U.S. patent application number 09/824258 was filed with the patent office on 2001-11-29 for vanilrep1 polynucleotides and vanilrep1 polypeptides.
Invention is credited to Davis, John Beresford, Duckworth, David Malcolm, Hayes, Philip David, Meadows, Helen Jane.
Application Number | 20010047090 09/824258 |
Document ID | / |
Family ID | 27269239 |
Filed Date | 2001-11-29 |
United States Patent
Application |
20010047090 |
Kind Code |
A1 |
Duckworth, David Malcolm ;
et al. |
November 29, 2001 |
VANILREP1 polynucleotides and VANILREP1 polypeptides
Abstract
VANILREP 1 polypeptides and polynucleotides and methods for
producing such polypeptides by recombinant techniques are
disclosed. Also disclosed are methods for utilizing VANILREP 1
polypeptides and polynucleotides in therapy, and diagnostic assays
for such.
Inventors: |
Duckworth, David Malcolm;
(Bishop's Stortford, GB) ; Hayes, Philip David;
(Cambridge, GB) ; Meadows, Helen Jane; (Upminster,
GB) ; Davis, John Beresford; (Bishop's Stortrord,
GB) |
Correspondence
Address: |
Ratner & Prestia
P.O. Box 980
Valley Forge
PA
19482-0980
US
|
Family ID: |
27269239 |
Appl. No.: |
09/824258 |
Filed: |
April 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09824258 |
Apr 2, 2001 |
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09197636 |
Nov 23, 1998 |
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6239267 |
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Current U.S.
Class: |
536/23.5 ;
424/760; 435/325; 435/455; 435/6.14; 435/69.1; 435/7.1;
530/350 |
Current CPC
Class: |
A61P 25/04 20180101;
A61P 9/00 20180101; A61P 9/10 20180101; A61P 29/00 20180101; C07K
14/705 20130101 |
Class at
Publication: |
536/23.5 ;
530/350; 435/69.1; 435/325; 435/455; 435/6; 435/7.1; 424/760 |
International
Class: |
C12Q 001/68; G01N
033/53; C07H 021/04; A61K 035/78; C12P 021/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 12, 1998 |
GB |
9805137.8 |
Jul 20, 1998 |
GB |
9815791.0 |
Sep 3, 1998 |
GB |
9819278.4 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
(i) an isolated polypeptide comprising an amino acid sequence
selected from the group having at least: (a) 90% identity; or (b)
95% identity to the amino acid sequence of SEQ ID NO:2 over the
entire length of SEQ ID NO:2; (ii) an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO:2; (iii) an
isolated polypeptide which is the amino acid sequence of SEQ ID
NO:2; (iv) an isolated polypeptide comprising an amino acid
sequence selected from the group having at least: (a) 90% identity;
or (b) 95% identity to the amino acid sequence of SEQ ID NO:8 over
the entire length of SEQ ID NO:8; (v) an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO:8 or (vi) an
isolated polypeptide which is the amino acid sequence of SEQ ID NO:
8.
2. An isolated polynucleotide selece from the group consisting of
(i) an isolated polynucleotide compriin a nuclecoide sequence
encoding a polypeptide that has at least (a) 90% identit; or (b)
95% identity; to the amino acid sequence of SEQ ID NO:2, over the
entire length of SEQ ID NO:2; (ii) an isolated polynucleotde
comprising a nucleotide sequence that has at least: (a) 85%
identitv; (b) 90% identity. or (c) 95% identity; over its entire
length to a nucleotide sequence encoding the polypeptide of SEQ ID
NO:2; (iii) an isolated polynucleotide comprising a nucleotide
sequence which has at least: (a) 85%identity; (b) 90% identity; or
(c) 95% identity; to that of SEQ ID NO: 1 over the entire length of
SEQ ID NO: 1; (iv) an isolated polynucleotide comprising a
nucleotide sequence encoding the polypeptide of SEQ ID NO:2; (v) an
isolated polynucleotide which is the polynucleotide of SEQ ID NO:
1; or (vi) an isolated polynucleotide obtainable by screeni an
appropriate library under stringent hybridization conditions with a
labeled probe having the sequence of SEQ ID NO: I or a fragment
thereof; or a nucleotide sequence complementary to said isolated
polynucleotide.
3. An isolated polynucleotide selected from the group consisting
of. (i) an isolated polynucleotide comprising a nucleotide sequence
encoding a polypeptide that has at least (a) 90%identity; or (b)
95%identity; to the anmino acid sequence of SEQ ID NO:8, over the
entire length of SEQ ID NO:8; (ii) an isolated polynucleotide
comprising a nucleotide sequence that has at least: (a)
85%identity; (b) 90% identity; or (c) 95% identity; over its entire
length to a nucleotide sequence encoding the polypeptide of SEQ ID
NO:8; (iii) an isolated polynucleotide comprising a nucleotide
sequence which has at least: (a) 85% identity; (b) 90% identity; or
(c) 95% identity; to that of SEQ ID NO:7 over the entire length of
SEQ ID NO:7; (iv) an isolated polynucleotide comprising a
nucleotide sequence encoding the polypeptide of SEQ ID NO:8; (v) an
isolated polynucleotide which is the polynucleotide of SEQ ID NO:7;
or (vi) an isolated polynucleotide obtainable by screening an
appropriate library under stringent hybridization conditions with a
labeled probe having the sequence of SEQ ID NO:7 or a fragment
thereof; or a nucleotide sequence complementary to said isolated
polynucleotide.
4. An antibody immunospecific for the polypeptide of claim 1.
5. A method for the treatment of a subject: (i) in need of enhanced
activity or expression of the polypeptide of claim 1 comprising:
(a) administering to the subject a therapeutically effective amount
of an agonist to said polypeptide; and/or (b) providing to the
subject an isolated polynucleotide comprising a nucleotide sequence
encoding said polypetide in a form so as to effect production of
said polypeptide activity in vivo; or (ii) having need to inhibit
activity or expression of the polypeptide of claim 1 comprising:
(a) administering to the subject a therapeutically effective amount
of an antagonist to said polypeptide; and/or (b) administering to
the subject a nucleic acid molecule that inhibits the expression of
a nucleotide sequence encoding said polypeptide; and/or (c)
administering to the subject a therapeutically effective amount of
a polypeptide that competes with said polypeptide for its ligand,
substrate , or receptor.
6. A process for diagnosing a disease or a susceptibility to a
disease in a subject related to expression or activity of the
polypeptide of claim 1 in a subject comprising: (a) determining the
presence or absence of a mutation in the nucleotide sequence
encoding said polypeptide in the genome of said subject; and/or (b)
analyzing for the presence or amount of said polypeptide expression
in a sample derived from said subject.
7. A method for screening to identify compounds which stimulate or
which inhibit the fliction of the polypeptide of claim 1 which
comprises a method selected from the group consisting of. (a)
measuring the binding of a candidate compound to the polypeptide
(or to the cells or membranes bearing the polypeptide) or a fusion
protein thereof by means of a label directly or indirectly
associated with the candidate compound; (b) measuring the binding
of a candidate compound to the polypeptide (or to the cells or
membranes bearing the polypeptide) or a fusion protein thereof in
the presense of a labeled competitior; (c) testing whether the
candidate compound results in a signal generated by activation or
inhibition of the polypeptide, using detection systems appropriate
to the cells or cell membranes bearing the polypeptide; (d) mixing
a candidate compound with a solution containing a polypeptide of
claim 1, to form a mixture, measuring activity of the polypeptide
in the mixture, and comparing the activity of the mixture to a
standard; or (e) detecting the effect of a candidate compound on
the production of mRNA encoding said polypeptide and said
polypeptide in cells, using for instance, an ELISA assay.
8. An agonist or an antagonist of the polypeptide of claim 1.
9. An expression system comprising a polynucleotide capable of
producing a polypeptide of claim 1 when said expression system is
present in a compatible host cell.
10. A process for producing a recombinant host cell comprising
transforming or transfecting a cell with the expression system of
claim 9 such the the host cell, under appropriate culture
conditions, produces a polypeptide selected from the group
consisting of: (a) a polypeptide comprising an amino acid sequence
having at least 90% identity to the amino acid sequence of SEQ ID
NO:2 over the entire length of SEQ ID NO:2; or (b) a polypeptide
comprising an amino acid sequence having at least 90% identity to
the amino acid sequence of SEQ ID NO:8 over the entire length of
SEQ ID NO:8.
11. A recombinant host cell produced by the process of claim
10.
12. A membrane of a recombinant host cell of claim 11 expressing a
polypeptide selected from the group consisting of: (a) a
polypeptide comprising an amino acid sequence having at least 90%
identity to the amino acid sequence of SEQ ID NO:2 over the entire
length of SEQ ID NO:2; or (b) a polypeptide comprising an amino
acid sequence having at least 90% identity to the amino acid
sequence of SEQ ID NO:8 over the entire length of SEQ ID NO:8.
13. A process for producing a polypeptide comprising culturing a
host cell of claim II under conditions sufficient for the
production of said polypeptide and recovering the polypeptide from
the culture.
14. An isolated polynucleotide selected form the group consisting
of (a) an isolated polynucleotide comprising a nucleotide sequence
which has at least 85%, 90%, 95%, 97% identity to SEQ ID NO:3 over
the entire length of SEQ ID NO:3; (b) an isolated polynucleotide
comprising the polynucleotide of SEQ ID NO:3; (c) the
polynucleotide of SEQ ID NO:3; (d) an isolated polynucleotide
comprising a nucleotide sequence encoding a polypeptide which has
at least 90%/o, 95%, 97-99% identity to the amino acid sequence of
SEQ ID NO:4, over the entire length of SEQ ID NO:4; (e) an isolated
polynucleotide comprising a nucleotide sequence which has at least
85%, 90%, 95%, 97% identity to SEQ ID NO:5 over the entire length
of SEQ ID NO:5; (f) an isolated polynucleotide comprising the
polynucleotide of SEQ ID NO:5; (g) the polynucleotide of SEQ ID
NO:5; or (h) an isolated polynucleotide comprising a nucleotide
sequence encoding a polypeptide which has at least 90%, 95%, 97-99%
identity to the amino acid sequence of SEQ ID NO:6, over the entire
length of SEQ ID NO:6.
15. A polypeptide selected from the group consisting of: (a) a
polypeptide which comprises an amino acid sequence which has at
least 90%, 95%, 97-99% identity to that of SEQ ID NO:4 over the
entire length of SEQ ID NO:4; (b) a polypeptide which has an amino
acid sequence which is at least 90%, 95%, 9799% identity to the
amino acid sequence of SEQ ID NO:4 over the entire length of SEQ ID
NO:4; (c) a polypeptide which comprises the amino acid of SEQ ID
NO:4; (d) a polypeptide which is the polypeptide of SEQ ID NO:4;
(e) a polypeptide which is encoded by a polynucleotide comprising
the sequence contained in SEQ ID NO:3; (f) a polypeptide which
comprises an amino acid sequence which has at least 90%, 95%,
97-99% identity to that of SEQ ID NO:6 over the entire length of
SEQ ID NO:6; (g) a polypeptide which has an amino acid sequence
which is at least 90%, 95%, 97-99% identity to the amino acid
sequence of SEQ ID NO:6 over the entire length of SEQ ID NO:6; (h)
a polypeptide which comprises the amino acid of SEQ ID NO:6; (i) a
polypeptide which is the polypeptide of SEQ ID NO:6; or (j) a
polypeptide which is encoded by a polynucleotide comprising the
sequence contained in SEQ ID NO:5.
Description
FIELD OF THE INVENTION
[0001] This invention relates to newly identified polypeptides and
polynucleotides encoding such polypeptides, to their use in therapy
and in identifying compounds which may be agonists, antagonists and
/or inhibitors which are potentially useful in therapy, and to
production of such polypeptides and polynucleotides.
BACKGROUND OF THE INVENTION
[0002] The drug discovery process is currently undergoing a
findamenal revolution as it embraces `functional genomics`, that
is, high throughput genome or genebased biology. This approach as a
means to identify genes and gne products as therapeutic targets is
rapidly superceding earlier approaches based on `positional
cloning`. A phenotype, that is a biological function or genetic
disease, would be identified and this would then be tracked back to
the responsible gene, based on its genetic map position.
[0003] Functional genomics relies heavily on highthroughput DNA
sequencing technologies and the various tools of bioinformatics to
identify gene sequences of potential interest from the many
molecular biology databases now available. There is a continuing
need to identify and characterise further genes and their related
polypeptides/proteins, as targets for drug discovery.
SUMMARY OF THE INVENTION
[0004] The present invention relates to VANILREP1, in particular
VANILREP1 polypeptides and VANILREP 1 polynucleotides, recombinant
materials and methods for their production. In another aspect, the
invention relates to methods for using such polypeptides and
polynucleotides, including the ieatrnent of pain, chronic pain,
neuropathic pain, postoperative pain, rheumatoid arthritic pain,
neuralgia, neuropathies, algesia, nerve injury, ischaemia,
neurodegeneration, stroke, incontinence and inflarnnatory
disorders, hereinaftr referred to as "the Diseases", amongst
others. In a further aspect, the invention relates to methods for
identifyg agonists and antagonists/inhibitors using the materials
provided by the invention, and treating conditions associated with
VANILREP 1 imbalance with the identified compounds. In a still
further aspect, the invention relates to diagnostic assays for
detecting diseases associated with inappropriate VANILREP 1
activity or levels.
DESCRIPTION OF THE INVENTION
[0005] In a first aspect, the present invention relates to VANILREP
1 polypeptides. These include the polypeptide of SEQ ID NO:2 and
polymorphic variants thereof, for example PVP1, the polypeptide of
SEQ ID NO:8. Such peptides include isolated polypeptides comprising
an amino acid sequence which has at least 90% identity, preferably
at least 95% identity, more preferably at least 97-99% identity, to
that of SEQ ID NO:2 or SEQ ID NO:8 over the entire length of SEQ ID
NO:2 or SEQ ID NO:8 respectively. Such polypeptides include those
comprising the amino acid of SEQ ID NO:2 or SEQ ID NO:8.
[0006] Further peptides of the present invention include isolated
polypeptides in which the amino acid sequence has at least 90%
identity, preferably at least 95% identity, more preferably at
least 97-99% identity, to the amino acid sequence of SEQ ID NO:2 or
SEQ ID NO:8, over the entire length of SEQ ID NO:2 or SEQ ID NO:8.
Such polypeptides include the polypeptide of SEQ ID NO:2 and SEQ ID
NO:8.
[0007] Further peptides of the present invention include isolated
polypeptides encoded by a polynucleotide comprising the sequence
contained in SEQ ID NO: 1 or SEQ ID NO:7.
[0008] Polypeptides of the present invention are believed to be
members of the ion channel family of polypeptides. They are
therefore of interest because they are associated with the
mechanism of action of capsaicin (a vanilloid compound), a
constituent of chilli peppers. Capsaicin elicits a senrtaion of
burning pain by selectively activating sensory neurons that convey
information about noxious stinuli to the central nervous system.
The channels are permeable to cations and exhibit a notable
preferance for divalent cations, particularly calcium ions. The
level of calcium ion permeability eceeds that observed for most
nonselective cation channels and is similar to values observed for
NMDAtype glutamate receptors and alpha7 nicotinic acetylcholine
receptors, both of uhich are noted for this property. These
properties are hereinafter referred to as "VANILREP 1 activity" or
"VANILREP 1 polypeptide activity" or "biological activity of
VANILREP 1". Also included amongst these activities are antigenic
and immunogenic activities of said VANILREP 1 polypeptides, in
particular the antigenic and immunogenic activities of the
polypeptides of SEQ ID NO:2 or SEQ ID NO:8. Preferably, a
polypeptide of the present invention exhibits at least one
biological activity of VANILREP 1.
[0009] The polypeptides of the present invention may be in the form
of the "mature" protein or may be a part of a larger protein such
as a precursor or a fusion protein. It is often advantageous to
include an additional arnino acid sequence which contains secretory
or leader sequences, pro sequences, sequences which aid in
purification such as multiple histidine residues, or an additional
sequence for stability during recombinant production.
[0010] The present invention also includes variants of the
aforementioned polypeptides, that is polypeptides that vary from
the referents by conservative amino acid substitutions, whereby a
residue is substituted by another with like characteristics.
Typical such substitutions are among Ala, Val, Leu and Ile; among
Ser and Thr; among the acidic residues Asp and Glu; among Asn and
Gln; and among the basic residues Lys and Arg; or aromatic residues
Phe and Tyr. Particularly preferred are variants in which several,
5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or
added in any combination.
[0011] Polypeptides of the present invention can be prepared in any
suitable manner. Such polypeptides include isolated naturally
occurring polypeptides, recombinantly produced polypeptides,
synthetically produced polypeptides, or polypeptides produced by a
combination of these methods. Means for preparing such polypeptides
are well understood in the art.
[0012] In a further aspect, the present invention relates to
VANILREP 1 polynucleotides. Such polynucleotides include isolated
polynucleotides comprising a nucleotide sequence encoding a
polypeptide which has at least 90% identity, preferably at least
95% identity, to the amino acid sequence of SEQ ID NO:2 or SEQ ID
NO:8, over the entire length of SEQ ID NO:2 or SEQ ID NO:8,
respectively. In this regard, polypeptides which have at least 97%
identity are highly preferred, whilst those with at least 98-99%
identity are more highly preferred, and those with at least 99%
identity are most highly preferred. Such polynucleotides include a
polynucleotide comprising the nucleotide sequence contained in SEQ
ID NO: 1 or SEQ ID NO:7, encoding the polypeptide of SEQ ID NO:2 or
SEQ ID NO:8, respectively.
[0013] Further polynucleotides of the present invention include
isolated polynucleotides comprising a nucleotide sequence that has
at least 85% identity, preferably at least 90% identity, more
preferably at least 95% identity, to a nucleotide sequence encoding
a polypeptide of SEQ ID NO:2 or SEQ ID NO:8, respectively, over the
entire coding region. In this regard, polynucleotides which have at
least 97% identity are highly preferred, whilst those with at least
98-99% identity are more highly preferred, and those with at least
99% identity are most highly preferred.
[0014] Further polyvnucleotides of the present invention include
isolated polynucleotides comprising a nucleotide sequence which has
at least 85% identity, preferably at least 90% identity, more
preferably at least 95 % identity, to SEQ ID NO: 1 or SEQ ID NO:7,
over the entire length of SEQ ID NO: 1 or SEQ ID NO:7,
respectively. In this regard, polynucleotides which have at least
97% identity are highly preferred, whilst those with at least
98-99% identiy are more highly preferred, and those with at least
99% identity are most highly preferred. Such polynucleotides
include a polynucleotide comprising the polynucleotide of SEQ ID
NO: 1 or SEQ ID NO:7, as well as the polknucleotide of SEQ ID NO: 1
and SEQ ID NO:7, respectively.
[0015] The invention also provides polynucleotides which are
complementary to all the above described polynucleotides.
[0016] The nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO:7
show homology with rat vanilloid receptor VRl(M. J. Caterina et
al., Nature 389: 816-824, 1997). The nucleotide sequence of SEQ ID
NO: 1 is a cDNA sequence and comprises a polypeptide encoding
sequence (nucleotide 864 to 3380) encoding a polypeptide of 839
amino acids, the polypeptide of SEQ ID NO:2. The nucleotide
sequence encoding the polypeptide of SEQ ID NO:2 may be identical
to the polypeptide encoding sequence contained in SEQ ID NO: 1 or
it may be a sequence other than the one contained in SEQ ID NO: 1,
which, as a result of the redundancy (degeneracy) of the genetic
code, also encodes the polypeptide of SEQ ID NO:2. The polypeptide
of the SEQ ID NO:2 is structurally related to other proteins of the
ion channel family, having homology and/or structural similarity
with rat vanilloid receptor VR(M. J. Caterinaetal., Nature 389:
816-824, 1997).
[0017] The nucleotide sequence of SEQ ID NO:7 is a cDNA sequence
and comprises a polypeptide encoding sequence (nucleotide 864 to
3380) encoding a polypeptide of 839 amino acids, the polypeptide of
SEQ ID NO: 8. The nucleotide sequence encoding the polypeptide of
SEQ ID NO:8 may be identical to the polypeptide encoding sequence
contained in SEQ ID NO:7 or it may be a sequence other than the one
contained in SEQ ID NO:7, which, as a result of the redundancy
(degeneracy) of the genetic code, also encodes the polypeptide of
SEQ ID NO:8. The polypeptide of the SEQ ID NO:8 is structurally
relted to other proteins of the ion channel fairly, having homology
and/or structural similarity pith rat vanilloid receptor VR1(M. J.
Caterina et al., Nature 389: 816-824, 1997).
[0018] Preferred polpeptides and polynucleotides of the present
invention are expected to have, inter alia, similar biological
finctions/properties to their homologous polypeptides and
polynucleotides. Furthermore, preferred polypeptides and
polynucleotides of the present invention have at least one VANILREP
1 activity.
[0019] The present imention also relates to partial or other
polynucleotide and polypeptide sequences which were first
identified prior to the determination of the corresponding full
length sequences of SEQ ID NO: 1, SEQ ID NO:7, SEQ ID NO:2 and SEQ
ID NO:8.
[0020] Accordingly, in a further aspect, the present invention
provides for an isolated polynucleotide which: (a) comprises a
nucleotide sequence which has at least 85% identity, preferably at
least 90% identity, more preferably at least 95% identity, yet more
preferably at least 97-99% identity to SEQ ID NO:3 or SEQ ID NO:5,
over the entire length of SEQ ID NO:3 or SEQ ID NO:5, respectively;
(b) has a nucleotide sequence which has at least 85% identity,
preferably at least 90% identity, more preferably at least 95%
identity, yet more preferably at least 97-99% identity, to SEQ ID
NO:3 or SEQ ID NO5, over the entire length of SEQ ID NO:3 or SEQ ID
NO:5, respectively; (c) the polynucleotide of SEQ ID NO:3 or SEQ ID
NO:5; or (d) a nucleotide sequence encoding a polypeptide which has
at least 90% identity, preferably at least 95% identity, more
preferably at least 97-99% identity, to the amino acid sequence of
SEQ ID NO:4 or SEQ ID NO:6, over the entire length of SEQ ID NO:4
or SEQ ID NO:6, respectively; as well as the polynucleotides of SEQ
ID NO:3 and SEQ ID) NO:5.
[0021] The present invention fiwther provides for a polypeptide
which: (a) comprises an amino acid sequence which has at least 90%
identity, preferably at least 95% identity, more preferably at
least 97-99% identity, to that of SEQ ID NO:4 or SEQ ID NO:6, over
the entire length of SEQ ID NO:4 or SEQ ID NO:6, respectively; (b)
has an amino acid sequence which has at least 90% identity,
preferably at least 95% identity, more preferably at least 97-99%
identity, to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6,
over the entire length of SEQ ID NO:4 or SEQ ID NO:6, respectively;
(c) comprises the amino acid of SEQ ID NO:4 or SEQ ID NO:6; and (d)
is the polypeptide of SEQ ID NO:4 or SEQ ID NO:6; as well as
polypeptides encoded by a polynucleotide comprising the sequence
contained in SEQ ID NO:3 or SEQ ID NO:5.
[0022] The nucleotide sequences of SEQ ID NO:3 and SEQ ID NO:5, and
the peptide sequences encoded thereby are derived from EST
(Expressed Sequence Tag) sequences. It is recognised by those
skilled in the art that there will inevitably be some nucleotide
sequence reading errors in EST sequences (see Adams, M. D. et al,
Nature 377 (supp) 3, 1995). Accordingly, the nucleotide sequences
of SEQ ID NO:3 and SEQ ID NO:5, and the peptide sequence encoded
therefrom, are therefore subject to the same inherent limitations
in sequence accuracy.
[0023] Polynucleotides of the present invention may be obtained,
using standard cloning and screening techniques, from a cDNA
library derived from MRNA in cells of human brain, cerebellum,
dorsal root ganglia, thymus, leukocytes, placenta, foetal liver
spleen and ovary, using the expressed sequence tag (EST) analysis
(Adams, M. D., etal Science (1991) 252:1651-1656; Adams, M. D.
etaL., Nature, (1992) 355:632-634; Adams, M. D., et al., Nature
(1995) 377 Supp:3-174). Polynucleotides of the invention can also
be obtained from natural sources such as genomic DNA libraries or
can be synthesized using well known and commercially available
techniques.
[0024] When polynucleotides of the present invention are used for
the recombinant production of polypeptides of the present
invention, the polynucleotide may include the coding sequence for
the mature polypeptide, by itself, or the coding sequence for the
mature polypeptide in reading frame with other coding sequences,
such as those encoding a leader or secretory sequence, a pre, or
pro or prepro protein sequence, or other fusion peptide portions.
For example, a marker sequence which facilitates purification of
the fuised polypeptide can be encoded. In certain preferred
embodiments of this aspect of the invention, the marker sequence is
a hexahistidine peptide, as provided in the pQE vector (Qiagen,
Inc.) and described in Gentz et al., Proc Natl Acad Sci USA (1989)
86:821-824, or is an HA tag. The polynucleotide may also contain
nonoding 5'and 3'sequences, such as transcribed, nontranslated
sequences, splicing and polyadenylation signals, ribosome binding
sites and sequences that stabilize mRNA.
[0025] Further embodiments of the present invention include
polynucleotides encoding polypeptide variants which comprise the
amino acid sequence of SEQ ID NO:2 or SEQ ID NO:8, respectively and
in which several for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2
or 1, amnino acid residues are substituted, deleted or added, in
any combination.
[0026] Polynucleotides which are identical or sufficiently
identical to a nucleotide sequence contained in SEQ ID NO: 1 or SEQ
ID NO:7, respectively, may be used as hybridization probes for cDNA
and genomic DNA or as primers for a nucleic acid amplification
(PCR) reaction, to isolate fulllength cDNAs and genomic clones
encoding polyamides of the present invention and to isolate cDNA
and genomic clones of other genes (including genes encoding
paralogs from human sources and orthologs and paralogs from species
other than human) that have a high sequence similarity to SEQ ID
NO: 1 or SEQ ID NO:7,. Typically these nucleotide sequences are 70%
identical, preferably 80% identical, more preferably 90% identical,
most preferably 95% identical to that of the referent. The probes
or primers will generally comprise at least 15 nucleotides
preferably, at least 30 nucleotides and may have at least 50
nucleotides. Particularly preferred probes will have between 30 and
50 nucleotides. Particularly preferred primers will have between 20
and 25 nucleotides.
[0027] A polynucleotide encoding a polypeptide of the present
invention, including homologs from species other than human, may be
obtained by a process which comprises the steps of screening an
appropriate library under stringent hybridization conditions with a
labeled probe having the sequence of SEQ ID NO: 1 or SEQ ID NO:7,
respectively or a fragment thereof; and isolating fulllength cDNA
and genornic clones containing said polynucleotide sequence. Such
hybridization techniques are well known to the skilled artisan.
Preferred stringent hiyrndization conditions include overnight
incubation at 42.degree. C. in a solution comprising: 50% for~mide,
5.times.SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium
phosphate (pH7.6), 5.times.Denhardtfs solution, 10 % dexran
sulfate, and 20 microgram/mi denatured, sheared salmon sperm DNA;
fou~ed by washing the filters in 0.1.times.SSC at about 65.degree.
C. Thus the present invention also includes pol,nucleotides
obtainable by screening an appropriate library under stingent
hybridization conditions with a labeled probe having the sequence
of SEQ ID NO:1 or SEQ ID NO:7, or a fragment thereof.
[0028] The skilled artisan will appreciate that, in many cases, an
isolated cDNA sequence will be incomplete, in that the region
coding for the polypeptide is short at the 5'end of the cDNA. This
is a consequence of reverse transcriptase, an enzyme with
inherently low `processivity` (a measure of the ability of the
enzyme to remain attached to the template during the polymerisation
reaction), failing to complete a DNA copy of the rnRNA template
during 1st strand cDNA synthesis.
[0029] There are several methods available and well known to those
skilled in the art to obtain full-length cDNAs, or extend short
cDNAs, for example those based on the method of Rapid Amplification
of cDNA ends (RACE) (see, for example, Frohman et al., PNAS USA 85,
8998-9002, 1988). Recent modifications of the technique,
exemplified by the Marathon.RTM. technology (Clontech Laboratories
Inc.) for example, have significantly simplified the search for
longer cDNAs. In the Marathon.RTM. technology, cDNAs have been
prepared from mRNA extracted from a chosen tissue and an `adaptor`
sequence ligated onto each end. Nucleic acid amplification (PCR) is
then carried out to amplify the `missing` 5'end of the cDNA using a
combination of gene specific and adaptor specific oligonucleotide
primers. The PCR reaction is then repeated using `nested` primers,
that is, primers designed to anneal within the amplified product
(typically an adaptor specific primer that anneals further 3'in the
adaptor sequence and a gene specific primer that anneals further
5'in the known gene sequence). The products of this reaction can
then be analysed by DNA sequencing and a full-length cDNA
constructed either by joining the product directly to the existing
cDNA to give a complete sequence, or carrying out a separate
full-length PCR using the new sequence information for the design
of the 5'primer.
[0030] Recombinant polypeptides of the present invention may be
prepared by processes well known in the art from genetically
engineered host cells comprising expression systems. Accordingly,
in a further aspect, the present invention relates to expression
systems which comprise a polynucleotide or polynucleotides of the
present invention, to host cells which are genetically engineered
with such expression sytems and to the production of polypeptides
of the invention by recombinant techniques. Cell-free translation
systems can also be employed to produce such proteins using RNAs
derived from the DNA constructs of the present invention.
[0031] For recombinant production host cells can be genetically
engineered to incorporate expression systems or portions thereof
for polynucleotides of the present invention. Introduction of
polynucleotides into host cells can be effected by methods
described in many standard laboratory manuals, such as Davis et al,
Basic Methods in Molecular Biology (1986) and Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989). Preferred such
methods include, for instnce, calcium phosphate transfection,
DEAEdexran mediated transfection, tansvection, microinjection,
cationic lipid-mediated tmnsfection, electroporation, transduction,
scrape loading, ballistic introduction or infection.
[0032] Representative examples of appropriate hosts include
bacterial cells, such as Streptococci, Staphylococci, E. coli,
Streptomyces and Bacillus subtilis cells; fingal cells, such as
yeast cells and Aspergillus cells; insect cells such as Drosophila
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa,
C127, 3T3, BHX, HEK 293 and Bowes melanoma cells; and plant
cells.
[0033] A great ninety of expression systems can be used, for
instance, chromosomal, episomal and virusderived sysrn, e.g.,
vectors derived from bacterial plasmids, from bacteriophage, from
transposons, from yeast episomes, from insertion elements, from
yeast chromosomal elements, from viruses such as bacutculovus,
papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl
pox viruses, pseudoraies viruses and retroviruses, and vectors
derived from combinations thereof, such as those derived from
plasmid and bacteriophage genetic elements, such as cosmids and
phagemids. The expression syste=s may contain control regions that
regulate as well as engender expression. Generally, any system or
ver which is able to naintain, propagate or express a
polynucleotide to produce a polypeptide in a o may be used. The
appropriate nucleotide sequence may be inserted into an expression
systems by any of a variety of wellknown and routine techniques,
such as, for example, those set forth in Sanbok et al., Molecular
Cloning, A Laboratory Manual (supra). Appropriate secretion signals
may be incorporated into the desired polypeptide to allow secretion
of the translated protein into the lumen of the =doplasmic
reticulum, the periplasmic space or the extracellular environment.
These signals may be erogenous to the polypeptide or they may be
heterologous signals.
[0034] If a polypeptide of the present invention is to be expressed
for use in screening assays, it is generally prefer that the
polypeptide be produced at the surface of the cell. In this event,
the cells may be harvest prior to use in the screening assay. If
the polypeptide is secreted into the medium, the mean can be
recovered in order to recover and purify the polypeptide. If
produced intracellularly, the cells must first be lysed before the
polypeptide is recovered.
[0035] Polypeprdes of the present invention can be recovered and
purified from recombinant cell cultures by welloNwn methods
including ammonium sulfte or ethanol precipitation, acid
extraction, anion or cation exhange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Most preferably, high
performance liquid chromatography is employed for purification.
Well known techniques for refolding proteins may be employed to
regenerate active conformation when the polypeptide is deuuured
during intracellular synthesis, isolation and or purification.
[0036] This invfiton also relates to the use of polynucleotides of
the present invention as diagnostic reagents. Detection of a
mutated form of the gene characterised by the polynucleotide of SEQ
ID NO:1 or SEQ ID NO:7. respectivelv which is associated with a
dysfunction will provide a diagnostic tool that can add to, or
define, a diagnosis of a disease, or susceptibility to a disease,
which results from under-expression, overression or altered spatial
or temporal expression of the gene. Individuals carrying mutations
in the gene may be detected at the DNA level by a variety of
techniques.
[0037] Nucleic rids for diagnosis may be obtained from a subject's
cells, such as from blood, urine, saliva, tissue biops or autopsy
material. The genornic DNA mav be used directly for detection or
may be amplified enzymatically by using PCR or other amplification
techniques prior to analysis. RNA or cDNA may also be used in
similar fashion. Deletions and insertions can be detected by a
change in size of the amplified product in comparison to the normal
genotype. Point mutations can be identified by hybridizing
amplified DNA to labeled VANILREP 1 nucleotide sequences. Perfectly
matched sequences can be distiushed from mismatched duplexes by
RNase digestion or by differences in melting temperatures. DNA
sequence differences may also be detected by alterations in
electrophoretic mobility of DNA fragments in gels, with or without
denaturing agents, or by direct DNA sequencing (ee, e.g., Myers et
al., Science (1985) 230:1242). Sequence changes at specific
locations may also be revealed by nuclease protection assays, such
as RNase and S1 protection or the chemical cleavage method (see
Cotton etal., Proc Nati Acad Sci USA (1985)85:4397-4401). In
another embodiment, an array of oligonucleotides probes comprising
VANILREP1 nucleotide sequence or fragments thereof can be
constructed to conduct efficient screening of e.g., genetic
mutations. Array technology methods are well known and have general
applicabilitv and can be used to address a variety of questions in
molecular genetics including gene expression, genetic linkage, and
genetic variability (see for example: M.Chee et al., Science, Vol
274, pp 610-613 (1996)).
[0038] The diagnostic assays offer a process for diagnosing or
determining a susceptibility to the Diseases through detection of
mutation in the VANILREP 1 gene by the methods described. In
addition, such diseases may be diagnosed by methods comprising
determining from a sample derived from a subject an abnormally
decreased or increased level of polypeptide or mRNA. Decreased or
increased expression can be measured at the RNA level using any of
the methods well known in the art for the quantitation of
polynucleotides, such as, for example, nucleic acid amplification,
for instance PCR, RT-PCR, RNase protection, Northern blotting and
other hybridization methods. Assay techniques that can be used to
determine levels of a protein, such as a polypeptide of the present
invention, in a sample derived from a host are wellknown to those
of skill in the art. Such assay methods include radioirnmunoassays,
competitivebinding assays, Western Blot analysis and ELISA
assays.
[0039] Thus in another aspect, the present invention relates to a
diagonostic kit which comprises: (a) a polynucleotide of the
present invention, preferably the nucleotide sequence of SEQ ID
NO:1 or SEQ ID NO:7, respectively or a fragment thereof; (b) a
nucleotide sequence complementary to that of (a); (c) a polypeptide
of the present invention, preferably the polypeptide of SEQ ID NO:2
or SEQ ID NO:8, respectively or a fragment thereof; or (d) an
antibody to a polypeptide of the present invention, preferably to
the polypeptide of SEQ ID NO:2 or SEQ ID NO:8, respectively.
[0040] It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component. Such a kit will be of
use in diagnosing a disease or suspectability to a disease,
particularly pain, chronic pain, neuropathic pain, postoperative
pain, rheumatoid arthritic pain, neuralgia nzropathies, algesia,
nerve injury, ischaemia, neurodegeneration, stroke, incontinence
and inflammicry disorders, amongst others.
[0041] The nucleotide sequences of the present invention are also
valuable for chromosome localisation. The sequece is specifically
targeted to, and can hybridize with, a particular location on an
individual human chnmosome. The mapping of relevant sequences to
chromosomes according to the present invention is an important
first step in correlating those sequences with gene associated
disease. Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromsome
can be correlated with genetic map data. Such data are found in,
for example, V. McKusicl Mendelian Inhentance in Man (available
online through Johns Hopkins University Welch Medical Library). The
relationship between genes and diseases that have been mapped to
the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0042] The differences in the cDNA or genomic sequence between
affected and unaffected individuals can also be determined. If a
mutation is observed in some or all of the affected individuals but
not in any normal individuals, then the mutation is likely to be
the causative agent of the disease.
[0043] The gene of the present invention maps to human chromosome
17p13. The nucleotide sequences of the present invention are also
valuable for tissue localisation. Such techniques allow the
determination of expression patterns of the human VANILREP 1
polypeptides in tissues by detection of the mRNAs that encode them.
These techniques include in situ hybridzizon techniques and
nucleotide amplification techniques, for example PCR. Such
techniques are well known in the art. Results from these studies
provide an indication of the normal functions of the polypeptides
in the organism. In addition, comparative studies of the normal
expression pattern of human VANILREP 1 mRNAs with that of mRNAs
encoded by a human VANILREP 1 gene provide valuable insights into
the role of mutant human VANILREP 1 polypeptdes, or that of
inappropriate expression of normal human VANILREP 1 polypeptides,
in disease. Such inappropriate expression may be of a temporal,
spatial or simply quantitative nature.
[0044] The polypeptides of the invention or their frgents or
atalogs thereof, or cells expressing them, can also be used as
immunogens to produce antibodies immunospecific for polypeptides of
the present invention. The term "immunospecific" means that the
antibodies have substantially greater affinity for the
polypeptetides of the invention than their affinity for other
related polypeptides in the prior art.
[0045] Antibodies generated against polypeptides of the present
invention may be obtained by administering the polypeptdes or
epitopebearing ents, analogs or cells to an animal, preferably a
non-human animal using routine protocols. For preparation of
monoclonal antibodies, any technique which provides antibodies
produced by continuous cell line cultures can be used. Examples
include the hybridoma technique (Kohler, G. and Mistein, C., Nature
(1975) 256:495-497), the trioma technique, the human Bell
hybridorna technique (Kozbor et al., Immunology Today (1983) 4:72)
and the EBV hybridorna technique (Cole et aL, Monoclonal Antibodies
and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985).
[0046] Techniques for the production of single chain antibodies,
such as those described in U.S. Pat. No. 4,946,778, can also be
adapted to produce single chain antibodies to polypeptides of this
invention. Also, transgenic mice, or other organisms, including
other maxnuals, may be used to express humanized antibodies.
[0047] The above-described antibodies may be employed to isolate or
to identify clones expressing the polypeptide or to purify the
polypeptides by affinity chromatography.
[0048] Antibodies against polypeptides of the present invention may
also be employed to treat the Diseases, amongst others.
[0049] In a further aspect, the present invention relates to
genetically engineered soluble fusion proteins comprising a
polypeptide of the present invention, or a fragment thereof, and
various portions of the constant regions of heavy or light chains
of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
Preferred as an immunoglobulin is the constant part of the heavy
chain of human IgG, particularly IgGI, where fusion takes place at
the hinge region. In a particular embodiment, the Fc part can be
removed simply by incorporation of a cleavage sequence which can be
cleaved with blood clotting factor Xa. Furthermore, this invention
relates to processes for the preparation of these fusion proteins
by genetic engineering, and to the use thereof for drug screening,
diagnosis and therapy. A further aspect of the invention also
relates to polynucleotides encoding such fusion proteins. Examples
of fusion protein technology can be found in International Patent
Application Nos. W094/29458 and W094/22914.
[0050] Another aspect of the invention relates to a method for
inducing an immunological response in a mammal which comprises
inoculating the mammal with a polypeptide of the present invention,
adequate to produce antibody and/or T cell immune response to
protect said animal from the Diseases hereinbefore mentioned,
amongst others. Yet another aspect of the invention relates to a
method of inducing immunological response in a mammal which
comprises, delivering a polypeptide of the present invention via a
vector directing expression of the polynucleotide and coding for
the polypeptide in vivo in order to induce such an immunological
response to produce antibody to protect said animal from
diseases.
[0051] A further aspect of the invention relates to an
immunological/vaccine formulation (composition) which, when
introduced into a mammalian host, induces an immunological response
in that mammal to a polypeptide of the present invention wherein
the composition comprises a polypeptide or polynucleotide ofthe
present invention. The vaccine formulation may further comprise a
suitable carrier. Since a polypeptide may be broken down in the
stomach, it is preferably administered parenterally (for instance,
subcutaneous, intramuscular, intravenous, or intradermal
injection). Formulations suitable for parenteral administration
include aqueous and nonaqueous sterile injection solutions which
may contain antioxidants, buffers, bacteriostats and solutes which
render the formulation instonic with the blood of the recipient;
and aqueous and non aqueous sterile suspensions which may include
suspending agents or thickening agents. The formulations may be
presented in unitdose or multidose containers, for example, sealed
ampoules and vials and may be stored in a freezedried condition
requiring only the addition of the sterile liquid carrier
immediately prior to use. The vaccine formulation may also include
adjuvant systems for enhancing the immunogenicity of the
formulation, such as oilin water systems and other systems known in
the art. The dosage will depend on the specific activity of the
vaccine and can be readilv determined by routine
experimentation.
[0052] Polypeptides of the present invention are responsible for
one or more biological functions, including one or more disease
states, in particular the Diseases hereinbefore mentioned. It is
therefore desirous to devise screening methods to identify
compounds which stimulate or which inhibit the function of the
polypeptide. Accordingly, in a further aspect, the present
invention provides for a method of screening compounds to identify
those which stimulate or which inhibit the function of the
polypeptide. In general, agonists or antagonists may be employed
for therapeutic and prophylactic purposes for such Diseases as
hereinbefore mentioned. Compounds may be identified from a variety
of sources, for example, cells, cell-free preparations, chemical
libraries, and natural product mixtures. Such agonists, antagonists
or inhibitors soidentified may be natural or modified substrates,
ligands, receptors, enzymes, etc., as the case may be, of the
polypeptide; or may be structural or flnctional mirnetics thereof
(see Coligan et al., Current Protocols in Emmunology 1(2):Chapter 5
(1991)).
[0053] The screening method may simply measure the binding of a
candidate compound to the polypeptide, or to cells or membranes
bearing the polypeptide, or a fusion protein thereof by means of a
label directly or indirectly associated with the candidate
compound. Alternatively, the screening method mav involve
competition with a labeled competitor. Further, these screening
methods may test whether the candidate compound results in a signal
generated by activation or inhibition of the polypeptide, using
detection systems appropriate to the cells bearing the polypeptide.
Inhibitors of activation are generally assayed in the presence of a
known agonist and the effect on activation by the agonist by the
presence of the candidate compound is observed. Constitutively
active polypeptides may be employed in screening methods for
inverse agonists or inhibitors, in the absence of an agonist or
inhibitor, by testing whether the candidate compound results in
inhibition of activation of the polypeptide. Further, the screening
methods may simply comprise the steps of mixing a candidate
compound with a solution containing a polypeptide of the present
invention, to form a mixture, measuring VANILREP 1 activity in the
mixture, and comparing the VANILREP 1 activity of the mixture to a
standard. Fusion proteins, such as those made from Fc portion and
VANILREP 1 polypeptide, as hereinbefore described, can also be used
for highthroughput screening assays to identify antagonists for the
polypeptide of the present invention (see D. Bennett et al., J Mol
Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,
270(16):9459-9471 (1995)).
[0054] The polynucleotides, polypeptides and antibodies to the
polypeptide of the present invention may also be used to configure
screening methods for detecting the effect of added compounds on
the production of MRNA and polypeptide in cells. For example, an
ELISA assay may be constructed for measuring secreted or cell
associated levels of polypeptide using monoclonal and polyclonal
antibodies by standard methods known in the art. This can be used
to discover agents which may inhibit or enhance the production of
polypeptide (also called antagonist or agonist, respectively) from
suitably manipulated cells or tissues.
[0055] The polypeptide may be used to identify membrane bound or
soluble receptors, if any, through standard receptor binding
techniques known in the art. These include, but are not limited to,
ligand binding and crosslinking assays in which the polypeptide is
labeled with a radioactive isotope (for instance, .sup.125I),
chemically modified (for instance, biotinylated), or fused to a
peptide sequence suitable for detection or purification, and
incubated with a source of the putative receptor (cells, cell
membranes, cell supernatants, tissue extracts, bodily fluids).
Other methods include biophysical techniques such as surface
plasmon resonance and spectroscopy. These screening methods may
also be used to identifi agonists and antagonists of the
polypeptide which compete with the binding of the polypeptide to
its receptors, if any. Standard methods for conducting such assays
are well understood in the art.
[0056] Examples of potential polypeptide antagonists include
antibodies or, in some cases, oligonucleotides or proteins which
are closely related to the ligands, substrates, receptors, enymes,
etc., as the case may be, of the polypeptide, e.g., a fraent of the
ligands, substrates, receptors, enzymes, etc.; or small molecules
which bind to the polypeptide of the present invention but do not
elicit a response, so that the activity of the polypeptide is
prevented.
[0057] Thus, in another aspect, the present invention relates to a
screening kit for identifyg agonists, antagonists, ligands,
receptors, substrates, enzymes, etc. for polypeptides of the
present invention; or compounds which decrease or enhance the
production of such polypeptides, which comprises: (a) a polypeptide
of the present invention; (b) a recombinant cell expressing a
polypeptide of the present invention; (c) a cell membrane
expressing a polypeptide of the present invention; or (d) antibody
to a polypeptide of the present invention; which polypeptide is
preferably that of SEQ ID NO:2 or SEQ ID NO:8.
[0058] It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component.
[0059] It will be readily appreciated by the skilled artisan that a
polypeptide of the present invention may also be used in a method
for the structurebased design of an agonist, antagonist or
inhibitor of the polypeptide, by: (a) determining in the first
instance the threedimensional structure of the polypeptide; (b)
deducing the threedimensional structure for the likely reactive or
binding site(s) of an agonist, antagonist or inhibitor; (c)
synthesing candidate compounds that are predicted to bind to or
react with the deduced binding or reactive site; and (d) testing
whether the candidate compounds are indeed agonists, antagonists or
inhibitors. It will be further appreciated that his will normally
be an iterative process.
[0060] In a furter aspect, the present invention provides methods
of treating abnormal conditions such as, for insance, pain, chronic
pain, neuropathic pain, postoperative pain, rheumatoid arthritic
pain, neuralgia, neuropathies, algesia, nerve injury, ischaemia,
neurodegeneration, stroke, incontinence and inflammatory disorders,
related to either an excess of, or an underxpression of, VANILREP 1
polypeptide activity.
[0061] If the activity of the polypeptide is in excess, several
approaches are available. One approach comprises administering to a
subject in need thereof an inhibitor compound (antagonist) as
hereinabove described, optionally in combination with a
phannaceutically acceptable carrier, in an amount effective to
inhibit the finction of the polypeptide, such as, for example, by
blocking the binding of ligands, substrates, receptors, enzymes,
etc., or by inhibiting a second signal, and thereby alleviating the
abnormal condition. In another approach, soluble forms of the
polypeptides still capable of binding the ligand, substrate,
enzymes, receptors, etc. in competition with endogenous polypeptide
may be administered. Typical examples of such competitors include
fragments of the VANILREP 1 polypeptide.
[0062] In still another approach, expression of the gene encoding
endogenous VANILREP 1 polypeptide can be inhibited using expression
blocking techniques. Known such techniques involve the use of
antisense sequences, either internally generated or externally
administered (see, for example, O'Connor, J Neurochem (1991) 56:560
in Oligodeoxynucleotides as Antisense ihibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988)). Alternatively,
oligonucleotides which form triple helices ("triplexes") with the
gene can be supplied (see, for example, Lee et al., Nucleic Acids
Res (1979) 6:3073; Cooney et aL, Science (1988) 241:456; Dervan
etal., Science (1991) 251:1360). These oligomers can be
administered per se or the relevant oligomers can be expressed in
vivo. Synthetic antisense or triplex oligonucleotides may comprise
modified bases or modified backbones. Examples of the latter
include methvlphosphonate, phosphorothioate or peptide nucleic acid
backbones. Such backbones are incorporated in the antisense or
triplex oligonucleotide in order to provide protection from
degradation bv nucleases and are well known in the art. Antisense
and triplex molecules synthesised with these or other modified
backbones also form part of the present invention.
[0063] In addition, expression of the human VANILREP 1 polypeptide
may be prevented by using ribozymes specific to the human VANILREP
1 mRNA sequence. Ribozymes are catalytically active RNAs that can
be natural or synthetic (see for example Usman, N, et al., Curr.
Opin. Struct. Biol (1996) 6(4), 527-33.) Synthetic ribozymes can be
designed to specifically cleave human VANILREP 1 mRNAs at selected
positions thereby preventing translation of the human VANILREP 1
mRNAs into functional polypeptide. Ribozyvmes may be synthesised
with a natural ribose phosphate backbone and natural bases, as
normally found in RNA molecules. Alternatively the ribosymes may be
synthesised with nonnatural backbones to provide protection from
ribonuclease degradation, for example, 2'-O-methyl RNA, and may
contain modified bases.
[0064] For treating abnormal conditions related to an
underexpression of VANILREP 1 and its activity, several approaches
are also available. One approach comprises administering to a
subject a therapeutically effective amount of a compound which
activates a polypeptide of the present invention, i.e., an agonist
as described above, in combination vith a pharmaceutically
acceptable carrier, to thereby alleviate the abnormal condition.
Altemnatively, gene therapy may be employed to effect the
endogenous production of VANILREP1 by the relevant cells in the
subject. For example, a polynucleotide of the invention may be
engineered for expression in a replication defective retroviral
vector, as discussed above. The retroviral expression construct may
then be isolated and introduced into a packaging cell transduced
with a retroviral plasnud vector containing RNA encoding a
polypeptide of the present invention such that the packaging cell
now produces infectious viral particles containing the gene of
interest. These producer cells may be administered to a subject for
engineering cells in vivo and expression of the polypeptide in
vivo. For an overview of gene therapy, see Chapter 20, Gene Therapy
and other Molecular Geneticbased Therapeutic Approaches, (and
references cited therein) in Humaan Molecular Genetics, T Strachan
and A P Read, BIOS Scientific Publishers Ltd (1996). Another
approach is to administer a therapeutic amount of a polypeptide of
the present invention in combination with a suitable pharmaceutical
carrier.
[0065] In a further aspect, the present invention provides for
pharmaceutical compositions comprising a therapeutically effective
amount of a polypeptide, such as the soluble form of a polypeptide
of the present invention, agonist/antagonist peptide or small
molecule compound, in combination with a pharmaceutically
acceptable carrier or excipient. Such carriers include, but are not
limited to, saline, buffered saline, dextrose, water, glycerol,
ethanol, and combinations thereof The invention further relates to
pharmaceutical packs and kits comprising one or more containers
filled with one or more of the ingredients of the aforementioned
compositions of the invention. Polypeptides and other compounds of
the present invention may be employed alone or in conjunction with
other compounds, such as therapeutic compounds.
[0066] The composition will be adapted to the route of
administraion, for instance by a systemic or an oral route.
Preferred forms of systemic acitnistraion include injection,
typically by intravenous injection. Other injection routes, such as
subcutaneous, intramuscular, or intraperitoneal, can be used.
Alternative means for systemic administration include transmucosal
and transdermal administration using penetrants such as bile salts
or fusidic acids or other detergents. In addition, if a polypeptide
or other compounds of the present invention can be formulated in an
enteric or an encapsulated formulation, oral administration mav
also be possible. Administr ion of these compounds may also be
topical and/or localized, in the form of salves, pastes, gels, and
the like.
[0067] The dosage range required depends on the choice of peptide
or other compounds of the present invention, the route of
administration, the nature of the formulation, the nature of the
subject's condition, and the judgment of the attending
practitioner. Suitable dosages, however, are in the range of
0.1-100 .mu.g/kg of subject Wide variations in the needed dosage,
however, are to be expected in view of the variety of compounds
available and the differing efficiencies of various routes of
administration. For example, oral administation would be expected
to require higher dosages than administration by intravenous
injection. Variations in these dosage levels can be adjusted using
standard empirical routines for optimization, as is well understood
in the art.
[0068] Polypeptides used in treatment can also be generated
endogenously in the subject, in treatment modalities often referred
to as "gene therapy" as described above. Thus, for example, cells
from a subject may be engineered with a polynucleotide, such as a
DNA or RNA, to encode a polypeptide ex vivo, and for example, by
the use of a retroviral plasmid vector. The cells are then
introduced into the subject.
[0069] Polynucleotide and polypeptide sequences form a valuable
information resource with which to identify further sequences of
similar homology. This is most easily facilitated by storing the
sequence in a computer readable medium and then using the stored
data to search a sequence database using well known searching
tools, such as those in the GCG and Lasergene software packages.
Accordingly, in a further aspect, the present invention provides
for a computer readable medium having stored thereon a
polynucleotide comprising the sequence of SEQ ID NO: 1 or SEQ ID
NO:7 and/or a polypeptide sequence encoded thereby.
[0070] The following definitions are provided to facilitate
understanding of certain terms used frequently hereinbefore.
[0071] "Antibodies" as used herein includes polyclonal and
monoclonal antibodies, chimeric, single chain, and humanized
antibodies, as well as Fab fragments, including the products of an
Fab or other immunoglobulin expression library.
[0072] "Isolated" means altered "by the hand of man" from the
natural state. If an "isolated" composition or substance occurs in
nature, it has been changed or removed from its original
environment, or both. For example, a polynucleotide or a
polypeptide naturally present in a living animal is not "isolated,"
but the same polynucleotide or polypeptide separated from the
coexisting materials of its natural state is "isolated", as the
term is employed herein.
[0073] "Polynucleotide" generally refers to any polyribonucleotide
or polydeoxribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. "Polynucleotides" include, without limitation,
single and doublestranded DNA, DNA that is a mixture of single and
double stranded regions, single and doublestranded RNA, and RNA
that is mixture of single and doublestranded regions, hybrid
molecules comprising DNA and RNA that may be singlestranded or,
more typically, doublestranded or a mixture of single and
doublestranded regions. In addition, "polynucleotide" refers to
triplestranded regions comprising RNA or DNA or both RNA and DNA.
The term "polynucleotide" also includes DNAs or RNAs containing one
or more modified bases and DNAs or RNAs with backbones modified for
stability or for other reasons. "Modified" bases include, for
example, tritylated bases and unusual bases such as inosine. A
variety of modifications may be made to DNA and RNA; thus,
"polynucleotide" embraces chemically, enzymatically or
metabolically modified forms of polynucleotides as typically found
in nature, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells. "Polynucleotide" also embraces
relatively short polynucleotides, often referred to as
oligonucleotides.
[0074] "Polypeptide" refers to any peptide or protein comprising
two or more amino acids joined to each other by peptide bonds or
modified peptide bonds, i.e., peptide isosteres. "Polypeptide"
refers to both short chains, commonly referred to as peptides,
oligopeptides or oligomers, and to longer chains, generally
referred to as proteins. Polypeptides may contain amino acids other
than the 20 gene-encoded amino acids. "Polypeptides" include amino
acid sequences modified either by natural processes, such as
post-translational processing, or by chemical modification
techniques which are well known in the art. Such modifications are
well described in basic texts and in more detailed monographs, as
well as in a voluminous research literature. Modifications may
occur anywhere in a polypeptide, including the peptide backbone,
the amino acid side-chains and the amino or carboxyl termini. It
will be appreciated that the same type of modification may be
present to the same or varying degrees at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched as a result of
ubiquitination, and they may be cyclic, with or without branching.
Cyclic, branched and branched cyclic polypeptides may result from
posttranslation natural processes or may be made by synthetic
methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, biotinylation, 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, crosslinking, cyclization, disulfide bond
formation, demethylation, formation of covalent crosslinks,
formation of cystine, formation of pyroglutamate, formylation,
gammacarboxylation, 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 (see,
for instance, Proteins-Structure and Molecular Properties, 2nd Ed.,
T. E. Creighton, W. H. Freeman and Company, New York, 1993; Wold,
F., Post-translational Protein Modifications: Perspectives and
Prospects, pgs. 1-12 in Post-translational Covalent Modification of
Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983;
Seifter et al, "Analysis for protein modifications and nonprotein
cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al.,
"Protein Synthesis: Post-translational Modifications and Aging",
Ann NY Acad Sci (1992) 663:48-62).
[0075] "Variant" refers to a polynucleotide or polypeptide that
differs from a reference polynucleotide or polypeptide, but retains
essential properties. A typical variant of a polynucleotide differs
in nucleotide sequence from another, reference polynucleotide.
Changes in the nucleotide sequence of the variant may or may not
alter the amino acid sequence of a polypeptide encoded by the
reference polynucleotide. Nucleotide changes may result in amino
acid substitutions, additions, deletions, fusions and truncations
in the polypeptide encoded by the reference sequence, as discussed
below. A typical variant of a polypeptide differs in amino acid
sequence from another, reference polypeptide. Generally,
differences are limited so that the sequences of the reference
polypeptide and the variant are closely similar overall and, in
many regions, identical. A variant and reference polypeptide may
differ in amino acid sequence by one or more substitutions,
additions, deletions in any combination. A substituted or inserted
amino acid residue may or may not be one encoded by the genetic
code. A variant of a polynucleotide or polypeptide may be a
naturally occurring such as an allelic variant, or it may be a
variant that is not known to occur naturally. Nonnaturally
occurring variants of polynucleotides and polypeptides may be made
by mutagenesis techniques or by direct synthesis.
[0076] "Identity," as known in the art, is a relationship between
two or more polypeptide sequences or two or more polynucleotide
sequences, as determined by comparing the sequences. In the art,
"identity" also means the degree of sequence relatedness between
polypeptide or polynucleotide sequences, as the case may be, as
determined by the match between strings of such sequences.
"Identity" and "similarity" can be readily calculated by known
methods, including but not limited to those described in
(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 I, 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; and Carillo, H., and
Lipman, D., SIAM J Applied Math., 48: 1073 (1988). Preferred
methods to determine identity are designed to give the largest
match between the sequences tested. Methods to determine identity
and similarity are codified in publicly available computer
programs. Preferred computer program methods to determine identity
and similarity between two sequences include, but are not limited
to, the GCG program package (Devereux, J., et al., Nucleic Acids
Research 12(I): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul,
S.F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X
program is publicly available from NCBI and other sources (BLAST
Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;
Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990). The well
known Smith Waterman algorithm may also be used to determine
identity.
[0077] Preferred parameters for polypeptide sequence comparison
include the following: 1) Algorithm: Needleman and Wunsch, J. Mol
Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 from
Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919
(1992) Gap Penalty: 12 Gap Length Penalty: 4
[0078] A program useful with these parameters is publicly available
as the "gap" program from Genetics Computer Group, Madison Wis. The
aforementioned parameters are the default parameters for peptide
comparisons (along with no penalty for end gaps).
[0079] Preferred parameters for polynucleotide comparison include
the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48:
443-453 (1970) Comparison matrix: matches=+10, mismatch=0 Gap
Penalty: 50 Gap Length Penalty: 3
[0080] Available as: The "gap" program from Genetics Computer
Group, Madison Wis. These are the default parameters for nucleic
acid comparisons.
[0081] By way of example, a polynucleotide sequence of the present
invention may be identical to 20 the reference sequence of SEQ ID
NO: 1, that is be 100% identical, or it may include up to a certain
integer number of nucleotide alterations as compared to the
reference sequence. Such alterations are selected from the group
consisting of at least one nucleotide deletion, substitution,
including transition and transversion, or insertion, and wherein
said alterations may occur at the 5'or 3'terminal positions of the
reference nucleotide sequence or anywhere between those terminal 25
positions, interspersed either individually among the nucleotides
in the reference sequence or in one or more contiguous groups
within the reference sequence. The number of nucleotide alterations
is determined by multiplying the total number of nucleotides in SEQ
ID NO: 1 by the numerical percent of the respective percent
identity (divided by 100) and subtracting that product from said
total number of nucleotides in SEQ ID NO: 1, or:
n.sub.n.ltoreq.x.sub.n-(x.sub.n.multidot.y),
[0082] wherein n.sub.n is the number of nucleotide alterations,
X.sub.n is the total number of nucleotides in SEQ ID NO:1, and y
is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90
for 90%, 0.95 for 95%,etc., and wherein any noninteger product of
x.sub.n and y is rounded down to the nearest integer prior to
subtracting it from x.sub.n. Alterations of a polynucleotide
sequence encoding the polypeptide of SEQ ID NO:2 may create
nonsense, missense or framneshift mutations in this coding sequence
and thereby alter the polypeptide encoded by the polynucleotide
following such alterations.
[0083] Similarly, a polypeptide sequence of the present invention
may be identical to the reference sequence of SEQ ID NO:2, that is
be 100% identical, or it may include up to a certain integer number
of amino acid alterations as compared to the reference sequence
such that the % identity is less than 100%. Such alterations are
selected from the group consisting of at least one amino acid
deletion, substitution, including conservative and non-conservative
substitution, or insertion, and wherein said alterations may occur
at the amino-or carboxy-terminal positions of the reference
polypeptide sequence or anywhere between those terminal positions,
interspersed either individually among the amino acids in the
reference sequence or in one or more contiguous groups within the
reference sequence. The number of amino acid alterations for a
given % identity is determined by multiplying the total number of
amino acids in SEQ ID NO:2 by the numerical percent of the
respective percent identity(divided by 100) and then subtracting
that product from said total number of amino acids in SEQ ID NO:2,
or:
n.sub.a.ltoreq.x.sub.a-(x.sub.a.multidot.y),
[0084] wherein n.sub.a is the number of amino acid alterations,
X.sub.a is the total number of amino acids in SEQ ID NO:2, and y
is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and
wherein any non-integer product of X.sub.a and y is rounded down to
the nearest integer prior to subtracting it from "Homolog" is a
generic term used in the art to indicate a polynucleotide or
polypeptide sequence possessing a high degree of sequence
relatedness to a subject sequence. Such relatedness may be
quantified by determining the degree of identity and/or similarity
between the sequences being compared as hereinbefore described.
Falling within this generic term are the terms "ortholog", meaning
a polynucleotide or polypeptide that is the functional equivalent
of a polynucleotide or polypeptide in another species, and
"paralog" meaning a functionally similar sequence when considered
within the same species.
[0085] "Fusion protein" refers to a protein encoded by two, often
unrelated, fused genes or fragments thereof. In one example, EP-A-0
464 discloses fusion proteins comprising various portions of
constant region of immunoglobulin molecules together with another
human protein or part thereof. In many cases, employing an
immunoglobulin Fc region as a part of a fusion protein is
advantageous for use in therapy and diagnosis resulting in, for
example, improved pharmacokinetic properties [see, e.g., EP-A 0232
262]. On the other hand, for some uses it would be desirable to be
able to delete the Fc part after the fusion protein has been
expressed, detected and purified.
[0086] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
EXAMPLE
[0087] Example 1 Electrophysiological Studies
[0088] Xenopus laevis oocyte removal and dissociation were
performed. Injections of cDNA for VANILREP1 were made into the
nuclei of defolliculated oocytes (1.5ng cDNA/oocyte). After
injection the oocytes were incubated between 19-22.degree. C. in
modified Barth's solution (MBS) plus gentamycin (0.1mg/ml, pH 7.4)
and used for electrophysiological recordings within 2-4 days.
[0089] For electrophysiological recordings oocytes were placed in a
recording chamber and continuously perfused with a solution
containing in mM: NaCl 88, KCI 1, NaHCO.sub.3 2.4, HEPES 15,
MgCI.sub.2 1, BaCI.sub.2 0.1. (14 ml min.sup.-1). Solution was
applied using large bore tubing (internal diameter 1.5mm) which
facilitated rapid solution exchange half-time 350 -1000 ms).
Oocytes were held under voltage clamp at -60mV using the
two-electrode voltage-clamp technique. Electrodes were low
resistance (0.5-3 M.OMEGA.) and were filled with 3 M KCI.
[0090] Currents were evoked in response to application of capsaicin
applied through the perfusion system until the maximum current
amplitude was reached. FIG. 1 shows currents evoked from a cell in
response to capsaicin over a range of concentrations. FIG. 2 shows
inhibition of the capsaicin evoked response (1uM) by capsazepine
(10uM). The inhibition is reversible with extended washing.
1 SEQ ID NO:1 CAGCGTCGGGTGCAGTTTGGCCGGAGGTTGCAGTGAGCAGAGATTG-
CCCCATTGCACTCT AGTCTGGGCGACAGGGTGAGACACACACACACAGACACACACA-
CACACACACACACACAC ACACAAGCCTAAACATTCRAGGCCAGGATGCTTGACAGAT-
GTTGATTCATAAAAATGACA AAAAGCACAAAATCCAAAATCTCGTATAAGCTCAGTG-
GCTGTGGCAGCGAGGTTGAAGAG CAAAGGCAGGCCAGGCACCTGGCTGATGATGTGT-
GGACCCGTTGCACAGCAGGGCCCCGC AGTGCGGTGTGGGTGTGGGTGTGGGTGGGCC-
AGTYTCTGCCGCTCACCCTATTCCAGGGA CACAGTCTGCTTGGCTCTTCTGGACTGA-
GCCATCCTCATCACCGAGATCCTCCCTGAATT CAGCCCACGACAGCCACCCCGGCCG-
TTTTCCTTGTTCTGTGTGGGGAGGGAGGCAGCGCG
GTGGTTATCAACCTCACCCTGCAGAGGAGGCACCTGAGGCCCAGAGACGAGGAGGGATGG
GTCTAACCCAGAACCACAGATGGCTCTGAGCCGGGGGCCTGTCCACCCTCCCAGGCCGAC
GTCAGTGGCCGCAGGACTGCCTGGGCCCTGCTAGGCCTGCTCACCTCTGAGGCCTCTGGG
GTGAGAGGTTCAGTCCTGGAAACACTTCAGTTCTAGGGGGCTGGGGGCAGCAGCAAGTTG
GAGTTTTGGGGTACCCTGCTTCACAGGGCCCTTGGCAAGGAGGGCAGGTGGGGTCTAAGG
ACAAGCAGTCCTTACTTTGGGAGTCAACCCCGGCGTGGTGGCTGCTGCAGGTTGCAC- ACT
GGGCCACAGAGGATCCAGCAAGGATGAAGAAATGGAGCAGCACAGACTTGGGGG- CAGCTG
CGGACCCACTCCAAAAGGACACCTGCCCAGACCCCCTGGATGGAGACCCTA- ACTCCAGGC
CACCTCCAGCCAAGCCCCAGCTCTCCACGGCCAAGAGCCGCACCCGGC- TCTTTGGGAAGG
GTGACTCGGAGGAGGCTTTCCCGGTGGATTGCCCTCACGAGGAAG- GTGAGCTGGACTCCT
GCCCGACCATCACAGTCAGCCCTGTTATCACCATCCAGAGGC- CAGGAGACGGCCCCACCG
GTGCCAGGCTGCCCTCCCAGGACTCTGTCGCCGCCAGCA- CCGAGAAGACCCTCAGGCTCT
ATGATCGCAGGAGTATCTTTGAAGCCGTTGCTCAGA- ATAACTGCCAGGATCTGGAGAGCC
TGCTGCTCTTCCTGCAGAAGAGCAAGAAGCACC- TCACAGACAACGAGTTCAAAGACCCTG
AGACAGGGAAGACCTGTCTGCTGAAAGCCA- TGCTCAACCTGCACGACGGACAGAACACCA
CCATCCCCCTGCTCCTGGAGATCGCGC- GGCAAACGGACAGCCTGAAGGAGCTTGTCAACG
CCAGCTACACGGACAGCTACTACA- AGGGCCAGACAGCACTGCACATCGCCATCGAGAGAC
GCAACATGGCCCTGGTGACCCTCCTGGTGGAGAACGGAGCAGACGTCCAGGCTGCGGCCC
ATGGGGACTTCTTTAAGAAAACCAAAGGGCGGCCTGGATTCTACTTCGGTGAACTGCCCC
TGTCCCTGGCCGTGCGCACCAACCAGCTGGGCATCGTGAAGTTCCTGCTGCAGAACTCCT
GGCAGACGGCCGACATCAGCGCCAGGGACTCGGTGGGCAACACGGTGCTGCACGCCCTGG
TGGAGGTGGCCGACAACACGGCCGACAACACGAAGTTTGTGACGAGCATGTACAATGAGA
TTCTGATCCTGGGGGCCAAACTGCACCCGACGCTGAAGCTGGAGGAGCTCACCAACA- AGA
AGGGAATGACGCGCCTGGCTCTGGCAGCTGGGACCGGGAAGATCGGGGTCTTGG- CCTATA
TTCTCCAGCGGGAGATCCAGGAGCCCGAGTGCAGGCACCTGTCCAGGAAGT- TCACCGAGT
GGGCCTACGGGCCCGTGCACTCCTCGCTGTACGACCTGTCCTGCATCG- ACACCTGCGAGA
AGAACTCGGTGCTGGAGGTGATCGCCTACAGCAGCAGCGAGACCC- CTAATCGCCACGACA
TGCTCTTGGTGGAGCCGCTGAACCGACTCCTGCAGGACAAGT- GGGACAGATTCGTCAAGC
GCATCTTCTACTTCAACTTCCTGGTCTACTGCCTGTACA- TGATCATCTTCACCATGGCTG
CCTACTACAGGCCCGTGGATGGCTTGCCTCCCTTTA- AGATGGAAAAAACTGGAGACTATT
TCCGAGTTACTGGAGAGATCCTGTCTGTGTTAG- GAGGAGTCTACTTCTTTTTCCGAGGGA
TTCAGTATTTCCGTCAGAGGCGGCCGTCGA- TGAAGACCCTGTTTGTGGACAGCTACAGTG
AGATGCTTTTCTTTCTGCAGTCACTGT- TCATGCTGGCCACCGTGGTGCTGTACTTCAGCC
ACCTCAAGGAGTATGTGGCTTCCA- TGGTATTCTCCCTGGCCTTGGGCTGGACCAACATGC
TCTACTACACCCGCGGTTTCCAGCAGATGGGCATCTATGCCGTCATGATAGAGAAGATGA
TCCTGAGAGACCTGTGCCGTTTCATGTTTGTCTACATCGTCTTCTTGTTCGGGTTTTCCA
CAGCGGTGGTGACGCTGATTGAAGACGGGAAGAATGACTCCCTGCCGTCTGAGTCCACGT
CGCACAGGTGGCGGGGGCCTGCCTGCAGGCCCCCCGATAGCTCCTACAACAGCCTCTACT
CCACCTGCCTGGAGCTGTTCAAGTTCACCATCGGCATGGGCGACCTGGAGTTCACTGAGA
ACTATGACTTCAAGGCTGTCTTCATCATCCTGCTGCTGGCCTATGTAATTCTCACCT- ACA
TCCTCCTGCTCAACATGCTCATCGCCCTCATGGGTGAGACTGTCAACAAGATCG- CACAGG
AGAGCAAGAACATCTGGAAGCTGCAGAGAGCCATCACCATCCTGGACACGG- AGAAGAGCT
TCCTTAAGTGCATGAGGAAGGCCTTCCGCTCAGGCAAGCTGCTGCAGG- TGGGGTACACAC
CTGATGGCAAGGACGACTACCGGTGGTGCFTCAGGGTGGACGAGG- TGAACTGGACCACCT
GGAACACCAACGTGGGCATCATCAACGAAGACCCGGGCAACT- GTGAGGGCGTCAAGCGCA
CCCTGAGCTTCTCCCTGCGGTCAAGCAGAGTTTCAGGCA- GACACTGGAAGAACTTTGCCC
TGGTCCCCCTTTTAAGAGAGGCAAGTGCTCGAGATA- GGCAGTCTGCTCAGCCCGAGGAAG
TTTATCTGCGACAGTTTTCAGGGTCTCTGAAGC- CAGAGGACGCTGAGGTCTTCAAGAGTC
CTGCCGCTTCCGGGGAGAAGTGAGGACGTC- ACGCAGACAGCACTGTCAACACTGGGCCTT
AGGAGACCCCGTTGCCACGGGGGGCTG- CTGAGGGAACACCAGTGCTCTGTCAGCAGCCTG
GCCTGGTCTGTGCCTGCCCAGCAT- GTTCCCAAATCTGTGCTGGACAAGCTGTGGGAAGCG
TTCTTGGAAGCATGGGGAGTGATGTACATCCAACCGTCACTGTCCCCAAGTGAATCTCCT
AACAGACTTTCAGGTTTTTACTCACTTTACTAAACAGTKTGGATGGTCAGTCTCTACTGG
GACATGTTAGGCCCTTGTTTTCTTTGATTTTATTCTTTTTTTTGAGACAGAATTTCACTC
TTCTCACCCAGGCTGGAATGCAGTGGCACAATTTTGGCTCCCTGCAACCTCCGCCTCCTG
GATTCCAGCAATTCTCCTGCCTCGGCTTCCCAAGTAGCTGGGATTACAGGCACGTGCCAC
CATGTCTGGCTAATTTTTTGTATTTTTTTAATAGATATGGGGTTTCGCCATGTTGGC- CAG
GCTGGTCTCGAACTCCTGACCTCAGGTGATCCGCCCACCTCGGCCTCCCAAAGT- GCTGGG
ATTACAGGTGTGAGCCTCCACACCTGGCTGTTTTCTTTGATTTTATTCTTT- TTTTTTTTT
TCTGTGAGACAGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAGTGG- TGTGATCTTGGC
TCACTGCAACTTCTGCCTCCCGGGTTCAAGCGATTCTTCTGCTTC- AGTCTCCCAAGTAGC
TTGGATTACAGGTGAGCACTACCACGCCCGGCTAATTTTTGT- ATTTTTAATARAGACGGG
GTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCTTGAC- CTCAGGTGATCTGCCCGCCTT
GGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCG- CTGCGCTCGGCCTTCTTTGATTTT
ATATTATTAGGAGCAAAAGTAAATGAAGCCCAG- GAAAACACCTTTGGGAACAAACTCTTC
CTTTGATGGAAAATGCAGAGGCCCTTCCTC- TCTGTGCCGTGCTTGCTCCTCTTACCTGCC
CGGGTGGTTTGGGGGTGTTGGTGTTTC- CTCCCTGGAGAAGATGGGGGAGGCTGTCCCACT
CCCAGCTCTGGCAGAATCAAGCTG- TTGCAGCAGTGCCTTCTTCATCCTTCCTTACGATCA
ATCACAGTCTCCAGAAGATCAGCTCAATTGCTGTGCAGGTTAAAACTACAGAACCACATC
CCAAAGGTACCTGGTAAGAATGTTTGAAAGATCTTCCATTTCTAGGAACCCCAGTCCTGC
TTCTCCGCAATGGCACATGCTTCCACTCCATCCATACTGGCATCCTCAAATAAACAGATA
TGTATACAATAAAAAAAAAAAAAAAAAAAARRGCGGCCGCTGAATTCTAGACCTGCCCGG
GCG
[0091]
2 SEQ ID NO:2 MKKWSSTDLGAAADPLQKDTCPDPLDGDPNSRPPPAKPQLSTAKSR-
TRLFGKGDSEEAFP VDCPHEEGELDSCPTITVSPVITIQRPGDGPTGARLLSQDSVA-
ASTEKTLRLYDRRSIFE AVAQNNCQDLESLLLFLQKSKKHLTDNEFKDPETGKTCLL-
KAMLNLHDGQNTTIPLLLEI ARQTDSLKELVNASYTDSYYKGQTALHIAIERRNMAL-
VTLLVENGADVQAAAHGDFFKKT KGRPGFYFGELPLSLAACTNQLGIVKFLLQNSWQ-
TADISARDSVGNTVLHALVEVADNTA DNTKFVTSMYNEILILGAKLHPTLKLEELTN-
KKGMTPLALAAGTGKIGVLAYILQREIQE PECRHLSRKFTEWAYGPVHSSLYDLSCI-
DTCEKNSVLEVIAYSSSETPNRHDMLLVEPLN RLLQDKWDRFVKRIFYFNFLVYCLY-
MIIFTMAAYYRPVDGLPPFKMEKTGDYFRVTGEIL
SVLGGVYFFFRGIQYFLQRRPSMKTLFVDSYSEMLFFLQSLFMLATVVLYFSHLKEYVAS
MVFSLALGWTNMLYYTRGFQQMGIYAVMIEICILRDLCRFMFVYIVFLFGFSTAVVTLIE
DGKNDSLPSESTSHRWRGPACRPPDSSYNSLYSTCLELFKFTIGMGDLEFTENYDFKAVF
IILLLAYVILTYILLLNMLIALMGETVNKIAQESKNIWKLQRAITILDTEKSFLKCMRKA
FRSGKLLQVGYTPDGKDDYRWCFRVDEVNWTTWNTNVGIINEDPGNCEGVKRTLSFSLRS
SRVSGRHWKNFALVPLLREASARDRQSAQPEEVYLRQFSGSLKPEDAEVFKSPAASG- EK
[0092]
3 SEQ ID NO:3 CAGCGTCGGGTGCAGTTTGGCCGGAGGTTGCAGTGAGCAGAGATTG-
CCCCATTGCACTCT AGTCTGGGCGACAGGGTGAGACACACACACACAGACACACACA-
CACACACACACACACAC ACACAAGCCTAAACATTCRAGGCCAGGATGCTTGACAGAT-
GTTGATTCATAAAAATGACA AAAAGCACAAAATCCAAAATCTCGTATAAGCTCAGTG-
GCTGTGGCAGCGAGGTTGAAGAG CAAAGGCAGGCCGGGCACCTGGCTGATGATGTGT-
GGACCCGTTGCACAGCAGGGCCCCGC AGTGCGGTGTGGGTGTGGGTGTGGGTGGGCC-
AGTYTCTGCCGCTCACCCTATTCCAGGGA CACAGTCTGCTTGGCTCTTCTGGACTGA-
GCCATCCTCATCACCGAGATCCTCCCTGAATT CAGCCCACGACAGCCACCCCGGCCG-
TTTTCCTTGTTCTGTGTGGGGAGGGAGGCAGCGCG
GTGGTTATCAACCTCACCCTGCAGAGGAGGCACCTGAGGCCCAGAGACGAGGAGGGATGG
GTCTAACCCAGAACCACAGATGGCTCTGAGCCGGGGGCCTGTCCACCCTCCCAGGCCGAC
GTCAGTGGCCGCAGGACTGCCTGGGCCCTGCTAGGCCTGCTCACCTCTGAGGCCTCTGGG
GTGAGAGGTTCAGTCCTGGAAACACTTCAGTTCTAGGGGGCTGGGGGCAGCAGCAAGTTG
GAGTTTTGGGGTACCCTGCTTCACAGGGCCCTTGGCAAGGAGGGCAGGTGGGGTCTAAGG
ACAAGCAGTCCTTACTTTGGGAGTCAACCCCGGCGTGGTGGCTGCTGCAGGTTGCAC- ACT
GGGCCACAGAGGATCCAGCAAGGATGAAGAAATGGAGCAGCACAGACTTGGGGG- CAGCTG
CGGACCCACTCCAAAAGGACACCTGCCCAGACCCCCTGGATGGAGACCCTA- ACTCCAGGC
CACCTCCAGCCAAGCCCCAGCTCTCCACGGCCAAGAGCCGCACCCGGC- TCTTTGGGAAGG
GTGACTCGGAGGAGGCTTTCCCGGTGGATTGCCCTCACGAGGAAG- GTGAGCTGGACTCCT
GCCCGACCATCACAGTCAGCCCTGTTATCACCATCCAGAGGC- CAGGAGACGGCCCCACCG
GTGCCAGGCTGCTGTCCCAGGACTCTGTCGCCGCCAGCA- CCGAGAAGACCCTCAGGCTCT
ATGATCGCAGGAGTATCTTTGAAGCCGTTGCTCAGA- ATAACTGCCAGGATCTGGAGAGCC
TGCTGCTCTTCCTGCAGAAGAGCAAGAAGCACY- TCACAGACAACGAGTTCAAAGACCCTG
AGACAGGGAAGACCTGTCTGCTGAAAGCCA- TGCTCAACCTGCACGACGGACAGAACACCA
CCATCCCCCTGCTCCTGGAGATCGCGC- GGCAAACGGACAGCCTGAAGGAGCTTGTCAACG
CCRGCTACACGGACAGSTACTACA- AGGGCCAGACAGCACTGCACATCGCCATCGAGAGAC
GCAACATGGCCCTGGTGACCCTCCTGGTGGAGAACGGAGCAGACGTCCAGGCTGCGGCCC
ATGGGGACTGCTTTAAGAAAACCAAAGGGCGGCCTGGATTCTACTTCGGTGAACTGCCCC
TGTCCCTGGCCGCGTGCACCAACCAGCTGGGCATCGTGAAGTTCCTGCTGCAGAACTCCT
GGCAGACGGCCGACATCAGCGCCAGGGACTCGGTGGGCAACACGGTGCTGCACGCCCTGG
TGGAGGTGGCCGACAACACGGCCGACAACACGAAGTTTGTGACGAGCATGTACAATGAGA
TTCTGATCCTGGGGGCCAAACTGCACCCGACGCTGAAGCTGGAGGAGCTCACCAACA- AGA
AGGGAATGACGCCGCTGGCTCTGGCAGCTGGGACCGGGAAGATCGGGGTCTTGG- CCTATA
TTCTCCAGCGGGAGATCCAGGAGCCCGAGTGCAGGCACCTGTCCAGGAAGT- TCACCGAGT
GGGCCTACGGGCCCGTGCACTCCTCGCTGTACGACCTGTCCTGCATCG- ACACCTGCGAGA
AGAACTCGGTGCTGGAGGTGATCGCCTACAGCAGCAGCGAGACCC- CTAATCGCCACGACA
TGCTCTTGGTGGAGCCGCTGAACCGACTCCTGCAGGAGAAGT- GGGACAGATTCGTCAAGC
GCATCTTCTACTTCAACTTCCTGGTCTACTGCCTGTACA- TGATCATCTTCACCATGGCTG
CCTACTACAGGCCCGTGGATGGCTTGCCTCCCTTTA- AGATGGAAAAAACTGGAGACTATT
TCCGAGTTACTGGAGAGATCCTGTCTGTGTTAG- GAGGAGTCTACTTCTTTTTCCGAGGGA
TTCAGTATTTCCTGCAGAGGCGGCCGTCGA- TGAAGACCCTGTTTGTGGACAGCTACAGTG
AGATGCTTTTCTTTCTGCAGTCACTGT- TCATGCTGGCCACCGTGGTGCTGTACTTCAGCC
ACCTCAAGGAGTATGTGGCTTCCA- TGGTATTCTCCCTGGCCTTGGGCTGGACCAACATGC
TCTACTACACCCGCGGTTTCCAGCAGATGGGCATCTATGCCGTCATGATAGAGAAGATGA
TCCTGAGAGACCTGTGCCGTTTCATGTTTGTCTACATCGTCTTCTTGTTCGGGTTTTCCA
CAGCGGTGGTGACGCTGATTGAAGACGGGAAGAATGACTCCCTGCCGTCTGAGTCCACGT
CGCACAGGTGGCGGGGGCCTGCCTGCAGGCCCCCCGATAGCTCCTACAACAGCCTGTACT
CCACCTGCCTGGAGCTGTTCAAGTTCACCATCGGCATGGGCGACCTGGAGTTCACTGAGA
ACTATGACTTCAAGGCTGTCTTCATCATCCTGCTGCTGGCCTATGTAATTCTCACCT- ACA
TCCTCCTGCTCAACATGCTCATCGCCCTCATGGGTGAGACTGTCAACAAGATCG- CACAGG
AGAGCAAGAACATCTGGAAGCTGCAGAGAGCCATCACCATCCTGGACACGG- AGAAGAGCT
TCCTTAAGTGCATGAGGAAGGCCTTCCGCTCAGGCAAGCTGCTGCAGG- TGGGGTACACAC
CTGATGGCAAGGACGACTACCGGTGGTGCTTCAGGGTGGACGAGG- TGAACTGGACCACCT
GGAACACCAACGTGGGCATCATCAACGAAGACCCGGGCAACT- GTGAGGGCGTCAAGCGCA
CCCTGAGCTTCTCCCTGCGGTCAAGCAGAGTTTCAGGCA- GACACTGGAAGAACTTTGCCC
TGGTCCCCCTTTTAAGAGAGGCAAGTGCTCGAGATA- GGCAGTCTGCTCAGCCCGAGGAAG
TTTATCTGCGACAGTTTTCAGGGTCTCTGAAGC- CAGAGGACGCTGAGGTCTTCAAGAGTC
CTGCCGCTTCCGGGGAGAAGTGAGGACGTC- ACGCAGACAGCACTGTCAACACTGGGCCTT
AGGAGACCCCGTTGCCACGGGGGGCTG- CTGAGGGAACACCAGTGCTCTGTCAGCAGCCTG
GCCTGGTCTGTGCCTGCCCAGCAT- GTTCCCAAATCTGTGCTGGACAAGCTGTGGGAAGCG
TTCTTGGAAGCATGGGGAGTGATGTACATCCAACCGTCACTGTCCCCAAGTGAATCTCCT
AACAGACTTTCAGGTTTTTACTCACTTTACTAAACAGTKTGGATGGTCAGTCTCTACTGG
GACATGTTAGGCCCTTGTTTTCTTTGATTTTATTCTTTTTTTTGAGACAGAATTTCACTC
TTCTCACCCAGGCTGGAATGCAGTGGCACAATTTTGGCTCCCTGCAACCTCCGCCTCCTG
GATTCCAGCAATTCTCCTGCCTCGGCTTCCCAAGTAGCTGGGATTACAGGCACGTGCCAC
CATGTCTGGCTAATTTTTTGTATTTTTTTAATAGATATGGGGTTTCGCCATGTTGGC- CAG
GCTGGTCTCGAACTCCTGACCTCAGGTGATCCGCCCACCTCGGCCTCCCAAAGT- GCTGGG
ATTACAGGTGTGAGCCTCCACACCTGGCTGTTTTCTTTGATTTTATTCTTT- TTTTTTTTT
TCTGTGAGACAGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAGTGG- TGTGATCTTGGC
TCACTGCAACTTCTGCCTCCCGGGTTCAAGCGATTCTTCTGCTTC- AGTCTCCCAAGTAGC
TTGGATTACAGGTGAGCACTACCACGCCCGGCTAATTTTTGT- ATTTTTAATARAGACGGG
GTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCTTGAC- CTCAGGTGATCTGCCCGCCTT
GGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCG- CTGCGCTCGGCCTTCTTTGATTTT
ATATTATTAGGAGCAAAAGTAAATGAAGCCCAG- GAAAACACCTTTGGGAACAAACTCTTC
CTTTGATGGAAAATGCAGAGGCCCTTCCTC- TCTGTGCCGTGCTTGCTCCTCTTACCTGCC
CGGGTGGTTTGGGGGTGTTGGTGTTTC- CTCCCTGGAGAAGATGGGGGAGGCTGTCCCACT
CCCAGCTCTGGCAGAATCAAGCTG- TTGCAGCAGTGCCTTCTTCATCCTTCCTTACGATCA
ATCACAGTCTCCAGAAGATCAGCTCAATTGCTGTGCAGGTTAAAACTACAGAACCACATC
CCAAAGGTACCTGGTAAGAATGTTTGAAAGATCTTCCATTTCTAGGAACCCCAGTCCTGC
TTCTCCGCAATGGCACATGCTTCCACTCCATCCATACTGGCATCCTCAAATAAACAGATA
TGTATACAATAAAAAAAAAAAAAAAAAAAARRGCGGCCGCTGAATTCTAGACCTGCCCGG
GCG
[0093]
4 SEQ ID NO:4 MKKWSSTDLGAAADPLQKDTCPDPLDGDPNSRPPPAKPQLSTAKSR-
TRLFGKGDSEEAFP VDCPHEEGELDSCPTITVSPVITIQRPGDGPTGARLLSQDSVA-
ASTEKTLRLYDRRSIFE AVAQNNCQDLESLLLFLQKSKKHXTDNEFKDPETGKTCLL-
KAMLNLHDGQNTTIPLLLEI ARQTDSLKELVNAXYTDXYYKGQTALHIAIERRNMAL-
VTLLVENGADVQAAAHGDFFKKT KGRPGFYFGELPLSLAACTNQLGIVKFLLQNSWQ-
TADISARDSVGNTVLHALVEVADNTA DNTKFVTSMYNEILILGAKLHPTLKLEELTN-
KKGMTPLALAAGTGKIGVLAYILQREIQE PECRHLSRKFTEWAYGPVHSSLYDLSCI-
DTCEKNSVLEVIAYSSSETPNRHDMLLVEPLN RLLQDKWDRFVKRIFYFNFLVYCLY-
MIIFTMAAYYRPVDGLPPFKMEKTGDYFRVTGEIL
SVLGGVYFFFRGIQYFLQRRPSMKTLFVDSYSEMLFFLQSLFMLATVVLYFSHLKEYVAS
MVFSLALGWTNMLYYTRGFQQMGIYAVMIEKMILRDLCRFMFVYIVFLFGFSTAVVTLIE
DGKNDSLPSESTSHRWRGPACRPPDSSYNSLYSTCLELFKFTIGMGDLEFTENYDFKAVF
IILLLAYVTLTYILLLNMLIALMGETVNKIAQESKNIWKLQRAITILDTEKSFLKCMRKA
FRSGKLLQVGYTPDGKDDYRWCFRVDEVNWTTWNTNVGIINEDPGNCEGVKRTLSFSLRS
SRVSGRHWKNFALVPLLREASARDRQSAQPEEVYLRQFSGSLKPEDAEVFKSPAASG- EK
[0094]
5 SEQ ID NO:5 GAGCTTCTCCCTGCGGTCAAGCAGAGTTTCAGGCAGACACTGGAAG-
AACTTTGCCCTGGT CCCCCTTTTAAGAGAGGCAAGTNCTCGANATAGGCAGTCTGCT-
CAGCCCGAGGAAGTTTA TCTGCGACAGTTTTCCAGGGTCTCTAAAGCCAGAGGACGC-
TGAGGTCTTCAAGAGTCCTC CGCTTCCGGGGAGAAGTGAGGACGTCACGCAGACAGC-
ACTGTCAACACTGGGCCTTAGGA GACCCCGTTGCCACGGGGGGCTGCTGAGGGAACA-
CCAGTGCTTTTTCAGCAGCCTTGCCT GGGTCTTTGCCTGCCCAGCATGTTCCCAAAT-
CTGTGCTGGACAAGCTGTGGGGAAGCGTT CTTGGGAAGCATGGGGGAGTGATGTTAC-
ATCCAACCGTCACTGTCCCCAAGTTGAATCTT CCTTAACAGATT
[0095]
6 SEQ ID NO:6 SFSLRSSRVSGRHWKNFALVPLLREASXRXRQSAQPEEVYLRQFSG-
SLKPEDAEVFKSPA ASGEK
[0096]
7 SEQ ID NO:7, PVP-1 (polymorphic variant of VANILREP1, A2625G)
CAGCGTCGGGTGCAGTTTGGCCGGAGGTTGCAGTGAGCAGAGATTGCCCCATTGCACTCT
AGTCTGGGCGACAGGGTGAGACACACACACACAGACACACACACACACACACACACACAC
ACACAAGCCTAAACATTCRAGGCCAGGATGCTTGACAGATGTTGATTCATAAAAATGACA
AAAAGCACAAAATCCAAAATCTCGTATAAGCTCAGTGGCTGTGGCAGCGAGGTTGAAGAG
CAAAGGCAGGCCGGGCACCTGGCTGATGATGTGTGGACCCGTTGCACAGCAGGGCCC- CGC
AGTGCGGTGTGGGTGTGGGTGTGGGTGGGCCAGTYTCTGCCGCTCACCCTATTC- CAGGGA
CACAGTCTGCTTGGCTCTTCTGGACTGAGCCATCCTCATCACCGAGATCCT- CCCTGAATT
CAGCCCACGACAGCCACCCCGGCCGTTTTCCTTGTTCTGTGTGGGGAG- GGAGGCAGCGCG
GTGGTTATCAACCTCACCCTGCAGAGGAGGCACCTGAGGCCCAGA- GACGAGGAGGGATGG
GTCTAACCCAGAACCACAGATGGCTCTGAGCCGGGGGCCTGT- CCACCCTCCCAGGCCGAC
GTCAGTGGCCGCAGGACTGCCTGGGCCCTGCTAGGCCTG- CTCACCTCTGAGGCCTCTGGG
GTGAGAGGTTCAGTCCTGGAAACACTTCAGTTCTAG- GGGGCTGGGGGCAGCAGCAAGTTG
GAGTTTTGGGGTACCCTGCTTCACAGGGCCCTT- GGCAAGGAGGGCAGGTGGGGTCTAAGG
ACAAGCAGTCCTTACTTTGGGAGTCAACCC- CGGCGTGGTGGCTGCTGCAGGTTGCACACT
GGGCCACAGAGGATCCAGCAAGGATGA- AGAAATGGAGCAGCACAGACTTGGGGGCAGCTG
CGGACCCACTCCAAAAGGACACCT- GCCCAGACCCCCTGGATGGAGACCCTAACTCCAGGC
CACCTCCAGCCAAGCCCCAGCTCTCCACGGCCAAGAGCCGCACCCGGCTCTTTGGGAAGG
GTGACTCGGAGGAGGCTTTCCCGGTGGATTGCCCTCACGAGGAAGGTGAGCTGGACTCCT
GCCCGACCATCACAGTCAGCCCTGTTATCACCATCCAGAGGCCAGGAGACGGCCCCACCG
GTGCCAGGCTGCTGTCCCAGGACTCTGTCGCCGCCAGCACCGAGAAGACCCTCAGGCTCT
ATGATCGCAGGAGTATCTTTGAAGCCGTTGCTCAGAATAACTGCCAGGATCTGGAGAGCC
TGCTGCTCTTCCTGCAGAAGAGCAAGAAGCACCTCACAGACAACGAGTTCAAAGACC- CTG
AGACAGGGAAGACCTGTCTGCTGAAAGCCATGCTCAACCTGCACGACGGACAGA- ACACCA
CCATCCCCCTGCTCCTGGAGATCGCGCGGCAAACGGACAGCCTGAAGGAGC- TTGTCAACG
CCAGCTACACGGACAGCTACTACAAGGGCCAGACAGATCTGCACATCG- CCATCGAGAGAC
GCAACATGGCCCTGGTGACCCTCCTGGTGGAGAACGGAGCAGACG- TCCAGGCTGCGGCCC
ATGGGGACTTCTTTAAGAAAACCAAAGGGCGGCCTGGATTCT- ACTTCGGTGAACTGCCCC
TGTCCCTGGCCGCGTGCACCAACCAGCTGGGCATCGTGA- AGTTCCTGCTGCAGAACTCCT
GGCAGACGGCCGACATCAGCGCCAGGGACTCGGTGG- GCAACACGGTGCTGCACGCCCTGG
TGGAGGTGGCCGACAACACGGCCGACAACACGA- AGTTTGTGACGAGCATGTACAATGAGA
TTCTGATCCTGGGGGCCAAACTGCACCCGA- CGCTGAAGCTGGAGGAGCTCACCAACAAGA
AGGGAATGACGCCGCTGGCTCTGGCAG- CTGGGACCGGGAAGATCGGGGTCTTGGCCTATA
TTCTCCAGCGGGAGATCCAGGAGC- CCGAGTGCAGGCACCTGTCCAGGAAGTTCACCGAGT
GGGCCTACGGGCCCGTGCACTCCTCGCTGTACGACCTGTCCTGCATCGACACCTGCGAGA
AGAACTCGGTGCTGGAGGTGATCGCCTACAGCAGCAGCGAGACCCCTAATCGCCACGACA
TGCTCTTGGTGGAGCCGCTGAACCGACTCCTGCAGGACAAGTGGGACAGATTCGTCAAGC
GCATCTTCTACTTCAACTTCCTGGTCTACTGCCTGTACATGATCATCTTCACCATGGCTG
CCTACTACAGGCCCGTGGATGGCTTGCCTCCCTTTAAGATGGAAAAAACTGGAGACTATT
TCCGAGTTACTGGAGAGATCCTGTCTGTGTTAGGAGGAGTCTACTTCTTTTTCCGAG- GGA
TTCAGTATTTCCTGCAGAGGCGGCCGTCGATGAAGACCCTGTTTGTGGACAGCT- ACAGTG
AGATGCTTTTCTTTCTGCAGTCACTGTTCATGCTGGCCACCGTGGTGCTGT- ACTTCAGCC
ACCTCAAGGAGTATGTGGCTTCCATGGTATTCTCCCTGGCCTTGGGCT- GGACCAACATGC
TCTACTACACCCGCGGTTTCCAGCAGATGGGCATCTATGCCGTCA- TGATAGAGAAGATGA
TCCTGAGAGACCTGTGCCGTTTCATGTTTGTCTACGTCGTCT- TCTTGTTCGGGTTTTCCA
CAGCGGTGGTGACGCTGATTGAAGACGGGAAGAATGACT- CCCTGCCGTCTGAGTCCACGT
CGCACAGGTGGCGGGGGCCTGCCTGCAGGCCCCCCG- ATAGCTCCTACAACAGCCTGTACT
CCACCTGCCTGGAGCTGTTCAAGTTCACCATCG- GCATGGGCGACCTGGAGTTCACTGAGA
ACTATGACTTCAAGGCTGTCTTCATCATCC- TGCTGCTGGCCTATGTAATTCTCACCTACA
TCCTCCTGCTCAACATGCTCATCGCCC- TCATGGGTGAGACTGTCAACAAGATCGCACAGG
AGAGCAAGAACATCTGGAAGCTGC- AGAGAGCCATCACCATCCTGGACACGGAGAAGAGCT
TCCTTAAGTGCATGAGGAAGGCCTTCCGCTCAGGCAAGCTGCTGCAGGTGGGGTACACAC
CTGATGGCAAGGACGACTACCGGTGGTGCTTCAGGGTGGACGAGGTGAACTGGACCACCT
GGAACACCAACGTGGGCATCATCAACGAAGACCCGGGCAACTGTGAGGGCGTCAAGCGCA
CCCTGAGCTTCTCCCTGCGGTCAAGCAGAGTTTCAGGCAGACACTGGAAGAACTTTGCCC
TGGTCCCCCTTTTAAGAGAGGCAAGTGCTCGAGATAGGCAGTCTGCTCAGCCCGAGGAAG
TTTATCTGCGACAGTTTTCAGGGTCTCTGAAGCCAGAGGACGCTGAGGTCTTCAAGA- GTC
CTGCCGCTTCCGGGGAGAAGTGAGGACGTCACGCAGACAGCACTGTCAACACTG- GGCCTT
AGGAGACCCCGTTGCCACGGGGGGCTGCTGAGGGAACACCAGTGCTCTGTC- AGCAGCCTG
GCCTGGTCTGTGCCTGCCCA
[0097] +D,2 !,1 SEQ ID NO:8, (PVIP-1 protein, I1585V)? !
!MKKWSSTDLGAAADPLQKDTCPDPLDGDPNSRPPPAKPQLSTAKSRTRLFGKGDSEEAFP? !
!VDCPHEEGELDSCPTITVSPVITIQRPGDGPTGARLLSQDSVAASTEKTLRLYDRRSIFE? !
!AVAQNNCQDLESLLLFLQKSKKHLTDNEFKDPETGKTCLLKAMLNLHDGQNTTIPLLLEI? !
!ARQTDSLKELVNASYTDSYYKGQTALHIAIERRNMALVTLLVENGADVQAAAHGDFFKKT? !
!KGRPGFYFGELPLSLAACTNQLGIVKFLLQNSWQTADISARDSVGNTVLHALVEVADNTA? !
!DNTKFVTSMYNEILILGAKLHPTLKLEELTNKKGMTPLALAAGTGKIGVLAYILQREIQE? !
!PECRHLSRKFTEWAYGPVHSSLYDLSCIDTCEKNSVLEVIAYSSSETPNRHDMLLVEPLN? !
!RLLQDKWDRFVKRIFYFNFLVYCLYMIIFTMAAYYRPVDGLPPFKMEKTGDYFRVTGEIL? !
!SVLGGVYFFFRGIQYFLQRRPSMKTLFVDSYSEMLFFLQSLFMLATVVLYFSHLKEYVAS? !
!MVFSLALGWTNNLYYTRGFQQMGIYAVMIEKMILRDLCRFMFVYVVFLFGFSTAVVTLIE? !
!DGKNDSLPSESTSHRWRGPACRPPDSSYNSLYSTCLELFKFTIGMGDLEFTENYDFKAVF? !
!IILLLAYVILTYILLLNNLIALMGETVNKIAQESKNIWKLQRAITILDTEKSFLKCMRKA? !
!FRSGKLLQVGYTPDGKDDYRWCFRVDEVNWTTWNTNVGIINEDPGNCEGVKRTLSFSLRS? !
!SRVSGRHWKNFAINPLLREASARDRQSAQPEEVYLRQFSGSLKPEDAEVFKSPAASGEK? !
[0098]
Sequence CWU 1
1
8 1 4803 DNA HOMO SAPIENS 1 cagcgtcggg tgcagtttgg ccggaggttg
cagtgagcag agattgcccc attgcactct 60 agtctgggcg acagggtgag
acacacacac acagacacac acacacacac acacacacac 120 acacaagcct
aaacattcra ggccaggatg cttgacagat gttgattcat aaaaatgaca 180
aaaagcacaa aatccaaaat ctcgtataag ctcagtggct gtggcagcga ggttgaagag
240 caaaggcagg ccgggcacct ggctgatgat gtgtggaccc gttgcacagc
agggccccgc 300 agtgcggtgt gggtgtgggt gtgggtgggc cagtytctgc
cgctcaccct attccaggga 360 cacagtctgc ttggctcttc tggactgagc
catcctcatc accgagatcc tccctgaatt 420 cagcccacga cagccacccc
ggccgttttc cttgttctgt gtggggaggg aggcagcgcg 480 gtggttatca
acctcaccct gcagaggagg cacctgaggc ccagagacga ggagggatgg 540
gtctaaccca gaaccacaga tggctctgag ccgggggcct gtccaccctc ccaggccgac
600 gtcagtggcc gcaggactgc ctgggccctg ctaggcctgc tcacctctga
ggcctctggg 660 gtgagaggtt cagtcctgga aacacttcag ttctaggggg
ctgggggcag cagcaagttg 720 gagttttggg gtaccctgct tcacagggcc
cttggcaagg agggcaggtg gggtctaagg 780 acaagcagtc cttactttgg
gagtcaaccc cggcgtggtg gctgctgcag gttgcacact 840 gggccacaga
ggatccagca aggatgaaga aatggagcag cacagacttg ggggcagctg 900
cggacccact ccaaaaggac acctgcccag accccctgga tggagaccct aactccaggc
960 cacctccagc caagccccag ctctccacgg ccaagagccg cacccggctc
tttgggaagg 1020 gtgactcgga ggaggctttc ccggtggatt gccctcacga
ggaaggtgag ctggactcct 1080 gcccgaccat cacagtcagc cctgttatca
ccatccagag gccaggagac ggccccaccg 1140 gtgccaggct gctgtcccag
gactctgtcg ccgccagcac cgagaagacc ctcaggctct 1200 atgatcgcag
gagtatcttt gaagccgttg ctcagaataa ctgccaggat ctggagagcc 1260
tgctgctctt cctgcagaag agcaagaagc acctcacaga caacgagttc aaagaccctg
1320 agacagggaa gacctgtctg ctgaaagcca tgctcaacct gcacgacgga
cagaacacca 1380 ccatccccct gctcctggag atcgcgcggc aaacggacag
cctgaaggag cttgtcaacg 1440 ccagctacac ggacagctac tacaagggcc
agacagcact gcacatcgcc atcgagagac 1500 gcaacatggc cctggtgacc
ctcctggtgg agaacggagc agacgtccag gctgcggccc 1560 atggggactt
ctttaagaaa accaaagggc ggcctggatt ctacttcggt gaactgcccc 1620
tgtccctggc cgcgtgcacc aaccagctgg gcatcgtgaa gttcctgctg cagaactcct
1680 ggcagacggc cgacatcagc gccagggact cggtgggcaa cacggtgctg
cacgccctgg 1740 tggaggtggc cgacaacacg gccgacaaca cgaagtttgt
gacgagcatg tacaatgaga 1800 ttctgatcct gggggccaaa ctgcacccga
cgctgaagct ggaggagctc accaacaaga 1860 agggaatgac gccgctggct
ctggcagctg ggaccgggaa gatcggggtc ttggcctata 1920 ttctccagcg
ggagatccag gagcccgagt gcaggcacct gtccaggaag ttcaccgagt 1980
gggcctacgg gcccgtgcac tcctcgctgt acgacctgtc ctgcatcgac acctgcgaga
2040 agaactcggt gctggaggtg atcgcctaca gcagcagcga gacccctaat
cgccacgaca 2100 tgctcttggt ggagccgctg aaccgactcc tgcaggacaa
gtgggacaga ttcgtcaagc 2160 gcatcttcta cttcaacttc ctggtctact
gcctgtacat gatcatcttc accatggctg 2220 cctactacag gcccgtggat
ggcttgcctc cctttaagat ggaaaaaact ggagactatt 2280 tccgagttac
tggagagatc ctgtctgtgt taggaggagt ctacttcttt ttccgaggga 2340
ttcagtattt cctgcagagg cggccgtcga tgaagaccct gtttgtggac agctacagtg
2400 agatgctttt ctttctgcag tcactgttca tgctggccac cgtggtgctg
tacttcagcc 2460 acctcaagga gtatgtggct tccatggtat tctccctggc
cttgggctgg accaacatgc 2520 tctactacac ccgcggtttc cagcagatgg
gcatctatgc cgtcatgata gagaagatga 2580 tcctgagaga cctgtgccgt
ttcatgtttg tctacatcgt cttcttgttc gggttttcca 2640 cagcggtggt
gacgctgatt gaagacggga agaatgactc cctgccgtct gagtccacgt 2700
cgcacaggtg gcgggggcct gcctgcaggc cccccgatag ctcctacaac agcctgtact
2760 ccacctgcct ggagctgttc aagttcacca tcggcatggg cgacctggag
ttcactgaga 2820 actatgactt caaggctgtc ttcatcatcc tgctgctggc
ctatgtaatt ctcacctaca 2880 tcctcctgct caacatgctc atcgccctca
tgggtgagac tgtcaacaag atcgcacagg 2940 agagcaagaa catctggaag
ctgcagagag ccatcaccat cctggacacg gagaagagct 3000 tccttaagtg
catgaggaag gccttccgct caggcaagct gctgcaggtg gggtacacac 3060
ctgatggcaa ggacgactac cggtggtgct tcagggtgga cgaggtgaac tggaccacct
3120 ggaacaccaa cgtgggcatc atcaacgaag acccgggcaa ctgtgagggc
gtcaagcgca 3180 ccctgagctt ctccctgcgg tcaagcagag tttcaggcag
acactggaag aactttgccc 3240 tggtccccct tttaagagag gcaagtgctc
gagataggca gtctgctcag cccgaggaag 3300 tttatctgcg acagttttca
gggtctctga agccagagga cgctgaggtc ttcaagagtc 3360 ctgccgcttc
cggggagaag tgaggacgtc acgcagacag cactgtcaac actgggcctt 3420
aggagacccc gttgccacgg ggggctgctg agggaacacc agtgctctgt cagcagcctg
3480 gcctggtctg tgcctgccca gcatgttccc aaatctgtgc tggacaagct
gtgggaagcg 3540 ttcttggaag catggggagt gatgtacatc caaccgtcac
tgtccccaag tgaatctcct 3600 aacagacttt caggttttta ctcactttac
taaacagtkt ggatggtcag tctctactgg 3660 gacatgttag gcccttgttt
tctttgattt tattcttttt tttgagacag aatttcactc 3720 ttctcaccca
ggctggaatg cagtggcaca attttggctc cctgcaacct ccgcctcctg 3780
gattccagca attctcctgc ctcggcttcc caagtagctg ggattacagg cacgtgccac
3840 catgtctggc taattttttg tattttttta atagatatgg ggtttcgcca
tgttggccag 3900 gctggtctcg aactcctgac ctcaggtgat ccgcccacct
cggcctccca aagtgctggg 3960 attacaggtg tgagcctcca cacctggctg
ttttctttga ttttattctt tttttttttt 4020 tctgtgagac agagtttcac
tcttgttgcc caggctggag tgcagtggtg tgatcttggc 4080 tcactgcaac
ttctgcctcc cgggttcaag cgattcttct gcttcagtct cccaagtagc 4140
ttggattaca ggtgagcact accacgcccg gctaattttt gtatttttaa taragacggg
4200 gtttcaccat gttggccagg ctggtctcga actcttgacc tcaggtgatc
tgcccgcctt 4260 ggcctcccaa agtgctggga ttacaggtgt gagccgctgc
gctcggcctt ctttgatttt 4320 atattattag gagcaaaagt aaatgaagcc
caggaaaaca cctttgggaa caaactcttc 4380 ctttgatgga aaatgcagag
gcccttcctc tctgtgccgt gcttgctcct cttacctgcc 4440 cgggtggttt
gggggtgttg gtgtttcctc cctggagaag atgggggagg ctgtcccact 4500
cccagctctg gcagaatcaa gctgttgcag cagtgccttc ttcatccttc cttacgatca
4560 atcacagtct ccagaagatc agctcaattg ctgtgcaggt taaaactaca
gaaccacatc 4620 ccaaaggtac ctggtaagaa tgtttgaaag atcttccatt
tctaggaacc ccagtcctgc 4680 ttctccgcaa tggcacatgc ttccactcca
tccatactgg catcctcaaa taaacagata 4740 tgtatacaat aaaaaaaaaa
aaaaaaaaaa rrgcggccgc tgaattctag acctgcccgg 4800 gcg 4803 2 839 PRT
HOMO SAPIENS 2 Met Lys Lys Trp Ser Ser Thr Asp Leu Gly Ala Ala Ala
Asp Pro Leu 1 5 10 15 Gln Lys Asp Thr Cys Pro Asp Pro Leu Asp Gly
Asp Pro Asn Ser Arg 20 25 30 Pro Pro Pro Ala Lys Pro Gln Leu Ser
Thr Ala Lys Ser Arg Thr Arg 35 40 45 Leu Phe Gly Lys Gly Asp Ser
Glu Glu Ala Phe Pro Val Asp Cys Pro 50 55 60 His Glu Glu Gly Glu
Leu Asp Ser Cys Pro Thr Ile Thr Val Ser Pro 65 70 75 80 Val Ile Thr
Ile Gln Arg Pro Gly Asp Gly Pro Thr Gly Ala Arg Leu 85 90 95 Leu
Ser Gln Asp Ser Val Ala Ala Ser Thr Glu Lys Thr Leu Arg Leu 100 105
110 Tyr Asp Arg Arg Ser Ile Phe Glu Ala Val Ala Gln Asn Asn Cys Gln
115 120 125 Asp Leu Glu Ser Leu Leu Leu Phe Leu Gln Lys Ser Lys Lys
His Leu 130 135 140 Thr Asp Asn Glu Phe Lys Asp Pro Glu Thr Gly Lys
Thr Cys Leu Leu 145 150 155 160 Lys Ala Met Leu Asn Leu His Asp Gly
Gln Asn Thr Thr Ile Pro Leu 165 170 175 Leu Leu Glu Ile Ala Arg Gln
Thr Asp Ser Leu Lys Glu Leu Val Asn 180 185 190 Ala Ser Tyr Thr Asp
Ser Tyr Tyr Lys Gly Gln Thr Ala Leu His Ile 195 200 205 Ala Ile Glu
Arg Arg Asn Met Ala Leu Val Thr Leu Leu Val Glu Asn 210 215 220 Gly
Ala Asp Val Gln Ala Ala Ala His Gly Asp Phe Phe Lys Lys Thr 225 230
235 240 Lys Gly Arg Pro Gly Phe Tyr Phe Gly Glu Leu Pro Leu Ser Leu
Ala 245 250 255 Ala Cys Thr Asn Gln Leu Gly Ile Val Lys Phe Leu Leu
Gln Asn Ser 260 265 270 Trp Gln Thr Ala Asp Ile Ser Ala Arg Asp Ser
Val Gly Asn Thr Val 275 280 285 Leu His Ala Leu Val Glu Val Ala Asp
Asn Thr Ala Asp Asn Thr Lys 290 295 300 Phe Val Thr Ser Met Tyr Asn
Glu Ile Leu Ile Leu Gly Ala Lys Leu 305 310 315 320 His Pro Thr Leu
Lys Leu Glu Glu Leu Thr Asn Lys Lys Gly Met Thr 325 330 335 Pro Leu
Ala Leu Ala Ala Gly Thr Gly Lys Ile Gly Val Leu Ala Tyr 340 345 350
Ile Leu Gln Arg Glu Ile Gln Glu Pro Glu Cys Arg His Leu Ser Arg 355
360 365 Lys Phe Thr Glu Trp Ala Tyr Gly Pro Val His Ser Ser Leu Tyr
Asp 370 375 380 Leu Ser Cys Ile Asp Thr Cys Glu Lys Asn Ser Val Leu
Glu Val Ile 385 390 395 400 Ala Tyr Ser Ser Ser Glu Thr Pro Asn Arg
His Asp Met Leu Leu Val 405 410 415 Glu Pro Leu Asn Arg Leu Leu Gln
Asp Lys Trp Asp Arg Phe Val Lys 420 425 430 Arg Ile Phe Tyr Phe Asn
Phe Leu Val Tyr Cys Leu Tyr Met Ile Ile 435 440 445 Phe Thr Met Ala
Ala Tyr Tyr Arg Pro Val Asp Gly Leu Pro Pro Phe 450 455 460 Lys Met
Glu Lys Thr Gly Asp Tyr Phe Arg Val Thr Gly Glu Ile Leu 465 470 475
480 Ser Val Leu Gly Gly Val Tyr Phe Phe Phe Arg Gly Ile Gln Tyr Phe
485 490 495 Leu Gln Arg Arg Pro Ser Met Lys Thr Leu Phe Val Asp Ser
Tyr Ser 500 505 510 Glu Met Leu Phe Phe Leu Gln Ser Leu Phe Met Leu
Ala Thr Val Val 515 520 525 Leu Tyr Phe Ser His Leu Lys Glu Tyr Val
Ala Ser Met Val Phe Ser 530 535 540 Leu Ala Leu Gly Trp Thr Asn Met
Leu Tyr Tyr Thr Arg Gly Phe Gln 545 550 555 560 Gln Met Gly Ile Tyr
Ala Val Met Ile Glu Lys Met Ile Leu Arg Asp 565 570 575 Leu Cys Arg
Phe Met Phe Val Tyr Ile Val Phe Leu Phe Gly Phe Ser 580 585 590 Thr
Ala Val Val Thr Leu Ile Glu Asp Gly Lys Asn Asp Ser Leu Pro 595 600
605 Ser Glu Ser Thr Ser His Arg Trp Arg Gly Pro Ala Cys Arg Pro Pro
610 615 620 Asp Ser Ser Tyr Asn Ser Leu Tyr Ser Thr Cys Leu Glu Leu
Phe Lys 625 630 635 640 Phe Thr Ile Gly Met Gly Asp Leu Glu Phe Thr
Glu Asn Tyr Asp Phe 645 650 655 Lys Ala Val Phe Ile Ile Leu Leu Leu
Ala Tyr Val Ile Leu Thr Tyr 660 665 670 Ile Leu Leu Leu Asn Met Leu
Ile Ala Leu Met Gly Glu Thr Val Asn 675 680 685 Lys Ile Ala Gln Glu
Ser Lys Asn Ile Trp Lys Leu Gln Arg Ala Ile 690 695 700 Thr Ile Leu
Asp Thr Glu Lys Ser Phe Leu Lys Cys Met Arg Lys Ala 705 710 715 720
Phe Arg Ser Gly Lys Leu Leu Gln Val Gly Tyr Thr Pro Asp Gly Lys 725
730 735 Asp Asp Tyr Arg Trp Cys Phe Arg Val Asp Glu Val Asn Trp Thr
Thr 740 745 750 Trp Asn Thr Asn Val Gly Ile Ile Asn Glu Asp Pro Gly
Asn Cys Glu 755 760 765 Gly Val Lys Arg Thr Leu Ser Phe Ser Leu Arg
Ser Ser Arg Val Ser 770 775 780 Gly Arg His Trp Lys Asn Phe Ala Leu
Val Pro Leu Leu Arg Glu Ala 785 790 795 800 Ser Ala Arg Asp Arg Gln
Ser Ala Gln Pro Glu Glu Val Tyr Leu Arg 805 810 815 Gln Phe Ser Gly
Ser Leu Lys Pro Glu Asp Ala Glu Val Phe Lys Ser 820 825 830 Pro Ala
Ala Ser Gly Glu Lys 835 3 4803 DNA HOMO SAPIENS 3 cagcgtcggg
tgcagtttgg ccggaggttg cagtgagcag agattgcccc attgcactct 60
agtctgggcg acagggtgag acacacacac acagacacac acacacacac acacacacac
120 acacaagcct aaacattcra ggccaggatg cttgacagat gttgattcat
aaaaatgaca 180 aaaagcacaa aatccaaaat ctcgtataag ctcagtggct
gtggcagcga ggttgaagag 240 caaaggcagg ccgggcacct ggctgatgat
gtgtggaccc gttgcacagc agggccccgc 300 agtgcggtgt gggtgtgggt
gtgggtgggc cagtytctgc cgctcaccct attccaggga 360 cacagtctgc
ttggctcttc tggactgagc catcctcatc accgagatcc tccctgaatt 420
cagcccacga cagccacccc ggccgttttc cttgttctgt gtggggaggg aggcagcgcg
480 gtggttatca acctcaccct gcagaggagg cacctgaggc ccagagacga
ggagggatgg 540 gtctaaccca gaaccacaga tggctctgag ccgggggcct
gtccaccctc ccaggccgac 600 gtcagtggcc gcaggactgc ctgggccctg
ctaggcctgc tcacctctga ggcctctggg 660 gtgagaggtt cagtcctgga
aacacttcag ttctaggggg ctgggggcag cagcaagttg 720 gagttttggg
gtaccctgct tcacagggcc cttggcaagg agggcaggtg gggtctaagg 780
acaagcagtc cttactttgg gagtcaaccc cggcgtggtg gctgctgcag gttgcacact
840 gggccacaga ggatccagca aggatgaaga aatggagcag cacagacttg
ggggcagctg 900 cggacccact ccaaaaggac acctgcccag accccctgga
tggagaccct aactccaggc 960 cacctccagc caagccccag ctctccacgg
ccaagagccg cacccggctc tttgggaagg 1020 gtgactcgga ggaggctttc
ccggtggatt gccctcacga ggaaggtgag ctggactcct 1080 gcccgaccat
cacagtcagc cctgttatca ccatccagag gccaggagac ggccccaccg 1140
gtgccaggct gctgtcccag gactctgtcg ccgccagcac cgagaagacc ctcaggctct
1200 atgatcgcag gagtatcttt gaagccgttg ctcagaataa ctgccaggat
ctggagagcc 1260 tgctgctctt cctgcagaag agcaagaagc acytcacaga
caacgagttc aaagaccctg 1320 agacagggaa gacctgtctg ctgaaagcca
tgctcaacct gcacgacgga cagaacacca 1380 ccatccccct gctcctggag
atcgcgcggc aaacggacag cctgaaggag cttgtcaacg 1440 ccrgctacac
ggacagstac tacaagggcc agacagcact gcacatcgcc atcgagagac 1500
gcaacatggc cctggtgacc ctcctggtgg agaacggagc agacgtccag gctgcggccc
1560 atggggactt ctttaagaaa accaaagggc ggcctggatt ctacttcggt
gaactgcccc 1620 tgtccctggc cgcgtgcacc aaccagctgg gcatcgtgaa
gttcctgctg cagaactcct 1680 ggcagacggc cgacatcagc gccagggact
cggtgggcaa cacggtgctg cacgccctgg 1740 tggaggtggc cgacaacacg
gccgacaaca cgaagtttgt gacgagcatg tacaatgaga 1800 ttctgatcct
gggggccaaa ctgcacccga cgctgaagct ggaggagctc accaacaaga 1860
agggaatgac gccgctggct ctggcagctg ggaccgggaa gatcggggtc ttggcctata
1920 ttctccagcg ggagatccag gagcccgagt gcaggcacct gtccaggaag
ttcaccgagt 1980 gggcctacgg gcccgtgcac tcctcgctgt acgacctgtc
ctgcatcgac acctgcgaga 2040 agaactcggt gctggaggtg atcgcctaca
gcagcagcga gacccctaat cgccacgaca 2100 tgctcttggt ggagccgctg
aaccgactcc tgcaggacaa gtgggacaga ttcgtcaagc 2160 gcatcttcta
cttcaacttc ctggtctact gcctgtacat gatcatcttc accatggctg 2220
cctactacag gcccgtggat ggcttgcctc cctttaagat ggaaaaaact ggagactatt
2280 tccgagttac tggagagatc ctgtctgtgt taggaggagt ctacttcttt
ttccgaggga 2340 ttcagtattt cctgcagagg cggccgtcga tgaagaccct
gtttgtggac agctacagtg 2400 agatgctttt ctttctgcag tcactgttca
tgctggccac cgtggtgctg tacttcagcc 2460 acctcaagga gtatgtggct
tccatggtat tctccctggc cttgggctgg accaacatgc 2520 tctactacac
ccgcggtttc cagcagatgg gcatctatgc cgtcatgata gagaagatga 2580
tcctgagaga cctgtgccgt ttcatgtttg tctacatcgt cttcttgttc gggttttcca
2640 cagcggtggt gacgctgatt gaagacggga agaatgactc cctgccgtct
gagtccacgt 2700 cgcacaggtg gcgggggcct gcctgcaggc cccccgatag
ctcctacaac agcctgtact 2760 ccacctgcct ggagctgttc aagttcacca
tcggcatggg cgacctggag ttcactgaga 2820 actatgactt caaggctgtc
ttcatcatcc tgctgctggc ctatgtaatt ctcacctaca 2880 tcctcctgct
caacatgctc atcgccctca tgggtgagac tgtcaacaag atcgcacagg 2940
agagcaagaa catctggaag ctgcagagag ccatcaccat cctggacacg gagaagagct
3000 tccttaagtg catgaggaag gccttccgct caggcaagct gctgcaggtg
gggtacacac 3060 ctgatggcaa ggacgactac cggtggtgct tcagggtgga
cgaggtgaac tggaccacct 3120 ggaacaccaa cgtgggcatc atcaacgaag
acccgggcaa ctgtgagggc gtcaagcgca 3180 ccctgagctt ctccctgcgg
tcaagcagag tttcaggcag acactggaag aactttgccc 3240 tggtccccct
tttaagagag gcaagtgctc gagataggca gtctgctcag cccgaggaag 3300
tttatctgcg acagttttca gggtctctga agccagagga cgctgaggtc ttcaagagtc
3360 ctgccgcttc cggggagaag tgaggacgtc acgcagacag cactgtcaac
actgggcctt 3420 aggagacccc gttgccacgg ggggctgctg agggaacacc
agtgctctgt cagcagcctg 3480 gcctggtctg tgcctgccca gcatgttccc
aaatctgtgc tggacaagct gtgggaagcg 3540 ttcttggaag catggggagt
gatgtacatc caaccgtcac tgtccccaag tgaatctcct 3600 aacagacttt
caggttttta ctcactttac taaacagtkt ggatggtcag tctctactgg 3660
gacatgttag gcccttgttt tctttgattt tattcttttt tttgagacag aatttcactc
3720 ttctcaccca ggctggaatg cagtggcaca attttggctc cctgcaacct
ccgcctcctg 3780 gattccagca attctcctgc ctcggcttcc caagtagctg
ggattacagg cacgtgccac 3840 catgtctggc taattttttg tattttttta
atagatatgg ggtttcgcca tgttggccag 3900 gctggtctcg aactcctgac
ctcaggtgat ccgcccacct cggcctccca aagtgctggg 3960 attacaggtg
tgagcctcca cacctggctg ttttctttga ttttattctt tttttttttt 4020
tctgtgagac agagtttcac tcttgttgcc caggctggag tgcagtggtg tgatcttggc
4080 tcactgcaac ttctgcctcc cgggttcaag cgattcttct gcttcagtct
cccaagtagc 4140 ttggattaca ggtgagcact accacgcccg gctaattttt
gtatttttaa taragacggg 4200 gtttcaccat gttggccagg ctggtctcga
actcttgacc tcaggtgatc tgcccgcctt 4260 ggcctcccaa agtgctggga
ttacaggtgt gagccgctgc gctcggcctt ctttgatttt 4320 atattattag
gagcaaaagt aaatgaagcc caggaaaaca cctttgggaa caaactcttc 4380
ctttgatgga aaatgcagag gcccttcctc tctgtgccgt gcttgctcct cttacctgcc
4440 cgggtggttt gggggtgttg gtgtttcctc cctggagaag atgggggagg
ctgtcccact 4500 cccagctctg gcagaatcaa gctgttgcag cagtgccttc
ttcatccttc cttacgatca 4560 atcacagtct ccagaagatc agctcaattg
ctgtgcaggt taaaactaca gaaccacatc 4620 ccaaaggtac ctggtaagaa
tgtttgaaag atcttccatt tctaggaacc ccagtcctgc 4680 ttctccgcaa
tggcacatgc ttccactcca tccatactgg catcctcaaa taaacagata 4740
tgtatacaat aaaaaaaaaa aaaaaaaaaa rrgcggccgc tgaattctag acctgcccgg
4800 gcg 4803 4 839 PRT HOMO SAPIENS UNSURE (144)(194)(198) 4 Met
Lys Lys Trp Ser Ser Thr Asp Leu Gly Ala Ala Ala Asp Pro Leu 1 5
10 15 Gln Lys Asp Thr Cys Pro Asp Pro Leu Asp Gly Asp Pro Asn Ser
Arg 20 25 30 Pro Pro Pro Ala Lys Pro Gln Leu Ser Thr Ala Lys Ser
Arg Thr Arg 35 40 45 Leu Phe Gly Lys Gly Asp Ser Glu Glu Ala Phe
Pro Val Asp Cys Pro 50 55 60 His Glu Glu Gly Glu Leu Asp Ser Cys
Pro Thr Ile Thr Val Ser Pro 65 70 75 80 Val Ile Thr Ile Gln Arg Pro
Gly Asp Gly Pro Thr Gly Ala Arg Leu 85 90 95 Leu Ser Gln Asp Ser
Val Ala Ala Ser Thr Glu Lys Thr Leu Arg Leu 100 105 110 Tyr Asp Arg
Arg Ser Ile Phe Glu Ala Val Ala Gln Asn Asn Cys Gln 115 120 125 Asp
Leu Glu Ser Leu Leu Leu Phe Leu Gln Lys Ser Lys Lys His Xaa 130 135
140 Thr Asp Asn Glu Phe Lys Asp Pro Glu Thr Gly Lys Thr Cys Leu Leu
145 150 155 160 Lys Ala Met Leu Asn Leu His Asp Gly Gln Asn Thr Thr
Ile Pro Leu 165 170 175 Leu Leu Glu Ile Ala Arg Gln Thr Asp Ser Leu
Lys Glu Leu Val Asn 180 185 190 Ala Xaa Tyr Thr Asp Xaa Tyr Tyr Lys
Gly Gln Thr Ala Leu His Ile 195 200 205 Ala Ile Glu Arg Arg Asn Met
Ala Leu Val Thr Leu Leu Val Glu Asn 210 215 220 Gly Ala Asp Val Gln
Ala Ala Ala His Gly Asp Phe Phe Lys Lys Thr 225 230 235 240 Lys Gly
Arg Pro Gly Phe Tyr Phe Gly Glu Leu Pro Leu Ser Leu Ala 245 250 255
Ala Cys Thr Asn Gln Leu Gly Ile Val Lys Phe Leu Leu Gln Asn Ser 260
265 270 Trp Gln Thr Ala Asp Ile Ser Ala Arg Asp Ser Val Gly Asn Thr
Val 275 280 285 Leu His Ala Leu Val Glu Val Ala Asp Asn Thr Ala Asp
Asn Thr Lys 290 295 300 Phe Val Thr Ser Met Tyr Asn Glu Ile Leu Ile
Leu Gly Ala Lys Leu 305 310 315 320 His Pro Thr Leu Lys Leu Glu Glu
Leu Thr Asn Lys Lys Gly Met Thr 325 330 335 Pro Leu Ala Leu Ala Ala
Gly Thr Gly Lys Ile Gly Val Leu Ala Tyr 340 345 350 Ile Leu Gln Arg
Glu Ile Gln Glu Pro Glu Cys Arg His Leu Ser Arg 355 360 365 Lys Phe
Thr Glu Trp Ala Tyr Gly Pro Val His Ser Ser Leu Tyr Asp 370 375 380
Leu Ser Cys Ile Asp Thr Cys Glu Lys Asn Ser Val Leu Glu Val Ile 385
390 395 400 Ala Tyr Ser Ser Ser Glu Thr Pro Asn Arg His Asp Met Leu
Leu Val 405 410 415 Glu Pro Leu Asn Arg Leu Leu Gln Asp Lys Trp Asp
Arg Phe Val Lys 420 425 430 Arg Ile Phe Tyr Phe Asn Phe Leu Val Tyr
Cys Leu Tyr Met Ile Ile 435 440 445 Phe Thr Met Ala Ala Tyr Tyr Arg
Pro Val Asp Gly Leu Pro Pro Phe 450 455 460 Lys Met Glu Lys Thr Gly
Asp Tyr Phe Arg Val Thr Gly Glu Ile Leu 465 470 475 480 Ser Val Leu
Gly Gly Val Tyr Phe Phe Phe Arg Gly Ile Gln Tyr Phe 485 490 495 Leu
Gln Arg Arg Pro Ser Met Lys Thr Leu Phe Val Asp Ser Tyr Ser 500 505
510 Glu Met Leu Phe Phe Leu Gln Ser Leu Phe Met Leu Ala Thr Val Val
515 520 525 Leu Tyr Phe Ser His Leu Lys Glu Tyr Val Ala Ser Met Val
Phe Ser 530 535 540 Leu Ala Leu Gly Trp Thr Asn Met Leu Tyr Tyr Thr
Arg Gly Phe Gln 545 550 555 560 Gln Met Gly Ile Tyr Ala Val Met Ile
Glu Lys Met Ile Leu Arg Asp 565 570 575 Leu Cys Arg Phe Met Phe Val
Tyr Ile Val Phe Leu Phe Gly Phe Ser 580 585 590 Thr Ala Val Val Thr
Leu Ile Glu Asp Gly Lys Asn Asp Ser Leu Pro 595 600 605 Ser Glu Ser
Thr Ser His Arg Trp Arg Gly Pro Ala Cys Arg Pro Pro 610 615 620 Asp
Ser Ser Tyr Asn Ser Leu Tyr Ser Thr Cys Leu Glu Leu Phe Lys 625 630
635 640 Phe Thr Ile Gly Met Gly Asp Leu Glu Phe Thr Glu Asn Tyr Asp
Phe 645 650 655 Lys Ala Val Phe Ile Ile Leu Leu Leu Ala Tyr Val Ile
Leu Thr Tyr 660 665 670 Ile Leu Leu Leu Asn Met Leu Ile Ala Leu Met
Gly Glu Thr Val Asn 675 680 685 Lys Ile Ala Gln Glu Ser Lys Asn Ile
Trp Lys Leu Gln Arg Ala Ile 690 695 700 Thr Ile Leu Asp Thr Glu Lys
Ser Phe Leu Lys Cys Met Arg Lys Ala 705 710 715 720 Phe Arg Ser Gly
Lys Leu Leu Gln Val Gly Tyr Thr Pro Asp Gly Lys 725 730 735 Asp Asp
Tyr Arg Trp Cys Phe Arg Val Asp Glu Val Asn Trp Thr Thr 740 745 750
Trp Asn Thr Asn Val Gly Ile Ile Asn Glu Asp Pro Gly Asn Cys Glu 755
760 765 Gly Val Lys Arg Thr Leu Ser Phe Ser Leu Arg Ser Ser Arg Val
Ser 770 775 780 Gly Arg His Trp Lys Asn Phe Ala Leu Val Pro Leu Leu
Arg Glu Ala 785 790 795 800 Ser Ala Arg Asp Arg Gln Ser Ala Gln Pro
Glu Glu Val Tyr Leu Arg 805 810 815 Gln Phe Ser Gly Ser Leu Lys Pro
Glu Asp Ala Glu Val Phe Lys Ser 820 825 830 Pro Ala Ala Ser Gly Glu
Lys 835 5 432 DNA HOMO SAPIENS UNSURE (83)(89) 5 gagcttctcc
ctgcggtcaa gcagagtttc aggcagacac tggaagaact ttgccctggt 60
ccccctttta agagaggcaa gtnctcgana taggcagtct gctcagcccg aggaagttta
120 tctgcgacag ttttcagggt ctctaaagcc agaggacgct gaggtcttca
agagtcctgc 180 cgcttccggg gagaagtgag gacgtcacgc agacagcact
gtcaacactg ggccttagga 240 gaccccgttg ccacgggggg ctgctgaggg
aacaccagtg ctttttcagc agccttgcct 300 gggtctttgc ctgcccagca
tgttcccaaa tctgtgctgg acaagctgtg gggaagcgtt 360 cttgggaagc
atgggggagt gatgttacat ccaaccgtca ctgtccccaa gttgaatctt 420
ccttaacaga tt 432 6 65 PRT HOMO SAPIENS UNSURE (28)(30) 6 Ser Phe
Ser Leu Arg Ser Ser Arg Val Ser Gly Arg His Trp Lys Asn 1 5 10 15
Phe Ala Leu Val Pro Leu Leu Arg Glu Ala Ser Xaa Arg Xaa Arg Gln 20
25 30 Ser Ala Gln Pro Glu Glu Val Tyr Leu Arg Gln Phe Ser Gly Ser
Leu 35 40 45 Lys Pro Glu Asp Ala Glu Val Phe Lys Ser Pro Ala Ala
Ser Gly Glu 50 55 60 Lys 65 7 3500 DNA HOMO SAPIENS 7 cagcgtcggg
tgcagtttgg ccggaggttg cagtgagcag agattgcccc attgcactct 60
agtctgggcg acagggtgag acacacacac acagacacac acacacacac acacacacac
120 acacaagcct aaacattcra ggccaggatg cttgacagat gttgattcat
aaaaatgaca 180 aaaagcacaa aatccaaaat ctcgtataag ctcagtggct
gtggcagcga ggttgaagag 240 caaaggcagg ccgggcacct ggctgatgat
gtgtggaccc gttgcacagc agggccccgc 300 agtgcggtgt gggtgtgggt
gtgggtgggc cagtytctgc cgctcaccct attccaggga 360 cacagtctgc
ttggctcttc tggactgagc catcctcatc accgagatcc tccctgaatt 420
cagcccacga cagccacccc ggccgttttc cttgttctgt gtggggaggg aggcagcgcg
480 gtggttatca acctcaccct gcagaggagg cacctgaggc ccagagacga
ggagggatgg 540 gtctaaccca gaaccacaga tggctctgag ccgggggcct
gtccaccctc ccaggccgac 600 gtcagtggcc gcaggactgc ctgggccctg
ctaggcctgc tcacctctga ggcctctggg 660 gtgagaggtt cagtcctgga
aacacttcag ttctaggggg ctgggggcag cagcaagttg 720 gagttttggg
gtaccctgct tcacagggcc cttggcaagg agggcaggtg gggtctaagg 780
acaagcagtc cttactttgg gagtcaaccc cggcgtggtg gctgctgcag gttgcacact
840 gggccacaga ggatccagca aggatgaaga aatggagcag cacagacttg
ggggcagctg 900 cggacccact ccaaaaggac acctgcccag accccctgga
tggagaccct aactccaggc 960 cacctccagc caagccccag ctctccacgg
ccaagagccg cacccggctc tttgggaagg 1020 gtgactcgga ggaggctttc
ccggtggatt gccctcacga ggaaggtgag ctggactcct 1080 gcccgaccat
cacagtcagc cctgttatca ccatccagag gccaggagac ggccccaccg 1140
gtgccaggct gctgtcccag gactctgtcg ccgccagcac cgagaagacc ctcaggctct
1200 atgatcgcag gagtatcttt gaagccgttg ctcagaataa ctgccaggat
ctggagagcc 1260 tgctgctctt cctgcagaag agcaagaagc acctcacaga
caacgagttc aaagaccctg 1320 agacagggaa gacctgtctg ctgaaagcca
tgctcaacct gcacgacgga cagaacacca 1380 ccatccccct gctcctggag
atcgcgcggc aaacggacag cctgaaggag cttgtcaacg 1440 ccagctacac
ggacagctac tacaagggcc agacagcact gcacatcgcc atcgagagac 1500
gcaacatggc cctggtgacc ctcctggtgg agaacggagc agacgtccag gctgcggccc
1560 atggggactt ctttaagaaa accaaagggc ggcctggatt ctacttcggt
gaactgcccc 1620 tgtccctggc cgcgtgcacc aaccagctgg gcatcgtgaa
gttcctgctg cagaactcct 1680 ggcagacggc cgacatcagc gccagggact
cggtgggcaa cacggtgctg cacgccctgg 1740 tggaggtggc cgacaacacg
gccgacaaca cgaagtttgt gacgagcatg tacaatgaga 1800 ttctgatcct
gggggccaaa ctgcacccga cgctgaagct ggaggagctc accaacaaga 1860
agggaatgac gccgctggct ctggcagctg ggaccgggaa gatcggggtc ttggcctata
1920 ttctccagcg ggagatccag gagcccgagt gcaggcacct gtccaggaag
ttcaccgagt 1980 gggcctacgg gcccgtgcac tcctcgctgt acgacctgtc
ctgcatcgac acctgcgaga 2040 agaactcggt gctggaggtg atcgcctaca
gcagcagcga gacccctaat cgccacgaca 2100 tgctcttggt ggagccgctg
aaccgactcc tgcaggacaa gtgggacaga ttcgtcaagc 2160 gcatcttcta
cttcaacttc ctggtctact gcctgtacat gatcatcttc accatggctg 2220
cctactacag gcccgtggat ggcttgcctc cctttaagat ggaaaaaact ggagactatt
2280 tccgagttac tggagagatc ctgtctgtgt taggaggagt ctacttcttt
ttccgaggga 2340 ttcagtattt cctgcagagg cggccgtcga tgaagaccct
gtttgtggac agctacagtg 2400 agatgctttt ctttctgcag tcactgttca
tgctggccac cgtggtgctg tacttcagcc 2460 acctcaagga gtatgtggct
tccatggtat tctccctggc cttgggctgg accaacatgc 2520 tctactacac
ccgcggtttc cagcagatgg gcatctatgc cgtcatgata gagaagatga 2580
tcctgagaga cctgtgccgt ttcatgtttg tctacgtcgt cttcttgttc gggttttcca
2640 cagcggtggt gacgctgatt gaagacggga agaatgactc cctgccgtct
gagtccacgt 2700 cgcacaggtg gcgggggcct gcctgcaggc cccccgatag
ctcctacaac agcctgtact 2760 ccacctgcct ggagctgttc aagttcacca
tcggcatggg cgacctggag ttcactgaga 2820 actatgactt caaggctgtc
ttcatcatcc tgctgctggc ctatgtaatt ctcacctaca 2880 tcctcctgct
caacatgctc atcgccctca tgggtgagac tgtcaacaag atcgcacagg 2940
agagcaagaa catctggaag ctgcagagag ccatcaccat cctggacacg gagaagagct
3000 tccttaagtg catgaggaag gccttccgct caggcaagct gctgcaggtg
gggtacacac 3060 ctgatggcaa ggacgactac cggtggtgct tcagggtgga
cgaggtgaac tggaccacct 3120 ggaacaccaa cgtgggcatc atcaacgaag
acccgggcaa ctgtgagggc gtcaagcgca 3180 ccctgagctt ctccctgcgg
tcaagcagag tttcaggcag acactggaag aactttgccc 3240 tggtccccct
tttaagagag gcaagtgctc gagataggca gtctgctcag cccgaggaag 3300
tttatctgcg acagttttca gggtctctga agccagagga cgctgaggtc ttcaagagtc
3360 ctgccgcttc cggggagaag tgaggacgtc acgcagacag cactgtcaac
actgggcctt 3420 aggagacccc gttgccacgg ggggctgctg agggaacacc
agtgctctgt cagcagcctg 3480 gcctggtctg tgcctgccca 3500 8 839 PRT
HOMO SAPIENS 8 Met Lys Lys Trp Ser Ser Thr Asp Leu Gly Ala Ala Ala
Asp Pro Leu 1 5 10 15 Gln Lys Asp Thr Cys Pro Asp Pro Leu Asp Gly
Asp Pro Asn Ser Arg 20 25 30 Pro Pro Pro Ala Lys Pro Gln Leu Ser
Thr Ala Lys Ser Arg Thr Arg 35 40 45 Leu Phe Gly Lys Gly Asp Ser
Glu Glu Ala Phe Pro Val Asp Cys Pro 50 55 60 His Glu Glu Gly Glu
Leu Asp Ser Cys Pro Thr Ile Thr Val Ser Pro 65 70 75 80 Val Ile Thr
Ile Gln Arg Pro Gly Asp Gly Pro Thr Gly Ala Arg Leu 85 90 95 Leu
Ser Gln Asp Ser Val Ala Ala Ser Thr Glu Lys Thr Leu Arg Leu 100 105
110 Tyr Asp Arg Arg Ser Ile Phe Glu Ala Val Ala Gln Asn Asn Cys Gln
115 120 125 Asp Leu Glu Ser Leu Leu Leu Phe Leu Gln Lys Ser Lys Lys
His Leu 130 135 140 Thr Asp Asn Glu Phe Lys Asp Pro Glu Thr Gly Lys
Thr Cys Leu Leu 145 150 155 160 Lys Ala Met Leu Asn Leu His Asp Gly
Gln Asn Thr Thr Ile Pro Leu 165 170 175 Leu Leu Glu Ile Ala Arg Gln
Thr Asp Ser Leu Lys Glu Leu Val Asn 180 185 190 Ala Ser Tyr Thr Asp
Ser Tyr Tyr Lys Gly Gln Thr Ala Leu His Ile 195 200 205 Ala Ile Glu
Arg Arg Asn Met Ala Leu Val Thr Leu Leu Val Glu Asn 210 215 220 Gly
Ala Asp Val Gln Ala Ala Ala His Gly Asp Phe Phe Lys Lys Thr 225 230
235 240 Lys Gly Arg Pro Gly Phe Tyr Phe Gly Glu Leu Pro Leu Ser Leu
Ala 245 250 255 Ala Cys Thr Asn Gln Leu Gly Ile Val Lys Phe Leu Leu
Gln Asn Ser 260 265 270 Trp Gln Thr Ala Asp Ile Ser Ala Arg Asp Ser
Val Gly Asn Thr Val 275 280 285 Leu His Ala Leu Val Glu Val Ala Asp
Asn Thr Ala Asp Asn Thr Lys 290 295 300 Phe Val Thr Ser Met Tyr Asn
Glu Ile Leu Ile Leu Gly Ala Lys Leu 305 310 315 320 His Pro Thr Leu
Lys Leu Glu Glu Leu Thr Asn Lys Lys Gly Met Thr 325 330 335 Pro Leu
Ala Leu Ala Ala Gly Thr Gly Lys Ile Gly Val Leu Ala Tyr 340 345 350
Ile Leu Gln Arg Glu Ile Gln Glu Pro Glu Cys Arg His Leu Ser Arg 355
360 365 Lys Phe Thr Glu Trp Ala Tyr Gly Pro Val His Ser Ser Leu Tyr
Asp 370 375 380 Leu Ser Cys Ile Asp Thr Cys Glu Lys Asn Ser Val Leu
Glu Val Ile 385 390 395 400 Ala Tyr Ser Ser Ser Glu Thr Pro Asn Arg
His Asp Met Leu Leu Val 405 410 415 Glu Pro Leu Asn Arg Leu Leu Gln
Asp Lys Trp Asp Arg Phe Val Lys 420 425 430 Arg Ile Phe Tyr Phe Asn
Phe Leu Val Tyr Cys Leu Tyr Met Ile Ile 435 440 445 Phe Thr Met Ala
Ala Tyr Tyr Arg Pro Val Asp Gly Leu Pro Pro Phe 450 455 460 Lys Met
Glu Lys Thr Gly Asp Tyr Phe Arg Val Thr Gly Glu Ile Leu 465 470 475
480 Ser Val Leu Gly Gly Val Tyr Phe Phe Phe Arg Gly Ile Gln Tyr Phe
485 490 495 Leu Gln Arg Arg Pro Ser Met Lys Thr Leu Phe Val Asp Ser
Tyr Ser 500 505 510 Glu Met Leu Phe Phe Leu Gln Ser Leu Phe Met Leu
Ala Thr Val Val 515 520 525 Leu Tyr Phe Ser His Leu Lys Glu Tyr Val
Ala Ser Met Val Phe Ser 530 535 540 Leu Ala Leu Gly Trp Thr Asn Met
Leu Tyr Tyr Thr Arg Gly Phe Gln 545 550 555 560 Gln Met Gly Ile Tyr
Ala Val Met Ile Glu Lys Met Ile Leu Arg Asp 565 570 575 Leu Cys Arg
Phe Met Phe Val Tyr Val Val Phe Leu Phe Gly Phe Ser 580 585 590 Thr
Ala Val Val Thr Leu Ile Glu Asp Gly Lys Asn Asp Ser Leu Pro 595 600
605 Ser Glu Ser Thr Ser His Arg Trp Arg Gly Pro Ala Cys Arg Pro Pro
610 615 620 Asp Ser Ser Tyr Asn Ser Leu Tyr Ser Thr Cys Leu Glu Leu
Phe Lys 625 630 635 640 Phe Thr Ile Gly Met Gly Asp Leu Glu Phe Thr
Glu Asn Tyr Asp Phe 645 650 655 Lys Ala Val Phe Ile Ile Leu Leu Leu
Ala Tyr Val Ile Leu Thr Tyr 660 665 670 Ile Leu Leu Leu Asn Met Leu
Ile Ala Leu Met Gly Glu Thr Val Asn 675 680 685 Lys Ile Ala Gln Glu
Ser Lys Asn Ile Trp Lys Leu Gln Arg Ala Ile 690 695 700 Thr Ile Leu
Asp Thr Glu Lys Ser Phe Leu Lys Cys Met Arg Lys Ala 705 710 715 720
Phe Arg Ser Gly Lys Leu Leu Gln Val Gly Tyr Thr Pro Asp Gly Lys 725
730 735 Asp Asp Tyr Arg Trp Cys Phe Arg Val Asp Glu Val Asn Trp Thr
Thr 740 745 750 Trp Asn Thr Asn Val Gly Ile Ile Asn Glu Asp Pro Gly
Asn Cys Glu 755 760 765 Gly Val Lys Arg Thr Leu Ser Phe Ser Leu Arg
Ser Ser Arg Val Ser 770 775 780 Gly Arg His Trp Lys Asn Phe Ala Leu
Val Pro Leu Leu Arg Glu Ala 785 790 795 800 Ser Ala Arg Asp Arg Gln
Ser Ala Gln Pro Glu Glu Val Tyr Leu Arg 805 810 815 Gln Phe Ser Gly
Ser Leu Lys Pro Glu Asp Ala Glu Val Phe Lys Ser 820 825 830 Pro Ala
Ala Ser Gly Glu Lys 835
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