U.S. patent application number 13/273808 was filed with the patent office on 2012-10-18 for compositions, kits, and methods for identification, assessment, prevention, and therapy of cervical cancer.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Yan Chen, Karen Glatt, Shubhangi Kamatkar, John E. Monahan, Xumei Zhao.
Application Number | 20120264625 13/273808 |
Document ID | / |
Family ID | 31946760 |
Filed Date | 2012-10-18 |
United States Patent
Application |
20120264625 |
Kind Code |
A1 |
Monahan; John E. ; et
al. |
October 18, 2012 |
COMPOSITIONS, KITS, AND METHODS FOR IDENTIFICATION, ASSESSMENT,
PREVENTION, AND THERAPY OF CERVICAL CANCER
Abstract
The invention relates to nucleic acid molecules and proteins
associated with cervical cancer including pre-malignant conditions
such as dysplasia. Compositions, kits, and methods for detecting,
characterizing, preventing, and treating human cervical cancers are
also provided.
Inventors: |
Monahan; John E.; (Walpole,
MA) ; Zhao; Xumei; (Wayland, MA) ; Chen;
Yan; (Acton, MA) ; Glatt; Karen; (Natick,
MA) ; Kamatkar; Shubhangi; (Newton, MA) |
Assignee: |
Millennium Pharmaceuticals,
Inc.
Cambridge
MA
|
Family ID: |
31946760 |
Appl. No.: |
13/273808 |
Filed: |
October 14, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12011459 |
Jan 24, 2008 |
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13273808 |
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10645756 |
Aug 20, 2003 |
7361511 |
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12011459 |
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60404770 |
Aug 20, 2002 |
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Current U.S.
Class: |
506/9 ; 435/6.11;
435/6.12; 435/6.14; 435/7.92; 436/501 |
Current CPC
Class: |
G01N 33/57488 20130101;
C12Q 2600/136 20130101; C12Q 2600/112 20130101; C07K 14/47
20130101; G01N 33/57411 20130101; G01N 33/5011 20130101; C12Q
2600/106 20130101; G01N 2800/56 20130101; A61P 35/00 20180101; G01N
2800/50 20130101; G01N 2800/52 20130101; C12Q 1/6886 20130101 |
Class at
Publication: |
506/9 ; 435/6.14;
435/7.92; 436/501; 435/6.12; 435/6.11 |
International
Class: |
G01N 33/574 20060101
G01N033/574; G01N 21/76 20060101 G01N021/76; G01N 21/64 20060101
G01N021/64; C12Q 1/68 20060101 C12Q001/68; C40B 30/04 20060101
C40B030/04 |
Claims
1. A method of assessing whether a patient is afflicted with
cervical cancer or has a pre-malignant condition, the method
comprising comparing: a) the level of expression of a M722 marker
in a patient sample, and b) the level of expression of the M722
marker in a normal control sample, wherein a difference between the
level of expression of the marker in the patient sample and the
normal control sample is an indication that the patient is
afflicted with cervical cancer or has a pre-malignant
condition.
2. The method of claim 1, wherein the patient has CIN or SIL.
3. The method of claim 1, wherein the sample comprises cells
obtained from the patient.
4. The method of claim 3, wherein the sample is a cervical
smear.
5. The method of claim 3, wherein the cells are in a fluid selected
from the group consisting of a fluid collected by peritoneal
rinsing, a fluid collected by uterine rinsing, a uterine fluid, a
uterine exudate, a pleural fluid, a cystic fluid, and an cervical
exudate.
6. The method of claim 1, wherein the level of expression of the
M722 marker in the sample is assessed by detecting the presence in
the sample of a protein corresponding to the marker.
7. The method of claim 6, wherein the presence of the protein is
detected using a reagent which specifically binds with the protein,
wherein the reagent is selected from the group consisting of an
antibody and an antigen binding fragment thereof.
8. The method of claim 1, wherein the level of expression of the
M722 marker in the sample is assessed by detecting the presence in
the sample of a transcribed polynucleotide or portion thereof,
wherein the transcribed polynucleotide comprises the M722
marker.
9. The method of claim 8, wherein the transcribed polynucleotide is
an mRNA.
10. The method of claim 8, wherein the transcribed polynucleotide
is a cDNA.
11. The method of claim 8, wherein the step of detecting further
comprises amplifying the transcribed polynucleotide.
12. The method of claim 1, wherein the level of expression of the
M722 marker in the sample differs from the level of expression of
the M722 marker in the control sample by a factor of at least about
2.
13. The method of claim 1, comprising comparing: a) the level of
expression in the sample of each of a plurality of markers
independently selected from M722 and at least one of the markers
listed in Table 1, and b) the level of expression of each of the
plurality of markers in a control sample, wherein a difference
between the level of expression of more than one of the markers in
the patient sample as compared to the control sample is an
indication that the patient is afflicted with cervical cancer or a
pre-malignant condition.
14. The method of claim 13, wherein the plurality comprises at
least three of the markers.
15. The method of claim 13, wherein the plurality comprises at
least five of the markers.
16. The method of claim 1, wherein the M722 marker is a polypeptide
encoded by a nucleic acid molecule comprising a nucleic acid
sequence at least 98% identical to SEQ ID NO:25.
17. The method of claim 1, wherein the M722 marker comprises the
nucleotide sequence of SEQ ID NO:25.
18. The method of claim 1, wherein the M722 marker is a polypeptide
comprising an amino acid sequence which is at least 95% identical
to SEQ ID NO:26.
19. The method of claim 1, wherein the M722 marker comprises the
amino acid sequence of SEQ ID NO:26.
20. The method of claim 1, wherein the level of expression of the
marker in the patient sample is assessed using a technique selected
from the group consisting of: Northern hybridization, polymerase
chain reaction analysis, RT-PCR, probe array and in situ
hybridization.
21. The method of claim 1, wherein the level of expression of the
marker in the patient sample is assessed using a technique selected
from the group consisting of: enzyme immunoassay, radioimmunoassay,
Western blot analysis, enzyme linked immuoabsorbant assay (ELISA),
immunoprecipitation and immunofluorescence.
Description
RELATED APPLICATION
[0001] The present application is a divisional of U.S. application
Ser. No. 12/011,459, filed on Jan. 24, 2008, which is a divisional
of U.S. application Ser. No. 10/645,756, filed on Aug. 20, 2003,
which claims priority to U.S. Provisional Application No.
60/404,770, filed on Aug. 20, 2002. The entire contents of each of
to the foregoing applications are expressly incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The field of the invention is cervical cancer, including
diagnosis, characterization, management, and therapy of cervical
cancer.
BACKGROUND OF THE INVENTION
[0003] The increased number of cancer cases reported in the United
States, and, indeed, around the world, is a major concern.
Currently there are only a handful of treatments available for
specific types of cancer, and these provide no absolute guarantee
of success. In order to be most effective, these treatments require
not only an early detection of the malignancy, but a reliable
assessment of the severity of the malignancy.
[0004] Cancer of the cervix is one of the most common malignancies
in women and remains a significant public health problem throughout
the world. In the United States alone, invasive cervical cancer
accounts for approximately 19% of all gynecological cancers. In
1996, it was estimated that there were 14,700 newly diagnosed cases
and 4900 deaths attributed to this disease (American Cancer
Society, Cancer Facts & Figures 1996, Atlanta, Ga.: American
Cancer Society, 1996). In many developing countries, where mass
screening programs are not widely available, the clinical problem
is more serious. Worldwide, the number of new cases is estimated to
be 471,000 with a four-year survival rate of only 40% (Munoz et
al., 1989, Epidemiology of Cervical Cancer In: "Human
Papillomavirus", New York, Oxford Press, pp 9-39; National
Institutes of Health, Consensus Development Conference Statement on
Cervical Cancer, Apr. 1-3, 1996).
[0005] In light of this, cervical cancer remains a highly
preventable form of cancer when pre-invasive lesions are detected
early. Cytological examination of Papanicolaou-stained cervical
smears (also referred to as Pap smears or Pap tests) is currently
the principle method for detecting cervical cancer and is the most
cost-effective cancer screening test developed to date (Greenberg,
M. D., et al., 1995, Clin Obstet Gynecol 38(3): 600-609). It has
dramatically decreased the incidence and mortality rates of
cervical cancer by more than 70% since it was introduced in the
United States and many other countries of the world (Eddy D. M.,
1990, Ann. Intern. Med. 113(3): 214-226). The abnormal morphologic
changes of Pap tests described by to The Bethesda System include
ASCUS (atypical squamous cells of undetermined significance), AGUS
(atypical glandular cells of undetermined significance), LSIL
(low-grade squamous intraepithelial lesion), HSIL (high-grade
squamous intraepithelial lesion), and squamous and adenocarcinoma
(National Cancer Institute Workshop: The 1988 Bethesda System for
reporting cervical/vaginal cytologic diagnosis. JAMA, 262(7):
931-934). The success of Pap tests is attributed mostly to the
diagnosis and treatment of precancerous lesions.
[0006] Currently, management of patients with HSIL and more
advanced diseases is relatively standard. Most women with such
lesions undergo colposcopy and appropriately directed biopsies. If
the histologic diagnosis is confirmed, ablative or excisional
treatment such as electrosurgical loop excision procedure (LEEP),
cryosurgery or conization is performed. However, management of
ambiguous or low-grade cytological results (ASCUS and LSIL) is very
controversial. This is mainly due to the nature of this
morphology-based test, which inevitably leads to interobserver
variability and some Pap test discordance with histological
follow-up. It was reported that the mean sensitivity of primary Pap
tests is approximately 58% and the accuracy of a repeat test is
only about 66% (Fahey M. T., et al., 1995, Am. J. Epidemiol. 141:
680-689). The low sensitivity and poor reproducibility have
complicated the management of ASCUS and LSIL patients. If an
"accelerated repeat Pap test" is recommended for the follow-up of
women with primary diagnosis of ASCUS or LSIL, patients will risk
delay in diagnosis of potential high-grade lesions. However, if
these patients are universally referred to colposcopy, the vast
majority of women will be over treated. Only 5-10% of women with
ASCUS have high-grade disease upon colposcopy, and more than 80% of
LSIL will regress to normal or stay in their current state (Cox, J.
T., 2000, Clinics in Laboratory Medicine. 20(2): 303-343, Ostor A.
G., 1993, Int. J. Gynecol. Pathol. 12(2): 186-192).
[0007] AGUS represents a much greater risk than ASCUS or LSIL
because cytology is less sensitive for this condition and the
disease progresses more rapidly (Anderson M. C., 1995, Baillieres
Clin. Obstet. Gynecol. 9:105). It was found that 9-54% of women
with AGUS have biopsy-confirmed cervical intraepithelial
neoplasias, 0-8% have biopsy-confirmed adenocarcinoma in situ
(AIS), and less than 1-9% have invasive carcinoma (Wright, T. C.,
et al., 2002, JAMA, 287(16): 2120-2129). Due to the greater risk,
all patients with AGUS are referred to colposcopy (Wright, T. C.,
et al., 2002).
[0008] The subjectivity of cervical cytology could be reduced by
objective markers that determine the presence and severity of
dysplastic cells. Since high-risk human papillomavirus (HPV)
infection is strongly associated with cervical cancer development
(Walboomers, J. M., et al., 1999, J. Pathol. 189: 12-19), HPV
testing using methods like Hybrid Capture II (Digene Diagnostics,
Silver Spring, Md.) or PCR appears to provide an objective
measurement (Wick, M. J., 2000, Clinics in Laboratory Medicine,
20(2): 271-287). However, since the vast majority of HPV infections
and the resulting squamous intraepithelial lesions regress
spontaneously, especially in young women, HPV testing cannot
specifically identify patients whose lesions will persist or
progress to invasive carcinoma (Sasieni, P. D., 2000, J. Am. Med.
Womens Assoc. 55(4): 216-219, Sasieni, P. D., 2000, Br. J. Cancer,
83(5): 561-565). As reported in the ASCUS-LSIL Triage Study (ALTS),
83% of woman with LSIL Pap results test positive for high-risk HPV
types, a level too high to be useful for triage (Human
papillomavirus testing for triage of women with cytologic evidence
of low-grade squamous intraepithelial lesions: baseline data from a
randomized trial. The Atypical Squamous Cells of Undetermined
Significance/Low-Grade Squamous Intraepithelial Lesions Triage
Study (ALTS) Group, 2000, J. Natl. Cancer Ist. 92:397-402).
Although triage using HPV testing significantly improved the
sensitivity for detecting HSIL in women with ASCUS Pap results, the
specificity was comparable to using conventional cytology (Solomon,
D., et al., 2001, J. Natl. Cancer Inst. 93(4): 293-299). A more
desirable cervical screening marker would identify all cervical
cancers, the majority of HSIL, and the small percentage of true
precancers amongst patients with LSIL and ASCUS on Pap.
[0009] It is now well accepted that cervical carcinogenesis occurs
in a step-wise fashion (Ried, T., et al., 1999, Genes Chromosomes
Cancer, 25(3): 195-204). The transition of normal epithelium to
preneoplastic lesions and invasive carcinoma occurs sequentially.
The morphologically defined steps of dysplastic and malignant
abnormalities are a reflection of cellular gene alterations during
tumorgenesis. It would thus be desirable to provide biomarkers
useful for the identification, assessment, prevention and therapy
of cervical cancer.
SUMMARY OF THE INVENTION
[0010] The invention relates to cancer markers (hereinafter
"markers" or "markers of the inventions"), which are listed in
Table 1. The invention provides nucleic acids and proteins that are
encoded by or correspond to the markers (hereinafter "marker
nucleic acids" and "marker proteins," respectively). Table 1
provides the sequence identifiers of the sequences of such marker
nucleic acids and proteins listed in the accompanying Sequence
Listing (SEQ ID NOs:1-44). Table 2 lists newly-identified
nucleotide and amino acid sequences. Table 3 lists newly-identified
nucleotide sequences. Tables 1-3 provide the sequence identifier
numbers of the sequences of such marker nucleic acids and proteins
listed in the accompanying Sequence Listing, and the gene names of
the markers. The invention further provides antibodies, antibody
derivatives and antibody fragments which bind specifically with
such proteins and/or fragments of the proteins.
[0011] The invention also relates to various methods, reagents and
kits for diagnosing, staging, prognosing, monitoring and treating
cervical cancer. "Cervical cancer" as used herein includes
carcinomas, (e.g., carcinoma in situ, invasive carcinoma,
metastatic carcinoma) and pre-malignant conditions, (e.g.,
dysplasia, including CIN or SIL). In one embodiment, the invention
provides a diagnostic method of assessing whether a patient has
cervical cancer or has higher than normal risk for developing
cervical cancer, comprising the steps of comparing the level of
expression of a marker of the invention in a patient sample and the
normal level of expression of the marker in a control, e.g., a
sample from a patient without cervical cancer. A significantly
higher level of expression of the marker in the patient sample as
compared to the normal level is an indication that the patient is
afflicted with cervical cancer or has higher than normal risk for
developing cervical cancer.
[0012] According to the invention, the markers are selected such
that the positive predictive value of the methods of the invention
is at least about 10%, preferably about 25%, more preferably about
50% and most preferably about 90%. Also preferred for use in the
methods of the invention are markers that are differentially
expressed, as compared to normal cervical cells, by at least
two-fold in at least about 20%, more preferably about 50% and most
preferably about 75% of any of the following conditions: stage 0
cervical cancer patients, stage I cervical cancer patients, stage
II cervical cancer patients, stage III cervical cancer patients,
stage 1V cervical cancer patients, grade I cervical cancer
patients, grade II cervical cancer patients, grade III cervical
cancer patients, squamous cell (epidermoid) cervical cancer
patients, cervical adenocarcinoma patients, cervical adenosquamous
carcinoma patients, small-cell cervical carcinoma patients,
malignant cervical cancer patients, patients with primary
carcinomas of the cervix, patients with primary malignant lymphomas
of the cervix and patients with secondary malignant lymphomas of
the cervix, and all other types of cancers, malignancies and
transformations associated with the cervix.
[0013] In one embodiment, the present invention provides a
diagnostic method of assessing whether a patient is afflicted with
cervical cancer (e.g., new detection ("screening"), detection of
recurrence, reflex testing), the method comprises comparing: [0014]
a) the level of expression of a marker of the invention in a
patient sample, and [0015] b) the normal level of expression of the
marker in a control non-cervical cancer sample. A significantly
higher level of expression of the marker in the patient sample as
compared to the normal level is an indication that the patient is
afflicted with cervical cancer.
[0016] In another embodiment, the invention provides a diagnostic
method of assessing whether a patient is afflicted with cervical
cancer (e.g., new detection ("screening"), detection of recurrence,
reflex testing), the method comprises comparing: [0017] a) the
level of expression of a marker set of the invention in a patient
sample, and [0018] b) the normal level of expression of the marker
set in a control non-cervical cancer sample. A significantly higher
level of expression of the marker set in the patient sample as
compared to the normal level is an indication that the patient is
afflicted with cervical cancer.
[0019] The invention also provides diagnostic methods for assessing
the efficacy of a therapy for inhibiting cervical cancer in a
patient. Such methods comprise comparing: [0020] a) expression of a
marker of the invention in a first sample obtained from the patient
prior to providing at least a portion of the therapy to the
patient, and [0021] b) expression of the marker in a second sample
obtained from the patient following provision of the portion of the
therapy. A significantly lower level of expression of the marker in
the second sample relative to that in the first sample is an
indication that the therapy is efficacious for inhibiting cervical
cancer in the patient.
[0022] It will be appreciated that in these methods the "therapy"
may be any therapy for treating cervical cancer including, but not
limited to, chemotherapy, radiation therapy, surgical removal of
tumor tissue, gene therapy and biologic therapy such as the
administering of antibodies and chemokines. Thus, the methods of
the invention may be used to evaluate a patient before, during and
after therapy, for example, to evaluate the reduction in tumor
burden.
[0023] In a preferred embodiment, the diagnostic methods are
directed to therapy using a chemical or biologic agent. These
methods comprise comparing: [0024] a) expression of a marker of the
invention in a first sample obtained from the patient and
maintained in the presence of the chemical or biologic agent, and
[0025] b) expression of the marker in a second sample obtained from
the patient and maintained in the absence of the agent. A
significantly lower level of expression of the marker in the second
sample relative to that in the first sample is an indication that
the agent is efficacious for inhibiting cervical cancer, in the
patient. In one embodiment, the first and second samples can be
portions of a single sample obtained from the patient or portions
of pooled samples obtained from the patient.
[0026] The invention additionally provides a monitoring method for
assessing the progression of cervical cancer in a patient, the
method comprising: [0027] a) detecting in a patient sample at a
first time point, the expression of a marker of the invention;
[0028] b) repeating step a) at a subsequent time point in time; and
[0029] c) comparing the level of expression detected in steps a)
and b), and therefrom monitoring the progression of cervical cancer
in the patient. A significantly higher level of expression of the
marker in the sample at the subsequent time point from that of the
sample at the first time point is an indication that the cervical
cancer has progressed, whereas a significantly lower level of
expression is an indication that the cervical cancer has
regressed.
[0030] The invention further provides a diagnostic method for
determining whether cervical cancer has metastasized or is likely
to metastasize in the future, the method comprising comparing:
[0031] a) the level of expression of a marker of the invention in a
patient sample, and [0032] b) the normal level (or non-metastatic
level) of expression of the marker in a control sample. A
significantly higher level of expression in the patient sample as
compared to the normal level (or non-metastatic level) is an
indication that the cervical cancer has metastasized or is likely
to metastasize in the future.
[0033] The invention moreover provides a test method for selecting
a composition for inhibiting cervical cancer in a patient. This
method comprises the steps of: [0034] a) obtaining a sample
comprising cancer cells from the patient; [0035] b) separately
maintaining aliquots of the sample in the presence of a plurality
of test compositions; [0036] c) comparing expression of a marker of
the invention in each of the aliquots; and [0037] d) selecting one
of the test compositions which significantly reduces the level of
expression of the marker in the aliquot containing that test
composition, relative to the levels of expression of the marker in
the presence of the other test compositions.
[0038] The invention additionally provides a test method of
assessing the cervical carcinogenic potential of a compound. This
method comprises the steps of: [0039] a) maintaining separate
aliquots of cervical cells in the presence and absence of the
compound; and [0040] b) comparing expression of a marker of the
invention in each of the aliquots. A significantly higher level of
expression of the marker in the aliquot maintained in the presence
of the compound, relative to that of the aliquot maintained in the
absence of the compound, is an indication that the compound
possesses cervical carcinogenic potential.
[0041] In addition, the invention further provides a method of
inhibiting cervical cancer in a patient. This method comprises the
steps of: [0042] a) obtaining a sample comprising cancer cells from
the patient; [0043] b) separately maintaining aliquots of the
sample in the presence of a plurality of compositions; [0044] c)
comparing expression of a marker of the invention in each of the
aliquots; and [0045] d) administering to the patient at least one
of the compositions which significantly lowers the level of
expression of the marker in the aliquot containing that
composition, relative to the levels of expression of the marker in
the presence of the other compositions.
[0046] In the aforementioned methods, the samples or patient
samples comprise cells obtained from the patient. The cells may be
found in a cervical smear collected, for example, by a cervical
brush. In another embodiment, the sample is a body fluid. Such
fluids include, for example, blood fluids, lymph, ascitic fluids,
gynecological fluids, urine, and fluids collected by vaginal
rinsing. In a further embodiment, the patient sample is in
vivo.
[0047] According to the invention, the level of expression of a
marker of the invention in a sample can be assessed, for example,
by detecting the presence in the sample of: [0048] the
corresponding marker protein (e.g., a protein having one of the
sequences set forth as "SEQ ID NO (AAs)" in Table 1, or a fragment
of the protein (e.g. by using a reagent, such as an antibody, an
antibody derivative, an antibody fragment or single-chain antibody,
which binds specifically with the protein or protein fragment)
[0049] the corresponding marker nucleic acid (e.g. a nucleotide
transcript having one of the nucleic acid sequences set forth as
"SEQ ID NO (nts)" in Table 1, or a complement thereof), or a
fragment of the nucleic acid (e.g. by contacting transcribed
polynucleotides obtained from the sample with a substrate having
affixed thereto one or more nucleic acids having the entire or a
segment of the nucleic acid sequence of any of the SEQ ID NO (nts),
or a complement thereof) [0050] a metabolite which is produced
directly (i.e., catalyzed) or indirectly by the corresponding
marker protein.
[0051] According to the invention, any of the aforementioned
methods may be performed using a plurality (e.g. 2, 3, 5, or 10 or
more) of cervical cancer markers, including cervical cancer markers
known in the art. In such methods, the level of expression in the
sample of each of a plurality of markers, at least one of which is
a marker of the invention, is compared with the normal level of
expression of each of the plurality of markers in samples of the
same type obtained from control humans not afflicted with cervical
cancer. A significantly altered (i.e., increased or decreased as
specified in the above-described methods using a single marker)
level of expression in the sample of one or more markers of the
invention, or some combination thereof, relative to that marker's
corresponding normal or control level, is an indication that the
patient is afflicted with cervical cancer. For all of the
aforementioned methods, the marker(s) are preferably selected such
that the positive predictive value of the method is at least about
10%.
[0052] In a further aspect, the invention provides an antibody, an
antibody derivative, or an antibody fragment, which binds
specifically with a marker protein (e.g., a protein having one of
the amino acid sequences set forth in the Sequence Listing) or a
fragment of the protein. The invention also provides methods for
making such antibody, antibody derivative, and antibody fragment.
Such methods may comprise immunizing a mammal with a protein or
peptide comprising the entirety, or a segment of 10 or more amino
acids, of a marker protein (e.g., a protein having one of the amino
acid sequences set forth in the Sequence Listing), wherein the
protein or peptide may be obtained from a cell or by chemical
synthesis. The methods of the invention also encompass producing
monoclonal and single-chain antibodies, which would further
comprise isolating splenocytes from the immunized mammal, fusing
the isolated splenocytes with an immortalized cell line to form
hybridomas, and screening individual hybridomas for those that
produce an antibody that binds specifically with a marker protein
or a fragment of the protein.
[0053] In another aspect, the invention relates to various
diagnostic and test kits. In one embodiment, the invention provides
a kit for assessing whether a patient is afflicted with cervical
cancer. The kit comprises a reagent for assessing expression of a
marker of the invention. In another embodiment, the invention
provides a kit for assessing the suitability of a chemical or
biologic agent for inhibiting cervical cancer in a patient. Such a
kit comprises a reagent for assessing expression of a marker of the
invention, and may also comprise one or more of such agents. In a
further embodiment, the invention provides kits for assessing the
presence of cervical cancer cells or treating cervical cancers.
Such kits comprise an antibody, an antibody derivative, or an
antibody fragment, which binds specifically with a marker protein,
or a fragment of the protein. Such kits may also comprise a
plurality of antibodies, antibody derivatives, or antibody
fragments wherein the plurality of such antibody agents binds
specifically with a marker protein, or a fragment of the
protein.
[0054] In an additional embodiment, the invention also provides a
kit for assessing the presence of cervical cancer cells, wherein
the kit comprises a nucleic acid probe that binds specifically with
a marker nucleic acid or a fragment of the nucleic acid. The kit
may also comprise a plurality of probes, wherein each of the probes
binds specifically with a marker nucleic acid, or a fragment of the
nucleic acid.
[0055] In a further aspect, the invention relates to methods for
treating a patient afflicted with cervical cancer or at risk of
developing cervical cancer. Such methods may comprise reducing the
expression and/or interfering with the biological function of a
marker of the invention. In one embodiment, the method comprises
providing to the patient an antisense oligonucleotide or
polynucleotide complementary to a marker nucleic acid, or a segment
thereof. For example, an antisense polynucleotide may be provided
to the patient through the delivery of a vector that expresses an
anti-sense polynucleotide of a marker nucleic acid or a fragment
thereof. In another embodiment, the method comprises providing to
the patient an antibody, an antibody derivative, or antibody
fragment, which binds specifically with a marker protein or a
fragment of the protein. In a preferred embodiment, the antibody,
antibody derivative or antibody fragment binds specifically with a
protein having one of the amino acid sequences set forth in the
Sequence Listing, or a fragment of the protein.
[0056] It will be appreciated that the methods and kits of the
present invention may also include known cancer markers including
known cervical cancer markers. It will further be appreciated that
the methods and kits may be used to identify cancers other than
cervical cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 depicts a cluster diagram of cervical tissue samples.
Dendrogram was created from hierarchical clustering of the
transcriptional profiles of 34 normal, LSIL, HSIL and cancerous
cervical tissue samples. Each sample was labeled by its tissue type
and an Id number. The abbreviations in FIG. 1 are defined as
follows: N.sub.ecto: normal ectocervix; N.sub.endo: normal
endocervix; LSIL: low-grade squamous intraepithelial lesion; HSIL:
high-grade squamous intraepithelial lesion; T.sub.scc: squamous
cell carcinoma; T.sub.aca: adenocarcinoma. The dashed line divides
the 34 samples into two major groups: control group and diseased
group. Filled circles indicate incorrectly clustered samples.
[0058] FIG. 2 depicts transcriptional profiles (TP) of MCM6 and
Claudin 1 in normal, dysplastic and cancerous cervical tissues by
cDNA microarray hybridization. Each data point represents the
average of duplicate microarray hybridizations. The TP intensity
was normalized by the median intensity of all spots on the array.
The abbreviations in FIG. 2 are defined as follows: Endo: normal
endocervical tissue; Ecto: normal ectocervical tissue; LSIL:
low-grade squamous intraepithelial lesion; HSIL: high-grade
squamous intraepithelial lesion; SCC: squamous cell carcinoma; ACA:
adenocarcinoma.
DETAILED DESCRIPTION OF THE INVENTION
[0059] The invention relates to newly discovered cancer markers set
forth in Table 1, associated with the cancerous state of cervical
cells. It has been discovered that the higher than normal level of
expression of any of these markers or combination of these markers
correlates with the presence of cervical cancer including
pre-malignant conditions such as dysplasia, in a patient. Methods
are provided for detecting the presence of cervical cancer in a
sample, the absence of cervical cancer in a sample, the stage of a
cervical cancer, and other characteristics of cervical cancer that
are relevant to prevention, diagnosis, characterization, and
therapy of cervical cancer in a patient. Methods of treating
cervical cancer are also provided.
[0060] Table 1 lists the markers of the invention, which are
over-expressed in cervical cancer cells compared to normal (i.e.,
non-cancerous) cervical cells and comprises markers listed in
Tables 2-13. Table 1 provides the sequence listing identifiers of
the cDNA sequence of a nucleotide transcript and the amino acid
sequence of a protein encoded by or corresponding to each marker,
as well as the location of the protein coding sequence within the
cDNA sequence. Table 2 lists newly-identified nucleotide and amino
acid sequences. Table 3 lists newly-identified nucleotide
sequences. Table 4 identifies markers of the present invention
which were selected by transcription profiling experiments and
their marker scores in SCC, ACA and HSIL. Table 5 identifies
markers of the present invention that are overexpressed in cervical
cancer by in situ hybridization and indicates the location of
marker expression. Table 6 identifies markers of the present
invention and the frequency of their expression using a cervical
tissue microarray. Table 7 identifies gene specific primers. Table
8 sets forth the scoring on a scale of 0-5 of ethidium bromide
agarose gel pictures of the end-point PCR on the tissue panel.
Tables 9-13 set forth expression of the target gene in each of the
tissues tested.
DEFINITIONS
[0061] As used herein, each of the following terms has the meaning
associated with it in this section.
[0062] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0063] A "marker" is a gene whose altered level of expression in a
tissue or cell from its expression level in normal or healthy
tissue or cell is associated with a disease state, such as cancer.
A "marker nucleic acid" is a nucleic acid (e.g., mRNA, cDNA)
encoded by or corresponding to a marker of the invention. Such
marker nucleic acids include DNA (e.g., cDNA) comprising the entire
or a partial sequence of any of the nucleic acid sequences set
forth in the Sequence Listing or the complement of such a sequence.
The marker nucleic acids also include RNA comprising the entire or
a partial sequence of any of the nucleic acid sequences set forth
in the Sequence Listing or the complement of such a sequence,
wherein all thymidine residues are replaced with uridine residues.
A "marker protein" is a protein encoded by or corresponding to a
marker of the invention. A marker protein comprises the entire or a
partial sequence of any of the sequences set forth in the Sequence
Listing. The terms "protein" and "polypeptide` are used
interchangeably.
[0064] A "marker set" is a group of more than one marker.
[0065] The term "probe" refers to any molecule which is capable of
selectively binding to a specifically intended target molecule, for
example, a nucleotide transcript or protein encoded by or
corresponding to a marker. Probes can be either synthesized by one
skilled in the art, or derived from appropriate biological
preparations. For purposes of detection of the target molecule,
probes may be specifically designed to be labeled, as described
herein. Examples of molecules that can be utilized as probes
include, but are not limited to, RNA, DNA, proteins, antibodies,
and organic molecules.
[0066] A "cervical-associated" body fluid is a fluid which, when in
the body of a patient, contacts or passes through cervical cells or
into which cells or proteins shed from cervical cells are capable
of passing. The cells may be found in a cervical smear collected,
for example, by a cervical brush. Exemplary cervical-associated
body fluids include blood fluids, lymph, ascitic fluids,
gynecological fluids, cystic fluid, urine, and fluids collected by
vaginal rinsing.
[0067] The "normal" level of expression of a marker is the level of
expression of the marker in cervical cells of a human subject or
patient not afflicted with cervical cancer.
[0068] An "over-expression" or "significantly higher level of
expression" of a marker refers to an expression level in a test
sample that is greater than the standard error of the assay
employed to assess expression, and is preferably at least twice,
and more preferably three, four, five or ten times the expression
level of the marker in a control sample (e.g., sample from a
healthy subjects not having the marker associated disease) and
preferably, the average expression level of the marker in several
control samples.
[0069] A "significantly lower level of expression" of a marker
refers to an expression level in a test sample that is at least
twice, and more preferably three, four, five or ten times lower
than the expression level of the marker in a control sample (e.g.,
sample from a healthy subject not having the marker associated
disease) and preferably, the average expression level of the marker
in several control samples.
[0070] As used herein, the term "promoter/regulatory sequence"
means a nucleic acid sequence which is required for expression of a
gene product operably linked to the promoter/regulatory sequence.
In some instances, this sequence may be the core promoter sequence
and in other instances, this sequence may also include an enhancer
sequence and other regulatory elements which are required for
expression of the gene product. The promoter/regulatory sequence
may, for example, be one which expresses the gene product in a
tissue-specific manner.
[0071] A "constitutive" promoter is a nucleotide sequence which,
when operably linked with a polynucleotide which encodes or
specifies a gene product, causes the gene product to be produced in
a living human cell under most or all physiological conditions of
the cell.
[0072] An "inducible" promoter is a nucleotide sequence which, when
operably linked with a polynucleotide which encodes or specifies a
gene product, causes the gene product to be produced in a living
human cell substantially only when an inducer which corresponds to
the promoter is present in the cell.
[0073] A "tissue-specific" promoter is a nucleotide sequence which,
when operably linked with a polynucleotide which encodes or
specifies a gene product, causes the gene product to be produced in
a living human cell substantially only if the cell is a cell of the
tissue type corresponding to the promoter.
[0074] A "transcribed polynucleotide" or "nucleotide transcript" is
a polynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such
RNA or cDNA) which is complementary to or homologous with all or a
portion of a mature mRNA made by transcription of a marker of the
invention and normal post-transcriptional processing (e.g.
splicing), if any, of the RNA transcript, and reverse transcription
of the RNA transcript.
[0075] "Complementary" refers to the broad concept of sequence
complementarity between regions of two nucleic acid strands or
between two regions of the same nucleic acid strand. It is known
that an adenine residue of a first nucleic acid region is capable
of forming specific hydrogen bonds ("base pairing") with a residue
of a second nucleic acid region which is antiparallel to the first
region if the residue is thymine or uracil. Similarly, it is known
that a cytosine residue of a first nucleic acid strand is capable
of base pairing with a residue of a second nucleic acid strand
which is antiparallel to the first strand if the residue is
guanine. A first region of a nucleic acid is complementary to a
second region of the same or a different nucleic acid if, when the
two regions are arranged in an antiparallel fashion, at least one
nucleotide residue of the first region is capable of base pairing
with a residue of the second region. Preferably, the first region
comprises a first portion and the second region comprises a second
portion, whereby, when the first and second portions are arranged
in an antiparallel fashion, at least about 50%, and preferably at
least about 75%, at least about 90%, or at least about 95% of the
nucleotide residues of the first portion are capable of base
pairing with nucleotide residues in the second portion. More
preferably, all nucleotide residues of the first portion are
capable of base pairing with nucleotide residues in the second
portion.
[0076] "Homologous" as used herein, refers to nucleotide sequence
similarity between two regions of the same nucleic acid strand or
between regions of two different nucleic acid strands. When a
nucleotide residue position in both regions is occupied by the same
nucleotide residue, then the regions are homologous at that
position. A first region is homologous to a second region if at
least one nucleotide residue position of each region is occupied by
the same residue. Homology between two regions is expressed in
terms of the proportion of nucleotide residue positions of the two
regions that are occupied by the same nucleotide residue. By way of
example, a region having the nucleotide sequence 5'-ATTGCC-3' and a
region having the nucleotide sequence 5'-TATGGC-3' share 50%
homology. Preferably, the first region comprises a first portion
and the second region comprises a second portion, whereby, at least
about 50%, and preferably at least about 75%, at least about 90%,
or at least about 95% of the nucleotide residue positions of each
of the portions are occupied by the same nucleotide residue. More
preferably, all nucleotide residue positions of each of the
portions are occupied by the same nucleotide residue.
[0077] A molecule is "fixed" or "affixed" to a substrate if it is
covalently or non-covalently associated with the substrate such the
substrate can be rinsed with a fluid (e.g. standard saline citrate,
pH 7.4) without a substantial fraction of the molecule dissociating
from the substrate.
[0078] As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in an organism found in nature.
[0079] A cancer is "inhibited" if at least one symptom of the
cancer is alleviated, terminated, slowed, or prevented. As used
herein, cervical cancer is also "inhibited" if recurrence or
metastasis of the cancer is reduced, slowed, delayed, or
prevented.
[0080] A kit is any manufacture (e.g. a package or container)
comprising at least one reagent, e.g. a probe, for specifically
detecting the expression of a marker of the invention. The kit may
be promoted, distributed, or sold as a unit for performing the
methods of the present invention.
[0081] "Proteins of the invention" encompass marker proteins and
their fragments; variant marker proteins and their fragments;
peptides and polypeptides comprising an at least 15 amino acid
segment of a marker or variant marker protein; and fusion proteins
comprising a marker or variant marker protein, or an at least 15
amino acid segment of a marker or variant marker protein.
[0082] Unless otherwise specified herewithin, the terms "antibody"
and "antibodies" broadly encompass naturally-occurring forms of
antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies
such as single-chain antibodies, chimeric and humanized antibodies
and multi-specific antibodies, as well as fragments and derivatives
of all of the foregoing, which fragments and derivatives have at
least an antigenic binding site. Antibody derivatives may comprise
a protein or chemical moiety conjugated to an antibody.
DESCRIPTION
[0083] The present invention is based, in part, on newly identified
markers which are over-expressed in cervical cancer cells as
compared to their expression in normal (i.e. non-cancerous)
cervical cells. The enhanced expression of one or more of these
markers in cervical cells is herein correlated with the cancerous
state of the tissue. The invention provides compositions, kits, and
methods for assessing the cancerous state of cervical cells (e.g.
cells obtained from a human, cultured human cells, archived or
preserved human cells and in vivo cells) as well as treating
patients afflicted with cervical cancer.
[0084] The compositions, kits, and methods of the invention have
the following uses, among others: [0085] 1) assessing whether a
patient is afflicted with cervical cancer; [0086] 2) assessing the
stage of cervical cancer in a human patient; [0087] 3) assessing
the grade of cervical cancer in a patient; [0088] 4) assessing the
benign or malignant nature of cervical cancer in a patient; [0089]
5) assessing the metastatic potential of cervical cancer in a
patient; [0090] 6) assessing the histological type of neoplasm
associated with cervical cancer in a patient; [0091] 7) making
antibodies, antibody fragments or antibody derivatives that are
useful for treating cervical cancer and/or assessing whether a
patient is afflicted with cervical cancer; [0092] 8) assessing the
presence of cervical cancer cells; [0093] 9) assessing the efficacy
of one or more test compounds for inhibiting cervical cancer in a
patient; [0094] 10) assessing the efficacy of a therapy for
inhibiting cervical cancer in a patient; [0095] 11) monitoring the
progression of cervical cancer in a patient; [0096] 12) selecting a
composition or therapy for inhibiting cervical cancer in a patient;
[0097] 13) treating a patient afflicted with cervical cancer;
[0098] 14) inhibiting cervical cancer in a patient; [0099] 15)
assessing the cervical carcinogenic potential of a test compound;
and [0100] 16) preventing the onset of cervical cancer in a patient
at risk for developing cervical cancer.
[0101] The invention thus includes a method of assessing whether a
patient is afflicted with cervical cancer which includes assessing
whether the patient has pre-metastasized cervical cancer. This
method comprises comparing the level of expression of a marker of
the invention (listed in Table 1) in a patient sample and the
normal level of expression of the marker in a control, e.g., a
non-cervical cancer sample. A significantly higher level of
expression of the marker in the patient sample as compared to the
normal level is an indication that the patient is afflicted with
cervical cancer.
[0102] Gene delivery vehicles, host cells and compositions (all
described herein) containing nucleic acids comprising the entirety,
or a segment of 15 or more nucleotides, of any of the nucleic acid
sequences set forth in the Sequence Listing, or the complement of
such sequences, and polypeptides comprising the entirety, or a
segment of 10 or more amino acids, of any of the amino acid
sequences set forth in the Sequence Listing, are also provided by
this invention.
[0103] As described herein, cervical cancer in patients is
associated with an increased level of expression of one or more
markers of the invention. While, as discussed above, some of these
changes in expression level result from occurrence of the cervical
cancer, others of these changes induce, maintain, and promote the
cancerous state of cervical cancer cells. Thus, cervical cancer
characterized by an increase in the level of expression of one or
more markers of the invention can be inhibited by reducing and/or
interfering with the expression of the markers and/or function of
the proteins encoded by those markers.
[0104] Expression of a marker of the invention can be inhibited in
a number of ways generally known in the art. For example, an
antisense oligonucleotide can be provided to the cervical cancer
cells in order to inhibit transcription, translation, or both, of
the marker(s). Alternately, a polynucleotide encoding an antibody,
an antibody derivative, or an antibody fragment which specifically
binds a marker protein, and operably linked with an appropriate
promoter/regulator region, can be provided to the cell in order to
generate intracellular antibodies which will inhibit the function
or activity of the protein. The expression and/or function of a
marker may also be inhibited by treating the cervical cancer cell
with an antibody, antibody derivative or antibody fragment that
specifically binds a marker protein. Using the methods described
herein, a variety of molecules, particularly including molecules
sufficiently small that they are able to cross the cell membrane,
can be screened in order to identify molecules which inhibit
expression of a marker or inhibit the function of a marker protein.
The compound so identified can be provided to the patient in order
to inhibit cervical cancer cells of the patient.
[0105] Any marker or combination of markers of the invention, as
well as any known markers in combination with the markers of the
invention, may be used in the compositions, kits, and methods of
the present invention. In general, it is preferable to use markers
for which the difference between the level of expression of the
marker in cervical cancer cells and the level of expression of the
same marker in normal cervical cells is as great as possible.
Although this difference can be as small as the limit of detection
of the method for assessing expression of the marker, it is
preferred that the difference be at least greater than the standard
error of the assessment method, and preferably a difference of at
least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 100-,
500-, 1000-fold or greater than the level of expression of the same
marker in normal cervical tissue.
[0106] It is recognized that certain marker proteins are secreted
from cervical cells (i.e. one or both of normal and cancerous
cells) to the extracellular space surrounding the cells. These
markers are preferably used in certain embodiments of the
compositions, kits, and methods of the invention, owing to the fact
that the such marker proteins can be detected in a
cervical-associated body fluid sample, which may be more easily
collected from a human patient than a tissue biopsy sample. In
addition, preferred in vivo techniques for detection of a marker
protein include introducing into a subject a labeled antibody
directed against the protein. For example, the antibody can be
labeled with a radioactive marker whose presence and location in a
subject can be detected by standard imaging techniques.
[0107] It is a simple matter for the skilled artisan to determine
whether any particular marker protein is a secreted protein. In
order to make this determination, the marker protein is expressed
in, for example, a mammalian cell, preferably a human cervical cell
line, extracellular fluid is collected, and the presence or absence
of the protein in the extracellular fluid is assessed (e.g. using a
labeled antibody which binds specifically with the protein).
[0108] The following is an example of a method which can be used to
detect secretion of a protein. About 8.times.10.sup.5 293T cells
are incubated at 37.degree. C. in wells containing growth medium
(Dulbecco's modified Eagle's medium {DMEM} supplemented with 10%
fetal bovine serum) under a 5% (v/v) CO.sub.2, 95% air atmosphere
to about 60-70% confluence. The cells are then transfected using a
standard transfection mixture comprising 2 micrograms of DNA
comprising an expression vector encoding the protein and 10
microliters of LipofectAMINE.TM. (GIBCO/BRL Catalog no. 18342-012)
per well. The transfection mixture is maintained for about 5 hours,
and then replaced with fresh growth medium and maintained in an air
atmosphere. Each well is gently rinsed twice with DMEM which does
not contain methionine or cysteine (DMEM-MC; ICN Catalog no.
16-424-54). About 1 milliliter of DMEM-MC and about 50 microcuries
of Trans-.sup.35S.TM. reagent (ICN Catalog no. 51006) are added to
each well. The wells are maintained under the 5% CO.sub.2
atmosphere described above and incubated at 37.degree. C. for a
selected period. Following incubation, 150 microliters of
conditioned medium is removed and centrifuged to remove floating
cells and debris. The presence of the protein in the supernatant is
an indication that the protein is secreted.
[0109] It will be appreciated that patient samples containing
cervical cells may be used in the methods of the present invention.
In these embodiments, the level of expression of the marker can be
assessed by assessing the amount (e.g. absolute amount or
concentration) of the marker in a cervical cell sample, e.g.,
cervical smear obtained from a patient. The cell sample can, of
course, be subjected to a variety of well-known post-collection
preparative and storage techniques (e.g., nucleic acid and/or
protein extraction, fixation, storage, freezing, ultrafiltration,
concentration, evaporation, centrifugation, etc.) prior to
assessing the amount of the marker in the sample. Likewise,
cervical smears may also be subjected to post-collection
preparative and storage techniques, e.g., fixation.
[0110] The compositions, kits, and methods of the invention can be
used to detect expression of marker proteins having at least one
portion which is displayed on the surface of cells which express
it. It is a simple matter for the skilled artisan to determine
whether a marker protein, or a portion thereof, is exposed on the
cell surface. For example, immunological methods may be used to
detect such proteins on whole cells, or well known computer-based
sequence analysis methods may be used to predict the presence of at
least one extracellular domain (i.e. including both secreted
proteins and proteins having at least one cell-surface domain).
Expression of a marker protein having at least one portion which is
displayed on the surface of a cell which expresses it may be
detected without necessarily lysing the cell (e.g. using a labeled
antibody which binds specifically with a cell-surface domain of the
protein).
[0111] Expression of a marker of the invention may be assessed by
any of a wide variety of well known methods for detecting
expression of a transcribed nucleic acid or protein. Non-limiting
examples of such methods include immunological methods for
detection of secreted, cell-surface, cytoplasmic, or nuclear
proteins, protein purification methods, protein function or
activity assays, nucleic acid hybridization methods, nucleic acid
reverse transcription methods, and nucleic acid amplification
methods.
[0112] In a preferred embodiment, expression of a marker is
assessed using an antibody (e.g. a radio-labeled,
chromophore-labeled, fluorophore-labeled, or enzyme-labeled
antibody), an antibody derivative (e.g. an antibody conjugated with
a substrate or with the protein or ligand of a protein-ligand pair
{e.g. biotin-streptavidin}), or an antibody fragment (e.g. a
single-chain antibody, an isolated antibody hypervariable domain,
etc.) which binds specifically with a marker protein or fragment
thereof, including a marker protein which has undergone all or a
portion of its normal post-translational modification.
[0113] In another preferred embodiment, expression of a marker is
assessed by preparing mRNA/cDNA (i.e. a transcribed polynucleotide)
from cells in a patient sample, and by hybridizing the mRNA/cDNA
with a reference polynucleotide which is a complement of a marker
nucleic acid, or a fragment thereof. cDNA can, optionally, be
amplified using any of a variety of polymerase chain reaction
methods prior to hybridization with the reference polynucleotide;
preferably, it is not amplified. Expression of one or more markers
can likewise be detected using quantitative PCR to assess the level
of expression of the marker(s). Alternatively, any of the many
known methods of detecting mutations or variants (e.g. single
nucleotide polymorphisms, deletions, etc.) of a marker of the
invention may be used to detect occurrence of a marker in a
patient.
[0114] In a related embodiment, a mixture of transcribed
polynucleotides obtained from the sample is contacted with a
substrate having fixed thereto a polynucleotide complementary to or
homologous with at least a portion (e.g. at least 7, 10, 15, 20,
25, 30, 40, 50, 100, 500, or more nucleotide residues) of a marker
nucleic acid. If polynucleotides complementary to or homologous
with are differentially detectable on the substrate (e.g.
detectable using different chromophores or fluorophores, or fixed
to different selected positions), then the levels of expression of
a plurality of markers can be assessed simultaneously using a
single substrate (e.g. a "gene chip" microarray of polynucleotides
fixed at selected positions). When a method of assessing marker
expression is used which involves hybridization of one nucleic acid
with another, it is preferred that the hybridization be performed
under stringent hybridization conditions.
[0115] Because the compositions, kits, and methods of the invention
rely on detection of a difference in expression levels of one or
more markers of the invention, it is preferable that the level of
expression of the marker is significantly greater than the minimum
detection limit of the method used to assess expression in at least
one of normal cervical cells and cancerous cervical cells.
[0116] It is understood that by routine screening of additional
patient samples using one or more of the markers of the invention,
it will be realized that certain of the markers are over-expressed
in cancers of various types, including specific cervical cancers,
as well as other cancers such as breast cancer, ovarian cancer,
etc. For example, it will be confirmed that some of the markers of
the invention are over-expressed in most (i.e. 50% or more) or
substantially all (i.e. 80% or more) of cervical cancer.
Furthermore, it will be confirmed that certain of the markers of
the invention are associated with cervical cancer of various stages
(i.e. stage 0, I, II, III, and IV cervical cancers, as well as
subclassifications IA1, IA2, IB, IB1, IB2, IIA, IIB, IIIA, IIIB,
IVA, and IVB, using the FIGO Stage Grouping system for primary
carcinoma of the cervix (see Gynecologic Oncology, 1991, 41:199 and
Cancer, 1992, 69:482)), and pre-malignant conditions (e.g.,
dysplasia including CIN or SIL), of various histologic subtypes
(e.g. squamous cell carcinomas and squamous cell carcinoma variants
such as verrucous carcinoma, lymphoepithelioma-like carcinoma,
papillary squamous neoplasm and spindle cell squamous cell
carcinoma (see Cervical Cancer and Preinvasive Neoplasia, 1996, pp.
90-91) serous, mucinous, endometrioid, and clear cell subtypes, as
well as subclassifications and alternate classifications
adenocarcinoma, papillary adenocarcinoma, papillary
cystadenocarcinoma, surface papillary carcinoma, malignant
adenofibroma, cystadenofibroma, adenocarcinoma, cystadenocarcinoma,
adenoacanthoma, endometrioid stromal sarcoma, mesodermal
{Mullerian} mixed tumor, malignant carcinoma, mixed epithelial
tumor, and undifferentiated carcinoma, using the WHO/FIGO system
for classification of malignant cervical tumors; Scully, Atlas of
Tumor Pathology, 3d series, Washington D.C.), and various grades
(i.e. grade I {well differentiated}, grade II {moderately well
differentiated}, and grade III {poorly differentiated from
surrounding normal tissue}). In addition, as a greater number of
patient samples are assessed for expression of the markers of the
invention and the outcomes of the individual patients from whom the
samples were obtained are correlated, it will also be confirmed
that altered expression of certain of the markers of the invention
are strongly correlated with malignant cancers and that altered
expression of other markers of the invention are strongly
correlated with benign tumors. The compositions, kits, and methods
of the invention are thus useful for characterizing one or more of
the stage, grade, histological type, and benign/malignant nature of
cervical cancer in patients.
[0117] When the compositions, kits, and methods of the invention
are used for characterizing one or more of the stage, grade,
histological type, and benign/malignant nature of cervical cancer
in a patient, it is preferred that the marker or panel of markers
of the invention is selected such that a positive result is
obtained in at least about 20%, and preferably at least about 40%,
60%, or 80%, and more preferably in substantially all patients
afflicted with a cervical cancer of the corresponding stage, grade,
histological type, or benign/malignant nature. Preferably, the
marker or panel of markers of the invention is selected such that a
positive predictive value (PPV) of greater than about 10% is
obtained for the general population (more preferably coupled with
an assay specificity greater than 80%).
[0118] When a plurality of markers of the invention are used in the
compositions, kits, and methods of the invention, the level of
expression of each marker in a patient sample can be compared with
the normal level of expression of each of the plurality of markers
in non-cancerous samples of the same type, either in a single
reaction mixture (i.e. using reagents, such as different
fluorescent probes, for each marker) or in individual reaction
mixtures corresponding to one or more of the markers. In one
embodiment, a significantly increased level of expression of more
than one of the plurality of markers in the sample, relative to the
corresponding normal levels, is an indication that the patient is
afflicted with cervical cancer. When a plurality of markers is
used, it is preferred that 2, 3, 4, 5, 8, 10, 12, 15, 20, 30, or 50
or more individual markers be used, wherein fewer markers are
preferred.
[0119] In order to maximize the sensitivity of the compositions,
kits, and methods of the invention (i.e. by interference
attributable to cells of non-cervical origin in a patient sample),
it is preferable that the marker of the invention used therein be a
marker which has a restricted tissue distribution, e.g., normally
not expressed in a non-cervical tissue.
[0120] Only a small number of markers are known to be associated
with cervical cancer (e.g. bcl-2, 15A8 antigen, cdc6, Mcm5, and
EGFR). These markers are not, of course, included among the markers
of the invention, although they may be used together with one or
more markers of the invention in a panel of markers, for example.
It is well known that certain types of genes, such as oncogenes,
tumor suppressor genes, growth factor-like genes, protease-like
genes, and protein kinase-like genes are often involved with
development of cancers of various types. Thus, among the markers of
the invention, use of those which correspond to proteins which
resemble known proteins encoded by known oncogenes and tumor
suppressor genes, and those which correspond to proteins which
resemble growth factors, proteases, and protein kinases are
preferred.
[0121] It is recognized that the compositions, kits, and methods of
the invention will be of particular utility to patients having an
enhanced risk of developing cervical cancer and their medical
advisors. Patients recognized as having an enhanced risk of
developing cervical cancer include, for example, patients having a
familial history of cervical cancer, patients identified as having
a mutant oncogene (i.e. at least one allele), and patients of
advancing age (i.e. women older than about 50 or 60 years).
[0122] The level of expression of a marker in normal (i.e.
non-cancerous) human cervical tissue can be assessed in a variety
of ways. In one embodiment, this normal level of expression is
assessed by assessing the level of expression of the marker in a
portion of cervical cells which appears to be non-cancerous and by
comparing this normal level of expression with the level of
expression in a portion of the cervical cells which is suspected of
being cancerous. Alternately, and particularly as further
information becomes available as a result of routine performance of
the methods described herein, population-average values for normal
expression of the markers of the invention may be used. In other
embodiments, the `normal` level of expression of a marker may be
determined by assessing expression of the marker in a patient
sample obtained from a non-cancer-afflicted patient, from a patient
sample obtained from a patient before the suspected onset of
cervical cancer in the patient, from archived patient samples, and
the like.
[0123] The invention includes compositions, kits, and methods for
assessing the presence of cervical cancer cells in a sample (e.g.
an archived tissue sample or a sample obtained from a patient).
These compositions, kits, and methods are substantially the same as
those described above, except that, where necessary, the
compositions, kits, and methods are adapted for use with samples
other than patient samples. For example, when the sample to be used
is a parafinized, archived human tissue sample, it can be necessary
to adjust the ratio of compounds in the compositions of the
invention, in the kits of the invention, or the methods used to
assess levels of marker expression in the sample. Such methods are
well known in the art and within the skill of the ordinary
artisan.
[0124] The invention includes a kit for assessing the presence of
cervical cancer cells (e.g. in a sample such as a patient sample).
The kit comprises a plurality of reagents, each of which is capable
of binding specifically with a marker nucleic acid or protein.
Suitable reagents for binding with a marker protein include
antibodies, antibody derivatives, antibody fragments, and the like.
Suitable reagents for binding with a marker nucleic acid (e.g. a
genomic DNA, an mRNA, a spliced mRNA, a cDNA, or the like) include
complementary nucleic acids. For example, the nucleic acid reagents
may include oligonucleotides (labeled or non-labeled) fixed to a
substrate, labeled oligonucleotides not bound with a substrate,
pairs of PCR primers, molecular beacon probes, and the like.
[0125] The kit of the invention may optionally comprise additional
components useful for performing the methods of the invention. By
way of example, the kit may comprise fluids (e.g. SSC buffer)
suitable for annealing complementary nucleic acids or for binding
an antibody with a protein with which it specifically binds, one or
more sample compartments, an instructional material which describes
performance of a method of the invention, a sample of normal
cervical cells, a sample of cervical cancer cells, and the
like.
[0126] The invention also includes a method of making an isolated
hybridoma which produces an antibody useful for assessing whether
patient is afflicted with a cervical cancer. In this method, a
protein or peptide comprising the entirety or a segment of a marker
protein is synthesized or isolated (e.g. by purification from a
cell in which it is expressed or by transcription and translation
of a nucleic acid encoding the protein or peptide in vivo or in
vitro using known methods). A vertebrate, preferably a mammal such
as a mouse, rat, rabbit, or sheep, is immunized using the protein
or peptide. The vertebrate may optionally (and preferably) be
immunized at least one additional time with the protein or peptide,
so that the vertebrate exhibits a robust immune response to the
protein or peptide. Splenocytes are isolated from the immunized
vertebrate and fused with an immortalized cell line to form
hybridomas, using any of a variety of methods well known in the
art. Hybridomas formed in this manner are then screened using
standard methods to identify one or more hybridomas which produce
an antibody which specifically binds with the marker protein or a
fragment thereof. The invention also includes hybridomas made by
this method and antibodies made using such hybridomas.
[0127] The invention also includes a method of assessing the
efficacy of a test compound for inhibiting cervical cancer cells.
As described above, differences in the level of expression of the
markers of the invention correlate with the cancerous state of
cervical cells. Although it is recognized that changes in the
levels of expression of certain of the markers of the invention
likely result from the cancerous state of cervical cells, it is
likewise recognized that changes in the levels of expression of
other of the markers of the invention induce, maintain, and promote
the cancerous state of those cells. Thus, compounds which inhibit a
cervical cancer in a patient will cause the level of expression of
one or more of the markers of the invention to change to a level
nearer the normal level of expression for that marker (i.e. the
level of expression for the marker in non-cancerous cervical
cells).
[0128] This method thus comprises comparing expression of a marker
in a first cervical cell sample and maintained in the presence of
the test compound and expression of the marker in a second cervical
cell sample and maintained in the absence of the test compound. A
significantly reduced expression of a marker of the invention in
the presence of the test compound is an indication that the test
compound inhibits cervical cancer. The cervical cell samples may,
for example, be aliquots of a single sample of normal cervical
cells obtained from a patient, pooled samples of normal cervical
cells obtained from a patient, cells of a normal cervical cell
line, aliquots of a single sample of cervical cancer cells obtained
from a patient, pooled samples of cervical cancer cells obtained
from a patient, cells of a cervical cancer cell line, or the like.
In one embodiment, the samples are cervical cancer cells obtained
from a patient and a plurality of compounds known to be effective
for inhibiting various cervical cancers are tested in order to
identify the compound which is likely to best inhibit the cervical
cancer in the patient.
[0129] This method may likewise be used to assess the efficacy of a
therapy for inhibiting cervical cancer in a patient. In this
method, the level of expression of one or more markers of the
invention in a pair of samples (one subjected to the therapy, the
other not subjected to the therapy) is assessed. As with the method
of assessing the efficacy of test compounds, if the therapy induces
a significantly lower level of expression of a marker of the
invention then the therapy is efficacious for inhibiting cervical
cancer. As above, if samples from a selected patient are used in
this method, then alternative therapies can be assessed in vitro in
order to select a therapy most likely to be efficacious for
inhibiting cervical cancer in the patient.
[0130] As described above, the cancerous state of human cervical
cells is correlated with changes in the levels of expression of the
markers of the invention. The invention includes a method for
assessing the human cervical cell carcinogenic potential of a test
compound. This method comprises maintaining separate aliquots of
human cervical cells in the presence and absence of the test
compound. Expression of a marker of the invention in each of the
aliquots is compared. A significantly higher level of expression of
a marker of the invention in the aliquot maintained in the presence
of the test compound (relative to the aliquot maintained in the
absence of the test compound) is an indication that the test
compound possesses human cervical cell carcinogenic potential. The
relative carcinogenic potentials of various test compounds can be
assessed by comparing the degree of enhancement or inhibition of
the level of expression of the relevant markers, by comparing the
number of markers for which the level of expression is enhanced or
inhibited, or by comparing both.
[0131] Various aspects of the invention are described in further
detail in the following subsections.
I. Isolated Nucleic Acid Molecules
[0132] One aspect of the invention pertains to isolated nucleic
acid molecules, including nucleic acids which encode a marker
protein or a portion thereof. Isolated nucleic acids of the
invention also include nucleic acid molecules sufficient for use as
hybridization probes to identify marker nucleic acid molecules, and
fragments of marker nucleic acid molecules, e.g., those suitable
for use as PCR primers for the amplification or mutation of marker
nucleic acid molecules. As used herein, the term "nucleic acid
molecule" is intended to include DNA molecules (e.g., cDNA or
genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA
or RNA generated using nucleotide analogs. The nucleic acid
molecule can be single-stranded or double-stranded, but preferably
is double-stranded DNA.
[0133] An "isolated" nucleic acid molecule is one which is
separated from other nucleic acid molecules which are present in
the natural source of the nucleic acid molecule. Preferably, an
"isolated" nucleic acid molecule is free of sequences (preferably
protein-encoding sequences) which naturally flank the nucleic acid
(i.e., sequences located at the 5' and 3' ends of the nucleic acid)
in the genomic DNA of the organism from which the nucleic acid is
derived. For example, in various embodiments, the isolated nucleic
acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1
kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank
the nucleic acid molecule in genomic DNA of the cell from which the
nucleic acid is derived. Moreover, an "isolated" nucleic acid
molecule, such as a cDNA molecule, can be substantially free of
other cellular material, or culture medium when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized.
[0134] A nucleic acid molecule of the present invention can be
isolated using standard molecular biology techniques and the
sequence information in the database records described herein.
Using all or a portion of such nucleic acid sequences, nucleic acid
molecules of the invention can be isolated using standard
hybridization and cloning techniques (e.g., as described in
Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd
ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989).
[0135] A nucleic acid molecule of the invention can be amplified
using cDNA, mRNA, or genomic DNA as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, nucleotides corresponding to all or a portion of a
nucleic acid molecule of the invention can be prepared by standard
synthetic techniques, e.g., using an automated DNA synthesizer.
[0136] In another preferred embodiment, an isolated nucleic acid
molecule of the invention comprises a nucleic acid molecule which
has a nucleotide sequence complementary to the nucleotide sequence
of a marker nucleic acid or to the nucleotide sequence of a nucleic
acid encoding a marker protein. A nucleic acid molecule which is
complementary to a given nucleotide sequence is one which is
sufficiently complementary to the given nucleotide sequence that it
can hybridize to the given nucleotide sequence thereby forming a
stable duplex.
[0137] Moreover, a nucleic acid molecule of the invention can
comprise only a portion of a nucleic acid sequence, wherein the
full length nucleic acid sequence comprises a marker nucleic acid
or which encodes a marker protein. Such nucleic acids can be used,
for example, as a probe or primer. The probe/primer typically is
used as one or more substantially purified oligonucleotides. The
oligonucleotide typically comprises a region of nucleotide sequence
that hybridizes under stringent conditions to at least about 7,
preferably about 15, more preferably about 25, 50, 75, 100, 125,
150, 175, 200, 250, 300, 350, or 400 or more consecutive
nucleotides of a nucleic acid of the invention.
[0138] Probes based on the sequence of a nucleic acid molecule of
the invention can be used to detect transcripts or genomic
sequences corresponding to one or more markers of the invention.
The probe comprises a label group attached thereto, e.g., a
radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as part of a diagnostic test kit
for identifying cells or tissues which mis-express the protein,
such as by measuring levels of a nucleic acid molecule encoding the
protein in a sample of cells from a subject, e.g., detecting mRNA
levels or determining whether a gene encoding the protein has been
mutated or deleted.
[0139] The invention further encompasses nucleic acid molecules
that differ, due to degeneracy of the genetic code, from the
nucleotide sequence of nucleic acids encoding a marker protein
(e.g., a protein having one of the amino acid sequences set forth
in the Sequence Listing), and thus encode the same protein.
[0140] It will be appreciated by those skilled in the art that DNA
sequence polymorphisms that lead to changes in the amino acid
sequence can exist within a population (e.g., the human
population). Such genetic polymorphisms can exist among individuals
within a population due to natural allelic variation. An allele is
one of a group of genes which occur alternatively at a given
genetic locus. In addition, it will be appreciated that DNA
polymorphisms that affect RNA expression levels can also exist that
may affect the overall expression level of that gene (e.g., by
affecting regulation or degradation).
[0141] As used herein, the phrase "allelic variant" refers to a
nucleotide sequence which occurs at a given locus or to a
polypeptide encoded by the nucleotide sequence.
[0142] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules comprising an open reading frame
encoding a polypeptide corresponding to a marker of the invention.
Such natural allelic variations can typically result in 1-5%
variance in the nucleotide sequence of a given gene. Alternative
alleles can be identified by sequencing the gene of interest in a
number of different individuals. This can be readily carried out by
using hybridization probes to identify the same genetic locus in a
variety of individuals. Any and all such nucleotide variations and
resulting amino acid polymorphisms or variations that are the
result of natural allelic variation and that do not alter the
functional activity are intended to be within the scope of the
invention.
[0143] In another embodiment, an isolated nucleic acid molecule of
the invention is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150,
200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200,
1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000,
4500, or more nucleotides in length and hybridizes under stringent
conditions to a marker nucleic acid or to a nucleic acid encoding a
marker protein. As used herein, the term "hybridizes under
stringent conditions" is intended to describe conditions for
hybridization and washing under which nucleotide sequences at least
60% (65%, 70%, preferably 75%) identical to each other typically
remain hybridized to each other. Such stringent conditions are
known to those skilled in the art and can be found in sections
6.3.1-6.3.6 of Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989). A preferred, non-limiting example of
stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50-65.degree. C.
[0144] In addition to naturally-occurring allelic variants of a
nucleic acid molecule of the invention that can exist in the
population, the skilled artisan will further appreciate that
sequence changes can be introduced by mutation thereby leading to
changes in the amino acid sequence of the encoded protein, without
altering the biological activity of the protein encoded thereby.
For example, one can make nucleotide substitutions leading to amino
acid substitutions at "non-essential" amino acid residues. A
"non-essential" amino acid residue is a residue that can be altered
from the wild-type sequence without altering the biological
activity, whereas an "essential" amino acid residue is required for
biological activity. For example, amino acid residues that are not
conserved or only semi-conserved among homologs of various species
may be non-essential for activity and thus would be likely targets
for alteration. Alternatively, amino acid residues that are
conserved among the homologs of various species (e.g., murine and
human) may be essential for activity and thus would not be likely
targets for alteration.
[0145] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding a variant marker protein that
contain changes in amino acid residues that are not essential for
activity. Such variant marker proteins differ in amino acid
sequence from the naturally-occurring marker proteins, yet retain
biological activity. In one embodiment, such a variant marker
protein has an amino acid sequence that is at least about 40%
identical, 50%, 60%, 70%, 80%, 90%, 95%, or 98% identical to the
amino acid sequence of a marker protein.
[0146] An isolated nucleic acid molecule encoding a variant marker
protein can be created by introducing one or more nucleotide
substitutions, additions or deletions into the nucleotide sequence
of marker nucleic acids, such that one or more amino acid residue
substitutions, additions, or deletions are introduced into the
encoded protein. Mutations can be introduced by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Preferably, conservative amino acid substitutions are
made at one or more predicted non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), non-polar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
Alternatively, mutations can be introduced randomly along all or
part of the coding sequence, such as by saturation mutagenesis, and
the resultant mutants can be screened for biological activity to
identify mutants that retain activity. Following mutagenesis, the
encoded protein can be expressed recombinantly and the activity of
the protein can be determined.
[0147] The present invention encompasses antisense nucleic acid
molecules, i.e., molecules which are complementary to a sense
nucleic acid of the invention, e.g., complementary to the coding
strand of a double-stranded marker cDNA molecule or complementary
to a marker mRNA sequence. Accordingly, an antisense nucleic acid
of the invention can hydrogen bond to (i.e. anneal with) a sense
nucleic acid of the invention. The antisense nucleic acid can be
complementary to an entire coding strand, or to only a portion
thereof, e.g., all or part of the protein coding region (or open
reading frame). An antisense nucleic acid molecule can also be
antisense to all or part of a non-coding region of the coding
strand of a nucleotide sequence encoding a marker protein. The
non-coding regions ("5' and 3' untranslated regions") are the 5'
and 3' sequences which flank the coding region and are not
translated into amino acids.
[0148] An antisense oligonucleotide can be, for example, about 5,
10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in
length. An antisense nucleic acid of the invention can be
constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used. Examples of modified nucleotides which can
be used to generate the antisense nucleic acid include
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been sub-cloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0149] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a marker protein to thereby inhibit expression of the
marker, e.g., by inhibiting transcription and/or translation. The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule which binds to DNA duplexes, through specific
interactions in the major groove of the double helix. Examples of a
route of administration of antisense nucleic acid molecules of the
invention includes direct injection at a tissue site or infusion of
the antisense nucleic acid into a cervical-associated body fluid.
Alternatively, antisense nucleic acid molecules can be modified to
target selected cells and then administered systemically. For
example, for systemic administration, antisense molecules can be
modified such that they specifically bind to receptors or antigens
expressed on a selected cell surface, e.g., by linking the
antisense nucleic acid molecules to peptides or antibodies which
bind to cell surface receptors or antigens. The antisense nucleic
acid molecules can also be delivered to cells using the vectors
described herein. To achieve sufficient intracellular
concentrations of the antisense molecules, vector constructs in
which the antisense nucleic acid molecule is placed under the
control of a strong pol II or pol III promoter are preferred.
[0150] An antisense nucleic acid molecule of the invention can be
an .alpha.-anomeric nucleic acid molecule. An .alpha.-anomeric
nucleic acid molecule forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .alpha.-units,
the strands run parallel to each other (Gaultier et al., 1987,
Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid
molecule can also comprise a 2'-o-methylribonucleotide (Inoue et
al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA
analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
[0151] The invention also encompasses ribozymes. Ribozymes are
catalytic RNA molecules with ribonuclease activity which are
capable of cleaving a single-stranded nucleic acid, such as an
mRNA, to which they have a complementary region. Thus, ribozymes
(e.g., hammerhead ribozymes as described in Haselhoff and Gerlach,
1988, Nature 334:585-591) can be used to catalytically cleave mRNA
transcripts to thereby inhibit translation of the protein encoded
by the mRNA. A ribozyme having specificity for a nucleic acid
molecule encoding a marker protein can be designed based upon the
nucleotide sequence of a cDNA corresponding to the marker. For
example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in which the nucleotide sequence of the active site is
complementary to the nucleotide sequence to be cleaved (see Cech et
al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.
5,116,742). Alternatively, an mRNA encoding a polypeptide of the
invention can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules (see, e.g.,
Bartel and Szostak, 1993, Science 261:1411-1418).
[0152] The invention also encompasses nucleic acid molecules which
form triple helical structures. For example, expression of a marker
of the invention can be inhibited by targeting nucleotide sequences
complementary to the regulatory region of the gene encoding the
marker nucleic acid or protein (e.g., the promoter and/or enhancer)
to form triple helical structures that prevent transcription of the
gene in target cells. See generally Helene (1991) Anticancer Drug
Des. 6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and
Maher (1992) Bioassays 14(12):807-15.
[0153] In various embodiments, the nucleic acid molecules of the
invention can be modified at the base moiety, sugar moiety or
phosphate backbone to improve, e.g., the stability, hybridization,
or solubility of the molecule. For example, the deoxyribose
phosphate backbone of the nucleic acids can be modified to generate
peptide nucleic acids (see Hyrup et al., 1996, Bioorganic &
Medicinal Chemistry 4(1): 5-23). As used herein, the terms "peptide
nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA
mimics, in which the deoxyribose phosphate backbone is replaced by
a pseudopeptide backbone and only the four natural nucleobases are
retained. The neutral backbone of PNAs has been shown to allow for
specific hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93:14670-675.
[0154] PNAs can be used in therapeutic and diagnostic applications.
For example, PNAs can be used as antisense or antigene agents for
sequence-specific modulation of gene expression by, e.g., inducing
transcription or translation arrest or inhibiting replication. PNAs
can also be used, e.g., in the analysis of single base pair
mutations in a gene by, e.g., PNA directed PCR clamping; as
artificial restriction enzymes when used in combination with other
enzymes, e.g., S1 nucleases (Hyrup (1996), supra; or as probes or
primers for DNA sequence and hybridization (Hyrup, 1996, supra;
Perry-O'Keefe et al., 1996, Proc. Natl. Acad. Sci. USA
93:14670-675).
[0155] In another embodiment, PNAs can be modified, e.g., to
enhance their stability or cellular uptake, by attaching lipophilic
or other helper groups to PNA, by the formation of PNA-DNA
chimeras, or by the use of liposomes or other techniques of drug
delivery known in the art. For example, PNA-DNA chimeras can be
generated which can combine the advantageous properties of PNA and
DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and
DNA polymerases, to interact with the DNA portion while the PNA
portion would provide high binding affinity and specificity.
PNA-DNA chimeras can be linked using linkers of appropriate lengths
selected in terms of base stacking, number of bonds between the
nucleobases, and orientation (Hyrup, 1996, supra). The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup (1996),
supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.
For example, a DNA chain can be synthesized on a solid support
using standard phosphoramidite coupling chemistry and modified
nucleoside analogs. Compounds such as
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite can be
used as a link between the PNA and the 5' end of DNA (Mag et al.,
1989, Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled
in a step-wise manner to produce a chimeric molecule with a 5' PNA
segment and a 3' DNA segment (Finn et al., 1996, Nucleic Acids Res.
24(17):3357-63). Alternatively, chimeric molecules can be
synthesized with a 5' DNA segment and a 3' PNA segment (Peterser et
al., 1975, Bioorganic Med. Chem. Lett. 5:1119-11124).
[0156] In other embodiments, the oligonucleotide can include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad.
Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad.
Sci. USA 84:648-652; PCT Publication No. WO 88/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134).
In addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (see, e.g., Krol et al.,
1988, Bio/Techniques 6:958-976) or intercalating agents (see, e.g.,
Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide
can be conjugated to another molecule, e.g., a peptide,
hybridization triggered cross-linking agent, transport agent,
hybridization-triggered cleavage agent, etc.
[0157] The invention also includes molecular beacon nucleic acids
having at least one region which is complementary to a nucleic acid
of the invention, such that the molecular beacon is useful for
quantitating the presence of the nucleic acid of the invention in a
sample. A "molecular beacon" nucleic acid is a nucleic acid
comprising a pair of complementary regions and having a fluorophore
and a fluorescent quencher associated therewith. The fluorophore
and quencher are associated with different portions of the nucleic
acid in such an orientation that when the complementary regions are
annealed with one another, fluorescence of the fluorophore is
quenched by the quencher. When the complementary regions of the
nucleic acid are not annealed with one another, fluorescence of the
fluorophore is quenched to a lesser degree. Molecular beacon
nucleic acids are described, for example, in U.S. Pat. No.
5,876,930.
II. Isolated Proteins and Antibodies
[0158] One aspect of the invention pertains to isolated marker
proteins and biologically active portions thereof, as well as
polypeptide fragments suitable for use as immunogens to raise
antibodies directed against a marker protein or a fragment thereof.
In one embodiment, the native marker protein can be isolated from
cells or tissue sources by an appropriate purification scheme using
standard protein purification techniques. In another embodiment, a
protein or peptide comprising the whole or a segment of the marker
protein is produced by recombinant DNA techniques. Alternative to
recombinant expression, such protein or peptide can be synthesized
chemically using standard peptide synthesis techniques.
[0159] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the protein is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of protein in which the protein is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. Thus, protein that is substantially free of
cellular material includes preparations of protein having less than
about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein
(also referred to herein as a "contaminating protein"). When the
protein or biologically active portion thereof is recombinantly
produced, it is also preferably substantially free of culture
medium, i.e., culture medium represents less than about 20%, 10%,
or 5% of the volume of the protein preparation. When the protein is
produced by chemical synthesis, it is preferably substantially free
of chemical precursors or other chemicals, i.e., it is separated
from chemical precursors or other chemicals which are involved in
the synthesis of the protein. Accordingly such preparations of the
protein have less than about 30%, 20%, 10%, 5% (by dry weight) of
chemical precursors or compounds other than the polypeptide of
interest.
[0160] Biologically active portions of a marker protein include
polypeptides comprising amino acid sequences sufficiently identical
to or derived from the amino acid sequence of the marker protein,
which include fewer amino acids than the full length protein, and
exhibit at least one activity of the corresponding full-length
protein. Typically, biologically active portions comprise a domain
or motif with at least one activity of the corresponding
full-length protein. A biologically active portion of a marker
protein of the invention can be a polypeptide which is, for
example, 10, 25, 50, 100 or more amino acids in length. Moreover,
other biologically active portions, in which other regions of the
marker protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the functional
activities of the native form of the marker protein.
[0161] Preferred marker proteins are encoded by nucleotide
sequences comprising the sequence of any of the sequences set forth
in the Sequence Listing. Other useful proteins are substantially
identical (e.g., at least about 40%, preferably 50%, 60%, 70%, 80%,
90%, 95%, or 99%) to one of these sequences and retain the
functional activity of the corresponding naturally-occurring marker
protein yet differ in amino acid sequence due to natural allelic
variation or mutagenesis.
[0162] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % identity=# of
identical positions/total # of positions (e.g., overlapping
positions).times.100). In one embodiment the two sequences are the
same length.
[0163] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. A preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in to
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
Such an algorithm is incorporated into the BLASTN and BLASTX
programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410.
BLAST nucleotide searches can be performed with the BLASTN program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to a nucleic acid molecules of the invention. BLAST protein
searches can be performed with the BLASTP program, score=50,
wordlength=3 to obtain amino acid sequences homologous to a protein
molecules of the invention. To obtain gapped alignments for
comparison purposes, a newer version of the BLAST algorithm called
Gapped BLAST can be utilized as described in Altschul et al. (1997)
Nucleic Acids Res. 25:3389-3402, which is able to perform gapped
local alignments for the programs BLASTN, BLASTP and BLASTX.
Alternatively, PSI-Blast can be used to perform an iterated search
which detects distant relationships between molecules. When
utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default
parameters of the respective programs (e.g., BLASTX and BLASTN) can
be used. See http://www.ncbi.nlm.nih.gov. Another preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of sequences is the algorithm of Myers and Miller,
(1988) CABIOS 4:11-17. Such an algorithm is incorporated into the
ALIGN program (version 2.0) which is part of the GCG sequence
alignment software package. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4 can be used. Yet
another useful algorithm for identifying regions of local sequence
similarity and alignment is the FASTA algorithm as described in
Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448.
When using the FASTA algorithm for comparing nucleotide or amino
acid sequences, a PAM120 weight residue table can, for example, be
used with a k-tuple value of 2.
[0164] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, only exact matches
are counted.
[0165] The invention also provides chimeric or fusion proteins
comprising a marker protein or a segment thereof. As used herein, a
"chimeric protein" or "fusion protein" comprises all or part
(preferably a biologically active part) of a marker protein
operably linked to a heterologous polypeptide (i.e., a polypeptide
other than the marker protein). Within the fusion protein, the term
"operably linked" is intended to indicate that the marker protein
or segment thereof and the heterologous polypeptide are fused
in-frame to each other. The heterologous polypeptide can be fused
to the amino-terminus or the carboxyl-terminus of the marker
protein or segment.
[0166] One useful fusion protein is a GST fusion protein in which a
marker protein or segment is fused to the carboxyl terminus of GST
sequences. Such fusion proteins can facilitate the purification of
a recombinant polypeptide of the invention.
[0167] In another embodiment, the fusion protein contains a
heterologous signal sequence at its amino terminus. For example,
the native signal sequence of a marker protein can be removed and
replaced with a signal sequence from another protein. For example,
the gp67 secretory sequence of the baculovirus envelope protein can
be used as a heterologous signal sequence (Ausubel et al., ed.,
Current Protocols in Molecular Biology, John Wiley & Sons, NY,
1992). Other examples of eukaryotic heterologous signal sequences
include the secretory sequences of melittin and human placental
alkaline phosphatase (Stratagene; La Jolla, Calif.). In yet another
example, useful prokaryotic heterologous signal sequences include
the phoA secretory signal (Sambrook et al., supra) and the protein
A secretory signal (Pharmacia Biotech; Piscataway, N.J.).
[0168] In yet another embodiment, the fusion protein is an
immunoglobulin fusion protein in which all or part of a marker
protein is fused to sequences derived from a member of the
immunoglobulin protein family. The immunoglobulin fusion proteins
of the invention can be incorporated into pharmaceutical
compositions and administered to a subject to inhibit an
interaction between a ligand (soluble or membrane-bound) and a
protein on the surface of a cell (receptor), to thereby suppress
signal transduction in vivo. The immunoglobulin fusion protein can
be used to affect the bioavailability of a cognate ligand of a
marker protein Inhibition of ligand/receptor interaction can be
useful therapeutically, both for treating proliferative and
differentiative disorders and for modulating (e.g. promoting or
inhibiting) cell survival. Moreover, the immunoglobulin fusion
proteins of the invention can be used as immunogens to produce
antibodies directed against a marker protein in a subject, to
purify ligands and in screening assays to identify molecules which
inhibit the interaction of the marker protein with ligands.
[0169] Chimeric and fusion proteins of the invention can be
produced by standard recombinant DNA techniques. In another
embodiment, the fusion gene can be synthesized by conventional
techniques including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor
primers which give rise to complementary overhangs between two
consecutive gene fragments which can subsequently be annealed and
re-amplified to generate a chimeric gene sequence (see, e.g.,
Ausubel et al., supra). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). A nucleic acid encoding a polypeptide of the
invention can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the polypeptide of the
invention.
[0170] A signal sequence can be used to facilitate secretion and
isolation of marker proteins. Signal sequences are typically
characterized by a core of hydrophobic amino acids which are
generally cleaved from the mature protein during secretion in one
or more cleavage events. Such signal peptides contain processing
sites that allow cleavage of the signal sequence from the mature
proteins as they pass through the secretory pathway. Thus, the
invention pertains to marker proteins, fusion proteins or segments
thereof having a signal sequence, as well as to such proteins from
which the signal sequence has been proteolytically cleaved (i.e.,
the cleavage products). In one embodiment, a nucleic acid sequence
encoding a signal sequence can be operably linked in an expression
vector to a protein of interest, such as a marker protein or a
segment thereof. The signal sequence directs secretion of the
protein, such as from a eukaryotic host into which the expression
vector is transformed, and the signal sequence is subsequently or
concurrently cleaved. The protein can then be readily purified from
the extracellular medium by art recognized methods. Alternatively,
the signal sequence can be linked to the protein of interest using
a sequence which facilitates purification, such as with a GST
domain.
[0171] The present invention also pertains to variants of the
marker proteins. Such variants have an altered amino acid sequence
which can function as either agonists (mimetics) or as antagonists.
Variants can be generated by mutagenesis, e.g., discrete point
mutation or truncation. An agonist can retain substantially the
same, or a subset, of the biological activities of the naturally
occurring form of the protein. An antagonist of a protein can
inhibit one or more of the activities of the naturally occurring
form of the protein by, for example, competitively binding to a
downstream or upstream member of a cellular signaling cascade which
includes the protein of interest. Thus, specific biological effects
can be elicited by treatment with a variant of limited function.
Treatment of a subject with a variant having a subset of the
biological activities of the naturally occurring form of the
protein can have fewer side effects in a subject relative to
treatment with the naturally occurring form of the protein.
[0172] Variants of a marker protein which function as either
agonists (mimetics) or as antagonists can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of the protein of the invention for agonist or antagonist
activity. In one embodiment, a variegated library of variants is
generated by combinatorial mutagenesis at the nucleic acid level
and is encoded by a variegated gene library. A variegated library
of variants can be produced by, for example, enzymatically ligating
a mixture of synthetic oligonucleotides into gene sequences such
that a degenerate set of potential protein sequences is expressible
as individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for phage display). There are a variety of
methods which can be used to produce libraries of potential
variants of the marker proteins from a degenerate oligonucleotide
sequence. Methods for synthesizing degenerate oligonucleotides are
known in the art (see, e.g., Narang, 1983, Tetrahedron 39:3;
Itakura et al., 1984, Annu. Rev. Biochem. 53:323; Itakura et al.,
1984, Science 198:1056; Ike et al., 1983 Nucleic Acid Res.
11:477).
[0173] In addition, libraries of segments of a marker protein can
be used to generate a variegated population of polypeptides for
screening and subsequent selection of variant marker proteins or
segments thereof. For example, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of the coding sequence of interest with a nuclease under
conditions wherein nicking occurs only about once per molecule,
denaturing the double stranded DNA, renaturing the DNA to form
double stranded DNA which can include sense/antisense pairs from
different nicked products, removing single stranded portions from
reformed duplexes by treatment with S1 nuclease, and ligating the
resulting fragment library into an expression vector. By this
method, an expression library can be derived which encodes amino
terminal and internal fragments of various sizes of the protein of
interest.
[0174] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. The most widely used techniques, which
are amenable to high through-put analysis, for screening large gene
libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a technique
which enhances the frequency of functional mutants in the
libraries, can be used in combination with the screening assays to
identify variants of a protein of the invention (Arkin and Yourvan,
1992, Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al.,
1993, Protein Engineering 6(3):327-331).
[0175] Another aspect of the invention pertains to antibodies
directed against a protein of the invention. In preferred
embodiments, the antibodies specifically bind a marker protein or a
fragment thereof. The terms "antibody" and "antibodies" as used
interchangeably herein refer to immunoglobulin molecules as well as
fragments and derivatives thereof that comprise an immunologically
active portion of an immunoglobulin molecule, (i.e., such a portion
contains an antigen binding site which specifically binds an
antigen, such as a marker protein, e.g., an epitope of a marker
protein). An antibody which specifically binds to a protein of the
invention is an antibody which binds the protein, but does not
substantially bind other molecules in a sample, e.g., a biological
sample, which naturally contains the protein. Examples of an
immunologically active portion of an immunoglobulin molecule
include, but are not limited to, single-chain antibodies (scAb),
F(ab) and F(ab').sub.2 fragments.
[0176] An isolated protein of the invention or a fragment thereof
can be used as an immunogen to generate antibodies. The full-length
protein can be used or, alternatively, the invention provides
antigenic peptide fragments for use as immunogens. The antigenic
peptide of a protein of the invention comprises at least 8
(preferably 10, 15, 20, or 30 or more) amino acid residues of the
amino acid sequence of one of the proteins of the invention, and
encompasses at least one epitope of the protein such that an
antibody raised against the peptide forms a specific immune complex
with the protein. Preferred epitopes encompassed by the antigenic
peptide are regions that are located on the surface of the protein,
e.g., hydrophilic regions. Hydrophobicity sequence analysis,
hydrophilicity sequence analysis, or similar analyses can be used
to identify hydrophilic regions. In preferred embodiments, an
isolated marker protein or fragment thereof is used as an
immunogen.
[0177] An immunogen typically is used to prepare antibodies by
immunizing a suitable (i.e. immunocompetent) subject such as a
rabbit, goat, mouse, or other mammal or vertebrate. An appropriate
immunogenic preparation can contain, for example,
recombinantly-expressed or chemically-synthesized protein or
peptide. The preparation can further include an adjuvant, such as
Freund's complete or incomplete adjuvant, or a similar
immunostimulatory agent. Preferred immunogen compositions are those
that contain no other human proteins such as, for example,
immunogen compositions made using a non-human host cell for
recombinant expression of a protein of the invention. In such a
manner, the resulting antibody compositions have reduced or no
binding of human proteins other than a protein of the
invention.
[0178] The invention provides polyclonal and monoclonal antibodies.
The term "monoclonal antibody" or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one species of an antigen binding site
capable of immunoreacting with a particular epitope. Preferred
polyclonal and monoclonal antibody compositions are ones that have
been selected for antibodies directed against a protein of the
invention. Particularly preferred polyclonal and monoclonal
antibody preparations are ones that contain only antibodies
directed against a marker protein or fragment thereof.
[0179] Polyclonal antibodies can be prepared by immunizing a
suitable subject with a protein of the invention as an immunogen
The antibody titer in the immunized subject can be monitored over
time by standard techniques, such as with an enzyme linked
immunosorbent assay (ELISA) using immobilized polypeptide. At an
appropriate time after immunization, e.g., when the specific
antibody titers are highest, antibody-producing cells can be
obtained from the subject and used to prepare monoclonal antibodies
(mAb) by standard techniques, such as the hybridoma technique
originally described by Kohler and Milstein (1975) Nature
256:495-497, the human B cell hybridoma technique (see Kozbor et
al., 1983, Immunol. Today 4:72), the EBV-hybridoma technique (see
Cole et al., pp. 77-96 In Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc., 1985) or trioma techniques. The technology for
producing hybridomas is well known (see generally Current Protocols
in Immunology, Coligan et al. ed., John Wiley & Sons, New York,
1994). Hybridoma cells producing a monoclonal antibody of the
invention are detected by screening the hybridoma culture
supernatants for antibodies that bind the polypeptide of interest,
e.g., using a standard ELISA assay.
[0180] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody directed against a protein of the
invention can be identified and isolated by screening a recombinant
combinatorial immunoglobulin library (e.g., an antibody phage
display library) with the polypeptide of interest. Kits for
generating and screening phage display libraries are commercially
available (e.g., the Pharmacia Recombinant Phage Antibody System,
Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display
Kit, Catalog No. 240612). Additionally, examples of methods and
reagents particularly amenable for use in generating and screening
antibody display library can be found in, for example, U.S. Pat.
No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No.
WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No.
WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No.
WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No.
WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et
al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989)
Science 246:1275-1281; Griffiths et al. (1993) EMBO J.
12:725-734.
[0181] The invention also provides recombinant antibodies that
specifically bind a protein of the invention. In preferred
embodiments, the recombinant antibodies specifically binds a marker
protein or fragment thereof. Recombinant antibodies include, but
are not limited to, chimeric and humanized monoclonal antibodies,
comprising both human and non-human portions, single-chain
antibodies and multi-specific antibodies. A chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human immunoglobulin constant region. (See,
e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al.,
U.S. Pat. No. 4,816,397, which are incorporated herein by reference
in their entirety.) Single-chain antibodies have an antigen binding
site and consist of a single polypeptide. They can be produced by
techniques known in the art, for example using methods described in
Ladner et. al U.S. Pat. No. 4,946,778 (which is incorporated herein
by reference in its entirety); Bird et al., (1988) Science
242:423-426; Whitlow et al., (1991) Methods in Enzymology 2:1-9;
Whitlow et al., (1991) Methods in Enzymology 2:97-105; and Huston
et al., (1991) Methods in Enzymology Molecular Design and Modeling:
Concepts and Applications 203:46-88. Multi-specific antibodies are
antibody molecules having at least two antigen-binding sites that
specifically bind different antigens. Such molecules can be
produced by techniques known in the art, for example using methods
described in Segal, U.S. Pat. No. 4,676,980 (the disclosure of
which is incorporated herein by reference in its entirety);
Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448;
Whitlow et al., (1994) Protein Eng. 7:1017-1026 and U.S. Pat. No.
6,121,424.
[0182] Humanized antibodies are antibody molecules from non-human
species having one or more complementarity determining regions
(CDRs) from the non-human species and a framework region from a
human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No.
5,585,089, which is incorporated herein by reference in its
entirety.) Humanized monoclonal antibodies can be produced by
recombinant DNA techniques known in the art, for example using
methods described in PCT Publication No. WO 87/02671; European
Patent Application 184,187; European Patent Application 171,496;
European Patent Application 173,494; PCT Publication No. WO
86/01533; U.S. Pat. No. 4,816,567; European Patent Application
125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987)
J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci.
USA 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005;
Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J.
Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science
229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat. No.
5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.
(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
[0183] More particularly, humanized antibodies can be produced, for
example, using transgenic mice which are incapable of expressing
endogenous immunoglobulin heavy and light chains genes, but which
can express human heavy and light chain genes. The transgenic mice
are immunized in the normal fashion with a selected antigen, e.g.,
all or a portion of a polypeptide corresponding to a marker of the
invention. Monoclonal antibodies directed against the antigen can
be obtained using conventional hybridoma technology. The human
immunoglobulin transgenes harbored by the transgenic mice rearrange
during B cell differentiation, and subsequently undergo class
switching and somatic mutation. Thus, using such a technique, it is
possible to produce therapeutically useful IgG, IgA and IgE
antibodies. For an overview of this technology for producing human
antibodies, see Lonberg and Huszar (1995) Int. Rev. Immunol.
13:65-93). For a detailed discussion of this technology for
producing human antibodies and human monoclonal antibodies and
protocols for producing such antibodies, see, e.g., U.S. Pat. No.
5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S.
Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. In addition,
companies such as Abgenix, Inc. (Freemont, Calif.), can be engaged
to provide human antibodies directed against a selected antigen
using technology similar to that described above.
[0184] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a murine antibody, is used to guide the selection
of a completely human antibody recognizing the same epitope
(Jespers et al., 1994, Bio/technology 12:899-903).
[0185] The antibodies of the invention can be isolated after
production (e.g., from the blood or serum of the subject) or
synthesis and further purified by well-known techniques. For
example, IgG antibodies can be purified using protein A
chromatography. Antibodies specific for a protein of the invention
can be selected or (e.g., partially purified) or purified by, e.g.,
affinity chromatography. For example, a recombinantly expressed and
purified (or partially purified) protein of the invention is
produced as described herein, and covalently or non-covalently
coupled to a solid support such as, for example, a chromatography
column. The column can then be used to affinity purify antibodies
specific for the proteins of the invention from a sample containing
antibodies directed against a large number of different epitopes,
thereby generating a substantially purified antibody composition,
i.e., one that is substantially free of contaminating antibodies.
By a substantially purified antibody composition is meant, in this
context, that the antibody sample contains at most only 30% (by dry
weight) of contaminating antibodies directed against epitopes other
than those of the desired protein of the invention, and preferably
at most 20%, yet more preferably at most 10%, and most preferably
at most 5% (by dry weight) of the sample is contaminating
antibodies. A purified antibody composition means that at least 99%
of the antibodies in the composition are directed against the
desired protein of the invention.
[0186] In a preferred embodiment, the substantially purified
antibodies of the invention may specifically bind to a signal
peptide, a secreted sequence, an extracellular domain, a
transmembrane or a cytoplasmic domain or cytoplasmic membrane of a
protein of the invention. In a particularly preferred embodiment,
the substantially purified antibodies of the invention specifically
bind to a secreted sequence or an extracellular domain of the amino
acid sequences of a protein of the invention. In a more preferred
embodiment, the substantially purified antibodies of the invention
specifically bind to a secreted sequence or an extracellular domain
of the amino acid sequences of a marker protein.
[0187] An antibody directed against a protein of the invention can
be used to isolate the protein by standard techniques, such as
affinity chromatography or immunoprecipitation. Moreover, such an
antibody can be used to detect the marker protein or fragment
thereof (e.g., in a cellular lysate or cell supernatant) in order
to evaluate the level and pattern of expression of the marker. The
antibodies can also be used diagnostically to monitor protein
levels in tissues or body fluids (e.g. in a cervical-associated
body fluid) as part of a clinical testing procedure, e.g., to, for
example, determine the efficacy of a given treatment regimen.
Detection can be facilitated by the use of an antibody derivative,
which comprises an antibody of the invention coupled to a
detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0188] Antibodies of the invention may also be used as therapeutic
agents in treating cancers. In a preferred embodiment, completely
human antibodies of the invention are used for therapeutic
treatment of human cancer patients, particularly those having an
cervical cancer. In another preferred embodiment, antibodies that
bind specifically to a marker protein or fragment thereof are used
for therapeutic treatment. Further, such therapeutic antibody may
be an antibody derivative or immunotoxin comprising an antibody
conjugated to a therapeutic moiety such as a cytotoxin, a
therapeutic agent or a radioactive metal ion. A cytotoxin or
cytotoxic agent includes any agent that is detrimental to cells.
Examples include taxol, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof.
[0189] Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0190] The conjugated antibodies of the invention can be used for
modifying a given biological response, for the drug moiety is not
to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as ribosome-inhibiting
protein (see Better et al., U.S. Pat. No. 6,146,631, the disclosure
of which is incorporated herein in its entirety), abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, .alpha.-interferon, .beta.-interferon, nerve
growth factor, platelet derived growth factor, tissue plasminogen
activator; or, biological response modifiers such as, for example,
lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"),
interleukin-6 ("IL-6"), granulocyte macrophase colony stimulating
factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"),
or other growth factors.
[0191] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982).
[0192] Accordingly, in one aspect, the invention provides
substantially purified antibodies, antibody fragments and
derivatives, all of which specifically bind to a protein of the
invention and preferably, a marker protein. In various embodiments,
the substantially purified antibodies of the invention, or
fragments or derivatives thereof, can be human, non-human, chimeric
and/or humanized antibodies. In another aspect, the invention
provides non-human antibodies, antibody fragments and derivatives,
all of which specifically bind to a protein of the invention and
preferably, a marker protein. Such non-human antibodies can be
goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies.
Alternatively, the non-human antibodies of the invention can be
chimeric and/or humanized antibodies. In addition, the non-human
antibodies of the invention can be polyclonal antibodies or
monoclonal antibodies. In still a further aspect, the invention
provides monoclonal antibodies, antibody fragments and derivatives,
all of which specifically bind to a protein of the invention and
preferably, a marker protein. The monoclonal antibodies can be
human, humanized, chimeric and/or non-human antibodies.
[0193] The invention also provides a kit containing an antibody of
the invention conjugated to a detectable substance, and
instructions for use. Still another aspect of the invention is a
pharmaceutical composition comprising an antibody of the invention.
In one embodiment, the pharmaceutical composition comprises an
antibody of the invention and a pharmaceutically acceptable
carrier.
III. Recombinant Expression Vectors and Host Cells
[0194] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding a
marker protein (or a portion of such a protein). As used herein,
the term "vector" refers to a nucleic acid molecule capable of
transporting another nucleic acid to which it has been linked. One
type of vector is a "plasmid", which refers to a circular double
stranded DNA loop into which additional DNA segments can be
ligated. Another type of vector is a viral vector, wherein
additional DNA segments can be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) are integrated
into the genome of a host cell upon introduction into the host
cell, and thereby are replicated along with the host genome.
Moreover, certain vectors, namely expression vectors, are capable
of directing the expression of genes to which they are operably
linked. In general, expression vectors of utility in recombinant
DNA techniques are often in the form of plasmids (vectors).
However, the invention is intended to include such other forms of
expression vectors, such as viral vectors (e.g., replication
defective retroviruses, adenoviruses and adeno-associated viruses),
which serve equivalent functions.
[0195] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell. This means that the recombinant
expression vectors include one or more regulatory sequences,
selected on the basis of the host cells to be used for expression,
which is operably linked to the nucleic acid sequence to be
expressed. Within a recombinant expression vector, "operably
linked" is intended to mean that the nucleotide sequence of
interest is linked to the regulatory sequence(s) in a manner which
allows for expression of the nucleotide sequence (e.g., in an in
vitro transcription/translation system or in a host cell when the
vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel,
Methods in Enzymology: Gene Expression Technology vol. 185,
Academic Press, San Diego, Calif. (1991). Regulatory sequences
include those which direct constitutive expression of a nucleotide
sequence in many types of host cell and those which direct
expression of the nucleotide sequence only in certain host cells
(e.g., tissue-specific regulatory sequences). It will be
appreciated by those skilled in the art that the design of the
expression vector can depend on such factors as the choice of the
host cell to be transformed, the level of expression of protein
desired, and the like. The expression vectors of the invention can
be introduced into host cells to thereby produce proteins or
peptides, including fusion proteins or peptides, encoded by nucleic
acids as described herein.
[0196] The recombinant expression vectors of the invention can be
designed for expression of a marker protein or a segment thereof in
prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells
{using baculovirus expression vectors}, yeast cells or mammalian
cells). Suitable host cells are discussed further in Goeddel,
supra. Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0197] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988, Gene 67:31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) which fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0198] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., 1988, Gene 69:301-315) and pET
11d (Studier et al., p. 60-89, In Gene Expression Technology:
Methods in Enzymology vol. 185, Academic Press, San Diego, Calif.,
1991). Target gene expression from the pTrc vector relies on host
RNA polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
co-expressed viral RNA polymerase (T7 gn1). This viral polymerase
is supplied by host strains BL21(DE3) or HMS174(DE3) from a
resident prophage harboring a T7 gn1 gene under the transcriptional
control of the lacUV 5 promoter.
[0199] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, p. 119-128, In Gene Expression Technology: Methods in
Enzymology vol. 185, Academic Press, San Diego, Calif., 1990.
Another strategy is to alter the nucleic acid sequence of the
nucleic acid to be inserted into an expression vector so that the
individual codons for each amino acid are those preferentially
utilized in E. coli (Wada et al., 1992, Nucleic Acids Res.
20:2111-2118). Such alteration of nucleic acid sequences of the
invention can be carried out by standard DNA synthesis
techniques.
[0200] In another embodiment, the expression vector is a yeast
expression vector. Examples of vectors for expression in yeast S.
cerevisiae include pYepSec1 (Baldari et al., 1987, EMBO J.
6:229-234), pMFa (Kurjan and Herskowitz, 1982, Cell 30:933-943),
pJRY88 (Schultz et al., 1987, Gene 54:113-123), pYES2 (Invitrogen
Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corp, San
Diego, Calif.).
[0201] Alternatively, the expression vector is a baculovirus
expression vector. Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-2165) and
the pVL series (Lucklow and Summers, 1989, Virology 170:31-39).
[0202] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, 1987, Nature 329:840) and pMT2PC (Kaufman et al., 1987, EMBO
J. 6:187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of Sambrook et al.,
supra.
[0203] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al., 1987, Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton,
1988, Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and to Baltimore, 1989, EMBO J. 8:729-733) and
immunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen and
Baltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle, 1989, Proc. Natl.
Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund
et al., 1985, Science 230:912-916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example the murine hox promoters (Kessel and Gruss, 1990, Science
249:374-379) and the .alpha.-fetoprotein promoter (Camper and
Tilghman, 1989, Genes Dev. 3:537-546).
[0204] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operably linked to a regulatory sequence in a manner
which allows for expression (by transcription of the DNA molecule)
of an RNA molecule which is antisense to the mRNA encoding a
polypeptide of the invention. Regulatory sequences operably linked
to a nucleic acid cloned in the antisense orientation can be chosen
which direct the continuous expression of the antisense RNA
molecule in a variety of cell types, for instance viral promoters
and/or enhancers, or regulatory sequences can be chosen which
direct constitutive, tissue-specific or cell type specific
expression of antisense RNA. The antisense expression vector can be
in the form of a recombinant plasmid, phagemid, or attenuated virus
in which antisense nucleic acids are produced under the control of
a high efficiency regulatory region, the activity of which can be
determined by the cell type into which the vector is introduced.
For a discussion of the regulation of gene expression using
antisense genes see Weintraub et al., 1986, Trends in Genetics,
Vol. 1(1).
[0205] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0206] A host cell can be any prokaryotic (e.g., E. coli) or
eukaryotic cell (e.g., insect cells, yeast or mammalian cells).
[0207] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (supra), and other
laboratory manuals.
[0208] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
for resistance to antibiotics) is generally introduced into the
host cells along with the gene of interest. Preferred selectable
markers include those which confer resistance to drugs, such as
G418, hygromycin and methotrexate. Cells stably transfected with
the introduced nucleic acid can be identified by drug selection
(e.g., cells that have incorporated the selectable marker will
survive, while the other cells die).
[0209] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce a marker
protein or a segment thereof. Accordingly, the invention further
provides methods for producing a marker protein or a segment
thereof using the host cells of the invention. In one embodiment,
the method comprises culturing the host cell of the invention (into
which a recombinant expression vector encoding a marker protein or
a segment thereof has been introduced) in a suitable medium such
that the is produced. In another embodiment, the method further
comprises isolating the marker protein or a segment thereof from
the medium or the host cell.
[0210] The host cells of the invention can also be used to produce
nonhuman transgenic animals. For example, in one embodiment, a host
cell of the invention is a fertilized oocyte or an embryonic stem
cell into which a sequences encoding a marker protein or a segment
thereof have been introduced. Such host cells can then be used to
create non-human transgenic animals in which exogenous sequences
encoding a marker protein of the invention have been introduced
into their genome or homologous recombinant animals in which
endogenous gene(s) encoding a marker protein have been altered.
Such animals are useful for studying the function and/or activity
of the marker protein and for identifying and/or evaluating
modulators of marker protein. As used herein, a "transgenic animal"
is a non-human animal, preferably a mammal, more preferably a
rodent such as a rat or mouse, in which one or more of the cells of
the animal includes a transgene. Other examples of transgenic
animals include non-human primates, sheep, dogs, cows, goats,
chickens, amphibians, etc. A transgene is exogenous DNA which is
integrated into the genome of a cell from which a transgenic animal
develops and which remains in the genome of the mature animal,
thereby directing the expression of an encoded gene product in one
or more cell types or tissues of the transgenic animal. As used
herein, an "homologous recombinant animal" is a non-human animal,
preferably a mammal, more preferably a mouse, in which an
endogenous gene has been altered by homologous recombination
between the endogenous gene and an exogenous DNA molecule
introduced into a cell of the animal, e.g., an embryonic cell of
the animal, prior to development of the animal.
[0211] A transgenic animal of the invention can be created by
introducing a nucleic acid encoding a marker protein into the male
pronuclei of a fertilized oocyte, e.g., by microinjection,
retroviral infection, and allowing the oocyte to develop in a
pseudopregnant female foster animal. Intronic sequences and
polyadenylation signals can also be included in the transgene to
increase the efficiency of expression of the transgene. A
tissue-specific regulatory sequence(s) can be operably linked to
the transgene to direct expression of the polypeptide of the
invention to particular cells. Methods for generating transgenic
animals via embryo manipulation and microinjection, particularly
animals such as mice, have become conventional in the art and are
described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009,
U.S. Pat. No. 4,873,191 and in Hogan, Manipulating the Mouse
Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1986. Similar methods are used for production of other
transgenic animals. A transgenic founder animal can be identified
based upon the presence of the transgene in its genome and/or
expression of mRNA encoding the transgene in tissues or cells of
the animals. A transgenic founder animal can then be used to breed
additional animals carrying the transgene. Moreover, transgenic
animals carrying the transgene can further be bred to other
transgenic animals carrying other transgenes.
[0212] To create an homologous recombinant animal, a vector is
prepared which contains at least a portion of a gene encoding a
marker protein into which a deletion, addition or substitution has
been introduced to thereby alter, e.g., functionally disrupt, the
gene. In a preferred embodiment, the vector is designed such that,
upon homologous recombination, the endogenous gene is functionally
disrupted (i.e., no longer encodes a functional protein; also
referred to as a "knock out" vector). Alternatively, the vector can
be designed such that, upon homologous recombination, the
endogenous gene is mutated or otherwise altered but still encodes
functional protein (e.g., the upstream regulatory region can be
altered to thereby alter the expression of the endogenous protein).
In the homologous recombination vector, the altered portion of the
gene is flanked at its 5' and 3' ends by additional nucleic acid of
the gene to allow for homologous recombination to occur between the
exogenous gene carried by the vector and an endogenous gene in an
embryonic stem cell. The additional flanking nucleic acid sequences
are of sufficient length for successful homologous recombination
with the endogenous gene. Typically, several kilobases of flanking
DNA (both at the 5' and 3' ends) are included in the vector (see,
e.g., Thomas and Capecchi, 1987, Cell 51:503 for a description of
homologous recombination vectors). The vector is introduced into an
embryonic stem cell line (e.g., by electroporation) and cells in
which the introduced gene has homologously recombined with the
endogenous gene are selected (see, e.g., Li et al., 1992, Cell
69:915). The selected cells are then injected into a blastocyst of
an animal (e.g., a mouse) to form aggregation chimeras (see, e.g.,
Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, Robertson, Ed., IRL, Oxford, 1987, pp. 113-152). A
chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal and the embryo brought to term.
Progeny harboring the homologously recombined DNA in their germ
cells can be used to breed animals in which all cells of the animal
contain the homologously recombined DNA by germline transmission of
the transgene. Methods for constructing homologous recombination
vectors and homologous recombinant animals are described further in
Bradley (1991) Current Opinion in Bio/Technology 2:823-829 and in
PCT Publication NOS. WO 90/11354, WO 91/01140, WO 92/0968, and WO
93/04169.
[0213] In another embodiment, transgenic non-human animals can be
produced which contain selected systems which allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a
recombinase system is the FLP recombinase system of Saccharomyces
cerevisiae (O'Gorman et al., 1991, Science 251:1351-1355). If a
cre/loxP recombinase system is used to regulate expression of the
transgene, animals containing transgenes encoding both the Cre
recombinase and a selected protein are required. Such animals can
be provided through the construction of "double" transgenic
animals, e.g., by mating two transgenic animals, one containing a
transgene encoding a selected protein and the other containing a
transgene encoding a recombinase.
[0214] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut
et al. (1997) Nature 385:810-813 and PCT Publication NOS. WO
97/07668 and WO 97/07669.
IV. Pharmaceutical Compositions
[0215] The nucleic acid molecules, polypeptides, and antibodies
(also referred to herein as "active compounds") of the invention
can be incorporated into pharmaceutical compositions suitable for
administration. Such compositions typically comprise the nucleic
acid molecule, protein, or antibody and a pharmaceutically
acceptable carrier. As used herein the language "pharmaceutically
acceptable carrier" is intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0216] The invention includes methods for preparing pharmaceutical
compositions for modulating the expression or activity of a marker
nucleic acid or protein. Such methods comprise formulating a
pharmaceutically acceptable carrier with an agent which modulates
expression or activity of a marker nucleic acid or protein. Such
compositions can further include additional active agents. Thus,
the invention further includes methods for preparing a
pharmaceutical composition by formulating a pharmaceutically
acceptable carrier with an agent which modulates expression or
activity of a marker nucleic acid or protein and one or more
additional active compounds.
[0217] The invention also provides methods (also referred to herein
as "screening assays") for identifying modulators, i.e., candidate
or test compounds or agents (e.g., peptides, peptidomimetics,
peptoids, small molecules or other drugs) which (a) bind to the
marker, or (b) have a modulatory (e.g., stimulatory or inhibitory)
effect on the activity of the marker or, more specifically, (c)
have a modulatory effect on the interactions of the marker with one
or more of its natural substrates (e.g., peptide, protein, hormone,
co-factor, or nucleic acid), or (d) have a modulatory effect on the
expression of the marker. Such assays typically comprise a reaction
between the marker and one or more assay components. The other
components may be either the test compound itself, or a combination
of test compound and a natural binding partner of the marker.
[0218] The test compounds of the present invention may be obtained
from any available source, including systematic libraries of
natural and/or synthetic compounds. Test compounds may also be
obtained by any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries; peptoid
libraries (libraries of molecules having the functionalities of
peptides, but with a novel, non-peptide backbone which are
resistant to enzymatic degradation but which nevertheless remain
bioactive; see, e.g., Zuckermann et al., 1994, J. Med. Chem.
37:2678-85); spatially addressable parallel solid phase or solution
phase libraries; synthetic library methods requiring deconvolution;
the `one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library and peptoid library approaches are limited to peptide
libraries, while the other four approaches are applicable to
peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam, 1997, Anticancer Drug Des. 12:145).
[0219] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med.
Chem. 37:1233.
[0220] Libraries of compounds may be presented in solution (e.g.,
Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991,
Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556),
bacteria and/or spores, (Ladner, U.S. Pat. No. 5,223,409), plasmids
(Cull et al, 1992, Proc Natl Acad Sci USA 89:1865-1869) or on phage
(Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science
249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci.
87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; Ladner,
supra.).
[0221] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a
protein encoded by or corresponding to a marker or biologically
active portion thereof. In another embodiment, the invention
provides assays for screening candidate or test compounds which
bind to a protein encoded by or corresponding to a marker or
biologically active portion thereof. Determining the ability of the
test compound to directly bind to a protein can be accomplished,
for example, by coupling the compound with a radioisotope or
enzymatic label such that binding of the compound to the marker can
be determined by detecting the labeled marker compound in a
complex. For example, compounds (e.g., marker substrates) can be
labeled with .sup.125I, .sup.35S, .sup.14C, or .sup.3H, either
directly or indirectly, and the radioisotope detected by direct
counting of radioemission or by scintillation counting.
Alternatively, assay components can be enzymatically labeled with,
for example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product.
[0222] In another embodiment, the invention provides assays for
screening candidate or test compounds which modulate the expression
of a marker or the activity of a protein encoded by or
corresponding to a marker, or a biologically active portion
thereof. In all likelihood, the protein encoded by or corresponding
to the marker can, in vivo, interact with one or more molecules,
such as but not limited to, peptides, proteins, hormones, cofactors
and nucleic acids. For the purposes of this discussion, such
cellular and extracellular molecules are referred to herein as
"binding partners" or marker "substrate".
[0223] One necessary embodiment of the invention in order to
facilitate such screening is the use of a protein encoded by or
corresponding to marker to identify the protein's natural in vivo
binding partners. There are many ways to accomplish this which are
known to one skilled in the art. One example is the use of the
marker protein as "bait protein" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al, 1993, Cell 72:223-232; Madura et al, 1993, J. Biol. Chem.
268:12046-12054; Bartel et al, 1993, Biotechniques 14:920-924;
Iwabuchi et al, 1993 Oncogene 8:1693-1696; Brent WO94/10300) in
order to identify other proteins which bind to or interact with the
marker (binding partners) and, therefore, are possibly involved in
the natural function of the marker. Such marker binding partners
are also likely to be involved in the propagation of signals by the
marker protein or downstream elements of a marker protein-mediated
signaling pathway. Alternatively, such marker protein binding
partners may also be found to be inhibitors of the marker
protein.
[0224] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that encodes a marker
protein fused to a gene encoding the DNA binding domain of a known
transcription factor (e.g., GAL-4). In the other construct, a DNA
sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming a marker-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be readily detected and cell colonies
containing the functional transcription factor can be isolated and
used to obtain the cloned gene which encodes the protein which
interacts with the marker protein.
[0225] In a further embodiment, assays may be devised through the
use of the invention for the purpose of identifying compounds which
modulate (e.g., affect either positively or negatively)
interactions between a marker protein and its substrates and/or
binding partners. Such compounds can include, but are not limited
to, molecules such as antibodies, peptides, hormones,
oligonucleotides, nucleic acids, and analogs thereof. Such
compounds may also be obtained from any available source, including
systematic libraries of natural and/or synthetic compounds. The
preferred assay components for use in this embodiment is an
cervical cancer marker protein identified herein, the known binding
partner and/or substrate of same, and the test compound. Test
compounds can be supplied from any source.
[0226] The basic principle of the assay systems used to identify
compounds that interfere with the interaction between the marker
protein and its binding partner involves preparing a reaction
mixture containing the marker protein and its binding partner under
conditions and for a time sufficient to allow the two products to
interact and bind, thus forming a complex. In order to test an
agent for inhibitory activity, the reaction mixture is prepared in
the presence and absence of the test compound. The test compound
can be initially included in the reaction mixture, or can be added
at a time subsequent to the addition of the marker protein and its
binding partner. Control reaction mixtures are incubated without
the test compound or with a placebo. The formation of any complexes
between the marker protein and its binding partner is then
detected. The formation of a complex in the control reaction, but
less or no such formation in the reaction mixture containing the
test compound, indicates that the compound interferes with the
interaction of the marker protein and its binding partner.
Conversely, the formation of more complex in the presence of
compound than in the control reaction indicates that the compound
may enhance interaction of the marker protein and its binding
partner.
[0227] The assay for compounds that interfere with the interaction
of the marker protein with its binding partner may be conducted in
a heterogeneous or homogeneous format. Heterogeneous assays involve
anchoring either the marker protein or its binding partner onto a
solid phase and detecting complexes anchored to the solid phase at
the end of the reaction. In homogeneous assays, the entire reaction
is carried out in a liquid phase. In either approach, the order of
addition of reactants can be varied to obtain different information
about the compounds being tested. For example, test compounds that
interfere with the interaction between the marker proteins and the
binding partners (e.g., by competition) can be identified by
conducting the reaction in the presence of the test substance,
i.e., by adding the test substance to the reaction mixture prior to
or simultaneously with the marker and its interactive binding
partner. Alternatively, test compounds that disrupt preformed
complexes, e.g., compounds with higher binding constants that
displace one of the components from the complex, can be tested by
adding the test compound to the reaction mixture after complexes
have been formed. The various formats are briefly described
below.
[0228] In a heterogeneous assay system, either the marker protein
or its binding partner is anchored onto a solid surface or matrix,
while the other corresponding non-anchored component may be
labeled, either directly or indirectly. In practice, microtitre
plates are often utilized for this approach. The anchored species
can be immobilized by a number of methods, either non-covalent or
covalent, that are typically well known to one who practices the
art. Non-covalent attachment can often be accomplished simply by
coating the solid surface with a solution of the marker protein or
its binding partner and drying. Alternatively, an immobilized
antibody specific for the assay component to be anchored can be
used for this purpose. Such surfaces can often be prepared in
advance and stored.
[0229] In related embodiments, a fusion protein can be provided
which adds a domain that allows one or both of the assay components
to be anchored to a matrix. For example,
glutathione-S-transferase/marker fusion proteins or
glutathione-S-transferase/binding partner can be adsorbed onto
glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or
glutathione derivatized microtiter plates, which are then combined
with the test compound or the test compound and either the
non-adsorbed marker or its binding partner, and the mixture
incubated under conditions conducive to complex formation (e.g.,
physiological conditions). Following incubation, the beads or
microtiter plate wells are washed to remove any unbound assay
components, the immobilized complex assessed either directly or
indirectly, for example, as described above. Alternatively, the
complexes can be dissociated from the matrix, and the level of
marker binding or activity determined using standard
techniques.
[0230] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either a marker protein or a marker protein binding partner can be
immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated marker protein or target molecules can be prepared
from biotin-NHS(N-hydroxy-succinimide) using techniques known in
the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,
Ill.), and immobilized in the wells of streptavidin-coated 96 well
plates (Pierce Chemical). In certain embodiments, the
protein-immobilized surfaces can be prepared in advance and
stored.
[0231] In order to conduct the assay, the corresponding partner of
the immobilized assay component is exposed to the coated surface
with or without the test compound. After the reaction is complete,
unreacted assay components are removed (e.g., by washing) and any
complexes formed will remain immobilized on the solid surface. The
detection of complexes anchored on the solid surface can be
accomplished in a number of ways. Where the non-immobilized
component is pre-labeled, the detection of label immobilized on the
surface indicates that complexes were formed. Where the
non-immobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the initially non-immobilized species
(the antibody, in turn, can be directly labeled or indirectly
labeled with, e.g., a labeled anti-Ig antibody). Depending upon the
order of addition of reaction components, test compounds which
modulate (inhibit or enhance) complex formation or which disrupt
preformed complexes can be detected.
[0232] In an alternate embodiment of the invention, a homogeneous
assay may be used. This is typically a reaction, analogous to those
mentioned above, which is conducted in a liquid phase in the
presence or absence of the test compound. The formed complexes are
then separated from unreacted components, and the amount of complex
formed is determined. As mentioned for heterogeneous assay systems,
the order of addition of reactants to the liquid phase can yield
information about which test compounds modulate (inhibit or
enhance) complex formation and which disrupt preformed
complexes.
[0233] In such a homogeneous assay, the reaction products may be
separated from unreacted assay components by any of a number of
standard techniques, including but not limited to: differential
centrifugation, chromatography, electrophoresis and
immunoprecipitation. In differential centrifugation, complexes of
molecules may be separated from uncomplexed molecules through a
series of centrifugal steps, due to the different sedimentation
equilibria of complexes based on their different sizes and
densities (see, for example, Rivas, G., and Minton, A. P., Trends
Biochem Sci 1993 August; 18(8):284-7). Standard chromatographic
techniques may also be utilized to separate complexed molecules
from uncomplexed ones. For example, gel filtration chromatography
separates molecules based on size, and through the utilization of
an appropriate gel filtration resin in a column format, for
example, the relatively larger complex may be separated from the
relatively smaller uncomplexed components. Similarly, the
relatively different charge properties of the complex as compared
to the uncomplexed molecules may be exploited to differentially
separate the complex from the remaining individual reactants, for
example through the use of ion-exchange chromatography resins. Such
resins and chromatographic techniques are well known to one skilled
in the art (see, e.g., Heegaard, 1998, J Mol. Recognit. 11:141-148;
Hage and Tweed, 1997, J. Chromatogr. B. Biomed. Sci. Appl.,
699:499-525). Gel electrophoresis may also be employed to separate
complexed molecules from unbound species (see, e.g., Ausubel et al
(eds.), In: Current Protocols in Molecular Biology, J. Wiley &
Sons, New York. 1999). In this technique, protein or nucleic acid
complexes are separated based on size or charge, for example. In
order to maintain the binding interaction during the
electrophoretic process, nondenaturing gels in the absence of
reducing agent are typically preferred, but conditions appropriate
to the particular interactants will be well known to one skilled in
the art. Immunoprecipitation is another common technique utilized
for the isolation of a protein-protein complex from solution (see,
e.g., Ausubel et al (eds.), In: Current Protocols in Molecular
Biology, J. Wiley & Sons, New York. 1999). In this technique,
all proteins binding to an antibody specific to one of the binding
molecules are precipitated from solution by conjugating the
antibody to a polymer bead that may be readily collected by
centrifugation. The bound assay components are released from the
beads (through a specific proteolysis event or other technique well
known in the art which will not disturb the protein-protein
interaction in the complex), and a second immunoprecipitation step
is performed, this time utilizing antibodies specific for the
correspondingly different interacting assay component. In this
manner, only formed complexes should remain attached to the beads.
Variations in complex formation in both the presence and the
absence of a test compound can be compared, thus offering
information about the ability of the compound to modulate
interactions between the marker protein and its binding
partner.
[0234] Also within the scope of the present invention are methods
for direct detection of interactions between the marker protein and
its natural binding partner and/or a test compound in a homogeneous
or heterogeneous assay system without further sample manipulation.
For example, the technique of fluorescence energy transfer may be
utilized (see, e.g., Lakowicz et al, U.S. Pat. No. 5,631,169;
Stavrianopoulos et al, U.S. Pat. No. 4,868,103). Generally, this
technique involves the addition of a fluorophore label on a first
`donor` molecule (e.g., marker or test compound) such that its
emitted fluorescent energy will be absorbed by a fluorescent label
on a second, `acceptor` molecule (e.g., marker or test compound),
which in turn is able to fluoresce due to the absorbed energy.
Alternately, the `donor` protein molecule may simply utilize the
natural fluorescent energy of tryptophan residues. Labels are
chosen that emit different wavelengths of light, such that the
`acceptor` molecule label may be differentiated from that of the
`donor`. Since the efficiency of energy transfer between the labels
is related to the distance separating the molecules, spatial
relationships between the molecules can be assessed. In a situation
in which binding occurs between the molecules, the fluorescent
emission of the `acceptor` molecule label in the assay should be
maximal. An FET binding event can be conveniently measured through
standard fluorometric detection means well known in the art (e.g.,
using a fluorimeter). A test substance which either enhances or
hinders participation of one of the species in the preformed
complex will result in the generation of a signal variant to that
of background. In this way, test substances that modulate
interactions between a marker and its binding partner can be
identified in controlled assays.
[0235] In another embodiment, modulators of marker expression are
identified in a method wherein a cell is contacted with a candidate
compound and the expression of marker mRNA or protein in the cell,
is determined. The level of expression of marker mRNA or protein in
the presence of the candidate compound is compared to the level of
expression of marker mRNA or protein in the absence of the
candidate compound. The candidate compound can then be identified
as a modulator of marker expression based on this comparison. For
example, when expression of marker mRNA or protein is greater
(statistically significantly greater) in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of marker mRNA or protein expression.
Conversely, when expression of marker mRNA or protein is less
(statistically significantly less) in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of marker mRNA or protein expression. The level of
marker mRNA or protein expression in the cells can be determined by
methods described herein for detecting marker mRNA or protein.
[0236] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a cell
free assay, and the ability of the agent to modulate the activity
of a marker protein can be further confirmed in vivo, e.g., in a
whole animal model for cellular transformation and/or
tumorigenesis.
[0237] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, an
agent identified as described herein (e.g., a marker modulating
agent, an antisense marker nucleic acid molecule, a marker-specific
antibody, or a marker-binding partner) can be used in an animal
model to determine the efficacy, toxicity, or side effects of
treatment with such an agent. Alternatively, an agent identified as
described herein can be used in an animal model to determine the
mechanism of action of such an agent. Furthermore, this invention
pertains to uses of novel agents identified by the above-described
screening assays for treatments as described herein.
[0238] It is understood that appropriate doses of small molecule
agents and protein or polypeptide agents depends upon a number of
factors within the knowledge of the ordinarily skilled physician,
veterinarian, or researcher. The dose(s) of these agents will vary,
for example, depending upon the identity, size, and condition of
the subject or sample being treated, further depending upon the
route by which the composition is to be administered, if
applicable, and the effect which the practitioner desires the agent
to have upon the nucleic acid or polypeptide of the invention.
Exemplary doses of a small molecule include milligram or microgram
amounts per kilogram of subject or sample weight (e.g. about 1
microgram per kilogram to about 500 milligrams per kilogram, about
100 micrograms per kilogram to about 5 milligrams per kilogram, or
about 1 microgram per kilogram to about 50 micrograms per
kilogram). Exemplary doses of a protein or polypeptide include
gram, milligram or microgram amounts per kilogram of subject or
sample weight (e.g. about 1 microgram per kilogram to about 5 grams
per kilogram, about 100 micrograms per kilogram to about 500
milligrams per kilogram, or about 1 milligram per kilogram to about
50 milligrams per kilogram). It is furthermore understood that
appropriate doses of one of these agents depend upon the potency of
the agent with respect to the expression or activity to be
modulated. Such appropriate doses can be determined using the
assays described herein. When one or more of these agents is to be
administered to an animal (e.g. a human) in order to modulate
expression or activity of a polypeptide or nucleic acid of the
invention, a physician, veterinarian, or researcher can, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific agent employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0239] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediamine-tetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampules, disposable syringes or multiple dose vials made of glass
or plastic.
[0240] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL (BASF; Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0241] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a polypeptide or antibody)
in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle which
contains a basic dispersion medium, and then incorporating the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying which yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0242] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
[0243] Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches, and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0244] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from a pressurized
container or dispenser which contains a suitable propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer.
[0245] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0246] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0247] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
having monoclonal antibodies incorporated therein or thereon) can
also be used as pharmaceutically acceptable carriers. These can be
prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811.
[0248] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0249] For antibodies, the preferred dosage is 0.1 mg/kg to 100
mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the
antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg
is usually appropriate. Generally, partially human antibodies and
fully human antibodies have a longer half-life within the human
body than other antibodies. Accordingly, lower dosages and less
frequent administration is often possible. Modifications such as
lipidation can be used to stabilize antibodies and to enhance
uptake and tissue penetration (e.g., into the cervical epithelium).
A method for lipidation of antibodies is described by Cruikshank et
al. (1997) J. Acquired Immune Deficiency Syndromes and Human
Retrovirology 14:193.
[0250] The invention also provides vaccine compositions for the
prevention and/or treatment of cervical cancer. The invention
provides cervical cancer vaccine compositions in which a protein of
a marker of Table 1, or a combination of proteins of the markers of
Table 1, are introduced into a subject in order to stimulate an
immune response against the cervical cancer. The invention also
provides cervical cancer vaccine compositions in which a gene
expression construct, which expresses a marker or fragment of a
marker identified in Table 1, is introduced into the subject such
that a protein or fragment of a protein encoded by a marker of
Table 1 is produced by transfected cells in the subject at a higher
than normal level and elicits an immune response.
[0251] In one embodiment, a cervical cancer vaccine is provided and
employed as an immunotherapeutic agent for the prevention of
cervical cancer. In another embodiment, a cervical cancer vaccine
is provided and employed as an immunotherapeutic agent for the
treatment of cervical cancer.
[0252] By way of example, a cervical cancer vaccine comprised of
the proteins of the markers of Table 1, may be employed for the
prevention and/or treatment of cervical cancer in a subject by
administering the vaccine by a variety of routes, e.g.,
intradermally, subcutaneously, or intramuscularly. In addition, the
cervical cancer vaccine can be administered together with adjuvants
and/or immunomodulators to boost the activity of the vaccine and
the subject's response. In one embodiment, devices and/or
compositions containing the vaccine, suitable for sustained or
intermittent release could be, implanted in the body or topically
applied thereto for the relatively slow release of such materials
into the body. The cervical cancer vaccine can be introduced along
with immunomodulatory compounds, which can alter the type of immune
response produced in order to produce a response which will be more
effective in eliminating the cancer.
[0253] In another embodiment, a cervical cancer vaccine comprised
of an expression construct of the markers of Table 1, may be
introduced by injection into muscle or by coating onto
microprojectiles and using a device designed for the purpose to
fire the projectiles at high speed into the skin. The cells of the
subject will then express the protein(s) or fragments of proteins
of the markers of Table 1 and induce an immune response. In
addition, the cervical cancer vaccine may be introduced along with
expression constructs for immunomodulatory molecules, such as
cytokines, which may increase the immune response or modulate the
type of immune response produced in order to produce a response
which will be more effective in eliminating the cancer.
[0254] The marker nucleic acid molecules can be inserted into
vectors and used as gene therapy vectors. Gene therapy vectors can
be delivered to a subject by, for example, intravenous injection,
local administration (U.S. Pat. No. 5,328,470), or by stereotactic
injection (see, e.g., Chen et al., 1994, Proc. Natl. Acad. Sci. USA
91:3054-3057). The pharmaceutical preparation of the gene therapy
vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is imbedded. Alternatively, where the complete
gene delivery vector can be produced intact from recombinant cells,
e.g. retroviral vectors, the pharmaceutical preparation can include
one or more cells which produce the gene delivery system.
[0255] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
V. Predictive Medicine
[0256] The present invention pertains to the field of predictive
medicine in which diagnostic assays, prognostic assays,
pharmacogenomics, and monitoring clinical trails are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. Accordingly, one aspect of the present invention
relates to diagnostic assays for determining the level of
expression of one or more marker proteins or nucleic acids, in
order to determine whether an individual is at risk of developing
cervical cancer. Such assays can be used for prognostic or
predictive purposes to thereby prophylactically treat an individual
prior to the onset of the cancer.
[0257] Yet another aspect of the invention pertains to monitoring
the influence of agents (e.g., drugs or other compounds
administered either to inhibit cervical cancer or to treat or
prevent any other disorder {i.e. in order to understand any
cervical carcinogenic effects that such treatment may have}) on the
expression or activity of a marker of the invention in clinical
trials. These and other agents are described in further detail in
the following sections.
[0258] A. Diagnostic Assays
[0259] An exemplary method for detecting the presence or absence of
a marker protein or nucleic acid in a biological sample involves
obtaining a biological sample (e.g. a cervical-associated body
fluid) from a test subject and contacting the biological sample
with a compound or an agent capable of detecting the polypeptide or
nucleic acid (e.g., mRNA, genomic DNA, or cDNA). The detection
methods of the invention can thus be used to detect mRNA, protein,
cDNA, or genomic DNA, for example, in a biological sample in vitro
as well as in vivo. For example, in vitro techniques for detection
of mRNA include Northern hybridizations and in situ hybridizations.
In vitro techniques for detection of a marker protein include
enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. In vitro techniques
for detection of genomic DNA include Southern hybridizations.
Furthermore, in vivo techniques for detection of a marker protein
include introducing into a subject a labeled antibody directed
against the protein or fragment thereof. For example, the antibody
can be labeled with a radioactive marker whose presence and
location in a subject can be detected by standard imaging
techniques.
[0260] A general principle of such diagnostic and prognostic assays
involves preparing a sample or reaction mixture that may contain a
marker, and a probe, under appropriate conditions and for a time
sufficient to allow the marker and probe to interact and bind, thus
forming a complex that can be removed and/or detected in the
reaction mixture. These assays can be conducted in a variety of
ways.
[0261] For example, one method to conduct such an assay would
involve anchoring the marker or probe onto a solid phase support,
also referred to as a substrate, and detecting target marker/probe
complexes anchored on the solid phase at the end of the reaction.
In one embodiment of such a method, a sample from a subject, which
is to be assayed for presence and/or concentration of marker, can
be anchored onto a carrier or solid phase support. In another
embodiment, the reverse situation is possible, in which the probe
can be anchored to a solid phase and a sample from a subject can be
allowed to react as an unanchored component of the assay.
[0262] There are many established methods for anchoring assay
components to a solid phase. These include, without limitation,
marker or probe molecules which are immobilized through conjugation
of biotin and streptavidin. Such biotinylated assay components can
be prepared from biotin-NHS (N-hydroxy-succinimide) using
techniques known in the art (e.g., biotinylation kit, Pierce
Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical). In certain
embodiments, the surfaces with immobilized assay components can be
prepared in advance and stored.
[0263] Other suitable carriers or solid phase supports for such
assays include any material capable of binding the class of
molecule to which the marker or probe belongs. Well-known supports
or carriers include, but are not limited to, glass, polystyrene,
nylon, polypropylene, nylon, polyethylene, dextran, amylases,
natural and modified celluloses, polyacrylamides, gabbros, and
magnetite.
[0264] In order to conduct assays with the above mentioned
approaches, the non-immobilized component is added to the solid
phase upon which the second component is anchored. After the
reaction is complete, uncomplexed components may be removed (e.g.,
by washing) under conditions such that any complexes formed will
remain immobilized upon the solid phase. The detection of
marker/probe complexes anchored to the solid phase can be
accomplished in a number of methods outlined herein.
[0265] In a preferred embodiment, the probe, when it is the
unanchored assay component, can be labeled for the purpose of
detection and readout of the assay, either directly or indirectly,
with detectable labels discussed herein and which are well-known to
one skilled in the art.
[0266] It is also possible to directly detect marker/probe complex
formation without further manipulation or labeling of either
component (marker or probe), for example by utilizing the technique
of fluorescence energy transfer (see, for example, Lakowicz et al.,
U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.
4,868,103). A fluorophore label on the first, `donor` molecule is
selected such that, upon excitation with incident light of
appropriate wavelength, its emitted fluorescent energy will be
absorbed by a fluorescent label on a second `acceptor` molecule,
which in turn is able to fluoresce due to the absorbed energy.
Alternately, the `donor` protein molecule may simply utilize the
natural fluorescent energy of tryptophan residues. Labels are
chosen that emit different wavelengths of light, such that the
`acceptor` molecule label may be differentiated from that of the
`donor`. Since the efficiency of energy transfer between the labels
is related to the distance separating the molecules, spatial
relationships between the molecules can be assessed. In a situation
in which binding occurs between the molecules, the fluorescent
emission of the `acceptor` molecule label in the assay should be
maximal. An FET binding event can be conveniently measured through
standard fluorometric detection means well known in the art (e.g.,
using a fluorimeter).
[0267] In another embodiment, determination of the ability of a
probe to recognize a marker can be accomplished without labeling
either assay component (probe or marker) by utilizing a technology
such as real-time Biomolecular Interaction Analysis (BIA) (see,
e.g., Sjolander, S, and Urbaniczky, C., 1991, Anal. Chem.
63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol.
5:699-705). As used herein, "BIA" or "surface plasmon resonance" is
a technology for studying biospecific interactions in real time,
without labeling any of the interactants (e.g., BIAcore). Changes
in the mass at the binding surface (indicative of a binding event)
result in alterations of the refractive index of light near the
surface (the optical phenomenon of surface plasmon resonance
(SPR)), resulting in a detectable signal which can be used as an
indication of real-time reactions between biological molecules.
[0268] Alternatively, in another embodiment, analogous diagnostic
and prognostic assays can be conducted with marker and probe as
solutes in a liquid phase. In such an assay, the complexed marker
and probe are separated from uncomplexed components by any of a
number of standard techniques, including but not limited to:
differential centrifugation, chromatography, electrophoresis and
immunoprecipitation. In differential centrifugation, marker/probe
complexes may be separated from uncomplexed assay components
through a series of centrifugal steps, due to the different
sedimentation equilibria of complexes based on their different
sizes and densities (see, for example, Rivas, G., and Minton, A.
P., 1993, Trends Biochem Sci. 18(8):284-7). Standard
chromatographic techniques may also be utilized to separate
complexed molecules from uncomplexed ones. For example, gel
filtration chromatography separates molecules based on size, and
through the utilization of an appropriate gel filtration resin in a
column format, for example, the relatively larger complex may be
separated from the relatively smaller uncomplexed components.
Similarly, the relatively different charge properties of the
marker/probe complex as compared to the uncomplexed components may
be exploited to differentiate the complex from uncomplexed
components, for example through the utilization of ion-exchange
chromatography resins. Such resins and chromatographic techniques
are well known to one skilled in the art (see, e.g., Heegaard, N.
H., 1998, J. Mol. Recognit. Winter 11(1-6):141-8; Hage, D. S., and
Tweed, S. A. J Chromatogr B Biomed Sci Appl 1997 Oct. 10;
699(1-2):499-525). Gel electrophoresis may also be employed to
separate complexed assay components from unbound components (see,
e.g., Ausubel et al., ed., Current Protocols in Molecular Biology,
John Wiley & Sons, New York, 1987-1999). In this technique,
protein or nucleic acid complexes are separated based on size or
charge, for example. In order to maintain the binding interaction
during the electrophoretic process, non-denaturing gel matrix
materials and conditions in the absence of reducing agent are
typically preferred. Appropriate conditions to the particular assay
and components thereof will be well known to one skilled in the
art.
[0269] In a particular embodiment, the level of marker mRNA can be
determined both by in situ and by in vitro formats in a biological
sample using methods known in the art. The term "biological sample"
is intended to include tissues, cells, biological fluids and
isolates thereof, isolated from a subject, as well as tissues,
cells and fluids present within a subject. Many expression
detection methods use isolated RNA. For in vitro methods, any RNA
isolation technique that does not select against the isolation of
mRNA can be utilized for the purification of RNA from cervical
cells (see, e.g., Ausubel et al., ed., Current Protocols in
Molecular Biology, John Wiley & Sons, New York 1987-1999).
Additionally, large numbers of tissue samples can readily be
processed using techniques well known to those of skill in the art,
such as, for example, the single-step RNA isolation process of
Chomczynski (1989, U.S. Pat. No. 4,843,155).
[0270] The isolated mRNA can be used in hybridization or
amplification assays that include, but are not limited to, Southern
or Northern analyses, polymerase chain reaction analyses and probe
arrays. One preferred diagnostic method for the detection of mRNA
levels involves contacting the isolated mRNA with a nucleic acid
molecule (probe) that can hybridize to the mRNA encoded by the gene
being detected. The nucleic acid probe can be, for example, a
full-length cDNA, or a portion thereof, such as an oligonucleotide
of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length
and sufficient to specifically hybridize under stringent conditions
to a mRNA or genomic DNA encoding a marker of the present
invention. Other suitable probes for use in the diagnostic assays
of the invention are described herein. Hybridization of an mRNA
with the probe indicates that the marker in question is being
expressed.
[0271] In one format, the mRNA is immobilized on a solid surface
and contacted with a probe, for example by running the isolated
mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as nitrocellulose. In an alternative format, the
probe(s) are immobilized on a solid surface and the mRNA is
contacted with the probe(s), for example, in an Affymetrix gene
chip array. A skilled artisan can readily adapt known mRNA
detection methods for use in detecting the level of mRNA encoded by
the markers of the present invention.
[0272] An alternative method for determining the level of mRNA
marker in a sample involves the process of nucleic acid
amplification, e.g., by rtPCR (the experimental embodiment set
forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain
reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193),
self sustained sequence replication (Guatelli et al., 1990, Proc.
Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification
system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA
86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988,
Bio/Technology 6:1197), rolling circle replication (Lizardi et al.,
U.S. Pat. No. 5,854,033) or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques well known to those of skill in the art. These detection
schemes are especially useful for the detection of nucleic acid
molecules if such molecules are present in very low numbers. As
used herein, amplification primers are defined as being a pair of
nucleic acid molecules that can anneal to 5' or 3' regions of a
gene (plus and minus strands, respectively, or vice-versa) and
contain a short region in between. In general, amplification
primers are from about 10 to 30 nucleotides in length and flank a
region from about 50 to 200 nucleotides in length. Under
appropriate conditions and with appropriate reagents, such primers
permit the amplification of a nucleic acid molecule comprising the
nucleotide sequence flanked by the primers.
[0273] For in situ methods, mRNA does not need to be isolated from
the cervical cells prior to detection. In such methods, a cell or
tissue sample is prepared/processed using known histological
methods. The sample is then immobilized on a support, typically a
glass slide, and then contacted with a probe that can hybridize to
mRNA that encodes the marker.
[0274] As an alternative to making determinations based on the
absolute expression level of the marker, determinations may be
based on the normalized expression level of the marker. Expression
levels are normalized by correcting the absolute expression level
of a marker by comparing its expression to the expression of a gene
that is not a marker, e.g., a housekeeping gene that is
constitutively expressed. Suitable genes for normalization include
housekeeping genes such as the actin gene, or epithelial
cell-specific genes. This normalization allows the comparison of
the expression level in one sample, e.g., a patient sample, to
another sample, e.g., a non-cervical cancer sample, or between
samples from different sources.
[0275] Alternatively, the expression level can be provided as a
relative expression level. To determine a relative expression level
of a marker, the level of expression of the marker is determined
for 10 or more samples of normal versus cancer cell isolates,
preferably 50 or more samples, prior to the determination of the
expression level for the sample in question. The mean expression
level of each of the genes assayed in the larger number of samples
is determined and this is used as a baseline expression level for
the marker. The expression level of the marker determined for the
test sample (absolute level of expression) is then divided by the
mean expression value obtained for that marker. This provides a
relative expression level.
[0276] Preferably, the samples used in the baseline determination
will be from cervical cancer or from non-cervical cancer cells of
cervical tissue. The choice of the cell source is dependent on the
use of the relative expression level. Using expression found in
normal tissues as a mean expression score aids in validating
whether the marker assayed is cervical specific (versus normal
cells). In addition, as more data is accumulated, the mean
expression value can be revised, providing improved relative
expression values based on accumulated data. Expression data from
cervical cells provides a means for grading the severity of the
cervical cancer state.
[0277] In another embodiment of the present invention, a marker
protein is detected. A preferred agent for detecting marker protein
of the invention is an antibody capable of binding to such a
protein or a fragment thereof, preferably an antibody with a
detectable label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment or derivative thereof
(e.g., Fab or F(ab').sub.2) can be used. The term "labeled", with
regard to the probe or antibody, is intended to encompass direct
labeling of the probe or antibody by coupling (i.e., physically
linking) a detectable substance to the probe or antibody, as well
as indirect labeling of the probe or antibody by reactivity with
another reagent that is directly labeled. Examples of indirect
labeling include detection of a primary antibody using a
fluorescently labeled secondary antibody and end-labeling of a DNA
probe with biotin such that it can be detected with fluorescently
labeled streptavidin.
[0278] Proteins from cervical cells can be isolated using
techniques that are well known to those of skill in the art. The
protein isolation methods employed can, for example, be such as
those described in Harlow and Lane (Harlow and Lane, 1988,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.).
[0279] A variety of formats can be employed to determine whether a
sample contains a protein that binds to a given antibody. Examples
of such formats include, but are not limited to, enzyme immunoassay
(EIA), radioimmunoassay (RIA), Western blot analysis and enzyme
linked immunoabsorbant assay (ELISA). A skilled artisan can readily
adapt known protein/antibody detection methods for use in
determining whether cervical cells express a marker of the present
invention.
[0280] In one format, antibodies, or antibody fragments or
derivatives, can be used in methods such as Western blots or
immunofluorescence techniques to detect the expressed proteins. In
such uses, it is generally preferable to immobilize either the
antibody or proteins on a solid support. Suitable solid phase
supports or carriers include any support capable of binding an
antigen or an antibody. Well-known supports or carriers include
glass, polystyrene, polypropylene, polyethylene, dextran, nylon,
amylases, natural and modified celluloses, polyacrylamides,
gabbros, and magnetite.
[0281] One skilled in the art will know many other suitable
carriers for binding antibody or antigen, and will be able to adapt
such support for use with the present invention. For example,
protein isolated from cervical cells can be run on a polyacrylamide
gel electrophoresis and immobilized onto a solid phase support such
as nitrocellulose. The support can then be washed with suitable
buffers followed by treatment with the detectably labeled antibody.
The solid phase support can then be washed with the buffer a second
time to remove unbound antibody. The amount of bound label on the
solid support can then be detected by conventional means.
[0282] The invention also encompasses kits for detecting the
presence of a marker protein or nucleic acid in a biological sample
(e.g., cervical smear). Such kits can be used to determine if a
subject is suffering from or is at increased risk of developing
cervical cancer. For example, the kit can comprise a labeled
compound or agent capable of detecting a marker protein or nucleic
acid in a biological sample and means for determining the amount of
the protein or mRNA in the sample (e.g., an antibody which binds
the protein or a fragment thereof, or an oligonucleotide probe
which binds to DNA or mRNA encoding the protein). Kits can also
include instructions for interpreting the results obtained using
the kit.
[0283] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) which
binds to a marker protein; and, optionally, (2) a second, different
antibody which binds to either the protein or the first antibody
and is conjugated to a detectable label.
[0284] For oligonucleotide-based kits, the kit can comprise, for
example: (1) an oligonucleotide, e.g., a detectably labeled
oligonucleotide, which hybridizes to a nucleic acid sequence
encoding a marker protein or (2) a pair of primers useful for
amplifying a marker nucleic acid molecule. The kit can also
comprise, e.g., a buffering agent, a preservative, or a protein
stabilizing agent. The kit can further comprise components
necessary for detecting the detectable label (e.g., an enzyme or a
substrate). The kit can also contain a control sample or a series
of control samples which can be assayed and compared to the test
sample. Each component of the kit can be enclosed within an
individual container and all of the various containers can be
within a single package, along with instructions for interpreting
the results of the assays performed using the kit.
[0285] B. Pharmacogenomics
[0286] The markers of the invention are also useful as
pharmacogenomic markers. As used herein, a "pharmacogenomic marker"
is an objective biochemical marker whose expression level
correlates with a specific clinical drug response or susceptibility
in a patient (see, e.g., McLeod et al. (1999) Eur. J. Cancer
35(12): 1650-1652). The presence or quantity of the pharmacogenomic
marker expression is related to the predicted response of the
patient and more particularly the patient's tumor to therapy with a
specific drug or class of drugs. By assessing the presence or
quantity of the expression of one or more pharmacogenomic markers
in a patient, a drug therapy which is most appropriate for the
patient, or which is predicted to have a greater degree of success,
may be selected. For example, based on the presence or quantity of
RNA or protein encoded by specific tumor markers in a patient, a
drug or course of treatment may be selected that is optimized for
the treatment of the specific tumor likely to be present in the
patient. The use of pharmacogenomic markers therefore permits
selecting or designing the most appropriate treatment for each
cancer patient without trying different drugs or regimes.
[0287] Another aspect of pharmacogenomics deals with genetic
conditions that alters the way the body acts on drugs. These
pharmacogenetic conditions can occur either as rare defects or as
polymorphisms. For example, glucose-6-phosphate dehydrogenase
(G6PD) deficiency is a common inherited enzymopathy in which the
main clinical complication is hemolysis after ingestion of oxidant
drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and
consumption of fava beans.
[0288] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, a PM will show no therapeutic
response, as demonstrated for the analgesic effect of codeine
mediated by its CYP2D6-formed metabolite morphine. The other
extreme are the so called ultra-rapid metabolizers who do not
respond to standard doses. Recently, the molecular basis of
ultra-rapid metabolism has been identified to be due to CYP2D6 gene
amplification.
[0289] Thus, the level of expression of a marker of the invention
in an individual can be determined to thereby select appropriate
agent(s) for therapeutic or prophylactic treatment of the
individual. In addition, pharmacogenetic studies can be used to
apply genotyping of polymorphic alleles encoding drug-metabolizing
enzymes to the identification of an individual's drug
responsiveness phenotype. This knowledge, when applied to dosing or
drug selection, can avoid adverse reactions or therapeutic failure
and thus enhance therapeutic or prophylactic efficiency when
treating a subject with a modulator of expression of a marker of
the invention.
[0290] C. Monitoring Clinical Trials
[0291] Monitoring the influence of agents (e.g., drug compounds) on
the level of expression of a marker of the invention can be applied
not only in basic drug screening, but also in clinical trials. For
example, the effectiveness of an agent to affect marker expression
can be monitored in clinical trials of subjects receiving treatment
for cervical cancer. In a preferred embodiment, the present
invention provides a method for monitoring the effectiveness of
treatment of a subject with an agent (e.g., an agonist, antagonist,
peptidomimetic, protein, peptide, nucleic acid, small molecule, or
other drug candidate) comprising the steps of (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of expression of one or more
selected markers of the invention in the pre-administration sample;
(iii) obtaining one or more post-administration samples from the
subject; (iv) detecting the level of expression of the marker(s) in
the post-administration samples; (v) comparing the level of
expression of the marker(s) in the pre-administration sample with
the level of expression of the marker(s) in the post-administration
sample or samples; and (vi) altering the administration of the
agent to the subject accordingly. For example, increased expression
of the marker gene(s) during the course of treatment may indicate
ineffective dosage and the desirability of increasing the dosage.
Conversely, decreased expression of the marker gene(s) may indicate
efficacious treatment and no need to change dosage.
[0292] D. Electronic Apparatus Readable Media and Arrays
[0293] Electronic apparatus readable media comprising a marker of
the present invention is also provided. As used herein, "electronic
apparatus readable media" refers to any suitable medium for
storing, holding or containing data or information that can be read
and accessed directly by an electronic apparatus. Such media can
include, but are not limited to: magnetic storage media, such as
floppy discs, hard disc storage medium, and magnetic tape; optical
storage media such as compact disc; electronic storage media such
as RAM, ROM, EPROM, EEPROM and the like; general hard disks and
hybrids of these categories such as magnetic/optical storage media.
The medium is adapted or configured for having recorded thereon a
marker of the present invention.
[0294] As used herein, the term "electronic apparatus" is intended
to include any suitable computing or processing apparatus or other
device configured or adapted for storing data or information.
Examples of electronic apparatus suitable for use with the present
invention include stand-alone computing apparatus; networks,
including a local area network (LAN), a wide area network (WAN)
Internet, Intranet, and Extranet; electronic appliances such as a
personal digital assistants (PDAs), cellular phone, pager and the
like; and local and distributed processing systems.
[0295] As used herein, "recorded" refers to a process for storing
or encoding information on the electronic apparatus readable
medium. Those skilled in the art can readily adopt any of the
presently known methods for recording information on known media to
generate manufactures comprising the markers of the present
invention.
[0296] A variety of software programs and formats can be used to
store the marker information of the present invention on the
electronic apparatus readable medium. For example, the marker
nucleic acid sequence can be represented in a word processing text
file, formatted in commercially-available software such as
WordPerfect and MicroSoft Word, or represented in the form of an
ASCII file, stored in a database application, such as DB2, Sybase,
Oracle, or the like, as well as in other forms. Any number of data
processor structuring formats (e.g., text file or database) may be
employed in order to obtain or create a medium having recorded
thereon the markers of the present invention.
[0297] By providing the markers of the invention in readable form,
one can routinely access the marker sequence information for a
variety of purposes. For example, one skilled in the art can use
the nucleotide or amino acid sequences of the present invention in
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. Search means are used to identify fragments or regions of
the sequences of the invention which match a particular target
sequence or target motif.
[0298] The present invention therefore provides a medium for
holding instructions for performing a method for determining
whether a subject has cervical cancer or a pre-disposition to
cervical cancer, wherein the method comprises the steps of
determining the presence or absence of a marker and based on the
presence or absence of the marker, determining whether the subject
has cervical cancer or a pre-disposition to cervical cancer and/or
recommending a particular treatment for cervical cancer or
pre-cervical cancer condition.
[0299] The present invention further provides in an electronic
system and/or in a network, a method for determining whether a
subject has cervical cancer or a pre-disposition to cervical cancer
associated with a marker wherein the method comprises the steps of
determining the presence or absence of the marker, and based on the
presence or absence of the marker, determining whether the subject
has cervical cancer or a pre-disposition to cervical cancer, and/or
recommending a particular treatment for the cervical cancer or
pre-cervical cancer condition. The method may further comprise the
step of receiving phenotypic information associated with the
subject and/or acquiring from a network phenotypic information
associated with the subject.
[0300] The present invention also provides in a network, a method
for determining whether a subject has cervical cancer or a
pre-disposition to cervical cancer associated with a marker, said
method comprising the steps of receiving information associated
with the marker receiving phenotypic information associated with
the subject, acquiring information from the network corresponding
to the marker and/or cervical cancer, and based on one or more of
the phenotypic information, the marker, and the acquired
information, determining whether the subject has a cervical cancer
or a pre-disposition to cervical cancer. The method may further
comprise the step of recommending a particular treatment for the
cervical cancer or pre-cervical cancer condition.
[0301] The present invention also provides a business method for
determining whether a subject has cervical cancer or a
pre-disposition to cervical cancer, said method comprising the
steps of receiving information associated with the marker,
receiving phenotypic information associated with the subject,
acquiring information from the network corresponding to the marker
and/or cervical cancer, and based on one or more of the phenotypic
information, the marker, and the acquired information, determining
whether the subject has cervical cancer or a pre-disposition to
cervical cancer. The method may further comprise the step of
recommending a particular treatment for the cervical cancer or
pre-cervical cancer condition.
[0302] The invention also includes an array comprising a marker of
the present invention. The array can be used to assay expression of
one or more genes in the array. In one embodiment, the array can be
used to assay gene expression in a tissue to ascertain tissue
specificity of genes in the array. In this manner, up to about 7600
genes can be simultaneously assayed for expression. This allows a
profile to be developed showing a battery of genes specifically
expressed in one or more tissues.
[0303] In addition to such qualitative determination, the invention
allows the quantitation of gene expression. Thus, not only tissue
specificity, but also the level of expression of a battery of genes
in the tissue is ascertainable. Thus, genes can be grouped on the
basis of their tissue expression per se and level of expression in
that tissue. This is useful, for example, in ascertaining the
relationship of gene expression between or among tissues. Thus, one
tissue can be perturbed and the effect on gene expression in a
second tissue can be determined. In this context, the effect of one
cell type on another cell type in response to a biological stimulus
can be determined. Such a determination is useful, for example, to
know the effect of cell-cell interaction at the level of gene
expression. If an agent is administered therapeutically to treat
one cell type but has an undesirable effect on another cell type,
the invention provides an assay to determine the molecular basis of
the undesirable effect and thus provides the opportunity to
co-administer a counteracting agent or otherwise treat the
undesired effect. Similarly, even within a single cell type,
undesirable biological effects can be determined at the molecular
level. Thus, the effects of an agent on expression of other than
the target gene can be ascertained and counteracted.
[0304] In another embodiment, the array can be used to monitor the
time course of expression of one or more genes in the array. This
can occur in various biological contexts, as disclosed herein, for
example development of cervical cancer, progression of cervical
cancer, and processes, such a cellular transformation associated
with cervical cancer.
[0305] The array is also useful for ascertaining the effect of the
expression of a gene on the expression of other genes in the same
cell or in different cells. This provides, for example, for a
selection of alternate molecular targets for therapeutic
intervention if the ultimate or downstream target cannot be
regulated.
[0306] The array is also useful for ascertaining differential
expression patterns of one or more genes in normal and abnormal
cells. This provides a battery of genes that could serve as a
molecular target for diagnosis or therapeutic intervention.
[0307] E. Surrogate Markers
[0308] The markers of the invention may serve as surrogate markers
for one or more disorders or disease states or for conditions
leading up to disease states, and in particular, cervical cancer.
As used herein, a "surrogate marker" is an objective biochemical
marker which correlates with the absence or presence of a disease
or disorder, or with the progression of a disease or disorder
(e.g., with the presence or absence of a tumor). The presence or
quantity of such markers is independent of the disease. Therefore,
these markers may serve to indicate whether a particular course of
treatment is effective in lessening a disease state or disorder.
Surrogate markers are of particular use when the presence or extent
of a disease state or disorder is difficult to assess through
standard methodologies (e.g., early stage tumors), or when an
assessment of disease progression is desired before a potentially
dangerous clinical endpoint is reached (e.g., an assessment of
cardiovascular disease may be made using cholesterol levels as a
surrogate marker, and an analysis of HIV infection may be made
using HIV RNA levels as a surrogate marker, well in advance of the
undesirable clinical outcomes of myocardial infarction or
fully-developed AIDS). Examples of the use of surrogate markers in
the art include: Koomen et al. (2000) J. Mass. Spectrom. 35:
258-264; and James (1994) AIDS Treatment News Archive 209.
[0309] The markers of the invention are also useful as
pharmacodynamic markers. As used herein, a "pharmacodynamic marker"
is an objective biochemical marker which correlates specifically
with drug effects. The presence or quantity of a pharmacodynamic
marker is not related to the disease state or disorder for which
the drug is being administered; therefore, the presence or quantity
of the marker is indicative of the presence or activity of the drug
in a subject. For example, a pharmacodynamic marker may be
indicative of the concentration of the drug in a biological tissue,
in that the marker is either expressed or transcribed or not
expressed or transcribed in that tissue in relationship to the
level of the drug. In this fashion, the distribution or uptake of
the drug may be monitored by the pharmacodynamic marker. Similarly,
the presence or quantity of the pharmacodynamic marker may be
related to the presence or quantity of the metabolic product of a
drug, such that the presence or quantity of the marker is
indicative of the relative breakdown rate of the drug in vivo.
Pharmacodynamic markers are of particular use in increasing the
sensitivity of detection of drug effects, particularly when the
drug is administered in low doses. Since even a small amount of a
drug may be sufficient to activate multiple rounds of marker
transcription or expression, the amplified marker may be in a
quantity which is more readily detectable than the drug itself.
Also, the marker may be more easily detected due to the nature of
the marker itself; for example, using the methods described herein,
antibodies may be employed in an immune-based detection system for
a protein marker, or marker-specific radiolabeled probes may be
used to detect a mRNA marker. Furthermore, the use of a
pharmacodynamic marker may offer mechanism-based prediction of risk
due to drug treatment beyond the range of possible direct
observations. Examples of the use of pharmacodynamic markers in the
art include: Matsuda et al. U.S. Pat. No. 6,033,862; Hattis et al.
(1991) Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J.
Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J.
Health-Syst. Pharm. 56 Suppl. 3: S16-S20.
Example 1
Identification of Cervical Cancer Markers by cDNA and Tissue
Microarrays
I. Materials and Methods
Sample Collection and RNA Preparation
[0310] Cervical tissues were collected and snap frozen in liquid
nitrogen. The histology and cellular composition of tissues were
confirmed before RNA extraction was performed. Total RNA was
extracted from the frozen tissues using Trizol Reagent (Life
Technologies) followed by a secondary clean up step with Qiagen's
RNeasy kit to increase RNA probe labeling efficiency (Qiagen,
Valencia Calif.). Only RNA with a 28S/18S ribosomal RNA ratio of at
least 1.0, calculated using Agilent Technologies 2100 Bioanalyzer
(Palo Alto, Calif.), was used in this study.
cDNA Microarray Hybridization
[0311] cDNA microarrays containing 30,732 Unigene clones from
Research Genetics (Hunstville, Ala.) were generated on nylon
filters. A total of 4-6 ug of total RNA was used as template to
generate radioactively labeled cDNA by reverse transcription with
.sup.33P-dCTP, oligo dT-30 primer and Superscript II Reverse
Transcriptase (Life Technologies). .sup.33P-labeled first strand
cDNA was preannealed with cot-1 DNA and poly-dA 40-60 (Pharmacia,
Peapack, N.J.) to reduce non-specific hybridization. Each filter
was hybridized at 65.degree. C. for 16 hours with approximately
6.times.10.sup.6 counts of labeled probe in a buffer containing 7%
sodium dodecyl sulfate (SDS), 250 mM Na.sub.3PO.sub.4 (pH 7.2), 1
mM EDTA, 0.5% Casein-Hammerstein and 0.1 mg/ml of denatured salmon
sperm DNA. After the filters were washed with 4% and 1% SDS wash
buffer (20 mM Na.sub.3PO.sub.4 (pH 7.2), 1 mM EDTA and 4% or 1%
SDS), they were exposed to Fuji Phosphoimager screens and scanned
using a Fuji scanner BAS 2500. Spots were quantitated using an
automated array analysis program, Grid Guru v1.0, developed at
Millennium Pharmaceuticals, Inc.
Marker Scoring Algorithm and Data Analysis
[0312] To correct for differences in hybridization efficiency, the
digitized data from each microarray filter was normalized by the
median intensity of all spots on that filter. Both array-based and
gene-based hierarchical clustering was performed and visualized
using Stanford's Gene Cluster and Tree View software.
Differentially expressed genes were ranked by calculating the
Marker Score for each gene.
[0313] To compute Marker Score, the samples were divided into
control and tester groups. The starting point for the Marker Score
is average fold change (ratio) of the tester samples above the
control samples. The score was designed to reflect both the degree
of change (the expression ratio) and the number of tester samples
showing differential expression, while not being dominated by a
small fraction of tester samples with very high values. To reduce
this "outlier" effect, genes were treated with expression ratios
greater than 10 as not meaningfully different from those with
ratios of 10. This desired performance from a Marker Score was
accomplished by transforming the tester:control expression ratio
using an asymptotic compression function before taking the average
fold-change across tester samples. A Marker Score has a value of 1
when the testers do not appear to be expressed more highly than the
controls, and a value greater than 1 otherwise. A Marker Score
cannot exceed a value of 10 for any gene.
[0314] The Marker Score S.sub.g for gene g is therefore computed as
the average of compressed tester:control ratios:
S.sub.g=(.SIGMA.S.sub.gs)/N.sub.tester [0315]
S.sub.gs=C(x.sub.gs/(k+x.sub.g.sup.Q)), where S.sub.gs represents
the Marker Score for gene g and the sample s, [0316] C(r) is the
compression function C(r)=A(1-e.sup.-r/A) for r.gtoreq.1, and
C(r)=1 for r<1, [0317] A is an upper asymptote on the
fold-change value (we used 10), [0318] x.sub.gs is the expression
value of gene g on sample s, [0319] x.sub.g.sup.Q is the Qth
percentile of the control samples' expression value; typically
Q=50, [0320] k is a constant reflecting the additive noise in the
data, i.e., the fixed component of the variance in repeated
measurements. A value of 0.25 was derived for this parameter from
calibration experiments using microarray technology. [0321]
N.sub.tester The number of tester samples
In Situ Hybridization of Tissue Microarrays
[0322] Formalin-fixed, paraffin embedded cervical tissue
microarrays containing tissue cores from normal, low-grade squamous
intraepithelial lesions (LSIL), high-grade squamous intraepithelial
lesions (HSIL), squamous cell carcinomas (SCC) and adenocarcinomas
(ACA) were provided. Prehybridization treatment was performed with
an automatic Tissue-Tek DRS 2000 Slide Stainer (Sakura, Torrance,
Calif.) using a previously described protocol (Duncan, L. M., et
al., 2001, J. Clin. Oncol. 19(2): 568-576). The cervical tissues
were deparaffinized, rehydrated and postfixed with 4%
paraformaldehyde in PBS for 15 minutes. After washing with PBS, the
tissue microarrays were digested with 2 ug/ml proteinase K at
37.degree. C. for 15 minutes and again incubated with 4%
paraformaldehyde/PBS for 10 minutes. Tissue sections were
subsequently incubated with 0.2N HCL for 10 minutes, 0.25% acetic
anhydride/0.1 mol/L triethanolamine for 10 minutes, and dehydrated
with graded ethanol. Antisense probes were labeled with
.sup.35S-UTP in an in vitro transcription reaction (Riboprobe
Combination System, Promega, Madison, Wis.) using 500 ng of
linearized plasmid DNA derived from IMAGE clones. Hybridizations
were performed at 50.degree. C. for 18 hours using probes labeled
at 5.times.10.sup.7 cpm/ml in 10 mM Tris-HCl (pH 7.6) buffer
containing 50% formamide, 10% dextran sulfate, 1.times.Denhardt's
solution, 0.6 M NaCl, 10 mM DTT, 0.25% SDS and 200 ug/ml tRNA.
After hybridization, slides were washed with 5.times. standard
saline citrate (SSC) at 50.degree. C. for 10 minutes, 50%
formamide/2.times.SSC at 50.degree. C. for 30 minutes, 10 mM
Tris-HCl (pH 7.6)/500 mM NaCl/1 mM EDTA (TNE) at 37.degree. C. for
10 minutes, incubated in 10 ug/ml Rnase A in TNE at 37.degree. C.
for 30 minutes, washed in TNE at 37.degree. C. for 10 minutes,
incubated once in 2.times.SSC at 50.degree. C. for 20 minutes,
twice in 0.2.times.SSC at 50.degree. C. for 20 minutes, and
dehydrated with graded ethanol. Localization of mRNA transcripts
was determined by dipping slides in Kodak NTB2 photoemulsion
(Eastman Kodak, Rochester, N.Y.) and exposing for 14-21 days at
4.degree. C. The slides were counterstained using Myers hematoxylin
and alcoholic eosin Y.
II. Results
[0323] Transcriptional Profiling of Cervical Tissues by cDNA
Microarrays
[0324] 12 normal cervical tissues (9 from ectocervix and 3 from
endocervix), 5 LSIL, 5 HSIL, 9 SCC and 3 ACA were profiled on cDNA
microarrays that contain 30,732 clones (30K microarray). To assess
the power of the data sets to discriminate between diseased and
normal tissue, a hierarchical clustering of the 34 sample data sets
was performed on the basis of overall similarity in gene expression
patterns (FIG. 1). The dendrogram shows that 10 of 12 normal
cervical tissues and all LSIL samples cluster in one group
(designated as "control group"), and 11 of 12 tumor samples and 3
of 5 HSIL samples cluster together in the other group (designated
as "diseased group"). This segregation indicates that global gene
expression profiles of normal ectocervical epithelium, normal
endocervical epithelium and LSIL are very similar, whereas the
expression profiles of 3/5 HSIL samples more closely resemble
cervical cancers. These findings indicate robust data sets that can
distinguish control tissues from diseased tissues despite the fact
that samples were taken from patients of different ages and from
different clinical sites.
Marker Selection
[0325] In order to identify gene markers that would differentiate
the control tissue group from the diseased group, marker scores
were calculated for each clone on the 30K cDNA microarray from
three marker selection paradigms: 9 SCC vs. control group (9
ectocervix, 3 endocervix and 5 LSIL), 5 HSIL vs. control group, and
3 ACA vs. control group. In order to discover new markers
associated with the transformation of cervical cells, up-regulated
genes related to an immune response (i.e. immunoglobulins, MHCs)
were excluded during marker selection. Clones with marker scores
ranked in the top 50 from SCC or ACA paradigms, and clones ranked
between 50 and 100 that were overexpressed in both SCC and ACA
samples were selected as top markers. Scores from the HSIL paradigm
were not used independently to select markers because increased
expression in tumors was considered essential for good marker
performance. Markers were selected and their scores in SCC, ACA and
HSIL paradigms are shown in Table 4. It was found that most of the
up-regulated genes from SCC samples were also elevated in ACA.
While many markers selected from the SCC and/or ACA paradigms have
scores.gtoreq.3.0, only a few of the HSIL markers had scores above
2.0, indicating increasing expression as lesions progress from
dysplasia to invasive carcinomas. FIG. 2 shows two genes from Table
4 that represent typical but distinct types of expression patterns
among normal, LSIL, HSIL, SCC and ACA tissues. MCM 6 was
overexpressed in HSILs, squamous cell carcinomas and
adenocarcinomas, while Claudin 1 was overexpressed only in squamous
cell carcinomas.
[0326] In an attempt to understand the characteristics of these
up-regulated genes, hierarchical clustering was performed based on
the expression profiles across all clinical samples. These
overexpressed genes were clustered into two main groups. One group
consists mainly of genes that encode either extracellular matrix
(ECM) proteins (collagen, laminin, fibronectin) or proteins
responsible for cell-ECM interaction or ECM degradation and
remodeling (e.g. osteonectin, matrix metalloproteinase, urokinase).
The other cluster contains many genes involved in cell replication
and proliferation. Examples include DNA replication licensing
factors (MCM 6), topoisomerase 2A, and the oncogene B-Myb.
Marker Confirmation by In Situ Hybridization (ISH)
[0327] Markers were also evaluated in clinical tissue samples by
ISH. ISH experiments were performed using tissue microarrays to
confirm transcriptional profiling results and to determine the cell
types responsible for increased mRNA expression. Depending on the
level of the paraffin block sectioned, 26-87 normal cervical tissue
cores (from ectocervix and endocervix), 2-10 LSIL, 5-33 HSIL and
10-21 cancer cores (including SCC, ACA and poorly differentiated
carcinomas) were examined. In general, the ISH signal was detected
in cervical epithelial cells (Table 5). Genes that are
overexpressed in epithelial cells are responsible for cell growth
and cell-ECM interactions. Several genes were differentially
expressed by the epithelial cells. This finding suggests
coordinated gene regulation between cervical epithelium and its
microenvironment during cancer progression.
[0328] Photomicrographs of a representative gene, claudin 1 were
taken. There was little or no detectable signal from Claudin 1
probes in normal endo-/ectocervical tissues and LSIL. Gene
expression was elevated in HSIL and increased further in cervical
tumors. Claudin 1 expression was limited to the epithelium and was
not significantly elevated in the 5 HSIL and 3 ACA samples that
were profiled on cDNA microarrays (FIG. 2). Without being limited
by theory, the increased sensitivity of ISH in this case could be
due to the focal nature of the signal. Such focal signals are
readily apparent by ISH but can be missed in RNA preparations of
whole tissue homogenates.
[0329] Since cervical screening evaluates morphological changes of
cells exfoliated from cervical epithelium, cells from stroma are
unlikely to be present in a Pap test sample. The marker selection
was therefore focused on those candidate markers that were
differentially expressed in the epithelial cells of cervical
dysplasias and invasive tumors. To understand the frequency with
which each marker was elevated in different types of cervical
lesions and tumors, a frequency calculation was performed using all
tissue cores on the microarray. The calculation was based on a
semi-quantitative, arbitrary scoring method. The signal was scored
on a scale from 0 to 3: 0--no signal; 1--weak, indeterminate
signal; 2--determinate, weak to moderate signal; 3--strong to very
strong signal. Table 6 shows the results of the scoring for markers
of the present invention. To be considered positive, a tissue core
had to have a signal score of .gtoreq.2. In cases where the
microarray contained more than one tissue core from a single
patient, a positive call required at least 50% of tissue cores to
be .gtoreq.2. To better visualize the results, the selected markers
are presented in the order of increasing frequency of positive
cores for normal cervical tissues. It was found that the frequency
of marker elevation is highly correlated with the stage of clinical
abnormality and varies in a broad range from marker to marker at
particular clinical stages. 1FI27, for example, had relatively high
(>20%) positive cores from normal cervical tissues, whereas
markers such as ITGB6 and CLDN1 were relatively lower in normals
and started to increase in LSIL and HSIL. The appearance of
positive cores for BST2 took place even later in the tumor
progression stage, at the transition from high-grade premalignant
lesions to invasive disease. These findings demonstrate the
existence of markers that identify sequential molecular changes
during cervical cancer development.
Example 2
Gene Expression Analysis
RNA Preparation
[0330] Total RNA was prepared from various human tissues by a
single step extraction method using TRIZOL Reagent according to the
manufacturer's instructions (Invitrogen). Each RNA preparation was
treated with DNase I (Ambion) at 37.degree. C. for 1 hour. DNAse I
treatment was determined to be complete if the sample required at
least 38 PCR amplification cycles to reach a threshold level of
fluorescence using .beta.-2 microglobulin as an internal amplicon
reference (or 35 PCR amplification cycles for 18s ribosome gene).
The integrity of the RNA samples following DNase I treatment was
confirmed by agarose gel electrophoresis and ethidium bromide
staining. After phenol extraction, cDNA was prepared from the
sample using the Taqman Reverse Transcription Reagents following
the manufacturer's instructions (Applied Biosytems). A negative
control of RNA without reverse transcriptase was mock reverse
transcribed for each RNA sample.
TAQMAN.RTM.
[0331] Gene expression was measured by TAQMAN.RTM. quantitative PCR
(Applied Biosystems) in cDNA prepared from a variety of normal and
diseased (e.g., cancerous) human tissues or cell lines.
Preparation of Probes
[0332] Probes were designed by PrimerExpress software (Applied
Biosystems) based on the sequence of the specific genes and their
related transcripts. Each target gene probe was labeled using FAM
(6-carboxyfluorescein), and the 18s reference probe was labeled
with a different fluorescent dye, VIC. The differential labeling of
the target gene and internal reference gene thus enabled
measurement in the same well. Primer and probes were checked for
their sensitivity and specificity for each transcript of the
specific gene. Forward and reverse primers and the probes for both
18s and the target gene were added to the TAQMAN.RTM. Universal PCR
Master Mix (Applied Biosystems). Although the final concentration
of primer and probe could vary, each was internally consistent
within a given experiment. A typical experiment contained 100 nM of
forward and reverse primers plus 200 nM probe for 18s and 900 nM
forward and reverse primers plus 250 nM probe for the target gene.
TAQMAN.RTM. matrix experiments were carried out on an ABI PRISM
7700 Sequence Detection System (Applied Biosystems). The thermal
cycler conditions were as follows: hold for 2 min at 50.degree. C.
and 10 min at 95.degree. C., followed by two-step PCR for 40 cycles
of 95.degree. C. for 15 sec followed by 60.degree. C. for 1 min
[0333] The following method was used to quantitatively calculate
gene expression in the various tissues relative to 18s expression
in the same tissue. The threshold cycle (Ct) value is defined as
the cycle at which a statistically significant increase in
fluorescence is detected. A lower Ct value is indicative of a
higher mRNA concentration. The Ct value of the gene is normalized
by subtracting the Ct value of the 18s ribosome gene to obtain a
.DELTA.Ct value using the following formula: .DELTA.Ct=Ct (target
transcript)-Ct (18s). Relative expression is then calculated using
the arithmetic formula given by 2-.DELTA.Ct. Expression of the
target gene in each of the tissues tested is then numerically
represented (Tables 9-13). Tables 9-13 identify the Sample (Sample
#), Tissue Stage, and Expression of the target gene. The marker
(set forth in Table 1) that was assayed is also identified along
with the variant, primer and probe (set forth in Table 7), if
applicable. For example, in Table 12, the data corresponding to
M30A[1] identifies Marker M30A using the forward 1 (F1), reverse 1
(R1) and probe 1 (P1) as identified in Table 7.
Gene Expression Analysis by End-Point PCR
[0334] Total RNA from different samples was pooled to be used as
template to generate first strand cDNA. The cervical panel
consisted of a cervical tumor pool, a cervical normal pool, an
`other normals` pool and an `other tumors` pool. The pools
consisted of equal amounts of each sample.
TABLE-US-00001 TYPE OF POOL CONSTITUENTS Cervical Tumor Pool 4
tumor samples (squamous cell carcinoma) Cervical Normal Pool 3
normal cervical samples Other Tumors Pool Cervical tumors - 4
squamous cell carcinoma samples Colon Tumors - 5 adenocarcinoma
samples Lung Tumors - 3 squamous cell carcinomas, 3
adenocarcinomas, 1 bronchioalveolar carcinoma and 1 large cell
undifferentiated carcinoma Ovarian Tumors - 2 serous carcinomas and
2 clear cell carcinomas Prostate Tumors - 5 adenocarcinomas Other
Normals Pool One sample each from normal heart, kidney, small
intestine, spleen, WBC, lung, liver, brain, bone marrow, and colon
tissues
[0335] ThermoScript RT-PCR System (Invitrogen, San Diego, Calif.)
was used to obtain cDNA. 1 .mu.g RNA was denatured at 65.degree. C.
for 5 min with 1 .mu.l of 50 .mu.M oligo (dT) 20 primer in a 100
volume according to the manufacturer's instructions. The reaction
was terminated by incubation at 85.degree. C. for 5 min. The final
product was diluted with water to a final volume of 100 .mu.l.
[0336] Gene specific primers were designed just outside or right at
the start of the Open Reading Frame (Table 7). The PCR conditions
were optimized for the primers and the size of the product
expected. 2 .mu.l of cDNA was used in a 20 .mu.l reaction with
touchdown cycling conditions. The products were run on an ethidium
bromide containing agarose gel. The gel picture was then
semi-quantitatively analyzed and scored.
[0337] The ethidium bromide agarose gel pictures of the end-point
PCR on the tissue panel were scored on a scale of 0-5 (Table 8).
Each picture was scored independently by 3 people and the results
were compiled. The scores were compared to make sure that there was
agreement on the relative intensities of the bands and
modifications were made where needed. The median of the 3 scores
was then recorded as the final score.
Summary of the Data Provided in the Tables
[0338] Tables 1 identifies markers of the invention (SEQ ID
NOs:1-44), which are designated with a name ("Marker"), the name
the gene is commonly known by, if applicable ("Gene Name"), the
Sequence Listing identifier of the cDNA sequence of a nucleotide
transcript encoded by or corresponding to the marker ("SEQ ID NO
(nts)"), the Sequence Listing identifier of the amino acid sequence
of a protein encoded by the nucleotide transcript ("SEQ ID NO
(AAs)"), and the location of the protein coding sequence within the
cDNA sequence ("CDS").
[0339] Tables 2 and 3 list newly-identified nucleotide and amino
acid sequences, which are designated with a name ("Marker"), the
name the gene is commonly known by, if applicable ("Gene Name"),
the Sequence Listing identifier of the cDNA sequence of a
nucleotide transcript encoded by or corresponding to the marker
("SEQ ID NO (nts)"), the Sequence Listing identifier of the amino
acid sequence of a protein encoded by the nucleotide transcript
("SEQ ID NO (AAs)"), and the location of the protein coding
sequence within the cDNA sequence ("CDS").
[0340] Table 4 identifies markers of the present invention and
their marker scores in SCC, ACA and HSIL. The markers of Table 4
are designated with a name ("Marker"), the name the gene is
commonly known by, if applicable ("Gene Name"), the marker score
from the squamous cell carcinomas paradigm ("Score SCC"), the
marker score from the adenocarcinomas paradigm ("Score ACA"), and
the marker score from the high-grade squamous intraepithelial
lesions paradigm ("Score HSIL").
[0341] Table 5 lists markers identified as overexpressed in
cervical cancer by in situ hybridization and indicates the location
of marker expression. The markers of Table 5 are designated with a
name ("Marker"), the name the gene is commonly known by, if
applicable ("Gene Name"), the in situ hybridization signal detected
in cervical epithelial cells ("Signal Location").
[0342] Table 6 sets forth the differential expression of the
markers in epithelial cells of cervical dysplasias and invasive
tumors. The markers of Table 6 are designated with a name
("Marker"), the name the gene is commonly known by, if applicable
("Gene Name"), and for each marker, the frequency of marker
elevation ("frequency") and the number of positives to the number
of patients ("# positives/# patients") in normal ectocervical and
endocervical cells ("Normal (EC+END)"), the frequency of marker
elevation ("frequency") and the number of positives to the number
of patients ("# positives/# patients") in low-grade squamous
intraepithelial lesions ("LSIL"), the frequency of marker elevation
("frequency") and the number of positives to the number of patients
("# positives/# patients") in high-grade squamous intraepithelial
lesions ("HSIL"), and the frequency of marker elevation
("frequency") and the number of positives to the number of patients
("# positives/# patients") in squamous cell carcinomas and
adenocarcinomas ("Tumor (SCC+ACA)"), is set forth.
[0343] Table 7 sets forth gene specific primers. Table 7 identifies
the marker, which are designated with a name ("Marker"), the gene
specific primers corresponding to matching positions for Taqman
Primer 1 ("Matching Positions: Taqman Primer 1"), the gene specific
primers corresponding to matching positions for Taqman Primer 2
("Matching Positions: Taqman Primer 2"), the gene specific primers
corresponding to matching positions for Taqman Probe ("Matching
Positions: Taqman Probe"), the gene specific primers corresponding
to matching positions for Endpoint PCR Primer 1 ("Matching
Positions: Endpoint PCR Primer 1"), and the gene specific primers
corresponding to matching positions for Endpoint PCR Primer 1
("Matching Positions: Endpoint PCR Primer 1"). Table 7 identifies
primers in the forward 1 direction ("F1"); the forward 2 direction
("F2"); the reverse 1 direction ("R1"); the reverse 2 direction
("R2"), as well as the probes ("P1" designates probe 1; and "P2"
designates probe 2).
[0344] Table 8 sets forth the scoring on a scale of 0-5 of ethidium
bromide agarose gel pictures of the end-point PCR on the tissue
panel. Table 8 identifies markers, which are designated with a name
("Marker"), and the samples used ("Cervical Normal" and "Cervical
Tumor").
[0345] Tables 9-13 identify the expression of the target gene in
each of the tissues tested. Tables 9-13 identify the Sample, which
is designated with a number ("Sample #"), the tissue stage of the
sample ("Tissue Stage"), and expression of the target gene ("Gene
Name"). Tables 9-13 also identify the marker name, corresponding to
the marker names set forth in Table 1, primer and probe (set forth
in Table 7), if applicable, that were assayed. For example, in
Table 12, the data corresponding to "M30A[1]" identifies Marker
M30A using the forward 1 primer (F1), reverse 1 primer (R1) and
probe 1 (P1) as identified in Table 7.
[0346] The markers obtained using the foregoing protocol should not
be construed as limiting. The contents of all references,
databases, patents and published patent applications cited
throughout this application are expressly incorporated herein by
reference.
Other Embodiments
[0347] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims:
TABLE-US-00002 TABLE 1 Sequence-Related Information Marker Gene
Name SEQ ID NO (nts) SEQ ID NO (AAs) CDS M1A APOL1: apolipoprotein
L1 1 2 162 . . . 1358 M718 APOL2: apolipoprotein L2 3 4 337 . . .
1350 OV3A AQP5: aquaporin 5, variant 1 5 6 517 . . . 1314 M719
AQP5: aquaporin 5, variant 2 7 8 517 . . . 1149 M720 AQP5:
aquaporin 5, variant 3 9 10 517 . . . 1185 M5A BST2: bone marrow
stromal cell antigen 2 11 12 78 . . . 620 M10A CLDN1: claudin-1,
senescence-associated epithelial membrane protein 1 13 14 221 . . .
856 M29A COTL1: coactosin-like 1 (Dictyostelium) 15 16 150 . . .
576 M30A IFI27: interferon, alpha-inducible protein 27, variant 1
17 18 120 . . . 488 M721 IFI27: interferon, alpha-inducible protein
27, variant 2 19 20 120 . . . 479 M488A ITGA3: integrin, alpha 3
(antigen CD49C, alpha 3 subunit of VLA-3 receptor) 21 22 240 . . .
3353 M35 ITGB6: integrin, beta 6, variant 1 23 24 195 . . . 2561
M722 ITGB6: integrin, beta 6, variant 2 25 26 241 . . . 2388 M723
ITGB6: integrin, beta 6, variant 3 27 28 195 . . . 2240 M666
KCNAB1: potassium voltage-gated channel, shaker-related subfamily,
beta member 29 30 89 . . . 1315 M489A MCM6: minichromosome
maintenance deficient (mis5, S. pombe) 6 31 32 56 . . . 2521 OV43A
MSLN: mesothelin, megakaryocyte potentiating factor 33 34 88 . . .
1956 M51A MYBL2: B-MYB, transcription factor (v-myb myeloblastosis
viral oncogene 35 36 128 . . . 2230 homolog M58 PLAU: plasminogen
activator, urokinase 37 38 77 . . . 1372 M22A RTP801;
hypoxia-inducible factor 1(HIF-1) responsive gene 39 40 198 . . .
896 M74A TOP2A: DNA topoisomerase II, alpha isozyme 41 42 127 . . .
4722 M78 ZNF-P66: C2H2 type zinc finger protein (66 kD) 43 44 45 .
. . 1343
TABLE-US-00003 TABLE 2 Sequence-Related Information Marker Gene
Name SEQ ID NO (nts) SEQ ID NO (AAs) CDS M1A APOL1: apolipoprotein
L1 1 2 162 . . . 1358 M719 AQP5: aquaporin 5, variant 2 7 8 517 . .
. 1149 M720 AQP5: aquaporin 5, variant 3 9 10 517 . . . 1185 M721
IFI27: interferon, alpha-inducible protein 27, variant 2 19 20 120
. . . 479 M488A ITGA3: integrin, alpha 3 (antigen CD49C, alpha 3
subunit of VLA-3 receptor) 21 22 240 . . . 3353 M722 ITGB6:
integrin, beta 6, variant 2 25 26 241 . . . 2388 M723 ITGB6:
integrin, beta 6, variant 3 27 28 195 . . . 2240 M78 ZNF-P66: C2H2
type zinc finger protein (66 kD) 43 44 45 . . . 1343
TABLE-US-00004 TABLE 3 Sequence-Related Information Marker Gene
Name SEQ ID NO (nts) SEQ ID NO (AAs) CDS M5A BST2: bone marrow
stromal cell antigen 2 11 12 78 . . . 620 M30A IFI27: interferon,
alpha-inducibte protein 27, variant 1 17 18 120 . . . 488 M35
ITGB6: integrin, beta 6, variant 1 23 24 195 . . . 2561 OV43A MSLN:
mesothelin, megakaryocyte potentiating factor 33 34 88 . . .
1956
TABLE-US-00005 TABLE 4 Marker Gene Name Score SCC Score ACA Score
HSIL M666 KCNAB1: potassium voltage-gated channel, 3.6 3.9 1.4
shaker-related subfamily, beta member 1 M10A CLDN1: claudin-1,
senescence-associated 3.3 1.0 1.3 epithelial membrane protein 1
M29A COTL1: coactosin-like 1 (Dictyostelium) 3.2 1.9 1.0 M5A BST2:
bone marrow stromal cell antigen 2 3.1 3.5 1.7 M78 ZNF-P66: C2H2
type zinc finger protein (66 kD) 3.0 3.1 1.4 M22A RTP801:
hypoxia-inducible factor 1(HIF-1) 2.9 3.0 1.4 responsive gene M30A
IFI27: interferon, alpha-inducible protein 27, 2.9 2.5 1.2 M721
variants 1 and 2 M1A APOL1: apolipoprotein L1 2.8 3.1 1.9 M488A
ITGA3: integrin, alpha 3 (antigen CD49C, alpha 3 2.7 3.7 1.1
subunit of VLA-3 receptor) M35 ITGB6: integrin, beta 6, variants 1,
2, and 3 2.4 3.9 1.0 M722 M723 M51A MYBL2: B-MYB, transcription
factor (v-myb 2.3 4.2 1.8 myeloblastosis viral oncogene homolog
(avian)- like 2) M489A MCM6: minichromosome maintenance deficient
2.3 3.2 1.5 (mis5, S. pombe) 6 M74A TOP2A: DNA topoisomerase II,
alpha isozyme 1.7 3.2 1.6 OV3A AQP5: aquaporin 5, variants 1, 2,
and 3 1.0 3.2 1.6 M719 M720
TABLE-US-00006 TABLE 5 Signal Marker Gene Name Location M666
KCNAB1: potassium voltage-gated channel, epithelium shaker-related
subfamily, beta member 1 M29A COTL1: coactosin-like 1
(Dictyostelium) epithelium M74A TOP2A: DNA topoisomerase II, alpha
isozyme epithelium M30A IFI27: interferon, alpha-inducible protein
27, epithelium M721 variants 1 and 2 M78 ZNF-P66: C2H2 type zinc
finger protein (66 kD) epithelium M488A ITGA3: integrin, alpha 3
(antigen CD49C, alpha 3 epithelium subunit of VLA-3 receptor) OV3A
AQP5: aquaporin 5, variants 1, 2, and 3 epithelium M719 M720 M5A
BST2: bone marrow stromal cell antigen 2 epithelium M22A RTP801:
hypoxia-inducible factor 1(HIF-1) epithelium responsive gene M51A
MYBL2: B-MYB, transcription factor (v-myb epithelium myeloblastosis
viral oncogens homolog (avian)-like 2) M35 ITGB6: integrin, beta 6,
variants 1, 2, and 3 epithelium M722 M723 M16 CRIP1: cysteine-rich
protein 1 (intestinal) epithelium M489A MCM6: minichromosome
maintenance deficient epithelium (mis5, S. pombe) 6 M10A CLDN1:
claudin-1, senescence-associated epithelium epithelial membrane
protein 1 M1A APOL1: apolipoprotein L1 epithelium
TABLE-US-00007 TABLE 6 ##STR00001## .sup.apositive tissue cores
were those which have ISH scores .gtoreq.2. .sup.bnormal
ectocervical and endocervical cells. .sup.cexpression in some
normal squamous epithelium restricted to basal/parabasal cells.
shaded cells indicate ISH scores .gtoreq.2 in at least 20% of the
patients.
TABLE-US-00008 TABLE 7 Taqman/PCR primer-related information
Matching positions: Matching positions: Matching positions:
Matching positions: Matching positions: Marker Taqman Primer 1
Taqman Primer 2 Taqman Probe Endpoint PCR Primer 1 Endpoint PCR
Primer 2 M1A 99-121 238-218 186-160 82-103 1673-1693 M718 166-188
251-231 190-217 143-164 1680-1700 OV3A 914-935 980-964 938-961
512-534 1432-1449 M719 842-857 912-895 869-894 512-534 1267-1284
M720 1097-1116 1174-1155 1154-1133 512-534 1512-1529 M5A 1-23;
34-56 628-647 M10A 164-182 888-908 M29A 123-139 592-610 M30A (F1)
208-228 // (F2) 257- (R1) 315-298 // (R2) 336- (P1) 260-242 // (P2)
277- 7-26 510-529 275 316 296 M721 (F1) 208-228 // (F2) 248- (R1)
306-289 // (R2) 327- (P1) 258-234 // (P2) 268- 7-26 501-520 266 307
287 M488A 187-209 3412-3434 M35 (F1) 1900-1920 // (F2) (R1)
1970-1950 // (R2) (P1) 1923-1945 // (P2) 188-208 2592-2616 628-648
698-672 670-650 M722 (F1) 1727-1747 // (F2) (R1) 1797-1777 // (R2)
(P1) 1750-1772 // (P2) 188-208 2419-2443 318-337 // (F3) 455-475
409-391 // (R3) 525-499 377-360 // (P3) 497-477 M723 (F1) 1796-1818
// (F2) (R1) 1891-1870 // (R2) (P1) 1869-1843 // (P2) 188-208
2271-2295 628-648 698-672 670-650 M666 89-108 1288-1312 M489A 21-39
2563-2580 OV43A 1198-1215 1272-1290 M51A 216-233 2291-2315 M58
52-70 1396-1415 M22A 139-159 997-1017 M74A M78 6-25 1393-1418
TABLE-US-00009 TABLE 8 Cervical Cervical Marker Normal Tumor M1A 1
5 M718 1 3 OV3A 1 4 M719 1 4 M720 1 4 M5A 3 3 M10A 3 5 M29A 2 5
M30A 4 5 M721 4 5 M488A 2 5 M35 0 5 M722 0 5 M723 0 5 M666 0 5
M489A 2 5 OV43A 0 4 M51A 0 5 M58 2 2 M22A 1 5 M74A M78 0 2
TABLE-US-00010 TABLE 9 Expression of Aquaporin 5 Sample # Tissue
Stage OV3A M719 M720 1 normal 0.37 0.01 0.00 2 normal 0.02 0.00
0.00 3 normal 0.98 0.01 0.02 4 normal 0.01 0.00 0.00 5 normal 0.39
0.01 0.01 6 normal 0.00 0.00 0.00 7 normal 1.59 0.07 0.01 8 normal
0.12 0.00 0.00 9 normal 0.00 0.00 0.00 10 normal 0.00 0.00 0.00 11
SCC 0.79 0.05 0.01 12 SCC 0.23 0.01 0.00 13 SCC 0.17 0.00 0.01 14
SCC 0.66 0.03 0.01 15 SCC 1.37 0.03 0.00 16 SCC 3.22 0.33 0.02 17
SCC 0.00 0.00 0.00 18 SCC/AIS 0.12 0.00 0.00 19 SSC 0.02 0.00 0.00
20 poorly diff. adenosquamous 0.18 0.01 0.00 21 SSC 0.01 0.00 0.00
22 Adenocarcinoma 0.02 0.00 0.00 23 Adenocarcinoma 0.78 0.03 0.01
24 SSC 0.12 0.01 0.00 25 SSC 0.00 0.00 0.00 26 SSC 0.00 0.00 0.00
27 SSC 0.00 0.00 0.00 28 SSC 0.08 0.01 0.00 29 SSC 1.59 0.06 0.02
30 SSC 0.07 0.00 0.00 31 Adenocarcinoma 0.27 0.01 0.00 32
Adenocarcinoma 1.29 0.03 0.03 33 SCC 0.03 0.00 0.00 34 SSC 0.01
0.00 0.00 35 SSC 6.92 0.11 0.05 36 SSC 0.03 0.00 0.00 37 SSC 0.15
0.00 0.00 38 SSC 0.00 0.00 0.00 39 SSC 0.01 0.00 0.00 40 SSC 0.06
0.00 0.00 41 SSC 0.02 0.00 0.00 42 tumor 0.13 0.00 0.00
TABLE-US-00011 TABLE 10 Expression of Apolipoprotein L1 Sample #
Tissue Stage M1A 1 normal 0.60 2 normal 0.14 3 normal 0.60 4 normal
0.48 5 normal 0.44 6 normal 0.24 7 normal 0.18 8 normal 0.34 9
normal 0.52 10 normal 0.62 11 SCC 1.56 12 SCC 2.02 13 SCC 2.50 14
SCC 3.15 15 SCC 1.14 16 SCC 3.42 17 SCC 2.51 18 SCC/AIS 17.88 19
SSC 1.18 20 poorly diff. adenosquamous 1.32 21 SSC 1.38 22
Adenocarcinoma 6.61 23 Adenocarcinoma 0.08 24 SCC 1.37 25 SSC 6.28
26 SSC 1.91 27 SSC 5.14 28 SSC 0.59 29 SSC 0.30 30 SSC 5.30 31
Adenocarcinoma 2.10 32 Adenocarcinoma 1.51 33 SCC 8.09 34 SSC 0.35
35 SSC 0.38 36 SSC 4.11 37 SSC 1.83 38 SSC 3.99 39 SSC 4.48 40 SSC
3.77 41 SSC 10.08 42 tumor 0.12
TABLE-US-00012 TABLE 11 Expression of Apolipoprotein L2 Sample #
Tissue Stage M718 1 normal 0.20 2 normal 0.06 3 normal 0.19 4
normal 0.15 5 normal 0.20 6 normal 0.15 7 normal 0.13 8 normal 0.26
9 normal 0.32 10 normal 0.34 11 SCC 1.15 12 SCC 0.42 13 SCC 0.67 14
SCC 0.93 15 SCC 0.51 16 SCC 0.69 17 SCC 0.54 18 SCC/AIS 0.75 19 SSC
0.36 20 poorly diff. adenosquamous 0.67 21 SSC 0.30 22
Adenocarcinoma 0.82 23 Adenocarcinoma 0.11 24 SCC 0.52 25 SSC 2.68
26 SSC 0.51 27 SSC 1.82 28 SSC 0.51 29 SSC 0.17 30 SSC 1.90 31
Adenocarcinoma 0.34 32 Adenocarcinoma 0.49 33 SCC 1.82 34 SSC 0.11
35 SSC 0.28 36 SSC 0.62 37 SSC 0.55 38 SSC 0.68 39 SSC 0.72 40 SSC
0.38 41 SSC 0.87 42 tumor 0.34
TABLE-US-00013 TABLE 12 Expression of Interferon, Alpha-Inducible
Protein 27 Sample M30A M721 M30A [2]/ # Tissue Stage [1] [1] M721
[2] 1 normal 1.75 1.77 3.84 2 normal 0.21 1.25 1.44 3 normal 2.73
4.15 9.46 4 normal 0.00 39.85 17.37 5 normal 14.62 29.54 62.89 6
normal 14.47 20.68 32.12 7 normal 1.04 12.95 8.31 8 normal 4.56
8.96 15.70 9 normal 18.02 23.27 46.52 10 normal 5.83 39.68 32.94 11
SCC 6.66 7.60 15.26 12 SCC 0.98 5.48 5.08 13 SCC 0.00 24.93 14.39
14 SCC 3.58 26.17 19.23 15 SCC 12.51 8.70 37.53 16 SCC 0.00 366.10
244.10 17 SCC 23.94 78.32 127.98 18 SCC/AIS 32.25 287.87 251.55 19
SSC 4.24 3.31 15.21 20 poorly diff. adenosquamous 6.88 6.04 24.17
21 SSC 6.51 5.44 17.83 22 Adenocarcinoma 14.72 74.02 110.70 23
Adenocarcinoma 0.06 0.05 0.25 24 SCC 11.61 7.58 32.57 25 SSC 0.00
117.40 71.70 26 SSC 0.00 73.80 35.81 27 SSC 11.76 6.31 31.11 28 SSC
14.72 9.34 31.94 29 SSC 0.67 0.42 2.69 30 SSC 34.47 33.49 107.11 31
Adenocarcinoma 0.00 10.66 5.03 32 Adenocarcinoma 6.97 5.66 16.30 33
SCC 17.92 97.36 101.33 34 SSC 11.51 7.52 22.49 35 SSC 6.89 42.12
38.96 36 SSC 2.73 35.04 25.04 37 SSC 13.85 7.68 34.26 38 SSC 0.00
28.34 18.79 39 SSC 20.60 15.88 94.41 40 SSC 0.00 13.33 9.11 41 SSC
10.09 12.91 40.59 42 tumor 0.41 0.68 2.13
TABLE-US-00014 TABLE 13 Expression of Integrin, Beta 6 M35 [2]/
Sample M35 [1]/ M722 M723 M722 [3]/ # Tissue Stage M722 [1] [2] [1]
M723 [2] 1 normal 0.45 0.0005 0.019 0.57 2 normal 0.21 0.0002 0.006
0.32 3 normal 0.09 0.0001 0.001 0.17 4 normal 0.13 0.0002 0.003
0.27 5 normal 0.11 0.0002 0.012 0.18 6 normal 0.55 0.0004 0.014
0.72 7 normal 0.11 0.0001 0.003 0.13 8 normal 0.08 0.0000 0.003
0.09 9 normal 0.27 0.0002 0.006 0.26 10 normal 0.56 0.0005 0.016
0.77 11 SCC 1.42 0.0016 0.058 1.90 12 SCC 0.25 0.0004 0.007 0.56 13
SCC 8.24 0.0033 0.333 10.81 14 SCC 0.26 0.0001 0.003 0.29 15 SCC
0.55 0.0003 0.014 0.62 16 SCC 1.22 0.0008 0.032 1.57 17 SCC 3.46
0.0048 0.181 5.36 18 SCC/AIS 1.58 0.0004 0.107 2.45 19 SSC 0.39
0.0004 0.023 0.66 20 poorly diff. 0.56 0.0005 0.022 0.91
adenosquamous 21 SSC 2.92 0.0013 0.092 3.95 22 Adenocarcinoma 0.48
0.0002 0.012 0.68 23 Adenocarcinoma 0.30 0.0003 0.002 0.56 24 SCC
1.75 0.0010 0.083 3.34 25 SSC 0.43 0.0003 0.029 0.79 26 SSC 0.31
0.0004 0.018 0.46 27 SSC 0.60 0.0009 0.026 0.89 28 SSC 3.30 0.0025
0.131 4.45 29 SSC 2.28 0.0037 0.124 4.37 30 SSC 0.54 0.0007 0.021
0.87 31 Adenocarcinoma 0.27 0.0002 0.007 0.58 32 Adenocarcinoma
0.27 0.0003 0.012 0.35 33 SCC 3.46 0.0036 0.127 4.77 34 SSC 0.67
0.0004 0.036 1.19 35 SSC 0.94 0.0003 0.047 1.06 36 SSC 1.66 0.0004
0.057 1.74 37 SSC 2.20 0.0016 0.086 3.34 36 SSC 0.46 0.0002 0.008
0.41 39 SSC 0.82 0.0004 0.030 0.74 40 SSC 0.41 0.0002 0.013 0.29 41
SSC 3.04 0.0021 0.076 2.69 42 tumor 0.07 0.0000 0.001 0.06
Sequence CWU 1
1
4412848DNAHomo sapiens 1actttccctt tcgaattcct cggtatatct tggggactgg
aggacctgtc tggttattat 60acagacgcat aactggaggt gggatccaca cagctcagaa
cagctggatc ttgctcagtc 120tctgccaggg gaagattcct tggaggaggc
cctgcagcga catggaggga gctgctttgc 180tgagagtctc tgtcctctgc
atctggatga gtgcactttt ccttggtgtg ggagtgaggg 240cagaggaagc
tggagcgagg gtgcaacaaa acgttccaag tgggacagat actggagatc
300ctcaaagtaa gcccctcggt gactgggctg ctggcaccat ggacccagag
agcagtatct 360ttattgagga tgccattaag tatttcaagg aaaaagtgag
cacacagaat ctgctactcc 420tgctgactga taatgaggcc tggaacggat
tcgtggctgc tgctgaactg cccaggaatg 480aggcagatga gctccgtaaa
gctctggaca accttgcaag acaaatgatc atgaaagaca 540aaaactggca
cgataaaggc cagcagtaca gaaactggtt tctgaaagag tttcctcggt
600tgaaaagtaa gcttgaggat aacataagaa ggctccgtgc ccttgcagat
ggggttcaga 660aggtccacaa aggcaccacc atcgccaatg tggtgtctgg
ctctctcagc atttcctctg 720gcatcctgac cctcgtcggc atgggtctgg
cacccttcac agagggaggc agccttgtac 780tcttggaacc tgggatggag
ttgggaatca cagcagcttt gaccgggatt accagcagta 840ccatagacta
cggaaagaag tggtggacac aagcccaagc ccacgacctg gtcatcaaaa
900gccttgacaa attgaaggag gtgaaggagt ttttgggtga gaacatatcc
aactttcttt 960ccttagctgg caatacttac caactcacac gaggcattgg
gaaggacatc cgtgccctca 1020gacgagccag agccaatctt cagtcagtac
cgcatgcctc agcctcacgc ccccgggtca 1080ctgagccaat ctcagctgaa
agcggtgaac aggtggagag agttaatgaa cccagcatcc 1140tggaaatgag
cagaggagtc aagctcacgg atgtggcccc tgtaagcttc tttcttgtgc
1200tggatgtagt ctacctcgtg tacgaatcaa agcacttaca tgagggggca
aagtcagaga 1260cagctgagga gctgaagaag gtggctcagg agctggagga
gaagctaaac attctcaaca 1320ataattataa gattctgcag gcggaccaag
aactgtgacc acagggcagg gcagccacca 1380ggagagatat gcctggcagg
ggccaggaca aaatgcaaac tttttttttt ttctgagaca 1440gagtcttgct
ctgtcgccaa gttggagtgc aatggtgcga tctcagctca ctgcaagctc
1500tgcctcccgt gttcaagcga ttctcctgcc ttggcctccc aagtagctgg
gactacaggc 1560gcctaccacc atgcccagct aatttttgta tttttaatag
agatggggtt tcaccatgtt 1620ggccaggatg gtctcgatct cctgacctct
tgatctgccc accttggcct cccaaagtgc 1680tgggattaca ggcgtgagcc
atcgcttttg acccaaatgc aaacatttta ttagggggat 1740aaagagggtg
aggtaaagtt tatggaactg agtgttaggg actttggcat ttccatagct
1800gagcacagca ggggaggggt taatgcagat ggcagtgcag caaggagaag
gcaggaacat 1860tggagcctgc aataagggaa aaatgggaac tggagagtgt
ggggaatggg aagaagcagt 1920ttactttaga ctaaagaata tattgggggg
ccgggtgtag tggctcatgc ctgtaatccg 1980agcactttgg gaggccaagg
cgggcggatc acgaggtcag gagatcaaga ccatcctggc 2040taacacagtg
aaaccccgtc tctactaaaa atacaaaaaa ttagccgggc atggtggcgg
2100gcgcctgtag ttccagctaa ctgggcggct gaggcaggag aatggcgtga
acctgggagg 2160tggagcttgc agtgagccga gatatcgcca ctgcactcca
gcctgggtga cagagcgaga 2220ctccatctca aaaaaaaaaa aaaaaagaat
atattgacgg aagaatagag aggaggcttg 2280aaggaaccag caatgagaag
gccaggaaaa gaaagagctg aaaatggaga aagcccaaga 2340gttagaacag
ttggatacag gagaagaaac agcggctcca ctacagaccc agccccaggt
2400tcaatgtcct ccgaagaatg aagtctttcc ctggtgatgg tcccctgccc
tgtctttcca 2460gcatccactc tcccttgtcc tcctgggggc atatctcagt
caggcagcgg cttcctgatg 2520atggtcgttg gggtggttgt catgtgatgg
gtcccctcca ggttactaaa gggtgcatgt 2580cccctgcttg aacactgaag
ggcaggtggt gggccatggc catggtcccc agctgaggag 2640caggtgtccc
tgagaaccca aacttcccag agagtatgtg agaaccaacc aatgaaaaca
2700gtcccatcgc tcttacccgg taagtaaaca gtcagaaaat tagcatgaaa
gcagtttagc 2760attgggagga agctcagatc tctagagctg tcttgtcgcc
gcccaggatt gacctgtgtg 2820taagtcccaa taaactcacc tactcatc
28482398PRTHomo sapiens 2Met Glu Gly Ala Ala Leu Leu Arg Val Ser
Val Leu Cys Ile Trp Met1 5 10 15Ser Ala Leu Phe Leu Gly Val Gly Val
Arg Ala Glu Glu Ala Gly Ala 20 25 30Arg Val Gln Gln Asn Val Pro Ser
Gly Thr Asp Thr Gly Asp Pro Gln 35 40 45Ser Lys Pro Leu Gly Asp Trp
Ala Ala Gly Thr Met Asp Pro Glu Ser 50 55 60Ser Ile Phe Ile Glu Asp
Ala Ile Lys Tyr Phe Lys Glu Lys Val Ser65 70 75 80Thr Gln Asn Leu
Leu Leu Leu Leu Thr Asp Asn Glu Ala Trp Asn Gly 85 90 95Phe Val Ala
Ala Ala Glu Leu Pro Arg Asn Glu Ala Asp Glu Leu Arg 100 105 110Lys
Ala Leu Asp Asn Leu Ala Arg Gln Met Ile Met Lys Asp Lys Asn 115 120
125Trp His Asp Lys Gly Gln Gln Tyr Arg Asn Trp Phe Leu Lys Glu Phe
130 135 140Pro Arg Leu Lys Ser Lys Leu Glu Asp Asn Ile Arg Arg Leu
Arg Ala145 150 155 160Leu Ala Asp Gly Val Gln Lys Val His Lys Gly
Thr Thr Ile Ala Asn 165 170 175Val Val Ser Gly Ser Leu Ser Ile Ser
Ser Gly Ile Leu Thr Leu Val 180 185 190Gly Met Gly Leu Ala Pro Phe
Thr Glu Gly Gly Ser Leu Val Leu Leu 195 200 205Glu Pro Gly Met Glu
Leu Gly Ile Thr Ala Ala Leu Thr Gly Ile Thr 210 215 220Ser Ser Thr
Ile Asp Tyr Gly Lys Lys Trp Trp Thr Gln Ala Gln Ala225 230 235
240His Asp Leu Val Ile Lys Ser Leu Asp Lys Leu Lys Glu Val Lys Glu
245 250 255Phe Leu Gly Glu Asn Ile Ser Asn Phe Leu Ser Leu Ala Gly
Asn Thr 260 265 270Tyr Gln Leu Thr Arg Gly Ile Gly Lys Asp Ile Arg
Ala Leu Arg Arg 275 280 285Ala Arg Ala Asn Leu Gln Ser Val Pro His
Ala Ser Ala Ser Arg Pro 290 295 300Arg Val Thr Glu Pro Ile Ser Ala
Glu Ser Gly Glu Gln Val Glu Arg305 310 315 320Val Asn Glu Pro Ser
Ile Leu Glu Met Ser Arg Gly Val Lys Leu Thr 325 330 335Asp Val Ala
Pro Val Ser Phe Phe Leu Val Leu Asp Val Val Tyr Leu 340 345 350Val
Tyr Glu Ser Lys His Leu His Glu Gly Ala Lys Ser Glu Thr Ala 355 360
365Glu Glu Leu Lys Lys Val Ala Gln Glu Leu Glu Glu Lys Leu Asn Ile
370 375 380Leu Asn Asn Asn Tyr Lys Ile Leu Gln Ala Asp Gln Glu
Leu385 390 39532545DNAHomo sapiens 3gtgctgggga gcagcgtgtt
tactgtgctt ggtcatgagc tgctgggaag ttgtgacttt 60cactttccct ttcgaattcc
agggtatatc tgggaggccg gaggacgtgt ctggttatta 120cacagatgca
cagctggacg tgggatccac acagctcaga acagttggat cttgctcagt
180ctctgtcaga ggaagatccc ttggacaaga ggaccctgcc ttggtgtgag
agtgagggaa 240gaggaagctg gaacgagggt taaggaaaac cttccagtct
ggacagtgac tggagagctc 300caaggaaagc ccctcggtaa cccagccgct
ggcaccatga acccagagag cagtatcttt 360attgaggatt accttaagta
tttccaggac caagtgagca gagagaatct gctacaactg 420ctgactgatg
atgaagcctg gaatggattc gtggctgctg ctgaactgcc cagggatgag
480gcagatgagc tccgtaaagc tctgaacaag cttgcaagtc acatggtcat
gaaggacaaa 540aaccgccacg ataaagacca gcagcacagg cagtggtttt
tgaaagagtt tcctcggttg 600aaaagggagc ttgaggatca cataaggaag
ctccgtgccc ttgcagagga ggttgagcag 660gtccacagag gcaccaccat
tgccaatgtg gtgtccaact ctgttggcac tacctctggc 720atcctgaccc
tcctcggcct gggtctggca cccttcacag aaggaatcag ttttgtgctc
780ttggacactg gcatgggtct gggagcagca gctgctgtgg ctgggattac
ctgcagtgtg 840gtagaactag taaacaaatt gcgggcacga gcccaagccc
gcaacttgga ccaaagcggc 900accaatgtag caaaggtgat gaaggagttt
gtgggtggga acacacccaa tgttcttacc 960ttagttgaca attggtacca
agtcacacaa gggattggga ggaacatccg tgccatcaga 1020cgagccagag
ccaaccctca gttaggagcg tatgccccac ccccgcatat cattgggcga
1080atctcagctg aaggcggtga acaggttgag agggttgttg aaggccccgc
ccaggcaatg 1140agcagaggaa ccatgatcgt gggtgcagcc actggaggca
tcttgcttct gctggatgtg 1200gtcagccttg catatgagtc aaagcacttg
cttgaggggg caaagtcaga gtcagctgag 1260gagctgaaga agcgggctca
ggagctggag gggaagctca actttctcac caagatccat 1320gagatgctgc
agccaggcca agaccaatga ccccagagca gtgcagccac cagggcagaa
1380atgccgggca caggccagga caaaatgcag actttttttt tttttttttt
ttttttttga 1440gatggagtct cgctctatcg cccaggatgg agtgcagtgg
ctcaatctcg gctcactgca 1500aactccgcct cccgggttca caccattctc
cggcctcagt ctcccgagta gctgggacta 1560caggcacctg ccaccacgcc
cggctaattt ttttgtattt tcactggaga cggggtttca 1620ctgtgttagc
cacgatggtc tccatctcct gacctcgtga tctgcccacc tcggcctccc
1680aaagtgctgg gattacaggc gtgagccacc gcgcctggcc aaaatgcaga
cattttatta 1740gggggataag gagggcaagg taaagcttat ggaactgagt
gttagtgact ttggcatttg 1800tgtagctgag cacagcaagg gaggggttaa
tgcagatggc aagtgcacca aggagaaggc 1860aggaacactg gagcctgcaa
taagggagga gagaggactg gagagtgtgg ggaatgggaa 1920gaagtagttt
actttggact aaagaatata ttgggcgaag aatagagggg gagcttgcag
1980gaaccagcaa tgagaaggcc aggaaaagaa agagctgaaa atggagaaaa
ccagagttag 2040aactgttgga tacaggagaa gaaacagcag ctccactacc
gacccccccc caggtttgat 2100gtccttccaa gaataaagtc tttccctggt
gatggtctct cgctctgtct ttccagcatc 2160cactctccct tgtccttctg
ggggtgtatc acagtcagcc agtggcttct tcatgatggt 2220ggttggggtg
gttgtcatgt gacgggtccc ctccaggtta ctaaagggtg catgtcccct
2280gcttgaaccc tgagaggcag gtggtaggcc atggccacaa tccccagctg
aggagcaggt 2340gtccctgaga acccaaactt cccagagagt atctgagaac
caaccaatga aaacagtccc 2400atcgctctta gccggtaagt aaacagtcag
aagattagca tgaaagcagt ttagcattgg 2460gaggaagcac agatctctag
agctgtcctg tcgctgccca ggattgacct gtgtgtaagt 2520cccaataaac
tcacctactc accaa 25454337PRTHomo sapiens 4Met Asn Pro Glu Ser Ser
Ile Phe Ile Glu Asp Tyr Leu Lys Tyr Phe1 5 10 15Gln Asp Gln Val Ser
Arg Glu Asn Leu Leu Gln Leu Leu Thr Asp Asp 20 25 30Glu Ala Trp Asn
Gly Phe Val Ala Ala Ala Glu Leu Pro Arg Asp Glu 35 40 45Ala Asp Glu
Leu Arg Lys Ala Leu Asn Lys Leu Ala Ser His Met Val 50 55 60Met Lys
Asp Lys Asn Arg His Asp Lys Asp Gln Gln His Arg Gln Trp65 70 75
80Phe Leu Lys Glu Phe Pro Arg Leu Lys Arg Glu Leu Glu Asp His Ile
85 90 95Arg Lys Leu Arg Ala Leu Ala Glu Glu Val Glu Gln Val His Arg
Gly 100 105 110Thr Thr Ile Ala Asn Val Val Ser Asn Ser Val Gly Thr
Thr Ser Gly 115 120 125Ile Leu Thr Leu Leu Gly Leu Gly Leu Ala Pro
Phe Thr Glu Gly Ile 130 135 140Ser Phe Val Leu Leu Asp Thr Gly Met
Gly Leu Gly Ala Ala Ala Ala145 150 155 160Val Ala Gly Ile Thr Cys
Ser Val Val Glu Leu Val Asn Lys Leu Arg 165 170 175Ala Arg Ala Gln
Ala Arg Asn Leu Asp Gln Ser Gly Thr Asn Val Ala 180 185 190Lys Val
Met Lys Glu Phe Val Gly Gly Asn Thr Pro Asn Val Leu Thr 195 200
205Leu Val Asp Asn Trp Tyr Gln Val Thr Gln Gly Ile Gly Arg Asn Ile
210 215 220Arg Ala Ile Arg Arg Ala Arg Ala Asn Pro Gln Leu Gly Ala
Tyr Ala225 230 235 240Pro Pro Pro His Ile Ile Gly Arg Ile Ser Ala
Glu Gly Gly Glu Gln 245 250 255Val Glu Arg Val Val Glu Gly Pro Ala
Gln Ala Met Ser Arg Gly Thr 260 265 270Met Ile Val Gly Ala Ala Thr
Gly Gly Ile Leu Leu Leu Leu Asp Val 275 280 285Val Ser Leu Ala Tyr
Glu Ser Lys His Leu Leu Glu Gly Ala Lys Ser 290 295 300Glu Ser Ala
Glu Glu Leu Lys Lys Arg Ala Gln Glu Leu Glu Gly Lys305 310 315
320Leu Asn Phe Leu Thr Lys Ile His Glu Met Leu Gln Pro Gly Gln Asp
325 330 335Gln52100DNAHomo sapiens 5agacgccccg aggtcggagt
gaagcgccgg gaccgagccc cgtctcccag ggagtccggg 60gcgcacggca ccgaggagag
cgcgggagcc aacctgggcg catcatgcgc agggcccggg 120acgctgggcc
ggtctacacc gccgcctggg tcacgtggcc cggacgggcc ggcggctgcc
180ccggccgggg ggcgggggtc gcgccggggt tgcgctggac gacggagagc
ggcgggcccg 240cagcggcctg gagcctccca acccgcgccg cgctggccct
cgagcgtagg agccgccccc 300tgcccccccg cgccggcccc gcgcccggcc
gcccgccccc tatatagcgc gccccagcag 360ggcccgcgcc aggccgccag
cctcggagtg ggcgcgggac agtgcgcggc gccccgcagc 420caggcccccg
cccccgccgc atccacctcc tccgccgcct gcgacccaac gggcgccccc
480cgccgcggca gctgccgccg ggcccccgcg gccaccatga agaaggaggt
gtgctccgtg 540gccttcctca aggccgtgtt cgcagagttc ttggccaccc
tcatcttcgt cttctttggc 600ctgggctcgg ccctcaagtg gccgtcggcg
ctgcctacca tcctgcagat cgcgctggcg 660tttggcctgg ccataggcac
gctggcccag gccctgggac ccgtgagcgg cggccacatc 720aaccccgcca
tcaccctggc cctcttggtg ggcaaccaga tctcgctgct ccgggctttc
780ttctacgtgg cggcccagct ggtgggcgcc attgccgggg ctggcatcct
ctacggtgtg 840gcaccgctca atgcccgggg caatctggcc gtcaacgcgc
tcaacaacaa cacaacgcag 900ggccaggcca tggtggtgga gctgattctg
accttccagc tggcactctg catcttcgcc 960tccactgact cccgccgcac
cagccctgtg ggctccccag ccctgtccat tggcctgtct 1020gtcaccctgg
gccaccttgt cggaatctac ttcactggct gctccatgaa cccagcccgc
1080tcttttggcc ctgcggtggt catgaatcgg ttcagccccg ctcactgggt
tttctgggta 1140gggcccatcg tgggggcggt cctggctgcc atcctttact
tctacctgct cttccccaac 1200tccctgagcc tgagtgagcg tgtggccatc
atcaaaggca cgtatgagcc tgacgaggac 1260tgggaggagc agcgggaaga
gcggaagaag accatggagc tgaccacccg ctgaccagtg 1320tcaggcaggg
gccagcccct cagcccctga gccaaggggg aaaagaagaa aaagtaccta
1380acacaagctt cctttttgca caaccggtcc tcttggctga ggaggaggag
ctggtcaccc 1440tggctgcaca gttagagagg ggagaaggaa cccatgatgg
gactcctggg gtaggggcca 1500ggggctgggg tctgctgggg acaggtctct
ctgggacaga cctcagagat tgtgaatgca 1560gtgccaagct cacaggctgc
aagggccagg ccagaaaagg gtgggcctgc agcctgcacc 1620ccccaccttc
cccaaccctt cctcaagagc tgaagggatc ccagccccta ggtgggcaga
1680ggcagaccct ccccagagct ccttaggaag aagacagact ggttcattga
atgccgcctt 1740atttatttct ggtgaggatg catgcgtggg gctgctggtg
tttagagtgg gggctaccca 1800ataaatcact gatactcaaa acaccagcag
accctcccca gagctcctta ggaagaagac 1860agactggttc attgaatgcc
gccttattta tttctggtga ggatgcatgc gtggggctgc 1920tggtgtttag
agtgggggct acccaataaa tcactgatac tcacattccg cctctgtctc
1980tcctcagagt gccttgagac actctggccc attgcctctc ctctttgtca
tcccacatcc 2040tccaccacga tctccacagg gtaccagggg accccaggac
aagtgctctg tgggaagaaa 21006265PRTHomo sapiens 6Met Lys Lys Glu Val
Cys Ser Val Ala Phe Leu Lys Ala Val Phe Ala1 5 10 15Glu Phe Leu Ala
Thr Leu Ile Phe Val Phe Phe Gly Leu Gly Ser Ala 20 25 30Leu Lys Trp
Pro Ser Ala Leu Pro Thr Ile Leu Gln Ile Ala Leu Ala 35 40 45Phe Gly
Leu Ala Ile Gly Thr Leu Ala Gln Ala Leu Gly Pro Val Ser 50 55 60Gly
Gly His Ile Asn Pro Ala Ile Thr Leu Ala Leu Leu Val Gly Asn65 70 75
80Gln Ile Ser Leu Leu Arg Ala Phe Phe Tyr Val Ala Ala Gln Leu Val
85 90 95Gly Ala Ile Ala Gly Ala Gly Ile Leu Tyr Gly Val Ala Pro Leu
Asn 100 105 110Ala Arg Gly Asn Leu Ala Val Asn Ala Leu Asn Asn Asn
Thr Thr Gln 115 120 125Gly Gln Ala Met Val Val Glu Leu Ile Leu Thr
Phe Gln Leu Ala Leu 130 135 140Cys Ile Phe Ala Ser Thr Asp Ser Arg
Arg Thr Ser Pro Val Gly Ser145 150 155 160Pro Ala Leu Ser Ile Gly
Leu Ser Val Thr Leu Gly His Leu Val Gly 165 170 175Ile Tyr Phe Thr
Gly Cys Ser Met Asn Pro Ala Arg Ser Phe Gly Pro 180 185 190Ala Val
Val Met Asn Arg Phe Ser Pro Ala His Trp Val Phe Trp Val 195 200
205Gly Pro Ile Val Gly Ala Val Leu Ala Ala Ile Leu Tyr Phe Tyr Leu
210 215 220Leu Phe Pro Asn Ser Leu Ser Leu Ser Glu Arg Val Ala Ile
Ile Lys225 230 235 240Gly Thr Tyr Glu Pro Asp Glu Asp Trp Glu Glu
Gln Arg Glu Glu Arg 245 250 255Lys Lys Thr Met Glu Leu Thr Thr Arg
260 26571935DNAHomo sapiens 7agacgccccg aggtcggagt gaagcgccgg
gaccgagccc cgtctcccag ggagtccggg 60gcgcacggca ccgaggagag cgcgggagcc
aacctgggcg catcatgcgc agggcccggg 120acgctgggcc ggtctacacc
gccgcctggg tcacgtggcc cggacgggcc ggcggctgcc 180ccggccgggg
ggcgggggtc gcgccggggt tgcgctggac gacggagagc ggcgggcccg
240cagcggcctg gagcctccca acccgcgccg cgctggccct cgagcgtagg
agccgccccc 300tgcccccccg cgccggcccc gcgcccggcc gcccgccccc
tatatagcgc gccccagcag 360ggcccgcgcc aggccgccag cctcggagtg
ggcgcgggac agtgcgcggc gccccgcagc 420caggcccccg cccccgccgc
atccacctcc tccgccgcct gcgacccaac gggcgccccc 480cgccgcggca
gctgccgccg ggcccccgcg gccaccatga agaaggaggt gtgctccgtg
540gccttcctca aggccgtgtt cgcagagttc ttggccaccc tcatcttcgt
cttctttggc 600ctgggctcgg ccctcaagtg gccgtcggcg ctgcctacca
tcctgcagat cgcgctggcg 660tttggcctgg ccataggcac gctggcccag
gccctgggac ccgtgagcgg cggccacatc 720aaccccgcca tcaccctggc
cctcttggtg ggcaaccaga tctcgctgct ccgggctttc 780ttctacgtgg
cggcccagct ggtgggcgcc attgccgggg ctggcatcct ctacggtgtg
840gcaccgctca atgcccgggg caatctggcc gtcaacgcga tctacttcac
tggctgctcc 900atgaacccag cccgctcttt tggccctgcg gtggtcatga
atcggttcag ccccgctcac 960tgggttttct gggtagggcc catcgtgggg
gcggtcctgg ctgccatcct ttacttctac 1020ctgctcttcc ccaactccct
gagcctgagt gagcgtgtgg ccatcatcaa aggcacgtat 1080gagcctgacg
aggactggga ggagcagcgg gaagagcgga agaagaccat ggagctgacc
1140acccgctgac cagtgtcagg caggggccag cccctcagcc cctgagccaa
gggggaaaag 1200aagaaaaagt acctaacaca agcttccttt ttgcacaacc
ggtcctcttg gctgaggagg 1260aggagctggt caccctggct gcacagttag
agaggggaga aggaacccat gatgggactc 1320ctggggtagg ggccaggggc
tggggtctgc tggggacagg tctctctggg acagacctca 1380gagattgtga
atgcagtgcc aagctcacag gctgcaaggg ccaggccaga aaagggtggg
1440cctgcagcct gcacccccca ccttccccaa cccttcctca agagctgaag
ggatcccagc 1500ccctaggtgg gcagaggcag accctcccca gagctcctta
ggaagaagac agactggttc 1560attgaatgcc gccttattta tttctggtga
ggatgcatgc gtggggctgc tggtgtttag 1620agtgggggct acccaataaa
tcactgatac tcaaaacacc agcagaccct ccccagagct 1680ccttaggaag
aagacagact ggttcattga atgccgcctt atttatttct ggtgaggatg
1740catgcgtggg gctgctggtg tttagagtgg gggctaccca ataaatcact
gatactcaca 1800ttccgcctct gtctctcctc agagtgcctt gagacactct
ggcccattgc ctctcctctt 1860tgtcatccca catcctccac cacgatctcc
acagggtacc aggggacccc aggacaagtg 1920ctctgtggga agaaa
19358210PRTHomo sapiens 8Met Lys Lys Glu Val Cys Ser Val Ala Phe
Leu Lys Ala Val Phe Ala1 5 10 15Glu Phe Leu Ala Thr Leu Ile Phe Val
Phe Phe Gly Leu Gly Ser Ala 20 25 30Leu Lys Trp Pro Ser Ala Leu Pro
Thr Ile Leu Gln Ile Ala Leu Ala 35 40 45Phe Gly Leu Ala Ile Gly Thr
Leu Ala Gln Ala Leu Gly Pro Val Ser 50 55 60Gly Gly His Ile Asn Pro
Ala Ile Thr Leu Ala Leu Leu Val Gly Asn65 70 75 80Gln Ile Ser Leu
Leu Arg Ala Phe Phe Tyr Val Ala Ala Gln Leu Val 85 90 95Gly Ala Ile
Ala Gly Ala Gly Ile Leu Tyr Gly Val Ala Pro Leu Asn 100 105 110Ala
Arg Gly Asn Leu Ala Val Asn Ala Ile Tyr Phe Thr Gly Cys Ser 115 120
125Met Asn Pro Ala Arg Ser Phe Gly Pro Ala Val Val Met Asn Arg Phe
130 135 140Ser Pro Ala His Trp Val Phe Trp Val Gly Pro Ile Val Gly
Ala Val145 150 155 160Leu Ala Ala Ile Leu Tyr Phe Tyr Leu Leu Phe
Pro Asn Ser Leu Ser 165 170 175Leu Ser Glu Arg Val Ala Ile Ile Lys
Gly Thr Tyr Glu Pro Asp Glu 180 185 190Asp Trp Glu Glu Gln Arg Glu
Glu Arg Lys Lys Thr Met Glu Leu Thr 195 200 205Thr Arg
21092180DNAHomo sapiens 9agacgccccg aggtcggagt gaagcgccgg
gaccgagccc cgtctcccag ggagtccggg 60gcgcacggca ccgaggagag cgcgggagcc
aacctgggcg catcatgcgc agggcccggg 120acgctgggcc ggtctacacc
gccgcctggg tcacgtggcc cggacgggcc ggcggctgcc 180ccggccgggg
ggcgggggtc gcgccggggt tgcgctggac gacggagagc ggcgggcccg
240cagcggcctg gagcctccca acccgcgccg cgctggccct cgagcgtagg
agccgccccc 300tgcccccccg cgccggcccc gcgcccggcc gcccgccccc
tatatagcgc gccccagcag 360ggcccgcgcc aggccgccag cctcggagtg
ggcgcgggac agtgcgcggc gccccgcagc 420caggcccccg cccccgccgc
atccacctcc tccgccgcct gcgacccaac gggcgccccc 480cgccgcggca
gctgccgccg ggcccccgcg gccaccatga agaaggaggt gtgctccgtg
540gccttcctca aggccgtgtt cgcagagttc ttggccaccc tcatcttcgt
cttctttggc 600ctgggctcgg ccctcaagtg gccgtcggcg ctgcctacca
tcctgcagat cgcgctggcg 660tttggcctgg ccataggcac gctggcccag
gccctgggac ccgtgagcgg cggccacatc 720aaccccgcca tcaccctggc
cctcttggtg ggcaaccaga tctcgctgct ccgggctttc 780ttctacgtgg
cggcccagct ggtgggcgcc attgccgggg ctggcatcct ctacggtgtg
840gcaccgctca atgcccgggg caatctggcc gtcaacgcgc tcaacaacaa
cacaacgcag 900ggccaggcca tggtggtgga gctgattctg accttccagc
tggcactctg catcttcgcc 960tccactgact cccgccgcac cagccctgtg
ggctccccag ccctgtccat tggcctgtct 1020gtcaccctgg gccaccttgt
cggaatctac ttcactggct gctccatgaa cccagcccgc 1080tcttttggcc
ctgcggtggt catgaatcgg ttcagccccg ctcactgggg tctgcttcta
1140tccctgcgtg gaggggacac gcgctctgtt catccgtctc tctgaggacc
cacgtgtccc 1200ctctgaaggt tttctgggta gggcccatcg tgggggcggt
cctggctgcc atcctttact 1260tctacctgct cttccccaac tccctgagcc
tgagtgagcg tgtggccatc atcaaaggca 1320cgtatgagcc tgacgaggac
tgggaggagc agcgggaaga gcggaagaag accatggagc 1380tgaccacccg
ctgaccagtg tcaggcaggg gccagcccct cagcccctga gccaaggggg
1440aaaagaagaa aaagtaccta acacaagctt cctttttgca caaccggtcc
tcttggctga 1500ggaggaggag ctggtcaccc tggctgcaca gttagagagg
ggagaaggaa cccatgatgg 1560gactcctggg gtaggggcca ggggctgggg
tctgctgggg acaggtctct ctgggacaga 1620cctcagagat tgtgaatgca
gtgccaagct cacaggctgc aagggccagg ccagaaaagg 1680gtgggcctgc
agcctgcacc ccccaccttc cccaaccctt cctcaagagc tgaagggatc
1740ccagccccta ggtgggcaga ggcagaccct ccccagagct ccttaggaag
aagacagact 1800ggttcattga atgccgcctt atttatttct ggtgaggatg
catgcgtggg gctgctggtg 1860tttagagtgg gggctaccca ataaatcact
gatactcaaa acaccagcag accctcccca 1920gagctcctta ggaagaagac
agactggttc attgaatgcc gccttattta tttctggtga 1980ggatgcatgc
gtggggctgc tggtgtttag agtgggggct acccaataaa tcactgatac
2040tcacattccg cctctgtctc tcctcagagt gccttgagac actctggccc
attgcctctc 2100ctctttgtca tcccacatcc tccaccacga tctccacagg
gtaccagggg accccaggac 2160aagtgctctg tgggaagaaa 218010222PRTHomo
sapiens 10Met Lys Lys Glu Val Cys Ser Val Ala Phe Leu Lys Ala Val
Phe Ala1 5 10 15Glu Phe Leu Ala Thr Leu Ile Phe Val Phe Phe Gly Leu
Gly Ser Ala 20 25 30Leu Lys Trp Pro Ser Ala Leu Pro Thr Ile Leu Gln
Ile Ala Leu Ala 35 40 45Phe Gly Leu Ala Ile Gly Thr Leu Ala Gln Ala
Leu Gly Pro Val Ser 50 55 60Gly Gly His Ile Asn Pro Ala Ile Thr Leu
Ala Leu Leu Val Gly Asn65 70 75 80Gln Ile Ser Leu Leu Arg Ala Phe
Phe Tyr Val Ala Ala Gln Leu Val 85 90 95Gly Ala Ile Ala Gly Ala Gly
Ile Leu Tyr Gly Val Ala Pro Leu Asn 100 105 110Ala Arg Gly Asn Leu
Ala Val Asn Ala Leu Asn Asn Asn Thr Thr Gln 115 120 125Gly Gln Ala
Met Val Val Glu Leu Ile Leu Thr Phe Gln Leu Ala Leu 130 135 140Cys
Ile Phe Ala Ser Thr Asp Ser Arg Arg Thr Ser Pro Val Gly Ser145 150
155 160Pro Ala Leu Ser Ile Gly Leu Ser Val Thr Leu Gly His Leu Val
Gly 165 170 175Ile Tyr Phe Thr Gly Cys Ser Met Asn Pro Ala Arg Ser
Phe Gly Pro 180 185 190Ala Val Val Met Asn Arg Phe Ser Pro Ala His
Trp Gly Leu Leu Leu 195 200 205Ser Leu Arg Gly Gly Asp Thr Arg Ser
Val His Pro Ser Leu 210 215 220111051DNAHomo sapiens 11cctaactcca
ggccagactc cttagcaccc tcccctaact ccaggccaga ctcctttcag 60ctaaaggggt
ggaattcatg gcatctactt cgtatgacta ttgcagagtg cccatggaag
120acggggataa gcgctgtaag cttctgctgg ggataggaat tctggtgctc
ctgatcatcg 180tgattctggg ggtgcccttg attatcttca ccatcaaggc
caacagcgag gcctgccggg 240acggccttcg ggcagtgatg gagtgtcgca
atgtcaccca tctcctgcaa caagagctga 300ccgaggccca gaagggcttt
caggatgtgg aggcccaggc cgccacctgc aaccacactg 360tgatggccct
aatggcttcc ctggatgcag agaaggccca aggacaaaag aaagtggagg
420agcttgaggg agagatcact acattaaacc ataagcttca ggacgcgtct
gcagaggtgg 480agcgactgag aagagaaaac caggtcttaa gcgtgagaat
cgcggacaag aagtactacc 540ccagctccca ggactccagc tccgctgcgg
cgccccagct gctgattgtg ctgctgggcc 600tcagcgctct gctgcagtga
gatcccagga agctggcaca tcttggaagg tccgtcctgc 660tcggcttttc
gcttgaacat tcccttgatc tcatcagttc tgagcgggtc atggggcaac
720acggttagcg gggagagcac ggggtagccg gagaagggcc tctggagcag
gtctggaggg 780gccatggggc agtcctgggt gtggggacac agtcgggttg
acccagggct gtctccctcc 840agagcctccc tccggacaat gagtcccccc
tcttgtctcc caccctgaga ttgggcatgg 900ggtgcggtgt ggggggcatg
tgctgcctgt tgttatgggt tttttttgcg gggggggttg 960cttttttctg
gggtctttga gctccaaaaa ataaacactt cctttgaggg agagcaaaaa
1020aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 105112180PRTHomo sapiens
12Met Ala Ser Thr Ser Tyr Asp Tyr Cys Arg Val Pro Met Glu Asp Gly1
5 10 15Asp Lys Arg Cys Lys Leu Leu Leu Gly Ile Gly Ile Leu Val Leu
Leu 20 25 30Ile Ile Val Ile Leu Gly Val Pro Leu Ile Ile Phe Thr Ile
Lys Ala 35 40 45Asn Ser Glu Ala Cys Arg Asp Gly Leu Arg Ala Val Met
Glu Cys Arg 50 55 60Asn Val Thr His Leu Leu Gln Gln Glu Leu Thr Glu
Ala Gln Lys Gly65 70 75 80Phe Gln Asp Val Glu Ala Gln Ala Ala Thr
Cys Asn His Thr Val Met 85 90 95Ala Leu Met Ala Ser Leu Asp Ala Glu
Lys Ala Gln Gly Gln Lys Lys 100 105 110Val Glu Glu Leu Glu Gly Glu
Ile Thr Thr Leu Asn His Lys Leu Gln 115 120 125Asp Ala Ser Ala Glu
Val Glu Arg Leu Arg Arg Glu Asn Gln Val Leu 130 135 140Ser Val Arg
Ile Ala Asp Lys Lys Tyr Tyr Pro Ser Ser Gln Asp Ser145 150 155
160Ser Ser Ala Ala Ala Pro Gln Leu Leu Ile Val Leu Leu Gly Leu Ser
165 170 175Ala Leu Leu Gln 180133445DNAHomo sapiens 13gagcaaccgc
agcttctagt atccagactc cagcgccgcc ccgggcgcgg accccaaccc 60cgacccagag
cttctccagc ggcggcgcag cgagcagggc tccccgcctt aacttcctcc
120gcggggccca gccaccttcg ggagtccggg ttgcccacct gcaaactctc
cgccttctgc 180acctgccacc cctgagccag cgcgggcgcc cgagcgagtc
atggccaacg cggggctgca 240gctgttgggc ttcattctcg ccttcctggg
atggatcggc gccatcgtca gcactgccct 300gccccagtgg aggatttact
cctatgccgg cgacaacatc gtgaccgccc aggccatgta 360cgaggggctg
tggatgtcct gcgtgtcgca gagcaccggg cagatccagt gcaaagtctt
420tgactccttg ctgaatctga gcagcacatt gcaagcaacc cgtgccttga
tggtggttgg 480catcctcctg ggagtgatag caatctttgt ggccaccgtt
ggcatgaagt gtatgaagtg 540cttggaagac gatgaggtgc agaagatgag
gatggctgtc attgggggtg cgatatttct 600tcttgcaggt ctggctattt
tagttgccac agcatggtat ggcaatagaa tcgttcaaga 660attctatgac
cctatgaccc cagtcaatgc caggtacgaa tttggtcagg ctctcttcac
720tggctgggct gctgcttctc tctgccttct gggaggtgcc ctactttgct
gttcctgtcc 780ccgaaaaaca acctcttacc caacaccaag gccctatcca
aaacctgcac cttccagcgg 840gaaagactac gtgtgacaca gaggcaaaag
gagaaaatca tgttgaaaca aaccgaaaat 900ggacattgag atactatcat
taacattagg accttagaat tttgggtatt gtaatctgaa 960gtatggtatt
acaaaacaaa caaacaaaca aaaaacccat gtgttaaaat actcagtgct
1020aaacatggct taatcttatt ttatcttctt tcctcaatat aggagggaag
atttttccat 1080ttgtattact gcttcccatt gagtaatcat actcaattgg
gggaaggggt gctccttaaa 1140tatatataga tatgtatata tacatgtttt
tctattaaaa atagacagta aaatactatt 1200ctcattatgt tgatactagc
atacttaaaa tatctctaaa ataggtaaat gtatttaatt 1260ccatattgat
gaagatgttt attggtatat tttctttttc gtctatatat acatatgtaa
1320cagtcaaata tcatttactc ttcttcatta gctttgggtg cctttgccac
aagacctagc 1380ctaatttacc aaggatgaat tctttcaatt cttcatgcgt
gcccttttca tatacttatt 1440ttatttttta ccataatctt atagcacttg
catcgttatt aagcccttat ttgttttgtg 1500tttcattggt ctctatctcc
tgaatctaac acatttcata gcctacattt tagtttctaa 1560agccaagaag
aatttattac aaatcagaac tttggaggca aatctttctg catgaccaaa
1620gtgataaatt cctgttgacc ttcccacaca atccctgtac tctgacccat
agcactcttg 1680tttgctttga aaatatttgt ccaattgagt agctgcatgc
tgttccccca ggtgttgtaa 1740cacaacttta ttgattgaat ttttaagcta
cttattcata gttttatatc cccctaaact 1800acctttttgt tccccattcc
ttaattgtat tgttttccca agtgtaatta tcatgcgttt 1860tatatcttcc
taataaggtg tggtctgttt gtctgaacaa agtgctagac tttctggagt
1920gataatctgg tgacaaatat tctctctgta gctgtaagca agtcacttaa
tctttctacc 1980tcttttttct atctgccaaa ttgagataat gatacttaac
cagttagaag aggtagtgtg 2040aatattaatt agtttatatt actctcattc
tttgaacatg aactatgcct atgtagtgtc 2100tttatttgct cagctggctg
agacactgaa gaagtcactg aacaaaacct acacacgtac 2160cttcatgtga
ttcactgcct tcctctctct accagtctat ttccactgaa caaaacctac
2220acacatacct tcatgtggtt cagtgccttc ctctctctac cagtctattt
ccactgaaca 2280aaacctacgc acataccttc atgtggctca gtgccttcct
ctctctacca gtctatttcc 2340attctttcag ctgtgtctga catgtttgtg
ctctgttcca ttttaacaac tgctcttact 2400tttccagtct gtacagaatg
ctatttcact tgagcaagat gatgtaatgg aaagggtgtt 2460ggcattggtg
tctggagacc tggatttgag tcttggtgct atcaatcacc gtctgtgttt
2520gagcaaggca tttggctgct gtaagcttat tgcttcatct gtaagcggtg
gtttgtaatt 2580cctgatcttc ccacctcaca gtgatgttgt ggggatccag
tgagatagaa tacatgtaag 2640tgtggttttg taatttaaaa agtgctatac
taagggaaag aattgaggaa ttaactgcat 2700acgttttggt gttgcttttc
aaatgtttga aaacaaaaaa aatgttaaga aatgggtttc 2760ttgccttaac
cagtctctca agtgatgaga cagtgaagta aaattgagtg cactaaacaa
2820ataagattct gaggaagtct tatcttctgc agtgagtatg gcccgatgct
ttctgtggct 2880aaacagatgt aatgggaaga aataaaagcc tacgtgttgg
taaatccaac agcaagggag 2940atttttgaat cataataact cataaggtgc
tatctgttca gtgatgccct cagagctctt 3000gctgttagct ggcagctgac
gctgctagga tagttagttt ggaaatggta cttcataata 3060aactacacaa
ggaaagtcag ccactgtgtc ttatgaggaa ttggacctaa taaattttag
3120tgtgccttcc aaacctgaga atatatgctt ttggaagtta aaatttaaat
ggcttttgcc 3180acatacatag atcttcatga tgtgtgagtg taattccatg
tggatatcag ttaccaaaca 3240ttacaaaaaa attttatggc ccaaaatgac
caacgaaatt gttacaatag aatttatcca 3300attttgatct ttttatattc
ttctaccaca cctggaaaca gaccaataga cattttgggg 3360ttttataata
ggaatttgta taaagcatta ctctttttca ataaattgtt ttttaattta
3420aaaaaaggaa aaaaaaaaaa aaaaa 344514211PRTHomo sapiens 14Met Ala
Asn Ala Gly Leu Gln Leu Leu Gly Phe Ile Leu Ala Phe Leu1 5 10 15Gly
Trp Ile Gly Ala Ile Val Ser Thr Ala Leu Pro Gln Trp Arg Ile 20 25
30Tyr Ser Tyr Ala Gly Asp Asn Ile Val Thr Ala Gln Ala Met Tyr Glu
35 40 45Gly Leu Trp Met Ser Cys Val Ser Gln Ser Thr Gly Gln Ile Gln
Cys 50 55 60Lys Val Phe Asp Ser Leu Leu Asn Leu Ser Ser Thr Leu Gln
Ala Thr65 70 75 80Arg Ala Leu Met Val Val Gly Ile Leu Leu Gly Val
Ile Ala Ile Phe 85 90 95Val Ala Thr Val Gly Met Lys Cys Met Lys Cys
Leu Glu Asp Asp Glu 100 105 110Val Gln Lys Met Arg Met Ala Val Ile
Gly Gly Ala Ile Phe Leu Leu 115 120 125Ala Gly Leu Ala Ile Leu Val
Ala Thr Ala Trp Tyr Gly Asn Arg Ile 130 135 140Val Gln Glu Phe Tyr
Asp Pro Met Thr Pro Val Asn Ala Arg Tyr Glu145 150 155 160Phe Gly
Gln Ala Leu Phe Thr Gly Trp Ala Ala Ala Ser Leu Cys Leu 165 170
175Leu Gly Gly Ala Leu Leu Cys Cys Ser Cys Pro Arg Lys Thr Thr Ser
180 185 190Tyr Pro Thr Pro Arg Pro Tyr Pro Lys Pro Ala Pro Ser Ser
Gly Lys 195 200 205Asp Tyr Val 210151850DNAHomo sapiens
15cgcgctcgca gctcgcaggc gccgcgtagc cgtcgccacc gccgccagcc cgtgcgccct
60cggcgcgtac ccgccgcgct cccatccccg ccgccggcca ggggcgcgct cggccgcccc
120ggacagtgtc ccgctgcggc tccgcggcga tggccaccaa gatcgacaaa
gaggcttgcc 180gggcggcgta caacctggtg cgcgacgacg gctcggccgt
catctgggtg acttttaaat 240atgacggctc caccatcgtc cccggcgagc
agggagcgga gtaccagcac ttcatccagc 300agtgcacaga tgacgtccgg
ttgtttgcct tcgtgcgctt caccaccggg gatgccatga 360gcaagaggtc
caagtttgcc ctcatcacgt ggatcggtga gaacgtcagc gggctgcagc
420gcgccaaaac cgggacggac aagaccctgg tgaaggaggt cgtacagaat
ttcgctaagg 480agtttgtgat cagtgatcgg aaggagctgg aggaagattt
catcaagagc gagctgaaga 540aggcgggggg agccaattac gacgcccaga
cggagtaacc ccagcccccg ccacaccacc 600ccttgccaaa gtcatctgcc
tgctccccgg gggagaggac cgccggcctc agctactagc 660ccaccagccc
accagggaga aaagaagcca tgagaggcag cgcccgccac cctgtgtcca
720cagcccccac cttcccgctt cccttagaac cctgccgtgt cctatctcat
gacgctcatg 780gaacctcttt ctttgatctt ctttttcttt tctccccctc
ttttttgttc taaagaaaag 840tcattttgat gcaaggtcct gcctgccatc
agatccgagg tgcctcctgc agtgacccct 900tttcctggca tttctcttcc
acgcgacgag gtctgcctag tgagatctgc atgacctcac 960gttgctttcc
agagcccggg cctattttgc catctcagtt ttcctggacc ctgcttcctg
1020tgtaccactg aggggcagct gggccaggag ctgtgcccgg tgcctgcagc
cttcataagc 1080acacacgtcc attccctact aaggcccaga cctcctggta
tctgccccgg gctccctcat 1140cccacctcca tccggagttg cctaagatgc
atgtccagca taggcaggat tgctcggtgg 1200tgagaaggtt aggtccggct
cagactgaat aagaagagat aaaatttgcc ttaaaactta 1260cctggcagtg
gctttgctgc acggtctgaa accacctgtt cccaccctct tgaccgaaat
1320ttccttgtga cacagagaag ggcaaaggtc tgagcccaga gttgacggag
ggagtatttc 1380agggttcact tcaggggctc ccaaagcgac aagatcgtta
gggagagagg cccagggtgg 1440ggactgggaa tttaaggaga gctgggaacg
gatcccttag gttcaggaag cttctgtgta 1500agctgcgagg atggcttggg
ccgaagggtt gctctgcccg ccgcgctagc tgtgagctga 1560gcaaagccct
gggctcacag caccccaaaa gcctgtggct tcagtcctgc gtctgcacca
1620cacattcaaa aggatcgttt tgttttgttt ttaaagaaag gtgagattgg
cttggttctt 1680catgagcaca tttgatatag ctctttttct gtttttcctt
gctcatttcg ttttggggaa 1740gaaatctgta ctgtattggg attgtaaaga
acatctctgc actcagacag tttacagaaa 1800taaatgtttt ttttgttttt
cagaaaaaaa aaaaaaaaaa aaaaaaaaaa 185016142PRTHomo sapiens 16Met Ala
Thr Lys Ile Asp Lys Glu Ala Cys Arg Ala Ala Tyr Asn Leu1 5 10 15Val
Arg Asp Asp Gly Ser Ala Val Ile Trp Val Thr Phe Lys Tyr Asp 20 25
30Gly Ser Thr Ile Val Pro Gly Glu Gln Gly Ala Glu Tyr Gln His Phe
35 40 45Ile Gln Gln Cys Thr Asp Asp Val Arg Leu Phe Ala Phe Val Arg
Phe 50 55 60Thr Thr Gly Asp Ala Met Ser Lys Arg Ser Lys Phe Ala Leu
Ile Thr65 70 75 80Trp Ile Gly Glu Asn Val Ser Gly Leu Gln Arg Ala
Lys Thr Gly Thr 85
90 95Asp Lys Thr Leu Val Lys Glu Val Val Gln Asn Phe Ala Lys Glu
Phe 100 105 110Val Ile Ser Asp Arg Lys Glu Leu Glu Glu Asp Phe Ile
Lys Ser Glu 115 120 125Leu Lys Lys Ala Gly Gly Ala Asn Tyr Asp Ala
Gln Thr Glu 130 135 14017662DNAHomo sapiens 17acacatccaa gcttaagacg
gtgaggtcag cttcacattc tcaggaactc tccttctttg 60ggtctagctg aagttgagga
tctcttactc tctaagccac ggaattaacc cgagcaggca 120tggaggcctc
tgctctcacc tcatcagcag tgaccagtgt ggccaaagtg gtcagggtgg
180cctctggctc tgccgtagtt ttgcccctgg ccaggattgc tacagttgtg
attggaggag 240ttgtggccat ggcggctgtg cccatggtgc tcagtgccat
gggcttcact gcggcgggaa 300tcgcctcgtc ctccatagca gccaagatga
tgtccgcggc ggccattgcc aatgggggtg 360gagttgcctc gggcagcctt
gtgggtactc tgcagtcact gggagcaact ggactctccg 420gattgaccaa
gttcatcctg ggctccattg ggtctgccat tgcggctgtc attgcgaggt
480tctactagct ccctgcccct cgccctgcag agaagagaac catgccaggg
gagaaggcac 540ccagccatcc tgacccagcg aggagccaac tatcccaaat
atacctgggt gaaatatacc 600aaattctgca tctccagagg aaaataagaa
ataaagatga attgttgcaa ctcttaaaaa 660aa 66218122PRTHomo sapiens
18Met Glu Ala Ser Ala Leu Thr Ser Ser Ala Val Thr Ser Val Ala Lys1
5 10 15Val Val Arg Val Ala Ser Gly Ser Ala Val Val Leu Pro Leu Ala
Arg 20 25 30Ile Ala Thr Val Val Ile Gly Gly Val Val Ala Met Ala Ala
Val Pro 35 40 45Met Val Leu Ser Ala Met Gly Phe Thr Ala Ala Gly Ile
Ala Ser Ser 50 55 60Ser Ile Ala Ala Lys Met Met Ser Ala Ala Ala Ile
Ala Asn Gly Gly65 70 75 80Gly Val Ala Ser Gly Ser Leu Val Gly Thr
Leu Gln Ser Leu Gly Ala 85 90 95Thr Gly Leu Ser Gly Leu Thr Lys Phe
Ile Leu Gly Ser Ile Gly Ser 100 105 110Ala Ile Ala Ala Val Ile Ala
Arg Phe Tyr 115 12019653DNAHomo sapiens 19acacatccaa gcttaagacg
gtgaggtcag cttcacattc tcaggaactc tccttctttg 60ggtctagctg aagttgagga
tctcttactc tctaagccac ggaattaacc cgagcaggca 120tggaggcctc
tgctctcacc tcatcagcag tgaccagtgt ggccaaagtg gtcagggtgg
180cctctggctc tgccgtagtt ttgcccctgg ccaggattgc tacagttgtg
attggaggag 240ttgtggctgt gcccatggtg ctcagtgcca tgggcttcac
tgcggcggga atcgcctcgt 300cctccatagc agccaagatg atgtccgcgg
cggccattgc caatgggggt ggagttgcct 360cgggcagcct tgtggctact
ctgcagtcac tgggagcaac tggactctcc ggattgacca 420agttcatcct
gggctccatt gggtctgcca ttgcggctgt cattgcgagg ttctactagc
480tccctgcccc tcgccctgca gagaagagaa ccatgccagg ggagaaggca
cccagccatc 540ctgacccagc gaggagccaa ctatcccaaa tatacctggg
tgaaatatac caaattctgc 600atctccagag gaaaataaga aataaagatg
aattgttgca actcttaaaa aaa 65320119PRTHomo sapiens 20Met Glu Ala Ser
Ala Leu Thr Ser Ser Ala Val Thr Ser Val Ala Lys1 5 10 15Val Val Arg
Val Ala Ser Gly Ser Ala Val Val Leu Pro Leu Ala Arg 20 25 30Ile Ala
Thr Val Val Ile Gly Gly Val Val Ala Val Pro Met Val Leu 35 40 45Ser
Ala Met Gly Phe Thr Ala Ala Gly Ile Ala Ser Ser Ser Ile Ala 50 55
60Ala Lys Met Met Ser Ala Ala Ala Ile Ala Asn Gly Gly Gly Val Ala65
70 75 80Ser Gly Ser Leu Val Ala Thr Leu Gln Ser Leu Gly Ala Thr Gly
Leu 85 90 95Ser Gly Leu Thr Lys Phe Ile Leu Gly Ser Ile Gly Ser Ala
Ile Ala 100 105 110Ala Val Ile Ala Arg Phe Tyr 115214755DNAHomo
sapiens 21gccgggggac ccctccctcc tgtcctcctt gcggtcgacc ggtgcgcttg
ccagatccgc 60cgcgaagccg ggatcgaagg cgacagcgcg gccaaggggg cgcggccggg
acaagctggg 120ggccggttgc ccggggcagg gacggcggcg acccggccgc
tggggaggca ggaagataga 180cccacggatc ttaggaaggg atccgagagc
gcagccgcgc gccccgcgcc ccacgcctga 240tgctctgtgc gctcgccttg
atggtggcgg ccggcggctg cgtcgtctcc gccttcaacc 300tggatacccg
attcctggta gtgaaggagg ccgggaaccc gggcagcctc ttcggctact
360cggtcgccct ccatcggcag acagagcggc agcagcgcta cctgctcctg
gctggtgccc 420cccgggagct cgctgtgccc gatggctaca ccaaccggac
tggtgctgtg tacctgtgcc 480cactcactgc ccacaaggat gactgtgagc
ggatgaacat cacagtgaaa aatgaccctg 540gccatcacat tattgaggac
atgtggcttg gagtgactgt ggccagccag ggccctgcag 600gcagagttct
ggtctgtgcc caccgctaca cccaggtgct gtggtcaggg tcagaagacc
660agcggcgcat ggtgggcaag tgctacgtgc gaggcaatga cctagagctg
gactccagtg 720atgactggca gacctaccac aacgagatgt gcaatagcaa
cacagactac ctggagacgg 780gcatgtgcca gctgggcacc agcggtggct
tcacccagaa cactgtgtac ttcggcgccc 840ccggtgccta caactggaaa
ggaaacagct acatgattca gcgcaaggag tgggacttat 900ctgagtatag
ttacaaggac ccagaggacc aaggaaacct ctatattggg tacacgatgc
960aggtaggcag cttcatcctg caccccaaaa acatcaccat tgtgacaggt
gccccacggc 1020accgacatat gggcgcggtg ttcttgctga gccaggaggc
aggcggagac ctgcggagga 1080ggcaggtgct ggagggctcg caggtgggcg
cctattttgg cagcgccatt gccctggcag 1140acctgaacaa tgatgggtgg
caggacctcc tggtgggcgc cccctactac ttcgagagga 1200aagaggaagt
agggggtgcc atctatgtct tcatgaacca ggcgggaacc tccttccctg
1260ctcacccctc actccttctt catggcccca gtggctctgc ctttggttta
tctgtggcca 1320gcattggtga catcaaccag gatggatttc aggatattgc
tgtgggagct ccgtttgaag 1380gcttgggcaa agtgtacatc tatcacagta
gctctaaggg gctccttaga cagccccagc 1440aggtaatcca tggagagaag
ctgggactgc ctgggttggc caccttcggc tattccctca 1500gtgggcagat
ggatgtggat gagaacttct acccagacct tctagtggga agcctgtcag
1560accacattgt gctgctgcgg gcccggcccg tcatcaacat cgtccacaag
accttggtgc 1620ccaggccagc tgtgctggac cctgcacttt gcacggccac
ctcttgtgtg caagtggagc 1680tgtgctttgc ttacaaccag agtgccggga
accccaacta caggcgaaac atcaccctgg 1740cctacactct ggaggctgac
agggaccgcc ggccgccccg gctccgcttt gccggcagtg 1800agtccgctgt
cttccacggc ttcttctcca tgcccgagat gcgctgccag aagctggagc
1860tgctcctgat ggacaacctc cgtgacaaac tccgccccat catcatctcc
atgaactact 1920ctttaccttt gcggatgccc gatcgccccc ggctggggct
gcggtccctg gacgcctacc 1980cgatcctcaa ccaggcacag gctctggaga
accacactga ggtccagttc cagaaggagt 2040gcgggcctga caacaagtgt
gagagcaact tgcagatgcg ggcagccttc gtgtcagagc 2100agcagcagaa
gctgagcagg ctccagtaca gcagagacgt ccggaaattg ctcctgagca
2160tcaacgtgac gaacacccgg acctcggagc gctccgggga ggacgcccac
gaggcgctgc 2220tcaccctggt ggtgcctccc gccctgctgc tgtcctcagt
gcgccccccc ggggcctgcc 2280aagctaatga gaccatcttt tgcgagctgg
ggaacccctt caaacggaac cagaggatgg 2340agctgctcat cgcctttgag
gtcatcgggg tgaccctgca cacaagggac cttcaggtgc 2400agctgcagct
ctccacgtcg agtcaccagg acaacctgtg gcccatgatc ctcactctgc
2460tggtggacta tacactccag acctcgctta gcatggtaaa tcaccggcta
caaagcttct 2520ttggggggac agtgatgggt gagtctggca tgaaaactgt
ggaggatgta ggaagccccc 2580tcaagtatga attccaggtg ggcccaatgg
gggaggggct ggtgggcctg gggaccctgg 2640tcctaggtct ggagtggccc
tacgaagtca gcaatggcaa gtggctgctg tatcccacgg 2700agatcaccgt
ccatggcaat gggtcctggc cctgccgacc acctggagac cttatcaacc
2760ctctcaacct cactctttct gaccctgggg acaggccatc atccccacag
cgcaggcggc 2820gacagctgga tccaggggga ggccagggcc ccccacctgt
cactctggct gctgccaaaa 2880aagccaagtc tgagactgtg ctgacctgtg
ccacagggcg tgcccactgt gtgtggctag 2940agtgccccat ccctgatgcc
cccgttgtca ccaacgtgac tgtgaaggca cgagtgtgga 3000acagcacctt
catcgaggat tacagagact ttgaccgagt ccgggtaaat ggctgggcta
3060ccctattcct ccgaaccagc atccccacca tcaacatgga gaacaagacc
acgtggttct 3120ctgtggacat tgactcggag ctggtggagg agctgccggc
cgaaatcgag ctgtggctgg 3180tgctggtggc cgtgggtgca gggctgctgc
tgctggggct gatcatcctc ctgctgtgga 3240agtgcggctt cttcaagcga
gcccgcactc gcgccctgta tgaagctaag aggcagaagg 3300cggagatgaa
gagccagccg tcagagacag agaggctgac cgacgactac tgagggggca
3360gccccccgcc cccggcccac ctggtgtgac ttctttaagc ggacccgcta
ttatcagatc 3420atgcccaagt accacgcagt gcggatccgg gaggaggagc
gctacccacc tccagggagc 3480accctgccca ccaagaagca ctgggtgacc
agctggcaga ctcgggacca atactactga 3540cgtcctccct gatcccaccc
cctcctcccc cagtgtcccc tttcttccta tttatcataa 3600gttatgcctc
tgacagtcca caggggccac cacctttggc tggtagcagc aggctcaggc
3660acatacacct cgtcaagagc atgcacatgc tgtctggccc tggggatctt
cccacaggag 3720ggccagcgct gtggacctta caacgccgag tgcactgcat
tcctgtgccc tagatgcacg 3780tggggcccac tgctcgtgga ctgtgctggt
gcatcacgga tggtgcatgg gctcgccgtg 3840tctcagcctc tgccagcgcc
aaaacaagcc aaagagcctc ccaccagagc cgggaggaaa 3900aggcccctgc
aatgtggtga cacctccccc tttcacactg gatccatctt gagccacagt
3960cactggattg actttgctgt caaaactact gacagggagc agcccccggg
ccgctggctg 4020gtgggccccc aatgacaccc atgccagaga ggtggggatc
ctgcctaagg ttgtctacgg 4080gggcacttgg aggacctggc gtgctcagac
ccaacagcaa aggaactaga aagaaggacc 4140cagaacggct tgctttcctg
catctctgtg aagcctctct ccttggccac agactgaact 4200cgcagggaat
gcagcaggaa ggaacaaaga caggcaaacg gcaacgtagc ctgggctcac
4260tgtgctgggg cacggcggga tcctccacag agaggagggg accaattctg
gacagacaga 4320tgttgggagg atacagagga gatgccactt ctcactcacc
actaccagcc agcctcagaa 4380ggccccagag agaccctgca agaccacgga
gggagcgaca cttgaatgta gaataggcag 4440ggggccctgc cccaccccat
ccagccagac cccacgctga ccatgcgtca ggggcctaga 4500ggtggagttc
ttagctatcc ttggctttca gagccagcct ggctctgccc cctcccccat
4560gggctgtgtc ctaaggccca tttgagaagc tgaggctagt tccagaaaac
ctctcctgac 4620ccctgcctgt tggcaggccc actccccagc cccagcccct
tccatggtac tgtagcaggg 4680gaattccctc cccctccttg tgccttcttt
gtatataggc ttctcacggc gaccaataaa 4740cagctcccag tttgt
4755221037PRTHomo sapiens 22Met Leu Cys Ala Leu Ala Leu Met Val Ala
Ala Gly Gly Cys Val Val1 5 10 15Ser Ala Phe Asn Leu Asp Thr Arg Phe
Leu Val Val Lys Glu Ala Gly 20 25 30Asn Pro Gly Ser Leu Phe Gly Tyr
Ser Val Ala Leu His Arg Gln Thr 35 40 45Glu Arg Gln Gln Arg Tyr Leu
Leu Leu Ala Gly Ala Pro Arg Glu Leu 50 55 60Ala Val Pro Asp Gly Tyr
Thr Asn Arg Thr Gly Ala Val Tyr Leu Cys65 70 75 80Pro Leu Thr Ala
His Lys Asp Asp Cys Glu Arg Met Asn Ile Thr Val 85 90 95Lys Asn Asp
Pro Gly His His Ile Ile Glu Asp Met Trp Leu Gly Val 100 105 110Thr
Val Ala Ser Gln Gly Pro Ala Gly Arg Val Leu Val Cys Ala His 115 120
125Arg Tyr Thr Gln Val Leu Trp Ser Gly Ser Glu Asp Gln Arg Arg Met
130 135 140Val Gly Lys Cys Tyr Val Arg Gly Asn Asp Leu Glu Leu Asp
Ser Ser145 150 155 160Asp Asp Trp Gln Thr Tyr His Asn Glu Met Cys
Asn Ser Asn Thr Asp 165 170 175Tyr Leu Glu Thr Gly Met Cys Gln Leu
Gly Thr Ser Gly Gly Phe Thr 180 185 190Gln Asn Thr Val Tyr Phe Gly
Ala Pro Gly Ala Tyr Asn Trp Lys Gly 195 200 205Asn Ser Tyr Met Ile
Gln Arg Lys Glu Trp Asp Leu Ser Glu Tyr Ser 210 215 220Tyr Lys Asp
Pro Glu Asp Gln Gly Asn Leu Tyr Ile Gly Tyr Thr Met225 230 235
240Gln Val Gly Ser Phe Ile Leu His Pro Lys Asn Ile Thr Ile Val Thr
245 250 255Gly Ala Pro Arg His Arg His Met Gly Ala Val Phe Leu Leu
Ser Gln 260 265 270Glu Ala Gly Gly Asp Leu Arg Arg Arg Gln Val Leu
Glu Gly Ser Gln 275 280 285Val Gly Ala Tyr Phe Gly Ser Ala Ile Ala
Leu Ala Asp Leu Asn Asn 290 295 300Asp Gly Trp Gln Asp Leu Leu Val
Gly Ala Pro Tyr Tyr Phe Glu Arg305 310 315 320Lys Glu Glu Val Gly
Gly Ala Ile Tyr Val Phe Met Asn Gln Ala Gly 325 330 335Thr Ser Phe
Pro Ala His Pro Ser Leu Leu Leu His Gly Pro Ser Gly 340 345 350Ser
Ala Phe Gly Leu Ser Val Ala Ser Ile Gly Asp Ile Asn Gln Asp 355 360
365Gly Phe Gln Asp Ile Ala Val Gly Ala Pro Phe Glu Gly Leu Gly Lys
370 375 380Val Tyr Ile Tyr His Ser Ser Ser Lys Gly Leu Leu Arg Gln
Pro Gln385 390 395 400Gln Val Ile His Gly Glu Lys Leu Gly Leu Pro
Gly Leu Ala Thr Phe 405 410 415Gly Tyr Ser Leu Ser Gly Gln Met Asp
Val Asp Glu Asn Phe Tyr Pro 420 425 430Asp Leu Leu Val Gly Ser Leu
Ser Asp His Ile Val Leu Leu Arg Ala 435 440 445Arg Pro Val Ile Asn
Ile Val His Lys Thr Leu Val Pro Arg Pro Ala 450 455 460Val Leu Asp
Pro Ala Leu Cys Thr Ala Thr Ser Cys Val Gln Val Glu465 470 475
480Leu Cys Phe Ala Tyr Asn Gln Ser Ala Gly Asn Pro Asn Tyr Arg Arg
485 490 495Asn Ile Thr Leu Ala Tyr Thr Leu Glu Ala Asp Arg Asp Arg
Arg Pro 500 505 510Pro Arg Leu Arg Phe Ala Gly Ser Glu Ser Ala Val
Phe His Gly Phe 515 520 525Phe Ser Met Pro Glu Met Arg Cys Gln Lys
Leu Glu Leu Leu Leu Met 530 535 540Asp Asn Leu Arg Asp Lys Leu Arg
Pro Ile Ile Ile Ser Met Asn Tyr545 550 555 560Ser Leu Pro Leu Arg
Met Pro Asp Arg Pro Arg Leu Gly Leu Arg Ser 565 570 575Leu Asp Ala
Tyr Pro Ile Leu Asn Gln Ala Gln Ala Leu Glu Asn His 580 585 590Thr
Glu Val Gln Phe Gln Lys Glu Cys Gly Pro Asp Asn Lys Cys Glu 595 600
605Ser Asn Leu Gln Met Arg Ala Ala Phe Val Ser Glu Gln Gln Gln Lys
610 615 620Leu Ser Arg Leu Gln Tyr Ser Arg Asp Val Arg Lys Leu Leu
Leu Ser625 630 635 640Ile Asn Val Thr Asn Thr Arg Thr Ser Glu Arg
Ser Gly Glu Asp Ala 645 650 655His Glu Ala Leu Leu Thr Leu Val Val
Pro Pro Ala Leu Leu Leu Ser 660 665 670Ser Val Arg Pro Pro Gly Ala
Cys Gln Ala Asn Glu Thr Ile Phe Cys 675 680 685Glu Leu Gly Asn Pro
Phe Lys Arg Asn Gln Arg Met Glu Leu Leu Ile 690 695 700Ala Phe Glu
Val Ile Gly Val Thr Leu His Thr Arg Asp Leu Gln Val705 710 715
720Gln Leu Gln Leu Ser Thr Ser Ser His Gln Asp Asn Leu Trp Pro Met
725 730 735Ile Leu Thr Leu Leu Val Asp Tyr Thr Leu Gln Thr Ser Leu
Ser Met 740 745 750Val Asn His Arg Leu Gln Ser Phe Phe Gly Gly Thr
Val Met Gly Glu 755 760 765Ser Gly Met Lys Thr Val Glu Asp Val Gly
Ser Pro Leu Lys Tyr Glu 770 775 780Phe Gln Val Gly Pro Met Gly Glu
Gly Leu Val Gly Leu Gly Thr Leu785 790 795 800Val Leu Gly Leu Glu
Trp Pro Tyr Glu Val Ser Asn Gly Lys Trp Leu 805 810 815Leu Tyr Pro
Thr Glu Ile Thr Val His Gly Asn Gly Ser Trp Pro Cys 820 825 830Arg
Pro Pro Gly Asp Leu Ile Asn Pro Leu Asn Leu Thr Leu Ser Asp 835 840
845Pro Gly Asp Arg Pro Ser Ser Pro Gln Arg Arg Arg Arg Gln Leu Asp
850 855 860Pro Gly Gly Gly Gln Gly Pro Pro Pro Val Thr Leu Ala Ala
Ala Lys865 870 875 880Lys Ala Lys Ser Glu Thr Val Leu Thr Cys Ala
Thr Gly Arg Ala His 885 890 895Cys Val Trp Leu Glu Cys Pro Ile Pro
Asp Ala Pro Val Val Thr Asn 900 905 910Val Thr Val Lys Ala Arg Val
Trp Asn Ser Thr Phe Ile Glu Asp Tyr 915 920 925Arg Asp Phe Asp Arg
Val Arg Val Asn Gly Trp Ala Thr Leu Phe Leu 930 935 940Arg Thr Ser
Ile Pro Thr Ile Asn Met Glu Asn Lys Thr Thr Trp Phe945 950 955
960Ser Val Asp Ile Asp Ser Glu Leu Val Glu Glu Leu Pro Ala Glu Ile
965 970 975Glu Leu Trp Leu Val Leu Val Ala Val Gly Ala Gly Leu Leu
Leu Leu 980 985 990Gly Leu Ile Ile Leu Leu Leu Trp Lys Cys Gly Phe
Phe Lys Arg Ala 995 1000 1005Arg Thr Arg Ala Leu Tyr Glu Ala Lys
Arg Gln Lys Ala Glu Met Lys 1010 1015 1020Ser Gln Pro Ser Glu Thr
Glu Arg Leu Thr Asp Asp Tyr1025 1030 1035234647DNAHomo sapiens
23gtagcctctg ttttcatttc agtcttaatg aaaactttct aacttatatc tcaagtttct
60tttcaaagca gtgtaagtag tatttaaaat gttatacttc aagaaagaaa gactttaacg
120atattcagcg ttggtcttgt aacgctgaag gtaattcatt ttttaatcgg
tctgcacagc 180aagaactgaa acgaatgggg attgaactgc tttgcctgtt
ctttctattt ctaggaagga 240atgatcacgt acaaggtggc tgtgccctgg
gaggtgcaga aacctgtgaa gactgcctgc 300ttattggacc tcagtgtgcc
tggtgtgctc aggagaattt tactcatcca tctggagttg 360gcgaaaggtg
tgatacccca gcaaaccttt tagctaaagg atgtcaatta aacttcatcg
420aaaaccctgt ctcccaagta gaaatactta aaaataagcc tctcagtgta
ggcagacaga 480aaaatagttc tgacattgtt cagattgcgc ctcaaagctt
gatccttaag ttgagaccag 540gtggtgcgca gactctgcag gtgcatgtcc
gccagactga ggactacccg gtggatttgt 600attacctcat ggacctctcc
gcctccatgg atgacgacct caacacaata aaggagctgg 660gctcccggct
ttccaaagag atgtctaaat taaccagcaa ctttagactg ggcttcggat
720cttttgtgga aaaacctgta tcccctttcg tgaaaacaac accagaagaa
attgccaacc 780cttgcagtag tattccatac ttctgtttac ctacatttgg
attcaagcac attttgccat 840tgacaaatga tgctgaaaga ttcaatgaaa
ttgtgaagaa tcagaaaatt tctgctaata 900ttgacacacc cgaaggtgga
tttgatgcaa ttatgcaagc tgctgtgtgt aaggaaaaaa 960ttggctggcg
gaatgactcc ctccacctcc tggtctttgt gagtgatgct gattctcatt
1020ttggaatgga cagcaaacta gcaggcatcg tcattcctaa tgacgggctc
tgtcacttgg 1080acagcaagaa tgaatactcc atgtcaactg tcttggaata
tccaacaatt ggacaactca 1140ttgataaact ggtacaaaac aacgtgttat
tgatcttcgc tgtaacccaa gaacaagttc 1200atttatatga gaattacgca
aaacttattc ctggagctac agtaggtcta cttcagaagg 1260actccggaaa
cattctccag ctgatcatct cagcttatga agaactgcgg tctgaggtgg
1320aactggaagt attaggagac actgaaggac tcaacttgtc atttacagcc
atctgtaaca 1380acggtaccct cttccaacac caaaagaaat gctctcacat
gaaagtggga gacacagctt 1440ccttcagcgt gactgtgaat atcccacact
gcgagagaag aagcaggcac attatcataa 1500agcctgtggg gctgggggat
gccctggaat tacttgtcag cccagaatgc aactgcgact 1560gtcagaaaga
agtggaagtg aacagctcca aatgtcacca cgggaacggc tctttccagt
1620gtggggtgtg tgcctgccac cctggccaca tggggcctcg ctgtgagtgt
ggcgaggaca 1680tgctgagcac agattcctgc aaggaggccc cagatcatcc
ctcctgcagc ggaaggggtg 1740actgctactg tgggcagtgt atctgccact
tgtctcccta tggaaacatt tatgggcctt 1800attgccagtg tgacaatttc
tcctgcgtga gacacaaagg gctgctctgc ggaggtaacg 1860gcgactgtga
ctgtggtgaa tgtgtgtgca ggagcggctg gactggcgag tactgcaact
1920gcaccaccag cacggactcc tgcgtctctg aagatggagt gctctgcagc
gggcgcgggg 1980actgtgtttg tggcaagtgt gtttgcacaa accctggagc
ctcaggacca acctgtgaac 2040gatgtcctac ctgtggtgac ccctgtaact
ctaaacggag ctgcattgag tgccacctgt 2100cagcagctgg ccaagcccga
gaagaatgtg tggacaagtg caaactagct ggtgcgacca 2160tcagtgaaga
agaagatttc tcaaaggatg gttctgtttc ctgctctctg caaggagaaa
2220atgaatgtct tattacattc ctaataacta cagataatga ggggaaaacc
atcattcaca 2280gcatcaatga aaaagattgt ccgaagcctc caaacattcc
catgatcatg ttaggggttt 2340ccctggctat tcttctcatc ggggttgtcc
tactgtgcat ctggaagcta ctggtgtcat 2400ttcatgatcg taaagaagtt
gccaaatttg aagcagaacg atcaaaagcc aagtggcaaa 2460cgggaaccaa
tccactctac agaggatcca caagtacttt taaaaatgta acttataaac
2520acagggaaaa acaaaaggta gacctttcca cagattgcta gaactacttt
atgcatgaaa 2580aaagtctgtt tcactgatat gaaatgttaa tgcactattt
aatttttttc tctttgttgc 2640ttcaaaatga ggttggttta agataataat
aggacatctg cagataagtc atcctctaca 2700tgaaggtgac agactgttgg
cagtttcaaa ataatcaaga agagaaatat ccttagcaaa 2760gagatgactt
tggggatcat ttgaggaata ctaactctgt tgcattaatg cttcaaaaaa
2820tcatcaaatg attcatgggg gcctgatttg catttgaaaa atgtttgaaa
ttagagtctc 2880atttgtttca ggaatgcagc tacctgagtt ttttgtctcg
gcaaagtcac aaagcccata 2940tactcacatt gtgtgtctat acttgccaat
taattctaaa cttgtaggaa atatgccctc 3000tcttaaagga gaattttttt
taaatctctg agaaatgaga ttctgagttt atttcagcta 3060aaaggttgca
attcttctga agatatctca aatataaggt tgaaagttaa gtgttaataa
3120tttttgtgaa tttatacaca cctaaacgtt aagtacacaa atattttatt
tgttttacaa 3180ataaggaata agtaatttat aaattaagaa gttacctata
aaaataaaaa gataacaacc 3240ctatcatata gcttattttt aaattacctg
aaaaacgata ttctacactg tttccttttt 3300gactctgagt tttcaaactg
ttacttctcc catatttctc aatccatttc actcagttgc 3360acagtctttt
aaaccctgta attgtcatac caaagtttct ttttaaaaaa aaattacttt
3420aaatgcttag tttattcaaa gagcgatcca ataatataaa aggaacatgt
gttaaacaca 3480ataaaatttt aaatggctct aaatcaagca catcaagagt
atacaagtct taaaggcttt 3540ttaatacata ctcttttccc atctatgtaa
cccaacttgc acatttcagc tgcatgtggt 3600gaatatgcat catatattta
ctttaagagg taagatttta cttgcaaaat acatgtgcaa 3660attaggatcc
atcagttgat ggaagagatg gactctagaa tattatttct tgtggttatt
3720actcctttac aaagcacttt cgtctcactt gatcctcata aggaaactaa
ggctcagaat 3780gagtagagct gggttcagaa tctagctctt ctaactccaa
gccatctcct ctttccactg 3840caggaaactg cctcttttgt cagtgaaata
atagaaagat tgtgttagtt aagtgataac 3900tgtcatttgt ttgaaaatgt
tcgagactga acaaatagca tttaaactgc tggcatatag 3960atgagatatt
gtacttttgt gcaatgttta ttacctttga ttaaattgta atgtgaagct
4020tttactaggt gaatagttca ttatgtagtg gaggcttcgt ggttgtccat
tgaattgtca 4080cagcaaaatc tataagtttc ttcaattcta caagatagat
ccatatacct ttgatcactt 4140ggagactctt tttttgctgg tttctagata
actcaggtaa atcagacctt tacagagtac 4200agggctaggt gaaagaatta
ctgaaaaatc accttgaaaa tccgaagggc tgatataccc 4260tttatgttcc
tgactgatgc gcagaacctg ggggaaatct acagcaatat acaggttgca
4320atgctgataa cacaacagca atcctctcct ctacgtggac ttactgttgt
ttttttaatt 4380attattggaa tgggatttta gaaaatagaa gttacctttg
tgtgtgtttt agggaaggta 4440gagaagaatc tgctctttct ctgaatactg
ttttgacccc aggcaggacc ttggaaaggc 4500caaaacatta acagtagtac
ttctgttcac tgaagagtta tgttacatga agataaaatg 4560gttttgtcgt
gtttattatt gtattttgtg ttgatataaa taaacatggt aatttaaaca
4620atgaaaaaaa aaaaaaaaaa aaaaaaa 464724788PRTHomo sapiens 24Met
Gly Ile Glu Leu Leu Cys Leu Phe Phe Leu Phe Leu Gly Arg Asn1 5 10
15Asp His Val Gln Gly Gly Cys Ala Leu Gly Gly Ala Glu Thr Cys Glu
20 25 30Asp Cys Leu Leu Ile Gly Pro Gln Cys Ala Trp Cys Ala Gln Glu
Asn 35 40 45Phe Thr His Pro Ser Gly Val Gly Glu Arg Cys Asp Thr Pro
Ala Asn 50 55 60Leu Leu Ala Lys Gly Cys Gln Leu Asn Phe Ile Glu Asn
Pro Val Ser65 70 75 80Gln Val Glu Ile Leu Lys Asn Lys Pro Leu Ser
Val Gly Arg Gln Lys 85 90 95Asn Ser Ser Asp Ile Val Gln Ile Ala Pro
Gln Ser Leu Ile Leu Lys 100 105 110Leu Arg Pro Gly Gly Ala Gln Thr
Leu Gln Val His Val Arg Gln Thr 115 120 125Glu Asp Tyr Pro Val Asp
Leu Tyr Tyr Leu Met Asp Leu Ser Ala Ser 130 135 140Met Asp Asp Asp
Leu Asn Thr Ile Lys Glu Leu Gly Ser Arg Leu Ser145 150 155 160Lys
Glu Met Ser Lys Leu Thr Ser Asn Phe Arg Leu Gly Phe Gly Ser 165 170
175Phe Val Glu Lys Pro Val Ser Pro Phe Val Lys Thr Thr Pro Glu Glu
180 185 190Ile Ala Asn Pro Cys Ser Ser Ile Pro Tyr Phe Cys Leu Pro
Thr Phe 195 200 205Gly Phe Lys His Ile Leu Pro Leu Thr Asn Asp Ala
Glu Arg Phe Asn 210 215 220Glu Ile Val Lys Asn Gln Lys Ile Ser Ala
Asn Ile Asp Thr Pro Glu225 230 235 240Gly Gly Phe Asp Ala Ile Met
Gln Ala Ala Val Cys Lys Glu Lys Ile 245 250 255Gly Trp Arg Asn Asp
Ser Leu His Leu Leu Val Phe Val Ser Asp Ala 260 265 270Asp Ser His
Phe Gly Met Asp Ser Lys Leu Ala Gly Ile Val Ile Pro 275 280 285Asn
Asp Gly Leu Cys His Leu Asp Ser Lys Asn Glu Tyr Ser Met Ser 290 295
300Thr Val Leu Glu Tyr Pro Thr Ile Gly Gln Leu Ile Asp Lys Leu
Val305 310 315 320Gln Asn Asn Val Leu Leu Ile Phe Ala Val Thr Gln
Glu Gln Val His 325 330 335Leu Tyr Glu Asn Tyr Ala Lys Leu Ile Pro
Gly Ala Thr Val Gly Leu 340 345 350Leu Gln Lys Asp Ser Gly Asn Ile
Leu Gln Leu Ile Ile Ser Ala Tyr 355 360 365Glu Glu Leu Arg Ser Glu
Val Glu Leu Glu Val Leu Gly Asp Thr Glu 370 375 380Gly Leu Asn Leu
Ser Phe Thr Ala Ile Cys Asn Asn Gly Thr Leu Phe385 390 395 400Gln
His Gln Lys Lys Cys Ser His Met Lys Val Gly Asp Thr Ala Ser 405 410
415Phe Ser Val Thr Val Asn Ile Pro His Cys Glu Arg Arg Ser Arg His
420 425 430Ile Ile Ile Lys Pro Val Gly Leu Gly Asp Ala Leu Glu Leu
Leu Val 435 440 445Ser Pro Glu Cys Asn Cys Asp Cys Gln Lys Glu Val
Glu Val Asn Ser 450 455 460Ser Lys Cys His His Gly Asn Gly Ser Phe
Gln Cys Gly Val Cys Ala465 470 475 480Cys His Pro Gly His Met Gly
Pro Arg Cys Glu Cys Gly Glu Asp Met 485 490 495Leu Ser Thr Asp Ser
Cys Lys Glu Ala Pro Asp His Pro Ser Cys Ser 500 505 510Gly Arg Gly
Asp Cys Tyr Cys Gly Gln Cys Ile Cys His Leu Ser Pro 515 520 525Tyr
Gly Asn Ile Tyr Gly Pro Tyr Cys Gln Cys Asp Asn Phe Ser Cys 530 535
540Val Arg His Lys Gly Leu Leu Cys Gly Gly Asn Gly Asp Cys Asp
Cys545 550 555 560Gly Glu Cys Val Cys Arg Ser Gly Trp Thr Gly Glu
Tyr Cys Asn Cys 565 570 575Thr Thr Ser Thr Asp Ser Cys Val Ser Glu
Asp Gly Val Leu Cys Ser 580 585 590Gly Arg Gly Asp Cys Val Cys Gly
Lys Cys Val Cys Thr Asn Pro Gly 595 600 605Ala Ser Gly Pro Thr Cys
Glu Arg Cys Pro Thr Cys Gly Asp Pro Cys 610 615 620Asn Ser Lys Arg
Ser Cys Ile Glu Cys His Leu Ser Ala Ala Gly Gln625 630 635 640Ala
Arg Glu Glu Cys Val Asp Lys Cys Lys Leu Ala Gly Ala Thr Ile 645 650
655Ser Glu Glu Glu Asp Phe Ser Lys Asp Gly Ser Val Ser Cys Ser Leu
660 665 670Gln Gly Glu Asn Glu Cys Leu Ile Thr Phe Leu Ile Thr Thr
Asp Asn 675 680 685Glu Gly Lys Thr Ile Ile His Ser Ile Asn Glu Lys
Asp Cys Pro Lys 690 695 700Pro Pro Asn Ile Pro Met Ile Met Leu Gly
Val Ser Leu Ala Ile Leu705 710 715 720Leu Ile Gly Val Val Leu Leu
Cys Ile Trp Lys Leu Leu Val Ser Phe 725 730 735His Asp Arg Lys Glu
Val Ala Lys Phe Glu Ala Glu Arg Ser Lys Ala 740 745 750Lys Trp Gln
Thr Gly Thr Asn Pro Leu Tyr Arg Gly Ser Thr Ser Thr 755 760 765Phe
Lys Asn Val Thr Tyr Lys His Arg Glu Lys Gln Lys Val Asp Leu 770 775
780Ser Thr Asp Cys785254474DNAHomo sapiens 25gtagcctctg ttttcatttc
agtcttaatg aaaactttct aacttatatc tcaagtttct 60tttcaaagca gtgtaagtag
tatttaaaat gttatacttc aagaaagaaa gactttaacg 120atattcagcg
ttggtcttgt aacgctgaag gtaattcatt ttttaatcgg tctgcacagc
180aagaactgaa acgaatgggg attgaactgc tttgcctgtt ctttctattt
ctaggaagga 240atgatcacgt acaaggtggc tgtgccctgg gaggtgcaga
aacctgtgaa gactgcctgc 300ttattggacc tcagtgtgcc tggtgtgctc
aggagaattt tactcatcca tctggagttg 360gcgaaaggtg gtgcgcagac
tctgcaggtg catgtccgcc agactgagga ctacccggtg 420gatttgtatt
acctcatgga cctctccgcc tccatggatg acgacctcaa cacaataaag
480gagctgggct cccggctttc caaagagatg tctaaattaa ccagcaactt
tagactgggc 540ttcggatctt ttgtggaaaa acctgtatcc cctttygtga
aaacaacacc agaagaaatt 600gccaaccctt gcagtagtat tccatacttc
tgtttaccta catttggatt caagcacatt 660ttgccattga caaatgatgc
tgaaagattc aatgaaattg tgaagaatca gaaaatttct 720gctaatattg
acacacccga aggtggattt gatgcaatta tgcaagctgc tgtgtgtaag
780gaaaaaattg gctggcggaa tgactccctc cacctcctgg tctttgtgag
tgatgctgat 840tctcattttg gaatggacag caaactagca ggcatcgtca
ttcctaatga cgggctctgt 900cacttggaca gcaagaatga atactccatg
tcaactgtct tggaatatcc aacaattgga 960caactcattg ataaactggt
acaaaacaac gtgttattga tcttcgctgt aacccaagaa 1020caagttcatt
tatatgagaa ttacgcaaaa cttattcctg gagctacagt aggtctactt
1080cagaaggact ccggaaacat tctccagctg atcatctcag cttatgaaga
actgcggtct 1140gaggtggaac tggaagtatt aggagacact gaaggactca
acttgtcatt tacagccatc 1200tgtaacaacg gtaccctctt ccaacaccaa
aagaaatgct ctcacatgaa agtgggagac 1260acagcttcct tcagcgtgac
tgtgaatatc ccacactgcg agagaagaag caggcacatt 1320atcataaagc
ctgtggggct gggggatgcc ctggaattac ttgtcagccc agaatgcaac
1380tgcgactgtc agaaagaagt ggaagtgaac agctccaaat gtcaccacgg
gaacggctct 1440ttccagtgtg gggtgtgtgc ctgccaccct ggccacatgg
ggcctcgctg tgagtgtggc 1500gaggacatgc tgagcacaga ttcctgcaag
gaggccccag atcatccctc ctgcagcgga 1560aggggtgact gctactgtgg
gcagtgtatc tgccacttgt ctccctatgg aaacatttat 1620gggccttatt
gccagtgtga caatttctcc tgcgtgagac acaaagggct gctctgcgga
1680ggtaacggcg actgtgactg tggtgaatgt gtgtgcagga gcggctggac
tggcgagtac 1740tgcaactgca ccaccagcac ggactcctgc gtctctgaag
atggagtgct ctgcagcggg 1800cgcggggact gtgtttgtgg caagtgtgtt
tgcacaaacc ctggagcctc aggaccaacc 1860tgtgaacgat gtcctacctg
tggtgacccc tgtaactcta aacggagctg cattgagtgc 1920cacctgtcag
cagctggcca agcccgagaa gaatgtgtgg acaagtgcaa actagctggt
1980gcgaccatca gtgaagaaga agatttctca aaggatggtt ctgtttcctg
ctctctgcaa 2040ggagaaaatg aatgtcttat tacattccta ataactacag
ataatgaggg gaaaaccatc 2100attcacagca tcaatgaaaa agattgtccg
aagcctccaa acattcccat gatcatgtta 2160ggggtttccc tggctattct
tctcatcggg gttgtcctac tgtgcatctg gaagctactg 2220gtgtcatttc
atgatcgtaa agaagttgcc aaatttgaag cagaacgatc aaaagccaag
2280tggcaaacgg gaaccaatcc actctacaga ggatccacaa gtacttttaa
aaatgtaact 2340tataaacaca gggaaaaaca aaaggtagac ctttccacag
attgctagaa ctactttatg 2400catgaaaaaa gtctgtttca ctgatatgaa
atgttaatgc actatttaat ttttttctct 2460ttgttgcttc aaaatgaggt
tggtttaaga taataatagg acatctgcag ataagtcatc 2520ctctacatga
aggtgacaga ctgttggcag tttcaaaata atcaagaaga gaaatatcct
2580tagcaaagag atgactttgg ggatcatttg aggaatacta actctgttgc
attaatgctt 2640caaaaaatca tcaaatgatt catgggggcc tgatttgcat
ttgaaaaatg tttgaaatta 2700gagtctcatt tgtttcagga atgcagctac
ctgagttttt tgtctcggca aagtcacaaa 2760gcccatatac tcacattgtg
tgtctatact tgccaattaa ttctaaactt gtaggaaata 2820tgccctctct
taaaggagaa ttttttttaa atctctgaga aatgagattc tgagtttatt
2880tcagctaaaa ggttgcaatt cttctgaaga tatctcaaat ataaggttga
aagttaagtg 2940ttaataattt ttgtgaattt atacacacct aaacgttaag
tacacaaata ttttatttgt 3000tttacaaata aggaataagt aatttataaa
ttaagaagtt acctataaaa ataaaaagat 3060aacaacccta tcatatagct
tatttttaaa ttacctgaaa aacgatattc tacactgttt 3120cctttttgac
tctgagtttt caaactgtta cttctcccat atttctcaat ccatttcact
3180cagttgcaca gtcttttaaa ccctgtaatt gtcataccaa agtttctttt
taaaaaaaaa 3240ttactttaaa tgcttagttt attcaaagag cgatccaata
atataaaagg aacatgtgtt 3300aaacacaata aaattttaaa tggctctaaa
tcaagcacat caagagtata caagtcttaa 3360aggcttttta atacatactc
ttttcccatc tatgtaaccc aacttgcaca tttcagctgc 3420atgtggtgaa
tatgcatcat atatttactt taagaggtaa gattttactt gcaaaataca
3480tgtgcaaatt aggatccatc agttgatgga agagatggac tctagaatat
tatttcttgt 3540ggttattact cctttacaaa gcactttcgt ctcacttgat
cctcataagg aaactaaggc 3600tcagaatgag tagagctggg ttcagaatct
agctcttcta actccaagcc atctcctctt 3660tccactgcag gaaactgcct
cttttgtcag tgaaataata gaaagattgt gttagttaag 3720tgataactgt
catttgtttg aaaatgttcg agactgaaca aatagcattt aaactgctgg
3780catatagatg agatattgta cttttgtgca atgtttatta cctttgatta
aattgtaatg 3840tgaagctttt actaggtgaa tagttcatta tgtagtggag
gcttcgtggt tgtccattga 3900attgtcacag caaaatctat aagtttcttc
aattctacaa gatagatcca tatacctttg 3960atcacttgga gactcttttt
ttgctggttt ctagataact caggtaaatc agacctttac 4020agagtacagg
gctaggtgaa agaattactg aaaaatcacc ttgaaaatcc gaagggctga
4080tatacccttt atgttcctga ctgatgcgca gaacctgggg gaaatctaca
gcaatataca 4140ggttgcaatg ctgataacac aacagcaatc ctctcctcta
cgtggactta ctgttgtttt 4200tttaattatt attggaatgg gattttagaa
aatagaagtt acctttgtgt gtgttttagg 4260gaaggtagag aagaatctgc
tctttctctg aatactgttt tgaccccagg caggaccttg 4320gaaaggccaa
aacattaaca gtagtacttc tgttcactga agagttatgt tacatgaaga
4380taaaatggtt ttgtcgtgtt tattattgta ttttgtgttg atataaataa
acatggtaat 4440ttaaacaatg aaaaaaaaaa aaaaaaaaaa aaaa
447426715PRTHomo sapiens 26Met Ile Thr Tyr Lys Val Ala Val Pro Trp
Glu Val Gln Lys Pro Val1 5 10 15Lys Thr Ala Cys Leu Leu Asp Leu Ser
Val Pro Gly Val Leu Arg Arg 20 25 30Ile Leu Leu Ile His Leu Glu Leu
Ala Lys Gly Gly Ala Gln Thr Leu 35 40 45Gln Val His Val Arg Gln Thr
Glu Asp Tyr Pro Val Asp Leu Tyr Tyr 50 55 60Leu Met Asp Leu Ser Ala
Ser Met Asp Asp Asp Leu Asn Thr Ile Lys65 70 75 80Glu Leu Gly Ser
Arg Leu Ser Lys Glu Met Ser Lys Leu Thr Ser Asn 85 90 95Phe Arg Leu
Gly Phe Gly Ser Phe Val Glu Lys Pro Val Ser Pro Phe 100 105 110Val
Lys Thr Thr Pro Glu Glu Ile Ala Asn Pro Cys Ser Ser Ile Pro 115 120
125Tyr Phe Cys Leu Pro Thr Phe Gly Phe Lys His Ile Leu Pro Leu Thr
130 135 140Asn Asp Ala Glu Arg Phe Asn Glu Ile Val Lys Asn Gln Lys
Ile Ser145 150 155 160Ala Asn Ile Asp Thr Pro Glu Gly Gly Phe Asp
Ala Ile Met Gln Ala 165 170 175Ala Val Cys Lys Glu Lys Ile Gly Trp
Arg Asn Asp Ser Leu His Leu 180 185 190Leu Val Phe Val Ser Asp Ala
Asp Ser His Phe Gly Met Asp Ser Lys 195 200 205Leu Ala Gly Ile Val
Ile Pro Asn Asp Gly Leu Cys His Leu Asp Ser 210 215 220Lys Asn Glu
Tyr Ser Met Ser Thr Val Leu Glu Tyr Pro Thr Ile Gly225 230 235
240Gln Leu Ile Asp Lys Leu Val Gln Asn Asn Val Leu Leu Ile Phe Ala
245 250 255Val Thr Gln Glu Gln Val His Leu Tyr Glu Asn Tyr Ala Lys
Leu Ile 260 265 270Pro Gly Ala Thr Val Gly Leu Leu Gln Lys Asp Ser
Gly Asn Ile Leu
275 280 285Gln Leu Ile Ile Ser Ala Tyr Glu Glu Leu Arg Ser Glu Val
Glu Leu 290 295 300Glu Val Leu Gly Asp Thr Glu Gly Leu Asn Leu Ser
Phe Thr Ala Ile305 310 315 320Cys Asn Asn Gly Thr Leu Phe Gln His
Gln Lys Lys Cys Ser His Met 325 330 335Lys Val Gly Asp Thr Ala Ser
Phe Ser Val Thr Val Asn Ile Pro His 340 345 350Cys Glu Arg Arg Ser
Arg His Ile Ile Ile Lys Pro Val Gly Leu Gly 355 360 365Asp Ala Leu
Glu Leu Leu Val Ser Pro Glu Cys Asn Cys Asp Cys Gln 370 375 380Lys
Glu Val Glu Val Asn Ser Ser Lys Cys His His Gly Asn Gly Ser385 390
395 400Phe Gln Cys Gly Val Cys Ala Cys His Pro Gly His Met Gly Pro
Arg 405 410 415Cys Glu Cys Gly Glu Asp Met Leu Ser Thr Asp Ser Cys
Lys Glu Ala 420 425 430Pro Asp His Pro Ser Cys Ser Gly Arg Gly Asp
Cys Tyr Cys Gly Gln 435 440 445Cys Ile Cys His Leu Ser Pro Tyr Gly
Asn Ile Tyr Gly Pro Tyr Cys 450 455 460Gln Cys Asp Asn Phe Ser Cys
Val Arg His Lys Gly Leu Leu Cys Gly465 470 475 480Gly Asn Gly Asp
Cys Asp Cys Gly Glu Cys Val Cys Arg Ser Gly Trp 485 490 495Thr Gly
Glu Tyr Cys Asn Cys Thr Thr Ser Thr Asp Ser Cys Val Ser 500 505
510Glu Asp Gly Val Leu Cys Ser Gly Arg Gly Asp Cys Val Cys Gly Lys
515 520 525Cys Val Cys Thr Asn Pro Gly Ala Ser Gly Pro Thr Cys Glu
Arg Cys 530 535 540Pro Thr Cys Gly Asp Pro Cys Asn Ser Lys Arg Ser
Cys Ile Glu Cys545 550 555 560His Leu Ser Ala Ala Gly Gln Ala Arg
Glu Glu Cys Val Asp Lys Cys 565 570 575Lys Leu Ala Gly Ala Thr Ile
Ser Glu Glu Glu Asp Phe Ser Lys Asp 580 585 590Gly Ser Val Ser Cys
Ser Leu Gln Gly Glu Asn Glu Cys Leu Ile Thr 595 600 605Phe Leu Ile
Thr Thr Asp Asn Glu Gly Lys Thr Ile Ile His Ser Ile 610 615 620Asn
Glu Lys Asp Cys Pro Lys Pro Pro Asn Ile Pro Met Ile Met Leu625 630
635 640Gly Val Ser Leu Ala Ile Leu Leu Ile Gly Val Val Leu Leu Cys
Ile 645 650 655Trp Lys Leu Leu Val Ser Phe His Asp Arg Lys Glu Val
Ala Lys Phe 660 665 670Glu Ala Glu Arg Ser Lys Ala Lys Trp Gln Thr
Gly Thr Asn Pro Leu 675 680 685Tyr Arg Gly Ser Thr Ser Thr Phe Lys
Asn Val Thr Tyr Lys His Arg 690 695 700Glu Lys Gln Lys Val Asp Leu
Ser Thr Asp Cys705 710 715274327DNAHomo sapiens 27gtagcctctg
ttttcatttc agtcttaatg aaaactttct aacttatatc tcaagtttct 60tttcaaagca
gtgtaagtag tatttaaaat gttatacttc aagaaagaaa gactttaacg
120atattcagcg ttggtcttgt aacgctgaag gtaattcatt ttttaatcgg
tctgcacagc 180aagaactgaa acgaatgggg attgaactgc tttgcctgtt
ctttctattt ctaggaagga 240atgatcacgt acaaggtggc tgtgccctgg
gaggtgcaga aacctgtgaa gactgcctgc 300ttattggacc tcagtgtgcc
tggtgtgctc aggagaattt tactcatcca tctggagttg 360gcgaaaggtg
tgatacccca gcaaaccttt tagctaaagg atgtcaatta aacttcatcg
420aaaaccctgt ctcccaagta gaaatactta aaaataagcc tctcagtgta
ggcagacaga 480aaaatagttc tgacattgtt cagattgcgc ctcaaagctt
gatccttaag ttgagaccag 540gtggtgcgca gactctgcag gtgcatgtcc
gccagactga ggactacccg gtggatttgt 600attacctcat ggacctctcc
gcctccatgg atgacgacct caacacaata aaggagctgg 660gctcccggct
ttccaaagag atgtctaaat taaccagcaa ctttagactg ggcttcggat
720cttttgtgga aaaacctgta tccccttttg tgaaaacaac accagaagaa
attgccaacc 780cttgcagtag tattccatac ttctgtttac ctacatttgg
attcaagcac attttgccat 840tgacaaatga tgctgaaaga ttcaatgaaa
ttgtgaagaa tcagaaaatt tctgctaata 900ttgacacacc cgaaggtgga
tttgatgcaa ttatgcaagc tgctgtgtgt aaggaaaaaa 960ttggctggcg
gaatgactcc ctccacctcc tggtctttgt gagtgatgct gattctcatt
1020ttggaatgga cagcaaacta gcaggcatcg tcattcctaa tgacgggctc
tgtcacttgg 1080acagcaagaa tgaatactcc atgtcaactg tcttggaata
tccaacaatt ggacaactca 1140ttgataaact ggtacaaaac aacgtgttat
tgatcttcgc tgtaacccaa gaacaagttc 1200atttatatga gaattacgca
aaacttattc ctggagctac agtaggtcta cttcagaagg 1260actccggaaa
cattctccag ctgatcatct cagcttatga agaactgcgg tctgaggtgg
1320aactggaagt attaggagac actgaaggac tcaacttgtc atttacagcc
atctgtaaca 1380acggtaccct cttccaacac caaaagaaat gctctcacat
gaaagtggga gacacagctt 1440ccttcagcgt gactgtgaat atcccacact
gcgagagaag aagcaggcac attatcataa 1500agcctgtggg gctgggggat
gccctggaat tacttgtcag cccagaatgc aactgcgact 1560gtcagaaaga
agtggaagtg aacagctcca aatgtcacca cgggaacggc tctttccagt
1620gtggggtgtg tgcctgccac cctggccaca tggggcctcg ctgtgagtgt
ggcgaggaca 1680tgctgagcac agattcctgc aaggaggccc cagatcatcc
ctcctgcagc ggaaggggtg 1740actgctactg tgggcagtgt atctgccact
tgtctcccta tggaaacatt tatgggcctt 1800attgccagtg tgacaatttc
tcctgcgtga gacacaaagg gctgctctgc ggagatttct 1860caaaggatgg
ttctgtttcc tgctctctgc aaggagaaaa tgaatgtctt attacattcc
1920taataactac agataatgag gggaaaacca tcattcacag catcaatgaa
aaagattgtc 1980cgaagcctcc aaacattccc atgatcatgt taggggtttc
cctggctatt cttctcatcg 2040gggttgtcct actgtgcatc tggaagctac
tggtgtcatt tcatgatcgt aaagaagttg 2100ccaaatttga agcagaacga
tcaaaagcca agtggcaaac gggaaccaat ccactctaca 2160gaggatccac
aagtactttt aaaaatgtaa cttataaaca cagggaaaaa caaaaggtag
2220acctttccac agattgctag aactacttta tgcatgaaaa aagtctgttt
cactgatatg 2280aaatgttaat gcactattta atttttttct ctttgttgct
tcaaaatgag gttggtttaa 2340gataataata ggacatctgc agataagtca
tcctctacat gaaggtgaca gactgttggc 2400agtttcaaaa taatcaagaa
gagaaatatc cttagcaaag agatgacttt ggggatcatt 2460tgaggaatac
taactctgtt gcattaatgc ttcaaaaaat catcaaatga ttcatggggg
2520cctgatttgc atttgaaaaa tgtttgaaat tagagtctca tttgtttcag
gaatgcagct 2580acctgagttt tttgtctcgg caaagtcaca aagcccatat
actcacattg tgtgtctata 2640cttgccaatt aattctaaac ttgtaggaaa
tatgccctct cttaaaagga gaattttttt 2700taaatctctg agaaatgaga
ttctgagttt atttcagcta aaaggttgca attcttctga 2760agatatctca
aatataaggt tgaaagttaa gtgttaataa tttttgtgaa tttatacaca
2820cctaaacgtt aagtacacaa atattttatt tgttttacaa ataaggaata
agtaatttat 2880aaattaagaa gttacctata aaaataaaaa gataacaacc
ctatcatata gcttattttt 2940aaattacctg aaaaacgata ttctacactg
tttccttttt gactctgagt tttcaaactg 3000ttacttctcc catatttctc
aatccatttc actcagttgc acagtctttt aaaccctgta 3060attgtcatac
caaagtttct ttttaaaaaa aaattacttt aaatgcttag tttattcaaa
3120gagcgatcca ataatataaa aggaacatgt gttaaacaca ataaaatttt
aaatggctct 3180aaatcaagca catcaagagt atacaagtct taaaggcttt
ttaatacata ctcttttccc 3240atctatgtaa cccaacttgc acatttcagc
tgcatgtggt gaatatgcat catatattta 3300ctttaagagg taagatttta
cttgcaaaat acatgtgcaa attaggatcc atcagttgat 3360ggaagagatg
gactctagaa tattatttct tgtggttatt actcctttac aaagcacttt
3420cgtctcactt gatcctcata aggaaactaa ggctcagaat gagtagagct
gggttcagaa 3480tctagctctt ctaactccaa gccatctcct ctttccactg
caggaaactg cctcttttgt 3540cagtgaaata atagaaagat tgtgttagtt
aagtgataac tgtcatttgt ttgaaaatgt 3600tcgagactga acaaatagca
tttaaactgc tggcatatag atgagatatt gtacttttgt 3660gcaatgttta
ttacctttga ttaaattgta atgtgaagct tttactaggt gaatagttca
3720ttatgtagtg gaggcttcgt ggttgtccat tgaattgtca cagcaaaatc
tataagtttc 3780ttcaattcta caagatagat ccatatacct ttgatcactt
ggagactctt tttttgctgg 3840tttctagata actcaggtaa atcagacctt
tacagagtac agggctaggt gaaagaatta 3900ctgaaaaatc accttgaaaa
tccgaagggc tgatataccc tttatgttcc tgactgatgc 3960gcagaacctg
ggggaaatct acagcaatat acaggttgca atgctgataa cacaacagca
4020atcctctcct ctacgtggac ttactgttgt ttttttaatt attattggaa
tgggatttta 4080gaaaatagaa gttacctttg tgtgtgtttt agggaaggta
gagaagaatc tgctctttct 4140ctgaatactg ttttgacccc aggcaggacc
ttggaaaggc caaaacatta acagtagtac 4200ttctgttcac tgaagagtta
tgttacatga agataaaatg gttttgtcgt gtttattatt 4260gtattttgtg
ttgatataaa taaacatggt aatttaaaca atgaaaaaaa aaaaaaaaaa 4320aaaaaaa
432728681PRTHomo sapiens 28Met Gly Ile Glu Leu Leu Cys Leu Phe Phe
Leu Phe Leu Gly Arg Asn1 5 10 15Asp His Val Gln Gly Gly Cys Ala Leu
Gly Gly Ala Glu Thr Cys Glu 20 25 30Asp Cys Leu Leu Ile Gly Pro Gln
Cys Ala Trp Cys Ala Gln Glu Asn 35 40 45Phe Thr His Pro Ser Gly Val
Gly Glu Arg Cys Asp Thr Pro Ala Asn 50 55 60Leu Leu Ala Lys Gly Cys
Gln Leu Asn Phe Ile Glu Asn Pro Val Ser65 70 75 80Gln Val Glu Ile
Leu Lys Asn Lys Pro Leu Ser Val Gly Arg Gln Lys 85 90 95Asn Ser Ser
Asp Ile Val Gln Ile Ala Pro Gln Ser Leu Ile Leu Lys 100 105 110Leu
Arg Pro Gly Gly Ala Gln Thr Leu Gln Val His Val Arg Gln Thr 115 120
125Glu Asp Tyr Pro Val Asp Leu Tyr Tyr Leu Met Asp Leu Ser Ala Ser
130 135 140Met Asp Asp Asp Leu Asn Thr Ile Lys Glu Leu Gly Ser Arg
Leu Ser145 150 155 160Lys Glu Met Ser Lys Leu Thr Ser Asn Phe Arg
Leu Gly Phe Gly Ser 165 170 175Phe Val Glu Lys Pro Val Ser Pro Phe
Val Lys Thr Thr Pro Glu Glu 180 185 190Ile Ala Asn Pro Cys Ser Ser
Ile Pro Tyr Phe Cys Leu Pro Thr Phe 195 200 205Gly Phe Lys His Ile
Leu Pro Leu Thr Asn Asp Ala Glu Arg Phe Asn 210 215 220Glu Ile Val
Lys Asn Gln Lys Ile Ser Ala Asn Ile Asp Thr Pro Glu225 230 235
240Gly Gly Phe Asp Ala Ile Met Gln Ala Ala Val Cys Lys Glu Lys Ile
245 250 255Gly Trp Arg Asn Asp Ser Leu His Leu Leu Val Phe Val Ser
Asp Ala 260 265 270Asp Ser His Phe Gly Met Asp Ser Lys Leu Ala Gly
Ile Val Ile Pro 275 280 285Asn Asp Gly Leu Cys His Leu Asp Ser Lys
Asn Glu Tyr Ser Met Ser 290 295 300Thr Val Leu Glu Tyr Pro Thr Ile
Gly Gln Leu Ile Asp Lys Leu Val305 310 315 320Gln Asn Asn Val Leu
Leu Ile Phe Ala Val Thr Gln Glu Gln Val His 325 330 335Leu Tyr Glu
Asn Tyr Ala Lys Leu Ile Pro Gly Ala Thr Val Gly Leu 340 345 350Leu
Gln Lys Asp Ser Gly Asn Ile Leu Gln Leu Ile Ile Ser Ala Tyr 355 360
365Glu Glu Leu Arg Ser Glu Val Glu Leu Glu Val Leu Gly Asp Thr Glu
370 375 380Gly Leu Asn Leu Ser Phe Thr Ala Ile Cys Asn Asn Gly Thr
Leu Phe385 390 395 400Gln His Gln Lys Lys Cys Ser His Met Lys Val
Gly Asp Thr Ala Ser 405 410 415Phe Ser Val Thr Val Asn Ile Pro His
Cys Glu Arg Arg Ser Arg His 420 425 430Ile Ile Ile Lys Pro Val Gly
Leu Gly Asp Ala Leu Glu Leu Leu Val 435 440 445Ser Pro Glu Cys Asn
Cys Asp Cys Gln Lys Glu Val Glu Val Asn Ser 450 455 460Ser Lys Cys
His His Gly Asn Gly Ser Phe Gln Cys Gly Val Cys Ala465 470 475
480Cys His Pro Gly His Met Gly Pro Arg Cys Glu Cys Gly Glu Asp Met
485 490 495Leu Ser Thr Asp Ser Cys Lys Glu Ala Pro Asp His Pro Ser
Cys Ser 500 505 510Gly Arg Gly Asp Cys Tyr Cys Gly Gln Cys Ile Cys
His Leu Ser Pro 515 520 525Tyr Gly Asn Ile Tyr Gly Pro Tyr Cys Gln
Cys Asp Asn Phe Ser Cys 530 535 540Val Arg His Lys Gly Leu Leu Cys
Gly Asp Phe Ser Lys Asp Gly Ser545 550 555 560Val Ser Cys Ser Leu
Gln Gly Glu Asn Glu Cys Leu Ile Thr Phe Leu 565 570 575Ile Thr Thr
Asp Asn Glu Gly Lys Thr Ile Ile His Ser Ile Asn Glu 580 585 590Lys
Asp Cys Pro Lys Pro Pro Asn Ile Pro Met Ile Met Leu Gly Val 595 600
605Ser Leu Ala Ile Leu Leu Ile Gly Val Val Leu Leu Cys Ile Trp Lys
610 615 620Leu Leu Val Ser Phe His Asp Arg Lys Glu Val Ala Lys Phe
Glu Ala625 630 635 640Glu Arg Ser Lys Ala Lys Trp Gln Thr Gly Thr
Asn Pro Leu Tyr Arg 645 650 655Gly Ser Thr Ser Thr Phe Lys Asn Val
Thr Tyr Lys His Arg Glu Lys 660 665 670Gln Lys Val Asp Leu Ser Thr
Asp Cys 675 680293176DNAHomo sapiens 29tgataaccca aggtattcac
agcaagatac agtgagtctt aaagttaagc accgtgcaat 60tagctttgct tccttgggtt
tttgaaacat gcatctgtat aaacctgcct gtgcagacat 120cccgagcccc
aagctgggtc tgccaaaatc cagtgaatcg gctctaaaat gtagatggca
180cctagcagtg accaagactc agcctcaggc ggcctgcaaa cctgtgaggc
ccagtggagc 240agccgaacag aaatatgtgg aaaagtttct acgtgttcat
ggaatttcgt tgcaggaaac 300caccagagca gagacgggca tggcatacag
gaatcttgga aaatcaggac tcagagtttc 360ttgcttgggt cttggaacat
gggtgacatt tggaggtcaa atttcagatg aggttgctga 420acggctgatg
accatcgcct atgaaagtgg tgttaacctc tttgatactg ccgaagtcta
480tgctgctgga aaggctgaag tgattctggg gagcatcatc aagaagaaag
gctggaggag 540gtccagtctg gtcataacaa ccaaactcta ctggggtgga
aaagctgaaa cagaaagagg 600gctgtcaaga aagcatatta ttgaaggatt
gaagggctcc ctccagaggc tgcagctcga 660gtatgtggat gtggtctttg
caaatcgacc ggacagtaac actcccatgg aagaaattgt 720ccgagccatg
acacatgtga taaaccaagg catggcgatg tactggggca cctcgagatg
780gagtgctatg gagatcatgg aagcctattc tgtagcaaga cagttcaata
tgatcccacc 840ggtctgtgaa caagctgagt accatctttt ccagagagag
aaagtggagg tccagctgcc 900agagctctac cacaaaatag gtgttggcgc
aatgacatgg tctccacttg cctgtggaat 960catctcagga aaatacggaa
acggggtgcc tgaaagttcc agggcttcac tgaagtgcta 1020ccagtggttg
aaagaaagaa ttgtaagtga agaagggaga aaacagcaaa acaagctaaa
1080agacctttcc ccaattgcgg agcgtctggg atgcacacta cctcagctag
ctgttgcgtg 1140gtgcctgaga aatgaaggtg tgagttctgt gctcctggga
tcatccactc ctgaacaact 1200cattgaaaac cttggtgcca ttcaggttct
cccaaagatg acatcacatg tggtaaatga 1260gattgataac atactgcgca
acaagcccta cagcaagaag gactatagat cataaggcaa 1320tgcatgaacc
acagaagctg catggttaaa atagcggcct gtgcccagta cagaaaggtg
1380ttactaacca gtcttttgaa tcacttagca gcttgctcgt caacctctag
tgtccctccc 1440tggattcttt gaggtgtctg ctgtcgctac cactgtgcac
atctgaaaac tcacaaccaa 1500gaaaatccat tctattttct tatcttggac
tggagtcacc tattcttgca ttgctgtata 1560cacctcatgc ttatgcaatg
ggaagaatat gggggccagg gggtgtggta ctaccttcag 1620gcatttggta
actcaaagaa ggctgtacag atatattttt tcaaaaagaa caaaatccac
1680agatgcaatg tgagttgcgt aagaaacaga gtagatagac taaattcagt
gaaggaaagg 1740aattgagaga tttttcttag taaatagatt attgttaagt
aaatagttat taaaaatata 1800tctcactgca aaaaaaaaaa aagcagtatc
ttcactcaaa agtcttgctt ggaagaataa 1860gcagaaagaa ttttatatat
tttttttcta ttttcacatt catactaaca agttttgttc 1920catttgttat
tcaataaaac aaaaatttct aggtatttgc tttattacct ttcaaatatt
1980tactgttgct tggccccaag aatggccttg tacaacttat ccagaatgtc
tattaggatt 2040ctaatgttat gtccacttac aagtagagac agtaaaagga
tgaataccca atctttagtg 2100acaatgcagc tgatttatga aagagagggc
tacactgcta tggaaactta gcttcaaaga 2160aaatgcaatg tatctgcaat
taggtgttca ttttttacta cattttatta aaacctgctt 2220tatactttca
actgcttgta ggcacaactt ctgcaagttt aaatatttga gctttacaaa
2280taaacataca catgctcagt ttttttaagt aaacctgtaa aatacccagg
aaggcaaatg 2340ttcattgttt aattagcact gggattttat aatataatgt
ttggtatttt tgaggcattg 2400ttaacatgaa agtcaaccac tggctttgtg
aaaaatgcta tgtcactatt cagaatatgc 2460tgggtaaatt aacttgccta
gtgaaaagca aaatgttaaa gaaagaactt ctggttctat 2520aatcatatta
tatgcactaa actatatgca tgaaagttct ttgcatggat taatggggct
2580tacccttgtt gcactcgaaa tctgaggtgt atctagccct gccactattg
gctacttacc 2640ctcattaata tcccacttga gaaaaattgt gagactatac
tgtgtcaata tctgtaaaaa 2700gagagaaaac atgttttttt ttttttgaag
ggggtggtgt gggagtggcc ctttaactcc 2760tatttggcta tctgaggatg
tacaaaattc tcatttaatt ttctggtcag caagttcccc 2820acacagaaat
cactctgagg tttacagaag aactgtaata ttattttaaa atgcgatttt
2880ctgtcattag ttctagatat gtacttcatg gttaaattct aaatctgaaa
atgctagtgg 2940gagatatcaa gaaattttct ttttgattac tagtacctgt
attctaacag agagtttgaa 3000ttttttgccc gtgttatcag aatgatggaa
attgatcatt ttcagttgtt cattgtgtat 3060tcaatccagc tgaactgctg
tatgtataga ggagcttgag gtgctgtcta atgggaaatg 3120tgatttgatt
gatttatttg cttagagtaa taaaagcatt ttgtgcattc aatctt 317630408PRTHomo
sapiens 30Met His Leu Tyr Lys Pro Ala Cys Ala Asp Ile Pro Ser Pro
Lys Leu1 5 10 15Gly Leu Pro Lys Ser Ser Glu Ser Ala Leu Lys Cys Arg
Trp His Leu 20 25 30Ala Val Thr Lys Thr Gln Pro Gln Ala Ala Cys Lys
Pro Val Arg Pro 35 40 45Ser Gly Ala Ala Glu Gln Lys Tyr Val Glu Lys
Phe Leu Arg Val His 50 55 60Gly Ile Ser Leu Gln Glu Thr Thr Arg Ala
Glu Thr Gly Met Ala Tyr65 70 75 80Arg Asn Leu Gly Lys Ser Gly Leu
Arg Val Ser Cys Leu Gly Leu Gly 85 90 95Thr Trp Val Thr Phe Gly Gly
Gln Ile Ser Asp
Glu Val Ala Glu Arg 100 105 110Leu Met Thr Ile Ala Tyr Glu Ser Gly
Val Asn Leu Phe Asp Thr Ala 115 120 125Glu Val Tyr Ala Ala Gly Lys
Ala Glu Val Ile Leu Gly Ser Ile Ile 130 135 140Lys Lys Lys Gly Trp
Arg Arg Ser Ser Leu Val Ile Thr Thr Lys Leu145 150 155 160Tyr Trp
Gly Gly Lys Ala Glu Thr Glu Arg Gly Leu Ser Arg Lys His 165 170
175Ile Ile Glu Gly Leu Lys Gly Ser Leu Gln Arg Leu Gln Leu Glu Tyr
180 185 190Val Asp Val Val Phe Ala Asn Arg Pro Asp Ser Asn Thr Pro
Met Glu 195 200 205Glu Ile Val Arg Ala Met Thr His Val Ile Asn Gln
Gly Met Ala Met 210 215 220Tyr Trp Gly Thr Ser Arg Trp Ser Ala Met
Glu Ile Met Glu Ala Tyr225 230 235 240Ser Val Ala Arg Gln Phe Asn
Met Ile Pro Pro Val Cys Glu Gln Ala 245 250 255Glu Tyr His Leu Phe
Gln Arg Glu Lys Val Glu Val Gln Leu Pro Glu 260 265 270Leu Tyr His
Lys Ile Gly Val Gly Ala Met Thr Trp Ser Pro Leu Ala 275 280 285Cys
Gly Ile Ile Ser Gly Lys Tyr Gly Asn Gly Val Pro Glu Ser Ser 290 295
300Arg Ala Ser Leu Lys Cys Tyr Gln Trp Leu Lys Glu Arg Ile Val
Ser305 310 315 320Glu Glu Gly Arg Lys Gln Gln Asn Lys Leu Lys Asp
Leu Ser Pro Ile 325 330 335Ala Glu Arg Leu Gly Cys Thr Leu Pro Gln
Leu Ala Val Ala Trp Cys 340 345 350Leu Arg Asn Glu Gly Val Ser Ser
Val Leu Leu Gly Ser Ser Thr Pro 355 360 365Glu Gln Leu Ile Glu Asn
Leu Gly Ala Ile Gln Val Leu Pro Lys Met 370 375 380Thr Ser His Val
Val Asn Glu Ile Asp Asn Ile Leu Arg Asn Lys Pro385 390 395 400Tyr
Ser Lys Lys Asp Tyr Arg Ser 405313744DNAHomo sapiens 31ccacgcgtcc
ggtggcggtc gagcgtggcg taggcgaatc ctcggcacta agcatatgga 60cctcgcggcg
gcagcggagc cgggcgccgg cagccagcac ctggaggtcc gcgacgaggt
120ggccgagaag tgccagaaac tgttcctgga cttcttggag gagtttcaga
gcagcgatgg 180agaaattaaa tacttgcaat tagcagagga actgattcgt
cctgagagaa acacattggt 240tgtgagtttt gtggacctgg aacaatttaa
ccagcaactt tccaccacca ttcaagagga 300gttctataga gtttaccctt
acctgtgtcg ggccttgaaa acattcgtca aagaccgtaa 360agagatccct
cttgccaagg atttttatgt tgcattccaa gacctgccta ccagacacaa
420gattcgagag ctcacctcat ccagaattgg tttgctcact cgcatcagtg
ggcaggtggt 480gcggactcac ccagttcacc cagagcttgt gagcggaact
tttctgtgct tggactgtca 540gacagtgatc agggatgtag aacagcagtt
caaatacaca cagccaaaca tctgccgaaa 600tccagtttgt gccaacagga
ggagattctt actggataca aataaatcaa gatttgttga 660ttttcaaaag
gttcgtattc aagagaccca agctgagctt cctcgaggga gtatcccccg
720cagtttagaa gtaattttaa gggctgaagc tgtggaatca gctcaagctg
gtgacaagtg 780tgactttaca gggacactga ttgttgtgcc tgacgtctcc
aagcttagca caccaggagc 840acgtgcagaa actaattccc gtgtcagtgg
tgttgatgga tatgagacag aaggcattcg 900aggactccgg gcccttggtg
ttagggacct ttcttatagg ctggtctttc ttgcctgctg 960tgttgcgcca
accaacccaa ggtttggggg gaaagagctc agagatgagg aacagacagc
1020tgagagcatt aagaaccaaa tgactgtgaa agaatgggag aaagtgtttg
agatgagtca 1080agataaaaat ctataccaca atctttgtac cagcctgttc
cctactatac atggcaatga 1140tgaagtaaaa cggggtgtcc tgctgatgct
ctttggtggc gttccaaaga caacaggaga 1200agggacctct cttcgagggg
acataaatgt ttgcattgtt ggtgacccaa gtacagctaa 1260gagccaattt
ctcaagcacg tggaggagtt cagccccaga gctgtctaca ccagtggtaa
1320agcgtccagt gctgctggct taacagcagc tgttgtgaga gatgaagaat
ctcatgagtt 1380tgtcattgag gctggagctt tgatgttggc tgataatggt
gtgtgttgta ttgatgaatt 1440tgataagatg gacgtgcggg atcaagttgc
tattcatgaa gctatggaac agcagaccat 1500atccatcact aaagcaggag
tgaaggctac tctgaacgcc cggacgtcca ttttggcagc 1560agcaaaccca
atcagtggac actatgacag atcaaaatca ttgaaacaga atataaattt
1620gtcagctccc atcatgtccc gattcgatct cttctttatc cttgtggatg
aatgtaatga 1680ggttacagat tatgccattg ccaggcgcat agtagatttg
cattcaagaa ttgaggaatc 1740aattgatcgt gtctattccc tcgatgatat
cagaagatat cttctctttg caagacagtt 1800taaacccaag atttccaaag
agtcagagga cttcattgtg gagcaatata aacatctccg 1860ccagagagat
ggttctggag tgaccaagtc ttcatggagg attacagtgc gacagcttga
1920gagcatgatt cgtctctctg aagctatggc tcggatgcac tgctgtgatg
aggtccaacc 1980taaacatgtg aaggaagctt tccggttact gaataaatca
atcatccgtg tggaaacacc 2040tgatgtcaat ctagatcaag aggaagagat
ccagatggag gtagatgagg gtgccggtgg 2100catcaatggt catgctgaca
gccctgctcc tgtgaacggg atcaatggct acaatgaaga 2160cataaatcaa
gagtctgctc ccaaagcctc cttaaggctg ggcttctctg agtactgccg
2220aatctctaac cttattgtgc ttcacctcag aaaggtggaa gaagaagagg
acgagtcagc 2280attaaagagg agcgagcttg ttaactggta cttgaaggaa
atcgaatcag agatagactc 2340tgaagaagaa cttataaata aaaaaagaat
catagagaaa gttattcatc gactcacaca 2400ctatgatcat gttctaattg
agctcaccca ggctggattg aaaggctcca cagagggaag 2460tgagagctat
gaagaagatc cctacttggt agttaaccct aactacttgc tcgaagattg
2520agatagtgaa agtaactgac cagagctgag gaactgtggc acagcacctc
gtggcctgga 2580gcctggctgg agctctgcta gggacagaag tgtttctgga
agtgatgctt ccaggatttg 2640ttttcagaaa caagaattga gttgatggtc
ctatgtgtca cattcatcac aggtttcata 2700ccaacacagg cttcagcact
tcctttggtg tgtttcctgt cccagtgaag ttggaaccaa 2760ataatgtgta
gtctctataa ccaatacctt tgttttcatg tgtaagaaaa ggcccattac
2820ttttaaggta tgtgctgtcc tattgagcaa ataacttttt ttcaattgcc
agctactgct 2880tttattcatc aaaataaaat aacttgttct gaagttgtct
attggatttc tttctactgt 2940accctgatta ttacttccat ctacttctga
atgtgagact ttcccttttt gcttaacctg 3000gagtgaagag gtagaactgt
ggtattatgg atgaggtttc tatgagaagg agtcattaga 3060gaactcatat
gaaagctaga ggccttagag atgactttcc aaggttaatt ccagtttttt
3120ttttttttaa gtttataaaa gtttattata cttttttaaa attactcttt
agtaatttat 3180tttacttctg tgtcctaagg gtaatttctc aggattgttt
tcaaattgct tttttagggg 3240aaataggtca tttgctatat tacaagcaat
ccccaaattt tatggtcttc caggaaaagt 3300tattaccgtt tatgatacta
acagttcctg agacttagct atgatcagta tgttcatgag 3360gtggagcagt
tcctgtgttg cagcttttaa caacagatgg cattcattaa atcacaaagt
3420atgttaaagg tcacaaaagc aaaataactg tctgaggcta aggcccacgt
gggacagtct 3480aatacccatg agtactcaac ttgccttgat gtctgagctt
tccagtgcaa tgtgaatttg 3540agcagccaga aatctattag tagaaagcaa
gacagattaa tataggttaa aacaatgatt 3600taaatatgtt tctcccaata
attatctctt tccctggaat caacttgtat gaaaccttgt 3660caaaatgtac
tccacaagta tgtacaatta agtattttaa aaataaatgg caaacattaa
3720aaaaaaaaaa aaaaaaaaaa aaaa 374432821PRTHomo sapiens 32Met Asp
Leu Ala Ala Ala Ala Glu Pro Gly Ala Gly Ser Gln His Leu1 5 10 15Glu
Val Arg Asp Glu Val Ala Glu Lys Cys Gln Lys Leu Phe Leu Asp 20 25
30Phe Leu Glu Glu Phe Gln Ser Ser Asp Gly Glu Ile Lys Tyr Leu Gln
35 40 45Leu Ala Glu Glu Leu Ile Arg Pro Glu Arg Asn Thr Leu Val Val
Ser 50 55 60Phe Val Asp Leu Glu Gln Phe Asn Gln Gln Leu Ser Thr Thr
Ile Gln65 70 75 80Glu Glu Phe Tyr Arg Val Tyr Pro Tyr Leu Cys Arg
Ala Leu Lys Thr 85 90 95Phe Val Lys Asp Arg Lys Glu Ile Pro Leu Ala
Lys Asp Phe Tyr Val 100 105 110Ala Phe Gln Asp Leu Pro Thr Arg His
Lys Ile Arg Glu Leu Thr Ser 115 120 125Ser Arg Ile Gly Leu Leu Thr
Arg Ile Ser Gly Gln Val Val Arg Thr 130 135 140His Pro Val His Pro
Glu Leu Val Ser Gly Thr Phe Leu Cys Leu Asp145 150 155 160Cys Gln
Thr Val Ile Arg Asp Val Glu Gln Gln Phe Lys Tyr Thr Gln 165 170
175Pro Asn Ile Cys Arg Asn Pro Val Cys Ala Asn Arg Arg Arg Phe Leu
180 185 190Leu Asp Thr Asn Lys Ser Arg Phe Val Asp Phe Gln Lys Val
Arg Ile 195 200 205Gln Glu Thr Gln Ala Glu Leu Pro Arg Gly Ser Ile
Pro Arg Ser Leu 210 215 220Glu Val Ile Leu Arg Ala Glu Ala Val Glu
Ser Ala Gln Ala Gly Asp225 230 235 240Lys Cys Asp Phe Thr Gly Thr
Leu Ile Val Val Pro Asp Val Ser Lys 245 250 255Leu Ser Thr Pro Gly
Ala Arg Ala Glu Thr Asn Ser Arg Val Ser Gly 260 265 270Val Asp Gly
Tyr Glu Thr Glu Gly Ile Arg Gly Leu Arg Ala Leu Gly 275 280 285Val
Arg Asp Leu Ser Tyr Arg Leu Val Phe Leu Ala Cys Cys Val Ala 290 295
300Pro Thr Asn Pro Arg Phe Gly Gly Lys Glu Leu Arg Asp Glu Glu
Gln305 310 315 320Thr Ala Glu Ser Ile Lys Asn Gln Met Thr Val Lys
Glu Trp Glu Lys 325 330 335Val Phe Glu Met Ser Gln Asp Lys Asn Leu
Tyr His Asn Leu Cys Thr 340 345 350Ser Leu Phe Pro Thr Ile His Gly
Asn Asp Glu Val Lys Arg Gly Val 355 360 365Leu Leu Met Leu Phe Gly
Gly Val Pro Lys Thr Thr Gly Glu Gly Thr 370 375 380Ser Leu Arg Gly
Asp Ile Asn Val Cys Ile Val Gly Asp Pro Ser Thr385 390 395 400Ala
Lys Ser Gln Phe Leu Lys His Val Glu Glu Phe Ser Pro Arg Ala 405 410
415Val Tyr Thr Ser Gly Lys Ala Ser Ser Ala Ala Gly Leu Thr Ala Ala
420 425 430Val Val Arg Asp Glu Glu Ser His Glu Phe Val Ile Glu Ala
Gly Ala 435 440 445Leu Met Leu Ala Asp Asn Gly Val Cys Cys Ile Asp
Glu Phe Asp Lys 450 455 460Met Asp Val Arg Asp Gln Val Ala Ile His
Glu Ala Met Glu Gln Gln465 470 475 480Thr Ile Ser Ile Thr Lys Ala
Gly Val Lys Ala Thr Leu Asn Ala Arg 485 490 495Thr Ser Ile Leu Ala
Ala Ala Asn Pro Ile Ser Gly His Tyr Asp Arg 500 505 510Ser Lys Ser
Leu Lys Gln Asn Ile Asn Leu Ser Ala Pro Ile Met Ser 515 520 525Arg
Phe Asp Leu Phe Phe Ile Leu Val Asp Glu Cys Asn Glu Val Thr 530 535
540Asp Tyr Ala Ile Ala Arg Arg Ile Val Asp Leu His Ser Arg Ile
Glu545 550 555 560Glu Ser Ile Asp Arg Val Tyr Ser Leu Asp Asp Ile
Arg Arg Tyr Leu 565 570 575Leu Phe Ala Arg Gln Phe Lys Pro Lys Ile
Ser Lys Glu Ser Glu Asp 580 585 590Phe Ile Val Glu Gln Tyr Lys His
Leu Arg Gln Arg Asp Gly Ser Gly 595 600 605Val Thr Lys Ser Ser Trp
Arg Ile Thr Val Arg Gln Leu Glu Ser Met 610 615 620Ile Arg Leu Ser
Glu Ala Met Ala Arg Met His Cys Cys Asp Glu Val625 630 635 640Gln
Pro Lys His Val Lys Glu Ala Phe Arg Leu Leu Asn Lys Ser Ile 645 650
655Ile Arg Val Glu Thr Pro Asp Val Asn Leu Asp Gln Glu Glu Glu Ile
660 665 670Gln Met Glu Val Asp Glu Gly Ala Gly Gly Ile Asn Gly His
Ala Asp 675 680 685Ser Pro Ala Pro Val Asn Gly Ile Asn Gly Tyr Asn
Glu Asp Ile Asn 690 695 700Gln Glu Ser Ala Pro Lys Ala Ser Leu Arg
Leu Gly Phe Ser Glu Tyr705 710 715 720Cys Arg Ile Ser Asn Leu Ile
Val Leu His Leu Arg Lys Val Glu Glu 725 730 735Glu Glu Asp Glu Ser
Ala Leu Lys Arg Ser Glu Leu Val Asn Trp Tyr 740 745 750Leu Lys Glu
Ile Glu Ser Glu Ile Asp Ser Glu Glu Glu Leu Ile Asn 755 760 765Lys
Lys Arg Ile Ile Glu Lys Val Ile His Arg Leu Thr His Tyr Asp 770 775
780His Val Leu Ile Glu Leu Thr Gln Ala Gly Leu Lys Gly Ser Thr
Glu785 790 795 800Gly Ser Glu Ser Tyr Glu Glu Asp Pro Tyr Leu Val
Val Asn Pro Asn 805 810 815Tyr Leu Leu Glu Asp 820332111DNAHomo
sapiens 33ggccggccac tcccgtctgc tgtgacgcgc ggacagagag ctaccggtgg
acccacggtg 60cctccctccc tgggatctac acagaccatg gccttgccaa cggctcgacc
cctgttgggg 120tcctgtggga cccccgccct cggcagcctc ctgttcctgc
tcttcagcct cggatgggtg 180cagccctcga ggaccctggc tggagagaca
gggcaggagg ctgcacccct ggacggagtc 240ctggccaacc cacctaacat
ttccagcctc tcccctcgcc aactccttgg cttcccgtgt 300gcggaggtgt
ccggcctgag cacggagcgt gtccgggagc tggctgtggc cttggcacag
360aagaatgtca agctctcaac agagcagctg cgctgtctgg ctcaccggct
ctctgagccc 420cccgaggacc tggacgccct cccattggac ctgctgctat
tcctcaaccc agatgcgttc 480tcggggcccc aggcctgcac ccgtttcttc
tcccgcatca cgaaggccaa tgtggacctg 540ctcccgaggg gggctcccga
gcgacagcgg ctgctgcctg cggctctggc ctgctggggt 600gtgcgggggt
ctctgctgag cgaggctgat gtgcgggctc tgggaggcct ggcttgcgac
660ctgcctgggc gctttgtggc cgagtcggcc gaagtgctgc taccccggct
ggtgagctgc 720ccgggacccc tggaccagga ccagcaggag gcagccaggg
cggctctgca gggcggggga 780cccccctacg gccccccgtc gacatggtct
gtctccacga tggacgctct gcggggcctg 840ctgcccgtgc tgggccagcc
catcatccgc agcatcccgc agggcatcgt ggccgcgtgg 900cggcaacgct
cctctcggga cccatcctgg cggcagcctg aacggaccat cctccggccg
960cggttccggc gggaagtgga gaagacagcc tgtccttcag gcaagaaggc
ccgcgagata 1020gacgagagcc tcatcttcta caagaagtgg gagctggaag
cctgcgtgga tgcggccctg 1080ctggccaccc agatggaccg cgtgaacgcc
atccccttca cctacgagca gctggacgtc 1140ctaaagcata aactggatga
gctctaccca caaggttacc ccgagtctgt gatccagcac 1200ctgggctacc
tcttcctcaa gatgagccct gaggacattc gcaagtggaa tgtgacgtcc
1260ctggagaccc tgaaggcttt gcttgaagtc aacaaagggc acgaaatgag
tcctcaggtg 1320gccaccctga tcgaccgctt tgtgaaggga aggggccagc
tagacaaaga caccctagac 1380accctgaccg ccttctaccc tgggtacctg
tgctccctca gccccgagga gctgagctcc 1440gtgcccccca gcagcatctg
ggcggtcagg ccccaggacc tggacacgtg tgacccaagg 1500cagctggacg
tcctctatcc caaggcccgc cttgctttcc agaacatgaa cgggtccgaa
1560tacttcgtga agatccagtc cttcctgggt ggggccccca cggaggattt
gaaggcgctc 1620agtcagcaga atgtgagcat ggacttggcc acgttcatga
agctgcggac ggatgcggtg 1680ctgccgttga ctgtggctga ggtgcagaaa
cttctgggac cccacgtgga gggcctgaag 1740gcggaggagc ggcaccgccc
ggtgcgggac tggatcctac ggcagcggca ggacgacctg 1800gacacgctgg
ggctggggct acagggcggc atccccaacg gctacctggt cctagacctc
1860agcgtgcaag aggccctctc ggggacgccc tgcctcctag gacctggacc
tgttctcacc 1920gtcctggcac tgctcctagc ctccaccctg gcctgagggc
cccactccct tgctggcccc 1980agccctgctg gggatccccg cctggccagg
agcaggcacg ggtgatcccc gttccacccc 2040aagagaactc gcgctcagta
aacgggaaca tgccccctgc agacacgtaa aaaaaaaaaa 2100aaaaaaaaaa a
211134622PRTHomo sapiens 34Met Ala Leu Pro Thr Ala Arg Pro Leu Leu
Gly Ser Cys Gly Thr Pro1 5 10 15Ala Leu Gly Ser Leu Leu Phe Leu Leu
Phe Ser Leu Gly Trp Val Gln 20 25 30Pro Ser Arg Thr Leu Ala Gly Glu
Thr Gly Gln Glu Ala Ala Pro Leu 35 40 45Asp Gly Val Leu Ala Asn Pro
Pro Asn Ile Ser Ser Leu Ser Pro Arg 50 55 60Gln Leu Leu Gly Phe Pro
Cys Ala Glu Val Ser Gly Leu Ser Thr Glu65 70 75 80Arg Val Arg Glu
Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu 85 90 95Ser Thr Glu
Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro 100 105 110Glu
Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu Asn Pro 115 120
125Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg Phe Phe Ser Arg Ile
130 135 140Thr Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu
Arg Gln145 150 155 160Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly
Val Arg Gly Ser Leu 165 170 175Leu Ser Glu Ala Asp Val Arg Ala Leu
Gly Gly Leu Ala Cys Asp Leu 180 185 190Pro Gly Arg Phe Val Ala Glu
Ser Ala Glu Val Leu Leu Pro Arg Leu 195 200 205Val Ser Cys Pro Gly
Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg 210 215 220Ala Ala Leu
Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp225 230 235
240Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly
245 250 255Gln Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala
Trp Arg 260 265 270Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro
Glu Arg Thr Ile 275 280 285Leu Arg Pro Arg Phe Arg Arg Glu Val Glu
Lys Thr Ala Cys Pro Ser 290 295 300Gly Lys Lys Ala Arg Glu Ile Asp
Glu Ser Leu Ile Phe Tyr Lys Lys305 310 315 320Trp Glu Leu Glu Ala
Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met 325 330 335Asp Arg Val
Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu 340 345 350Lys
His Lys Leu Asp Glu Leu Tyr Pro Gln Gly
Tyr Pro Glu Ser Val 355 360 365Ile Gln His Leu Gly Tyr Leu Phe Leu
Lys Met Ser Pro Glu Asp Ile 370 375 380Arg Lys Trp Asn Val Thr Ser
Leu Glu Thr Leu Lys Ala Leu Leu Glu385 390 395 400Val Asn Lys Gly
His Glu Met Ser Pro Gln Val Ala Thr Leu Ile Asp 405 410 415Arg Phe
Val Lys Gly Arg Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr 420 425
430Leu Thr Ala Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu
435 440 445Leu Ser Ser Val Pro Pro Ser Ser Ile Trp Ala Val Arg Pro
Gln Asp 450 455 460Leu Asp Thr Cys Asp Pro Arg Gln Leu Asp Val Leu
Tyr Pro Lys Ala465 470 475 480Arg Leu Ala Phe Gln Asn Met Asn Gly
Ser Glu Tyr Phe Val Lys Ile 485 490 495Gln Ser Phe Leu Gly Gly Ala
Pro Thr Glu Asp Leu Lys Ala Leu Ser 500 505 510Gln Gln Asn Val Ser
Met Asp Leu Ala Thr Phe Met Lys Leu Arg Thr 515 520 525Asp Ala Val
Leu Pro Leu Thr Val Ala Glu Val Gln Lys Leu Leu Gly 530 535 540Pro
His Val Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val Arg545 550
555 560Asp Trp Ile Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly
Leu 565 570 575Gly Leu Gln Gly Gly Ile Pro Asn Gly Tyr Leu Val Leu
Asp Leu Ser 580 585 590Val Gln Glu Ala Leu Ser Gly Thr Pro Cys Leu
Leu Gly Pro Gly Pro 595 600 605Val Leu Thr Val Leu Ala Leu Leu Leu
Ala Ser Thr Leu Ala 610 615 620352731DNAHomo sapiens 35gcgcttggcg
ggagatagaa aagtgcttca acccgcgccg gcggcgactg cagttcctgc 60gagcgaggag
cgcgggacct gctgacacgc tgacgccttc gagcgcggcc cggggcccgg
120agcggccgga gcagcccggg tcctgacccc ggcccggctc ccgctccggg
ctctgccggc 180gggcgggcga gcgcggcgcg gtccgggccg gggggatgtc
tcggcggacg cgctgcgagg 240atctggatga gctgcactac caggacacag
attcagatgt gccggagcag agggatagca 300agtgcaaggt caaatggacc
catgaggagg acgagcagct gagggccctg gtgaggcagt 360ttggacagca
ggactggaag ttcctggcca gccacttccc taaccgcact gaccagcaat
420gccagtacag gtggctgaga gttttgaatc cagaccttgt caaggggcca
tggaccaaag 480aggaagacca aaaagtcatc gagctggtta agaagtatgg
cacaaagcag tggacactga 540ttgccaagca cctgaagggc cggctgggga
agcagtgccg tgaacgctgg cacaaccacc 600tcaaccctga ggtgaagaag
tcttgctgga ccgaggagga ggaccgcatc atctgcgagg 660cccacaaggt
gctgggcaac cgctgggccg agatcgccaa gatgttgcca gggaggacag
720acaatgctgt gaagaatcac tggaactcta ccatcaaaag gaaggtggac
acaggaggct 780tcttgagcga gtccaaagac tgcaagcccc cagtgtactt
gctgctggag ctcgaggaca 840aggacggcct ccagagtgcc cagcccacgg
aaggccaggg aagtcttctg accaactggc 900cctccgtccc tcctaccata
aaggaggagg aaaacagtga ggaggaactt gcagcagcca 960ccacatcgaa
ggaacaggag cccatcggta cagatctgga cgcagtgcga acaccagagc
1020ccttggagga attcccgaag cgtgaggacc aggaaggctc cccaccagaa
acgagcctgc 1080cttacaagtg ggtggtggag gcagctaacc tcctcatccc
cgctgtgggt tctagcctct 1140ctgaagccct ggacttgatc gagtcggacc
ctgatgcttg gtgtgacctg agtaaatttg 1200acctccctga ggaaccatct
gcagaggaca gtatcaacaa cagcctagtg cagctgcaag 1260cgtcacatca
gcagcaagtc ctgccacccc gccagccttc cgccctggtg cccagtgtga
1320ccgagtaccg cctggatggc cacaccatct cagacctgag ccggagcagc
cggggcgagc 1380tgatccccat ctcccccagc actgaagtcg ggggctctgg
cattggcaca ccgccctctg 1440tgctcaagcg gcagaggaag aggcgtgtgg
ctctgtcccc tgtcactgag aatagcacca 1500gtctgtcctt cctggattcc
tgtaacagcc tcacgcccaa gagcacacct gttaagaccc 1560tgcccttctc
gccctcccag tttctgaact tctggaacaa acaggacaca ttggagctgg
1620agagcccctc gctgacatcc accccagtgt gcagccagaa ggtggtggtc
accacaccac 1680tgcaccggga caagacaccc ctgcaccaga aacatgctgc
gtttgtaacc ccagatcaga 1740agtactccat ggacaacact ccccacacgc
caaccccgtt caagaacgcc ctggagaagt 1800acggacccct gaagcccctg
ccacagaccc cgcacctgga ggaggacttg aaggaggtgc 1860tgcgttctga
ggctggcatc gaactcatca tcgaggacga catcaggccc gagaagcaga
1920agaggaagcc tgggctgcgg cggagcccca tcaagaaagt ccggaagtct
ctggctcttg 1980acattgtgga tgaggatgtg aagctgatga tgtccacact
gcccaagtct ctatccttgc 2040cgacaactgc cccttcaaac tcttccagcc
tcaccctgtc aggtatcaaa gaagacaaca 2100gcttgctcaa ccagggcttc
ttgcaggcca agcccgagaa ggcagcagtg gcccagaagc 2160cccgaagcca
cttcacgaca cctgccccta tgtccagtgc ctggaagacg gtggcctgcg
2220gggggaccag ggaccagctt ttcatgcagg agaaagcccg gcagctcctg
ggccgcctga 2280agcccagcca cacatctcgg accctcatct tgtcctgagg
tgttgagggt gtcacgagcc 2340cattctcatg tttacagggg ttgtgggggc
agagggggtc tgtgaatctg agagtcattc 2400aggtgacctc ctgcagggag
ccttctgcca ccagcccctc cccagactct caggtggagg 2460caacagggcc
atgtgctgcc ctgttgccga gcccagctgt gggcggctcc tggtgctaac
2520aacaaagttc cacttccagg tctgcctggt tccctcccca aggccacagg
gagctccgtc 2580agcttctccc aagcccacgt caggcctggc ctcatctcag
accctgctta ggatggggga 2640tgtggccagg ggtgctcctg tgctcaccct
ctcttggtgc atttttttgg aagaataaaa 2700ttgcctctct cttaaaaaaa
aaaaaaaaaa a 273136700PRTHomo sapiens 36Met Ser Arg Arg Thr Arg Cys
Glu Asp Leu Asp Glu Leu His Tyr Gln1 5 10 15Asp Thr Asp Ser Asp Val
Pro Glu Gln Arg Asp Ser Lys Cys Lys Val 20 25 30Lys Trp Thr His Glu
Glu Asp Glu Gln Leu Arg Ala Leu Val Arg Gln 35 40 45Phe Gly Gln Gln
Asp Trp Lys Phe Leu Ala Ser His Phe Pro Asn Arg 50 55 60Thr Asp Gln
Gln Cys Gln Tyr Arg Trp Leu Arg Val Leu Asn Pro Asp65 70 75 80Leu
Val Lys Gly Pro Trp Thr Lys Glu Glu Asp Gln Lys Val Ile Glu 85 90
95Leu Val Lys Lys Tyr Gly Thr Lys Gln Trp Thr Leu Ile Ala Lys His
100 105 110Leu Lys Gly Arg Leu Gly Lys Gln Cys Arg Glu Arg Trp His
Asn His 115 120 125Leu Asn Pro Glu Val Lys Lys Ser Cys Trp Thr Glu
Glu Glu Asp Arg 130 135 140Ile Ile Cys Glu Ala His Lys Val Leu Gly
Asn Arg Trp Ala Glu Ile145 150 155 160Ala Lys Met Leu Pro Gly Arg
Thr Asp Asn Ala Val Lys Asn His Trp 165 170 175Asn Ser Thr Ile Lys
Arg Lys Val Asp Thr Gly Gly Phe Leu Ser Glu 180 185 190Ser Lys Asp
Cys Lys Pro Pro Val Tyr Leu Leu Leu Glu Leu Glu Asp 195 200 205Lys
Asp Gly Leu Gln Ser Ala Gln Pro Thr Glu Gly Gln Gly Ser Leu 210 215
220Leu Thr Asn Trp Pro Ser Val Pro Pro Thr Ile Lys Glu Glu Glu
Asn225 230 235 240Ser Glu Glu Glu Leu Ala Ala Ala Thr Thr Ser Lys
Glu Gln Glu Pro 245 250 255Ile Gly Thr Asp Leu Asp Ala Val Arg Thr
Pro Glu Pro Leu Glu Glu 260 265 270Phe Pro Lys Arg Glu Asp Gln Glu
Gly Ser Pro Pro Glu Thr Ser Leu 275 280 285Pro Tyr Lys Trp Val Val
Glu Ala Ala Asn Leu Leu Ile Pro Ala Val 290 295 300Gly Ser Ser Leu
Ser Glu Ala Leu Asp Leu Ile Glu Ser Asp Pro Asp305 310 315 320Ala
Trp Cys Asp Leu Ser Lys Phe Asp Leu Pro Glu Glu Pro Ser Ala 325 330
335Glu Asp Ser Ile Asn Asn Ser Leu Val Gln Leu Gln Ala Ser His Gln
340 345 350Gln Gln Val Leu Pro Pro Arg Gln Pro Ser Ala Leu Val Pro
Ser Val 355 360 365Thr Glu Tyr Arg Leu Asp Gly His Thr Ile Ser Asp
Leu Ser Arg Ser 370 375 380Ser Arg Gly Glu Leu Ile Pro Ile Ser Pro
Ser Thr Glu Val Gly Gly385 390 395 400Ser Gly Ile Gly Thr Pro Pro
Ser Val Leu Lys Arg Gln Arg Lys Arg 405 410 415Arg Val Ala Leu Ser
Pro Val Thr Glu Asn Ser Thr Ser Leu Ser Phe 420 425 430Leu Asp Ser
Cys Asn Ser Leu Thr Pro Lys Ser Thr Pro Val Lys Thr 435 440 445Leu
Pro Phe Ser Pro Ser Gln Phe Leu Asn Phe Trp Asn Lys Gln Asp 450 455
460Thr Leu Glu Leu Glu Ser Pro Ser Leu Thr Ser Thr Pro Val Cys
Ser465 470 475 480Gln Lys Val Val Val Thr Thr Pro Leu His Arg Asp
Lys Thr Pro Leu 485 490 495His Gln Lys His Ala Ala Phe Val Thr Pro
Asp Gln Lys Tyr Ser Met 500 505 510Asp Asn Thr Pro His Thr Pro Thr
Pro Phe Lys Asn Ala Leu Glu Lys 515 520 525Tyr Gly Pro Leu Lys Pro
Leu Pro Gln Thr Pro His Leu Glu Glu Asp 530 535 540Leu Lys Glu Val
Leu Arg Ser Glu Ala Gly Ile Glu Leu Ile Ile Glu545 550 555 560Asp
Asp Ile Arg Pro Glu Lys Gln Lys Arg Lys Pro Gly Leu Arg Arg 565 570
575Ser Pro Ile Lys Lys Val Arg Lys Ser Leu Ala Leu Asp Ile Val Asp
580 585 590Glu Asp Val Lys Leu Met Met Ser Thr Leu Pro Lys Ser Leu
Ser Leu 595 600 605Pro Thr Thr Ala Pro Ser Asn Ser Ser Ser Leu Thr
Leu Ser Gly Ile 610 615 620Lys Glu Asp Asn Ser Leu Leu Asn Gln Gly
Phe Leu Gln Ala Lys Pro625 630 635 640Glu Lys Ala Ala Val Ala Gln
Lys Pro Arg Ser His Phe Thr Thr Pro 645 650 655Ala Pro Met Ser Ser
Ala Trp Lys Thr Val Ala Cys Gly Gly Thr Arg 660 665 670Asp Gln Leu
Phe Met Gln Glu Lys Ala Arg Gln Leu Leu Gly Arg Leu 675 680 685Lys
Pro Ser His Thr Ser Arg Thr Leu Ile Leu Ser 690 695
700372304DNAHomo sapiens 37gtccccgcag cgccgtcgcg ccctcctgcc
gcaggccacc gaggccgccg ccgtctagcg 60ccccgacctc gccaccatga gagccctgct
ggcgcgcctg cttctctgcg tcctggtcgt 120gagcgactcc aaaggcagca
atgaacttca tcaagttcca tcgaactgtg actgtctaaa 180tggaggaaca
tgtgtgtcca acaagtactt ctccaacatt cactggtgca actgcccaaa
240gaaattcgga gggcagcact gtgaaataga taagtcaaaa acctgctatg
aggggaatgg 300tcacttttac cgaggaaagg ccagcactga caccatgggc
cggccctgcc tgccctggaa 360ctctgccact gtccttcagc aaacgtacca
tgcccacaga tctgatgctc ttcagctggg 420cctggggaaa cataattact
gcaggaaccc agacaaccgg aggcgaccct ggtgctatgt 480gcaggtgggc
ctaaagccgc ttgtccaaga gtgcatggtg catgactgcg cagatggaaa
540aaagccctcc tctcctccag aagaattaaa atttcagtgt ggccaaaaga
ctctgaggcc 600ccgctttaag attattgggg gagaattcac caccatcgag
aaccagccct ggtttgcggc 660catctacagg aggcaccggg ggggctctgt
cacctacgtg tgtggaggca gcctcatcag 720cccttgctgg gtgatcagcg
ccacacactg cttcattgat tacccaaaga aggaggacta 780catcgtctac
ctgggtcgct caaggcttaa ctccaacacg caaggggaga tgaagtttga
840ggtggaaaac ctcatcctac acaaggacta cagcgctgac acgcttgctc
accacaacga 900cattgccttg ctgaagatcc gttccaagga gggcaggtgt
gcgcagccat cccggactat 960acagaccatc tgcctgccct cgatgtataa
cgatccccag tttggcacaa gctgtgagat 1020cactggcttt ggaaaagaga
attctaccga ctatctctat ccggagcagc tgaaaatgac 1080tgttgtgaag
ctgatttccc accgggagtg tcagcagccc cactactacg gctctgaagt
1140caccaccaaa atgctatgtg ctgctgaccc ccaatggaaa acagattcct
gccagggaga 1200ctcaggggga cccctcgtct gttccctcca aggccgcatg
actttgactg gaattgtgag 1260ctggggccgt ggatgtgccc tgaaggacaa
gccaggcgtc tacacgagag tctcacactt 1320cttaccctgg atccgcagtc
acaccaagga agagaatggc ctggccctct gagggtcccc 1380agggaggaaa
cgggcaccac ccgctttctt gctggttgtc atttttgcag tagagtcatc
1440tccatcagct gtaagaagag actgggaaga taggctctgc acagatggat
ttgcctgtgg 1500caccaccagg gtgaacgaca atagctttac cctcacggat
aggcctgggt gctggctgcc 1560cagaccctct ggccaggatg gaggggtggt
cctgactcaa catgttactg accagcaact 1620tgtctttttc tggactgaag
cctgcaggag ttaaaaaggg cagggcatct cctgtgcatg 1680ggctcgaagg
gagagccagc tcccccgacc ggtgggcatt tgtgaggccc atggttgaga
1740aatgaataat ttcccaatta ggaagtgtaa gcagctgagg tctcttgagg
gagcttagcc 1800aatgtgggag cagcggtttg gggagcagag acactaacga
cttcagggca gggctctgat 1860attccatgaa tgtatcagga aatatatatg
tgtgtgtatg tttgcacact tgttgtgtgg 1920gctgtgagtg taagtgtgag
taagagctgg tgtctgattg ttaagtctaa atatttcctt 1980aaactgtgtg
gactgtgatg ccacacagag tggtctttct ggagaggtta taggtcactc
2040ctggggcctc ttgggtcccc cacgtgacag tgcctgggaa tgtacttatt
ctgcagcatg 2100acctgtgacc agcactgtct cagtttcact ttcacataga
tgtccctttc ttggccagtt 2160atcccttcct tttagcctag ttcatccaat
cctcactggg tggggtgagg accactcctt 2220acactgaata tttatatttc
actattttta tttatatttt tgtaatttta aataaaagtg 2280atcaataaaa
tgtgattttt ctga 230438431PRTHomo sapiens 38Met Arg Ala Leu Leu Ala
Arg Leu Leu Leu Cys Val Leu Val Val Ser1 5 10 15Asp Ser Lys Gly Ser
Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp 20 25 30Cys Leu Asn Gly
Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile 35 40 45His Trp Cys
Asn Cys Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile 50 55 60Asp Lys
Ser Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly65 70 75
80Lys Ala Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser
85 90 95Ala Thr Val Leu Gln Gln Thr Tyr His Ala His Arg Ser Asp Ala
Leu 100 105 110Gln Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro
Asp Asn Arg 115 120 125Arg Arg Pro Trp Cys Tyr Val Gln Val Gly Leu
Lys Pro Leu Val Gln 130 135 140Glu Cys Met Val His Asp Cys Ala Asp
Gly Lys Lys Pro Ser Ser Pro145 150 155 160Pro Glu Glu Leu Lys Phe
Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg 165 170 175Phe Lys Ile Ile
Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp 180 185 190Phe Ala
Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val 195 200
205Cys Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile Ser Ala Thr His
210 215 220Cys Phe Ile Asp Tyr Pro Lys Lys Glu Asp Tyr Ile Val Tyr
Leu Gly225 230 235 240Arg Ser Arg Leu Asn Ser Asn Thr Gln Gly Glu
Met Lys Phe Glu Val 245 250 255Glu Asn Leu Ile Leu His Lys Asp Tyr
Ser Ala Asp Thr Leu Ala His 260 265 270His Asn Asp Ile Ala Leu Leu
Lys Ile Arg Ser Lys Glu Gly Arg Cys 275 280 285Ala Gln Pro Ser Arg
Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr 290 295 300Asn Asp Pro
Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys305 310 315
320Glu Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met Thr Val
325 330 335Val Lys Leu Ile Ser His Arg Glu Cys Gln Gln Pro His Tyr
Tyr Gly 340 345 350Ser Glu Val Thr Thr Lys Met Leu Cys Ala Ala Asp
Pro Gln Trp Lys 355 360 365Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly
Pro Leu Val Cys Ser Leu 370 375 380Gln Gly Arg Met Thr Leu Thr Gly
Ile Val Ser Trp Gly Arg Gly Cys385 390 395 400Ala Leu Lys Asp Lys
Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu 405 410 415Pro Trp Ile
Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 420 425
430391760DNAHomo sapiens 39gcagcaggcc aagggggagg tgcgagcgtg
gacctgggac gggtctgggc ggctctcggt 60ggttggcacg ggttcgcaca cccattcaag
cggcaggacg cacttgtctt agcagttctc 120gctgaccgcg ctagctgcgg
cttctacgct ccggcactct gagttcatca gcaaacgccc 180tggcgtctgt
cctcaccatg cctagccttt gggaccgctt ctcgtcgtcg tccacctcct
240cttcgccctc gtccttgccc cgaactccca ccccagatcg gccgccgcgc
tcagcctggg 300ggtcggcgac ccgggaggag gggtttgacc gctccacgag
cctggagagc tcggactgcg 360agtccctgga cagcagcaac agtggcttcg
ggccggagga agacacggct tacctggatg 420gggtgtcgtt gcccgacttc
gagctgctca gtgaccctga ggatgaacac ttgtgtgcca 480acctgatgca
gctgctgcag gagagcctgg cccaggcgcg gctgggctct cgacgccctg
540cgcgcctgct gatgcctagc cagttggtaa gccaggtggg caaagaacta
ctgcgcctgg 600cctacagcga gccgtgcggc ctgcgggggg cgctgctgga
cgtctgcgtg gagcagggca 660agagctgcca cagcgtgggc cagctggcac
tcgaccccag cctggtgccc accttccagc 720tgaccctcgt gctgcgcctg
gactcacgac tctggcccaa gatccagggg ctgtttagct 780ccgccaactc
tcccttcctc cctggcttca gccagtccct gacgctgagc actggcttcc
840gagtcatcaa gaagaagctg tacagctcgg aacagctgct cattgaggag
tgttgaactt 900caacctgagg gggccgacag tgccctccaa gacagagacg
actgaacttt tggggtggag 960actagaggca ggagctgagg gactgattcc
agtggttgga aaactgaggc agccacctaa 1020ggtggaggtg ggggaatagt
gtttcccagg aagctcattg agttgtgtgc gggtggctgt 1080gcattgggga
cacatacccc tcagtactgt agcatggaac aaaggcttag gggccaacaa
1140ggcttccagc tggatgtgtg tgtagcatgt accttattat ttttgttact
gacagttaac 1200agtggtgtga catccagaga gcagctgggc tgctcccgcc
ccagcctggc ccagggtgaa 1260ggaagaggca cgtgctcctc agagcagccg
gagggagggg ggaggtcgga ggtcgtggag 1320gtggtttgtg tatcttactg
gtctgaaggg accaagtgtg
tttgttgttt gttttgtatc 1380ttgtttttct gatcggagca tcactactga
cctgttgtag gcagctatct tacagacgca 1440tgaatgtaag agtaggaagg
ggtgggtgtc agggatcact tgggatcttt gacacttgaa 1500aaattacacc
tggcagctgc gtttaagcct tcccccatcg tgtactgcag agttgagctg
1560gcaggggagg ggctgagagg gtgggggctg gaacccctcc ccgggaggag
tgccatctgg 1620gtcttccatc tagaactgtt tacatgaaga taagatactc
actgttcatg aatacacttg 1680atgttcaagt attaagacct atgcaatatt
ttttactttt ctaataaaca tgtttgttaa 1740aacaaaaaaa aaaaaaaaaa
176040232PRTHomo sapiens 40Met Pro Ser Leu Trp Asp Arg Phe Ser Ser
Ser Ser Thr Ser Ser Ser1 5 10 15Pro Ser Ser Leu Pro Arg Thr Pro Thr
Pro Asp Arg Pro Pro Arg Ser 20 25 30Ala Trp Gly Ser Ala Thr Arg Glu
Glu Gly Phe Asp Arg Ser Thr Ser 35 40 45Leu Glu Ser Ser Asp Cys Glu
Ser Leu Asp Ser Ser Asn Ser Gly Phe 50 55 60Gly Pro Glu Glu Asp Thr
Ala Tyr Leu Asp Gly Val Ser Leu Pro Asp65 70 75 80Phe Glu Leu Leu
Ser Asp Pro Glu Asp Glu His Leu Cys Ala Asn Leu 85 90 95Met Gln Leu
Leu Gln Glu Ser Leu Ala Gln Ala Arg Leu Gly Ser Arg 100 105 110Arg
Pro Ala Arg Leu Leu Met Pro Ser Gln Leu Val Ser Gln Val Gly 115 120
125Lys Glu Leu Leu Arg Leu Ala Tyr Ser Glu Pro Cys Gly Leu Arg Gly
130 135 140Ala Leu Leu Asp Val Cys Val Glu Gln Gly Lys Ser Cys His
Ser Val145 150 155 160Gly Gln Leu Ala Leu Asp Pro Ser Leu Val Pro
Thr Phe Gln Leu Thr 165 170 175Leu Val Leu Arg Leu Asp Ser Arg Leu
Trp Pro Lys Ile Gln Gly Leu 180 185 190Phe Ser Ser Ala Asn Ser Pro
Phe Leu Pro Gly Phe Ser Gln Ser Leu 195 200 205Thr Leu Ser Thr Gly
Phe Arg Val Ile Lys Lys Lys Leu Tyr Ser Ser 210 215 220Glu Gln Leu
Leu Ile Glu Glu Cys225 230415698DNAHomo sapiens 41aggttcaagt
ggagctctcc taaccgacgc gcgtctgtgg agaagcggct tggtcggggg 60tggtctcgtg
gggtcctgcc tgtttagtcg ctttcagggt tcttgagccc cttcacgacc
120gtcaccatgg aagtgtcacc attgcagcct gtaaatgaaa atatgcaagt
caacaaaata 180aagaaaaatg aagatgctaa gaaaagactg tctgttgaaa
gaatctatca aaagaaaaca 240caattggaac atattttgct ccgcccagac
acctacattg gttctgtgga attagtgacc 300cagcaaatgt gggtttacga
tgaagatgtt ggcattaact atagggaagt cacttttgtt 360cctggtttgt
acaaaatctt tgatgagatt ctagttaatg ctgcggacaa caaacaaagg
420gacccaaaaa tgtcttgtat tagagtcaca attgatccgg aaaacaattt
aattagtata 480tggaataatg gaaaaggtat tcctgttgtt gaacacaaag
ttgaaaagat gtatgtccca 540gctctcatat ttggacagct cctaacttct
agtaactatg atgatgatga aaagaaagtg 600acaggtggtc gaaatggcta
tggagccaaa ttgtgtaaca tattcagtac caaatttact 660gtggaaacag
ccagtagaga atacaagaaa atgttcaaac agacatggat ggataatatg
720ggaagagctg gtgagatgga actcaagccc ttcaatggag aagattatac
atgtatcacc 780tttcagcctg atttgtctaa gtttaaaatg caaagcctgg
acaaagatat tgttgcacta 840atggtcagaa gagcatatga tattgctgga
tccaccaaag atgtcaaagt ctttcttaat 900ggaaataaac tgccagtaaa
aggatttcgt agttatgtgg acatgtattt gaaggacaag 960ttggatgaaa
ctggtaactc cttgaaagta atacatgaac aagtaaacca caggtgggaa
1020gtgtgtttaa ctatgagtga aaaaggcttt cagcaaatta gctttgtcaa
cagcattgct 1080acatccaagg gtggcagaca tgttgattat gtagctgatc
agattgtgac taaacttgtt 1140gatgttgtga agaagaagaa caagggtggt
gttgcagtaa aagcacatca ggtgaaaaat 1200cacatgtgga tttttgtaaa
tgccttaatt gaaaacccaa cctttgactc tcagacaaaa 1260gaaaacatga
ctttacaacc caagagcttt ggatcaacat gccaattgag tgaaaaattt
1320atcaaagctg ccattggctg tggtattgta gaaagcatac taaactgggt
gaagtttaag 1380gcccaagtcc agttaaacaa gaagtgttca gctgtaaaac
ataatagaat caagggaatt 1440cccaaactcg atgatgccaa tgatgcaggg
ggccgaaact ccactgagtg tacgcttatc 1500ctgactgagg gagattcagc
caaaactttg gctgtttcag gccttggtgt ggttgggaga 1560gacaaatatg
gggttttccc tcttagagga aaaatactca atgttcgaga agcttctcat
1620aagcagatca tggaaaatgc tgagattaac aatatcatca agattgtggg
tcttcagtac 1680aagaaaaact atgaagatga agattcattg aagacgcttc
gttatgggaa gataatgatt 1740atgacagatc aggaccaaga tggttcccac
atcaaaggct tgctgattaa ttttatccat 1800cacaactggc cctctcttct
gcgacatcgt tttctggagg aatttatcac tcccattgta 1860aaggtatcta
aaaacaagca agaaatggca ttttacagcc ttcctgaatt tgaagagtgg
1920aagagttcta ctccaaatca taaaaaatgg aaagtcaaat attacaaagg
tttgggcacc 1980agcacatcaa aggaagctaa agaatacttt gcagatatga
aaagacatcg tatccagttc 2040aaatattctg gtcctgaaga tgatgctgct
atcagcctgg cctttagcaa aaaacagata 2100gatgatcgaa aggaatggtt
aactaatttc atggaggata gaagacaacg aaagttactt 2160gggcttcctg
aggattactt gtatggacaa actaccacat atctgacata taatgacttc
2220atcaacaagg aacttatctt gttctcaaat tctgataacg agagatctat
cccttctatg 2280gtggatggtt tgaaaccagg tcagagaaag gttttgttta
cttgcttcaa acggaatgac 2340aagcgagaag taaaggttgc ccaattagct
ggatcagtgg ctgaaatgtc ttcttatcat 2400catggtgaga tgtcactaat
gatgaccatt atcaatttgg ctcagaattt tgtgggtagc 2460aataatctaa
acctcttgca gcccattggt cagtttggta ccaggctaca tggtggcaag
2520gattctgcta gtccacgata catctttaca atgctcagct ctttggctcg
attgttattt 2580ccaccaaaag atgatcacac gttgaagttt ttatatgatg
acaaccagcg tgttgagcct 2640gaatggtaca ttcctattat tcccatggtg
ctgataaatg gtgctgaagg aatcggtact 2700gggtggtcct gcaaaatccc
caactttgat gtgcgtgaaa ttgtaaataa catcaggcgt 2760ttgatggatg
gagaagaacc tttgccaatg cttccaagtt acaagaactt caagggtact
2820attgaagaac tggctccaaa tcaatatgtg attagtggtg aagtagctat
tcttaattct 2880acaaccattg aaatctcaga gcttcccgtc agaacatgga
cccagacata caaagaacaa 2940gttctagaac ccatgttgaa tggcaccgag
aagacacctc ctctcataac agactatagg 3000gaataccata cagataccac
tgtgaaattt gttgtgaaga tgactgaaga aaaactggca 3060gaggcagaga
gagttggact acacaaagtc ttcaaactcc aaactagtct cacatgcaac
3120tctatggtgc tttttgacca cgtaggctgt ttaaagaaat atgacacggt
gttggatatt 3180ctaagagact tttttgaact cagacttaaa tattatggat
taagaaaaga atggctccta 3240ggaatgcttg gtgctgaatc tgctaaactg
aataatcagg ctcgctttat cttagagaaa 3300atagatggca aaataatcat
tgaaaataag cctaagaaag aattaattaa agttctgatt 3360cagaggggat
atgattcgga tcctgtgaag gcctggaaag aagcccagca aaaggttcca
3420gatgaagaag aaaatgaaga gagtgacaac gaaaaggaaa ctgaaaagag
tgactccgta 3480acagattctg gaccaacctt caactatctt cttgatatgc
ccctttggta tttaaccaag 3540gaaaagaaag atgaactctg caggctaaga
aatgaaaaag aacaagagct ggacacatta 3600aaaagaaaga gtccatcaga
tttgtggaaa gaagacttgg ctacatttat tgaagaattg 3660gaggctgttg
aagccaagga aaaacaagat gaacaagtcg gacttcctgg gaaagggggg
3720aaggccaagg ggaaaaaaac acaaatggct gaagttttgc cttctccgcg
tggtcaaaga 3780gtcattccac gaataaccat agaaatgaaa gcagaggcag
aaaagaaaaa taaaaagaaa 3840attaagaatg aaaatactga aggaagccct
caagaagatg gtgtggaact agaaggccta 3900aaacaaagat tagaaaagaa
acagaaaaga gaaccaggta caaagacaaa gaaacaaact 3960acattggcat
ttaagccaat caaaaaagga aagaagagaa atccctggtc tgattcagaa
4020tcagatagga gcagtgacga aagtaatttt gatgtccctc cacgagaaac
agagccacgg 4080agagcagcaa caaaaacaaa attcacaatg gatttggatt
cagatgaaga tttctcagat 4140tttgatgaaa aaactgatga tgaagatttt
gtcccatcag atgctagtcc acctaagacc 4200aaaacttccc caaaacttag
taacaaagaa ctgaaaccac agaaaagtgt cgtgtcagac 4260cttgaagctg
atgatgttaa gggcagtgta ccactgtctt caagccctcc tgctacacat
4320ttcccagatg aaactgaaat tacaaaccca gttcctaaaa agaatgtgac
agtgaagaag 4380acagcagcaa aaagtcagtc ttccacctcc actaccggtg
ccaaaaaaag ggctgcccca 4440aaaggaacta aaagggatcc agctttgaat
tctggtgtct ctcaaaagcc tgatcctgcc 4500aaaaccaaga atcgccgcaa
aaggaagcca tccacttctg atgattctga ctctaatttt 4560gagaaaattg
tttcgaaagc agtcacaagc aagaaatcca agggggagag tgatgacttc
4620catatggact ttgactcagc tgtggctcct cgggcaaaat ctgtacgggc
aaagaaacct 4680ataaagtacc tggaagagtc agatgaagat gatctgtttt
aaaatgtgag gcgattattt 4740taagtaatta tcttaccaag cccaagactg
gttttaaagt tacctgaagc tcttaacttc 4800ctcccctctg aatttagttt
ggggaaggtg tttttagtac aagacatcaa agtgaagtaa 4860agcccaagtg
ttctttagct ttttataata ctgtctaaat agtgaccatc tcatgggcat
4920tgttttcttc tctgctttgt ctgtgttttg agtctgcttt cttttgtctt
taaaacctga 4980tttttaagtt cttctgaact gtagaaatag ctatctgatc
acttcagcgt aaagcagtgt 5040gtttattaac catccactaa gctaaaacta
gagcagtttg atttaaaagt gtcactcttc 5100ctccttttct actttcagta
gatatgagat agagcataat tatctgtttt atcttagttt 5160tatacataat
ttaccatcag atagaacttt atggttctag tacagatact ctactacact
5220cagcctctta tgtgccaagt ttttctttaa gcaatgagaa attgctcatg
ttcttcatct 5280tctcaaatca tcagaggcca aagaaaaaca ctttggctgt
gtctataact tgacacagtc 5340aatagaatga agaaaattag agtagttatg
tgattatttc agctcttgac ctgtcccctc 5400tggctgcctc tgagtctgaa
tctcccaaag agagaaacca atttctaaga ggactggatt 5460gcagaagact
cggggacaac atttgatcca agatcttaaa tgttatattg ataaccatgc
5520tcagcaatga gctattagat tcattttggg aaatctccat aatttcaatt
tgtaaacttt 5580gttaagacct gtctacattg ttatatgtgt gtgacttgag
taatgttatc aacgtttttg 5640taaatattta ctatgttttt ctattagcta
aattccaaca attttgtact ttaataaa 5698421531PRTHomo sapiens 42Met Glu
Val Ser Pro Leu Gln Pro Val Asn Glu Asn Met Gln Val Asn1 5 10 15Lys
Ile Lys Lys Asn Glu Asp Ala Lys Lys Arg Leu Ser Val Glu Arg 20 25
30Ile Tyr Gln Lys Lys Thr Gln Leu Glu His Ile Leu Leu Arg Pro Asp
35 40 45Thr Tyr Ile Gly Ser Val Glu Leu Val Thr Gln Gln Met Trp Val
Tyr 50 55 60Asp Glu Asp Val Gly Ile Asn Tyr Arg Glu Val Thr Phe Val
Pro Gly65 70 75 80Leu Tyr Lys Ile Phe Asp Glu Ile Leu Val Asn Ala
Ala Asp Asn Lys 85 90 95Gln Arg Asp Pro Lys Met Ser Cys Ile Arg Val
Thr Ile Asp Pro Glu 100 105 110Asn Asn Leu Ile Ser Ile Trp Asn Asn
Gly Lys Gly Ile Pro Val Val 115 120 125Glu His Lys Val Glu Lys Met
Tyr Val Pro Ala Leu Ile Phe Gly Gln 130 135 140Leu Leu Thr Ser Ser
Asn Tyr Asp Asp Asp Glu Lys Lys Val Thr Gly145 150 155 160Gly Arg
Asn Gly Tyr Gly Ala Lys Leu Cys Asn Ile Phe Ser Thr Lys 165 170
175Phe Thr Val Glu Thr Ala Ser Arg Glu Tyr Lys Lys Met Phe Lys Gln
180 185 190Thr Trp Met Asp Asn Met Gly Arg Ala Gly Glu Met Glu Leu
Lys Pro 195 200 205Phe Asn Gly Glu Asp Tyr Thr Cys Ile Thr Phe Gln
Pro Asp Leu Ser 210 215 220Lys Phe Lys Met Gln Ser Leu Asp Lys Asp
Ile Val Ala Leu Met Val225 230 235 240Arg Arg Ala Tyr Asp Ile Ala
Gly Ser Thr Lys Asp Val Lys Val Phe 245 250 255Leu Asn Gly Asn Lys
Leu Pro Val Lys Gly Phe Arg Ser Tyr Val Asp 260 265 270Met Tyr Leu
Lys Asp Lys Leu Asp Glu Thr Gly Asn Ser Leu Lys Val 275 280 285Ile
His Glu Gln Val Asn His Arg Trp Glu Val Cys Leu Thr Met Ser 290 295
300Glu Lys Gly Phe Gln Gln Ile Ser Phe Val Asn Ser Ile Ala Thr
Ser305 310 315 320Lys Gly Gly Arg His Val Asp Tyr Val Ala Asp Gln
Ile Val Thr Lys 325 330 335Leu Val Asp Val Val Lys Lys Lys Asn Lys
Gly Gly Val Ala Val Lys 340 345 350Ala His Gln Val Lys Asn His Met
Trp Ile Phe Val Asn Ala Leu Ile 355 360 365Glu Asn Pro Thr Phe Asp
Ser Gln Thr Lys Glu Asn Met Thr Leu Gln 370 375 380Pro Lys Ser Phe
Gly Ser Thr Cys Gln Leu Ser Glu Lys Phe Ile Lys385 390 395 400Ala
Ala Ile Gly Cys Gly Ile Val Glu Ser Ile Leu Asn Trp Val Lys 405 410
415Phe Lys Ala Gln Val Gln Leu Asn Lys Lys Cys Ser Ala Val Lys His
420 425 430Asn Arg Ile Lys Gly Ile Pro Lys Leu Asp Asp Ala Asn Asp
Ala Gly 435 440 445Gly Arg Asn Ser Thr Glu Cys Thr Leu Ile Leu Thr
Glu Gly Asp Ser 450 455 460Ala Lys Thr Leu Ala Val Ser Gly Leu Gly
Val Val Gly Arg Asp Lys465 470 475 480Tyr Gly Val Phe Pro Leu Arg
Gly Lys Ile Leu Asn Val Arg Glu Ala 485 490 495Ser His Lys Gln Ile
Met Glu Asn Ala Glu Ile Asn Asn Ile Ile Lys 500 505 510Ile Val Gly
Leu Gln Tyr Lys Lys Asn Tyr Glu Asp Glu Asp Ser Leu 515 520 525Lys
Thr Leu Arg Tyr Gly Lys Ile Met Ile Met Thr Asp Gln Asp Gln 530 535
540Asp Gly Ser His Ile Lys Gly Leu Leu Ile Asn Phe Ile His His
Asn545 550 555 560Trp Pro Ser Leu Leu Arg His Arg Phe Leu Glu Glu
Phe Ile Thr Pro 565 570 575Ile Val Lys Val Ser Lys Asn Lys Gln Glu
Met Ala Phe Tyr Ser Leu 580 585 590Pro Glu Phe Glu Glu Trp Lys Ser
Ser Thr Pro Asn His Lys Lys Trp 595 600 605Lys Val Lys Tyr Tyr Lys
Gly Leu Gly Thr Ser Thr Ser Lys Glu Ala 610 615 620Lys Glu Tyr Phe
Ala Asp Met Lys Arg His Arg Ile Gln Phe Lys Tyr625 630 635 640Ser
Gly Pro Glu Asp Asp Ala Ala Ile Ser Leu Ala Phe Ser Lys Lys 645 650
655Gln Ile Asp Asp Arg Lys Glu Trp Leu Thr Asn Phe Met Glu Asp Arg
660 665 670Arg Gln Arg Lys Leu Leu Gly Leu Pro Glu Asp Tyr Leu Tyr
Gly Gln 675 680 685Thr Thr Thr Tyr Leu Thr Tyr Asn Asp Phe Ile Asn
Lys Glu Leu Ile 690 695 700Leu Phe Ser Asn Ser Asp Asn Glu Arg Ser
Ile Pro Ser Met Val Asp705 710 715 720Gly Leu Lys Pro Gly Gln Arg
Lys Val Leu Phe Thr Cys Phe Lys Arg 725 730 735Asn Asp Lys Arg Glu
Val Lys Val Ala Gln Leu Ala Gly Ser Val Ala 740 745 750Glu Met Ser
Ser Tyr His His Gly Glu Met Ser Leu Met Met Thr Ile 755 760 765Ile
Asn Leu Ala Gln Asn Phe Val Gly Ser Asn Asn Leu Asn Leu Leu 770 775
780Gln Pro Ile Gly Gln Phe Gly Thr Arg Leu His Gly Gly Lys Asp
Ser785 790 795 800Ala Ser Pro Arg Tyr Ile Phe Thr Met Leu Ser Ser
Leu Ala Arg Leu 805 810 815Leu Phe Pro Pro Lys Asp Asp His Thr Leu
Lys Phe Leu Tyr Asp Asp 820 825 830Asn Gln Arg Val Glu Pro Glu Trp
Tyr Ile Pro Ile Ile Pro Met Val 835 840 845Leu Ile Asn Gly Ala Glu
Gly Ile Gly Thr Gly Trp Ser Cys Lys Ile 850 855 860Pro Asn Phe Asp
Val Arg Glu Ile Val Asn Asn Ile Arg Arg Leu Met865 870 875 880Asp
Gly Glu Glu Pro Leu Pro Met Leu Pro Ser Tyr Lys Asn Phe Lys 885 890
895Gly Thr Ile Glu Glu Leu Ala Pro Asn Gln Tyr Val Ile Ser Gly Glu
900 905 910Val Ala Ile Leu Asn Ser Thr Thr Ile Glu Ile Ser Glu Leu
Pro Val 915 920 925Arg Thr Trp Thr Gln Thr Tyr Lys Glu Gln Val Leu
Glu Pro Met Leu 930 935 940Asn Gly Thr Glu Lys Thr Pro Pro Leu Ile
Thr Asp Tyr Arg Glu Tyr945 950 955 960His Thr Asp Thr Thr Val Lys
Phe Val Val Lys Met Thr Glu Glu Lys 965 970 975Leu Ala Glu Ala Glu
Arg Val Gly Leu His Lys Val Phe Lys Leu Gln 980 985 990Thr Ser Leu
Thr Cys Asn Ser Met Val Leu Phe Asp His Val Gly Cys 995 1000
1005Leu Lys Lys Tyr Asp Thr Val Leu Asp Ile Leu Arg Asp Phe Phe Glu
1010 1015 1020Leu Arg Leu Lys Tyr Tyr Gly Leu Arg Lys Glu Trp Leu
Leu Gly Met1025 1030 1035 1040Leu Gly Ala Glu Ser Ala Lys Leu Asn
Asn Gln Ala Arg Phe Ile Leu 1045 1050 1055Glu Lys Ile Asp Gly Lys
Ile Ile Ile Glu Asn Lys Pro Lys Lys Glu 1060 1065 1070Leu Ile Lys
Val Leu Ile Gln Arg Gly Tyr Asp Ser Asp Pro Val Lys 1075 1080
1085Ala Trp Lys Glu Ala Gln Gln Lys Val Pro Asp Glu Glu Glu Asn Glu
1090 1095 1100Glu Ser Asp Asn Glu Lys Glu Thr Glu Lys Ser Asp Ser
Val Thr Asp1105 1110 1115 1120Ser Gly Pro Thr Phe Asn Tyr Leu Leu
Asp Met Pro Leu Trp Tyr Leu 1125 1130 1135Thr Lys Glu Lys Lys Asp
Glu Leu Cys Arg Leu Arg Asn Glu Lys Glu 1140 1145 1150Gln Glu Leu
Asp Thr Leu Lys Arg Lys Ser Pro Ser Asp Leu Trp Lys 1155 1160
1165Glu Asp Leu Ala Thr Phe Ile Glu Glu Leu Glu Ala Val Glu Ala Lys
1170 1175 1180Glu Lys Gln Asp Glu Gln Val Gly Leu Pro Gly Lys Gly
Gly Lys Ala1185 1190 1195 1200Lys Gly Lys Lys Thr Gln Met Ala Glu
Val Leu Pro Ser Pro Arg Gly 1205 1210 1215Gln Arg Val Ile Pro Arg
Ile Thr Ile Glu Met Lys
Ala Glu Ala Glu 1220 1225 1230Lys Lys Asn Lys Lys Lys Ile Lys Asn
Glu Asn Thr Glu Gly Ser Pro 1235 1240 1245Gln Glu Asp Gly Val Glu
Leu Glu Gly Leu Lys Gln Arg Leu Glu Lys 1250 1255 1260Lys Gln Lys
Arg Glu Pro Gly Thr Lys Thr Lys Lys Gln Thr Thr Leu1265 1270 1275
1280Ala Phe Lys Pro Ile Lys Lys Gly Lys Lys Arg Asn Pro Trp Ser Asp
1285 1290 1295Ser Glu Ser Asp Arg Ser Ser Asp Glu Ser Asn Phe Asp
Val Pro Pro 1300 1305 1310Arg Glu Thr Glu Pro Arg Arg Ala Ala Thr
Lys Thr Lys Phe Thr Met 1315 1320 1325Asp Leu Asp Ser Asp Glu Asp
Phe Ser Asp Phe Asp Glu Lys Thr Asp 1330 1335 1340Asp Glu Asp Phe
Val Pro Ser Asp Ala Ser Pro Pro Lys Thr Lys Thr1345 1350 1355
1360Ser Pro Lys Leu Ser Asn Lys Glu Leu Lys Pro Gln Lys Ser Val Val
1365 1370 1375Ser Asp Leu Glu Ala Asp Asp Val Lys Gly Ser Val Pro
Leu Ser Ser 1380 1385 1390Ser Pro Pro Ala Thr His Phe Pro Asp Glu
Thr Glu Ile Thr Asn Pro 1395 1400 1405Val Pro Lys Lys Asn Val Thr
Val Lys Lys Thr Ala Ala Lys Ser Gln 1410 1415 1420Ser Ser Thr Ser
Thr Thr Gly Ala Lys Lys Arg Ala Ala Pro Lys Gly1425 1430 1435
1440Thr Lys Arg Asp Pro Ala Leu Asn Ser Gly Val Ser Gln Lys Pro Asp
1445 1450 1455Pro Ala Lys Thr Lys Asn Arg Arg Lys Arg Lys Pro Ser
Thr Ser Asp 1460 1465 1470Asp Ser Asp Ser Asn Phe Glu Lys Ile Val
Ser Lys Ala Val Thr Ser 1475 1480 1485Lys Lys Ser Lys Gly Glu Ser
Asp Asp Phe His Met Asp Phe Asp Ser 1490 1495 1500Ala Val Ala Pro
Arg Ala Lys Ser Val Arg Ala Lys Lys Pro Ile Lys1505 1510 1515
1520Tyr Leu Glu Glu Ser Asp Glu Asp Asp Leu Phe 1525
1530434797DNAHomo sapiens 43gcagtgaaca caacctttcc cctgagccac
tggaattgga cagaatgccc cattctcctc 60tgatctccat tcctcatgtg tggtgtcacc
cagaagagga ggaaagaatg catgatgaac 120ttctacaagc agtatccaag
gggccggtga tgttcaggga tgtttccata gacttctctc 180aagaggaatg
ggaatgcctg gacgctgatc agatgaattt atacaaagaa gtgatgttgg
240agaatttcag caacctggtt tcagtgggac tttccaattc taagccagct
gtgatctcct 300tattggaaca aggaaaagag ccctggatgg ttgatagaga
gctgactaga ggcctgtgtt 360cagatctgga atcaatgtgt gagaccaaaa
tattatctct aaagaagaga catttcagtc 420aagtaataat tacccgtgaa
gacatgtcta cttttattca gcccacattt cttattccac 480ctcaaaaaac
tatgagtgaa gagaaaccat gggaatgtaa gatatgtgga aagaccttta
540atcaaaactc acaatttatc caacatcaga gaattcattt tggtgaaaaa
cactatgaat 600ctaaggagta tgggaagtcc tttagtcgtg gctcactcgt
tactcgacat cagaggattc 660acactggtaa aaaaccctat gaatgtaagg
aatgtggcaa ggcttttagt tgtagttcat 720atttttctca acatcagagg
attcacactg gtgagaaacc ctatgaatgt aaggaatgtg 780gaaaagcctt
taagtattgc tcaaacctta atgatcatca gagaattcac actggtgaga
840aaccctatga atgtaaagta tgtggaaaag cctttactaa aagttcacaa
ctttttctac 900atctgagaat tcatactggt gagaaacctt atgaatgtaa
agaatgtggg aaagccttta 960ctcaacactc aaggcttatt cagcatcaga
gaatgcatac tggtgagaaa ccttatgaat 1020gtaagcagtg tgggaaggcc
tttaatagtg cctcaacact tactaaccat cacagaattc 1080atgctggtga
gaagctctat gaatgtgaag aatgtagaaa ggcctttatt cagagctcag
1140aacttattca acatcagaga atccatacag atgaaaaacc atatgaatgt
aatgaatgtg 1200ggaaggcctt taataaaggc tcaaatctta ctcgacatca
gagaattcac actggtgaga 1260aaccctatga ctgtaaggaa tgtggaaagg
cttttggtag tcgctctgac ctcattcgcc 1320atgagggaat tcatactggt
tgaatgacag taaagtaaga ccattttgtt aacctttata 1380ataatttttt
taaaacaggt aaggagaaca aattaggata catattatca aaggttctcc
1440tatgtattcg tttttaaacg atacgataac aaagtaccaa gtaccaaaac
cttggtggct 1500taaaacaaga gaaatttatt ctctcatagt ttagagcctg
gaaatctaaa ctcaagggtg 1560ctgatcgttt tggttccttc tgaggactct
gaggatctgt tctatgcctt tttcctaacc 1620tctgttaaca gctggcagtc
cttggcattc catggctttt acatacacca ttccaatctc 1680tgcctccatc
ttcacattgc attctcgctg tgtatctctg tgtatgtctt ttatttggac
1740accagtcagg ttagattggg gctacctggt gacctcatct taacttgatt
atatctgcca 1800agaccctgtt tccaagtaag gtcacattta ccggtaccag
gggttaggac ttcagcatat 1860ctttttaggg gatacagttc aacccataat
accctgttag aatgattttg tctaatatat 1920ttgtaatttc cttttataca
taagttgtta gtcaaattta ttttatttta ttttattttg 1980agacagagtc
tcgctctgtt gcccaggctg gagtgcagtg gtgtgatctc agctcactgc
2040aacctccagc tcctgagttc aagcgattct tgtgcctcag cctctcaagt
agttgggatt 2100acaggcatgc gccaccatgc ccggctaatt tttttttttt
tttttttgta tttttagtag 2160cgacggggtt tcaccatgtt ggccaggctg
gtcttgaact cctgacttca agtgatctgc 2220ccgcctcagc ctcccaaagt
gctgggatta cagacgtgag ccaccgtgat ggccaaaaca 2280gactttatac
caacaaaaat taaaaaggac aaagaaggtc atttataatg ataaaggata
2340aattcaacaa gaagataaaa caatcctaaa tatgtatgca cccaacactg
caacacccag 2400atccataaca cagatactac tagacctaag aaaagagata
gacagcaata caacaatagc 2460aggggacttc accactccat tgacagcact
agacagatca ctgggacaga aatcaacaaa 2520gaaactctgg acttaaattg
gactctacac caaatggacc caacagacat ctgaagaaca 2580ttctacccaa
caaccacaga atatatactc ttctcttctg tgcatggaac attctcaaaa
2640ataggtcata tactggacca caaagcaagt atcaataaat tttaaaaaaa
caaaatcata 2700tctaacatct tctctgacca tagtggaata aaactagata
tcaataccaa gaggaactct 2760caaaacagat acatggaatt taaacagctt
gctcctgaat gatttttgga tcaatgatga 2820aactaaggtg gaaatttaaa
attttttgaa ataaatgaaa atagagacaa aacacatgaa 2880aacatctgag
atacagcaaa agcagtgcta agagaggatt ttatagcatt aaatgcctac
2940accaaaaaga tagaaaaatc tcaaatgaat agcctaacgt cacatctcaa
ggaactagga 3000aaaaacaaaa caaactcaac ccaaagctgg cagaagaaaa
gcaataacaa atatcagagc 3060aggcaaaaat gagactgaga acaaaggaat
gcaaaagatc aataaaagaa aaagttggtt 3120ctttgtaaag ataaaactga
cagaccacta gctagattaa ccaagaaaaa aagaagattc 3180aaataaatac
aatcagaaat gataaggtga tattataact gataacacag acatataaaa
3240tatcagcaga aactatatgc acatattaga aaacctagag gaagtggata
aattcctaga 3300aacacataac cttccaagat tgaaccaggg agaaatagga
atcctcaaca gactactgag 3360tattgaaatt gaatcagtaa tagaaaaaaa
tcttgcaaaa acaaaaagcc caggaccaga 3420cagattcaca gctgaattct
actagacatg caaggaagaa ctagtaacag cactattgaa 3480actattccaa
aaattatagg agggaatcct ccctaactca ttctacaaag ccagtatcat
3540cctgatactg aagccaggca aggataaaac acacaaaaaa actacaagcc
aatatccctg 3600atgaaaatag acacaaaaat cttcagcaaa atactagcaa
accaaatcaa acagtacata 3660aaaaagatag taacagcaca gtcaagtgga
ttttattcct ggggtgtaag gatggctcaa 3720catatgcaac tcaatacatg
attcatcaca tacacagaat taaaaataag ccaggcactc 3780acacctgtaa
tcccagcact ttgcaaggcc aaggcgggca gatcacatga tgtcaagagt
3840ttgagaccag tctggctgac atggcgaaac cctgtctcta ctaaaaatag
aaaaattggc 3900tgggcatggt ggcaggcact gtagtcccag ctacttggga
ggctgaggca ggagaattac 3960ttgaacctga gaagcggagg ttgcagtgag
ctgagatagt gccattgcac tccagcctgg 4020gcaacagagc aaattgcttg
aatgtgggag gtggaggttg cagtgagccg agattatgcc 4080attgcactcc
agccggggga gcaacaaagc cagactccat ctcaaaaaaa aaccaaaaaa
4140aatcctattt agtacaaggt acattattta ggtaatgagt ccattaaaag
ccaacacttt 4200ccccactaca ctatatgtgt atgtaacaca actgcccttg
taacttccta aacctataat 4260taagaaacaa taaaaggcaa attaagaatg
cttttttaaa aggtgggggc attatgctaa 4320taagttactg tggatttcag
agtgcagagt agaaagatca caagaattta gtgtggtagg 4380tgggaacaga
aaatgggtgt ataaatttta ttgacgtggg agtactggat attgtagaga
4440cagatatcat cagggcaagg agattaaaga tttttgcatt gacggtttga
cactatattg 4500tggtaataac actgtatgtg ttgggagata gaacaggaaa
catcttccct ggaatatgta 4560tactattaaa tgttttatca aacttttgat
caaacaagac agcacaattt ataatttcat 4620ttctatttct atgttatgag
aaactgatca tttattcaaa tgtttaacag gcatgttcat 4680gttactataa
actcttctgt ttctccatca cgttgttggt catctttact gattacaaat
4740ttctttacat atttaagaaa tatatatatt tctttatata ttaaaaaaaa aaaaaaa
479744432PRTHomo sapiens 44Met Pro His Ser Pro Leu Ile Ser Ile Pro
His Val Trp Cys His Pro1 5 10 15Glu Glu Glu Glu Arg Met His Asp Glu
Leu Leu Gln Ala Val Ser Lys 20 25 30Gly Pro Val Met Phe Arg Asp Val
Ser Ile Asp Phe Ser Gln Glu Glu 35 40 45Trp Glu Cys Leu Asp Ala Asp
Gln Met Asn Leu Tyr Lys Glu Val Met 50 55 60Leu Glu Asn Phe Ser Asn
Leu Val Ser Val Gly Leu Ser Asn Ser Lys65 70 75 80Pro Ala Val Ile
Ser Leu Leu Glu Gln Gly Lys Glu Pro Trp Met Val 85 90 95Asp Arg Glu
Leu Thr Arg Gly Leu Cys Ser Asp Leu Glu Ser Met Cys 100 105 110Glu
Thr Lys Ile Leu Ser Leu Lys Lys Arg His Phe Ser Gln Val Ile 115 120
125Ile Thr Arg Glu Asp Met Ser Thr Phe Ile Gln Pro Thr Phe Leu Ile
130 135 140Pro Pro Gln Lys Thr Met Ser Glu Glu Lys Pro Trp Glu Cys
Lys Ile145 150 155 160Cys Gly Lys Thr Phe Asn Gln Asn Ser Gln Phe
Ile Gln His Gln Arg 165 170 175Ile His Phe Gly Glu Lys His Tyr Glu
Ser Lys Glu Tyr Gly Lys Ser 180 185 190Phe Ser Arg Gly Ser Leu Val
Thr Arg His Gln Arg Ile His Thr Gly 195 200 205Lys Lys Pro Tyr Glu
Cys Lys Glu Cys Gly Lys Ala Phe Ser Cys Ser 210 215 220Ser Tyr Phe
Ser Gln His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr225 230 235
240Glu Cys Lys Glu Cys Gly Lys Ala Phe Lys Tyr Cys Ser Asn Leu Asn
245 250 255Asp His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys
Lys Val 260 265 270Cys Gly Lys Ala Phe Thr Lys Ser Ser Gln Leu Phe
Leu His Leu Arg 275 280 285Ile His Thr Gly Glu Lys Pro Tyr Glu Cys
Lys Glu Cys Gly Lys Ala 290 295 300Phe Thr Gln His Ser Arg Leu Ile
Gln His Gln Arg Met His Thr Gly305 310 315 320Glu Lys Pro Tyr Glu
Cys Lys Gln Cys Gly Lys Ala Phe Asn Ser Ala 325 330 335Ser Thr Leu
Thr Asn His His Arg Ile His Ala Gly Glu Lys Leu Tyr 340 345 350Glu
Cys Glu Glu Cys Arg Lys Ala Phe Ile Gln Ser Ser Glu Leu Ile 355 360
365Gln His Gln Arg Ile His Thr Asp Glu Lys Pro Tyr Glu Cys Asn Glu
370 375 380Cys Gly Lys Ala Phe Asn Lys Gly Ser Asn Leu Thr Arg His
Gln Arg385 390 395 400Ile His Thr Gly Glu Lys Pro Tyr Asp Cys Lys
Glu Cys Gly Lys Ala 405 410 415Phe Gly Ser Arg Ser Asp Leu Ile Arg
His Glu Gly Ile His Thr Gly 420 425 430
* * * * *
References