U.S. patent application number 10/800665 was filed with the patent office on 2004-11-04 for novel nucleic acid and amino acid sequences.
This patent application is currently assigned to Compugen Ltd.. Invention is credited to Azar, Idit, David, Anat, Mintz, Liat, Savitzky, Kinneret.
Application Number | 20040219583 10/800665 |
Document ID | / |
Family ID | 27271903 |
Filed Date | 2004-11-04 |
United States Patent
Application |
20040219583 |
Kind Code |
A1 |
Savitzky, Kinneret ; et
al. |
November 4, 2004 |
Novel nucleic acid and amino acid sequences
Abstract
The invention concerns novel nucleic acid sequences, amino acid
sequences coded thereby and method of detection using the above.
The novel nucleic acid sequences are naturally occurring splice
variants of a prostate specific antigen sequence (PSA) or of the
KLK-2 gene.
Inventors: |
Savitzky, Kinneret; (Tel
Aviv, IL) ; Mintz, Liat; (Ramat Hasharon, IL)
; David, Anat; (Givataim, IL) ; Azar, Idit;
(Rehovot, IL) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER
LLP
1300 I STREET, NW
WASHINGTON
DC
20005
US
|
Assignee: |
Compugen Ltd.
|
Family ID: |
27271903 |
Appl. No.: |
10/800665 |
Filed: |
March 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10800665 |
Mar 16, 2004 |
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09755100 |
Jan 8, 2001 |
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6740516 |
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09755100 |
Jan 8, 2001 |
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09701238 |
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09701238 |
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PCT/IL00/00102 |
Feb 18, 2000 |
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Current U.S.
Class: |
435/6.14 ;
435/320.1; 435/325; 435/7.23; 530/350; 530/388.8; 536/23.2 |
Current CPC
Class: |
C12N 9/6445
20130101 |
Class at
Publication: |
435/006 ;
435/007.23; 435/320.1; 435/325; 530/350; 530/388.8; 536/023.2 |
International
Class: |
C12Q 001/68; G01N
033/574; C07H 021/04; C07K 014/705 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 18, 1999 |
IL |
128587 |
Apr 14, 1999 |
IL |
129439 |
Aug 11, 1999 |
IL |
131363 |
Claims
1-35 (Cancelled).
36. An amino acid selected from the group consisting of: (i) an
amino acid having the sequence of SEQ ID NO:12; (ii) fragments of
the amino acid of (i) having at least 10 amino acids; (iii)
analogues of the amino acids of (i) or (ii) in which one or more
amino acids has been added, deleted, replaced, or chemically
modified without substantially altering the biological activity of
the parent amino acid.
37. A method for identifying candidate compounds capable of binding
to a KLK-2 variant product and modulating its activity the method
comprising: (i) providing an amino acid as claimed in claim 36;
(ii) contacting a candidate compound with said amino acid; (iii)
determining the effect of said candidate compound on the biological
activity of said amino acid; and (iv) selecting those compounds
that show a significant effect on said biological activity.
38. A method according to claim 37, wherein the compound is an
activator and the measured effect is increase in the biological
activity.
39. A method according to claim 37, wherein the compound is a
deactivator and the effect is decrease in the biological
activity.
40. A method for determining the level of amino acid sequences of
KLK-2 variants in a biological sample comprising: (a) providing a
biological sample containing an amino acid as claimed in claim 36;
(b) contacting said biological sample with an antibody specific for
the amino acid, thereby forming an antibody-antigen complex; (c)
detecting the amount of antibody-antigen complex; and (d)
normalizing the amount to provide the level of the amino acid
sequences of KLK-2 variants in the sample.
41. A method for determining the ratio between the level of any one
of the amino acids of claim 36 of the KLK-2 variant present in a
first biological sample and the level of the native KLK-2 sequence
from which the variant has been varied by alternative splicing, in
a second biological sample comprising: (a) determining the level of
the KLK-2 variant amino acid sequence in the first biological
sample according to the method of claim 40; (b) determining the
level of the KLK-2 native sequence in the second biological sample;
and (c) comparing the levels obtained in (a) and (b) to give said
ratio.
42. A method according to claim 41, wherein said first and said
second biological samples are the same sample.
43. A method according to any one of claims 40 to 42 for detecting
the presence of prostate cancer or detecting pre-disposition to
prostate cancer, or for detection of the malignancy of prostate
cancer.
Description
FIELD OF THE INVENTION
[0001] The present invention concerns novel nucleic acid sequences,
vectors and host cells containing them, amino acid sequences
encoded by said sequences, and antibodies reactive with said amino
acid sequences, as well as pharmaceutical compositions comprising
any of the above. The present invention further concerns methods
for screening for candidate activator or deactivators utilizing
said amino acid sequences.
BACKGROUND OF THE INVENTION
[0002] Prostate-specific antigen (PSA) is the most important tumor
marker for early detection, staging, and monitoring of men with
prostate cancer today. PSA testing has appreciable false-positive
and false-negative results, particularly in the 2.5-10 ng/ml range.
Measurement of the percentage of non-protein-bound (i.e. free) PSA
in serum, which is lower in patients with prostate cancer, have
been evaluated as a method for increasing the accuracy of PSA
testing.
[0003] Thus measurement of PSA in serum, has been postulated as
having potential clinical utility for increasing the sensitivity
and specificity of PSA testing. Cutoff figures are affected by
total PSA levels at prostate value. The prevalence rate of cancer
in the screened population, depending on age, race, previous biopsy
history etc., also influences the screening cutoffs. It has also
been postulated that the percentage of free PSA may also correlate
with a potential aggressiveness of early-stage prostate cancer.
Thus, the level of free PSA may not only be used in order to
diagnose prostate cancer, but also to predict the course of
development of this cancer, and the patient's prognosis, and decide
on a suitable treatment regime.
[0004] Human kallikrein-2 gene (termed herein after: "KLK" which is
also known as KLK-2) is transcribed from the same locus as the PSA
and is also known to be prostate specific. It has been speculated
that both PSA and KLK have common expression control such as common
enhancer and/or promoter and both function as serine proteases.
GLOSSARY
[0005] In the following description and claims use will be made, at
times, with a variety of terms, and the meaning of such terms as
they should be construed in accordance with the invention is as
follows:
[0006] "Prostate specific antigen (PSA) variant"--the sequence
shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6, sequences having
at least 70% identity to said sequence and fragments of the above
sequences of least 20 b.p. long. SEQ ID NO: 1 to ID NO:5 are
nucleic acid sequences which resulted from alternative splicing of
the native and known PSA sequence appearing in HSPSAR and
HUMPSANTIG (GenBank Acc. X05332 and M24543, respectively). It
should be emphasized that the PSA variants of the invention are
naturally occurring sequences resulting from the alternative
splicing of the RNA transcribed from the PSA gene and not merely
truncated or mutated forms of the gene. SEQ ID NO: 6 is an
alternative splice variant of the human kallikrein-2 gene (KLK-2)
appearing in GenBank as KLK2 under Accession Number
NM.sub.--005551.
[0007] SEQ ID NO: 1--(PSAL.sub.--0): The nucleic acid sequence
starting in position 4364 of the HUMSANTIG up to position 7305,
then a different sequence. The coded peptide (SEQ ID NO:7) starting
identically to the original PSA for 16 aa, then a different
sequence which is trancribed from the PSA intron between exons 1
and 2. SEQ ID NO:2-(PSAL.sub.--1):
[0008] Nucleic acid sequence identical to SEQ 1.
[0009] Peptide (SEQ ID NO: 8)--Starting in a Methionine 114 aa
upstream from the original PSA, and has the same 16 aa identity and
3' end as PSAL.sub.--0.
[0010] SEQ ID NO:3--(PSAL.sub.--2):
[0011] Nucleic acid sequence which starts in same place as
PSAL.sub.--0 but goes up to position 6336 of the HUMPSANTIG, then
continues in a different sequence.
[0012] Peptide (SEQ ID NO: 9)--Identical to PSAL.sub.--1
peptide.
[0013] SEQ ID NO: 4--(PSAL.sub.--5):
[0014] Nucleic acid sequence which starts in same place as
PSAL.sub.--0, goes up to position 6069 of HUMPSANTIG and end there
(original intron).
[0015] Peptide (SEQ ID NO:10)--Has same starting place as
PSAL.sub.--1, the same 16 aa identity to PSA, then a different
intron region translated.
[0016] SEQ ID NO: 5--(PSAL.sub.--6):
[0017] Nucleic acid sequence starts in the same place as
PSAL.sub.--0, goes up to position 5913 of HUMPSANTIG, then enter
the original PSA exon # 2 and continues. Peptide (SEQ ID NO:11) has
same starting place as PSAL.sub.--1, then enters the same identity
region and continues as the original PSA until the end.
[0018] SEQ ID NO:6 is a splice variant of the KLK-2 that includes
coding region from the original KLK-2 intron between exons 1 and 2.
The term of "PSA variant" in the context of the present invention
concerns splice variants of the known PSA gene as well as splice
variants of the KLK-2 gene, which is also known to code for
antigens specific to the prostate.
[0019] "Prostate specific antigen variant product (PSA variant
product)--also referred at times as the "PSA variant protein" or
"PSA variant polypeptide"--an amino acid sequence coded by said PSA
variant nucleic acid sequence. The amino acid sequence may be a
peptide, a protein, as well as peptides or proteins having
chemically modified amino acids (see below) such as a glycopeptide
or glycoprotein. An example of a PSA variant product is shown in
any one of SEQ ID NO: 7 to SEQ ID NO: 12, and includes also
analogues of said sequences in which one or more amino acids has
been added, deleted, substituted (see below) or chemically modified
(see below) as well as fragments of this sequence having at least
10 amino acids. The products may be membrane associated or present
in a free form in body fluids, for example in the serum.
[0020] "Nucleic acid sequence"--a sequence composed of DNA
nucleotides, RNA nucleotides or a combination of both types and may
includes natural nucleotides, chemically modified nucleotides and
synthetic nucleotides.
[0021] "Amino acid sequence"--a sequence composed of any one of the
20 naturally appearing amino acids, amino acids which have been
chemically modified (see below), or composed of synthetic amino
acids.
[0022] "Fragment of PSA variant product"--a sequence which is the
same as part of but not all of the amino acid sequence of the PSA
variant product.
[0023] "Fragments of PSA variant nucleic acid sequence" a
continuous portion, preferably of about 20 nucleic acid sequences
of the PSA variant nucleic acid sequence (see below), which
sequence does not appear in the original PSA.
[0024] "Conservative substitution"--refers to the substitution of
an amino acid in one class by an amino acid of the same class,
where a class is defined by common physicochemical amino acid side
chain properties and high substitution frequencies in homologous
proteins found in nature, as determined, for example, by a standard
Dayhoff frequency exchange matrix or BLOSUM matrix. [Six general
classes of amino acid side chains have been categorized and
include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class
III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile,
Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example,
substitution of an Asp for another class III residue such as Asn,
Gln, or Glu, is a conservative substitution.
[0025] "Non-conservative substitution"--refers to the substitution
of an amino acid in one class with an amino acid from another
class; for example, substitution of an Ala, a class II residue,
with a class III residue such as Asp, Asn, Glu, or Gln.
[0026] "Chemically modified"--when referring to the product of the
invention, means a product (protein) where at least one of its
amino acid resides is modified either by natural processes, such as
processing or other post-translational modifications, or by
chemical modification techniques which are well known in the art.
Among the numerous known modifications typical, but not exclusive
examples include: acetylation, acylation, amidation,
ADP-ribosylation, glycosylation, GPI anchor formation, covalent
attachment of a lipid or lipid derivative, methylation,
myristlyation, pegylation, prenylation, phosphorylation,
ubiqutination, or any similar process.
[0027] "Biologically active"--refers to a PSA variant product which
has the ability to serve as a marker of cancer, of predisposition
to cancer, or of malignancy of a tumor.
[0028] "Immunologically active" defines the capability of a
natural, recombinant or synthetic PSA variant product, or any
fragment thereof, to induce a specific immune response in
appropriate animals or cells and to bind with specific antibodies.
Thus, for example, a biologically active fragment of PSA variant
product denotes a fragment which retains some or all of the
biological properties of the PSA variant product, e.g the ability
to serve as a marker for prostate cancer; an immunologically active
fragment is a fragment which can bind specific anti-PSA variant
product antibodies or "distinguishing antibodies" (see below) which
can elicit an immune response which will generate such antibodies
or cause proliferation of PSA variant product-specific immune
cells. The fragment will also be denoted hereinafter as
"distinguishing amino acid sequence".
[0029] "Optimal alignment"--is defined as an alignment giving the
highest percent identity score. Such alignment can be performed
using a variety of commercially available sequence analysis
programs, such as the local alignment program LALIGN using a ktup
of 1, default parameters and the default PAM. A preferred alignment
is the one performed using the CLUSTAL-W program from MacVector
(.TM.), operated with an open cap penalty of 10.0, an extended gap
penalty of 0.1, and a BLOSUM similarity matrix. If a gap needs to
be inserted into a first sequence to optimally align it with a
second sequence, the percent identity is calculated using only the
residues that are paired with a corresponding amino acid residue
(i.e., the calculation does not consider residues in the second
sequences that are in the "gap" of the first sequence).
[0030] "Having at least X% identity"--with respect to two amino
acid or nucleic acid sequence sequences, refers to the percentage
of residues that are identical in the two sequences when the
sequences are optimally aligned. Thus, 90% amino acid sequence
identity means that 90% of the amino acids in two or more optimally
aligned polypeptide sequences are identical.
[0031] "Isolated nucleic acid molecule having an PSA variant
nucleic acid sequence" --is a nucleic acid molecule that includes
the coding PSA variant nucleic acid sequences. Said isolated
nucleic acid molecule may include the PSA variant nucleic acid
sequence as an independent insert; may include the PSA variant
nucleic acid sequence fused to an additional coding sequences,
encoding together a fusion protein in which the PSA variant coding
sequence is the dominant coding sequence (for example, the
additional coding sequence may code for a signal peptide); the PSA
variant nucleic acid sequence may be in combination with non-coding
sequences, e.g., introns or control elements, such as promoter and
terminator elements or 5' and/or 3' untranslated regions, effective
for expression of the coding sequence in a suitable host; or may be
a vector in which the PSA variant protein coding sequence is a
heterologous.
[0032] "Expression vector"--refers to vectors that have the ability
to incorporate and express heterologous DNA fragments in a foreign
cell. Many prokaryotic and eukaryotic expression vectors are known
and/or commercially available. Selection of appropriate expression
vectors is within the knowledge of those having skill in the
art.
[0033] "Deletion"--is a change in either nucleotide or amino acid
sequence in which one or more nucleotides or amino acid residues,
respectively, are absent.
[0034] "Insertion" or "addition"--is that change in a nucleotide or
amino acid sequence which has resulted in the addition of one or
more nucleotides or amino acid residues, respectively, as compared
to the naturally occurring sequence.
[0035] "Substitution"--replacement of one or more nucleotides or
amino acids by different nucleotides or amino acids, respectively.
As regards amino acid sequences the substitution may be
conservative or non- conservative.
[0036] "Antibody"--refers to IgG, IgM, IgD, IgA, and IgG antibody.
The definition includes polyclonal antibodies or monoclonal
antibodies. This term refers to whole antibodies or fragments of
the antibodies comprising the antigen-binding domain of the
anti-PSA variant product antibodies, e.g. antibodies without the Fc
portion, single chain antibodies, fragments consisting of
essentially only the variable, antigen-binding domain of the
antibody, etc.
[0037] "Distinguishing antibody"--an antibody capable of binding
only to the novel PSA variant product of the invention while not
binding to the original PSA product, i.e. an antibody recognizing
an additional amino acid sequence which appears only in the variant
product of the invention and not in the original PSA sequence. This
term may also refer at times to antibodies which binda sequence
present in the original PSA and not present in the PSA variant
product.
[0038] "Distinguishing amino acid sequence"--an amino acid sequence
of at least two amino acids which are present only in the PSA
variant of the invention and not in the original PSA of which are
used to prepare the above distinguishing antibodies.
[0039] "Activator"--as used herein, refers to a molecule which
mimics the effect of the natural PSA variant product or at times
even increases or prolongs the duration of the biological activity
of said product, as compared to that induced by the natural
product. The mechanism may be by binding to the PSA variant
receptor, by prolonging the lifetime of the PSA variant, by
increasing the activity of the PSA variant on its target, by
increasing the affinity of PSA variant to its receptor, etc.
Activators may be polypeptides, nucleic acids, carbohydrates,
lipids, or derivatives thereof, or any other molecules which can
bind to and activate the PSA variant product.
[0040] "Deactivator"--refers to a molecule which modulates the
activity of the PSA variant product in an opposite manner to that
of the activator, by decreasing or shortening the duration of the
biological activity of the PSA variant product. This may be done by
blocking the binding of the PSA variant to its receptor,
competitive or non competitive inhibitor, by causing rapid
degradation of the PSA variant, etc. Deactivators may be
polypeptides, nucleic acids, carbohydrates, lipids, or derivatives
thereof, or any other molecules which bind to and modulate the
activity of said product.
[0041] "Treating a disease"--refers to administering a therapeutic
substance effective to ameliorate symptoms associated with a
disease, to lessen the severity or cure the disease, or to prevent
the disease from occurring. In the context of the invention the
disease is typically cancer and in particular prostate cancer.
[0042] "Detection"--refers to a method of detection of a disease,
such as prostate cancer. May be detection of an active disease or
detection of a predisposition to a disease. By another alternative
the detection may be capable of distinguishing between benign and
malignant conditions. This term may also be used in connection with
a method for evaluating the aggressiveness of a malignant state in
order to correctly predict the prognosis of the patient, and in
that case the detection may be used to assess the stage of the
tumor.
[0043] "Probe"--the PSA variant nucleic acid sequence, or a
sequence (including fragments) complementary therewith, when used
to detect presence of other similar sequences in a sample. The
detection is carried out by identification of hybridization
complexes between the probe and the assayed sequence. The probe may
be attached to a solid support or to a detectable label. The probe
may be a fragment of any one of the SEQ ID NO: 1 to SEQ ID NO: 6
(including a is fragment of the non-coding region) which is of
sufficient length to hybridize to the PSA variants at a level
significantly different from the binding to the original PSA
sequence. The probes may also be used to detect the polymorphisms
described in the nucleic acid for the purpose of determining
predisposition to cancer, especially prostate cancer in healthy
individuals, and for detecting loss of heterozigosity in prostate
tissues as part of a malignant transformation. The probes may be
used in any method of performing this assay, including
primer-specific PCR, allele-specific oligonucleotide assay,
restriction fragment length differences, and mini-sequencing.
[0044] "Targeting,"--directing a compound or drug to a desired cell
population.
[0045] Targeting is carried out by conjugating to the compound or
drug an agent capable of binding specifically to the desired cell
population, while not binding to non-desired cell populations. A
specific example is targeting cytotoxic drugs directed only to
tumor cells, more specifically directed to prostate tumor cells,
for example, by conjugating the drug to an antibody of the
invention.
[0046] "Original PSA sequence"--the known sequence of PSA as
appears in GenBank HSPSAR locus and Acc # X05332, as well as to the
known KLK-2 sequence as appears in GenBank KLK2
(NM.sub.--005551).
SUMMARY OF THE INVENTION
[0047] The present invention provides by its first aspect, a novel
isolated nucleic acid molecule comprising or consisting of the
coding sequence of any one of SEQ ID NO:1 to SEQ ID NO: 6,
fragments of said coding sequence having at least 20 nucleic acids,
or a molecule comprising a sequence having at 90% identity to any
one of SEQ ID NO:1 to SEQ ID NO: 6. Preferably, the fragments
should be such that they comprise sequences present in the PSA
variants of the invention and not a sequence present in the
original PSA (the term "original PSA" also includes the KLK-2
sequence).
[0048] These sequences are novel splice variants which results from
alternative splicing of the original PSA sequence (this term
according to the glossary refers also to the KLK-2sequence).
[0049] The present invention further provides a protein or
polypeptide comprising or consisting of an amino acid sequence
encoded by any of the above nucleic acid sequences, termed herein
"PSA variant product", for example, an amino acid sequence having
the sequence as depicted in any one of SEQ ID NO: 7 to SEQ ID NO:
12 fragments of the above amino acid sequence having a length of at
least 10 amino acids, in particular fragments comprising sequences
which do not appear in the original PSA sequence, as well as
homologues of the amino acid sequences SEQ ID NO.:7 to SEQ ID NO:
12 in which one or more of the amino acid residues has been
substituted (by conservative or non-conservative substitution)
added, deleted, or chemically modified.
[0050] The novel PSA variant products of the invention may have the
same physiological activity as the original PSA peptide (this term
refers also to the KLK-2 product) from which they are varied
(although perhaps at a different level); may have an opposite
physiological activity from the activity featured by the original
peptide from which they are varied; may have a completely
different, unrelated activity to the activity of the original from
which they are varied; or alternatively may have no activity at all
and this may lead to various diseases or pathological
conditions.
[0051] The novel variants of the invention whether being nucleic
acid or amino acid sequences may serve for detection purposes, i.e.
their presence or level may be indicative of prostate cancer,
predisposition to prostate cancer, malignancy of the cancer, stage
of the cancer, or may be indicative to normal condition.
Alternatively the ratio between the level of each variant and the
level original PSA sequence from which it has been varied; the
ratio of each variant to the or other variants; the total amount
(sum) of two or more variants either by itself or compared to other
variants; or the sum of two or more variants, may be indicative of
cancer or predisposition to cancer in general, and prostate cancer
or predisposition to prostate cancer in particular, as well as
indicative of the malignancy of the cancer, its stage of
development or of normal condition. The variants may be detected in
blood or serum or in the prostate gland, the ovary, breast or
salivary glands, which may share gene properties with the prostate
gland. The variant products may be soluble or membrane bound.
[0052] For example, for detection purposes, it is possible to
establish differential expression of the various variants in
various tissues. A certain variant may be expressed mainly in one
tissue, while the original PSA sequence may be expressed mainly in
another tissue such as the prostate. Understanding of the
distribution of the variants in various tissues may be helpful in
basic research, for understanding the physiological function of the
genes as well as may help in targeting pharmaceuticals or
developing pharmaceuticals.
[0053] The study of the variants may also be helpful to distinguish
various stages in the life cycles of the same type of cells which
may also be helpful for development of pharmaceuticals for various
pathological conditions in which cell cycles is un-normal, notably
cancer. For example, various stages in the development of prostate
cancer may be characterized by expression, or change in level of
individual PSA variants of the invention.
[0054] Thus the detection may by determination of the presence or
the level of expression of the variant within a specific cell
population, comprising said presence or level between various cell
types in a tissue, between different tissues and between
individuals.
[0055] The present invention further provides nucleic acid molecule
comprising or consisting of a sequence which encodes the above
amino acid sequences, (including the fragments and analogs of the
amino acid sequences). Due to the degenerative nature of the
genetic code, a plurality of alternative nucleic acid sequences,
beyond those of SEQ ID NO:1 to SEQ ID NO: 6, can code for the amino
acid sequences of the invention. Those alternative nucleic acid
sequences which code for the amino acid sequences codes by any one
of the sequence SEQ ID NO: 1 to SEQ ID NO: 6 are also an aspect of
the of the present invention.
[0056] The present invention farther provides expression vectors
and cloning vectors comprising any of the above nucleic acid
sequences, as well as host cells transfected by said vectors.
[0057] The present invention still further provides pharmaceutical
compositions comprising, as an active ingredient, said nucleic acid
molecules, said expression vectors, or said protein or
polypeptide.
[0058] These pharmaceutical compositions are suitable for the
treatment of diseases and pathological conditions, which can be
ameliorated or cured by raising the level of the PSA variant
product, for example for the treatment of prostate cancer, or for
inhibiting the transformation from prostate hyperplasia to
malignancy. By another aspect, the present invention provides a
nucleic acid molecule comprising or consisting of a non-coding
sequence which is complementary to that of any one of SEQ ID NO:1
to SEQ ID NO: 6, or complementary to a sequence having at least 90%
identity to said sequence or a fragment of said two sequences. The
complementary sequence may be a DNA sequence which hybridizes with
any one of the SEQ of ID NO:1 to SEQ ID NO: 6 or hybridizes to a
portion of that sequence having a length sufficient to inhibit the
transcription of the complementary sequence. The complementary
sequence may be a DNA sequence which can be transcribed into an
mRNA being an antisense to the mRNA transcribed from SEQ ID NO:1 to
SEQ ID NO: 6 or into an mRNA which is an antisense to a fragment of
the mRNA transcribed from SEQ ID NO.:1 to SEQ ID NO: 6 which has a
length sufficient to hybridize with the mRNA transcribed from SEQ
ID NO:. 1 to SEQ ID NO:6, so as to inhibit its translation. The
complementary sequence may also be the mRNA or the fragment of the
mRNA itself.
[0059] The nucleic acids of the invention may be used for
therapeutic or diagnostic applications for example for detection of
the expression of PSA variant in various tissues which may be
indicative to the presence of prostate cancer, indicative of
pre-disposition to prostate cancer, as well as indicative of the
malignancy and hence the prognosis of the prostate cancer. The
variants of the invention may also be indicative of other types of
cancer from glands binding physiological similarity to the prostate
gland such as ovary, breast, and salivary gland.
[0060] The present invention also provides expression vectors
comprising any one of the above defined complementary nucleic acid
sequences and host cells transfected with said nucleic acid
sequences or vectors, being complementary to those specified in the
first aspect of the invention.
[0061] The invention also provides anti-PSA variant product
antibodies, namely antibodies directed against the PSA variant
product which specifically bind to said PSA variant product. Said
antibodies are useful both for diagnostic and therapeutic purposes.
For example said antibody may be used to detect the presence of
prostate specific antigen-variant product in various tissues which
may be indicative of the presence of prostate cancer of a
predisposition for having prostate cancer, or of the malignancy of
prostate cancer.
[0062] The present invention further concerns distinguishing
antibodies which can bind only to a sequence present in the
variants of the invention which is not present (as a continuous
sequence) in the original PSA sequence. The present invention
further concerns amino acid sequences for producing said
distinguishing antibodies termed "distinguishing amino acid
sequences" which are sequences present in the novel PSA variant and
not present (as a continuous sequence) in the original PSA An
example of such a sequence is the sequence of positions 33-51 in
SEQ ID NO:7 being:
[0063]
Cys-Gln-Ala-Glu-Leu-Ser-Pro-Pro-Thr-Gln-His-Pro-Ser-Pro-Asp-Arg-Glu-
-Leu
[0064] The present invention also provides pharmaceutical
compositions comprising, as an active ingredient, the nucleic acid
molecules which comprise or consist of said complementary
sequences, or of a vector comprising said complementary sequences.
Alternatively, the pharmaceutical composition can comprise, as an
active ingredient, said anti-PSA variant product antibodies, or
said distinguishing antibodies.
[0065] The pharmaceutical compositions comprising said anti-PSA
variant product antibodies, said distinguishing antibodies or the
nucleic acid molecule comprising said complementary sequence, are
suitable for the treatment of diseases and pathological conditions
where a therapeutically beneficial effect may be achieved by
neutralizing at least one of the PSA variants or decreasing the
amount of the PSA variant product or blocking its binding to the
receptor, for example, by the neutralizing effect of the
antibodies, or by the effect of the antisense mRNA in decreasing
expression level of the PSA variant product. An example of such a
disease is prostate cancer. Furthermore, where the PSA variant is
membrane bound, the anti-PSA variant antibodies may be used to
target cytotoxic or cytostatic compounds to the tumor cells, in
particular to prostate tumor cells. Since PSA variants may be
produced specifically by prostate tumor cells, (and not normal
prostate cells) and since this protein may be membrane associated,
conjugates of anti-PSA variant antibodies and a drug can be
targeted only to tumor cells and not harm healthy cells.
[0066] According to the third aspect of the invention the present
invention provides methods for detecting the level of the
transcript (mRNA) of said PSA variant product in a body fluid
sample, or in a specific tissue sample, for example by use of
probes comprising or consisting of said sequences (which may be a
coding or uncoding sequence), as well as methods for detecting
levels of expression of said product in tissue, e.g. by the use of
antibodies capable of specifically reacting with the above amino
acid sequences.
[0067] The method, according to this latter aspect, for detection
of a nucleic acid sequence which encodes the PSA variant product in
a biological sample, comprises the steps of:
[0068] (a) providing a probe comprising at least one of the nucleic
acid sequence defined above;
[0069] (b) contacting the biological sample with said probe under
conditions allowing hybridization of nucleic acid sequences thereby
enabling formation of hybridization complexes;
[0070] (c) detecting hybridization complexes, wherein the presence
of the complex indicates the presence of nucleic acid sequence
encoding the PSA variant product in the biological sample.
[0071] The method as described above is qualitative, i.e. indicates
whether the transcript is present in or absent from the sample. The
method can also be quantitative, by determining the level of
hybridization complexes and then calibrating said levels to
determining levels of transcripts of the desired PSA variant in the
sample.
[0072] Both qualitative and quantitative determination methods can
be used for diagnostic, prognostic and therapy planning
purposes.
[0073] By a preferred embodiment the probe is part of a nucleic
acid chip used for detection purposes, i.e. the probe is a part of
an array of probes each present in a known location on a solid
support.
[0074] As indicated above the method may be utilized for detecting
the presence of prostate cancer, detecting predisposition to
prostate cancer, or evaluating the malignancy of prostate cancer,
or assessing the development stage of the cancer.
[0075] The nucleic acid sequence used in the above method may be a
DNA sequence, an RNA sequence, etc; it may be a coding or a
sequence, or a non-coding sequence, or a sequence complementary
thereto (for respective detection of RNA transcripts or coding-DNA
sequences). By quantization of the level of hybridization complexes
and calibrating the quantified results it is possible also to
detect the level of the transcript in the sample.
[0076] The probes of the invention may be used to detect
polymorphisms (in a specific individual or while screening a
population) specifically for pre-disposition to cancer (especially
prostate cancer) and loss-of-heterozigosity may be important for
monitoring the development of the disease. Detection of disease
predisposition or loss of heterozigosity in prostate tissue may be
performed on either the coding or non-coding DNA sequence. One
example of such a test is the determination of the exact sequence
before position 5620 in GenBank HUMPSANTIG/1257 of SEQ ID 1 5620
(which is non coding and which contains an additional inserted A as
compared to the native PSA), or testing a possible A to G
substitution in position 5573 of HUMPSANTIG/1210 of SEQ ID 1. Both
these sites may be indicative of cancer risk and useful in
prognosis.
[0077] Methods for detecting mutations in the region coding for the
PSA variant product are also provided, which may be methods
carried-out in a binary fashion, namely merely detecting whether
there is any mismatches between the normal PSA variant nucleic acid
sequence and the one present in the sample, or carried-out by
specifically detecting the nature and location of the mutation.
[0078] The present invention also concerns a method for detecting
PSA variant product in a biological sample, comprising the steps
of:
[0079] (a) contacting with said biological sample the antibody of
the invention, thereby forming an antibody-antigen complex; and
[0080] (b) detecting said antibody-antigen complex
[0081] wherein the presence of said antibody-antigen complex
correlates with the presence of PSA variant product in said
biological sample.
[0082] As indicated above, the method can be quantitized to
determine the level or the amount of the PSA variant in the sample,
alone or in comparison to the level of the original PSA amino acid
sequence from which it was varied, and qualitative and quantitative
results may be used for diagnostic, prognostic and therapy planning
purposes.
[0083] By yet another aspect the invention also provides a method
for identifying candidate compounds capable of binding to the PSA
variant product and modulating its activity (being either
activators or deactivators). The method includes:
[0084] (i) providing a protein or polypeptide comprising an amino
acid sequence substantially as depicted in any one of SEQ ID NO: 7
to SEQ ID NO: 12, or a fragment of such a sequence;
[0085] (ii) contacting a candidate compound with said amino acid
sequence;
[0086] (iii) measuring the physiological effect of said candidate
compound on the activity of the amino acid sequences and selecting
those compounds which show a significant effect on said
physiological activity.
[0087] The activity of the amino acid which should be changed by
the modulator (being either the activator or deactivator) may be
for example the binding of the amino acid (PSA variant product) to
its native, receptor. Any modulator which changes such an activity
has an intersecting potential
[0088] The present invention also concerns compounds identified by
the above methods described above, which compound may either be an
activator of the serotonin-receptor like product or a deactivator
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0089] In order to understand the invention and to see how it may
be carried out in practice, a preferred embodiment will now be
described, by way of non-limiting example only, with reference to
the accompanying drawings, in which:
[0090] FIG. 1 shows the sequence of the original PSA as depicted in
HUMPSANTIG. (This is the genomic sequence, which is the same in PSA
and PSAL);
[0091] FIG. 2 shows the translation of the original PSA
sequence;
[0092] FIG. 3 shows multiple alignment between the original PSA
sequence (termed "HUMPSANTIG") and the sequences of the invention
(PSAL-O is SEQ ID NO:1; PSAL-1 is SEQ ID NO:2, PSAL-2 is SEQ ID
NO:3-; PSAL-5 is SEQ ID NO:4 and PSAL-6 is SEQ ID NO.5);
[0093] FIG. 4 shows a multiple alignment between the 5 PSA splice
variant products and the direct translation of the genomic PSA
region, depicted here as HUMPSANTIG;
[0094] FIG. 5 shows the specific region (signal peptide) of the
original PSA that is common with all the splice variants of
PSA;
[0095] FIG. 6 shows a schematic representation of the common locus
of the KLK-2 of PSA genes;
[0096] FIG. 7 shows a Northern Blot analysis of RNA obtained from
various tissues and tested with probes for PSA (left) and probes
obtained from SEQ ID NO:2, (termed PSALM in the Figure)
(right);
[0097] FIG. 8 shows a Western Blot analysis of proteins obtained
from prostate glands of several patients tested for PSA protein
(left) and the PSA variant protein depicted in SEQ ID NO: 2
(right);
[0098] FIG. 9 shows immune-histochemical labeling of human prostate
gland with serum of rabbit immunized with PSAL variant peptide of
the invention (right) or unimmunized rabbit;
[0099] FIG. 10 shows a cross-section of in-situ hybridization of
sense and anti-sense probes of PSA (termed "PSALM") to tissue
obtained from a prostate cancer;
[0100] FIG. 11 shows a Western blot analysis of 2 different
serum-samples tested for PSA protein PSA (termed "PSALM"). The
results indicate that the PSA is secreted to the serum. In the
right lane there is a recombinant PSA;
[0101] FIG. 12 shows immuno-histochemical labeling of human
prostate gland with serum of rabbit immunized with the peptide of
the invention (derived from alternative splicing of the KLK-2 gene)
(right) or unimmunized rabbit; and
[0102] FIG. 13 shows a Western blot analysis of proteins obtained
from prostate glands of 2 different patients (prostate #1 and #2),
and from other tissues tested for the protein of the invention
derived from the KLK-2 gene.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
Example I
PSA Variant--Nucleic Acid Sequence
[0103] The nucleic acid sequences of the invention include nucleic
acid sequences which encode PSA variant product and fragments and
analogs thereof. The nucleic acid sequences may alternatively be
sequences complementary to the above coding sequence, or to a
region of said coding sequence. The length of the complementary
sequence is sufficient to avoid the expression of the coding
sequence. The nucleic acid sequences may be in the form of RNA or
in the form of DNA, and include messenger RNA, synthetic RNA and
DNA, cDNA, and genomic DNA. The DNA may be double-stranded or
single-stranded, and if single-stranded may be the coding strand or
the non-coding (anti-sense, complementary) strand. The nucleic acid
sequences may also both include dNTPs, rNTPs as well as non
naturally occurring sequences. The sequence may also be a part of a
hybrid between an amino acid sequence and a nucleic acid
sequence.
[0104] In a general embodiment, the nucleic acid sequence has at
least 70%, preferably 80% or 90% sequence identity with the
sequence identified as SEQ ID NO:1 to SEQ ID NO:6.
[0105] The nucleic acid sequences may include the coding sequence
by itself. By another alternative the coding region may be in
combination with additional coding sequences, such as those coding
for fusion protein or signal peptides, in combination with
non-coding sequences, such as introns and control elements,
promoter and terminator elements or 5' and/or 3' untranslated
regions, effective for expression of the coding sequence in a
suitable host, and/or in a vector or host environment in which the
PSA variant nucleic acid sequence is introduced as a heterologous
sequence.
[0106] The nucleic acid sequences of the present invention may also
have the product coding sequence fused in-frame to a marker
sequence which allows for purification of the PSA variant product.
The marker sequence may be, for example, a hexahistidine tag to
provide for purification of the mature polypeptide fused to the
marker in the case of a bacterial host, or, the marker sequence may
be a hemagglutinin (HA) tag when a mamnualian host, e.g. COS-7
cells, is used. The HA tag corresponds to an epitope derived from
the influenza hemagglutinin protein (Wilson, I., et al. Cell 37:767
(1984)).
[0107] Also included in the scope of the invention are fragments
also referred to herein as oligonucleotides, typically having at
least 20 bases, preferably 20-30 bases corresponding to a region of
the coding-sequence nucleic acid sequence. The fragments may be
used as probes, primers, and when complementary also as antisense
agents, and the like, according to known methods.
[0108] As indicated above, the nucleic acid sequence may be
substantially a depicted in SEQ ID NO:1 to SEQ ID NO:6 or fragments
thereof or sequences having at least 70%, preferably 70-80%, most
preferably 90% identity to the above sequence. Alternatively, due
to the degenerative nature of the genetic code, the sequence may be
a sequence coding the amino acid sequence of SEQ ID NO:6 to SEQ ID
NO:12, or fragments or analogs of said amino acid sequence.
[0109] A. Preparation of Nucleic Acid Sequences
[0110] The nucleic acid sequences may be obtained by screening cDNA
libraries using oligonucleotide probes which can hybridize to or
PCR-amplify nucleic acid sequences which encode the PSA variant
products disclosed above. cDNA libraries prepared from a variety of
tissues are commercially available and procedures for screening and
isolating cDNA clones are well-known to those of skill in the art.
Such techniques are described in, for example, Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual (2nd Edition), Cold
Spring Harbor Press, Plainview, N.Y. and Ausubel FM et al. (1989)
Current Protocols in Molecular Biology, John Wiley & Sons, New
York, N.Y.
[0111] The nucleic acid sequences may be extended to obtain
upstream and downstream sequences such as promoters, regulatory
elements, and 5' and 3' untranslated regions (UTRs). Extension of
the available transcript sequence may be performed by numerous
methods known to those of skill in the art, such as PCR or primer
extension (Sambrook et al., supra), or by the RACE method using,
for example, the Marathon RACE kit (Clontech, Cat. #K1802-1).
[0112] Alternatively, the technique of "restriction-site" PCR
(Gobinda et al. PCR Methods Applic. 2:318-22, (1993)), which uses
universal primers to retrieve flanking sequence adjacent a known
locus, may be employed. First, genomic DNA is amplified in the
presence of primer to a linker sequence and a primer specific to
the known region. The amplified sequences are subjected to a second
round of PCR with the same linker primer and another specific
primer internal to the first one. Products of each round of PCR are
transcribed with an appropriate RNA polymerase and sequenced using
reverse transcriptase.
[0113] Inverse PCR can be used to amplify or extend sequences using
divergent primers based on a known region (Triglia, T. et al.,
Nucleic Acids Res. 16:8186, (1988)). The primers may be designed
using OLIGO(R) 4.06 Primer Analysis Sofvare (1992; National
Biosciences Inc, Plymouth, Minn.), or another appropriate program,
to be 22-30 nucleotides in length to have a GC content of 50% or
more, and to anneal to the target sequence at temperatures about
68-72.degree. C. The method uses several restriction enzymes to
generate a suitable fragment in the known region of a gene. The
fragment is then circularized by intramolecular ligation and used
as a PCR template.
[0114] Capture PCR (Lagerstrom, M. et al., PCR Methods Applic.
1:111-19, (1991)) is a method for PCR amplification of DNA
fragments adjacent to a known sequence in human and yeast
artificial chromosome DNA. Capture PCR also requires multiple
restriction enzyme digestions and ligations to place an engineered
double-stranded sequence into a flanking part of the DNA molecule
before PCR.
[0115] Another method which may be used to retrieve fianklnz
sequences is that of Parker. J. D., et al, Nucleic Acids Res.,
19:3055-60, (1991)). Additionally, one can use PCR, nested primers
and PromoterFinder.TM. libraries to "walk in" genomic DNA
(PromoterFinder.TM.; Clontech, Palo Alto, Calif.). This process
avoids the need to screen libraries and is useful in finding,
intron/exon junctions. Preferred libraries for screening for full
length cDNAs are ones that have been size-selected to include
larger cDNAs. Also, random primed libraries are preferred in that
they will contain more sequences which contain the 5' and upstream
regions of genes.
[0116] A randomly primed library may be particularly useful if an
oligo d(T) library does not yield a full-length cDNA. Genomic
libraries are useful for extension into the 5' nontranslated
regulatory region.
[0117] The nucleic acid sequences and oligonucleotides of the
invention can also be prepared by solid-phase methods, according to
known synthetic methods. Typically, fragments of up to about 100
bases are individually synthesized, then joined to form continuous
sequences up to several hundred bases.
[0118] B. Use of PSA Variant Nucleic Acid Sequence for the
Production of PSA Variant Products
[0119] In accordance with the present invention, nucleic acid
sequences specified above may be used as recombinant DNA molecules
that direct the expression of PSA variant products.
[0120] As will be understood by those of skill in the art, it many
be advantageous to produce PSA variant product-encoding nucleotide
sequences possessing codons other than those which appear in any
one of SEQ ID NO:1 to SEQ ID NO:6 which are those which naturally
occur in the human genome. Codons preferred by a particular
prokaryotic or eukaryotic host (Murray, E. et al. Nuc Acids Res.,
17:477-508, (1989)) can be selected, for example, to increase the
rate of PSA variant product expression or to produce recombinant
RNA transcripts having desirable properties, such as a longer
half-life, than transcripts produced from naturally occurring
sequence.
[0121] The nucleic acid sequences of the present invention can be
engineered in order to alter a PSA variant product coding sequence
for a variety of reasons, including but not limited to, alterations
which modify the cloning, processing and/or expression of the
product. For example, alterations may be introduced using
techniques which are well known in the art, e.g., site-directed
mutagenesis, to insert new restriction sites, to alter
glycosylation patterns, to change codon preference, to produce
splice variant, etc.
[0122] The present invention also includes recombinant constructs
comprising one or more of the sequences as broadly described above.
The constructs comprise a vector, such as a plasmid or viral
vector, into which a nucleic acid sequence of the invention has
been inserted, in a forward or reverse orientation. In a preferred
aspect of this embodiment, the construct further comprises
regulatory sequences, including, for example, a promoter, operably
linked to the sequence. Large numbers of suitable vectors and
promoters are known to those of skill in the art, and are
commercially available. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are also described in
Sambrook, et al., (supra).
[0123] The present invention also relates to host cells which are
genetically engineered with vectors of the invention, and the
production of the product of the invention by recombinant
techniques. Host cells are genetically engineered (i.e.,
transduced, transformed or transfected) with the vectors of this
invention which may be, for example, cloning vectors or expression
vectors. The vector may be, for example, in the form of a plasmid,
a viral particle, a phage, etc. The engineered host cells can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying the
expression of the PSA variant nucleic acid sequence. The culture
conditions, such as temperature, pH and the like, are those
previously used with the host cell selected for expression, and
will be apparent to those skilled in the art.
[0124] The nucleic acid sequences of the present invention may be
included in any one of a variety of expression vectors for
expressing a product. Such vectors include chromosomal,
nonchromosomal and synthetic DNA sequences, e.g., derivatives of
SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids;
vectors derived from combinations of plasmids and phage DNA, viral
DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be used as long as it is replicable
and viable in the host. The appropriate DNA sequence may be
inserted into the vector by a variety of procedures. In general,
the DNA sequence is inserted into an appropriate restriction
endonuclease site(s) by procedures known in the art. Such
procedures and related sub-cloning procedures are deemed to be
within the scope of those skilled in the art.
[0125] The DNA sequence in the expression vector is operatively
linked to an appropriate transcription control sequence (promoter)
to direct mRNA synthesis. Examples of such promoters include: LTR
or SV40 promoter, the E.coli lac or trp promoter, the phage lambda
PL promoter, and other promoters known to control expression of
genes in prokaryotic or eukaryotic cells or their viruses.
[0126] The expression vector also contains a ribosome binding site
for translation initiation, and a transcription terminator. The
vector may also include appropriate sequences for amplifying
expression. In addition, the expression vectors preferably contain
one or more selectable marker genes to provide a phenotypic trait
for selection of transformed host cells such as dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture, or
such as tetracycline or ampicillin resistance in E.coli.
[0127] The vector containing the appropriate DNA sequence as
described above, as well as an appropriate promoter or control
sequence, may be employed to transform an appropriate host to
permit the host to express the protein. Examples of appropriate
expression hosts include: bacterial cells, such as E.coli,
Streptomyces, Salmonella typhimurium; fungal cells, such as yeast;
insect cells such as Drosophila and Spodoptera Sf9; animal cells
such as CHO, COS, HEK 293 or Bowes melanoma; adenoviruses; plant
cells, etc. The selection of an appropriate host is deemed to be
within the scope of those skilled in the art from the teachings
herein. The invention is not limited by the host cells
employed.
[0128] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for the PSA variant
product. For example, when large quantities of PSA variant product
are needed for the induction of antibodies, vectors which direct
high level expression of fusion proteins that are readily purified
may be desirable. Such vectors include, but are not limited to,
multifunctional E.coli cloning and expression vectors such as
Bluescript.RTM. (Stratagene), in which the PSA variant polypeptide
coding sequence may be ligated into the vector in-frame with
sequences for the amino-terminal Met and the subsequent 7 residues
of beta-galactosidase so that a hybrid protein is produced; pIN
vectors (Van Heeke & Schuster J. Biol. Chem. 264:5503-5509,
(1989)); pET vectors (Novagen, Madison Wis.); and the like.
[0129] In the yeast Saccharomyces cerevisiae a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase and PGH may be used. For reviews, see
Ausubel et al. (supra) and Grant et al., (Methods in Enzymology
153:516-544, (1987)).
[0130] In cases where plant expression vectors are used, the
expression of a sequence encoding PSA variant product may be driven
by any of a number of promoters. For example, viral promoters such
as the 35S and 19S promoters of CaMV (Brisson et al., Nature
310:511-514. (1984)) may be used alone or in combination with the
omega leader sequence from TMV (Takamnatsu et al., EMBO J.,
6:307-311, (1987)). Alternatively, plant promoters such as the
small subunit of RUBISCO (Coruzzi et al., EMBO J. 3:1671-1680,
(1984); Broglie et al., Science 224:838-843, (1984)); or heat shock
promoters (Winter J and Sinibaldi R. M., Results Probl. Cell
Differ., 17:85-105, (1991)) may be used. These constructs can be
introduced into plant cells by direct DNA transformation or
pathogen-mediated transfection. For reviews of such techniques, see
Hobbs S. or Murry L. E. (1992) in McGraw Hill Yearbook of Science
and Technology, McGraw Hill, New York, N.Y., pp 191-196; or
Weissbach and Weissbach (1988) Methods for Plant Molecular Biology,
Academic Press, New York, N.Y., pp 421-463.
[0131] PSA variant product may also be expressed in an insect
system. In one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The
PSA variant product coding sequence may be cloned into a
nonessential region of the virus, such as the polyhedrin gene, and
placed under control of the polyhedrin promoter. Successful
insertion of PSA variant coding sequence will render the polyhedrin
gene inactive and produce recombinant virus lacking coat protein
coat. The recombinant viruses are then used to infect S. frugiperda
cells or Trichoplusia larvae in which PSA variant protein is
expressed (Smith et al, J. Virol. 46:584, (1983); Engelhard, E. K.
et al., Proc. Nat. Acad. Sci. 91:3224-7, (1994)).
[0132] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, a PSA variant product coding sequence may be
ligated into an adenovirus transcription/translation complex
consisting of the late promoter and tripartite leader sequence.
Insertion in a nonessential E1 or E3 region of the viral genome
will result in a viable virus capable of expressing PSA variant
protein in infected host cells (Logan and Shenk, Proc. Natl. Acad.
Sci. 81:3655-59, (1984). In addition, transcription enhancers, such
as the Rous sarcoma virus (RSV) enhancer, may be used to increase
expression in mammalian host cells.
[0133] Specific initiation signals may also be required for
efficient translation of a PSA variant protein coding sequence.
These signals include the ATG initiation codon and adjacent
sequences. In cases where PSA variant product coding sequence, its
initiation codon and upstream sequences are inserted into the
appropriate expression vector, no additional translational control
signals may be needed. However, in cases where only coding
sequence, or a portion thereof, is inserted, exogenous
transcriptional control signals including the ATG initiation codon
must be provided. Furthermore, the initiation codon must be in the
correct reading frame to ensure transcription of the entire insert.
Exogenous transcriptional elements and initiation codons can be of
various origins, both natural and synthetic. The efficiency of
expression may be enhanced by the inclusion of enhancers
appropriate to the cell system in use (Scharf, D. et al., (1994)
Results Probl. Cell Differ., 20:125-62, (1994); Bittner et al.,
Methods in Enzymol 153:516-544, (1987)).
[0134] In a further embodiment, the present invention relates to
host cells containing the above-described constructs. The host cell
can be a higher eukaryotic cell, such as a mamnualian cell, or a
lower eukaryotic cell, such as a yeast cell, or the host cell can
be a prokaryotic cell, such as a bacterial cell.
[0135] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-Dextran mediated
transfection, or electroporation (Davis, L., Dibner, M., and
Battey, I. (1986) Basic Methods in Molecular Biology). Cell-free
translation systems can also be employed to produce polypeptides
using RNAs derived from the DNA constructs of the present
invention.
[0136] A host cell strain may be chosen for its ability to modulate
the expression of the inserted sequences or to process the
expressed protein in the desired fashion. Such modifications of the
protein include, but are not limited to, acetylation,
carboxylation, glycosylation, phosphorylation, lipidation and
acylation. Post-translational processing which cleaves a "pre-pro"
form of the protein may also be important for correct insertion,
folding and/or function. Different host cells such as CHO, HeLa,
MDCK, 293, WI38, etc. have specific cellular machinery and
characteristic mechanisms for such post-translational activities
and may be chosen to ensure the correct modification and processing
of the introduced, foreign protein.
[0137] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express PSA variant product may be transformed using
expression vectors which contain viral origins of replication or
endogenous expression elements and a selectable marker gene.
Following the introduction of the vector, cells may be allowed to
throw for 1-2 days in an enriched media before they are switched to
selective media. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows growth and
recovery of cells which successfully express the introduced
sequences. Resistant clumps of stably transformed cells can be
proliferated using tissue culture techniques appropriate to the
cell type.
[0138] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler M., et al., Cell
11:223-32, (1977)) and adenine phosphoribosyltransferase (Lowy I.,
et al., Cell 22:817-23, (1980)) genes which can be employed in tk-
or aprt- cells, respectively. Also, antimetabolite, antibiotic or
herbicide resistance can be used as the basis for selection; for
example, dhfr which confers resistance to methotrexate (Wigler M.,
et al, Proc. Natl. Acad Sci. 77:3567-70, (1980)); npt, which
confers resistance to the aminoglycosides neomycin and G-418
(Colbere-Garapin, F. et al, J. Mol. Biol., 150:1-14, (1981)) and
als or pat, which confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described, for example, trpB,
which allows cells to utilize indole in place of tryptophan, or
hisD, which allows cells to utilize histinol in place of histidine
(Hartman S. C. and R. C. Mulligan, Proc. Natl. Acad. Sci.
85:8047-51, (1988)). The use of visible markers has gained
popularity with such markers as anthocyanins, beta-glucuronidase
and its substrate, GUS, and luciferase and its substrates,
luciferin and ATP, being widely used not only to identify
transformants, but also to quantify the amount of transient or
stable protein expression attributable to a specific vector system
(Rhodes, C. A. et. al., Methods Mol. Biol., 55:121-131,
(1995)).
[0139] Host cells transformed with a nucleotide sequence encoding
PSA variant product may be cultured under conditions suitable for
the expression and recovery of the encoded protein from cell
culture. The product produced by a recombinant cell may be secreted
or contained intracellularly depending on the sequence and/or the
vector used. As will be understood by those of skill in the art,
expression vectors containing nucleic acid sequences encoding PSA
variant product can be designed with signal sequences which direct
secretion of PSA variant product through a prokaryotic or
eukaryotic cell membrane.
[0140] PSA variant product may also be expressed as a recombinant
protein with one or more additional polypeptide domains added to
facilitate protein purification. Such purification facilitating
domains include, but are not limited to, metal chelating peptides
such as histidine-tryptophan modules that allow purification on
immobilized metals, protein A domains that allow purification on
immobilized immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp, Seattle,
Wash.). The inclusion of a protease-cleavable polypeptide linker
sequence between the purification domain and PSA variant protein is
useful to facilitate purification.
[0141] One such expression vector provides for expression of a
fusion protein compromising a PSA variant polypeptide fused to a
polyhistidine region separated by an enterokinase cleavage site.
The histidine residues facilitate purification on IMIAC
(immobilized metal ion affinity chromatography, as described in
Porath, et al., Protein Expression and Purification, 3:263-281,
(1992)) while the enterokinase cleavage site provides a means for
isolating PSA variant polypeptide from the fusion protein. pGEX
vectors (Promega, Madison, Wis.) may also be used to express
foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
ligand-agarose beads (e.g., glutathione-agarose in the case of
GST-fusions) followed by elution in the presence of free
ligand.
[0142] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for
an additional period. Cells are typically harvested by
centrifugation, disrupted by physical or chemical means, and the
resulting crude extract retained for further purification.
Microbial cells employed in expression of proteins can be disrupted
by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents, or
other methods, which are well know to those skilled in the art.
[0143] The PSA variant products can be recovered and purified from
recombinant cell cultures by any of a number of methods well known
in the art, including ammonium sulfate or ethanol precipitation,
acid extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography, and lectin chromatography. Protein refolding steps
can be used, as necessary, in completing configuration of the
mature protein. Finally, high performance liquid chromatography
(HPLC) can be employed for final purification steps.
[0144] C. Diagnostic Applications Utilizing Nucleic Acid
Sequences
[0145] The nucleic acid sequences of the present invention may be
used for a variety of diagnostic purposes. The nucleic acid
sequences may be used to detect and quantitate expression of PSA
variant in patient's cells, e.g. biopsied tissues, by detecting the
presence of mRNA coding for PSA variant product. Alternatively, the
assay may be used to detect free PSA variant in the serum or blood.
This assay typically involves obtaining total mRNA from the tissue
or serum and contacting the mRNA with a nucleic acid probe. The
probe is a nucleic acid molecule of at least 20 nucleotides,
preferably 20-30 nucleotides, capable of specifically hybridizing
with a sequence included within the sequence of a nucleic acid
molecule encoding PSA variant under hybridizing conditions,
detecting the presence of mRNA hybridized to the probe, and thereby
detecting the expression of PSA variant. This assay can be used to
distinguish between absence, presence, and excess expression of PSA
variant product and to monitor levels of PSA variant expression
during therapeutic intervention.
[0146] The invention also contemplates the use of the nucleic acid
sequences as a diagnostic for diseases resulting from inherited
defective PSA variant sequences. These sequences can be detected by
comparing the sequences of the defective (i.e., mutant) PSA variant
coding region with that of a normal coding region. Association of
the sequence coding for mutant PSA variant product with abnormal
PSA variant product activity may be verified. In addition,
sequences encoding mutant PSA variant products can be inserted into
a suitable vector for expression in a functional assay system
(e.g., calorimetric assay, complementation experiments in a PSA
variant protein deficient strain of HEK293 cells) as yet another
means to verify or identify mutations. Once mutant genes have been
identified, one can then screen populations of interest for
carriers of the mutant gene.
[0147] Individuals carrying mutations in the nucleic acid sequence
of the present invention may be detected at the DNA level by a
variety of techniques. Nucleic acids used for diagnosis may be
obtained from a patient's cells, including but not limited to such
as from blood, urine, saliva, placenta, tissue biopsy and autopsy
material and in particular tissue obtained from the prostate gland.
Genomic DNA may be used directly for detection or may be amplified
enzymatically by using PCR (Saiki, et al., Nature 324:163-166,
(1986)) prior to analysis. RNA or cDNA may also be used for the
same purpose. As an example, PCR primers complementary to the
nucleic acid of the present invention can be used to identify and
analyze mutations in the gene of the present invention. Deletions
and insertions can be detected by a change in size of the amplified
product in comparison to the normal genotype.
[0148] Point mutations can be identified by hybridizing amplified
DNA to radiolabeled RNA of the invention or alternatively,
radiolabeled antisense DNA sequences of the invention. Sequence
changes at specific locations may also be revealed by nuclease
protection assays, such RNase and S 1 protection or the chemical
cleavage method (e.g. Cotton, et al Proc. Natl. Acad Sci. USA,
85:4397-4401, (1985)), or by differences in melting temperatures.
"Molecular beacons" (Kostrikis L. G. et a!., Science 279:1228-1229,
(1998)), hairpin-shaped, single-stranded synthetic oligo-
nucleotides containing probe sequences which are complementary to
the nucleic acid of the present invention, may also be used to
detect point mutations or other sequence changes as well as monitor
expression levels of PSA variant product.
[0149] Another method for detecting mutations uses two DNA probes
which are designed to hybridize to adjacent regions of a target,
with abutting bases, where the region of known or suspected
mutation(s) is at or near the abutting bases. The two probes may be
joined at the abutting bases, e.g., in the presence of a ligase
enzyme, but only if both probes are correctly base paired in the
region of probe junction. The presence or absence of mutations is
then detectable by the presence or absence of ligated probe.
[0150] Also suitable for detecting mutations in the PSA variant
product coding sequence are oligonucleotide array methods based on
sequencing by hybridization (SBH), as described, for example, in
U.S. Pat. No. 5,547,839. In a typical method, the DNA target
analyte is hybridized with an array of oligonucleotides formed on a
microchip. The sequence of the target can then be "read" from the
pattern of target binding to the array.
[0151] D. In Situ Hybridization Using Probes of PSA
[0152] In-situ hybridisation was carried out according to the
procedure described in the Boehringer-Mannheim's publication
"Non-Radioative In-Situ Hybridization Application Manual", 2.sup.nd
edition, 1996. Labelling was carried out according to Chapter 4,
section V, and hybridization according to Chapter 5, 25 section IV.
Slides were prepared in paraffin and treated according to the
procedures described in Chapter 2. The probe used was derived from
the PSAL-1 sequence (SEQ ID NO: 2). The anti-sense probe was used
to detect the presence of PSA variant mRNA, and the sense probe was
used as control. Results in FIG. 10 indicates high-level expression
of the PSA variant mRNA in prostate epithelial lumen cells.
[0153] E. Therapeutic Applications of Nucleic Acid Sequences
[0154] Nucleic acid sequences of the invention may also be used for
therapeutic purposes. Turning first to the anti-PSA variant aspect,
expression of PSA variant product may be modulated through
antisense technology, which controls gene expression through
hybridization of complementary nucleic acid sequences, i.e.
antisense DNA or RNA, to the control, 5' or regulatory regions of
the gene encoding PSA variant product. For example, the 5' coding
portion of the nucleic acid sequence sequence which codes for the
product of the present invention is used-to design an antisense
oligonucleotide of from about 10 to 40 base pairs in length.
Oligonucleotides derived from the transcription start site, e.g.
between positions -10 and +10 from the start site, are preferred.
An antisense DNA oligonucleotide is designed to be complementary to
a region of the nucleic acid sequence involved in transcription
(Lee et al, Nucl. Acids, Res., 6:3073, (1979); Cooney et al.,
Science 241:456, (1988); and Dervan et al., Science 251:1360, is
(1991)), thereby preventing transcription and the production of the
PSA variant products. An antisense RNA oligonucleotide hybridizes
to the mRNA in vivo and blocks translation of the mRNA molecule
into the PSA variant products (Okano J. Neurochem. 56:560, (1991)).
The antisense constructs can be delivered to cells by procedures
known in the art such that the antisense RNA or DNA may be
expressed in vivo. The antisense may be antisense mRNA or DNA
sequence capable of coding such antisense mRNA. The antisense mRNA
or the DNA coding thereof can be complementary to the full sequence
of nucleic acid sequences coding to the PSA variant protein or to a
fragment of such a sequence which is sufficient to inhibit
production of a protein product.
[0155] Turning now to the PSA variant aspect, expression of PSA
variant product may be increased by providing coding sequences for
coding for said product under the control of suitable control
elements ending its expression in the desired host.
[0156] The nucleic acid sequences of the invention may be employed
in combination with a suitable pharmaceutical carrier. Such
compositions comprise a therapeutically effective amount of the
compound, and a pharmaceutically acceptable carrier or excipient.
Such a carrier includes but is not limited to saline, buffered
saline, dextrose, water, glycerol, ethanol, and combinations
thereof. The formulation should suit the mode of
administration.
[0157] The polypeptides, and activator and deactivator compounds
(see below), which are polypeptides, may also be employed in
accordance with the present invention by expression of such
polypeptides in vivo, which is often referred to as "gene therapy."
Cells from a patient may be engineered with a nucleic acid sequence
(DNA or RNA) encoding a polypeptide ex vivo, with the engineered
cells then being provided to a patient to be treated with the
polypeptide. Such methods are well-known in the art. For example,
cells may be engineered by procedures known in the art by use of a
retroviral particle containing RNA encoding a polypeptide of the
present invention.
[0158] Similarly, cells may be engineered in vivo for expression of
a polypeptide in vivo by procedures known in the art. As known in
the art, a producer cell for producing a retroviral particle
containing RNA encoding the polypeptide of the present invention
may be administered to a patient for engineering cells in vivo and
expression of the polypeptide in vivo. These and other methods for
administering a product of the present invention by such method
should be apparent to those skilled in the art from the teachings
of the present invention. For example, the expression vehicle for
engineering cells may be other than a retrovirus, for example, an
adenovirus which -may be used to engineer cells in vivo after
combination with a suitable delivery vehicle.
[0159] Retroviruses from which the retroviral plasmid vectors
mentioned above may be derived include, but are not limited to,
Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses
such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis
virus, gibbon ape leukemia virus, human immunodeficiency virus,
adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor
virus.
[0160] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, psi-2, psi-AM, PA12, T19-14X,
VT-19-17-H2, psi-CRE, psi-CRIP, GP+E-86, GP+envAm12, and DAN cell
lines as described in Miller (Human Gene Therapy, Vol. 1. pg. 5-14,
(1990)). The vector may transduce the packaging cells through any
means known in the art. Such means include, but are not limited to,
electroporation, the use of liposomes, and CaPO.sub.4
precipitation. In one alternative, the retroviral plasmid vector
may be encapsulated into a liposome, or coupled to a lipid, and
then administered to a host.
[0161] The producer cell line generates infectious retroviral
vector particles which include the nucleic acid sequence(s)
encoding the polypeptides. Such retroviral vector particles then
may be employed, to transduce eukaryotic cells, either in vitro or
in vivo. The transduced eukaryotic cells will express the nucleic
acid sequence(s) encoding the polypeptide. Eukaryotic cells which
may be transduced include, but are not limited to, embryonic stem
cells, embryonic carcinoma cells, as well as hematopoietic stem
cells, hepatocytes, fibroblasts, myoblasts, keratinocytes,
endothelial cells, and bronchial epithelial cells.
[0162] The genes introduced into cells may be placed under the
control of inducible promoters, such as the radiation-inducible
Egr-1 promoter, (Maceri, H. J., et al., Cancer Res., 56(19):4311
(1996)), to stimulate PSA variant production or antisense
inhibition in response to radiation, eg., radiation therapy for
treating tumors.
[0163] F. Northern Blot Analysis
[0164] RNA samples were obtained from spleen, thymus, prostate,
testis, ovary, small intestine, colon and leukocytes
electrophoresed through a 1.5% agarose gel containing formaldehyde
and transfered onto nylon (Hybond-N, Amersham) paper (Thomas,
1980). Prehybridization was for 2 hours in a buffer containing 10%
Dextrane Sulfate, 1M NaCl and 1% SDS, at 65?C. Hybridization was in
the same buffer with 5X106 cpm of the appropriate probe at
65.degree. C. for 18 hours. After one wash in 2XSSC, 0.1% SDS for
15 minutes at 65.degree. C. and several washes in 0.2XSSC, 0.1% SDS
at 65.degree. C. the filter was exposed to an X-ray film.
Phosphorimager analysis was performed as well. The results are
shown in FIG. 7. As can be seen with the PSA probe (left), a single
band was detected, while with the probe of the invention (termed
"PSAL") (derived from SEQ ID 2, in the common region with SEQ IDs
3,4, and 5) several bands were detected in prostate tissue, which
indicates the presence of different PSA splice variants.
Example II
PSA Variant Product
[0165] The substantially purified PSA variant product of the
invention has been defined above as the product coded from the
nucleic acid sequence of the invention. Preferably the amino acid
sequence is an amino acid sequence having at least 90% identity to
the sequence identified as any one of SEQ ID NO:7 to SEQ. ID NO.
12. The protein may be in mature and/or modified form, also as
defined above. Also contemplated are protein fragments having at
least 10 contiguous amino acid residues, preferably at least 10-20
residues, derived from the PSA variant protein.
[0166] The sequence variations are preferably those that are
considered conserved substitutions, as defined above. Thus, for
example, a protein with a sequence having at least 80% sequence
identity with the protein identified as SEQ ID NO:7 to SEQ ID NO:
12; preferably by utilizing conserved substitutions as defined
above. In a more specific embodiment, the protein has or contains
the sequence identified SEQ ID NO:7 to SEQ. ID NO: 12. The PSA
variant product may be (i) one in which one or more of the amino
acid residues in a sequence listed above are substituted with a
conserved or non-conserved amino acid residue (preferably a
conserved amino acid residue), or (ii) one in which one or more of
the amino acid residues includes a substituent group, or (iii) one
in which the PSA variant product is fused with another compound,
such as a compound to increase the half-life of the protein (for
example, polyethylene glycol (PEG)), or a moiety which serves as
targeting means to direct the protein to its target tissue or
target cell population (such as an antibody), or (iv) one in which
additional amino acids are fused to the PSA variant product. Such
fragments, variant and derivatives are deemed to be within the
scope of those skilled in the art from the teachings herein.
[0167] A. Preparation of PSA Variant Product
[0168] Recombinant methods for producing and isolating the PSA
variant product, and fragments of the protein are described
above.
[0169] In addition to recombinant production, fragments and
portions of PSA variant product may be produced by direct peptide
synthesis using solid-phase techniques (cf. Stewart et al., (1969)
Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco;
Merrifield J., J. Am. Chem. Soc., 85:2149-2154, (1963)). In vitro
peptide synthesis may be performed using manual techniques or by
automation. Automated synthesis may be achieved, for example, using
Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster
City, Calif.) in accordance with the instructions provided by the
manufacturer. Fragments of PSA variant product may be chemically
synthesized separately and combined using chemical methods to
produce the full length molecule.
[0170] B. Western Blot Analysis
[0171] Western Blot analysis was performed according to procedures
well known in the art, that are described in the Maniatis
Laboratory Manual. Post processing was performed using PIERCE
SuperSignal staining kit.
[0172] CG-47, CG-30, CG-23, and CG-35 are all hyperplastic prostate
tissue sample. CG-37 is a normal prostate sample. The results
indicate that the original PSA is expressed in hyperplastic, but
not normal, prostate tissue (right) while the PSA variant (denoted
PSALM) is weakly present hyperplastic prostate and highly expressed
in normal tissue. Neither molecule is present in detectable levels
in the spleen control.
[0173] C. Therapeutic Uses and Compositions Utilizing the PSA
Variant Product
[0174] The PSA variant product of the invention is generally useful
in treating diseases and disorders which are characterized by a
lower than normal level of PSA variant expression, and or diseases
which can be cured or ameliorated by raising the level of the PSA
variant product, even if the level is normal.
[0175] PSA variant products or fragments may be administered by any
of a number of routes and methods designed to provide a consistent
and predictable concentration of compound at the target organ or
tissue. The product-containing compositions may be administered
alone or in combination with other agents, such as stabilizing
compounds, and/or in combination with other pharmaceutical agents
such as drugs or hormones.
[0176] PSA variant product-containing compositions may be
administered by a number of routes including, but not limited to
oral, intravenous, intramuscular, transdermal, subcutaneous,
topical, sublingual, or rectal means as well as by nasal
application. PSA variant product-containing compositions may also
be administered via liposomes. Such administration routes and
appropriate formulations are generally known to those of skill in
the art.
[0177] The product can be given via intravenous or intraperitoneal
injection. Similarly, the product may be injected to other
localized regions of the body. The product may also be administered
via nasal insufflation. Enteral administration is also possible.
For such administration, the product should be formulated into an
appropriate capsule or elixir for oral administration, or into a
suppository for rectal administration.
[0178] The foregoing exemplary administration modes will likely
require that the product be formulated into an appropriate carrier,
including ointments, gels, suppositories. Appropriate formulations
are well known to persons skilled in the art.
[0179] Dosage of the product will vary, depending upon the potency
and therapeutic index of the particular polypeptide selected.
[0180] A therapeutic composition for use in the treatment method
can include the product in a sterile injectable solution, the
polypeptide in an oral delivery vehicle, the product in an aerosol
suitable for nasal administration, or the product in a nebulized
form, all prepared according to well known methods. Such
compositions comprise a therapeutically effective amount of the
compound, and a pharmaceutically acceptable carrier or excipient.
Such a carrier includes but is not limited to saline, buffered
saline, dextrose, water, glycerol, ethanol, and combinations
thereof.
Example III
Screening Methods for Activators and Deactivators
[0181] The present invention also includes an assay for identifying
molecules, such as synthetic drugs, antibodies, peptides, or other
molecules, which have a modulating effect on the activity of the
PSA variant product, e.g. activators or deactivators of the PSA
variant product of the present invention. Such an assay comprises
the steps of providing an PSA variant product encoded by the
nucleic acid sequences of the present invention, contacting the PSA
variant protein with one or more candidate molecules to determine
the candidate molecules modulating effect on the activity of the
PSA variant product, and selecting from the molecules a candidate's
molecule capable of modulating PSA variant product physiological
activity.
[0182] PSA variant product, its catalytic or immunogenic fragments
or oligopeptides thereof, can be used for screening therapeutic
compounds in any of a variety of drug screening techniques. The
fragment employed in such a test may be free in solution, affixed
to a solid support, borne on a cell membrane or located
intracellularly. The formation of binding complexes, between PSA
variant product and the agent being tested, may be measured.
Alternatively, the activator or deactivator may work by serving as
agonist or antagonist, respectively, of the PSA variant receptor
and their effect may be determined in connection with the
receptor.
[0183] Another technique for drug screening which may be used
provides for high throughput screening of compounds having suitable
binding affinity to the PSA variant product is described in detail
by Geysen in PCT Application WO 84/03564, published on Sep. 13,
1984. In summary, large numbers of different small peptide test
compounds are synthesized on a solid substrate, such as plastic
pins or some other surface. The peptide test compounds are reacted
with the full PSA variant product or with fragments of PSA variant
product and washed. Bound PSA variant product is then detected by
methods well known in the art. Substantially purified PSA variant
product can also be coated directly onto plates for use in the
aforementioned drug screening techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and
immobilize it on a solid support.
[0184] Antibodies to the PSA variant product, as described in
Example IV below, may also be used in screening assays according to
methods well known in the art. For example, a "sandwich" assay may
be performed, in which an anti-PSA variant antibody is affixed to a
solid surface such as a microtiter plate and PSA variant product is
added. Such an assay can be used to capture compounds which bind to
the PSA variant product. Alternatively, such an assay may be used
to measure the ability of compounds to influence with the binding
of PSA variant product to the PSA variant receptor, and then select
those compounds which effect the binding.
EXAMPLE IV
Anti-PSA Variant Antibodies
[0185] A. Synthesis
[0186] In still another aspect of the invention, the purified PSA
variant product is used to produce anti-PSA variant antibodies
which have diagnostic and therapeutic uses related to the activity,
distribution, and expression of the PSA variant product, in
particular diagnostic application in identification of prostate
cancer, (distinguishing between malignant and benign states) and as
targeting means for delivery of cytotoxic compounds to tumor
cells.
[0187] Antibodies to PSA variant product may be generated by
methods well known in the art. Such antibodies may include, but are
not limited to, polyclonal, monoclonal, chimeric, humanized, single
chain, Fab fragments and fragments produced by an Fab expression
library. Antibodies, i.e., those which inhibit dimer formation, are
especially preferred for therapeutic use.
[0188] PSA variant product for antibody induction does not require
biological activity; however, the protein fragment or oligopeptide
must be antigenic. Peptides used to induce specific antibodies may
have an amino acid sequence consisting of at least five amino
acids, preferably at least 10 amino acids of the sequences
specified in any of the 7 to 12 SEQ ID NO. Preferably they should
mimic a portion of the amino acid sequence of the natural protein
and may contain the entire amino acid sequence of a small,
naturally occurring molecule. The antibodies may also distinguish
antibodies, i.e. antibodies which bind to an amino acid sequence
present in the PSA variant and not in the original PSA sequence.
For the production of said distinguishing antibodies
"distinguishing amino acid sequences" may be used for example
having the sequence CQAELSPPTQHPSPDREL.
[0189] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening recombinant
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in Orlandi et al. (Proc. Natl. Acad. Sci.
86:3833-3837, 1989)), and Winter G and Milstein C., (Nature
349:293-299, (1991)).
[0190] Antibody fragments which contain specific binding sites for
PSA variant protein may also be generated. For example, such
fragments include, but are not limited to, the F(ab')2 fragments
which can be produced by pepsin digestion of the antibody molecule
and the Fab fragments which can be generated by reducing the
disulfide bridges of the F(ab')2 fragments. Alternatively, Fab
expression libraries may be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity (Huse W. D. et al., Science 256:1275-1281, (1989)).
Production of Antibodies
[0191] Human and mouse cDNA fragments were subcloned into the
pET-28(a-c) vectors (Novagen,USA). DNA was prepared from positive
clones and introduced into the E.coli strain DE3 according to the
manufacturer's recommendations. After induction, extracts were
electrophoresed through a 10% SDS-PAGE. Extracts were prepared from
clones that expressed the expected size protein and loaded on a
nickel-agarose column. The His containing proteins were isolated
from the column according to the manufacturer's recommendations and
used in injections. Polyclonal antibodies against human PSAL
peptide were prepared by immunizing rabbits with 3-4 injections of
0.5 mg of the purified protein at 1-2 week intervals. Animals were
bled 10 days after the final booster. Serum was separated from the
blood and stored at -80C. The peptide defined above as
"distinguishing amino acid sequence" was used for immunization.
[0192] B. Diagnostic Applications of Antibodies
[0193] A variety of protocols for competitive binding or
immunoradiometric assays using either polyclonal or monoclonal
antibodies with established specificities are well known in the
art. Such immunoassays typically involve the formation of complexes
between PSA variant product and its specific antibody and the
measurement of complex formation. A two-site, monoclonal-based
immunoassay utilizing monoclonal antibodies reactive to two
non-interfering epitopes on a specific PSA variant product is
preferred, but a competitive binding assay may also be employed.
These assays are described in Maddox D. E., et al., (J. Exp. Med.
158:1211, (1983)).
[0194] Antibodies which specifically bind PSA variant product are
useful for the diagnosis of conditions or diseases characterized by
expression of PSA variant protein, in particular prostate cancer.
Alternatively, such antibodies may be used in assays to monitor
patients being treated with PSA variant product, its activators, or
its deactivators. Diagnostic assays for PSA variant protein include
methods utilizing the antibody and a label to detect PSA variant
product in human body fluids or extracts of cells or tissues. The
products and antibodies of the present invention may be used with
or without modification. Frequently, the proteins and antibodies
will be labeled by joining them, either covalently or
noncovalently, with a reporter molecule. A wide variety of reporter
molecules are known in the art.
[0195] A variety of protocols for measuring PSA variant product,
using either polyclonal or monoclonal antibodies specific for the
respective protein are known in the art. Examples include
enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),
and fluorescent activated cell sorting (FACS). As noted above, a
two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two non-interfering epitopes on PSA variant
product is preferred, but a competitive binding assay may be
employed. These assays are described, among other places, in
Maddox, et al. (supra). Such protocols provide a basis for
diagnosing altered or abnormal levels of PSA variant product
expression. Normal or standard values for PSA variant product
expression are established by combining body fluids or cell
extracts taken from normal subjects, preferably human, with
antibody to PSA variant product under conditions suitable for
complex formation which are well known in the art. The amount of
standard complex formation may be quantified by various methods,
preferably by photometric methods. Then, standard values obtained
from normal samples may be compared with values obtained from
samples from subjects potentially affected by disease. Deviation
between standard and subject values establishes the presence of
disease state.
[0196] The antibody assays are useful to determine the level of PSA
variant present in a body fluid sample, or in a particular tissue,
e.g., biopsied tumor tissue, for example from the prostate gland,
as an indication of whether PSA variant is being overexpressed or
underexpressed in the tissue, or as an indication of how PSA
variant levels are responding to drug treatment.
[0197] C. Immunohistochemical Staining
[0198] Human prostate micron sections were prepared using a R. Gung
microtome and fixed on slides pretreated with 2% Tespa (Sigma,
USA). Deparaffinization was performed for 30 minutes at 80.degree.
C. Hydration was executed by immersing the slides twice in xylene
(5 minutes each), twicein 100% ethanol (5 minutes each), twice in
95% ethanol (5 minutes each),once in 70% ethanol (5 minutes), and
once in PBS pH7.4 (10 minutes). After incubation in 50 ?l/slide of
1.5 mg/ml hyaluronidase in PBS pH6.5 for 1 hour at 37.degree. C.
the slides were washed in PBS pH 7.4 (10 minutes). 50 .mu.l/slide
of 0.3% H.sub.2O.sub.2 in PBS pH 7.4 were added for 10 minutes
after which they were washed in PBS pH 7.4 (10 minutes).
[0199] Blocking was performed by addition of 50 .mu.l/slide of
normal goat serum containing 20% trasylol at 37.degree. C. for 10
minutes. Rabbit anti-testilin antibodies were diluted 1:50in 10%
blocking solution (normal goat serum containing 20% trasylol(Bayer,
Germany) and interacted with the slides for 18 hours at 4.degree.
C. Then the slides were washed 3 times in PBS pH 7.4 and immersed
for 10 minutes in PBS pH 7.4. Horse raddish peroxidase (HPR)
conjugated goatanti rabbit antibodies (Sigma,USA) in PBS pH7.4
containing 20% trasylol(Bayer, Germany) diluted 1:40, were added
for 30 minutes at room temperature, in the dark, following by wash
in PBS pH7.4 for 10 minutes. For HRP reaction 0.4 mg/ml of the
substrate (3'3' diaminbenzoidin) was added in the dark for 10
minutes. Following 3 washes in PBS pH 7.4 and immersion for 10
minutes in PBS pH 7.4, staining of the slides was performed with 1%
methylene blue in PBS pH 7.4 for 5 min. Following two washes in
water, dehydration was carried out by immersing the slides 3 times
in 70% ethanol, 3 times in 95% ethanol, 6 times in 100% ethanol and
6 times in xylene. Mounting was performed with Mercoglass
(Merk,USA). The results are shown in FIG. 9. The result indicate a
high presence level of the PSA variant protein in the lumen-lining
of prostate epithelial cells (color gold, left picture, vs. the
pre-immune serum control on the right picture, where no gold color
is detectable.)
[0200] D. Therapeutic Uses of Antibodies
[0201] In addition to their diagnostic use the antibodies may have
a therapeutical utility in blocking or decreasing the activity of
the PSA variant product in pathological conditions where its
activity or concentration are too high, for example in prostate
cancer. In addition, the antibodies may be conjugated to cytotoxic
compounds and thus may serve as means for targeting the cytotoxic
moiety only to cancer cells which express membrane-bond PSA variant
product.
[0202] The antibody employed is preferably a humanized monoclonal
antibody, or a human Mab produced by known globulin-gene library
methods. The antibody is administered typically as a sterile
solution by IV injection, although other parenteral routes may be
suitable. Typically, the antibody is administered in an amount
between about 1-15 mg/kg body weight of the subject. Treatment is
continued, e.g., with dosing every 1-7 days, until a therapeutic
improvement is seen.
[0203] Although the invention has been described with reference to
specific methods and embodiments, it is appreciated that various
modifications and changes may be made without departing from the
invention.
EXAMPLE V
Immuno-Histochemistry
[0204] Immunohistochemical staining was performed using Histostain
sp kit (Zymed Laboratories INC.).
[0205] Human prostate micron sections were prepared using a R. Gung
microtome and fixed on superfrost slides with 2% Tespa.
Deparaffinization was performed for 10 mins. at xylen. Hydration
three times 100% ethanol and once 95% ethanol. The slides were
washed in Ddw, following incubation with 3% H.sub.2O.sub.2 for 5
mins. After incubation the slides were washed twice in ddw, and
twice in 0.05M Tris Hel Ph 7.6 (optimax wash buffer, BioGenex).
[0206] Blocking was performed with serum blocking solution (ready
to use, reagent A, Zymed) 100 ul each slide incubate 10 mins.
Primary antibody was diluted 1:50 in antibody diluent reagent
solution (Zymed), and incubated in moist chamber with the slides
for 1 hour.
[0207] Following washing (three times in optimax buffer) the slides
were incubated with 100 .mu.l biotinylated second antibody ready to
use (reagent B Zymed), for 10 mins, then washed three more times in
optimax buffer. The slides were incubated with 100 .mu.l enzyme
conjugate HRP-streptavidin ready to use (reagent C, Zymed) for 10
mins, and washed twice in optimax buffer. Then 100 .mu.l substrate
(liquid DAB substrate, Zymed) were added for 3 mins.
[0208] Following the incubation with the substrate, the slides were
washed twice in ddw and stained with Hematoxylen solution (Zymed)
for 2 mins. Then the slides were washed in tap water for I hour.
The dehydration was carried out by immersing the slides 2 times in
95% ethanol, 3 times in 100% ethanol, 3 times in Zylen. Mounting
was performed with mounting solution (Zymed).
[0209] The results are shown in FIG. 12. The results indicate a
high presence level of the protein (derived by alternative splicing
from the KLK-2 gene) in the lumen-lining of prostate epithelial
cells (gold color, left picture, vs. the pre-immune serum control
on the right picture, where no gold color is detectable).
Sequence CWU 1
1
16 1 4661 DNA Homo sapiens 1 attttgcatg ccaccttaat cttttttttt
ttttttttaa atcgaggttt cagtctcatt 60 ctatttccca ggctggagtt
caatagcgtg atcacagctc actgtagcct tgaactcctg 120 gccttaagag
attctcctgc ttcggtctcc caatagctaa gactacagta gtccaccacc 180
atatccagat aatttttaaa ttttttgggg ggccgggcac agtggctcac gcctgtaatc
240 ccaacaccat gggaggctga gatgggtgga tcacgaggtc aggagtttga
gaccagcctg 300 accaacatgg tgaaactctg tctctactaa aaaaaaaaaa
aatagaaaaa ttagccgggc 360 gtggtggcac acggcacctg taatcccagc
tactgaggag gctgaggcag gagaatcact 420 tgaacccaga aggcagaggt
tgcaatgagc cgagattgcg ccactgcact ccagcctggg 480 tgacagagtg
agactctgtc tcaaaaaaaa aaaatttttt tttttttttt gtagagatgg 540
atcttgcttt gtttctctgg ttggccttga actcctggct tcaagtgatc ctcctacctt
600 ggcctcggaa agtgttggga ttacaggcgt gagccaccat gactgacctg
tcgttaatct 660 tgaggtacat aaacctggct cctaaaggct aaaggctaaa
tatttgttgg agaaggggca 720 ttggattttg catgaggatg attctgacct
gggagggcag gtcagcaggc atctctgttg 780 cacagataga gtgtacaggt
ctggagaaca aggagtgggg ggttattgga attccacatt 840 gtttgctgca
cgttggattt tgaaatgcta gggaactttg ggagactcat atttctgggc 900
tagaggatct gtggaccaca agatcttttt atgatgacag tagcaatgta tctgtggagc
960 tggattctgg gttgggagtg caaggaaaag aatgtactaa atgccaagac
atctatttca 1020 ggagcatgag gaataaaagt tctagtttct ggtctcagag
tggtgcaggg atcagggagt 1080 ctcacaatct cctgagtgct ggtgtcttag
ggcacactgg gtcttggagt gcaaaggatc 1140 taggcacgtg aggctttgta
tgaagaatcg gggatcgtac ccaccccctg tttctgtttc 1200 atcctgggcg
tgtctcctct gcctttgtcc cctagatgaa gtctccatga gctacagggc 1260
ctggtgcatc cagggtgatc tagtaattgc agaacagcaa gtactagctc tccctcccct
1320 tccacagctc tgggtgtggg agggggttgt ccagcctcca gcagcatggg
gagggccttg 1380 gtcagcctct gggtgccagc agggcagggg cggagtcctg
gggaatgaag gttttatagg 1440 gctcctgggg gaggctcccc agccccaagc
ttaccacctg cacccggaga gctgtgtcac 1500 catgtgggtc ccggttgtct
tcctcaccct gtccgtgacg tggattggtg agaggggcca 1560 tggttggggg
gatgcaggag agggagccag ccctgactgt caagctgagg ctctttcccc 1620
cccaacccag caccccagcc cagacaggga gctgggctct tttctgtctc tcccagcccc
1680 actccaagcc cataccccca gcccctccat attgcaacag tcctcactcc
cacaccaggt 1740 ccccgctccc tcccacttac cccagaactt tctccccatt
gcccagccag ctccctgctc 1800 ccagctgctt tactaaaggg gaagttcctg
ggcatctccg tgtttctctt tgtggggctc 1860 aaaacctcca aggacctctc
tcaatgccat tggttccttg gaccgtatca ctggtccacc 1920 tcctgaggcc
ctcaatccta tcacagtcta ctgacttttc ccattcagct gtgagtgccc 1980
aaccctatcc cagagacctt gatgcttggc ctcccaatct tgccctagga tacccagatg
2040 ccaaccagac acctccttct tcctagccag gctatctggc ctgagacaac
aaatgggtcc 2100 ctcagtctgg caatgggact ctgagaactc ctcattccct
gactcttagc cccagactct 2160 tcattcagtg gcccacattt tccttaggaa
aaacatgagc atccccagcc acaactgcca 2220 gctctctgat tccccaaatc
tgcatccttt tcaaaaccta aaaacaaaaa gaaaaacaaa 2280 taaaacaaaa
ccaactcaga ccagaactgt tttctcaacc tgggacttcc taaactttcc 2340
aaaaccttcc tcttccagca actgaacctc gccataaggc acttatccct ggttcctagc
2400 accccttatc ccctcagaat ccacaacttg taccaagttt cccttctccc
agtccaagac 2460 cccaaatcac cacaaaggac ccaatcccca gactcaagat
atggtctggg cgctgtcttg 2520 tgtctcctac cctgatccct gggttcaact
ctgctcccag agcatgaagc ctctccacca 2580 gcaccagcca ccaacctgca
aacctaggga agattgacag aattcccagc ctttcccagc 2640 tccccctgcc
catgtcccag gactcccagc cttggttctc tgcccccgtg tcttttcaaa 2700
cccacatcct aaatccatct cctatccgag tcccccagtt cctcctgtca accctgattc
2760 ccctgatcta gcaccccctc tgcaggtgct gcacccctca tcctgtctcg
gattgtggga 2820 ggctgggagt gcgagaagca ttcccaaccc tggcaggtgc
ttgtggcctc tcgtggcagg 2880 gcagtctgcg gcggtgttct ggtgcacccc
cagtgggtcc tcacagctgc ccactgcatc 2940 aggaacaaaa gcgtgatctt
gctgggtcgg cacagcctgt ttcatcctga agacacaggc 3000 caggtatttc
aggtcagcca cagcttccca cacccgctct acgatatgag cctcctgaag 3060
aatcgattcc tcaggccagg tgatgactcc agccacgacc tcatgctgct ccgcctgtca
3120 gagcctgccg agctcacgga tgctgtgaag gtcatggacc tgcccaccca
ggagccagca 3180 ctggggacca cctgctacgc ctcaggctgg ggcagcattg
aaccagagga gttcttgacc 3240 ccaaagaaac ttcagtgtgt ggacctccat
gttatttcca atgacgtgtg tgcgcaagtt 3300 caccctcaga aggtgaccaa
gttcatgctg tgtgctggac gctggacagg gggcaaaagc 3360 acctgctcgg
gtgattctgg gggcccactt gtctgtaatg gtgtgcttca aggtatcacg 3420
tcatggggca gtgaaccatg tgccctgccc gaaaggcctt ccctgtacac caaggtggtg
3480 cattaccgga agtggatcaa ggacaccatc gtggccaacc cctgagcacc
cctatcaact 3540 ccctattgta gtaaacttgg aaccttggaa atgaccaggc
caagactcaa gcctccccag 3600 ttctactgac ctttgtcctt aggtgtgagg
tccagggttg ctaggaaaag aaatcagcag 3660 acacaggtgt agaccagagt
gtttcttaaa tggtgtaatt ttgtcctctc tgtgtcctgg 3720 ggaatactgg
ccatgcctgg agacatatca ctcaatttct ctgaggacac agataggatg 3780
gggtgtctgt gttatttgtg ggrtacagag atgaaagagg ggtgggwwcc acactgagag
3840 agtggagagt gacatgtgct ggacactgtc catgaagcac tgagcagaag
ctggaggcac 3900 aacgcaccag acactcacag caaggatgga gctgaaaaca
taacccactc tgtcctggag 3960 gcactgggaa gcctagagaa ggctgtgagc
caaggaggga gggtcttcct ttggcatggg 4020 atggggatga agtaaggaga
gggactggac cccctggaag ctgattcact atggggggag 4080 gtgtattgaa
gtcctccaga caaccctcag atttgatgat ttcctagtag aactcacaga 4140
aataaagagc tsttatacgt ggtttattct ggtttgttac attgacagga gacacactga
4200 aatcagcaaa ggaaacaggc atctaagtgg ggatgtgaag aaaacaggga
aaatctttca 4260 gttgttttct cccagtgggg tgttgtggac agcacttaaa
tcacacagaa gtgatgtgtg 4320 accttgtgta tgaagtattt ccaactaagg
aagctcacct gagccttagt gtccagagtt 4380 cttattgggg gtctgtagga
taggcatggg gtactggaat agctgacctt aacttctcag 4440 acctgaggtt
cccaagagtt caagcagata cagcatggcc tagagcctca gatgtacaaa 4500
aacaggcatt catcatgaat cgcactgtta gcatgaatca tctggcacgg cccaaggccc
4560 caggtatacc aaggcacttg ggccgaatgt tccaagggat taaatgtcat
ctcccaggag 4620 ttattcaagg gtgagccctg tacttggaac gttcaggctt t 4661
2 4661 DNA Homo sapiens 2 attttgcatg ccaccttaat cttttttttt
ttttttttaa atcgaggttt cagtctcatt 60 ctatttccca ggctggagtt
caatagcgtg atcacagctc actgtagcct tgaactcctg 120 gccttaagag
attctcctgc ttcggtctcc caatagctaa gactacagta gtccaccacc 180
atatccagat aatttttaaa ttttttgggg ggccgggcac agtggctcac gcctgtaatc
240 ccaacaccat gggaggctga gatgggtgga tcacgaggtc aggagtttga
gaccagcctg 300 accaacatgg tgaaactctg tctctactaa aaaaaaaaaa
aatagaaaaa ttagccgggc 360 gtggtggcac acggcacctg taatcccagc
tactgaggag gctgaggcag gagaatcact 420 tgaacccaga aggcagaggt
tgcaatgagc cgagattgcg ccactgcact ccagcctggg 480 tgacagagtg
agactctgtc tcaaaaaaaa aaaatttttt tttttttttt gtagagatgg 540
atcttgcttt gtttctctgg ttggccttga actcctggct tcaagtgatc ctcctacctt
600 ggcctcggaa agtgttggga ttacaggcgt gagccaccat gactgacctg
tcgttaatct 660 tgaggtacat aaacctggct cctaaaggct aaaggctaaa
tatttgttgg agaaggggca 720 ttggattttg catgaggatg attctgacct
gggagggcag gtcagcaggc atctctgttg 780 cacagataga gtgtacaggt
ctggagaaca aggagtgggg ggttattgga attccacatt 840 gtttgctgca
cgttggattt tgaaatgcta gggaactttg ggagactcat atttctgggc 900
tagaggatct gtggaccaca agatcttttt atgatgacag tagcaatgta tctgtggagc
960 tggattctgg gttgggagtg caaggaaaag aatgtactaa atgccaagac
atctatttca 1020 ggagcatgag gaataaaagt tctagtttct ggtctcagag
tggtgcaggg atcagggagt 1080 ctcacaatct cctgagtgct ggtgtcttag
ggcacactgg gtcttggagt gcaaaggatc 1140 taggcacgtg aggctttgta
tgaagaatcg gggatcgtac ccaccccctg tttctgtttc 1200 atcctgggcg
tgtctcctct gcctttgtcc cctagatgaa gtctccatga gctacagggc 1260
ctggtgcatc cagggtgatc tagtaattgc agaacagcaa gtactagctc tccctcccct
1320 tccacagctc tgggtgtggg agggggttgt ccagcctcca gcagcatggg
gagggccttg 1380 gtcagcctct gggtgccagc agggcagggg cggagtcctg
gggaatgaag gttttatagg 1440 gctcctgggg gaggctcccc agccccaagc
ttaccacctg cacccggaga gctgtgtcac 1500 catgtgggtc ccggttgtct
tcctcaccct gtccgtgacg tggattggtg agaggggcca 1560 tggttggggg
gatgcaggag agggagccag ccctgactgt caagctgagg ctctttcccc 1620
cccaacccag caccccagcc cagacaggga gctgggctct tttctgtctc tcccagcccc
1680 actccaagcc cataccccca gcccctccat attgcaacag tcctcactcc
cacaccaggt 1740 ccccgctccc tcccacttac cccagaactt tctccccatt
gcccagccag ctccctgctc 1800 ccagctgctt tactaaaggg gaagttcctg
ggcatctccg tgtttctctt tgtggggctc 1860 aaaacctcca aggacctctc
tcaatgccat tggttccttg gaccgtatca ctggtccacc 1920 tcctgaggcc
ctcaatccta tcacagtcta ctgacttttc ccattcagct gtgagtgccc 1980
aaccctatcc cagagacctt gatgcttggc ctcccaatct tgccctagga tacccagatg
2040 ccaaccagac acctccttct tcctagccag gctatctggc ctgagacaac
aaatgggtcc 2100 ctcagtctgg caatgggact ctgagaactc ctcattccct
gactcttagc cccagactct 2160 tcattcagtg gcccacattt tccttaggaa
aaacatgagc atccccagcc acaactgcca 2220 gctctctgat tccccaaatc
tgcatccttt tcaaaaccta aaaacaaaaa gaaaaacaaa 2280 taaaacaaaa
ccaactcaga ccagaactgt tttctcaacc tgggacttcc taaactttcc 2340
aaaaccttcc tcttccagca actgaacctc gccataaggc acttatccct ggttcctagc
2400 accccttatc ccctcagaat ccacaacttg taccaagttt cccttctccc
agtccaagac 2460 cccaaatcac cacaaaggac ccaatcccca gactcaagat
atggtctggg cgctgtcttg 2520 tgtctcctac cctgatccct gggttcaact
ctgctcccag agcatgaagc ctctccacca 2580 gcaccagcca ccaacctgca
aacctaggga agattgacag aattcccagc ctttcccagc 2640 tccccctgcc
catgtcccag gactcccagc cttggttctc tgcccccgtg tcttttcaaa 2700
cccacatcct aaatccatct cctatccgag tcccccagtt cctcctgtca accctgattc
2760 ccctgatcta gcaccccctc tgcaggtgct gcacccctca tcctgtctcg
gattgtggga 2820 ggctgggagt gcgagaagca ttcccaaccc tggcaggtgc
ttgtggcctc tcgtggcagg 2880 gcagtctgcg gcggtgttct ggtgcacccc
cagtgggtcc tcacagctgc ccactgcatc 2940 aggaacaaaa gcgtgatctt
gctgggtcgg cacagcctgt ttcatcctga agacacaggc 3000 caggtatttc
aggtcagcca cagcttccca cacccgctct acgatatgag cctcctgaag 3060
aatcgattcc tcaggccagg tgatgactcc agccacgacc tcatgctgct ccgcctgtca
3120 gagcctgccg agctcacgga tgctgtgaag gtcatggacc tgcccaccca
ggagccagca 3180 ctggggacca cctgctacgc ctcaggctgg ggcagcattg
aaccagagga gttcttgacc 3240 ccaaagaaac ttcagtgtgt ggacctccat
gttatttcca atgacgtgtg tgcgcaagtt 3300 caccctcaga aggtgaccaa
gttcatgctg tgtgctggac gctggacagg gggcaaaagc 3360 acctgctcgg
gtgattctgg gggcccactt gtctgtaatg gtgtgcttca aggtatcacg 3420
tcatggggca gtgaaccatg tgccctgccc gaaaggcctt ccctgtacac caaggtggtg
3480 cattaccgga agtggatcaa ggacaccatc gtggccaacc cctgagcacc
cctatcaact 3540 ccctattgta gtaaacttgg aaccttggaa atgaccaggc
caagactcaa gcctccccag 3600 ttctactgac ctttgtcctt aggtgtgagg
tccagggttg ctaggaaaag aaatcagcag 3660 acacaggtgt agaccagagt
gtttcttaaa tggtgtaatt ttgtcctctc tgtgtcctgg 3720 ggaatactgg
ccatgcctgg agacatatca ctcaatttct ctgaggacac agataggatg 3780
gggtgtctgt gttatttgtg ggrtacagag atgaaagagg ggtgggwwcc acactgagag
3840 agtggagagt gacatgtgct ggacactgtc catgaagcac tgagcagaag
ctggaggcac 3900 aacgcaccag acactcacag caaggatgga gctgaaaaca
taacccactc tgtcctggag 3960 gcactgggaa gcctagagaa ggctgtgagc
caaggaggga gggtcttcct ttggcatggg 4020 atggggatga agtaaggaga
gggactggac cccctggaag ctgattcact atggggggag 4080 gtgtattgaa
gtcctccaga caaccctcag atttgatgat ttcctagtag aactcacaga 4140
aataaagagc tsttatacgt ggtttattct ggtttgttac attgacagga gacacactga
4200 aatcagcaaa ggaaacaggc atctaagtgg ggatgtgaag aaaacaggga
aaatctttca 4260 gttgttttct cccagtgggg tgttgtggac agcacttaaa
tcacacagaa gtgatgtgtg 4320 accttgtgta tgaagtattt ccaactaagg
aagctcacct gagccttagt gtccagagtt 4380 cttattgggg gtctgtagga
taggcatggg gtactggaat agctgacctt aacttctcag 4440 acctgaggtt
cccaagagtt caagcagata cagcatggcc tagagcctca gatgtacaaa 4500
aacaggcatt catcatgaat cgcactgtta gcatgaatca tctggcacgg cccaaggccc
4560 caggtatacc aaggcacttg ggccgaatgt tccaagggat taaatgtcat
ctcccaggag 4620 ttattcaagg gtgagccctg tacttggaac gttcaggctt t 4661
3 3846 DNA Homo sapiens 3 attttgcatg ccaccttaat cttttttttt
ttttttttaa atcgaggttt cagtctcatt 60 ctatttccca ggctggagtt
caatagcgtg atcacagctc actgtagcct tgaactcctg 120 gccttaagag
attctcctgc ttcggtctcc caatagctaa gactacagta gtccaccacc 180
atatccagat aatttttaaa ttttttgggg ggccgggcac agtggctcac gcctgtaatc
240 ccaacaccat gggaggctga gatgggtgga tcacgaggtc aggagtttga
gaccagcctg 300 accaacatgg tgaaactctg tctctactaa aaaaaaaaaa
aatagaaaaa ttagccgggc 360 gtggtggcac acggcacctg taatcccagc
tactgaggag gctgaggcag gagaatcact 420 tgaacccaga aggcagaggt
tgcaatgagc cgagattgcg ccactgcact ccagcctggg 480 tgacagagtg
agactctgtc tcaaaaaaaa aaaatttttt tttttttttt gtagagatgg 540
atcttgcttt gtttctctgg ttggccttga actcctggct tcaagtgatc ctcctacctt
600 ggcctcggaa agtgttggga ttacaggcgt gagccaccat gactgacctg
tcgttaatct 660 tgaggtacat aaacctggct cctaaaggct aaaggctaaa
tatttgttgg agaaggggca 720 ttggattttg catgaggatg attctgacct
gggagggcag gtcagcaggc atctctgttg 780 cacagataga gtgtacaggt
ctggagaaca aggagtgggg ggttattgga attccacatt 840 gtttgctgca
cgttggattt tgaaatgcta gggaactttg ggagactcat atttctgggc 900
tagaggatct gtggaccaca agatcttttt atgatgacag tagcaatgta tctgtggagc
960 tggattctgg gttgggagtg caaggaaaag aatgtactaa atgccaagac
atctatttca 1020 ggagcatgag gaataaaagt tctagtttct ggtctcagag
tggtgcaggg atcagggagt 1080 ctcacaatct cctgagtgct ggtgtcttag
ggcacactgg gtcttggagt gcaaaggatc 1140 taggcacgtg aggctttgta
tgaagaatcg gggatcgtac ccaccccctg tttctgtttc 1200 atcctgggcg
tgtctcctct gcctttgtcc cctagatgaa gtctccatga gctacagggc 1260
ctggtgcatc cagggtgatc tagtaattgc agaacagcaa gtactagctc tccctcccct
1320 tccacagctc tgggtgtggg agggggttgt ccagcctcca gcagcatggg
gagggccttg 1380 gtcagcctct gggtgccagc agggcagggg cggagtcctg
gggaatgaag gttttatagg 1440 gctcctgggg gaggctcccc agccccaagc
ttaccacctg cacccggaga gctgtgtcac 1500 catgtgggtc ccggttgtct
tcctcaccct gtccgtgacg tggattggtg agaggggcca 1560 tggttggggg
gatgcaggag agggagccag ccctgactgt caagctgagg ctctttcccc 1620
cccaacccag caccccagcc cagacaggga gctgggctct tttctgtctc tcccagcccc
1680 actccaagcc cataccccca gcccctccat attgcaacag tcctcactcc
cacaccaggt 1740 ccccgctccc tcccacttac cccagaactt tctccccatt
gcccagccag ctccctgctc 1800 ccagctgctt tactaaaggg gaagttcctg
ggcatctccg tgtttctctt tgtggggctc 1860 aaaacctcca aggacctctc
tcaatgccat tggttccttg gaccgtatca ctggtccacc 1920 tcctgaggcc
ctcaatccta tcacagtcta ctgacttttc ccattcagct gtgctgcacc 1980
cctcatcctg tctcggattg tgggaggctg ggagtgcgag aagcattccc aaccctggca
2040 ggtgcttgtg gcctctcgtg gcagggcagt ctgcggcggt gttctggtgc
acccccagtg 2100 ggtcctcaca gctgcccact gcatcaggaa caaaagcgtg
atcttgctgg gtcggcacag 2160 cctgtttcat cctgaagaca caggccaggt
atttcaggtc agccacagct tcccacaccc 2220 gctctacgat atgagcctcc
tgaagaatcg attcctcagg ccaggtgatg actccagcca 2280 cgacctcatg
ctgctccgcc tgtcagagcc tgccgagctc acggatgctg tgaaggtcat 2340
ggacctgccc acccaggagc cagcactggg gaccacctgc tacgcctcag gctggggcag
2400 cattgaacca gaggagttct tgaccccaaa gaaacttcag tgtgtggacc
tccatgttat 2460 ttccaatgac gtgtgtgcgc aagttcaccc tcagaaggtg
accaagttca tgctgtgtgc 2520 tggacgctgg acagggggca aaagcacctg
ctcgggtgat tctgggggcc cacttgtctg 2580 taatggtgtg cttcaaggta
tcacgtcatg gggcagtgaa ccatgtgccc tgcccgaaag 2640 gccttccctg
tacaccaagg tggtgcatta ccggaagtgg atcaaggaca ccatcgtggc 2700
caacccctga gcacccctat caactcccta ttgtagtaaa cttggaacct tggaaatgac
2760 caggccaaga ctcaagcctc cccagttcta ctgacctttg tccttaggtg
tgaggtccag 2820 ggttgctagg aaaagaaatc agcagacaca ggtgtagacc
agagtgtttc ttaaatggtg 2880 taattttgtc ctctctgtgt cctggggaat
actggccatg cctggagaca tatcactcaa 2940 tttctctgag gacacagata
ggatggggtg tctgtgttat ttgtgggrta cagagatgaa 3000 agaggggtgg
gwwccacact gagagagtgg agagtgacat gtgctggaca ctgtccatga 3060
agcactgagc agaagctgga ggcacaacgc accagacact cacagcaagg atggagctga
3120 aaacataacc cactctgtcc tggaggcact gggaagccta gagaaggctg
tgagccaagg 3180 agggagggtc ttcctttggc atgggatggg gatgaagtaa
ggagagggac tggaccccct 3240 ggaagctgat tcactatggg gggaggtgta
ttgaagtcct ccagacaacc ctcagatttg 3300 atgatttcct agtagaactc
acagaaataa agagctstta tacgtggttt attctggttt 3360 gttacattga
caggagacac actgaaatca gcaaaggaaa caggcatcta agtggggatg 3420
tgaagaaaac agggaaaatc tttcagttgt tttctcccag tggggtgttg tggacagcac
3480 ttaaatcaca cagaagtgat gtgtgacctt gtgtatgaag tatttccaac
taaggaagct 3540 cacctgagcc ttagtgtcca gagttcttat tgggggtctg
taggataggc atggggtact 3600 ggaatagctg accttaactt ctcagacctg
aggttcccaa gagttcaagc agatacagca 3660 tggcctagag cctcagatgt
acaaaaacag gcattcatca tgaatcgcac tgttagcatg 3720 aatcatctgg
cacggcccaa ggccccaggt ataccaaggc acttgggccg aatgttccaa 3780
gggattaaat gtcatctccc aggagttatt caagggtgag ccctgtactt ggaacgttca
3840 ggcttt 3846 4 1709 DNA Homo sapiens 4 attttgcatg ccaccttaat
cttttttttt ttttttttaa atcgaggttt cagtctcatt 60 ctatttccca
ggctggagtt caatagcgtg atcacagctc actgtagcct tgaactcctg 120
gccttaagag attctcctgc ttcggtctcc caatagctaa gactacagta gtccaccacc
180 atatccagat aatttttaaa ttttttgggg ggccgggcac agtggctcac
gcctgtaatc 240 ccaacaccat gggaggctga gatgggtgga tcacgaggtc
aggagtttga gaccagcctg 300 accaacatgg tgaaactctg tctctactaa
aaaaaaaaaa aatagaaaaa ttagccgggc 360 gtggtggcac acggcacctg
taatcccagc tactgaggag gctgaggcag gagaatcact 420 tgaacccaga
aggcagaggt tgcaatgagc cgagattgcg ccactgcact ccagcctggg 480
tgacagagtg agactctgtc tcaaaaaaaa aaaatttttt tttttttttt gtagagatgg
540 atcttgcttt gtttctctgg ttggccttga actcctggct tcaagtgatc
ctcctacctt 600 ggcctcggaa agtgttggga ttacaggcgt gagccaccat
gactgacctg tcgttaatct 660 tgaggtacat aaacctggct cctaaaggct
aaaggctaaa tatttgttgg agaaggggca 720 ttggattttg catgaggatg
attctgacct gggagggcag gtcagcaggc atctctgttg 780 cacagataga
gtgtacaggt ctggagaaca aggagtgggg ggttattgga attccacatt 840
gtttgctgca cgttggattt tgaaatgcta gggaactttg ggagactcat atttctgggc
900 tagaggatct gtggaccaca agatcttttt atgatgacag tagcaatgta
tctgtggagc 960 tggattctgg gttgggagtg caaggaaaag aatgtactaa
atgccaagac atctatttca 1020 ggagcatgag gaataaaagt tctagtttct
ggtctcagag tggtgcaggg atcagggagt 1080 ctcacaatct cctgagtgct
ggtgtcttag ggcacactgg gtcttggagt gcaaaggatc 1140 taggcacgtg
aggctttgta tgaagaatcg gggatcgtac ccaccccctg tttctgtttc 1200
atcctgggcg tgtctcctct gcctttgtcc cctagatgaa gtctccatga gctacagggc
1260 ctggtgcatc cagggtgatc tagtaattgc agaacagcaa gtactagctc
tccctcccct 1320 tccacagctc tgggtgtggg agggggttgt ccagcctcca
gcagcatggg gagggccttg 1380 gtcagcctct gggtgccagc agggcagggg
cggagtcctg gggaatgaag gttttatagg 1440 gctcctgggg gaggctcccc
agccccaagc ttaccacctg cacccggaga gctgtgtcac 1500 catgtgggtc
ccggttgtct tcctcaccct gtccgtgacg tggattggtg agaggggcca 1560
tggttggggg gatgcaggag agggagccag ccctgactgt caagctgagg ctctttcccc
1620 cccaacccag caccccagcc cagacaggga gctgggctct tttctgtctc
tcccagcccc 1680 actccaactc cctgctccca
gctgcttaa 1709 5 3423 DNA Homo sapiens 5 attttgcatg ccaccttaat
cttttttttt ttttttttaa atcgaggttt cagtctcatt 60 ctatttccca
ggctggagtt caatagcgtg atcacagctc actgtagcct tgaactcctg 120
gccttaagag attctcctgc ttcggtctcc caatagctaa gactacagta gtccaccacc
180 atatccagat aatttttaaa ttttttgggg ggccgggcac agtggctcac
gcctgtaatc 240 ccaacaccat gggaggctga gatgggtgga tcacgaggtc
aggagtttga gaccagcctg 300 accaacatgg tgaaactctg tctctactaa
aaaaaaaaaa aatagaaaaa ttagccgggc 360 gtggtggcac acggcacctg
taatcccagc tactgaggag gctgaggcag gagaatcact 420 tgaacccaga
aggcagaggt tgcaatgagc cgagattgcg ccactgcact ccagcctggg 480
tgacagagtg agactctgtc tcaaaaaaaa aaaatttttt tttttttttt gtagagatgg
540 atcttgcttt gtttctctgg ttggccttga actcctggct tcaagtgatc
ctcctacctt 600 ggcctcggaa agtgttggga ttacaggcgt gagccaccat
gactgacctg tcgttaatct 660 tgaggtacat aaacctggct cctaaaggct
aaaggctaaa tatttgttgg agaaggggca 720 ttggattttg catgaggatg
attctgacct gggagggcag gtcagcaggc atctctgttg 780 cacagataga
gtgtacaggt ctggagaaca aggagtgggg ggttattgga attccacatt 840
gtttgctgca cgttggattt tgaaatgcta gggaactttg ggagactcat atttctgggc
900 tagaggatct gtggaccaca agatcttttt atgatgacag tagcaatgta
tctgtggagc 960 tggattctgg gttgggagtg caaggaaaag aatgtactaa
atgccaagac atctatttca 1020 ggagcatgag gaataaaagt tctagtttct
ggtctcagag tggtgcaggg atcagggagt 1080 ctcacaatct cctgagtgct
ggtgtcttag ggcacactgg gtcttggagt gcaaaggatc 1140 taggcacgtg
aggctttgta tgaagaatcg gggatcgtac ccaccccctg tttctgtttc 1200
atcctgggcg tgtctcctct gcctttgtcc cctagatgaa gtctccatga gctacagggc
1260 ctggtgcatc cagggtgatc tagtaattgc agaacagcaa gtactagctc
tccctcccct 1320 tccacagctc tgggtgtggg agggggttgt ccagcctcca
gcagcatggg gagggccttg 1380 gtcagcctct gggtgccagc agggcagggg
cggagtcctg gggaatgaag gttttatagg 1440 gctcctgggg gaggctcccc
agccccaagc ttaccacctg cacccggaga gctgtgtcac 1500 catgtgggtc
ccggttgtct tcctcaccct gtccgtgacg tggattggtg ctgcacccct 1560
catcctgtct cggattgtgg gaggctggga gtgcgagaag cattcccaac cctggcaggt
1620 gcttgtggcc tctcgtggca gggcagtctg cggcggtgtt ctggtgcacc
cccagtgggt 1680 cctcacagct gcccactgca tcaggaacaa aagcgtgatc
ttgctgggtc ggcacagcct 1740 gtttcatcct gaagacacag gccaggtatt
tcaggtcagc cacagcttcc cacacccgct 1800 ctacgatatg agcctcctga
agaatcgatt cctcaggcca ggtgatgact ccagccacga 1860 cctcatgctg
ctccgcctgt cagagcctgc cgagctcacg gatgctgtga aggtcatgga 1920
cctgcccacc caggagccag cactggggac cacctgctac gcctcaggct ggggcagcat
1980 tgaaccagag gagttcttga ccccaaagaa acttcagtgt gtggacctcc
atgttatttc 2040 caatgacgtg tgtgcgcaag ttcaccctca gaaggtgacc
aagttcatgc tgtgtgctgg 2100 acgctggaca gggggcaaaa gcacctgctc
gggtgattct gggggcccac ttgtctgtaa 2160 tggtgtgctt caaggtatca
cgtcatgggg cagtgaacca tgtgccctgc ccgaaaggcc 2220 ttccctgtac
accaaggtgg tgcattaccg gaagtggatc aaggacacca tcgtggccaa 2280
cccctgagca cccctatcaa ctccctattg tagtaaactt ggaaccttgg aaatgaccag
2340 gccaagactc aagcctcccc agttctactg acctttgtcc ttaggtgtga
ggtccagggt 2400 tgctaggaaa agaaatcagc agacacaggt gtagaccaga
gtgtttctta aatggtgtaa 2460 ttttgtcctc tctgtgtcct ggggaatact
ggccatgcct ggagacatat cactcaattt 2520 ctctgaggac acagatagga
tggggtgtct gtgttatttg tgggrtacag agatgaaaga 2580 ggggtgggww
ccacactgag agagtggaga gtgacatgtg ctggacactg tccatgaagc 2640
actgagcaga agctggaggc acaacgcacc agacactcac agcaaggatg gagctgaaaa
2700 cataacccac tctgtcctgg aggcactggg aagcctagag aaggctgtga
gccaaggagg 2760 gagggtcttc ctttggcatg ggatggggat gaagtaagga
gagggactgg accccctgga 2820 agctgattca ctatgggggg aggtgtattg
aagtcctcca gacaaccctc agatttgatg 2880 atttcctagt agaactcaca
gaaataaaga gctsttatac gtggtttatt ctggtttgtt 2940 acattgacag
gagacacact gaaatcagca aaggaaacag gcatctaagt ggggatgtga 3000
agaaaacagg gaaaatcttt cagttgtttt ctcccagtgg ggtgttgtgg acagcactta
3060 aatcacacag aagtgatgtg tgaccttgtg tatgaagtat ttccaactaa
ggaagctcac 3120 ctgagcctta gtgtccagag ttcttattgg gggtctgtag
gataggcatg gggtactgga 3180 atagctgacc ttaacttctc agacctgagg
ttcccaagag ttcaagcaga tacagcatgg 3240 cctagagcct cagatgtaca
aaaacaggca ttcatcatga atcgcactgt tagcatgaat 3300 catctggcac
ggcccaaggc cccaggtata ccaaggcact tgggccgaat gttccaaggg 3360
attaaatgtc atctcccagg agttattcaa gggtgagccc tgtacttgga acgttcaggc
3420 ttt 3423 6 1261 DNA Homo sapiens 6 gggcggggtc ctggagaatg
aaggctttat agggctcctc agggaggccc cccagcccca 60 aactgcacca
cctggccgtg gacacctgtg tcagcatgtg ggacctggtt ctctccatcg 120
ccttgtctgt ggggtgcact ggtgagattg gggggataaa ggaagggggg cgggttctga
180 ctcttatgct gaagcccttt tcctcccacc cagtgcccca gcctcgtccc
ttcagcccac 240 agttcagccc agacaatgtg cccctgactc ttccacattg
caatagtcct catgcccaca 300 ctaggtcccc gctccctccc acttacctca
gacctttctc tccattgccc agccaaatcc 360 ctgctcccag ctgctttact
aaagagcaag ttcctaggca tctctgtgtt tctctttatg 420 gggttcaaaa
cctttcaagg acctctctcc atgccactgg ttccttggac cctatcactg 480
ggctgcctcc tgagcccctc agtcctacca cagtctactg acttttccca ttcagctgtg
540 agcattcaac cctgtcccct ggaccttgac acctggctcc ccaaccctgt
cccaggaaac 600 ccagattcca ccagacactt ccttcttccc ccccgaggct
atctggcctg agacaacaaa 660 tgctgcctcc caccctgagt ctggcactgg
gactttcaga actcctcctt ccctgactct 720 ttgccccaga cccgtcattc
aatggctagc tttttccatg ggaagaagaa caacgagcac 780 ccccaaccac
aacggccagt tctctgattc cctaaatccg cacccttttc aaaacctcaa 840
aaacaaaaca aaacaaaaca aagcaagaaa caactcaggc aaaacttgtt gcttaacctt
900 ggacatggta aaccatccaa aaccttcctc tcccagcaac taaacctctc
cactgggcac 960 ttaacctttg gtttcttgga acctcttaat ctcttagaac
ccacagctgc caccacatgc 1020 ccttctccca atgtaagacc ccaaatcact
ccaaatgacc caacccccaa cccatgcctc 1080 cttcagatat ttcccatgtc
ccctactctg atctctgggg tcagctccgt tctcgagagc 1140 atgaagcctc
ccgacctggt ccagccacca acccgctaac gcagggaata gctacagaat 1200
tgccagccct cccaggaccc cttgcttgtg tcctggactc ccagtcctgg tcctctgccc
1260 c 1261 7 104 PRT Homo sapiens 7 Met Trp Val Pro Val Val Phe
Leu Thr Leu Ser Val Thr Trp Ile Gly 1 5 10 15 Glu Arg Gly His Gly
Trp Gly Asp Ala Gly Glu Gly Ala Ser Pro Asp 20 25 30 Cys Gln Ala
Glu Ala Leu Ser Pro Pro Thr Gln His Pro Ser Pro Asp 35 40 45 Arg
Glu Leu Gly Ser Phe Leu Ser Leu Pro Ala Pro Leu Gln Ala His 50 55
60 Thr Pro Ser Pro Ser Ile Leu Gln Gln Ser Ser Leu Pro His Gln Val
65 70 75 80 Pro Ala Pro Ser His Leu Pro Gln Asn Phe Leu Pro Ile Ala
Gln Pro 85 90 95 Ala Pro Cys Ser Gln Leu Leu Tyr 100 8 218 PRT Homo
sapiens 8 Met Lys Asn Arg Gly Ser Tyr Pro Pro Pro Val Ser Val Ser
Ser Trp 1 5 10 15 Ala Cys Leu Leu Cys Leu Cys Pro Leu Asp Glu Val
Ser Met Ser Tyr 20 25 30 Arg Ala Trp Cys Ile Gln Gly Asp Leu Val
Ile Ala Glu Gln Gln Val 35 40 45 Leu Ala Leu Pro Pro Leu Pro Gln
Leu Trp Val Trp Glu Gly Val Val 50 55 60 Gln Pro Pro Ala Ala Trp
Gly Gly Pro Trp Ser Ala Ser Gly Cys Gln 65 70 75 80 Gln Gly Arg Gly
Gly Val Leu Gly Asn Glu Gly Phe Ile Gly Leu Leu 85 90 95 Gly Glu
Ala Pro Gln Pro Gln Ala Tyr His Leu His Pro Glu Ser Cys 100 105 110
Val Thr Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp 115
120 125 Ile Gly Glu Arg Gly His Gly Trp Gly Asp Ala Gly Glu Gly Ala
Ser 130 135 140 Pro Asp Cys Gln Ala Glu Ala Leu Ser Pro Pro Thr Gln
His Pro Ser 145 150 155 160 Pro Asp Arg Glu Leu Gly Ser Phe Leu Ser
Leu Pro Ala Pro Leu Gln 165 170 175 Ala His Thr Pro Ser Pro Ser Ile
Leu Gln Gln Ser Ser Leu Pro His 180 185 190 Gln Val Pro Ala Pro Ser
His Leu Pro Gln Asn Phe Leu Pro Ile Ala 195 200 205 Gln Pro Ala Pro
Cys Ser Gln Leu Leu Tyr 210 215 9 218 PRT Homo sapiens 9 Met Lys
Asn Arg Gly Ser Tyr Pro Pro Pro Val Ser Val Ser Ser Trp 1 5 10 15
Ala Cys Leu Leu Cys Leu Cys Pro Leu Asp Glu Val Ser Met Ser Tyr 20
25 30 Arg Ala Trp Cys Ile Gln Gly Asp Leu Val Ile Ala Glu Gln Gln
Val 35 40 45 Leu Ala Leu Pro Pro Leu Pro Gln Leu Trp Val Trp Glu
Gly Val Val 50 55 60 Gln Pro Pro Ala Ala Trp Gly Gly Pro Trp Ser
Ala Ser Gly Cys Gln 65 70 75 80 Gln Gly Arg Gly Gly Val Leu Gly Asn
Glu Gly Phe Ile Gly Leu Leu 85 90 95 Gly Glu Ala Pro Gln Pro Gln
Ala Tyr His Leu His Pro Glu Ser Cys 100 105 110 Val Thr Met Trp Val
Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp 115 120 125 Ile Gly Glu
Arg Gly His Gly Trp Gly Asp Ala Gly Glu Gly Ala Ser 130 135 140 Pro
Asp Cys Gln Ala Glu Ala Leu Ser Pro Pro Thr Gln His Pro Ser 145 150
155 160 Pro Asp Arg Glu Leu Gly Ser Phe Leu Ser Leu Pro Ala Pro Leu
Gln 165 170 175 Ala His Thr Pro Ser Pro Ser Ile Leu Gln Gln Ser Ser
Leu Pro His 180 185 190 Gln Val Pro Ala Pro Ser His Leu Pro Gln Asn
Phe Leu Pro Ile Ala 195 200 205 Gln Pro Ala Pro Cys Ser Gln Leu Leu
Tyr 210 215 10 183 PRT Homo sapiens 10 Met Lys Asn Arg Gly Ser Tyr
Pro Pro Pro Val Ser Val Ser Ser Trp 1 5 10 15 Ala Cys Leu Leu Cys
Leu Cys Pro Leu Asp Glu Val Ser Met Ser Tyr 20 25 30 Arg Ala Trp
Cys Ile Gln Gly Asp Leu Val Ile Ala Glu Gln Gln Val 35 40 45 Leu
Ala Leu Pro Pro Leu Pro Gln Leu Trp Val Trp Glu Gly Val Val 50 55
60 Gln Pro Pro Ala Ala Trp Gly Gly Pro Trp Ser Ala Ser Gly Cys Gln
65 70 75 80 Gln Gly Arg Gly Gly Val Leu Gly Asn Glu Gly Phe Ile Gly
Leu Leu 85 90 95 Gly Glu Ala Pro Gln Pro Gln Ala Tyr His Leu His
Pro Glu Ser Cys 100 105 110 Val Thr Met Trp Val Pro Val Val Phe Leu
Thr Leu Ser Val Thr Trp 115 120 125 Ile Gly Glu Arg Gly His Gly Trp
Gly Asp Ala Gly Glu Gly Ala Ser 130 135 140 Pro Asp Cys Gln Ala Glu
Ala Leu Ser Pro Pro Thr Gln His Pro Ser 145 150 155 160 Pro Asp Arg
Glu Leu Gly Ser Phe Leu Ser Leu Pro Ala Pro Leu Gln 165 170 175 Leu
Pro Ala Pro Ser Cys Leu 180 11 375 PRT Homo sapiens 11 Met Lys Asn
Arg Gly Ser Tyr Pro Pro Pro Val Ser Val Ser Ser Trp 1 5 10 15 Ala
Cys Leu Leu Cys Leu Cys Pro Leu Asp Glu Val Ser Met Ser Tyr 20 25
30 Arg Ala Trp Cys Ile Gln Gly Asp Leu Val Ile Ala Glu Gln Gln Val
35 40 45 Leu Ala Leu Pro Pro Leu Pro Gln Leu Trp Val Trp Glu Gly
Val Val 50 55 60 Gln Pro Pro Ala Ala Trp Gly Gly Pro Trp Ser Ala
Ser Gly Cys Gln 65 70 75 80 Gln Gly Arg Gly Gly Val Leu Gly Asn Glu
Gly Phe Ile Gly Leu Leu 85 90 95 Gly Glu Ala Pro Gln Pro Gln Ala
Tyr His Leu His Pro Glu Ser Cys 100 105 110 Val Thr Met Trp Val Pro
Val Val Phe Leu Thr Leu Ser Val Thr Trp 115 120 125 Ile Gly Ala Ala
Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu 130 135 140 Cys Glu
Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg Gly 145 150 155
160 Arg Ala Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr
165 170 175 Ala Ala His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu Gly
Arg His 180 185 190 Ser Leu Phe His Pro Glu Asp Thr Gly Gln Val Phe
Gln Val Ser His 195 200 205 Ser Phe Pro His Pro Leu Tyr Asp Met Ser
Leu Leu Lys Asn Arg Phe 210 215 220 Leu Arg Pro Gly Asp Asp Ser Ser
His Asp Leu Met Leu Leu Arg Leu 225 230 235 240 Ser Glu Pro Ala Glu
Leu Thr Asp Ala Val Lys Val Met Asp Leu Pro 245 250 255 Thr Gln Glu
Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly 260 265 270 Ser
Ile Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val 275 280
285 Asp Leu His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln
290 295 300 Lys Val Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly
Gly Lys 305 310 315 320 Ser Thr Cys Ser Gly Asp Ser Gly Gly Pro Leu
Val Cys Asn Gly Val 325 330 335 Leu Gln Gly Ile Thr Ser Trp Gly Ser
Glu Pro Cys Ala Leu Pro Glu 340 345 350 Arg Pro Ser Leu Tyr Thr Lys
Val Val His Tyr Arg Lys Trp Ile Lys 355 360 365 Asp Thr Ile Val Ala
Asn Pro 370 375 12 141 PRT Homo sapiens 12 Met Trp Asp Leu Val Leu
Ser Ile Ala Leu Ser Val Gly Cys Thr Gly 1 5 10 15 Glu Ile Gly Gly
Ile Lys Glu Gly Gly Arg Val Leu Thr Leu Met Leu 20 25 30 Lys Pro
Phe Ser Ser His Pro Val Pro Gln Pro Arg Pro Phe Ser Pro 35 40 45
Gln Phe Ser Pro Asp Asn Val Pro Leu Thr Leu Pro His Cys Asn Ser 50
55 60 Pro His Ala His Thr Arg Ser Pro Leu Pro Pro Thr Tyr Leu Arg
Pro 65 70 75 80 Phe Ser Pro Leu Pro Ser Gln Ile Pro Ala Pro Ser Cys
Phe Thr Lys 85 90 95 Glu Gln Val Pro Arg His Leu Cys Val Ser Leu
Tyr Gly Val Gln Asn 100 105 110 Leu Ser Arg Thr Ser Leu His Ala Thr
Gly Ser Leu Asp Pro Ile Thr 115 120 125 Gly Leu Pro Pro Glu Pro Leu
Ser Pro Thr Thr Val Tyr 130 135 140 13 10574 DNA Homo sapiens 13
aagcttctag ttttcttttc ccggtgacat cgtggaaagc actagcatct ctaagcaatg
60 atctgtgaca atattcacag tgtaatgcca tccagggaac tcaactgagc
cttgatgtcc 120 agagattttt gtgttttttt ctgagactga gtctcgctct
gtgccaggct ggagtgcagt 180 ggtgcaacct tggctcactg caagctccgc
ctcctgggtt cacgccattc tcctgcctca 240 gcctcctgag tagctgggac
tacaggcacc cgccaccacg cctggctaat ttttttgtat 300 ttttagtaga
gatggggttt cactgtgtta gccaggatgg tctcagtctc ctgacctcgt 360
gatctgccca ccttggcctc ccaaagtgct gggatgacag gcgtgagcca ccgcgcctgg
420 ccgatatcca gagatttttt ggggggctcc atcacacaga catgttgact
gtcttcatgg 480 ttgactttta gtatccagcc cctctagaaa tctagctgat
atagtgtggc tcaaaacctt 540 cagcacaaat cacaccgtta gactatctgg
tgtggcccaa accttcaggt gaacaaaggg 600 actctaatct ggcaggatat
tccaaagcat tagagatgac ctcttgcaaa gaaaaagaaa 660 tggaaaagaa
aaagaaagaa aggaaaaaaa aaaaaaaaaa gagatgacct ctcaggctct 720
gaggggaaac gcctgaggtc tttgagcaag gtcagtcctc tgttgcacag tctccctcac
780 agggtcattg tgacgatcaa atgtggtcac gtgtatgagg caccagcaca
tgcctggctc 840 tggggagtgc cgtgtaagtg tatgcttgca ctgctgaatg
cttgggatgt gtcagggatt 900 atcttcagca cttacagatg ctcatctcat
cctcacagca tcactatggg atgggtatta 960 ctggcctcat ttgatggaga
aagtggctgt ggctcagaaa ggggggacca ctagaccagg 1020 gacactctgg
atgctgggga ctccagagac catgaccact caccaactgc agagaaatta 1080
attgtggcct gatgtccctg tcctggagag ggtggaggtg gaccttcact aacctcctac
1140 cttgaccctc tcttttaggg ctctttctga cctccaccat ggtactagga
ccccattgta 1200 ttctgtaccc tcttgactct atgaccccca ctgcccactg
catccagctg ggtcccctcc 1260 tatctctatt cccagctggc cagtgcagtc
tcagtgccca cctgtttgtc agtaactctg 1320 aaggggctga cattttactg
acttgcaaac aaataagcta actttccaga gttttgtgaa 1380 tgctggcaga
gtccatgaga ctcctgagtc agaggcaaag gcttttactg ctcacagctt 1440
agcagacagc atgaggttca tgttcacatt agtacacctt gcccccccca aatcttgtag
1500 ggtgaccaga gcagtctagg tggatgctgt gcagaagggg tttgtgccac
tggtgagaaa 1560 cctgagatta ggaatcctca atcttatact gggacaactt
gcaaacctgc tcagcctttg 1620 tctctgatga agatattatc ttcatgatct
tggattgaaa acagacctac tctggaggaa 1680 catattgtat cgattgtcct
tgacagtaaa caaatctgtt gtaagagaca ttatctttat 1740 tatctaggac
agtaagcaag cctggatctg agagagatat catcttgcaa ggatgcctgc 1800
tttacaaaca tccttgaaac aacaatccag aaaaaaaaag gtgttgctgt ctttgctcag
1860 aagacacaca gatacgtgac agaaccatgg agaattgcct cccaacgctg
ttcagccaga 1920 gccttccacc cttgtctgca ggacagtctc aacgttccac
cattaaatac ttcttctatc 1980 acatcctgct tctttatgcc taaccaaggt
tctaggtccc gatcgactgt gtctggcagc 2040 actccactgc caaacccaga
ataaggcagc gctcaggatc ccgaaggggc atggctgggg 2100 atcagaactt
ctgggtttga gtgaggagtg ggtccaccct cttgaatttc aaaggaggaa 2160
gaggctggat gtgaaggtac tgggggaggg aaagtgtcag ttccgaactc ttaggtcaat
2220 gagggaggag actggtaagg tcccagctcc cgaggtactg atgtgggaat
ggcctaagaa 2280 tctcatatcc tcaggaagaa ggtgctggaa tcctgagggg
tagagttctg ggtatatttg 2340 tggcttaagg ctctttggcc cctgaaggca
gaggctggaa ccattaggtc cagggtttgg 2400 ggtgatagta atgggatctc
ttgattcctc aagagtctga
ggatcgaggg ttgcccattc 2460 ttccatcttg ccacctaatc cttactccac
ttgagggtat caccagccct tctagctcca 2520 tgaaggtccc ctgggcaagc
acaatctgag catgaaagat gccccagagg ccttgggtgt 2580 catccactca
tcatccagca tcacactctg agggtgtggc cagcaccatg acgtcatgtt 2640
gctgtgacta tccctgcagc gtgcctctcc agccacctgc caaccgtaga gctgcccatc
2700 ctcctctggt gggagtggcc tgcatggtgc caggctgagg cctagtgtca
gacagggagc 2760 ctggaatcat agggatccag gactcaaaag tgctagagaa
tggccatatg tcaccatcca 2820 tgaaatctca agggcttctg ggtggagggc
acagggacct gaacttatgg tttcccaagt 2880 ctattgctct cccaagtgag
tctcccagat acgaggcact gtgccagcat cagccttatc 2940 tccaccacat
cttgtaaaag gactacccag ggccctgatg aacaccatgg tgtgtacagg 3000
agtagggggt ggaggcacgg actcctgtga ggtcacagcc aagggagcat catcatgggt
3060 ggggaggagg caatggacag gcttgagaac ggggatgtgg ttgtatttgg
ttttctttgg 3120 ttagataaag tgctgggtat aggattgaga gtggagtatg
aagaccagtt aggatggagg 3180 atcagattgg agttgggtta gataaagtgc
tgggtatagg attgagagtg gagtatgaag 3240 accagttagg atggaggatc
agattggagt tgggttagag atggggtaaa attgtgctcc 3300 ggatgagttt
gggattgaca ctgtggaggt ggtttgggat ggcatggctt tgggatggaa 3360
atagatttgt tttgatgttg gctcagacat ccttggggat tgaactgggg atgaagctgg
3420 gtttgatttt ggaggtagaa gacgtggaag tagctgtcag atttgacagt
ggccatgagt 3480 tttgtttgat ggggaatcaa acaatggggg aagacataag
ggttggcttg ttaggttaag 3540 ttgcgttggg ttgatggggt cggggctgtg
tataatgcag ttggattggt ttgtattaaa 3600 ttgggttggg tcaggttttg
gttgaggatg agttgaggat atgcttgggg acaccggatc 3660 catgaggttc
tcactggagt ggagacaaac ttcctttcca ggatgaatcc agggaagcct 3720
taattcacgt gtaggggagg tcaggccact ggctaagtat atccttccac tccagctcta
3780 agatggtctt aaattgtgat tatctatatc cacttctgtc tccctcactg
tgcttggagt 3840 ttacctgatc actcaactag aaacagggga agattttatc
aaattctttt tttttttttt 3900 ttttttttga gacagagtct cactctgttg
cccaggctgg agtgcagtgg cgcagtctcg 3960 gctcactgca acctctgcct
cccaggttca agtgattctc ctgcctcagc ctcctgagtt 4020 gctgggatta
caggcatgca gcaccatgcc cagctaattt ttgtattttt agtagagatg 4080
gggtttcacc aatgtttgcc aggctggcct cgaactcctg acctggtgat ccacctgcct
4140 cagcctccca aagtgctggg attacaggcg tcagccaccg cgcccagcca
cttttgtcaa 4200 attcttgaga cacagctcgg gctggatcaa gtgagctact
ctggttttat tgaacagctg 4260 aaataaccaa ctttttggaa attgatgaaa
tcttacggag ttaacagtgg aggtaccagg 4320 gctcttaaga gttcccgatt
ctcttctgag actacaaatt gtgattttgc atgccacctt 4380 aatctttttt
tttttttttt taaatcgagg tttcagtctc attctatttc ccaggctgga 4440
gttcaatagc gtgatcacag ctcactgtag ccttgaactc ctggccttaa gagattctcc
4500 tgcttcggtc tcccaatagc taagactaca gtagtccacc accatatcca
gataattttt 4560 aaattttttg gggggccggg cacagtggct cacgcctgta
atcccaacac catgggaggc 4620 tgagatgggt ggatcacgag gtcaggagtt
tgagaccagc ctgaccaaca tggtgaaact 4680 ctgtctctac taaaaaaaaa
aaaaatagaa aaattagccg ggcgtggtgg cacacggcac 4740 ctgtaatccc
agctactgag gaggctgagg caggagaatc acttgaaccc agaaggcaga 4800
ggttgcaatg agccgagatt gcgccactgc actccagcct gggtgacaga gtgagactct
4860 gtctcaaaaa aaaaaaattt tttttttttt tttgtagaga tggatcttgc
tttgtttctc 4920 tggttggcct tgaactcctg gcttcaagtg atcctcctac
cttggcctcg gaaagtgttg 4980 ggattacagg cgtgagccac catgactgac
ctgtcgttaa tcttgaggta cataaacctg 5040 gctcctaaag gctaaaggct
aaatatttgt tggagaaggg gcattggatt ttgcatgagg 5100 atgattctga
cctgggaggg caggtcagca ggcatctctg ttgcacagat agagtgtaca 5160
ggtctggaga acaaggagtg gggggttatt ggaattccac attgtttgct gcacgttgga
5220 ttttgaaatg ctagggaact ttgggagact catatttctg ggctagagga
tctgtggacc 5280 acaagatctt tttatgatga cagtagcaat gtatctgtgg
agctggattc tgggttggga 5340 gtgcaaggaa aagaatgtac taaatgccaa
gacatctatt tcaggagcat gaggaataaa 5400 agttctagtt tctggtctca
gagtggtgca gggatcaggg agtctcacaa tctcctgagt 5460 gctggtgtct
tagggcacac tgggtcttgg agtgcaaagg atctaggcac gtgaggcttt 5520
gtatgaagaa tcggggatcg tacccacccc ctgtttctgt ttcatcctgg gcatgtctcc
5580 tctgcctttg tcccctagat gaagtctcca tgagctacag ggcctggtgc
atccagggtg 5640 atctagtaat tgcagaacag caagtgctag ctctccctcc
ccttccacag ctctgggtgt 5700 gggagggggt tgtccagcct ccagcagcat
ggggagggcc ttggtcagcc tctgggtgcc 5760 agcagggcag gggcggagtc
ctggggaatg aaggttttat agggctcctg ggggaggctc 5820 cccagcccca
agcttaccac ctgcacccgg agagctgtgt caccatgtgg gtcccggttg 5880
tcttcctcac cctgtccgtg acgtggattg gtgagagggg ccatggttgg ggggatgcag
5940 gagagggagc cagccctgac tgtcaagctg aggctctttc ccccccaacc
cagcacccca 6000 gcccagacag ggagctgggc tcttttctgt ctctcccagc
cccactccaa gcccataccc 6060 ccagcccctc catattgcaa cagtcctcac
tcccacacca ggtccccgct ccctcccact 6120 taccccagaa ctttctcccc
atttgcccag ccagctccct gctcccagct gctttactaa 6180 aggggaagtt
cctgggcatc tccgtgtttc tctttgtggg gctcaaaacc tccaaggacc 6240
tctctcaatg ccattggttc cttggaccgt atcactggtc cacctcctga gcccctcaat
6300 cctatcacag tctactgact tttccattca gctgtgagtg cccaacccta
tcccagagac 6360 cttgatgctt ggcctcccaa tcttgcccta ggatacccag
atgccaacca gacacctcct 6420 tcttcctagc caggctatct ggctgagaca
acaaatgggt ccctcagtct ggcaatggga 6480 ctctgagaac tcctcattcc
ctgactctta gccccagact cttcattcag tggcccacat 6540 tttccttagg
aaaaacatga gcatccccag ccacaactgc cagctctctg attccccaaa 6600
tctgcatcct tttcaaaacc taaaaacaaa aagaaaaaca aataaaacaa aaccaactca
6660 gaccagaact gttttctcaa cctgggactt cctaaacttt ccaaaacctt
cctcttccag 6720 caactgaacc tcccgataag gcacttatcc ctggttccta
gcaccgctta tcccctcaga 6780 atccacaact tgtaccaagt ttcccttctc
ccagtccaag accccaaatc accacaaagg 6840 acccaatccc cagactcaag
atatggtctg gggctgtctt gtgtctccta ccctgatccc 6900 tgggttcaac
tctgtcccag agcatgaagc ctctccacca gcaccagcca ccaacctgca 6960
aacctaggga agattgacag aattcccagc ctttcccagc tccccctgcc catgtcccag
7020 gactcccagc cttggttctc tgcccccgtg tcttttcaaa cccacatcct
aaatccatct 7080 cctatccgag tcccccagtt cctcctgtca accctgattc
ccctgatcta gcaccccctc 7140 tgcaggtgct gcacccctca tcctgtctcg
gattgtggga ggctgggagt gcgagaagca 7200 ttcccaaccc tggcaggtgc
ttgtggcctc tcgtggcagg gcagtctgcg gcggtgttct 7260 ggtgcacccc
cagtgggtcc tcacagctgc ccactgcatc aggaagtgag taggggcctg 7320
gggtctgggg agcaggtgtc tgtgtccaga ggaataacag ctgggcattt tccccaggat
7380 aacctctaag gccagccttg ggactggggg agagagggaa agttctggtt
caggtcacat 7440 ggggaggcag ggttggggct ggaccaccct ccccatggct
gcctgggtct ccatctgtgt 7500 tcctctatgt ctctttgtgt cgctttcatt
atgtctcttg gtaactggct tcggttgtgt 7560 ctctccgtgt gactattttg
ttctctctct ccctctcttc tctgtcttca gtctccatat 7620 ctccccctct
ctctgtcctt ctctggtccc tctctagcca gtgtgtctca ccctgtatct 7680
ctctgccagg ctctgtctct cggtctctgt ctcacctgtg ccttctccct actgagcaca
7740 cgcatgggat gggcctgggg ggaccctgag aaaaggaagg gctttggctg
ggcgcggtgg 7800 ctcacacctg taatcccagc actttgggag gccaaggcag
gtagatcacc tgaggtcagg 7860 agttcgagac cagcctggcc aactggtgaa
accccatctc tactaaaaat acaaaaaatt 7920 agccaggcgt ggtcggcgca
tgcctgtagt cccagctact caggaggctg agggaggaga 7980 attgcttgaa
cctgggaggt ggaggttgca gtgagccgag acgtgccact gcactccagc 8040
ctgggtgaca gagtgagact ccgcctcaaa aaaaaaaaaa aaaaaaaaga aaagaaaaga
8100 aaagaaaagg aagtgtttta tccctgatgt gtgtgggtat gagggtatga
gagggcccct 8160 ctcactccat tccttctcca ggacatccct ccactcttgg
gagacacaga gaagggctgg 8220 ttcagctgga gctgggaggg gcaattgagg
gaggaggaag gagaaggggg aaggaaaaca 8280 gggtatgggg gaaaggaccc
tggggagcga agtggaggat acaaccttgg gcctgcaggc 8340 caggctacct
acccacttgg aaacccacgc caaagccgca tctacagctg agccactctg 8400
aggcctcccc tccccagcgg tccccactca gctccaaagt ctctctccct tttctctccc
8460 acactctatc atcccccgga ttcctctcta cttggttctc attcttcctt
tgacttcctg 8520 cttccctttc tcattcatct gtttctcact ttctgcctgg
ttttgttctt ctctctctct 8580 ttctctggcc catgtctgtt tctctatgtt
tctgtctttt ctttctcatc ctgtgtattt 8640 tcggctcacc ttgtttgtca
ctgttctccc ctctgccctt tcattctctc tgtcctttta 8700 ccctcttcct
ttttcccttg gtttctctca gtttctgtat ctgcccttca ccctctcaca 8760
ctgctgtttc ccaactcgtt gtctgtattt ttggcctgaa catgtgtctt ccccaaccct
8820 gtgtttttct cactgtttct ttttctcttt tggagcctcc tccttgctcc
tctgtccctt 8880 ctctctttcc ttatcatcct cgctcctcat tcctgcgtct
gcttcctccc cagcaaaagc 8940 gtgatcttgc tgggtcggca cagcctgttt
catcctgaag acacaggcca ggtatttcag 9000 gtcagccaca gcttcccaca
cccgctctac gatatgagcc tcctgaagaa tcgattcctc 9060 aggccaggtg
atgactccag ccacgacctc atgctgctcc gcctgtcaga gcctgccgag 9120
ctcacggatg ctgtgaaggt catggacctg cccacccagg agccagcact ggggaccacc
9180 tgctacgcct caggctgggg cagcattgaa ccagaggagt gtacgcctgg
gccagatggt 9240 gcagccggga gcccagatgc ctgggtctga gggaggaggg
gacaggactc ctaggtctga 9300 gggaggaggg ccaaggaacc aggtggggtc
cagcccacaa cagtgttttt tgcctggccc 9360 gtagtcttga ccccaaagaa
acttcagtgt gtggacctcc atgttatttc caatgacgtg 9420 tgtgcgcaag
ttcaccctca gaaggtgacc aagttcatgc tgtgtgctgg acgctggaca 9480
gggggcaaaa gcacctgctc ggtgagtcat ccctactccc aagatcttga ggggaaaggt
9540 gagtggggac cttaattctg ggctggggtc tagaagccaa caagcatctg
cctcccctgc 9600 tccccagctg tagccatgcc acctccccgt gtctcatctc
attccctcct tccctcttct 9660 ttgactccct caaggcaata ggttattctt
acagcacaac tcatctgttc ctgcgttcag 9720 cacacggtta ctaggcacct
gctatgcacc cagcactgcc ctagagcctg gacatagcag 9780 tgaacagaca
gagagcagcc cctcccttct gtagccccca agccagtgag gggcacaggc 9840
aggaacaggg accacaacac agaaaagctg gagggtgtca ggaggtgatc aggctctcgg
9900 ggagggagaa ggggtgggga gtgtgactgg gaggagacat cctgcagaag
gcgggagtga 9960 gcaaacacct gccgcagggg aggggagggc ctgcggcacc
tgggggagca gagggaacag 10020 catctggcca ggcctgggag gaggggccta
gagggcgtca ggagcagaga ggaggttgcc 10080 tggctggagt gaaggatcgg
ggcagggtgc gagagggaag aaggacccct cctgcagggc 10140 ctcacctggg
ccacaggagg acactgcttt tcctctgagg agtcaggaac tgtggatggt 10200
gctggacaga agcaggacag ggcctggctc aggtgtccag aggctgccgc tggcctccct
10260 atgggatcag actgcaggga gggagggcag cagggatgtg gagggagtga
tgatggggct 10320 gacctggggg tggctccagg cattgtcccc acctgggccc
ttacccagcc tccctcacag 10380 gctcctggcc ctcagtctct cccctccact
ccattctcca cctacccaca gtgggtcatt 10440 ctgatcaccg aactgaccat
gccagccctg ccgatggtcc tccatggctc cctagtgccc 10500 tggagaggag
gtgtctagtc agagagtagt cctggaaggt ggcctctgtg aggagccacg 10560
gggacagcat cctg 10574 14 262 PRT Homo sapiens 14 Met Trp Val Pro
Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1 5 10 15 Ala Ala
Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu 20 25 30
Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala 35
40 45 Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala
Ala 50 55 60 His Cys Ile Arg Lys Cys Lys Ser Val Ile Leu Leu Gly
Arg His Ser 65 70 75 80 Leu Phe His Pro Glu Asp Thr Gly Gln Val Phe
Gln Val Ser His Ser 85 90 95 Phe Pro His Pro Leu Tyr Asp Met Ser
Leu Leu Lys Asn Arg Phe Leu 100 105 110 Arg Pro Gly Asp Asp Ser Ser
His Asp Leu Met Leu Leu Arg Leu Ser 115 120 125 Glu Pro Ala Glu Leu
Thr Asp Ala Val Lys Val Met Asp Leu Pro Thr 130 135 140 Gln Glu Pro
Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser 145 150 155 160
Ile Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp 165
170 175 Leu His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln
Lys 180 185 190 Val Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly
Gly Lys Ser 195 200 205 Thr Cys Ser Gly Asp Ser Gly Gly Pro Leu Val
Cys Asn Gly Val Leu 210 215 220 Gln Gly Ile Thr Ser Trp Gly Ser Glu
Pro Cys Ala Leu Pro Glu Arg 225 230 235 240 Pro Ser Leu Tyr Thr Lys
Val Val His Tyr Arg Lys Trp Ile Lys Asp 245 250 255 Thr Ile Val Ala
Asn Pro 260 15 18 PRT Homo sapiens 15 Met Trp Asp Leu Val Leu Ser
Ile Ala Leu Ser Val Gly Cys Thr Gly 1 5 10 15 Ala Val 16 18 PRT
Homo sapiens 16 Cys Gln Ala Glu Leu Ser Pro Pro Thr Gln His Pro Ser
Pro Asp Arg 1 5 10 15 Glu Leu
* * * * *