Leu Val Asp Val Phe Gly Arg Ala 85 90 95 Ser Pro Lys Thr Ile
His Glu Val Asn Val Gln Gly Thr Arg Asn Val 100 105 110 Ile Glu Ala
Cys Val Gln Thr Gly Thr Arg Phe Leu Val Tyr Thr Ser 115 120 125 Ser
Met Glu Val Val Gly Pro Asn Thr Lys Gly His Pro Phe Tyr Arg 130 135
140 Gly Asn Glu Asp Thr Pro Tyr Glu Ala Val His Arg His Pro Tyr Pro
145 150 155 160 Cys Ser Lys Ala Leu Ala Glu Trp Leu Val Leu Glu Ala
Asn Gly Arg 165 170 175 Lys Val Arg Gly Gly Leu Pro Leu Val Thr Cys
Ala Leu Arg Pro Thr 180 185 190 Gly Ile Tyr Gly Glu Gly His Gln Ile
Met Arg Asp Phe Tyr Arg Gln 195 200 205 Gly Leu Arg Leu Gly Gly Trp
Leu Phe Arg Ala Ile Pro Ala Ser Val 210 215 220 Glu His Gly Arg Val
Tyr Val Gly Asn Val Ala Trp Met His Val Leu 225 230 235 240 Ala Ala
Arg Glu Leu Glu Gln Arg Ala Ala Leu Met Gly Gly Gln Val 245 250 255
Tyr Phe Cys Tyr Asp Gly Ser Pro His Arg Ser Tyr Glu Asp Phe Asn 260
265 270 Met Glu Phe Leu Gly Pro Cys Gly Leu Arg Leu Val Gly Ala Arg
Pro 275 280 285 Leu Leu Pro Tyr Trp Leu Leu Val Phe Leu Ala Ala Leu
Asn Ala Leu 290 295 300 Leu Gln Trp Leu Leu Arg Pro Leu Val Leu Tyr
Ala Pro Leu Leu Asn 305 310 315 320 Pro Tyr Thr Leu Ala Val Ala Asn
Ala Thr Phe Thr Val Ser Thr Asp 325 330 335 Lys Ala Gln Arg His Phe
Gly Tyr Glu Pro Leu Phe Ser Trp Glu Asp 340 345 350 Ser Arg Thr Arg
Thr Ile Leu Trp Val Gln Ala Ala Thr Gly Ser Ala 355 360 365 Gln
* * * * *
References