U.S. patent application number 11/730664 was filed with the patent office on 2007-08-09 for isolated human enzyme proteins, nucleic acid molecules encoding human enzyme proteins, and uses thereof.
This patent application is currently assigned to APPLERA CORPORATION. Invention is credited to Ellen M. Beasley, Valentina Di Francesco, Ming-Hui Wei, Chunhua Yan.
Application Number | 20070184053 11/730664 |
Document ID | / |
Family ID | 25216714 |
Filed Date | 2007-08-09 |
United States Patent
Application |
20070184053 |
Kind Code |
A1 |
Wei; Ming-Hui ; et
al. |
August 9, 2007 |
Isolated human enzyme proteins, nucleic acid molecules encoding
human enzyme proteins, 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) ; Di
Francesco; Valentina; (Rockville, MD) ; Beasley;
Ellen M.; (Darnestown, MD) |
Correspondence
Address: |
CELERA;ATTN: Victor Lee, Vice President
45 WEST GUDE DRIVE
C1-1#316
ROCKVILLE
MD
20850
US
|
Assignee: |
APPLERA CORPORATION
Norwalk
CT
06856-5435
|
Family ID: |
25216714 |
Appl. No.: |
11/730664 |
Filed: |
April 3, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10785106 |
Feb 25, 2004 |
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11730664 |
Apr 3, 2007 |
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09815048 |
Mar 23, 2001 |
6737261 |
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10785106 |
Feb 25, 2004 |
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Current U.S.
Class: |
424/146.1 ;
435/183; 435/320.1; 435/325; 435/69.1; 530/388.26 |
Current CPC
Class: |
C12N 9/90 20130101; A01K
2217/05 20130101 |
Class at
Publication: |
424/146.1 ;
435/069.1; 435/183; 435/320.1; 435/325; 530/388.26 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12P 21/06 20060101 C12P021/06; C12N 9/00 20060101
C12N009/00; C07K 16/40 20060101 C07K016/40 |
Claims
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: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.
3. An isolated antibody that selectively binds to a peptide of
claim 2.
4. 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.
5. 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.
6. 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.
7. 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.
8. The method of claim 7, wherein said agent is administered to a
host cell comprising an expression vector that expresses said
peptide.
9. 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.
10. A pharmaceutical composition comprising an agent identified by
the method of claim 9 and a pharmaceutically acceptable carrier
therefor.
11. 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 9.
12. 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.
13. 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.
14. A peptide according to claim 13 that shares at least 90 percent
homology with an amino acid sequence shown in SEQ ID NO:2.
Description
FIELD OF THE INVENTION
[0001] The present invention is in the field of enzyme proteins
that are related to the lanosterol synthase enzyme 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 lanosterol synthase
subfamily.
[0003] Lanosterol Synthase
[0004] The novel human protein, and encoding gene, provided by the
present invention is related to lanosterol synthase enzymes.
Specifically, the protein provided by the present invention is 11
amino acids shorter in exon 4 compared with the art-known
lanosterol synthase protein provided in Genbank gi4505027 (see the
amino acid sequence alignment of the protein of the present
invention against gi4505027 provided in FIG. 2).
[0005] Lanosterol synthase enzymes are important for catalyzing the
cyclization of squalene-2,3-epoxide lanosterol, which is the
parental compound of all mammalian steroids (Young et al., Human
Genet 1996 May; 97(5):620-4). Baker et al. (Biochem Biophys Res
Commun 1995 Aug. 4; 213(1):154-60) cloned and characterized the
human lanosterol synthase gene and found that it encoded a
predicted 83 kDa protein of 732 amino acids; this amino acid
sequence shared 36-40% identity with yeast and plant homologues and
83% identity with Rattus norvegicus lanosterol synthase. For a
further review of the lanosterol synthase gene/protein, see Sung et
al., Biol Pharm Bull 1995 October; 18(10):1459-61.
[0006] Enzyme proteins, particularly members of the lanosterol
synthase enzyme 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 lanosterol
synthase enzyme subfamily. These novel compositions are useful in
the diagnosis, prevention and treatment of biological processes
associated with human diseases.
SUMMARY OF THE INVENTION
[0007] 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 lanosterol synthase enzyme 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 teratocarcinoma, ovary,
uterus, muscle, brain, colon, and hippocampus.
DESCRIPTION OF THE FIGURE SHEETS
[0008] FIG. 1 provides the nucleotide sequence of a cDNA molecule
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
teratocarcinoma, ovary, uterus, muscle, brain, colon, and
hippocampus.
[0009] 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.
[0010] 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 56 different nucleotide positions.
DETAILED DESCRIPTION OF THE INVENTION
[0011] General Description
[0012] 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 lanosterol synthase enzyme 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 lanosterol synthase enzyme 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.
[0013] 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 lanosterol synthase enzyme subfamily and the
expression pattern observed. Experimental data as provided in FIG.
1 indicates expression in humans in teratocarcinoma, ovary, uterus,
muscle, brain, colon, and hippocampus. 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 lanosterol synthase family or
subfamily of enzyme proteins.
Specific Embodiments
[0014] Peptide Molecules
[0015] 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 lanosterol
synthase enzyme 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.
[0016] 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.
[0017] 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).
[0018] 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.
[0019] 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.
[0020] 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 teratocarcinoma, ovary, uterus,
muscle, brain, colon, and hippocampus. 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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 comparison 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.
[0030] 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.com), using a NWSgapdna.CMP matrix and a gap weight
of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or
6. 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.
[0031] 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.
[0032] 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. The gene encoding the novel enzyme of the
present invention is located on a genome component that has been
mapped to human chromosome 21 (as indicated in FIG. 3), which is
supported by multiple lines of evidence, such as STS and BAC map
data.
[0033] 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.
The gene encoding the novel enzyme of the present invention is
located on a genome component that has been mapped to human
chromosome 21 (as indicated in FIG. 3), which is supported by
multiple lines of evidence, such as STS and BAC map data. 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.
[0034] FIG. 3 provides information on SNPs that have been found in
the gene encoding the enzyme of the present invention. SNPs were
identified at 56 different nucleotide positions, including a
non-synonymous coding SNP at position 36001 (protein position 631).
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. Some of these SNPs, located outside the ORF and in
introns, may affect gene transcription.
[0035] 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 to a enzyme peptide encoding nucleic
acid molecule under moderate to stringent conditions as more fully
described below.
[0036] 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.
[0037] 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).
[0038] 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.
[0039] 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.
[0040] 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)).
[0041] 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.
[0042] 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.
[0043] 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).
[0044] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, 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.
[0045] 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)).
[0046] 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 or a
pro-protein sequence.
[0047] Protein/Peptide Uses
[0048] 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.
[0049] 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.
[0050] 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
teratocarcinoma, ovary, uterus, muscle, brain, and colon, as
indicated by virtual northern blot analysis. In addition, PCR-based
tissue screening panels indicate expression in the hioppocampus. A
large percentage of pharmaceutical agents are being developed that
modulate the activity of enzyme proteins, particularly members of
the lanosterol synthase 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 teratocarcinoma, ovary, uterus, muscle, brain, colon, and
hippocampus. Such uses can readily be determined using the
information provided herein, that which is known in the art, and
routine experimentation.
[0051] 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 lanosterol synthase 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 teratocarcinoma, ovary, uterus, muscle,
brain, and colon, as indicated by virtual northern blot analysis.
In addition, PCR-based tissue screening panels indicate expression
in the hioppocampus.
[0052] 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
teratocarcinoma, ovary, uterus, muscle, brain, colon, and
hippocampus. In an alternate embodiment, cell-based assays involve
recombinant host cells expressing the enzyme protein.
[0053] 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.
[0054] 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.
[0055] 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).
[0056] 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.
[0057] 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.
[0058] 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 teratocarcinoma, ovary, uterus, muscle,
brain, and colon, as indicated by virtual northern blot analysis.
In addition, PCR-based tissue screening panels indicate expression
in the hioppocampus.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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-5-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
supernatant 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.
[0063] 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.
[0064] 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 teratocarcinoma, ovary,
uterus, muscle, brain, colon, and hippocampus. 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.
[0065] 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.
[0066] 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.
[0067] 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 agent. 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.
[0068] 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 teratocarcinoma, ovary, uterus, muscle, brain, colon, and
hippocampus. 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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 teratocarcinoma, ovary, uterus,
muscle, brain, colon, and hippocampus. Accordingly, methods for
treatment include the use of the enzyme protein or fragments.
[0074] Antibodies
[0075] 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.
[0076] 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.
[0077] 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).
[0078] 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.
[0079] 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.
[0080] 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).
[0081] 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, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0082] Antibody Uses
[0083] 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
teratocarcinoma, ovary, uterus, muscle, brain, and colon, as
indicated by virtual northern blot analysis. In addition, PCR-based
tissue screening panels indicate expression in the hioppocampus.
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.
[0084] 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 teratocarcinoma, ovary,
uterus, muscle, brain, colon, and hippocampus. 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.
[0085] 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 teratocarcinoma, ovary, uterus, muscle,
brain, colon, and hippocampus. 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.
[0086] 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.
[0087] The antibodies are also useful for tissue typing.
Experimental data as provided in FIG. 1 indicates expression in
humans in teratocarcinoma, ovary, uterus, muscle, brain, colon, and
hippocampus. 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.
[0088] 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.
[0089] 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.
[0090] Nucleic Acid Molecules
[0091] 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.
[0092] 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 3' 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.
[0093] 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.
[0094] 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.
[0095] Accordingly, the present invention provides nucleic acid
molecules that consist of the nucleotide sequence shown in FIG. 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.
[0096] The present invention further provides nucleic acid
molecules that consist essentially of the nucleotide sequence shown
in FIG. 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.
[0097] The present invention further provides nucleic acid
molecules that comprise the nucleotide sequences shown in FIG. 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.
[0098] 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 gene
regulatory 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.
[0099] 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 than one 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.
[0100] 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.
[0101] 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).
[0102] 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), paralogs (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, inversions and
insertions. Variation can occur in either or both the coding and
non-coding regions. The variations can produce both conservative
and non-conservative amino acid substitutions.
[0103] 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.
[0104] A fragment comprises a contiguous nucleotide sequence
greater than 12 or more nucleotides. Further, a fragment could at
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.
[0105] 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.
[0106] 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. The gene encoding
the novel enzyme of the present invention is located on a genome
component that has been mapped to human chromosome 21 (as indicated
in FIG. 3), which is supported by multiple lines of evidence, such
as STS and BAC map data.
[0107] FIG. 3 provides information on SNPs that have been found in
the gene encoding the enzyme of the present invention. SNPs were
identified at 56 different nucleotide positions, including a
non-synonymous coding SNP at position 36001 (protein position 631).
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. Some of these SNPs, located outside the ORF and in
introns, may affect gene transcription.
[0108] 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 hybridized
to 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 are 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.
[0109] Nucleic Acid Molecule Uses
[0110] 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 56 different nucleotide
positions.
[0111] 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' noncodin.g 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.
[0112] 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.
[0113] 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.
[0114] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0115] 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. The gene encoding the
novel enzyme of the present invention is located on a genome
component that has been mapped to human chromosome 21 (as indicated
in FIG. 3), which is supported by multiple lines of evidence, such
as STS and BAC map data.
[0116] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0117] 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.
[0118] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0119] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0120] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0121] 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 teratocarcinoma, ovary, uterus, muscle, brain, and
colon, as indicated by virtual northern blot analysis. In addition,
PCR-based tissue screening panels indicate expression in the
hioppocampus. 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.
[0122] 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.
[0123] 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 teratocarcinoma, ovary, uterus, muscle,
brain, and colon, as indicated by virtual northern blot analysis.
In addition, PCR-based tissue screening panels indicate expression
in the hioppocampus.
[0124] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate enzyme nucleic acid
expression.
[0125] 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 teratocarcinoma, ovary, uterus,
muscle, brain, colon, and hippocampus. 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.
[0126] 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.
[0127] 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.
[0128] 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
teratocarcinoma, ovary, uterus, muscle, brain, and colon, as
indicated by virtual northern blot analysis. In addition, PCR-based
tissue screening panels indicate expression in the hioppocampus.
Modulation includes both up-regulation (i.e. activation or
agonization) or down-regulation (suppression or antagonization) or
nucleic acid expression.
[0129] 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 teratocarcinoma, ovary,
uterus, muscle, brain, colon, and hippocampus.
[0130] 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.
[0131] 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.
[0132] 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 56 different nucleotide positions, including a non-synonymous
coding SNP at position 36001 (protein position 631). 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. Some of these
SNPs, located outside the ORF and in introns, may affect gene
transcription. The gene encoding the novel enzyme of the present
invention is located on a genome component that has been mapped to
human chromosome 21 (as indicated in FIG. 3), which is supported by
multiple lines of evidence, such as STS and BAC map data. 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.
[0133] Alternatively, mutations in a enzyme gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0134] Further, sequence-specific ribozymes (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.
[0135] 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)).
[0136] 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. 217:286-295 (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.
[0137] The nucleic acid molecules are also useful for testing 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 56 different nucleotide
positions, including a non-synonymous coding SNP at position 36001
(protein position 631). 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. Some of these SNPs, located outside the
ORF and in introns, may affect gene transcription.
[0138] 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.
[0139] 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.
[0140] 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.
[0141] 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.
[0142] 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
teratocarcinoma, ovary, uterus, muscle, brain, and colon, as
indicated by virtual northern blot analysis. In addition, PCR-based
tissue screening panels indicate expression in the hioppocampus.
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.
[0143] Nucleic Acid Arrays
[0144] 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).
[0145] 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 WO95/11995 (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.
[0146] 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.
[0147] 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 situations 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.
[0148] 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 WO95/251116 (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.
[0149] 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.
[0150] 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 56 different nucleotide positions, including a
non-synonymous coding SNP at position 36001 (protein position 631).
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. Some of these SNPs, located outside the ORF and in
introns, may affect gene transcription.
[0151] 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 (1982), 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).
[0152] 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.
[0153] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0154] 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.
[0155] 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.
[0156] Vectors/Host Cells
[0157] 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.
[0158] A vector can be maintained in the host cell as an
extrachromosomal 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.
[0159] 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).
[0160] 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 trans-acting factor interacting with the
cis-regulatory control region to allow transcription of the nucleic
acid molecules from the vector. Alternatively, a trans-acting
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.
[0161] 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 .lamda., 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.
[0162] 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.
[0163] 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).
[0164] 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 bacteriophage
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).
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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 enteroenzyme. 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)).
[0169] 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. 20:2111-2118 (1992)).
[0170] 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 (Kurjan et al.,
Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0171] 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., Sf 9 cells) include the pAc series
(Smith et al., Mol. Cell. Biol. 3:2156-2165 (1983)) and the pVL
series (Lucklow et al., Virology 170:31-39 (1989)).
[0172] 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
(Kaufman et al., EMBO J. 6:187-195 (1987)).
[0173] 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.
[0174] The invention also encompasses vectors in which the nucleic
acid sequences 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).
[0175] 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.
[0176] 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).
[0177] 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
trans-acting 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.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] Uses of Vectors and Host Cells
[0185] 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.
[0186] 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.
[0187] 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 are 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.
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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 can then 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.
[0192] In 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.
[0193] 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.
[0194] 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.
[0195] 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 4239 DNA Homo sapiens 1 tggcagtggg cggcgtagag cactgcagca
gcaatgacgg agggcacgtg tctgcggcgc 60 cgagggggcc cctacaagac
cgagcccgcc accgacctcg gccgctggcg actcaactgc 120 gagaggggcc
ggcagacgtg gacctacctg caggacgagc gcgccggccg cgagcagacc 180
ggcctggaag cctacgccct ggggctggac accaagaatt actttaagga cttgcccaaa
240 gcccacaccg cctttgaggg ggctctgaac gggatgacat tttacgtggg
gctgcaggct 300 gaggatgggc actggacggg tgattatggt ggcccacttt
tcctcctgcc aggcctcctg 360 atcacttgcc acgtggcacg catccctctg
ccagccggat acagagaaga gattgtgcgg 420 tacctgcggc acattgagga
taagtccacc gtgtttggga ctgcgctcaa ctatgtgtct 480 ctcagaattc
tgggtgttgg gcctgacgat cctgacctgg tacgagcccg gaacattctt 540
cacaagaaag gtggtgctgt ggccatcccc tcctggggga agttctggct ggctgtcctg
600 aatgtttaca gctgggaagg cctcaatacc ctgttcccag agatgtggct
gtttcctgac 660 tgggcaccgg cacacccctc cacactctgg tgccactgcc
ggcaggtgta cctgcccatg 720 agctactgct acgccgttcg gctgagtgcc
gcggaagacc cgctggtcca gagcctccgc 780 caggagctct atgtggagga
cttcgccagc attgactggc tggcgcagag gaacaacgtg 840 gcccccgacg
agctgtacac gccgcacagc tggctgctcc gcgtggtata tgcgctcctc 900
aacctgtatg agcaccacca cagtgcccac ctgcggcagc gggccgtgca gaagctgtat
960 gaacacattg tggccgacga ccgattcacc aagagcatca gcatcggccc
gatctcgaaa 1020 accatcaaca tgcttgtgcg ctggtatgtg gacgggcccg
cctccactgc cttccaggag 1080 catgtctcca gaatcccgga ctatctctgg
atgggccttg acggcatgaa aatgcagggc 1140 accaacggct cacagatctg
ggacaccgca ttcgccatcc aggctctgct tgaggcgggc 1200 gggcaccaca
ggcccgagtt ttcgtcctgc ctgcagaagg ctcatgagtt cctgaggctc 1260
tcacaggtcc cagataaccc tcccgactac cagaagtact accgccagat gcgcaagggt
1320 ggcttctcct tcagtacgct ggactgcggc tggatcgttt ctgactgcac
ggctgaggcc 1380 ttgaaggctg tgctgctcct gcaggagaag tgtccccatg
tcaccgagca catccccaga 1440 gaacggctct gcgatgctgt ggctgtgctg
ctgaacatga gaaatccaga tggagggttc 1500 gccacctatg agaccaagcg
tggggggcac ttgctggagc tgctgaaccc ctcggaggtc 1560 ttcggggaca
tcatgattga ctacacctat gtggagtgca cctcagccgt gatgcaggcg 1620
cttaagtatt tccacaagcg tttcccggag cacagggcag cggagatccg ggagaccctc
1680 acgcagggct tagagttctg tcggcggcag cagagggccg atggctcctg
ggaaggctcc 1740 tggggagttt gcttcaccta cggcacctgg tttggcctgg
aggccttcgc ctgtatgggg 1800 cagacctacc gagatgggac tgcctgtgca
gaggtctccc gggcctgtga cttcctgctg 1860 tcccggcaga tggcagacgg
aggctggggg gaggactttg agtcctgcga ggagcggcgt 1920 tatgtgcaga
gtgcccagtc ccagatccac aacacatgct gggccatgat ggggctgatg 1980
gccgttcggc atcctgacat cgaggcccag gagagaggag tccggtgtct acttgagaaa
2040 cagctcccca atggcgactg gccgcaggaa aacattgctg gggtcttcaa
caagtcctgt 2100 gccatctcct acacgagcta caggaacatc ttccccatct
gggccctcgg ccgcttctcc 2160 cagctgtacc ctgagagagc ccttgctggc
cacccctgag aacatgccta cctgctgggt 2220 gccgtctgtg cgttccagtg
aggccaaggg gtcctggccg ggttggggag ccctcccata 2280 accctgtctt
gggctccaac ccctcaacct ctatctcata gatgtgaatc tgggggccag 2340
gctggaggca gggatgggga cagggtgggt ggcttagact cttgattttt actgtaggtt
2400 catttctgaa agtagcttgt cgggcttggg tgaggaaggg ggcacaggag
ccgtgacccc 2460 tgaggaggca cagcgccttc tgccacctct gggcacggcc
tcaaggtagt gaggctagga 2520 ggttttttct gaccaatagc tgagttcttg
ggagaggagc agctgtgcct gtgtgattcc 2580 ttagtgtcga gtgggctctg
ggctggggtc ggccctgggc aggcttctcc tgcacctttt 2640 gtctgctggg
ctgagggaca cgagggcaac cctgtgacaa tggcaggtag tgtgcatccg 2700
tgaatagccc agtgcggggg ttgctcatgg agcatcctga ggccgtgcag cagggagccc
2760 catgcccctg ggtcgtgagc ttgcctgcgt atggggtggt gtcatggagc
ctcatgcccc 2820 tgggtcgtga gctcgcctga gtatggggtg gtgtcatgga
gccgcatacc cctgggttgt 2880 gagctcgcct gcatatgcag ggtctgtcat
ggaacatccc aagtctgtgc agcagggagc 2940 cccatgcccc tgggacatga
acccacctgc gtggaatgct gtttgtgagg tgtctacagg 3000 gtttatagta
gtcttgtgga cacagaaatg cacaggggac acttacggac acagaaatgc 3060
acaggggagg ccgagcataa ccaggggtga ggggcaggca gcagttgtag ttactgccgc
3120 ggggcactgc tatgtgcagg gacagccagc gcccagccca tcaccactcc
ctgggctggc 3180 tggcaggtat ggcaccctgg gagcccggca tatacccagg
gcacccctac ggctgccgcc 3240 agtctcatgc ccaggtgggt gctctgggct
ggagcgaggg ccaggttttg ggccgaggct 3300 tccccaggca atcctgtgag
ctcccttcta gcctctgacc cagtctggtc tggcttgcat 3360 ggatgtaggg
cttggggtgg gaagttcagg tcctggcttt gcctttgcct gatgtggatg 3420
agcagctcac atgctcaggg ccacctgaga ctgtcactgc tctcccctgg ctactgggag
3480 gagtcactga gagcttcgtt acccctgctg ccttgcccag ggcacaccct
atacctcctc 3540 atctgctctt cccctccctg ccgccttctg ggcaggtagc
agtccctggc ctctccccct 3600 ggctgatcac tctccctcag gcagtggaga
tctgcgtctg gacaccctca gatcctgtca 3660 ttgcctgccc agagtccttc
aggggcaccc ctctgccttg gtgtgcggtc cagggctctc 3720 acccaggtgc
cgcaccctct ggggtcttct gtccagctcc cttgccccat gtgctgtcac 3780
tgactctcct tgggactcgc ctgcctgctc agagccctgc agggcttggt cagctgcctg
3840 ttcagtgtca acacttccct gcacatctta aaactgggct ttattttcgc
tgaaggaact 3900 gtgttgggac ccttgacatc tgtcaggttt gcacatgctg
tttttttttc tcagcccacg 3960 tgttctcccc cacgtggggt agcagcagga
cagacagtga atcacagagt ctgccctgag 4020 cagaggctgc tgtccctggg
actcctagcc atggtcagac tgtacaaaac ggttttccag 4080 aaatgaaatg
taaatccatt tttatactga aaatgttact gaaagtcact tttatgagca 4140
tctgccttaa taaacagaca ttgattccct taaaaaaaaa aaaaaaaaaa aaaaaaaaaa
4200 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 4239 2 721 PRT
Human 2 Met Thr Glu Gly Thr Cys Leu Arg Arg Arg Gly Gly Pro Tyr Lys
Thr 1 5 10 15 Glu Pro Ala Thr Asp Leu Gly Arg Trp Arg Leu Asn Cys
Glu Arg Gly 20 25 30 Arg Gln Thr Trp Thr Tyr Leu Gln Asp Glu Arg
Ala Gly Arg Glu Gln 35 40 45 Thr Gly Leu Glu Ala Tyr Ala Leu Gly
Leu Asp Thr Lys Asn Tyr Phe 50 55 60 Lys Asp Leu Pro Lys Ala His
Thr Ala Phe Glu Gly Ala Leu Asn Gly 65 70 75 80 Met Thr Phe Tyr Val
Gly Leu Gln Ala Glu Asp Gly His Trp Thr Gly 85 90 95 Asp Tyr Gly
Gly Pro Leu Phe Leu Leu Pro Gly Leu Leu Ile Thr Cys 100 105 110 His
Val Ala Arg Ile Pro Leu Pro Ala Gly Tyr Arg Glu Glu Ile Val 115 120
125 Arg Tyr Leu Arg His Ile Glu Asp Lys Ser Thr Val Phe Gly Thr Ala
130 135 140 Leu Asn Tyr Val Ser Leu Arg Ile Leu Gly Val Gly Pro Asp
Asp Pro 145 150 155 160 Asp Leu Val Arg Ala Arg Asn Ile Leu His Lys
Lys Gly Gly Ala Val 165 170 175 Ala Ile Pro Ser Trp Gly Lys Phe Trp
Leu Ala Val Leu Asn Val Tyr 180 185 190 Ser Trp Glu Gly Leu Asn Thr
Leu Phe Pro Glu Met Trp Leu Phe Pro 195 200 205 Asp Trp Ala Pro Ala
His Pro Ser Thr Leu Trp Cys His Cys Arg Gln 210 215 220 Val Tyr Leu
Pro Met Ser Tyr Cys Tyr Ala Val Arg Leu Ser Ala Ala 225 230 235 240
Glu Asp Pro Leu Val Gln Ser Leu Arg Gln Glu Leu Tyr Val Glu Asp 245
250 255 Phe Ala Ser Ile Asp Trp Leu Ala Gln Arg Asn Asn Val Ala Pro
Asp 260 265 270 Glu Leu Tyr Thr Pro His Ser Trp Leu Leu Arg Val Val
Tyr Ala Leu 275 280 285 Leu Asn Leu Tyr Glu His His His Ser Ala His
Leu Arg Gln Arg Ala 290 295 300 Val Gln Lys Leu Tyr Glu His Ile Val
Ala Asp Asp Arg Phe Thr Lys 305 310 315 320 Ser Ile Ser Ile Gly Pro
Ile Ser Lys Thr Ile Asn Met Leu Val Arg 325 330 335 Trp Tyr Val Asp
Gly Pro Ala Ser Thr Ala Phe Gln Glu His Val Ser 340 345 350 Arg Ile
Pro Asp Tyr Leu Trp Met Gly Leu Asp Gly Met Lys Met Gln 355 360 365
Gly Thr Asn Gly Ser Gln Ile Trp Asp Thr Ala Phe Ala Ile Gln Ala 370
375 380 Leu Leu Glu Ala Gly Gly His His Arg Pro Glu Phe Ser Ser Cys
Leu 385 390 395 400 Gln Lys Ala His Glu Phe Leu Arg Leu Ser Gln Val
Pro Asp Asn Pro 405 410 415 Pro Asp Tyr Gln Lys Tyr Tyr Arg Gln Met
Arg Lys Gly Gly Phe Ser 420 425 430 Phe Ser Thr Leu Asp Cys Gly Trp
Ile Val Ser Asp Cys Thr Ala Glu 435 440 445 Ala Leu Lys Ala Val Leu
Leu Leu Gln Glu Lys Cys Pro His Val Thr 450 455 460 Glu His Ile Pro
Arg Glu Arg Leu Cys Asp Ala Val Ala Val Leu Leu 465 470 475 480 Asn
Met Arg Asn Pro Asp Gly Gly Phe Ala Thr Tyr Glu Thr Lys Arg 485 490
495 Gly Gly His Leu Leu Glu Leu Leu Asn Pro Ser Glu Val Phe Gly Asp
500 505 510 Ile Met Ile Asp Tyr Thr Tyr Val Glu Cys Thr Ser Ala Val
Met Gln 515 520 525 Ala Leu Lys Tyr Phe His Lys Arg Phe Pro Glu His
Arg Ala Ala Glu 530 535 540 Ile Arg Glu Thr Leu Thr Gln Gly Leu Glu
Phe Cys Arg Arg Gln Gln 545 550 555 560 Arg Ala Asp Gly Ser Trp Glu
Gly Ser Trp Gly Val Cys Phe Thr Tyr 565 570 575 Gly Thr Trp Phe Gly
Leu Glu Ala Phe Ala Cys Met Gly Gln Thr Tyr 580 585 590 Arg Asp Gly
Thr Ala Cys Ala Glu Val Ser Arg Ala Cys Asp Phe Leu 595 600 605 Leu
Ser Arg Gln Met Ala Asp Gly Gly Trp Gly Glu Asp Phe Glu Ser 610 615
620 Cys Glu Glu Arg Arg Tyr Val Gln Ser Ala Gln Ser Gln Ile His Asn
625 630 635 640 Thr Cys Trp Ala Met Met Gly Leu Met Ala Val Arg His
Pro Asp Ile 645 650 655 Glu Ala Gln Glu Arg Gly Val Arg Cys Leu Leu
Glu Lys Gln Leu Pro 660 665 670 Asn Gly Asp Trp Pro Gln Glu Asn Ile
Ala Gly Val Phe Asn Lys Ser 675 680 685 Cys Ala Ile Ser Tyr Thr Ser
Tyr Arg Asn Ile Phe Pro Ile Trp Ala 690 695 700 Leu Gly Arg Phe Ser
Gln Leu Tyr Pro Glu Arg Ala Leu Ala Gly His 705 710 715 720 Pro 3
42450 DNA Homo sapiens 3 tcatgactgc ccctagaagc ttaactgtgt
caattctcag acgtagttta cagctttttc 60 ttttctttca gacattaaaa
agagcggatt attttactca taaaaagtcc agtccattaa 120 gatatcaaaa
ctcaaactct tatccagttg aaacctcttc cctcacctag ctttgccagg 180
ttcagtgtga gattccatcc aggctgaagc cccttatccc tattcttcat gtttctacat
240 ggaggaactt acctggagaa aaacttccag cctctttctg cttccagaga
agtagagtga 300 ctcatttgat tgaatttcag agaacagata gggtggagtg
tgctcaggct cctctgggta 360 ctctttctgg ggtctgtggg ttgactggag
gggtgtcttc tggtgggcac tcaattgcat 420 agtgcttggt gaggcagttt
catggcctag aggctggggg atatgtttgt ctgacttacg 480 ggtgatttag
tagcttgccc tcttgcttgc agatttaagc cttgtccttc aagctaggtt 540
tttaatttgt ggcaaagctg atattttgat acccacccat cttattgctg tgtctttttc
600 atccgtttct gaactgggat aggaagaggt gattatcctt gattgtctaa
aaccccgcta 660 ttccactgtg gggaaggtgc ctgtgggtat tcttttgtcc
actctctctt ccaactttct 720 cctccggctt gctgtggctc accgcccctt
cgaagttagg ctgggggtag gaattgagga 780 gtgggtgccg aaatgctcac
taggctgggg cagttgtaac tggatgtcag ggcttctgtg 840 ggccaggtga
agacatgctg gggtcttctg tgggtccttg acctgactta gggaccactg 900
gctgcagcct ccagacgtca gccatgtttc caacagtcag acgccccctg ccctgttgcg
960 cccggctgtc ccttccaagt tcggtcactc gctctgcctc catcttcctc
ttccctctgc 1020 tgctaaggct tttcaccttt aatttctcct ggggccaccc
ccaactccag cgaccccgtg 1080 agcagctgag gctctaccgc gctcggtcct
ggccagcgac gcagcccttc cctggcgggg 1140 ctccagggct tctggcccct
gtggtccgcc aggtgtgggg gcccacggcc tcaccgcgcc 1200 taccccactc
cccccggcga agctacgcgg cgctcagctt cccagggacg ccggcggcgc 1260
cctcggctcc tccgctccgc cccgccctcc ccctggtctc gcactggagc cgacggcccg
1320 cgcccacctc acctcagggc ggcctcccgc ccccaccccc ggccccggcg
tccgggcaaa 1380 tcctgcagcg cgagagcaat tccctgccac ccgaccttcg
cactcgctgt cgctcgctcg 1440 agcctcgctc cccacgtcct tccttccgac
ccgcggctgg accctcctca caaatttctc 1500 agagaggctc acctcaaagc
gcggcgcacg aggccgggct cccgggacgc aagcctctag 1560 agggcgcgcg
agaggccccg cccccgccct tcggccccac ccaccagccc cgcccccacc 1620
cgcacccacc aggccccgcc cccacctccc cacccaccag ccccgccccc acctccccac
1680 ccaccagccc cgcccctcat gccccgccaa taaggcccca cccgcctccc
ccgtcccgtc 1740 gccttcaccc accatccccg ctccctcagg ccccgcccca
cgccgcatgg ggcaccaagc 1800 gctccaccac tgtggtcgcc tggcacaccc
cggggtcacg ctcgcggcgc tctgattggt 1860 tgcgtgggcg tcggcccacc
taagcctgag cgcctgccga ggcctgcgcc tgcgtagtgc 1920 gcgcgggagg
ggcgggaggg gcgggagggg cgggaggggc ggggctgggc ggcaggtccc 1980
gggtgcggac atctggcagc tggcagtggg cggcgtagag cactgcagca gcaatgacgg
2040 agggcacgtg agtcccctcg ccccgggctc ctgacgaatg cggggtggtc
ctaggtgctg 2100 aggagagcgc gactggggca gtgggccggc ggccggcgtt
ggggcggggc ctgggtcgct 2160 gatggccggt ggtcctcagg tgtctgcggc
gccgaggggg cccctacaag accgagcccg 2220 ccaccgacct cggccgctgg
cgactcaact gcgagagggg ccggcagacg tggacctacc 2280 tgcaggacga
gcgcgccggc cgcgagcaga ccggcctgga agcctacgcc ctggggctgg 2340
acaccgtaag ttgcttccgc ggagcgtcag cgagctcggg accctgaggg gtgagccgtg
2400 aggagcacgt tttctctcag aaaggcgggt gggaggaccc ggccagcgac
gcccatcccc 2460 aaggcgagcg cccacgggaa ctgcgttcgc gggcccctcc
gcttcagccc cttcatctct 2520 aaaccacgca taggagactc ctaatgtttt
attttttagc accttatttt gagataattt 2580 ttgacttata ggagagttgc
aaagatagtt gtaactttgt ttttattcac aaaaagtgtt 2640 tggatccact
gtcttagttg tgtgcattgt aagagatttt ggtcgtcaga gtctgcagtg 2700
taaacagggt ctcctgccga gccccggcca ccgagggaaa ggctgtgccg ccccttgggc
2760 cctctttgag aggcccgagt cccaggccca ggtcggcacc cgtgccccac
cctacagtct 2820 gggtgcctgg tttattccag acatcttgga gaagttgtga
agaatacatg actggcaaat 2880 aaagcaacga aaatgtgcag ctgttctttt
actttgctga ggtgtgatgc tctcatcaaa 2940 gagtttcaga cttttgatgg
aaacagctga aacttttaaa gtaatttaca ttcactgttt 3000 tgacttgggc
tgtatgtgaa gagggttcct ctggccgggc aacagtcccg tcagctatct 3060
cttttttttt ttttcgatct ctttgcagaa gaattacttt aaggacttgc ccaaagccca
3120 caccgccttt gagggggctc tgaacgggat gacattttac gtggggctgc
aggctgagga 3180 tgggcactgg acgggtgatt atggtggccc acttttcctc
ctgccaggta ggagtatgct 3240 gccccagcct gatggtatgg ccaccctgga
tcacccttgg gatcctggcc cagcctggtc 3300 tagggttttg atgaagcagg
tgaaaatcca ggggctcaca agaaaagggc tggcaaactc 3360 tgccctatgt
cagagtcgtc ctgctattgg tctaggggat cagctagcct tgccagtgta 3420
gggtgacagg ctctctgata agagaagcaa gtggttctct agggctctgt gttgccttga
3480 gggaggagga aggtgggctt tgaagtctca gtacaggatg ggatggacat
tccaggtgga 3540 aggcccagcc tatgccaagg ggctgtaggt gggcagagtg
gtgggtgggg agctgatatc 3600 tgctgtgaac ttcctcgggg ctattgcagg
agagcttcag gttcaggctg gtgagtagga 3660 ggagcatagc agttggactg
cctgggtatt gaactgattt ggctacacaa gactattttg 3720 catcctggga
gtgtttctct acagaaatcc tcagccttgt aaaatgggaa attccctcct 3780
atgaatttat gcaataggac ttttttccct agtgacttgt aatcacattg tttcaatgac
3840 gtgaattcct acataaatag gttttgtttc tgtgataact cttactgata
catcattttc 3900 ttttactacg ctgactttgt aatagataga aagtccttat
atacctttgt tgcctttctt 3960 tttaaaacat ctcttacctg tgtctattca
tttactcatc caaattgcct ttatcctgat 4020 tttgtcccag acttgaaatg
aagttgcaat aggcttatat gttagtttgg gaagagttgg 4080 cctttaacgt
taaaaacagt tccatggtgt ttactgtagg ccaagccctg ctcaaggcct 4140
gttcttcttt tagtccttag aataagccta atgagataca ttagaaagct gaggcacatt
4200 tattccaggt aaccagacta gcaggaggag cactgggatc cccatctctg
ctttgacttc 4260 tagccctgct gccacctgga ctgtacagca ttgagttttt
ctgtcctggg atttgagggc 4320 ctgtccttag gggaagtcaa ggtgctcttc
ttcccttggc cccatcaggg cctgtttaga 4380 ctgttctcag ggctcgtggt
aaggcaatga catagagttg gtcaggagat gggtcagccc 4440 cactttgcct
ctgtagcctg acctgtgaca ggattggaat caggtttggt catgtgcaca 4500
gtgtcaggca tgcagtggtg cttggtcagt ggggattact gtgttgtttg ttcttgctgc
4560 tttggctctg ggcttagctg gctgggaccc ttcctgtggg ctggctgtga
gttggagttt 4620 ttttgtattt tttttttttt tttgagacag cgttcgctct
tgttccccag gctggagtgc 4680 aatggcacaa ttttggctcg ttgcagcctc
tgcctcctgg gtgcaagtga ttctcctgcc 4740 tcagcctcct gtagggtcca
gccccacagg gtcggtaggt ttttctccct gtgtgcggag 4800 atgagagatt
gtagaaataa agacacaaga cagagagatg aaagaaaaga cagctgggcc 4860
ccgggggacc actaccacca agacgtggaa accggtagtg gccctgaatg ccaggctgcg
4920 ctgatattta ttggatacaa gacaaagggg cagggtaagg agtgtgagcc
atctccaatg 4980 ataggtaagg tcacatgggt cacgtgtcca ctggacagtg
ggcccttccc tgcctggcag 5040 ccgaggcaga gagtgggaga gagagagaga
gagacagctt atgccattat ttctgcatat 5100 cagagacttt tagtactttc
actaattttg ctactgttat ctaaaaggca gagccaggtg 5160 tacagggtgg
aacatgaaag tggactagga gcgtgaccac tgaagcacag catcacaggg 5220
agatggttag gcctccggat aactgcgggt gggcctgact gatgtcaggc cgtcccacaa
5280 gaggtggagg agtagagtct tctctaaact cccccgggga aagggagatt
ccctttcccg 5340 gtatgctaag tagcgggtgt ttttccttga cactgacgct
accgctagac cacggttggg 5400 tccgcttggc aacgggcctc ttcccagatg
ctggcgttac cgctagacca aggagccctc 5460 tagtggcctt gtccgggctt
aacagaaggc tctcactctt gtcttctggt cacttctcac 5520 tatgtctctt
cagctcctat ctctgtatgg cctggttttt cctaggttat gattgtagag 5580
cgaggattat tataatattg gaataaagag taattgctac aaactaatga ttaatgatat
5640 tcatatataa tcatatgtat gatctagatc tagtataact cttgttgttt
tatatatttt 5700 attatactgg aacagctcgt gccctcggtc tcttgccttg
gcaccaaggt ggcttgccac 5760 ccacagcctc tcgagtagct gggattacag
ccatgtgcca ccatgcctgg ctaatttttg 5820 tatttttggt agagacaggt
tttcaccttg ttggtcaggc tggtctcgaa ctcctgacct 5880 cgtgatcccc
caccccccac ccccagcctc ccaaagtgct gggattacag gcgtgagcca 5940
ctgcacctgg ctgagttgga gcttttcttc cctctttttg gactttggaa aatgctcttg
6000 gtccatgatg ctatgtagac agctcccgtt gactgtggcc tgtgcggcat
tgggcagcac 6060 tctggtgaac actgaatcgg gtctgacctc ctagccccac
catttactgg ctgagcctca 6120 gtttccttgc ctgtaaaatc aggaagatgc
tggctctgct cctctctgca catttccccg 6180 tcctaacaac attataactg
ttaggaaaga gacgggcttg ttttgggatg gctcatttta 6240 tgtgaccctg
tgcgctgtct ctgagtccat ctgcccttct tccagggtgt agggaccagc 6300
cccacagggt cggtgggtct ctccctgtgt gcggcgatga gagagtgtag aaataaagac
6360 acaagacaaa gagataaaag acagctgggc ccgggggacc actgccacca
atgcatggag 6420 accagtagtg gccccgaatg tctggctgtg ctgttattta
ttggatacaa agcaaaaggg 6480 gcagggtaaa gagtgtgagt catctccagt
gataggtaag gtcacatggg tcacgtgtcc 6540 actgggacag ggggcccttc
cctgcctggc agccgaggca gagagaggag acacagagaa 6600 agaaaactta
tgccattatt tctgcatatc agagactttt agtactttca ctaattgact 6660
actgctatct agaaggcaga gccaggtgta caggatggaa catgaaggcg gactaggagc
6720 gtgaccactg aagcacagca tcacagggag acaggcctcc ggataactgc
gggcaggtct 6780 gactaatgtg aggccctcca caagaggtgg aggagcagag
tcttctctaa attcccccgg 6840 ggaaagggag cctccctttc ccggtctgct
aagtagcggg tgttgttcct tgacactttt 6900 cgctaccgct agaccaccgt
ccgctcggca acgggcgtct tcccagacgc tggcgttacc 6960 actagaccaa
ggagcccttt tgctggcccc gtccgggcat aacagaaggc tcgcactcct 7020
gtcttctggt cacacctcac tatgtcccct cagctcctat ctctgtatgg cctggttttt
7080 cctaggttat gattgtagag cgaggattat tataatattg ggataaagag
taattactac 7140 aaactaatga ttaatgatat tcatatatct ctaagatcta
tatctggtat aactattctt 7200 gttttatatt ttattatact ggaacagctc
gtgtcctcgg tctcttgcct tggcgcctgg 7260 gtggcttgcc gcccacacag
ggcatgtctg gatggtttga acactagggc ttctgatgct 7320 ctaagccaga
gtcaggtatt cattccatgg cacatgtggc tggggtctgc cctgagacct 7380
gtcccgtgcc aggctctggg ggcacatggc tgatggaacc aagcatgggg agtgaaggtg
7440 gagggtggcc tgtgagcacc atgcctgaga ggaccaggct ggggacggaa
ggttcttagt 7500 ggataatatt tattgtctct gcctcccccc tgacatttgc
aaagcggcat atgcttgtaa 7560 aaaaattttg aaacagaaaa atataaataa
ataagtaggt attaccacat gcaagggtga 7620 ccaattttgt atttttcttc
ccagcagatg ttaaagcaag accaacagtc tcccctcatg 7680 gaaggcccac
tgatctaaaa tgctggttcc ttttggacct tcagggcact tgggggagac 7740
cttcctgagg tgctgtgcag tgtctggtgt ttctcagacc caggtggtca tgggagccag
7800 gcgtggctga gtgggctcta caggccctag gcagggagca tcgcctgtgc
tgtggctgac 7860 gttccttctg gccctgttcc caaagttccc catgggggcc
tgggaggaat ggcctttcca 7920 gggggtgttt ttatgagaag gaggtagctc
cctgttggag tgaggtgctc aggaggaaag 7980 gggcctggtc ttagcagtca
tgaccacctg tccccagtga ggaacatctc tcctgccaca 8040 caggcctcct
gatcacttgc cacgtggcac gcatccctct gccagccgga tacagagaag 8100
agattgtgcg gtacctgcgg tcagtgcagc tccctgacgg tggctggggc ctgtgagtgt
8160 gcctgcccct gtgtcactgc acatgtgcat gtgtgtgttc tcatgatgta
ggagatgctt 8220 gggtttccag gcagctgcca ggggttagga gtgattgcag
ctgtgggtgt ggggtgggtg 8280 agggagagac tagcaggcgg ggagtgggct
gaaggccatg caggtggggc ctcggcttca 8340 catcttttgt taaatggatt
ttgtggctgt tacgacactc ttgagaccca catgtgaaaa 8400 ctgtcagtct
gttatcactt aagacagaag aaaattgccc ttgactctgg gctggcagca 8460
ggtggagaca aggcctgaca gctttcctgc catgtggcac acactttggg agcagagcca
8520 tagcccaaag tggaccgccc ttgagctaga agtgttgact caggcgtggg
aaggtgtaga 8580 gcaggcgggt cacggtgagg aaggagtggg gggctcagtt
gtcatgggag gtgcatgaat 8640 tcgtactgca gagtggctgc tcaggggtct
cctgtgttga catgttatgt caggttaagc 8700 cattttagca ttcttagttt
tctgaggaaa ctccacagaa agttttgctt tatttcttag 8760 aagtaaggac
agataccggt ttctcacctg tcctctgctc ctgtaggcac attgaggata 8820
agtccaccgt gtttgggact gcgctcaact atgtgtctct cagaattctg ggtgttgggc
8880 ctgacgatcc tgacctggta cgagcccgga acattcttca caagaaaggt
acggcatgtg 8940 cagcatgtgc tgggccaggg gttcgtgtca actcgataat
gagctctcac aaacgagata 9000 cagaaagatg cacttgcagc tgaaacagtg
ggcaaaagca catgagcagg gaatttgtca 9060 aagcagaagt aggcagacac
tgtttaacct aggcatcatt ttttaaaaaa gcaaattaag 9120 agccaggcac
agtgagtggc tcacgcctgc aattccagca ctttgggaga ctgaggtaga 9180
aggaccactt caacctaaga gttcgaggcc agcctgggca acatagtgag acctggtctc
9240 tacaaaaaca ataaaatatt agccaggtgt gatgatatgc acctgtagtc
tcagctactt 9300 ggaggctagt aaggcaggag gatcacttga gcccaggagt
tctgggttgc aatgagctgg 9360 ttgtactact gcactctagc ctgggtgaca
gagtgcgacc ctgtctctaa taaaataaaa 9420 aagccaagca aactaagaca
accaggtaat tctgtttgtt tcctgaattg gcaaaaactt 9480 aaacgaaccg
tgttaatatg tccaccttct ggggggcagc ctggctgcag gcaagagcag 9540
ccctggagct tgcaccttcc aagctgatcg tctacctctc caagcccggg gctgtccacc
9600 tctccaagcc cggggctgtc cacctctcca agcccggggc tgtccacctc
tccaagcccc 9660 gggctgtcca cctctccaag ccccgggttg tcttacctct
ccaagcccca actgtctact 9720 tctccaagcc ctggtctggc tacctctcca
agccctgggc tgtccacctc tccaagcgcc 9780 aactatcttt ctctccaagc
cctggcctgg ctacctctcc aagccccagg ctgtccacct 9840 ctccaagccc
caactgtcta cctctccaag ccccggcctg gctacctctc caagcccctg 9900
gctacctctc catgcccggc ctggctacct ctcctcttgc ctataggccc tgaggggcaa
9960 ttccagccca agggaatcca tggctcctgc tgctccaaga aaacctagtt
tatgttgtgg 10020 ctctgcagag cctggcctgg tcttgtcctc tgtgtttcac
agaccttccg tagccagtcc 10080 cacctgccct gctctctgct gcatgcgcag
gggcctcctg tcagctcctc agagaccctt 10140 attatcccag ggctcgccat
gcactgcctc cttcgcctgg agcctcttac cttccactcc 10200 tgccccgctg
gctcacactt tacgtgttcc ttctttgagg acctcttcct gacctaccgt 10260
gccaggtgga gtgtcctgtt acgcattctc atgagatcct gccttctttc ttggtgagct
10320 tgtcactatt gtcctcagtt cactgtcagc ctttggtgtc gttgatgctg
cgtccccaag 10380 gctgctgtcc ggttcccacc acactcctgg cgcctgcctg
gtgaaggaac gtgtttaggc 10440 tgcactttgc ctagtagctt tgtgggtctt
tattgacttt tgcatacctt ttggggtttg 10500 gagcagggac tcctcagaag
catgtttaga tggtgtggct gtgccaggac tgctgctgct 10560 gaagtggctc
tggcatgggg ccagcgtgct ggagctactc tggagtctag ggtcgtcttt 10620
gttcccatac aggaccagtc tgccaagtgg agatgacaca gactggggca gctcaggctt
10680 ggctcagagg gcgaggctga gtgtgcgctg tcacttcccc accttgcctt
ctccaggcgc 10740 atgtgcacct gggcccctcg ctcacctgag cactgaggtg
tccctggacc ttcccaggta 10800 gctgtcttca tgtgctcctt cctggggcca
ggggttgcaa acacctctcc tggggctgga 10860 cacacacact cccaggaaag
ccactggttc cacctagggg gccgtgtatc caggcaagtt 10920 ctcagcactc
tggaacctgc ttcgcacatg ggggtcgcaa gatccacatg aggctgccct 10980
tgcctcatgg agaggggcac acgtgactcc cagagggtga agcttcccag ctagaggcag
11040 tgcagacttt gctgacagga agcagatgac gtgggcctat tctctccccg
ctcaggtggt 11100 gctgtggcca tcccctcctg ggggaagttc tggctggctg
tcctgaatgt ttacagctgg 11160 gaaggcctca ataccctgtt cccagagatg
tggtatgtct gctgttgatt gggttgttgg 11220 gtcgctgctg ctgtcccggg
gagtagagtg acagggaccg tgggtcaggt gcaggctgtg 11280 acagcagaga
ggggtgggca ttctgtgggt gggtggagtt aggctcctgg cagaggccct 11340
gatcaagctt gagtcctgta ggggtacaga aagggggagg ttcccaattg agcaggaaga
11400 aggctgtgcc atggatggag gtaccccgag tcaggctgca ggcagggctg
ggtggcttcc 11460 ctcttgctgt ggaagactca gcatctgtag aagtgggggg
gtgcccctcc cccagcctgc 11520 acaggggcgt cctgtgttgc tgctgctgcg
tttgtctcct ttgctggtga atgtgaagtg 11580 tgtcccgacg tgacacctca
cctgtggact cagcgtgtgt gcctttaaaa gatcagtgtc 11640 tgtggccagg
tggggtggct catgcctgta atcccagcac ttcgggaggc cgaggcgggc 11700
agatcacgag gtcaagggat cgagaccatc ctggccaaca tagtgaaatc ccgtctctac
11760 taaaaataca aaaattagct gggcgtggcg gcgcgtgcct ctagtttcca
gctactcggg 11820 aggctgaggc aggagaatca cttgaccctg ggaggcagag
gttaccgtga gccgagatcg 11880 tgccaccata ttccagcctg gcgacggagt
gagactctgt ctcaaaaaaa aaaaaaaaaa 11940 gatcagtgtt tgttttttta
aacagaacca catactgttt aaatacccag caaaatcaac 12000 attaatttct
tattatctgg tgtgtgtttt ttttgttttg ttttgagacg gagtctagct 12060
ttgtcactca ggctggagtg cagtggcgtg atttggggtc actgcaacct ccgcctcccg
12120 gattcaagca attctcctgc ctctgcctcc cgagtagctg ggattacagt
ctcaggccat 12180 cacgcccagc taattgttgt atttttagta gagacagggt
ttcactatgt tggccaggat 12240 ggtctcaaac tcctgacctc aggtgatccg
cctgccttgg cctcccaaaa gtgctgggag 12300 ccatgagcca ctgctcccgg
ccttatgtgg tgtctttaac cagtgtcttg taacatttta 12360 tggctatcta
ttgaaagcag tggacatctc cccagaaaac actcgtgcat atgagtttac 12420
cccgttatgc attttgggaa gtgagaccct ggaaccacac agagcccctg ctggcttcct
12480 tgagtgttgt gggaaccctg gtgggggtgt cccctacaga gctatcatca
gggctggggg 12540 ggtcccttgt gttagatgac tttggtgcgg gggtgggggg
tggggggtca agttagggga 12600 ggcaggaagt gaaggggccg ctcaagaaag
gacagcagca gtgtcctgat gcaaaggccg 12660 ggggcttaac cccggaagcc
agtttgggtg gtgacgggga ggcacaggga tggtgagatc 12720 accccgggag
ggtagacaga gataccagag tagggggcag ggttagggtg ccgctacctg 12780
aggcgggccg tagagcacat aggttgggag gtgtcctggg gccattcaaa tgcccgctgg
12840 actctgcgcc tcgcccgtgt gtaatgagcg gcagaggaag gactgagacg
gcagtcagca 12900 cagctgccag ggcaggaggg gtgtgggttc cacacgctgg
tgctggtgag ggcgtctcat 12960 ctgccccact tgggggggcc gtcggtcagt
gctgccgcat gggcacgcca gggtgctgct 13020 tgtctttgct ggagttgctt
ggagggtggg ttgggaggtg aaaggaggac cacagacctg 13080 aaccactcca
gctgcgaaat gctggaagtg taacccaaaa tgtgagaaaa aaaaacaccc 13140
ttttaagtaa gtgggtgtga aagtgggcca aggcctgatg ccacagtcag ggagcaggga
13200 aggctcagca ttgctcaccc tcacttaagg atggggctag catcacataa
ggcatcacat 13260 aaggatgggg gctagcaggg aaagggagag aaaacacatg
aggcacacac agaccctggg 13320 aagctggtgg agctgtgcta acgtcagcag
accagtgatc aaagacccag gccttgggga 13380 gattccacag acctacagac
ctacagtttc ttttttcttt ctttttcttt tttttttttt 13440 ttttgagaca
gagtctctcg ctctgtcacc aggctgtgtg cagtggcaca atctcggctc 13500
actgcaacct cctcccaggt tcaagcgatt ctcctgcctc agcctcccga gtagctggga
13560 ctagaggcac acaccaccat gcctggctta tttttgtatt tttagtagag
atggggtttc 13620 gccatgttgg tcaggctggt ctcaaactcc tgacctcaag
tgatccacca gcctcggcct 13680 cccaaagtgc tagggttaca ggcgtgagcc
accgtgcccc tcctaaagtt tcttaaatac 13740 acttttaaaa gtaaacttta
aattttggag tagtttccaa tttctggaaa agttgcaaag 13800 atagccaaga
gtgttccctg gggccctcac accatatccc cattgttgat gttttatgtt 13860
accaaggtac gtttgttgta gctaagaaac ccacggacaa tcctaagcat ttaggagctc
13920 catcacctgg ttttaggatg caaaatgctg accgaactag gaggtgcagc
tcctcagagg 13980 gtgcacctat ggttcaactg tgcccctcag gagcacggtt
gggaaatgcc cgcagatgca 14040 ctgacgtggt ggggaatagc catccaccag
tgttcgtgct tgaaagggcc caaggtatgg 14100 atgctggcgg agggggcagg
cttgagtctt ggggtctccc actgactcct gctgttgccc 14160 caggctgttt
cctgactggg caccggcaca cccctccaca ctctggtgcc actgccggca 14220
ggtgtacctg cccatgagct actgctacgc cgttcggctg agtgccgcgg aagacccgct
14280 ggtccagagc ctccgccagg taggacctca tcagggaaca aagtgaaggc
ctctggggct 14340 gggacccaca gggcctgggg cttctggaat ctaaccacac
ctgtccactc acctggtggc 14400 cctgtggagc ggagagccct gtggagcaga
gcctccacct tcctccatcc tataataaac 14460 agtgagcaag ctctgcccag
aggggacttg tgctatggga cagtcagtag ctgtagccca 14520 gggttcctgg
gggggacttc caggactcaa gggatgcagg aggcagatgt gcactgtgtc 14580
ctctggaagc aggcctgagg cgaggtttga ggtgcaggat gtttatcagg cctgccatgg
14640 ggaagaagga ggggcagagg gaggaaatga gcttctgggc agacctggga
ctcatggagc 14700 tggggagctc ctcagagcgg tcctcccata gggggccttc
atgtgccctc ggggtcagtt 14760 gctggaggga cccccaccca ggaagggact
ggcccagggc cctgagggcg gatggtggga 14820 ggccacccct cctggtttga
gccaggccta ccaggtgctc ccaggcccca aggctcagac 14880 actgccccta
ccaggagctc tatgtggagg acttcgccag cattgactgg ctggcgcaga 14940
ggaacaacgt ggcccccgac gagctgtaca cgccgcacag ctggctgctc cgcgtggtat
15000 atggtgagcg cctcctgagg ggccggcagg gcagcccagg gtcagggtca
gggtgtcgcc 15060 cactcattca cgcactcatc ccctgccagc ggcactgggc
cacctcctct gtgccaggcc 15120 ccagggggcg ggatctcatc gccctgcccc
tccaccctga gaaccagctg gtcttctact 15180 ctcaggagtc caccctgtgc
aagggtgtgt ggtaggaggt gtggggcagc ccctcctggg 15240 cagggaagga
ggagctcaga gaccaggcct gggggtgggt gggaggggga aaccctgggg 15300
aagggcaagt ccaggcgttg cagtgcatgg agctccaggc tgaggccagt gtcatggtgt
15360 ctggcatcca ctgacccctg tcccctgtag cgctcctcaa cctgtatgag
caccaccaca 15420 gtgcccacct gcggcagcgg gccgtgcaga agctgtatga
acacattgtg gccgacgacc 15480 gattcaccaa gagcatcagc atcggcccgg
tcagtgcccc tgcccggcct ctgactgcag 15540 cccctggggg ttgaggtccg
aaagtgaagt cctagaggcc gggctgtgag ctgggagtgg 15600 ggtttcctgg
agcctggtgt acctccattt gggaggtggc cctctgatcg cacaagtgtc 15660
tgagggcttc tgtcctggac ccctgcacgc ccagctcagt gaagttgccc caccacactc
15720 gaccccccgc ttccgtcccc caccggctct tgtcctcagt gtgcctggac
actctcctag 15780 aggcccctcc ctgagatctt gctggctagc tggctagctg
ggaggggtgc tttttcctca 15840 cttggttccc tctccccaaa cagttcatca
ttcgccattc tcccgtgggg tttagacatg 15900 cccagggtgg gtgggagtag
caggtgccac tcctgattcc tcctgcctag ctagggactt 15960 ggagctctca
cctctgtggg gcctgcaggg gtccaggtgt ggccagttca gtgaccttag 16020
agggtgcaat ccccgggctg tgctggtgcg tggccgcctc ctgacagagt cagcaggccc
16080 tgggctgtgc tgcagctgct gccgtagctg tgcgcgtagc tgctgcggtg
tagtgggttg 16140 gcttaggcat tctctggaca tacccaggtg gcactgggcc
actgagtccc accctgacac 16200 tgcatctcgg attttctggg cctcatgcca
cctcagtgga tcacaaatcc tgactgaccc 16260 tgcagcgggt cccttgtttt
ttgctcagca gtgatgtggt tctttgtggg ttttggttta 16320 atcccatata
gagcacatct gtactaaacg cattagaaac atgcttgcaa ttggatcttg 16380
acttgtgaga tgcataagta aaaagttggg ggcctctgga acattctgtt ctgaggaaga
16440 aggggggcaa gtggtcccta ctgctacagt cctgtcttcg catctcttcc
tgggcccctc 16500 aggccctgtc ctctgtcccc tgtgttgtct ctaaggcacc
tggtagccca tgcccctctg 16560 gtttctcctg gaacccctcg cttctccctg
gtggagtgct gctccttctc acagcctaag 16620 gcaggctgtg gccttggccg
acactgcctc tgtctgagtt gggtcctggg gacacagttg 16680 ttgcccatcc
tcgctcagga aatgcctgtt agagcagaag gcccctgtcc tggccctgag 16740
tgatctgcac ggcactttat gcctgggggc tgctgtggat ctggacgaga ccttgtccct
16800 ggaggctgct gtgggtctgg agcggagcct tgacagggct gtctctcctg
cagatctcga 16860 aaaccatcaa catgcttgtg cgctggtatg tggacgggcc
cgcctccact gccttccagg 16920 agcatgtctc cagaatcccg gactatctct
ggtgagtgtg gctgggatat gctggcgggg 16980 cctctcacga agactggatc
tgagccccag ctgcatccca gtgagggggc ccccacggtg 17040 ccatctggga
atactgccag ggaatacctc caggaaccag cagtgtcagg gcttgtggaa 17100
gccactgagg gttgtctttg aattggaaga tttgccaccc agtggaagtg tggggtgttc
17160 ccagaaggta gagtgaggaa gggggtggta ggtagcaggg caggttcagg
ttggcatcag 17220 gaggcctgtg gacaagggga gcttgtcagc catggactgt
gccctggagg tggggcccct 17280 gtcatggagg gcagagagcc gtcccatggt
gggaagcttc cgctgtacag gcctcttcct 17340 ctggtgcctc agcactgcac
gagggcggca gggctggcac agcctggggt cggggagcct 17400 cccgctgccc
cttgccttgg gtgtggccct tctgggtgag tgtgtcctgt tttccataga 17460
gtgtggccct cacccccagg agcccagcag cccagctggg gtggcatcca ggccagtgcc
17520 aggcctcggg aggggacaga cggcctctct gggaccctcc tgagtgcagg
gtctgggtag 17580 cagctgggct tccagctttc tccttgcacc tgacttgggc
ttttttctcc tcacaggatg 17640 ggccttgacg gcatgaaaat gcaggtaagg
gctgcgggac tgcggctgca tgcttccttt 17700 gcaatcatgt ctccccttta
ttatttttcc tttggggttc agaaataact cctcctggac 17760 caggtcccgg
cagcgtgcga ctagaggctg agtcagttga ggcctctggc cgtgtccctg 17820
tgggtgctgt tggtctctgt gtgggtgccc accgttctcg atgtctgtct gcagctgtcc
17880 tgtttgcttt ttgccctgat gatctgagtg ggctcagctg tgtaacgaca
gacccagagc 17940 tgcagaagct ctcatcttgt tactgtggca ggaggtggct
ctggttagtg ggggcttctc 18000 ctccatgcac tcttaattta aggggcttct
tcttaaaggt cctgggtgga caggacagga 18060 gcctggagga ccgtggtggc
gtgtggccgg gcctgggagc tccccgtgga cttggcctga 18120 gtgggctgga
acccagtcat gaggggcacc aagcacaagg agaggggagg ccgggtggat 18180
cctggctgac cctggtcctg tcctggctct gggggccctg tagaccgcag tcctgtccga
18240 ctgggctgag cctgcgcccc tctgtgcgtg tcagaagccc agacagtgtt
gccctgtgtc 18300 ttgtggtcta aggagggtta cgccctgcgg tgcctgtctt
ctgtccccca cctgattcag 18360 tgtggaaatg tggagtctcc agaggtgtcc
tgggtgtcac atttgggatg gatacacgtg 18420 ggcccagcac tgcccgcccc
agggctaccc ttggtgccag gtgcccccag ccacgagctt 18480 ttacccagct
ggccttgagc tccccagagg ctccccggac actgtccgtg ttttgtgaaa 18540
aggttttcaa aacacatgta aagtggaggt gagtagcaag ccctagagca ggccctggcc
18600 tccctgcccc tccctgtccc ctccctgccc ctccctgccc agcgctccct
cagcaccgac 18660 tcatcagtgc acctcaagct gatgagggcg tctgtgtttt
gacaaaattg ctctgaggtt 18720 gtcacaccca acaaacttat gacggttcct
gagtgtagtc ctcacgttgt ggctggtgtt 18780 tgtgaatcag gattcaggcc
aggcctgcac aggccttcag ttgttggtct ttgagctcct 18840 gttagtccag
ccgtctctcg tggtctcttt tctcctcctg gaaggtttgt tcctgaaggg 18900
cttcacattg cagatctgac tggttgcttc ttatgttccc tgagtttttg taaactggcc
18960 aggccctgag gctcgatccc attgtgtttc tttggcgaga atgcttttct
ggtggtccct 19020 gccttgtccc tccagtgcac gatgtctgga tgcctctgcc
acacaccacc ccctgcccag 19080 tccccatgtc tgtctggtca gtgcccagct
ctgtctcact agggtttggt caccggccct 19140 ttgaactgag accaggctgt
gtacctgtga gcccagctcg gggtgagatt tgaggtggag 19200 ccttcccagc
cctgtgcaga attcccatca cctccaggtg tactcagaaa tggggatcat 19260
tggccaggtg cggtggctca cgcctgtaat ccctacactt tgggaggcca aggtgggcgg
19320 atcacaaggt caggagatag agaccatcct ggctaacacg gtgaaacccc
gatgctacta 19380 aaaaatacaa aaaaaattag ctggatgtgc tggcaggagc
ctgtaatccc agctactccg 19440 gaggctgagg caggagaatg gcgtgaaccc
aggaggcgga gcttgcagcg agctgagatc 19500 acgccactgc actccagcct
gggcaacaga gcgagacttc atctcaaaaa aaaaagaaat 19560 ggggtcattt
ccaggcatca ccatgactga ggtgcgccac tgtcattggg tgagagcagc 19620
tggatgctct atgtgtaggt gctggagcct ctgagggatc gtccagtcct agaagtgtcc
19680 tcagagggac actgtcctgc ctggtggccc atgaagaaag ggagggctcc
ctgagtctcc 19740 ctgacgtgtg tctgcctgca gggctcagcc ttctctgagg
cccttgtcag ccatgagggg 19800 tgcccagggc tcagagcctg aggctgagcg
ttggctgggt gggagccccc acacctggcc 19860 ctcaggcgcc cattggatcc
tggaggcagt ggctgggagt gggaggggct gcatctgctg 19920 ctgtaacacc
atcctttgtg tgtagggcac caacggctca cagatctggg acaccgcatt 19980
cgccatccag gctctgcttg aggttcgtgg ctccttctct tttctcagcc tcagctgacc
20040 ttcctgtgca cgtaagccca cgcatccacc tgagggcagc actgctggcc
acacacttgc 20100 cactcctcga tacttccagt gacctgggct ctggcctctg
gcttcagagg gtcgtgctgt 20160 ggagggggcg gccttggcca gcagccttgg
gtgttgggct gggtcggggg ccttgggagg 20220 gcaggggctg gaggctgtgt
gagaagggga gtctggtgaa ggctgtttct gagagtgcag 20280 gcaggagtgg
gactccaggc tcttcttaga actggaactg cttgggccag gcacggtggc 20340
tcacacctgt aatcccagca ctttgggagg ccgaggaggg tggatcacga ggtcaggagt
20400 tcaagaccag cctggccaag atggtgaaac cccgtctcta ctaaaagtac
acaaaaatta 20460 gccaagcgtg gtggcgggca cctgtaatcc cagctacttg
ggaggctgag gcagagaatt 20520 gcttgaaccc gggaagtgga gggtgcagcg
agccgagatt gtgccactgc actccagcct 20580 gggtgacaga gagaggctcc
gtctcaaaaa aaaaaaaaaa aaaaaagaac tggaactgtt 20640 tgttatgggc
attctcgagc cagtactgga gaaaaacgag agtggatttt tatgccggtg 20700
ggaatgaggt aggtgggatt ctgaaggtgt ttctggagag ccctgagggc tgggccacgc
20760 aaagggcctg cctacacagg gtgctggaga ccctctgggc atggatgctg
gccaggcagg 20820 ggggtgctgg catccataaa tggtctcctg cgcccttcca
tcttcagtca tatctcatgg 20880 acttttgctg ttttgtcttt aaaggtaagt
gcagcaggag accctggcac tctctggaga 20940 tgtctgctgg tttgattctg
gtccccggtt ggggcaggat gtggccagga ccatcgggaa 21000 accagcgcag
ccatgctggc cgtgcaaggg cagctgagcc tctctgtcct gctgtctctt 21060
ccaggcgggc gggcaccaca ggcccgagtt ttcgtcctgc ctgcagaagg ctcatgagtt
21120 cctgaggctc tcacaggtga ggccggtgcc tggggctctg agggggctga
agagggggat 21180 cagggctggg agctcctgca ggcagaagtg cccacctcac
ctccaccctg ccctatttcc 21240 tgcactggtg tttcagggtc acccccaccc
tcccatcccc tccctagccc ctgctccatc 21300 caccggtcct cctcgggctg
gcctcacctg gggcagttct ctgaggcctg cagggtgctg 21360
ggggtgctgg cagtttctgc gtcctgctca tgttggagcc actgtgtgca agggccaggc
21420 acgggcaggg gctgtgtacc ctgagctgca cagcctacac ggcacctcca
tgtctctgaa 21480 gcaccttctg cccatggagg tgacgccagc ctgtggactt
gccctcctga gactgtttgc 21540 agcaaaagcc ccggtccctc ctgccagatc
agctgcccac agaccctgcc cgagcccata 21600 gtttgacctc agtgtctctc
acacgtgcct gcaccccagt ctgcagccac agtcatccca 21660 tacatgcgcc
ccaacctccc gtgtctccca caccctgtcc cggccacggc ctcagccagt 21720
gtccctctgc ctggaaccgc tgccccccag ccccgtctcc ctcccttcag ctctcactag
21780 gacattgttc tgcagggctt ctgggtcttc ctggcctctg tgtggccaag
gctggcaccc 21840 atcttgggct caagcagagg aggggcattg tcctgctgtg
cctggcccaa tggcggcctg 21900 ctcctgctcc tgcctcctgc ccaggacttg
ctctgggtga tggggacttg gggaggctga 21960 ctgaacccta cggcactcca
ggcctcttcc cttctcactg aggtgagaga ggcagccaga 22020 agctgaggtt
gttcaggagg cattgggggc gcctggcaca gagcacaccc gcagagacct 22080
gggccccctc cctgccttct ggccggtggg gagatcacag gggagtcagg tgctgactcc
22140 cagtcccgtc tgggctggtt tgagccctcg ctggccagtc acgtttccca
gcagctgtgg 22200 gtggtgagct aaacaggtgc aggccctcgc gcgcctcgca
gcaccagtgg tggctgtggc 22260 cggcagagta agctcccagg cacgttctgc
ctctccagtc ctgcccagtc tgtctcagcg 22320 atgtcccaga tggggacgtc
ccgtggtgac gtgttctctg cttccacatt tgccctcgat 22380 gctgcccagg
tcccagataa ccctcccgac taccagaagt actaccgcca gatgcgcaag 22440
gtatgcggga gccagcccca tccctgtccc gtcccccagg ggaggccgcc ctcagcaggg
22500 tgggtccttc cctctgaagg gggggctcct ccctggggga ctcctccctt
ggcgtttttg 22560 ggtgtcctgc tgtggtggat gcctggccta ggggctcatg
cttcatgttg ctgagctgcc 22620 tggcacatgg aggcacagtt ggcttgcaca
cacagccgtg cctcagagca gttccagtgg 22680 tcacggcaca cacaggcttc
agaaggacag ccgaagtgta gccagtgtgt ccggggaagg 22740 cagaggaaag
aagtagacct cagagccggt gtgggctgtg accacaggtg cagactgtga 22800
aattaggcat ggacccagct gctgctgcct gtttacaatg ggggtggggg gcacctgggc
22860 cccatcctgt ccgtcgtgag atctgcaggt gttgagggtg tgagctgcac
ccctgagggt 22920 ccctgtgctg gaagctggag gtctgtctgg atgtacccag
cttggggccc tggctgcacc 22980 cacacctttg gtggctgggc ccctgccctg
accgggtgct ctgtggtggg gagggatgcg 23040 tgcggctgtg gggaggttct
gagaactggg gtgtggacac ccccagcctg gagtcatggc 23100 ttgtgctctg
cagggtggct tctccttcag tacgctggac tgcggctgga tcgtttctga 23160
ctgcacggct gaggccttga aggctgtgct gctcctgcag gagaagtgtc cccatgtcac
23220 cgagcacatc cccagagaac ggctctgcga tgctgtggct gtggtaaggc
tgtggtccca 23280 gcagccccgt ccatacctcg tgtcctgcag atgagctgcg
tgctcacttc cactcctgtg 23340 ggctccagcc cagcacacag tccggccagg
ccgtaggagc ttgtccttgg atggtgtcta 23400 tatgtggaga actgtgagct
ctggctggac ccctaggggc cttgctgggc tgtgtgcaca 23460 gggccctgca
ctgcggagct ggtgtccagc ccagccaccg atacttgggg gagccggcgt 23520
ggcccccaag gtttctctct ggtggtttcc actgggtgtc tgaagaggga atttgttggt
23580 gttggttttg gtgccacatc ctttcagcac atctggcttt tgtgtgtgtt
tcccagtgga 23640 gaccctgccc ttttctggca gcacagactt ggtttctaag
tcatgggcac gtgtgggggc 23700 atgttccctg gtggctgtgc atggaggccc
tgacagatga ggttgcagct gctgcttggg 23760 gcacccgagg gcttggttaa
cgtggaaatc agctctccgc cccctgttcc tgccccatcg 23820 gttgtcagcc
ctagtgttgc ctctagagag ttccgctgtg ccctgggcgc ctgtgtgtgc 23880
tcagcacatg ggcgagttct agggtgctct ctgtgatttc agctgctgaa catgagaaat
23940 ccagatggag ggttcgccac ctatgagacc aagcgtgggg ggcacttgct
ggagctgctg 24000 aacccctcgg aggtcttcgg tgagtggtcg gccagcactg
cggcgcgcaa acccggggct 24060 ggctagcact gtggtacaca aacctggggg
ccagcttttc ccccttgccc gaggctgcaa 24120 gggcccaggt tcaccggcag
atctgtctgg agccctccct cagcccaggc tgttctgcgc 24180 tcctccatcc
cccggggtgg caggatcctt gtgttgtgga taggagggca tcaggtcaga 24240
cctaggggac agtggagggt tccagtgaga tccacagcct gggctggttc ctgctcagtc
24300 cacagggctt gtgttctgtg gaggctgctg tgtatccaga gcgcctgcag
ggaggtgtct 24360 ttggggactg tggggactgt ggggacccat gccatgggca
gtaggctgct gtgtgtgcat 24420 ggttgccacc gtactggtct tgggggagga
tctcagccct ggtccacctc tgggcacctc 24480 acatacccgc cttcctggtc
ccctccacat cacacatggc tttttggggt ggggtcgcag 24540 ctttctgctg
tgttcccctc atcttcgctc tcaggtagca caggtgtgtg tcctggacca 24600
gccggcgttt gctctggagg ttggtcaggg aggcagcgtc cgggcccggg ctcactgcaa
24660 cactcttgct tgttgtggct ttgcctgagc tgcagagcct gggcagccag
ggtgaaaccc 24720 aacacttggt tcttccctcc ctttcccagg ggacatcatg
attgactaca cctatgtgga 24780 gtgcacctca gccgtgatgc aggcgcttaa
gtatttccac aagcgtttcc cggagcacag 24840 ggcagcggag atccggtaag
gagggtctca gccattcagt gtgggcgctg ccaagtcggg 24900 ggccaagacc
cagacgcatc attctgtgac acggccctgg tggcccatct cagaagcgaa 24960
actcatggaa acatgcaaga ggcttcggat gttgtggaat ccagtcatat gccctaaagc
25020 atacaaaata tctgttaggg gctcagaata gcacagttat gatacaaaaa
tggattttct 25080 ctctctttta ataatgttaa gaagacatca catacctgac
tccaccggtg tcccagaaac 25140 ggtttttaag taacctttcc tgttgaaggg
tagcaagtat tcagaaaagt gtacaggttg 25200 gtcttcttga agcaaacagg
aagcgaacag tgccagcatt agacatggtg acaccaccag 25260 agccctcggc
ccgccccatg acggggccgc ccacatgcct gccaggtcgt gggtgtctgt 25320
tgctcgcttt ggatcttgtc taggtggact cctgaggtgt ggaattcgtg ttgccttctc
25380 ctgctctcct gctctcctgc tcgcggttag tcaggtggct cgggtaacag
cagcgttctc 25440 tccctcgggc cttcggttga acacagaatg ccgcgctatc
cagctgcctg ttctcagcac 25500 ctgggaggat ttcagtctgg gttattatga
agcatctact gtgaacactc ttgtacttat 25560 cttttggggg cacctgggta
cccatttctc atggtcacgt acctaggagt ggcattgctg 25620 tgttagaggg
tacgttatag ggtatgtgat ttttgtaggt tcttctttat cctatcacga 25680
ttacattttt ttacttttgt tcaacctggt gtagactcac cttggtcaca atgcactgtc
25740 ctttttatat attgctagat tcaatttgaa gaatattttg ttaggatttt
agcaactctg 25800 gttacaagag acgctggtct ataatttttt tttctttata
atgtttttgt caggttttcc 25860 tgttaagatg atgctggact tagaaaagca
gttggaaaat gcttttaaaa tactctttgg 25920 aagaatttat gtaatattca
taatatttct gccttaaatg tttgggaaaa attaccggaa 25980 atgccagttg
ggcctggaga tttctttgag gaaagttttt aaattagaag ttcaatttct 26040
ttctttcttt ctttctttct tttttttttg agatgggttc tagctctctc actcaggctg
26100 gagtgcggtg taatttcttt aatagtttat aggactgagc agattttcca
tttttgtatc 26160 agtctgggga gtcttcccat ttccactcag ctttacactg
attcatgcaa agttgttcag 26220 tgtcctctta gatggctctg agcccaacgc
tgacatcctc ctcttccttc tgagaatctt 26280 atactgatct tttgaaaaaa
aaaaaatctt agtctttgat tctgttttta aagagacttt 26340 atttttggtt
tcatcaattt ctattgtttg ttattttctt tctttcttaa tttttttgag 26400
atggagtctt gctctgttgc tgaggttggg gagcagtggc gtgatctcag ttcactgcaa
26460 cctccgtttc cggggttcaa gcgattctcc tgcctcagcc tcccgagtag
ctgggactac 26520 aggtgctgac caccatgact ggccaatttt ttggtatttt
tattagagac agggttttac 26580 catgttgtcc aagctggtct tgaactcctg
acctcaggtg atccaccttc cttggcctcc 26640 cagagtgctg ggattacagg
tgtgagccac cacactggcc tttgctattt tctttctcct 26700 ttatttttct
aacttgaata cttagatatt tgattttcag gcttttattg aaatatgaat 26760
ttgaggctat aaatgagttt tgagatatca ttcagttaaa tgtgtgttct ggtgcttgct
26820 gtggtagcac agatactaaa agtgttttct gtttctactg ttcttctctg
gcccatgagt 26880 tatgtgggag tatgctgctt catttacaat ctgagaatgt
tctggtgtgg tttttttgga 26940 agccgtggat ggagcagggg ttttcttgtg
cttcacaggt gcagctagga gggcactgtg 27000 tccagggtct tctgtcggcc
tggcgtggcc cttggccatg tgctgctctg cggcatgagg 27060 tgggcgtgag
ttgtcctcag ccacatttag agaattggcc ttttaaaaaa tagatcatct 27120
tttaaaaatc actgtaataa aagtaaagca ggttctttgc aaacaagact tgcaaaatac
27180 agagaagcgc aaagaagaag ctaagtcgcc cctcctcgcc cctgaaggag
aatctgctgt 27240 tgctgtttgg tctccacatt tccatggcgg cttgctgccc
ctttcacgcc tggcccactt 27300 tgtgcctggt gaggtttcta aaagccccac
ccttgagcgc gctcctccag cacgagcagt 27360 aatggcacag gtgttgtgtc
attttactca gtagcctctg ggttattttt cagttttcct 27420 tgttgttttt
tagcttttcc ccattttaac cttaactggt attttcttgt taaatattta 27480
ttcatgacca ttattattcc ctagagccac atggcttggg gtccacctgc ctgggtccgc
27540 ccccatccct gccccttctg gctgtctgac ctggcctggt gacttctctt
ctctgctcat 27600 ctctctccct gcctgagtgg gcaagagtac agcctcacag
agtggtggga ttgtgtgaga 27660 tgccacaggg aagcacatgt cagttgttgt
cactgtgtag aacaatgagt cccggatgtg 27720 gcccgcaggg gagcaatggt
gacttaatcg cgggcttcct ctgcatttct ttggtgactt 27780 ccaagctaga
acattctttt tttgtttatt tgtttgaagc agggtctcac tctgttacct 27840
aggctggagt gcagtagcaa aatcatggct caccacagtc tcaaacttcc gggctcaagc
27900 aatcctccca cctcagcctc ctgagtagct gggactacag gtgcatacca
tcacctgtgg 27960 ctaatttttt aaatgttttg tattttttaa atgttgctca
ggctggtctt gaactgctgg 28020 gctcaagcaa tcctcccacc tcggcctccc
caaatgctgg gattacagag tgagccacca 28080 cacccagcca tttttaaaat
tttcaccagg aagttttttc tttcattttt aagcacagta 28140 agtatttgtg
tattatgtta cagatatttt cccctcaatt tctttgttct ttttatctct 28200
ttagggagta tgaacataag tttttaactt ttaaatggtt aaatatatta gtgtgatttt
28260 tatattaaga ttttatttta tttatttttt ttttttttga gacggagtct
cgctctgtcg 28320 cccaggctgg agtgcagtgg cgggatctcg gctcactgca
agctccgcct cccgggttca 28380 cgccattctc ctgcctcagc ctcccaagta
gctgggacta caggcgcccg ccactacgcc 28440 cggctaattt tttgtatttt
tagtagagac ggggtttcac cgttttagcc aggatggtct 28500 cgatctcctg
acctcgtgat ccgcccgcct cggcctccca aagtgctggg attacaggcg 28560
tgagccaccg cgcccggcct atattaagat tttaaacttg ccgggcgcag tggctgacgc
28620 ctgtaatccc agcactttgg gaggccgagg cgggtggatc acaaggtcag
gagatcgaga 28680 ccatcctggc taacacggtg aaaccttgtc tactaaaaat
acaaaaatta gccgggcgtg 28740 gtggcgggcg cttgtagtcc cagctactcg
ggaggctgag gcaggagaat ggcgtgaacc 28800 cgggaggtgg agcttgcagt
gagccgagat ggtgccactg cactccagcc taggcgagag 28860 tgcaagacac
cgtctcaaaa aaaaaaaaaa gattttaaac ttacctggag agtttttgag 28920
atacagtttg gagttgcaag ttactttaac actatttata tggaatattc tattttacta
28980 gacagactta aattctccct taaattcaca aatttataga aaagttacaa
aaatactgaa 29040 aagtgctcct gtttactctg actagaattc tttagtgggt
ggcaccctac cctgagggct 29100 tcatgacctg tcctcccaca tgatccaggc
tctaccctca gggcttcatg acctgtcctc 29160 ccacatgatc caggctctac
cctcagggct tcatgacctg tcctcccacg tgatccaggc 29220 tctaccctca
gggcttcatg acctgtcctc ccacatgatc caggctctac cctcagggct 29280
tcatgacctg tcctcccacg tgatccaggc tctaccctca gggcttcatg acctgtcctc
29340 ccacgtgatc caggctctac cctcagggct tcatgacctg tcctcccacg
tgatccaggc 29400 tctaccctca gggcttcatg acccttcctc ccacatgatc
caggctctac cctgaggact 29460 tcatgacctg tcctcccacg tgatccaggc
tctaccctca gggcttcatg acctgtcctc 29520 ccacatgatc caggctctac
cctcagggct tcattacctg tcttcccaca tgatccaggc 29580 ccattctttc
ttgaaccatt ggaaaggaat ttgcagatag gatgtgtacc cctaactgcc 29640
tgagtatttc ttagcaggtg tattcttttg tgcaagtgta agtcagaatg ttaatgttga
29700 tgaaatacta atctgcaggc ctaattgttc caataatgtc ctttatggca
aaatcttccc 29760 ctcaagcttt aatccaatat catgggttgc atttgtttat
ttttaattat ttttcttttc 29820 tttttctgtt ttccttaccc ttctcactgt
gcacatgggt tgcatttagt tatcacattg 29880 acaccctttt aacctggaat
aattccttaa tctttccttg tgtttgatga ctttgtcatt 29940 tttgaattgt
tccacaagtt attttgtaga atatcctcag tgtttttttt tttctggtgt 30000
ctcgtgatta gattcaggtt atgaaactac atttttgtca ggaagattgc agaagaaatg
30060 gggccttctc ctgcacctta ccaggaagca cacaccaact ttgatccctt
gattaaggtg 30120 atgaccgctg tacttagttt ctccgctatg aagttgcttt
ttttttcttt gtggggagac 30180 agttttagac tatgtaaacg tcctatttct
catcatactt atacctgtta gtgttagcat 30240 ttgatgatga ttcttgcctg
aatcaattat ttgcataatg cttacaaaat tatcattcct 30300 tccatatgta
ttagttggtg ttcttccaaa agcttttcct ttgcctctgt ttatttactt 30360
atttatcact gtggacgcat agattcttac gcaattgatt ttgatactga tctcatagct
30420 agacaatttt gctaaacttt taaaaaaatt tatgtacttt atcttttata
gcagctttaa 30480 atttacagaa aatttgagtg gaagatgcag tgttcccata
aagccgctaa ctcctcgcac 30540 cttccctcaa gtttccccag tactaacatc
ttgcattcaa gtggtgcgtt tgcaacattc 30600 ataaattatt atcgtccaga
gtccattgtt tacattcagc ttcctcttca tgttgttcat 30660 tctgtggttt
cacagatgtg tgatgcatgt gcccaccact gcagtgtcac acaggatctc 30720
actgccccgg agtcctctgc gctgtccccg cctccagaac cccttagtag caaacactga
30780 tatttttact gtctccatag ttttgccttt tcagactgac ctatttcact
tagtaagaag 30840 catttaagat tcctgagtct ctttctatgg ctcaatagca
catttctttt tagtgctgaa 30900 taatattcca ttgtctggat gtaccacagt
ttattcattc acctactaag gtgaatgtct 30960 tgcttgcttc caagttttgg
caactatgaa taaagttgct atcaatgtta gcgtgcacat 31020 aagttttcag
ctcatttggg taaatgccaa gaagcatgat tgcgggatcc tatggtaaga 31080
gtgtgtttag ttctgtaaga agctgccaaa ctgtatctta agtggctgca ccatttgcgt
31140 ttccaccagc aatgatgagc gttttgttgc tccacatcct caccagcatt
tgctgttgtg 31200 ttttgggttt tagcctttct aagaggtgtg tagtggtatc
tccttgtttc aatttgcaat 31260 tccctaatga cattatgtta aaatcttgtc
atatagttat ttgccatctg tgtatctttt 31320 tcagtgatgt gtcctttaaa
gtctttggct catttttaaa ttaaattttc ttattgttga 31380 gttttagttc
ttcatatatt ttggctgcca gtcctttatc agatatgtct ttcgcaaata 31440
ttttctgcct gtgtcttgtc ttttcattct attaacagta tcttttgcag agccagtttt
31500 catttcaagg aagtccagct tatcaatgtt ctctttcatg tatcatgttt
ttggtgttgt 31560 atctaaaaag ttactgccaa gcccaagggt acctagattt
tttcctgtgt tatattctag 31620 gatttttaaa gttttgcatt ttacatctag
gtccatgatt cattttgagt taacttttgt 31680 gaagggttta tggtttgtgt
ctagattttt tttttttttt tttttttgca tgtggatgtc 31740 cagttgtttt
ggtaccatct gtcaagaaga ctctttttgg gtcattttgt tgcctttgtt 31800
tctttgtaaa aaatcagttg actgcatttg catgggtcta tttctgagct ctctgttcca
31860 ttgcattgat ctgtttgttc ttctcagcaa tcccacactg tcttggttcc
tgtagctctg 31920 tagtaggcct tgcagtcagt taccgcccct gttctcactt
cagtgttctc ttcaatagtg 31980 ttttgactat tctaggtttt ttccctctcc
atatacattt tagagtcagt ttgtcaatag 32040 tttacaaaat aacttgctga
gactttgatt gggattacat tgaatctgta gctcaagttg 32100 gaaagatctt
ttatttcttt catcagaatt ttgtagtttt catcatatat agatcttgta 32160
catattttgt tgtttatacc taaggatttc atttttttgg tgctaatgta aatggcgttg
32220 tgttttaaat gtcaaaatct aattgttcat tgctggtagg aaaacaactg
accctttttt 32280 ttttttttaa gggacgcagt cttactctgt tgcccaggca
gagtgcagtg gtgccatcat 32340 agctcactgc agcctcaaac tcctgggctt
aaggaatcct cctgtctcag cctcctgagc 32400 agctaggacc acaggcatgt
gccactacgt tcagctaatt tttcaatttt tttgtagaga 32460 tgggatcttg
ctctgttgcc caggctggtc tcaaactccc gtctgctttg agatgattat 32520
atatttgtgt cctttgttaa tttagaggat tattatggat ttttctaatg ttaagacacc
32580 tttgtatttc tgagatcgac cttagtattg gtctatattt aagacagtat
tcagtttctc 32640 agttgttttt tgttttttgg tttttttttt tgagacagag
tctctgtctc ccaggctgga 32700 gtccagtggc acaatctcag ctcaccgcaa
gctctgcctc ccggattcac gccattctcc 32760 tgcctcagcc tcccgagtag
ctgggactac aggcgcctgt catcatgccc agctaatttt 32820 ttgtattttt
agtagagacg gggtttcacc atgttagcca gggtggtctc aatctcctga 32880
cctcgtgatc tgcccacctc gatctcccaa agtgctggga ttacaaggcg tgagccactg
32940 cgcccggcag cagtttctca gttttaattt ggagttttgc atctgtgttc
atgagtgagc 33000 ctgaaatttt cacttttcca tatcttattt ctctgggttc
ctagaatgag ctagagagtg 33060 ttcctccttt ctgttctctg gaagagtttg
tgtgagatta gaatgagtgt gtctgataat 33120 ttagttgcat tcatttataa
aattcctagg cctagagttt tttttctggg aaaagtttac 33180 attttgactc
atttttttag tagttttagg actgtttagg ttctctattt cttgattgag 33240
ccagttttga taagttaatc tttctaattt gtagatattt tctctaagtt tgcaaatgta
33300 atacataaaa ctttcttgtc atttctcacc atatctgtag ttctatcttt
ttattgctaa 33360 tattactaat ttgtactttg actatttgta tttgttacct
gttgccgagt aacaatatta 33420 gtacaaacct agtggcttag aacaacacac
attgattact tcaccgtttc tgtgtgtcag 33480 aagtccaggc gcggcctcgc
aggtcgtcct ctgcctcagg gtctctccgg gcttcagtca 33540 gggtgttagc
caggaccggg gtctcgcctg agcttccagt gaggaaggat ctgcctctga 33600
gcacacaggg tcctcggcac gatcccattc ctcagctgga agctgccgac tgccgtctgc
33660 tgcggggcct ctctagatgg catcttcaca aaagcgagaa gggagagttg
gtagagggag 33720 tctgctagca ccatgggagt cgcggtcaca cagacctcgg
tcccaggacc cgcacccatc 33780 aaccctgccg tgatctgctg gttaaagaca
agtcccacgt cccacagggt gacactggag 33840 tagacacttc gctctggcct
tttcagagaa ctggttattt tttggaaata tcagttagat 33900 gtaggatggg
tcttgtcttc taaatctatt gttttctctc taattgattt tttcctgttt 33960
ttatttagtt cactttgttg ggtttgctca agcctgggtc actggatctc agggatgctg
34020 ctcctgtttg cagctgtgtc tgcaggggct tcccaaggcc ttgctttccc
ctcacgtccc 34080 tttctcagac tctgccaatc cgcttcccgc tctggtgtcc
tgtggttgct tctttttaaa 34140 accctcatcg gtctgtgtaa actgtttatt
tttatgtggt ttttaaggga gaccattctc 34200 attcttttga gaccctggaa
aggatggaat tgggataggt aaactgctgt tttaccagaa 34260 tgttcactgg
accaatctcg tgttccaggg agaccctcac gcagggctta gagttctgtc 34320
ggcggcagca gagggccgat ggctcctggg aagggtgagt gagcctccac tcgtgagtgc
34380 agagatgcat gggatccaga ggtttctgct ctcacacact gcgttcataa
atgttggctt 34440 gtatgttgtt gctacaccag aagtttctgg aagtgagctg
ccagcccgtg acttctgggg 34500 gacctcgttc ctttgtggca tgcgtggcct
ttgccccggt ggaaattgct cagtacgttg 34560 ctgggcgcag ccgggctgct
gggagcgcgc tgtagcctga gcgtggctat tccctccacc 34620 ctttctgctt
gctcttaggg tccagcagac agagctgctg tcttccacgg ccttaatgcc 34680
tgaggcactg gagttggtgg gctggctggg gcacgtgtga ttgttgcaga atgcgtgttg
34740 tttcacacac cggctgtgaa cagggtggaa gggctgaggc tctccctgtt
tccctccagc 34800 tcctggggag tttgcttcac ctacggcacc tggtttggcc
tggaggcctt cgcctgtatg 34860 gggcagacct accgagatgg gtgagtgagt
gcctgtcctc tggtgggtgg gggttctcaa 34920 cccaatgctc tgtcatgagt
gttttttgct ttgacatttg gttttagggt ttgtttgttt 34980 gtttgtttgt
ttttgagacg gagtctcgct ctgtcaaccg ggctgacatg cagtggcatg 35040
atcctagctc actgcagtct caaactcgtg ggctcaagcg atcctcccga gtagctggga
35100 tcacaggtgc acgccaccac cccgggctaa tcttttaaaa cttttatgta
gagatggagt 35160 cttgctgtgt tgctcacact ggtttgggct caagcagtct
tcctacctcg gccttccaaa 35220 gtgctggggt tacaggcatg agccaatgtg
cctggcctgt ttttaatatt tttaaacagt 35280 gagataagat ccccggttga
aatgaagatg tttccctggt cccacagctc tctggagctt 35340 cctgacatgt
atgctggagg gacgcttctg gtctccggcc cctccaggca tacagatgcc 35400
tcccaaccct gagtaggaag attagggtcc acggcctcgc tggagcgggt tagaaggcag
35460 gagatctccg gtcccagccg tgtctccagc cgccggactc tctcccagcc
ctgtctccag 35520 ctgccccact gtctcccaga gtctgccgtg tggatgttta
gaggtgggga gcaccgtgct 35580 tggctgagtg cagcttgtga gacgctgctc
ccaagcactg cagacctcac tcagcctgac 35640 gcgtccgtga ggccatcctc
ggtactcgca tgtccctttg tcttcccagc gactctggga 35700 ggcaggagta
tctgttccca gttcacatct gcaaaagtca agctcgggtt tcagtagtgg 35760
cccatggccc ttaggtaggg tggccccatc gtgcaggctc ctccccgtac cccaaggcag
35820 cctgctgggg tgagaagcca ggggtctggg accttccttg gtgtgatggt
gtctcctgtc 35880 tctggtcttt gcaggactgc ctgtgcagag gtctcccggg
cctgtgactt cctgctgtcc 35940 cggcagatgg cagacggagg ctggggggag
gactttgagt cctgcgagga gcggcgttat 36000 ttgcagagtg cccagtccca
gatccataac acatgctggg ccatgatggg gctgatggcc 36060 gttcggtggg
gacgacggga ccgtccctga gccttgggtt tgggtagagg agggacactc 36120
agctgtgagc cggtggcctg ggctgagtga atgtagagag gaggggaggc ctgtgggcca
36180 ggtcagctgc cactctggga acagacacct acaagagcca catgcctggt
tcctggggca 36240 agaacgtggg ctgctctgac caagtggggc cctgcagaga
ggctcgcctc ttagaagtga 36300 accacccacc attagccatg tcagtggaag
agcaagcaca tcagggaccc atggaaacag 36360 cgaggtgggc tgcgatgagg
atgctgcttc ctggtgtggt agtgatgacg gtcacagcag 36420
ctgctctctg tggccctact gtgttcacag ctggtgctga gccacatatg tgccaggtgc
36480 acacacacgc agacgcatgc aggcaggcat cagtgtacac actgatgtgc
acacacagat 36540 gtacatggag acagatgcac acacaggcct atgcacacac
gtacgcatgc ccacacaggc 36600 acctgtgtcc acacacatac agatgcaccc
acagcatccc atctgtgcca cacactgaca 36660 taggtacatg gagacagatg
cacacacagg tctgtgcaca cacgtatgca tgcacaggca 36720 cctgtgtaca
cacacgtaca gatgcaccca caggatccca tctgtgccac acacagacgt 36780
aggtacatgg agacagatgc acacacaggt ctgtgcacac acatacatac gcatgcacag
36840 gcacctgtgt acacacatgc agatacaccc acagcatccc atctgtgcca
cacacagaca 36900 taggtacatg gagacagatg cacacacagg tctatgcaca
cacatacgca tgcacaggca 36960 cctgtgtaca cacacgtaca gatgcaccca
caggatccca tctgtgccac acacagacgt 37020 aggtacatgg agacagatgc
acacacaggt ctgtgcacac acatacatac gcatgcacag 37080 gcacctgtgt
acacacacgc agatacaccc acagcatacc atctgtgaca cacacagacg 37140
taggtacatg gagacagatg cacacacatg tctgtgcaca cacatacata cgcatgcaca
37200 ggcacgtgtg tacacacatg cagatacacc cacagcatgc catctgtgac
acacacagac 37260 gtaggtacat ggagacacat gcacacacag gtctgtgcac
acacatacgc atgcacaggc 37320 acctatgtac acacatgcag atacacccac
agcatcccat ctgtgccaca cacagacata 37380 ggtacatgaa gacagatgca
cacacaggtc tatgcacaca cgtatgcatg cacaggcacc 37440 tgtgtacaca
catgcagatg cacccacagt atcccatctg tgccacacac agacatacgt 37500
acatggagac agatgcacat acaggtctat gcacacatgt acacatgcac aggcacctgt
37560 gtacacacat gcagatgcac ccgcagtatc ccatctgtgc catacacaga
catacgtaca 37620 tggagacaga tgcacataca ggtctatgca cacatgtaca
catgcacagg cacctgtgca 37680 cacatatgca gatgcacccg cagtatccca
tctgtgccac acacagacat acgtacatgg 37740 agacagatgt acacacaggt
ctatgcacac atgtacacat gcacaggcac ctgtgtacac 37800 acatgcagat
gcacccgcag tatcccatct gtgccacaca cagacatacg tacatggaga 37860
cagatgcaca cacaggtcta tgcacacatg tacacatgca caggcacctg tgcacacata
37920 tgcagatgca cccgcagtat cgcatctgtg ccacacagac atacgtacat
ggagacagat 37980 gtacatacag gtctatgcac acatgtacac atgcacaggc
acctgtgcac acatacatac 38040 agatgcaccc gcaacatccc gtctgtgctg
ccctattagg tttgtggcca tttggggaat 38100 cttcctaaaa ccctaaaagc
tagggcaggt ctgcttgagc aggagcagca gggtctgggg 38160 gacccctgag
ggcaggacag tcagggaccc acagttgagc tgggcccgct gagccctgga 38220
tccttcttgg tgtcttatcc tggccagcaa gcaagtgtga gctcctgtgg gtctccagag
38280 gcccatgagg accagtgggc cagttgggaa caaggcttgg cgtcctcttc
aggggggaac 38340 accagggcag gcctgaggag gcctgtgtcc ccagcctgtc
attgctgtgg ctccgcttct 38400 cagggagcct aggaagaagg tgtggcaaga
gcccgaggcg ctggctgcac ctggcggggc 38460 ctgtgggcgt cagtttagac
ccatccattc tcactgcagc attccagggt ttgcccttat 38520 gctcggctgt
gtgagggtga ggatgatgct gtgggggcat gcatgctggg tgtgtttcag 38580
ccttctcttc caccaggcat cctgacatcg aggcccagga gagaggagtc cggtgtctac
38640 ttgagaaaca gctccccaat ggcgactggc cgcaggtatg ccgccaggga
cctgagcgca 38700 caaggcccag cactgacctc cagcgtgcat ggctgtttcc
acgtccccct gctctgtgtc 38760 ctttttgggg tactttggac acttgggagg
cgtcacctct gccagtgaat gccacagttg 38820 gtggcaggtc tgtggcaggt
ggtcgggtcc taaagtccag atcttgctgt tgtttcaagt 38880 gatgctctgg
gtgggggagg agctggatgg gagaagccag tgggcgggaa gcctttttgc 38940
tgcaggacag accctcccac tccagatgac ctagtggccc ctcactgagc cagaagtccc
39000 tgtggtgtgg gtgtcatgag gtcatgtgag gccaaccgcc ctcccctggg
atgaggctga 39060 gttggtggaa gctgatgtgg ttgtgagggg ctggtgaccc
tggcttaggg tttgctgcag 39120 ggcggggagt ctgagctggg ctgatggtgc
catgactgat gcgggatgga ctacttgctt 39180 tcctatgctc ttgcttaatt
agccctttcc aggctgactc acccacaagc cagccaagcc 39240 aacagccagg
gctccagttc agggactagc cctcagctga ctggtgaagc ctttgtgttt 39300
atttctctgt gttcttttag gaaaacattg ctggggtctt caacaagtcc tgtgccatct
39360 cctacacgag ctacaggaac atcttcccca tctgggccct cggccgcttc
tcccagctgt 39420 accctgagag agcccttgct ggccacccct gagaacatgc
ctacctgctg ggtgccgtct 39480 gtgcgttcca gtgaggccaa ggggtcctgg
ccgggttggg gagccctccc ataaccctgt 39540 cttgggctcc aacccctcaa
cctctatctc atagatgtga atctgggggc caggctggag 39600 gcagggatgg
ggacagggtg ggtggcttag actcttgatt tttactgtag gttcatttct 39660
gaaagtagct tgtcgggctt gggtgaggaa gggggcacag gagccgtgac ccctgaggag
39720 gcacagcgcc ttctgccacc tctgggcacg gcctcaaggt agtgaggcta
ggaggttttt 39780 tctgaccaat agctgagttc ttgggagagg agcagctgtg
cctgtgtgat tccttagtgt 39840 cgagtgggct ctgggctggg gtcggccctg
ggcaggcttc tcctgcacct tttgtctgct 39900 gggctgaggg acacgagggc
aaccctgtga caatggcagg tagtgtgcat ccgtgaatag 39960 cccagtgcgg
gggttgctca tggagcatcc tgaggccgtg cagcagggag ccccatgccc 40020
ctgggtcgtg agcttgcctg cgtatggggt ggtgtcatgg agcctcatgc ccctgggtcg
40080 tgagctcgcc tgagtatggg gtggtgtcat ggagccgcat acccctgggt
tgtgagctcg 40140 cctgcatatg cagggtctgt catggaacat cccaagtctg
tgcagcaggg gagccccatg 40200 cccctgggac atgaacccac ctgcgtggaa
tgctgtttgt gaggtgtcta cagggtttat 40260 agtagtcttg tggacacaga
aatgcacagg ggacacttac ggacacagaa atgcacaggg 40320 gaggccgagc
ataaccaggg gtgaggggca ggcagcagtt gtagttactg ccgcggggca 40380
ctgctatgtg cagggacagc cagcgcccag cccatcacca ctccctgggc tggctggcag
40440 gtatggcacc ctgggagccc ggcatatacc cagggcaccc ctacggctgc
cgccagtctc 40500 atgcccaggt gggtgctctg ggctggagcg agggccaggt
tttgggccga ggcttcccca 40560 ggcaatcctg tgagctccct tctagcctct
gacccagtct ggtctggctt gcatggatgt 40620 agggcttggg gtgggaagtt
caggtcctgg ctttgccttt gcctgatgtg gatgagcagc 40680 tcacatgctc
agggccacct gagactgtca ctgctctccc ctggctactg ggaggagtca 40740
ctgagagctt cgttacccct gctgccttgc ccagggcaca ccctatacct cctcatctgc
40800 tcttcccctc cctgccgcct tctgggcagg tagcagtccc tggcctctcc
ccctggctga 40860 tcactctccc tcaggcagtg gagatctgcg tctggacacc
ctcagatcct gtcattgcct 40920 gcccagagtc cttcaggggc acccctctgc
cttggtgtgc ggtccagggc tctcacccag 40980 gtgccgcacc ctctggggtc
ttctgtccag ctcccttgcc ccatgtgctg tcactgactc 41040 tccttgggac
tcgcctgcct gctcagagcc ctgcagggct tggtcagctg cctgttcagt 41100
gtcaacactt ccctgcacat cttaaaactg ggctttattt tcgctgaagg aactgtgttg
41160 ggacccttga catctgtcag gtttgcacat gctgtttttt tttctcagcc
cacgtgttct 41220 cccccacgtg gggtagcagc aggacagaca gtgaatcaca
gagtctgccc tgagcagagg 41280 ctgctgtccc tgggactcct agccatggtc
agactgtaca aaacggtttt ccagaaatga 41340 aatgtaaatc catttttata
ctgaaaatgt tactgaaagt cacttttatg agcatctgcc 41400 ttaataaaca
gacattgatt cccttatcag aagcctgtca cactgtgttt cgtttcatcc 41460
tggggagaac tgcagatttg gggtttctgg ctgtcatacg tcacctgcct gtggggcgag
41520 tgggaggccc agcctggttt agggaacaag agtgacgtga ggagtagcag
ggtgcgtctc 41580 cagttacctg agggaaaaca gatattttaa gagataatag
catagcctat tttaatatgt 41640 tttaaaggcc ataagcatat ccaggaagat
aaataaacgt gatacaatgt ccacatagga 41700 ggaactttct ttcactgcat
tgttttcctt cacagtggcc ttcaagtcac aggacgcagc 41760 gattccctgc
cctcttcggt gttattacac aggcaggact tcagtgtcag tatccctgcc 41820
ttcagtcttc tttagaaatc acatctgtgt tcaatccatt gtttagaggg agtgtatttt
41880 tcctgttcca cgaagaggac tttttgttca caattggatc acaatgcaga
ggagtctgtt 41940 cctcccccgt cggcttctcg gtgctgggag ggtgacctgt
cccagatgac tcatcaccct 42000 gacatgctct tgacaaagga caccaccaag
aggagatggc agctgtaccg gtgcagcctc 42060 tgtctgaggg ggatatttgc
ctcagtgtga ttaaaaatca gtcatgaaag atttttgaat 42120 tcagattatt
tttatcagga acagattttg aacatcctga aatcttttcc ctggcatcat 42180
attaggtttt ctttgttcac tatgatgtaa agtttcagac tcttgatatt tttaatatca
42240 acatagacgg taggacaagg aacggtacca gaaatgagta aagagacaat
aatgataaga 42300 tcgatttatc aagacataac aaccccaaat gtatatgcac
taaataacag cttcaaaata 42360 catgaagcaa aatggcagaa ttgaagagaa
tgagataaaa acagaatttt aacgggtgct 42420 ttccgtactt tgtaactgac
agacatgaga 42450 4 732 PRT Homo sapiens 4 Met Thr Glu Gly Thr Cys
Leu Arg Arg Arg Gly Gly Pro Tyr Lys Thr 1 5 10 15 Glu Pro Ala Thr
Asp Leu Gly Arg Trp Arg Leu Asn Cys Glu Arg Gly 20 25 30 Arg Gln
Thr Trp Thr Tyr Leu Gln Asp Glu Arg Ala Gly Arg Glu Gln 35 40 45
Thr Gly Leu Glu Ala Tyr Ala Leu Gly Leu Asp Thr Lys Asn Tyr Phe 50
55 60 Lys Asp Leu Pro Lys Ala His Thr Ala Phe Glu Gly Ala Leu Asn
Gly 65 70 75 80 Met Thr Phe Tyr Val Gly Leu Gln Ala Glu Asp Gly His
Trp Thr Gly 85 90 95 Asp Tyr Gly Gly Pro Leu Phe Leu Leu Pro Gly
Leu Leu Ile Thr Cys 100 105 110 His Val Ala Arg Ile Pro Leu Pro Ala
Gly Tyr Arg Glu Glu Ile Val 115 120 125 Arg Tyr Leu Arg Ser Val Gln
Leu Pro Asp Gly Gly Trp Gly Leu His 130 135 140 Ile Glu Asp Lys Ser
Thr Val Phe Gly Thr Ala Leu Asn Tyr Val Ser 145 150 155 160 Leu Arg
Ile Leu Gly Val Gly Pro Asp Asp Pro Asp Leu Val Arg Ala 165 170 175
Arg Asn Ile Leu His Lys Lys Gly Gly Ala Val Ala Ile Pro Ser Trp 180
185 190 Gly Lys Phe Trp Leu Ala Val Leu Asn Val Tyr Ser Trp Glu Gly
Leu 195 200 205 Asn Thr Leu Phe Pro Glu Met Trp Leu Phe Pro Asp Trp
Ala Pro Ala 210 215 220 His Pro Ser Thr Leu Trp Cys His Cys Arg Gln
Val Tyr Leu Pro Met 225 230 235 240 Ser Tyr Cys Tyr Ala Val Arg Leu
Ser Ala Ala Glu Asp Pro Leu Val 245 250 255 Gln Ser Leu Arg Gln Glu
Leu Tyr Val Glu Asp Phe Ala Ser Ile Asp 260 265 270 Trp Leu Ala Gln
Arg Asn Asn Val Ala Pro Asp Glu Leu Tyr Thr Pro 275 280 285 His Ser
Trp Leu Leu Arg Val Val Tyr Ala Leu Leu Asn Leu Tyr Glu 290 295 300
His His His Ser Ala His Leu Arg Gln Arg Ala Val Gln Lys Leu Tyr 305
310 315 320 Glu His Ile Val Ala Asp Asp Arg Phe Thr Lys Ser Ile Ser
Ile Gly 325 330 335 Pro Ile Ser Lys Thr Ile Asn Met Leu Val Arg Trp
Tyr Val Asp Gly 340 345 350 Pro Ala Ser Thr Ala Phe Gln Glu His Val
Ser Arg Ile Pro Asp Tyr 355 360 365 Leu Trp Met Gly Leu Asp Gly Met
Lys Met Gln Gly Thr Asn Gly Ser 370 375 380 Gln Ile Trp Asp Thr Ala
Phe Ala Ile Gln Ala Leu Leu Glu Ala Gly 385 390 395 400 Gly His His
Arg Pro Glu Phe Ser Ser Cys Leu Gln Lys Ala His Glu 405 410 415 Phe
Leu Arg Leu Ser Gln Val Pro Asp Asn Pro Pro Asp Tyr Gln Lys 420 425
430 Tyr Tyr Arg Gln Met Arg Lys Gly Gly Phe Ser Phe Ser Thr Leu Asp
435 440 445 Cys Gly Trp Ile Val Ser Asp Cys Thr Ala Glu Ala Leu Lys
Ala Val 450 455 460 Leu Leu Leu Gln Glu Lys Cys Pro His Val Thr Glu
His Ile Pro Arg 465 470 475 480 Glu Arg Leu Cys Asp Ala Val Ala Val
Leu Leu Asn Met Arg Asn Pro 485 490 495 Asp Gly Gly Phe Ala Thr Tyr
Glu Thr Lys Arg Gly Gly His Leu Leu 500 505 510 Glu Leu Leu Asn Pro
Ser Glu Val Phe Gly Asp Ile Met Ile Asp Tyr 515 520 525 Thr Tyr Val
Glu Cys Thr Ser Ala Val Met Gln Ala Leu Lys Tyr Phe 530 535 540 His
Lys Arg Phe Pro Glu His Arg Ala Ala Glu Ile Arg Glu Thr Leu 545 550
555 560 Thr Gln Gly Leu Glu Phe Cys Arg Arg Gln Gln Arg Ala Asp Gly
Ser 565 570 575 Trp Glu Gly Ser Trp Gly Val Cys Phe Thr Tyr Gly Thr
Trp Phe Gly 580 585 590 Leu Glu Ala Phe Ala Cys Met Gly Gln Thr Tyr
Arg Asp Gly Thr Ala 595 600 605 Cys Ala Glu Val Ser Arg Ala Cys Asp
Phe Leu Leu Ser Arg Gln Met 610 615 620 Ala Asp Gly Gly Trp Gly Glu
Asp Phe Glu Ser Cys Glu Glu Arg Arg 625 630 635 640 Tyr Leu Gln Ser
Ala Gln Ser Gln Ile His Asn Thr Cys Trp Ala Met 645 650 655 Met Gly
Leu Met Ala Val Arg His Pro Asp Ile Glu Ala Gln Glu Arg 660 665 670
Gly Val Arg Cys Leu Leu Glu Lys Gln Leu Pro Asn Gly Asp Trp Pro 675
680 685 Gln Glu Asn Ile Ala Gly Val Phe Asn Lys Ser Cys Ala Ile Ser
Tyr 690 695 700 Thr Ser Tyr Arg Asn Ile Phe Pro Ile Trp Ala Leu Gly
Arg Phe Ser 705 710 715 720 Gln Leu Tyr Pro Glu Arg Ala Leu Ala Gly
His Pro 725 730
* * * * *
References