U.S. patent application number 12/719554 was filed with the patent office on 2010-11-18 for nucleic acid molecules and proteins for the identification, assessment, prevention, and therapy of ovarian cancer.
This patent application is currently assigned to MILLENNIUM PHARMACEUTICALS, INC.. Invention is credited to Robert C. Bast, JR., Wilson O. Endege, Donna Ford, Manjula Gannavarapu, Karen Glatt, Sebastian Hoersch, Shubhangi Kamatkar, Karen Lu, Gordon B. Mills, John E. Monahan, Robert Schlegel, Yong Yao Xu, Xumei Zhao.
Application Number | 20100291068 12/719554 |
Document ID | / |
Family ID | 34434947 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100291068 |
Kind Code |
A1 |
Endege; Wilson O. ; et
al. |
November 18, 2010 |
NUCLEIC ACID MOLECULES AND PROTEINS FOR THE IDENTIFICATION,
ASSESSMENT, PREVENTION, AND THERAPY OF OVARIAN CANCER
Abstract
The invention relates to newly discovered nucleic acid molecules
and proteins associated with ovarian cancer. Compositions, kits,
and methods for detecting, characterizing, preventing, and treating
human ovarian cancers are provided.
Inventors: |
Endege; Wilson O.; (Norwood,
MA) ; Ford; Donna; (Plainville, MA) ;
Gannavarapu; Manjula; (Acton, MA) ; Glatt; Karen;
(Natick, MA) ; Hoersch; Sebastian; (Arlington,
MA) ; Kamatkar; Shubhangi; (Newton, MA) ;
Monahan; John E.; (Walpole, MA) ; Schlegel;
Robert; (Auburndale, MA) ; Xu; Yong Yao;
(Belmont, MA) ; Zhao; Xumei; (Wayland, MA)
; Bast, JR.; Robert C.; (Houston, TX) ; Mills;
Gordon B.; (Houston, TX) ; Lu; Karen;
(Houston, TX) |
Correspondence
Address: |
MCCARTER & ENGLISH, LLP BOSTON
265 Franklin Street
Boston
MA
02110
US
|
Assignee: |
MILLENNIUM PHARMACEUTICALS,
INC.
Cambridge
MA
Board of Regents, The University of Texas System
Austin
TX
|
Family ID: |
34434947 |
Appl. No.: |
12/719554 |
Filed: |
March 8, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10961139 |
Oct 7, 2004 |
7799518 |
|
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12719554 |
|
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60509171 |
Oct 7, 2003 |
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Current U.S.
Class: |
424/130.1 ;
435/29; 435/6.11; 435/6.14; 435/7.1; 530/350; 530/387.9;
536/23.1 |
Current CPC
Class: |
A61P 35/00 20180101;
G01N 33/57449 20130101; G01N 33/5011 20130101; C12Q 2600/158
20130101; C12Q 2600/136 20130101; C07K 14/47 20130101; C12Q 1/6886
20130101 |
Class at
Publication: |
424/130.1 ;
435/29; 435/6; 435/7.1; 536/23.1; 530/350; 530/387.9 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12Q 1/02 20060101 C12Q001/02; C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53; C07H 21/04 20060101
C07H021/04; C07K 14/425 20060101 C07K014/425; C07K 16/18 20060101
C07K016/18; A61P 35/00 20060101 A61P035/00 |
Claims
1. A method of assessing whether a patient is afflicted with
ovarian cancer, the method comprising the steps of: a) determining
the level of expression of a marker in a patient sample, wherein
the marker is selected from the group consisting of the markers
listed in Table 1; b) determining the level of expression of the
marker in a sample from a control subject having no ovarian cancer
or no ovarian tumor; and c) comparing the level of expression of
the marker in the patient sample and in the sample from the control
subject wherein a significant difference between the level of
expression of the marker in the patient sample and the sample from
the control subject is an indication that the patient is afflicted
with breast cancer.
2. The method of claim 1, wherein the level of expression from the
control subject is determined from ovarian cells from said patient
which appear to be non-cancerous.
3. The method of claim 1, wherein the level of expression from the
control subject is predetermined using an average of the levels of
expression from a population of subjects having no ovarian
cancer.
4. The method of claim 1, wherein the marker corresponds to a
secreted protein.
5. The method of claim 1, wherein the marker corresponds to a
transcribed polynucleotide or portion thereof, wherein the
polynucleotide comprises the marker.
6. The method of claim 1, wherein the sample comprises cells
obtained from the patient.
7. The method of claim 6, wherein the cells are in a fluid selected
from the group consisting of blood fluids, lymph, ascites,
gynecological fluids, cystic fluid, urine, and fluids collected by
peritoneal rinsing.
8. The method of claim 1, wherein the level of expression of the
marker in the sample is assessed by detecting the presence in the
sample of a protein corresponding to the marker.
9. The method of claim 8, wherein the presence of the protein is
detected using a reagent which specifically binds with the
protein.
10. The method of claim 9, wherein the reagent is selected from the
group consisting of an antibody, an antibody derivative, and an
antibody fragment.
11. The method of claim 1, wherein the level of expression of the
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 marker.
12. The method of claim 11, wherein the transcribed polynucleotide
is a mRNA or a cDNA.
13. The method of claim 11, wherein the step of detecting further
comprises amplifying the transcribed polynucleotide.
14. The method of claim 1, wherein the level of expression of the
marker in the sample is assessed by detecting the presence in the
sample of a transcribed polynucleotide which anneals with the
marker or anneals with a portion of a polynucleotide wherein the
polynucleotide comprises the marker, under stringent hybridization
conditions.
15. The method of claim 1, wherein the level of expression of the
marker in the sample differs from the normal level of expression of
the marker in a patient not afflicted with ovarian cancer by a
factor of at least about 2.
16. The method of claim 1, wherein the level of expression of the
marker in the sample differs from the normal level of expression of
the marker in a patient not afflicted with ovarian cancer by a
factor of at least about 5.
17. A method of assessing whether a patient is afflicted with
ovarian cancer, the method comprising the steps of: a) determining
the level of expression in the sample of each of a plurality of
markers independently selected from the markers listed in Table 1;
b) determining the level of expression of each of the plurality of
markers in a sample from a control subject having no ovarian
cancer; and c) comparing the level of expression of the marker in
the patient sample and in the sample from the control subject;
wherein the level of expression of more than one of the markers is
significantly altered, relative to the corresponding control levels
of expression of the markers, is an indication that the patient is
afflicted with ovarian cancer.
18. The method of claim 17, wherein the levels of expression from a
control subject are determined from ovarian cells from said patient
which appear to be non-cancerous.
19. The method of claim 17, wherein the levels of expression from a
control subject are predetermined using an average of the levels of
expression from a population of subjects having no ovarian
cancer.
20. The method of claim 17, wherein the plurality comprises at
least three of the markers.
21. The method of claim 17, wherein the plurality comprises at
least five of the markers.
22. A method for determining whether a patient has ovarian cancer
that has metastasized or is likely to metastasize, the method
comprising comparing: a) the level of expression of a marker listed
in Table 1 in a sample from the patient, and b) the level of
expression of the marker in a sample from a control subject having
a non-metastasized ovarian cancer or no ovarian cancer, c) wherein,
a significantly higher level of expression in the patient sample as
compared to the level in the sample from the control subject is an
indication that the ovarian cancer has metastasized or is likely to
metastasize.
23. A method of assessing the efficacy of a test compound for
inhibiting ovarian cancer in a patient, the method comprising
comparing: a) expression of a marker in a first sample obtained
from the patient and exposed to the test compound, wherein the
marker is selected from the group consisting of the markers listed
in Table 1, and b) expression of the marker in a second sample
obtained from the patient, wherein the sample is not exposed to the
test compound, c) wherein a significantly lower level of expression
of the marker in the first sample, relative to the second sample,
is an indication that the test compound is efficacious for
inhibiting ovarian cancer in the patient.
24. The method of claim 23, wherein the first and second samples
are portions of a single sample obtained from the patient.
25. The method of claim 23, wherein the first and second samples
are portions of pooled samples obtained from the patient.
26. A method of assessing the efficacy of a therapy for inhibiting
ovarian cancer in a patient, the method comprising comparing: a)
expression of a marker in a first sample obtained from the patient
prior to administering at least a portion of the therapy to the
patient, wherein the marker is selected from the group consisting
of the markers listed in Table 1, and b) expression of the marker
in a second sample obtained from the patient subsequent to
administering the portion of the therapy, c) wherein a
significantly lower level of expression of the marker in the second
sample, relative to the first sample, is an indication that the
therapy is efficacious for inhibiting ovarian cancer in the
patient.
27. A method of selecting a composition for inhibiting ovarian
cancer in a patient, the method comprising: a) obtaining a sample
comprising cancer cells from the patient; b) separately exposing
aliquots of the sample in the presence of a plurality of test
compositions; c) comparing expression of a marker in each of the
aliquots, wherein the marker is selected from the group consisting
of the markers listed in Table 1; and d) selecting at least one of
the test compositions which induces a lower level of expression of
the marker in the aliquot containing that test composition,
relative to the other test compositions.
28. A method of inhibiting ovarian cancer in a patient, the method
comprising: a) obtaining a sample comprising cancer cells from the
patient; b) separately maintaining aliquots of the sample in the
presence of a plurality of test compositions; c) comparing
expression of a marker in each of the aliquots, wherein the marker
is selected from the group consisting of the markers listed in
Table 1; and administering to the patient at least one of the test
compositions which induces a lower level of expression of the
marker in the aliquot containing that test composition, relative to
the other test compositions.
29. A method for assessing the ovarian cell carcinogenic potential
of a test composition, the method comprising; a) maintaining
separate aliquots of ovarian cancer cells in the presence and
absence of the test composition; and b) comparing expression of a
marker in each of the aliquots, wherein the marker is selected from
the group consisting of the markers listed in Table 1, wherein a
significantly enhanced level of expression of the marker in the
aliquot maintained in the presence of the test composition, is an
indication that the test composition possesses human ovarian cell
carcinogenic potential.
30. A kit for assessing whether a patient is afflicted with ovarian
cancer, the kit comprising reagents for assessing expression of a
plurality of markers selected from the group consisting of the
markers listed in Table 1.
31. A kit for assessing the presence of ovarian cancer cells, the
kit comprising a plurality of nucleic acid probes wherein the
probes specifically bind with transcribed polynucleotides
corresponding to a plurality of markers selected from the group
consisting of the markers listed in Table 1.
32. A kit for assessing the presence of ovarian cancer cells, the
kit comprising a plurality of antibodies, wherein the antibodies
specifically bind with proteins corresponding to a plurality of
markers selected from the group consisting of the markers listed in
Table 1.
33. A kit for assessing the suitability of each of a plurality of
compounds for inhibiting ovarian cancer in a patient, the kit
comprising: a plurality of compounds; and a reagent for assessing
expression of a marker selected from the group consisting of the
markers listed in Table 1.
34. A method of inhibiting ovarian cancer in a patient at risk for
developing ovarian cancer, the method comprising inhibiting
expression of a gene corresponding to a marker selected from the
markers listed in Table 1.
35. An isolated nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:31,
SEQ ID NO:39 or SEQ ID NO:41.
36. An isolated polypeptide comprising the amino acid sequence of
SEQ ID NO:10, SEQ ID NO:32, SEQ ID NO:40 or SEQ ID NO:42.
37. An antibody which selectively binds to the polypeptide of claim
36.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. application Ser.
No. 10/961,139 filed Oct. 7, 2004, which claims the benefit of U.S.
Provisional Application Ser. No. 60/509,171 filed Oct. 7, 2003. The
entire contents of each of the foregoing applications are hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] The field of the invention is ovarian cancer, including
diagnosis, characterization, management, and therapy of ovarian
cancer.
BACKGROUND OF THE INVENTION
[0003] Ovarian cancer is responsible for significant morbidity and
mortality in populations around the world. Ovarian cancer is
classified, on the basis of clinical and pathological features, in
three groups, namely epithelial ovarian cancer (EOC; >90% of
ovarian cancer in Western countries), germ cell tumors (circa 2-3%
of ovarian cancer), and stromal ovarian cancer (circa 5% of ovarian
cancer; Ozols et al., 1997, Cancer Principles and Practice of
Oncology, 5th ed., DeVita et al., Eds. pp. 1502). Relative to EOC,
germ cell tumors and stromal ovarian cancers are more easily
detected and treated at an early stage, translating into
higher/better survival rates for patients afflicted with these two
types of ovarian cancer.
[0004] There are numerous types of ovarian tumors, some of which
are benign, and others of which are malignant. Treatment (including
non-treatment) options and predictions of patient outcome depend on
accurate classification of the ovarian cancer. Ovarian cancers are
named according to the type of cells from which the cancer is
derived and whether the ovarian cancer is benign or malignant.
Recognized histological tumor types include, for example, serous,
mucinous, endometrioid, and clear cell tumors. In addition, ovarian
cancers are classified according to recognized grade and stage
scales.
[0005] In grade I, the tumor tissue is well differentiated. In
grade II, tumor tissue is moderately well differentiated. In grade
III, the tumor tissue is poorly differentiated. This grade
correlates with a less favorable prognosis than grades I and II.
Stage I is generally confined within the capsule surrounding one
(stage IA) or both (stage IB) ovaries, although in some stage I
(i.e. stage IC) cancers, malignant cells may be detected in
ascites, in peritoneal rinse fluid, or on the surface of the
ovaries. Stage II involves extension or metastasis of the tumor
from one or both ovaries to other pelvic structures. In stage IIA,
the tumor extends or has metastasized to the uterus, the fallopian
tubes, or both. Stage IIB involves extension of the tumor to the
pelvis. Stage IIC is stage IIA or IIB in which malignant cells may
be detected in ascites, in peritoneal rinse fluid, or on the
surface of the ovaries. In stage III, the tumor comprises at least
one malignant extension to the small bowel or the omentum, has
formed extrapelvic peritoneal implants of microscopic (stage IIIA)
or macroscopic (<2 centimeter diameter, stage IIIB; >2
centimeter diameter, stage IIIC) size, or has metastasized to a
retroperitoneal or inguinal lymph node (an alternate indicator of
stage IIIC). In stage IV, distant (i.e. non-peritoneal) metastases
of the tumor can be detected.
[0006] The durations of the various stages of ovarian cancer are
not presently known, but are believed to be at least about a year
each (Richart et al., 1969, Am. J. Obstet. Gynecol. 105:386).
Prognosis declines with increasing stage designation. For example,
5-year survival rates for patients diagnosed with stage I, II, III,
and IV ovarian cancer are 80%, 57%, 25%, and 8%, respectively.
[0007] Despite being the third most prevalent gynecological cancer,
ovarian cancer is the leading cause of death among those afflicted
with gynecological cancers. The disproportionate mortality of
ovarian cancer is attributable to a substantial absence of symptoms
among those afflicted with early-stage ovarian cancer and to
difficulty diagnosing ovarian cancer at an early stage. Patients
afflicted with ovarian cancer most often present with non-specific
complaints, such as abnormal vaginal bleeding, gastrointestinal
symptoms, urinary tract symptoms, lower abdominal pain, and
generalized abdominal distension. These patients rarely present
with paraneoplastic symptoms or with symptoms which clearly
indicate their affliction. Presently, less than about 40% of
patients afflicted with ovarian cancer present with stage I or
stage II. Management of ovarian cancer would be significantly
enhanced if the disease could be detected at an earlier stage, when
treatments are much more generally efficacious.
[0008] Ovarian cancer may be diagnosed, in part, by collecting a
routine medical history from a patient and by performing physical
examination, x-ray examination, and chemical and hematological
studies on the patient. Hematological tests which may be indicative
of ovarian cancer in a patient include analyses of serum levels of
proteins designated CA125 and DF3 and plasma levels of
lysophosphatidic acid (LPA). Palpation of the ovaries and
ultrasound techniques (particularly including endovaginal
ultrasound and color Doppler flow ultrasound techniques) can aid
detection of ovarian tumors and differentiation of ovarian cancer
from benign ovarian cysts. However, a definitive diagnosis of
ovarian cancer typically requires performing exploratory laparotomy
of the patient.
[0009] Potential tests for the detection of ovarian cancer (e.g.,
screening, reflex or monitoring) may be characterized by a number
of factors. The "sensitivity" of an assay refers to the probability
that the test will yield a positive result in an individual
afflicted with ovarian cancer. The "specificity" of an assay refers
to the probability that the test will yield a negative result in an
individual not afflicted with ovarian cancer. The "positive
predictive value" (PPV) of an assay is the ratio of true positive
results (i.e. positive assay results for patients afflicted with
ovarian cancer) to all positive results (i.e. positive assay
results for patients afflicted with ovarian cancer+positive assay
results for patients not afflicted with ovarian cancer). It has
been estimated that in order for an assay to be an appropriate
population-wide screening tool for ovarian cancer the assay must
have a PPV of at least about 10% (Rosenthal et al., 1998, Sem.
Oncol. 25:315-325). It would thus be desirable for a screening
assay for detecting ovarian cancer in patients to have a high
sensitivity and a high PPV. Monitoring and reflex tests would also
require appropriate specifications.
[0010] Owing to the cost, limited sensitivity, and limited
specificity of known methods of detecting ovarian cancer, screening
is not presently performed for the general population. In addition,
the need to perform laparotomy in order to diagnose ovarian cancer
in patients who screen positive for indications of ovarian cancer
limits the desirability of population-wide screening, such that a
PPV even greater than 10% would be desirable.
[0011] Prior use of serum CA125 level as a diagnostic marker for
ovarian cancer indicated that this method exhibited insufficient
specificity for use as a general screening method. Use of a refined
algorithm for interpreting CA125 levels in serial retrospective
samples obtained from patients improved the specificity of the
method without shifting detection of ovarian cancer to an earlier
stage (Skakes, 1995, Cancer 76:2004). Screening for LPA to detect
gynecological cancers including ovarian cancer exhibited a
sensitivity of about 96% and a specificity of about 89%. However,
CA125-based screening methods and LPA-based screening methods are
hampered by the presence of CA125 and LPA, respectively, in the
serum of patients afflicted with conditions other than ovarian
cancer. For example, serum CA125 levels are known to be associated
with menstruation, pregnancy, gastrointestinal and hepatic
conditions such as colitis and cirrhosis, pericarditis, renal
disease, and various non-ovarian malignancies. Serum LPA is known,
for example, to be affected by the presence of non-ovarian
gynecological malignancies. A screening method having a greater
specificity for ovarian cancer than the current screening methods
for CA125 and LPA could provide a population-wide screening for
early stage ovarian cancer.
[0012] Presently greater than about 60% of ovarian cancers
diagnosed in patients are stage III or stage IV cancers. Treatment
at these stages is largely limited to cytoreductive surgery (when
feasible) and chemotherapy, both of which aim to slow the spread
and development of metastasized tumor. Substantially all late stage
ovarian cancer patients currently undergo combination chemotherapy
as primary treatment, usually a combination of a platinum compound
and a taxane. Median survival for responding patients is about one
year. Combination chemotherapy involving agents such as
doxorubicin, cyclophosphamide, cisplatin, hexamethylmelamine,
paclitaxel, and methotrexate may improve survival rates in these
groups, relative to single-agent therapies. Various
recently-developed chemotherapeutic agents and treatment regimens
have also demonstrated usefulness for treatment of advanced ovarian
cancer. For example, use of the topoisomerase I inhibitor topectan,
use of amifostine to minimize chemotherapeutic side effects, and
use of intraperitoneal chemotherapy for patients having
peritoneally implanted tumors have demonstrated at least limited
utility. Presently, however, the 5-year survival rate for patients
afflicted with stage III ovarian cancer is 25%, and the survival
rate for patients afflicted with stage IV ovarian cancer is 8%.
[0013] In summary, the earlier ovarian cancer is detected, the
aggressiveness of therapeutic intervention and the side effects
associated with therapeutic intervention are minimized. More
importantly, the earlier the cancer is detected, the survival rate
and quality of life of ovarian cancer patients is enhanced. Thus, a
pressing need exists for methods of detecting ovarian cancer as
early as possible. There also exists a need for methods of
detecting recurrence of ovarian cancer as well as methods for
predicting and monitoring the efficacy of treatment. There further
exists a need for new therapeutic methods for treating ovarian
cancer. The present invention satisfies these needs.
DESCRIPTION OF THE INVENTION
[0014] 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). The invention
further provides antibodies, antibody derivatives and antibody
fragments which bind specifically with such proteins and/or
fragments of the proteins.
[0015] In one aspect, the invention relates to various diagnostic,
monitoring, test and other methods related to ovarian cancer
detection and therapy. In one embodiment, the invention provides a
diagnostic method of assessing whether a patient has ovarian cancer
or has higher than normal risk for developing ovarian 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 ovarian 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
ovarian cancer or has higher than normal risk for developing
ovarian cancer.
[0016] In a preferred embodiment of the diagnostic method, the
marker is over-expressed by at least two-fold in at least about 20%
of stage I ovarian cancer patients, stage II ovarian cancer
patients, stage III ovarian cancer patients, stage IV ovarian
cancer patients, grade I ovarian cancer patients, grade II ovarian
cancer patients, grade III ovarian cancer patients, epithelial
ovarian cancer patients, stromal ovarian cancer patients, germ cell
ovarian cancer patients, malignant ovarian cancer patients, benign
ovarian cancer patients, serous neoplasm ovarian cancer patients,
mucinous neoplasm ovarian cancer patients, endometrioid neoplasm
ovarian cancer patients and/or clear cell neoplasm ovarian cancer
patients.
[0017] The diagnostic methods of the present invention are
particularly useful for patients with an identified pelvic mass or
symptoms associated with ovarian cancer. The methods of the present
invention can also be of particular use with patients having an
enhanced risk of developing ovarian cancer (e.g., patients having a
familial history of ovarian cancer, patients identified as having a
mutant oncogene, and patients at least about 50 years of age).
[0018] In a preferred diagnostic method of assessing whether a
patient is afflicted with ovarian cancer (e.g., new detection
("screening"), detection of recurrence, reflex testing), the method
comprises 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 non-ovarian 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 ovarian cancer.
[0019] The invention also provides diagnostic methods for assessing
the efficacy of a therapy for inhibiting ovarian cancer in a
patient. Such methods comprise comparing 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
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 ovarian cancer in the
patient.
[0020] It will be appreciated that in these methods the "therapy"
may be any therapy for treating ovarian 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.
[0021] In a preferred embodiment, the diagnostic methods of the
present invention are directed to therapy using a chemical or
biologic agent. These methods comprise comparing 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 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 first sample
relative to that in the second sample is an indication that the
agent is efficacious for inhibiting ovarian 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.
[0022] The invention additionally provides a monitoring method for
assessing the progression of ovarian cancer in a patient, the
method comprising detecting in a patient sample at a first time
point, the expression of a marker of the invention; repeating the
detection at a subsequent time point in time; and comparing the
level of expression detected, and therefrom monitoring the
progression of ovarian 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 ovarian cancer has progressed,
whereas a significantly lower level of expression is an indication
that the ovarian cancer has regressed.
[0023] The invention further provides a diagnostic method for
determining whether ovarian cancer has metastasized or is likely to
metastasize in the future, the method comprising comparing the
level of expression of a marker of the invention in a patient
sample, and 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
ovarian cancer has metastasized or is likely to metastasize in the
future.
[0024] The invention moreover provides a test method for selecting
a composition for inhibiting ovarian cancer in a patient. This
method comprises obtaining a sample comprising cancer cells from
the patient; separately maintaining aliquots of the sample in the
presence of a plurality of test compositions; comparing expression
of a marker of the invention in each of the aliquots; and then
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.
[0025] The invention additionally provides a test method of
assessing the ovarian carcinogenic potential of a compound. This
method comprises maintaining separate aliquots of ovarian cells in
the presence and absence of the compound; and 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 ovarian carcinogenic
potential.
[0026] In addition, the invention further provides a method of
inhibiting ovarian cancer in a patient. This method comprises
obtaining a sample comprising cancer cells from the patient;
separately maintaining aliquots of the sample in the presence of a
plurality of compositions; then comparing expression of a marker of
the invention in each of the aliquots; and lastly 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.
[0027] In the aforementioned methods, the samples or patient
samples comprise cells obtained from the patient. The cells may be
found in an ovarian tissue sample collected, for example, by an
ovarian tissue biopsy or histology section. In one embodiment, the
patient sample is an ovary-associated body fluid. Such fluids
include, for example, blood fluids, lymph, ascites fluids,
gynecological fluids, cystic fluids, urine, and fluids collected by
peritoneal rinsing. In another embodiment, the sample comprises
cells obtained from the patient. In this embodiment, the cells may
be found 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, and
an ovarian exudate. In a further embodiment, the patient sample is
in vivo.
[0028] 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:
the corresponding marker protein (e.g., a protein having one of the
sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ
ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:
26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ
ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID
NO: 44) 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); the corresponding marker nucleic acid (e.g., a
nucleic acid having one of the sequences of SEQ ID NO: 1, SEQ ID
NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ
ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:
21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ
ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO:
39, SEQ ID NO: 41, or SEQ ID NO: 43 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 sequence or a
complement thereof); or a metabolite which is produced directly
(i.e., catalyzed) or indirectly by the corresponding marker
protein.
[0029] 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 ovarian cancer markers, including ovarian 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 or control
level of expression of each of the plurality of markers in samples
of the same type obtained from control humans not afflicted with
ovarian 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 levels, is an indication that
the patient is afflicted with ovarian 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%.
[0030] 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 the
sequence of any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID
NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,
SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID
NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,
SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ
ID NO: 44; 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 or more, of a marker protein, 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
(e.g., a protein having the sequence of any of SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID
NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,
SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID
NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44; or a fragment of the
protein.
[0031] 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 ovarian
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 an ovarian cancer in a patient. Such
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 ovarian cancer cells or treating ovarian 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.
[0032] In an additional embodiment, the invention also provides a
kit for assessing the presence of ovarian 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.
[0033] In a further aspect, the invention relates to methods for
treating a patient afflicted with ovarian cancer or at risk of
developing ovarian 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
antisense 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 the sequence of any of the markers listed in Table
1, or a fragment of such a protein.
[0034] It will be appreciated that the methods and kits of the
present invention may also include known cancer markers including
known ovarian cancer markers. It will further be appreciated that
the methods and kits may be used to identify cancers other than
ovarian cancer.
[0035] In another aspect the invention features nucleic acid
molecules which encode marker proteins or marker polypeptides,
e.g., a biologically active portion of the marker protein. In a
preferred embodiment, the isolated nucleic acid molecules encode
marker polypeptides having the amino acid sequence of SEQ ID NO: 2,
SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:
12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ
ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:
30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ
ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44. In other embodiments,
the invention provides isolated marker nucleic acid molecules
having the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO:
3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID
NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,
SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID
NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,
SEQ ID NO: 41, or SEQ ID NO: 43. In still other embodiments, the
invention provides nucleic acid molecules that are substantially
identical (e.g., naturally occurring allelic variants) to the
nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID
NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,
SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID
NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,
SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID
NO: 41, or SEQ ID NO: 43. In other embodiments, the invention
provides nucleic acid molecules which hybridize under stringent
hybridization condition as described herein to nucleic acid
molecules comprising the nucleotide sequence of SEQ ID NO: 1, SEQ
ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,
SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID
NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29,
SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID
NO: 39, SEQ ID NO: 41, or SEQ ID NO: 43, wherein the nucleic acid
encodes a full length marker protein or an active fragment
thereof.
[0036] In a related aspect, the invention further provides nucleic
acid constructs which include marker nucleic acid molecules
described herein. In certain embodiments, the nucleic acid
molecules of the invention are operatively linked to native or
heterologous regulatory sequences. Also included are vectors and
host cells containing marker nucleic acid molecules of the
invention e.g., vectors and host cells suitable for producing
polypeptides.
[0037] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection of marker-encoding nucleic acids.
[0038] In still another related aspect, isolated nucleic acid
molecules that are antisense to a marker encoding nucleic acid
molecule are provided.
[0039] In other embodiments, the invention provides marker
polypeptides, e.g., marker polypeptide having the amino acid
sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID
NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,
SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID
NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,
SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ
ID NO: 44; an amino acid sequence that is substantially identical
to the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, or SEQ ID NO: 44; or amino acid sequences encoded by
nucleic acid molecules having a nucleotide sequence which
hybridizes under a stringent hybridization condition as described
herein to nucleic acid molecules comprising the nucleotide
sequences of SEQ ID NO:(nts), wherein the nucleic acid encodes a
full length marker protein or an active fragment thereof.
[0040] In a related aspect, the invention further provides nucleic
acid constructs which include marker nucleic acid molecule
described herein.
[0041] In a related aspect, the invention provides marker
polypeptides or fragments operatively linked to non-marker
polypeptides to form fusion proteins.
[0042] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically or selectively bind marker
polypeptides.
[0043] The invention relates to newly discovered markers,
identified in Table 1 that are associated with the cancerous state
of ovarian 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 ovarian cancer in
a patient. Methods are provided for detecting the presence of
ovarian cancer in a sample, the absence of ovarian cancer in a
sample, the stage of an ovarian cancer, assessing whether a breast
cancer has metastasized, predicting the likely clinical outcome of
a breast cancer patient and with other characteristics of ovarian
cancer that are relevant to prevention, diagnosis,
characterization, and therapy of ovarian cancer in a patient.
Methods of treating ovarian cancer are also provided.
[0044] Table 1 lists all of the markers of the invention, which are
over-expressed in ovarian cancer cells compared to normal (i.e.,
non-cancerous) ovarian cells. In this Table the markers are
identified 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").
TABLE-US-00001 TABLE 1 Ovarian Cancer Markers SEQ ID SEQ ID Marker
Gene Name NO (nts) NO (AAs) CDS M138 CTHRC1: collagen triple helix
repeat 1 2 27 . . . 863 containing 1 M437 FLJ10546: hypothetical
protein FLJ10546 3 4 28 . . . 1815 M445 FLJ23499: hypothetical
protein FLJ23499 5 6 21 . . . 473 M452A IMP-2: IGF-II mRNA-binding
protein 2, 7 8 65 . . . 1735 variant 1 M712 IMP-2: IGF-II
mRNA-binding protein 2, 9 10 65 . . . 1603 variant 2 OV32A KLK10:
kallikrein 10 11 12 220 . . . 1050 OV33A KLK6: kallikrein 6
(neurosin, zyme) 13 14 246 . . . 980 M472 MAL2: T-cell
differentiation protein 2 15 16 88 . . . 618 M590 FLJ90687:
hypothetical protein FLJ90687 17 18 21 . . . 404 OV52A MMP7: matrix
metalloproteinase 7 19 20 48 . . . 851 (matrilysin, uterine) OV51A
PTGS1: prostaglandin-endoperoxide 21 22 136 . . . 1935 synthase 1
(prostaglandin G/H synthase and cyclooxygenase), transcript variant
1 M713 PTGS1: prostaglandin-endoperoxide 23 24 136 . . . 1824
synthase 1 (prostaglandin G/H synthase and cyclooxygenase),
transcript variant 2 OV55 S100A1: S100 calcium-binding protein A1
25 26 114 . . . 398 M458 SCGB2A1: secretoglobin, family 2A, 27 28
65 . . . 352 member 1 M714 SLC39A4: solute carrier family 39 (zinc
29 30 101 . . . 2044 transporter), member 4, variant 1 M715
SLC39A4: solute carrier family 39 (zinc 31 32 101 . . . 1762
transporter), member 4, variant 2 M185A SLPI: secretory leukocyte
protease 33 34 23 . . . 421 inhibitor (antileukoproteinase) OV65
SPON1: VSGP/F-spondin 35 36 25 . . . 2448 M476 TACSTD2:
tumor-associated calcium 37 38 616 . . . 1587 signal transducer 2
M716 WFDC2: WAP four-disulfide core domain 39 40 28 . . . 402 2,
variant 1 M717 WFDC2: WAP four-disulfide core domain 41 42 67 . . .
288 2, variant 2 M724 MGC13057: hypothetical protein 43 44 339 . .
. 626 MGC13057
DEFINITIONS
[0045] As used herein, each of the following terms has the meaning
associated with it in this section.
[0046] 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.
[0047] 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 can include DNA (e.g., cDNA) comprising the
entire or a partial sequence of any of SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:
13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ
ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:
31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ
ID NO: 41, or SEQ ID NO: 43. The marker nucleic acids also can
include RNA comprising the entire or a partial sequence
corresponding to any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,
SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID
NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,
SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID
NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41,
or SEQ ID NO: 43, 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 partial
sequence of any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID
NO: 8, SEQ ID NO: 10. SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,
SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID
NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,
SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ
ID NO: 44. The terms "protein" and "polypeptide` are used
interchangeably.
[0048] A "marker set" is a group of more than one marker.
[0049] 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.
[0050] "Ovarian cancer" as used herein includes carcinomas, (e.g.,
carcinoma in situ, invasive carcinoma, metastatic carcinoma) and
pre-malignant conditions.
[0051] An "ovary-associated" body fluid is a fluid which, when in
the body of a patient, contacts or passes through ovarian cells or
into which cells or proteins shed from ovarian cells e.g., ovarian
epithelium, are capable of passing. Exemplary ovary-associated body
fluids include blood fluids, lymph, ascites, gynecological fluids,
cystic fluid, urine, and fluids collected by peritoneal
rinsing.
[0052] A "sample" or "patient sample" comprises cells obtained from
the patient, e.g., a lump biopsy, body fluids including blood
fluids, lymph and cystic fluids, as well as nipple aspirates. In a
further embodiment, the patient sample is in vivo.
[0053] The "normal" level of expression of a marker is the level of
expression of the marker in ovarian cells of a human subject or
patient not afflicted with ovarian cancer
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] "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.
[0061] "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.
[0062] 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.
[0063] 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.
[0064] A cancer is "inhibited" if at least one symptom of the
cancer is alleviated, terminated, slowed, or prevented. As used
herein, ovarian cancer is also "inhibited" if recurrence or
metastasis of the cancer is reduced, slowed, delayed, or
prevented.
[0065] 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.
[0066] "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.
[0067] 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 moiety.
[0068] The present invention is based, in part, on newly identified
markers which are over-expressed in ovarian cancer cells as
compared to their expression in normal (i.e. non-cancerous) ovarian
cells. The enhanced expression of one or more of these markers in
ovarian cells is herein correlated with the cancerous state of the
tissue. The invention provides compositions, kits, and methods for
assessing the cancerous state of ovarian 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 ovarian cancer.
[0069] The compositions, kits, and methods of the invention have
the following uses, among others: assessing whether a patient is
afflicted with ovarian cancer; assessing the stage of ovarian
cancer in a human patient; assessing the grade of ovarian cancer in
a patient; assessing the benign or malignant nature of ovarian
cancer in a patient; assessing the metastatic potential of ovarian
cancer in a patient; determining if breast cancer has metastasized;
predicting the clinical outcome of a breast cancer patient;
assessing the histological type of neoplasm (e.g. serous, mucinous,
endometroid, or clear cell neoplasm) associated with ovarian cancer
in a patient; making antibodies, antibody fragments or antibody
derivatives that are useful for treating ovarian cancer and/or
assessing whether a patient is afflicted with ovarian cancer;
assessing the presence of ovarian cancer cells; assessing the
efficacy of one or more test compounds for inhibiting ovarian
cancer in a patient; assessing the efficacy of a therapy for
inhibiting ovarian cancer in a patient; monitoring the progression
of ovarian cancer in a patient; selecting a composition or therapy
for inhibiting ovarian cancer in a patient; treating a patient
afflicted with ovarian cancer; inhibiting ovarian cancer in a
patient; assessing the ovarian carcinogenic potential of a test
compound; and preventing the onset of ovarian cancer in a patient
at risk for developing ovarian cancer.
[0070] The invention thus includes a method of assessing whether a
patient is afflicted with ovarian cancer which includes assessing
whether the patient has pre-metastasized ovarian 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-ovarian 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
ovarian cancer.
[0071] 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 sequences of
SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:
9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ
ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:
27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ
ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 43 or the
complement of such sequences, and polypeptides comprising the
entirety, or a segment of 10 or more amino acids, of any of the
sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10. SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ
ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:
26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ
ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO:
44 are also provided by this invention.
[0072] As described herein, ovarian 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 ovarian
cancer, others of these changes induce, maintain, and promote the
cancerous state of ovarian cancer cells. Thus, ovarian 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.
[0073] 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 ovarian 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 ovarian 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 ovarian cancer cells of the patient.
[0074] 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 ovarian cancer cells and the level of expression of the
same marker in normal ovarian 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 ovarian tissue.
[0075] It is recognized that certain marker proteins are secreted
from ovarian 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 such marker
proteins can be detected in an ovary-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.
[0076] 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 ovarian 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).
[0077] 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.
[0078] Examples of ovary-associated body fluids include blood
fluids (e.g. whole blood, blood serum, blood having platelets
removed therefrom, etc.), lymph, ascitic fluids, gynecological
fluids (e.g. ovarian, fallopian, and uterine secretions, menses,
vaginal douching fluids, fluids used to rinse ovarian cell samples,
etc.), cystic fluid, urine, and fluids collected by peritoneal
rinsing (e.g. fluids applied and collected during laparoscopy or
fluids instilled into and withdrawn from the peritoneal cavity of a
human patient). 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 protein in an
ovary-associated body fluid obtained from a patient. The fluid can,
of course, be subjected to a variety of well-known post-collection
preparative and storage techniques (e.g. storage, freezing,
ultrafiltration, concentration, evaporation, centrifugation, etc.)
prior to assessing the amount of the marker in the fluid.
[0079] Many ovary-associated body fluids (i.e. usually excluding
urine) can have ovarian cells, e.g. ovarian epithelium, therein,
particularly when the ovarian cells are cancerous, and, more
particularly, when the ovarian cancer is metastasizing.
Cell-containing fluids which can contain ovarian cancer cells
include, but are not limited to, peritoneal ascites, fluids
collected by peritoneal rinsing, fluids collected by uterine
rinsing, uterine fluids such as uterine exudate and menses, pleural
fluid, and ovarian exudates. Thus, 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 (e.g. the SIGNALP
program; Nielsen et al., 1997, Protein Engineering 10:1-6) 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).
[0080] 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.
[0081] 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.) or derivative 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.
[0082] 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.
[0083] 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 several marker nucleic acids 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.
[0084] 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 ovarian cells and cancerous ovarian cells.
[0085] 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 ovarian cancers, as
well as other cancers such as breast cancer, cervical 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 ovarian cancer.
Furthermore, it will be confirmed that certain of the markers of
the invention are associated with ovarian cancer of various stages
(i.e. stage I, II, III, and IV ovarian cancers, as well as
subclassifications IA, IB, IC, IIA, IIB, IIC, IIIA, IIIB, and IIIC,
using the FIGO Stage Grouping system for primary carcinoma of the
ovary; 1987, Am. J. Obstet. Gynecol. 156:236), of various
histologic subtypes (e.g. serous, mucinous, endometroid, 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, mesonephroid tumor, malignant carcinoma,
Brenner tumor, mixed epithelial tumor, and undifferentiated
carcinoma, using the WHO/FIGO system for classification of
malignant ovarian 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 altered 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 ovarian cancer in
patients. In addition, these compositions, kits, and methods can be
used to detect and differentiate epithelial, stromal, and germ cell
ovarian cancers.
[0086] 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 ovarian 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 an ovarian 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 99.5%).
[0087] 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 ovarian 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.
[0088] In order to maximize the sensitivity of the compositions,
kits, and methods of the invention (i.e. by interference
attributable to cells of non-ovarian 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-epithelial tissue, and more preferably a
marker which is normally not expressed in a non-ovarian tissue.
[0089] Only a small number of markers are known to be associated
with ovarian cancers (e.g. AKT2, Ki-RAS, ERBB2, c-MYC, RB1, and
TP53; Lynch, supra). 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 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.
[0090] 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 ovarian cancer and their medical
advisors. Patients recognized as having an enhanced risk of
developing ovarian cancer include, for example, patients having a
familial history of ovarian 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).
[0091] The level of expression of a marker in normal (i.e.
non-cancerous) human ovarian 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 ovarian 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 ovarian cells which is suspected of
being cancerous. For example, when laparoscopy or other medical
procedure, reveals the presence of a lump on one portion of a
patient's ovary, but not on another portion of the same ovary or on
the other ovary, the normal level of expression of a marker may be
assessed using one or both or the non-affected ovary and a
non-affected portion of the affected ovary, and this normal level
of expression may be compared with the level of expression of the
same marker in an affected portion (i.e. the lump) of the affected
ovary. 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 ovarian cancer in the
patient, from archived patient samples, and the like.
[0092] The invention includes compositions, kits, and methods for
assessing the presence of ovarian 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.
[0093] The invention includes a kit for assessing the presence of
ovarian 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.
[0094] 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
ovarian cells, a sample of ovarian cancer cells, and the like.
[0095] The invention also includes a method of making an isolated
hybridoma which produces an antibody useful for assessing whether
patient is afflicted with an ovarian 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.
[0096] The invention also includes a method of assessing the
efficacy of a test compound for inhibiting ovarian cancer cells. As
described above, differences in the level of expression of the
markers of the invention correlate with the cancerous state of
ovarian 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 ovarian 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 an
ovarian 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 ovarian
cells).
[0097] This method thus comprises comparing expression of a marker
in a first ovarian cell sample and maintained in the presence of
the test compound and expression of the marker in a second ovarian
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 ovarian cancer. The ovarian cell samples may, for
example, be aliquots of a single sample of normal ovarian cells
obtained from a patient, pooled samples of normal ovarian cells
obtained from a patient, cells of a normal ovarian cell line,
aliquots of a single sample of ovarian cancer cells obtained from a
patient, pooled samples of ovarian cancer cells obtained from a
patient, cells of an ovarian cancer cell line, or the like. In one
embodiment, the samples are ovarian cancer cells obtained from a
patient and a plurality of compounds known to be effective for
inhibiting various ovarian cancers are tested in order to identify
the compound which is likely to best inhibit the ovarian cancer in
the patient.
[0098] This method may likewise be used to assess the efficacy of a
therapy for inhibiting ovarian 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 ovarian
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 ovarian cancer in the patient.
[0099] As described above, the cancerous state of human ovarian
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 ovarian cell carcinogenic potential of a test
compound. This method comprises maintaining separate aliquots of
human ovarian 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 ovarian 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.
[0100] Various aspects of the invention are described in further
detail in the following subsections.
Isolated Nucleic Acid Molecules
[0101] 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.
[0102] 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.
[0103] 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).
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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 and
thus encode the same protein.
[0109] 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).
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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).
[0118] 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 an ovary-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.
[0119] 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).
[0120] 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).
[0121] 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.
[0122] 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.
[0123] 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).
[0124] 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).
[0125] 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 bather (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.
[0126] 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.
Isolated Proteins and Antibodies
[0127] 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.
[0128] 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.
[0129] 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.
[0130] Preferred marker proteins are encoded by nucleotide
sequences comprising the sequences listed in Table 1. 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.
[0131] 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.
[0132] 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
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. 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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.).
[0137] 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.
[0138] 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.
[0139] 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.
[0140] 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.
[0141] 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).
[0142] 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.
[0143] 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).
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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 single polypeptides. 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.
[0151] 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.
[0152] 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.
[0153] 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).
[0154] 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.
[0155] 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.
[0156] 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 an ovary-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.
[0157] 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
ovarian 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. 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).
[0158] 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.
[0159] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon 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).
[0160] 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.
[0161] 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
and a pharmaceutically acceptable carrier. In preferred
embodiments, the pharmaceutical composition contains an antibody of
the invention, a therapeutic moiety, and a pharmaceutically
acceptable carrier.
Recombinant Expression Vectors and Host Cells
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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.).
[0169] 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).
[0170] 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.
[0171] 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 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).
[0172] 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).
[0173] 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.
[0174] A host cell can be any prokaryotic (e.g., E. coli) or
eukaryotic cell (e.g., insect cells, yeast or mammalian cells).
[0175] 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.
[0176] 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 gene will
survive, while the other cells die).
[0177] 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 a marker protein or a segment thereof from
the medium or the host cell.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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.
[0182] 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.
Pharmaceutical Compositions
[0183] 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.
[0184] 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.
[0185] 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.
[0186] 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).
[0187] 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.
[0188] 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.).
[0189] 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.
[0190] 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".
[0191] 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.
[0192] 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.
[0193] 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 ovarian
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.
[0194] 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.
[0195] 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.
[0196] 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, microtiter
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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.), as described 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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., an marker modulating
agent, an antisense marker nucleic acid molecule, an
marker-specific antibody, or an 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.
[0206] 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.
[0207] 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.
[0208] 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.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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.
[0214] 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.
[0215] 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.
[0216] 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.
[0217] 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 ovarian epithelium).
A method for lipidation of antibodies is described by Cruikshank et
al. (1997) J. Acquired Immune Deficiency Syndromes and Human
Retrovirology 14:193.
[0218] The invention also provides vaccine compositions for the
prevention and/or treatment of ovarian cancer. The invention
provides ovarian 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 ovarian cancer. The invention also
provides ovarian 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.
[0219] In one embodiment, an ovarian cancer vaccine is provided and
employed as an immunotherapeutic agent for the prevention of
ovarian cancer. In another embodiment, an ovarian cancer vaccine is
provided and employed as an immunotherapeutic agent for the
treatment of ovarian cancer.
[0220] By way of example, an ovarian cancer vaccine comprised of
the proteins of the markers of Table 1, may be employed for the
prevention and/or treatment of ovarian cancer in a subject by
administering the vaccine by a variety of routes, e.g.,
intradermally, subcutaneously, or intramuscularly. In addition, the
ovarian 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 ovarian 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.
[0221] In another embodiment, an ovarian 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 ovarian 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.
[0222] The marker nucleic acid molecules of the present invention
can also 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.
[0223] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
Predictive Medicine
[0224] 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
ovarian cancer. Such assays can be used for prognostic or
predictive purposes to thereby prophylactically treat an individual
prior to the onset of the cancer.
[0225] Yet another aspect of the invention pertains to monitoring
the influence of agents (e.g., drugs or other compounds
administered either to inhibit ovarian cancer or to treat or
prevent any other disorder {i.e. in order to understand any ovarian
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.
[0226] A. Diagnostic Assays
[0227] 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. an ovary-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.
[0228] 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.
[0229] 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.
[0230] 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.
[0231] 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.
[0232] 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.
[0233] 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.
[0234] 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).
[0235] 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.
[0236] 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.
[0237] 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 ovarian 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).
[0238] 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.
[0239] 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.
[0240] 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.
[0241] For in situ methods, mRNA does not need to be isolated from
the ovarian 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.
[0242] 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-ovarian cancer sample, or between
samples from different sources.
[0243] 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.
[0244] Preferably, the samples used in the baseline determination
will be from ovarian cancer or from non-ovarian cancer cells of
ovarian 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 ovarian 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
ovarian cells provides a means for grading the severity of the
ovarian cancer state.
[0245] 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 derivatives
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.
[0246] Proteins from ovarian 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.).
[0247] 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 immunoabsorbent assay (ELISA). A skilled artisan can readily
adapt known protein/antibody detection methods for use in
determining whether ovarian cells express a marker of the present
invention.
[0248] 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.
[0249] 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 ovarian 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.
[0250] The invention also encompasses kits for detecting the
presence of a marker protein or nucleic acid in a biological sample
(e.g. an ovary-associated body fluid such as a urine sample). Such
kits can be used to determine if a subject is suffering from or is
at increased risk of developing ovarian 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.
[0251] 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.
[0252] 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.
[0253] B. Pharmacogenomics
[0254] Agents or modulators which have a stimulatory or inhibitory
effect on expression of a marker of the invention can be
administered to individuals to treat (prophylactically or
therapeutically) ovarian cancer in the patient. In conjunction with
such treatment, the pharmacogenomics (i.e., the study of the
relationship between an individual's genotype and that individual's
response to a foreign compound or drug) of the individual may be
considered. Differences in metabolism of therapeutics can lead to
severe toxicity or therapeutic failure by altering the relation
between dose and blood concentration of the pharmacologically
active drug. Thus, the pharmacogenomics of the individual permits
the selection of effective agents (e.g., drugs) for prophylactic or
therapeutic treatments based on a consideration of the individual's
genotype. Such pharmacogenomics can further be used to determine
appropriate dosages and therapeutic regimens. Accordingly, 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.
[0255] Pharmacogenomics deals with clinically significant
variations in the response to drugs due to altered drug disposition
and abnormal action in affected persons. See, e.g., Linder (1997)
Clin. Chem. 43(2):254-266. In general, two types of pharmacogenetic
conditions can be differentiated. Genetic conditions transmitted as
a single factor altering the way drugs act on the body are referred
to as "altered drug action." Genetic conditions transmitted as
single factors altering the way the body acts on drugs are referred
to as "altered drug metabolism". 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.
[0256] 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.
[0257] 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.
[0258] C. Monitoring Clinical Trials
[0259] 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 ovarian 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 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.
[0260] D. Electronic Apparatus Readable Media and Arrays
[0261] 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.
[0262] 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.
[0263] 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.
[0264] 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.
[0265] 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.
[0266] The present invention therefore provides a medium for
holding instructions for performing a method for determining
whether a subject has ovarian cancer or a pre-disposition to
ovarian 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 ovarian cancer or a pre-disposition to ovarian cancer and/or
recommending a particular treatment for ovarian cancer or
pre-ovarian cancer condition.
[0267] The present invention further provides in an electronic
system and/or in a network, a method for determining whether a
subject has ovarian cancer or a pre-disposition to ovarian 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 ovarian cancer or a pre-disposition to ovarian cancer, and/or
recommending a particular treatment for the ovarian cancer or
pre-ovarian 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.
[0268] The present invention also provides in a network, a method
for determining whether a subject has ovarian cancer or a
pre-disposition to ovarian 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 ovarian cancer, and based on one or more of
the phenotypic information, the marker, and the acquired
information, determining whether the subject has a ovarian cancer
or a pre-disposition to ovarian cancer. The method may further
comprise the step of recommending a particular treatment for the
ovarian cancer or pre-ovarian cancer condition.
[0269] The present invention also provides a business method for
determining whether a subject has ovarian cancer or a
pre-disposition to ovarian 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
ovarian cancer, and based on one or more of the phenotypic
information, the marker, and the acquired information, determining
whether the subject has ovarian cancer or a pre-disposition to
ovarian cancer. The method may further comprise the step of
recommending a particular treatment for the ovarian cancer or
pre-ovarian cancer condition.
[0270] 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.
[0271] 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.
[0272] 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 ovarian cancer, progression of ovarian
cancer, and processes, such a cellular transformation associated
with ovarian cancer.
[0273] 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.
[0274] 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.
[0275] E. Surrogate Markers
[0276] 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, ovarian 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.
[0277] 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 Ovarian Cancer Markers by cDNA and Tissue
Microarrays
Materials and Methods
[0278] Sample collection and RNA preparation
[0279] Ovarian 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 from ethidium staining of the RNA after
electrophoresis on agarose gels, was used in this study.
cDNA Microarray Hybridization
[0280] 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 pre-annealed 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
[0281] 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.
[0282] 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.
[0283] The Marker Score Sg for gene g is therefore computed as the
average of the ratios of weighted intensities of the individual
testers and a control level as follows:
S.sub.g=(.SIGMA.S.sub.gs)/N.sub.tester
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, [0284] 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, [0285] A is an upper asymptote on the
fold-change value (we used 10), [0286] x.sub.gs is the expression
value of gene g on sample s, [0287] x.sub.g.sup.Q is the Qth
percentile of the control samples' expression value; typically
Q=50, [0288] 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. [0289]
N.sub.tester The number of tester samples
Results
Marker Selection
[0290] All of the markers listed in Table 1 were identified by
transcription profiling as defined in the materials and methods
section. mRNA from markers M138, M437, M445, M452A, M712, M472,
M590A, M713, M458, M714, M715, M185A, M476, M716, M717 and M724 was
obtained from 67 ovarian tumors of various histotypes and stages
and 96 non-ovarian tumor tissues including normal ovarian
epithelium, benign conditions, other normal tissues and other
abnormal tissues. Clones having expression at least three-fold
higher in at least 10% of ovarian tumors, as compared to their
expression in non-ovarian tumor tissue, were designated as ovarian
cancer specific markers. These cDNA clones were selected to have
their protein-encoding transcript sequences determined mRNA from
markers OV32A, OV33A, OV52A, OV51A, OV55 and OV65 was obtained from
9 normal ovarian epithelial, 11 stage I/II ovarian cancer tumors
and 25 stage III/IV ovarian cancer tumors. Clones having expression
of at least two-fold higher in ovarian tumors as compared to their
expression in non-ovarian tumor tissues in at least 4 tumor samples
were selected to have their protein-encoding transcript sequence
determined.
[0291] In order to determine the full-length protein-encoding
transcripts for the selected cDNA clones, the sequence(s) of the
selected clones were used to query the public and proprietary
sequence databases in order to identify other EST sequences or
clusters with significant overlap. Briefly, BLAST analysis, against
both public and proprietary sequence databases, of EST sequences
known to be associated with each clone was performed, either
directly or in the context of automatically, high-stringency
assembled contiguous sequences. An identification of protein
sequence corresponding to the clone was accomplished by obtaining
one of the following:
1) a direct match between the protein sequence and at least one EST
sequence in one of its 6 possible translations; 2) a direct match
between the nucleotide sequence for the mRNA corresponding to the
protein sequence and at least one EST sequence; 3) a match between
the protein sequence and a contiguous assembly (contig) of the EST
sequences with other available EST sequences in the databases in
one of its 6 possible translations; or 4) a match between the
nucleotide sequence for the mRNA corresponding to the protein
sequence and a contiguous assembly of the EST sequences with other
available EST sequences in the databases in one of its 6 possible
translations. Thus, contiguous EST sequences and/or clusters were
assembled into protein-encoding transcripts. Alternative transcript
analysis for all of the claimed markers was undertaken as follows:
1) Using existing mappings of known nucleotide sequences for any
given marker gene to the human genome sequence and by additionally
mapping novel nucleotide sequences for any given marker gene onto
the human genome sequence (e.g. using resources like the "UCSC
genome browser" or in-house resources of similar functionality in
conjunction with algorithms like BLAT that allow a rapid and
precise mapping of search sequences onto genomic sequence), the
exon-intron structure of a marker gene was established, taking
additionally into account EST sequences matching the same gene. 2)
PCR primers were designed to amplify the coding sequence of a given
marker gene from the tissue of interest and control samples. Any
alternative 5' or 3' ends of a marker gene arising from this
analysis with the potential to alter the coding sequence led to the
design of an additional primer specific for this alternative end.
3) PCR products obtained with cDNA templates derived from ovarian
tumor specimens were cloned into a plasmid vector and characterized
by DNA sequence analysis. Typically, 96 clones were analyzed by
restriction digestion and gel electrophoresis of the PCR products
or by DNA sequence analysis. 4) Clones representative of
alternative gene transcripts occurring at a frequency of 2% or
greater were sequenced. 5) The differential gene expression of the
identified alternative transcripts was confirmed by TAQMAN.RTM.
quantitative PCR (Applied Biosystems) in cDNA prepared from the
patient tissue specimens. Splice-form specific TaqMan primers and
probe regent sets were developed for each transcript and similar
amplification efficiencies were obtained with all reagents sets for
each gene. 6) The identification of protein sequence corresponding
to these alternative transcripts was accomplished by the
identification of the open reading frame (ORF) contained within a
manually curated assembly (contig) based on all available
sequences.
EXAMPLE 2
Gene Expression Analysis by End-Point PCR
Materials and Methods
[0292] Briefly, total RNA from different samples was pooled to be
used as template to generate first strand cDNA. The ovarian panel
consisted of patient samples of a "ovarian tumor pool" (4 tumor
samples containing seous and clear cell ovarian tumors) and a
"ovarian normal pool" (3 normal ovarian epithelia).
[0293] Total RNA was prepared from patient samples 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. RNA from
each patient sample was pooled into one of the two pools, e.g.,
ovarian tumor pool and ovarian normal pool. ThermoScript RT-PCR
System (Invitrogen, San Diego, Calif.) was used to obtain cDNA from
each of the pools. Briefly, 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 10
.mu.l 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.
[0294] Gene specific primers were designed just outside the Open
Reading Frame (as shown in Table 2 categories "Endpoint PCR Primer
1" and "Endpoint PCR Primer 2"). 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.
[0295] The ethidium bromide agarose gel pictures of the end-point
PCR on the tissue panel were scored on a scale of 1-5. Each picture
was scored independently by three people based on visual band
intensity and the results were compiled. The scores were compared
to confirm all three agreed on the relative intensities of the
bands and modifications were made where needed. The median of the
three scores was then recorded as the final score.
[0296] As shown in Table 2 every marker of the invention tested in
End-point PCR was expressed at higher levels in the ovarian tumor
pool when compared to the ovarian normal pool.
Results
TABLE-US-00002 [0297] TABLE 2 Endpoint PCR Data. Endpoint Endpoint
Ovarian Ovarian PCR PCR Tumor Normal Marker Primer 1 Primer 2 Pool
Pool M138 62-81 898-920 1 0 OV32A 87-108 1107-1128 3 0 OV33A
237-254 1082-1103 5 1 M472 62-80 458-480 2 0 M590A 751-772
1114-1135 5 1 OV52A 2-23 927-948 5 0 OV51A 91-108 1968-1989 4 0
M713 91-108 1857-1878 4 0 OV55 17-36 485-504 4 0 M458 1-22 373-394
5 0 M185A 13-33 519-538 3 1 OV65 106-123 2444-2465 5 0 M476 152-171
1742-1761 2 0 M716 20-39 414-432 1 0 M717 95-114 403-422 3 1 M724
275-296 638-659 5 1
Other Embodiments
[0298] 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:
Sequence CWU 1
1
4411257DNAHomo sapienCDS(27)...(863) 1gaggcgcgcg ggtgaaaggc gcattg
atg cag cct gcg gcg gcc tcg gag cgc 53 Met Gln Pro Ala Ala Ala Ser
Glu Arg 1 5ggc gga gca gac gct gac cac gtt cct ctc ctc ggt ctc ctc
cgc ctc 101Gly Gly Ala Asp Ala Asp His Val Pro Leu Leu Gly Leu Leu
Arg Leu10 15 20 25cag ctc cgc gct gcc cgg cag ccg gga gcc atg cga
ccc cag ggc ccc 149Gln Leu Arg Ala Ala Arg Gln Pro Gly Ala Met Arg
Pro Gln Gly Pro 30 35 40gcc gcc tcc ccg cag cgg ctc cgc ggc ctc ctg
ctg ctc ctg ctg ctg 197Ala Ala Ser Pro Gln Arg Leu Arg Gly Leu Leu
Leu Leu Leu Leu Leu 45 50 55cag ctg ccc gcg ccg tcg agc gcc tct gag
atc ccc aag ggg aag caa 245Gln Leu Pro Ala Pro Ser Ser Ala Ser Glu
Ile Pro Lys Gly Lys Gln 60 65 70aag gcg cag ctc cgg cag agg gag gtg
gtg gac ctg tat aat gga atg 293Lys Ala Gln Leu Arg Gln Arg Glu Val
Val Asp Leu Tyr Asn Gly Met 75 80 85tgc tta caa ggg cca gca gga gtg
cct ggt cga gac ggg agc cct ggg 341Cys Leu Gln Gly Pro Ala Gly Val
Pro Gly Arg Asp Gly Ser Pro Gly90 95 100 105gcc aat ggc att ccg ggt
aca cct ggg atc cca ggt cgg gat gga ttc 389Ala Asn Gly Ile Pro Gly
Thr Pro Gly Ile Pro Gly Arg Asp Gly Phe 110 115 120aaa gga gaa aag
ggg gaa tgt ctg agg gaa agc ttt gag gag tcc tgg 437Lys Gly Glu Lys
Gly Glu Cys Leu Arg Glu Ser Phe Glu Glu Ser Trp 125 130 135aca ccc
aac tac aag cag tgt tca tgg agt tca ttg aat tat ggc ata 485Thr Pro
Asn Tyr Lys Gln Cys Ser Trp Ser Ser Leu Asn Tyr Gly Ile 140 145
150gat ctt ggg aaa att gcg gag tgt aca ttt aca aag atg cgt tca aat
533Asp Leu Gly Lys Ile Ala Glu Cys Thr Phe Thr Lys Met Arg Ser Asn
155 160 165agt gct cta aga gtt ttg ttc agt ggc tca ctt cgg cta aaa
tgc aga 581Ser Ala Leu Arg Val Leu Phe Ser Gly Ser Leu Arg Leu Lys
Cys Arg170 175 180 185aat gca tgc tgt cag cgt tgg tat ttc aca ttc
aat gga gct gaa tgt 629Asn Ala Cys Cys Gln Arg Trp Tyr Phe Thr Phe
Asn Gly Ala Glu Cys 190 195 200tca gga cct ctt ccc att gaa gct ata
att tat ttg gac caa gga agc 677Ser Gly Pro Leu Pro Ile Glu Ala Ile
Ile Tyr Leu Asp Gln Gly Ser 205 210 215cct gaa atg aat tca aca att
aat att cat cgc act tct tct gtg gaa 725Pro Glu Met Asn Ser Thr Ile
Asn Ile His Arg Thr Ser Ser Val Glu 220 225 230gga ctt tgt gaa gga
att ggt gct gga tta gtg gat gtt gct atc tgg 773Gly Leu Cys Glu Gly
Ile Gly Ala Gly Leu Val Asp Val Ala Ile Trp 235 240 245gtt ggc act
tgt tca gat tac cca aaa gga gat gct tct act gga tgg 821Val Gly Thr
Cys Ser Asp Tyr Pro Lys Gly Asp Ala Ser Thr Gly Trp250 255 260
265aat tca gtt tct cgc atc att att gaa gaa cta cca aaa taa 863Asn
Ser Val Ser Arg Ile Ile Ile Glu Glu Leu Pro Lys * 270 275atgctttaat
tttcatttgc tacctctttt tttattatgc cttggaatgg ttcacttaaa
923tgacatttta aataagttta tgtatacatc tgaatgaaaa gcaaagctaa
atatgtttac 983agaccaaagt gtgatttcac actgttttta aatctagcat
tattcatttt gcttcaatca 1043aaagtggttt caatattttt tttagttggt
tagaatactt tcttcatagt cacattctct 1103caacctataa tttggaatat
tgttgtggtc ttttgttttt tctcttagta tagcattttt 1163aaaaaaatat
aaaagctacc aatctttgta caatttgtaa atgttaagaa ttttttttat
1223atctgttaaa taaaaattat ttccaacaac ctta 12572278PRTHomo sapien
2Met Gln Pro Ala Ala Ala Ser Glu Arg Gly Gly Ala Asp Ala Asp His1 5
10 15Val Pro Leu Leu Gly Leu Leu Arg Leu Gln Leu Arg Ala Ala Arg
Gln 20 25 30Pro Gly Ala Met Arg Pro Gln Gly Pro Ala Ala Ser Pro Gln
Arg Leu 35 40 45Arg Gly Leu Leu Leu Leu Leu Leu Leu Gln Leu Pro Ala
Pro Ser Ser 50 55 60Ala Ser Glu Ile Pro Lys Gly Lys Gln Lys Ala Gln
Leu Arg Gln Arg65 70 75 80Glu Val Val Asp Leu Tyr Asn Gly Met Cys
Leu Gln Gly Pro Ala Gly 85 90 95Val Pro Gly Arg Asp Gly Ser Pro Gly
Ala Asn Gly Ile Pro Gly Thr 100 105 110Pro Gly Ile Pro Gly Arg Asp
Gly Phe Lys Gly Glu Lys Gly Glu Cys 115 120 125Leu Arg Glu Ser Phe
Glu Glu Ser Trp Thr Pro Asn Tyr Lys Gln Cys 130 135 140Ser Trp Ser
Ser Leu Asn Tyr Gly Ile Asp Leu Gly Lys Ile Ala Glu145 150 155
160Cys Thr Phe Thr Lys Met Arg Ser Asn Ser Ala Leu Arg Val Leu Phe
165 170 175Ser Gly Ser Leu Arg Leu Lys Cys Arg Asn Ala Cys Cys Gln
Arg Trp 180 185 190Tyr Phe Thr Phe Asn Gly Ala Glu Cys Ser Gly Pro
Leu Pro Ile Glu 195 200 205Ala Ile Ile Tyr Leu Asp Gln Gly Ser Pro
Glu Met Asn Ser Thr Ile 210 215 220Asn Ile His Arg Thr Ser Ser Val
Glu Gly Leu Cys Glu Gly Ile Gly225 230 235 240Ala Gly Leu Val Asp
Val Ala Ile Trp Val Gly Thr Cys Ser Asp Tyr 245 250 255Pro Lys Gly
Asp Ala Ser Thr Gly Trp Asn Ser Val Ser Arg Ile Ile 260 265 270Ile
Glu Glu Leu Pro Lys 27534039DNAHomo sapienCDS(28)...(1815)
3cgccatcgtt ccctggctgc ttactaa gtt gga tcc gga att tct ttt caa ccg
54 Val Gly Ser Gly Ile Ser Phe Gln Pro 1 5gga gcc att ggt gtc gaa
gtg tct gca atg aac ccc gtg aat gct act 102Gly Ala Ile Gly Val Glu
Val Ser Ala Met Asn Pro Val Asn Ala Thr10 15 20 25gct ctc tac att
tcc gcg agc cgc cta gtg ctc aac tac gac ccc gga 150Ala Leu Tyr Ile
Ser Ala Ser Arg Leu Val Leu Asn Tyr Asp Pro Gly 30 35 40gac ccc aag
gcg ttt act gag att aac agg ctc ttg cct tac ttc cga 198Asp Pro Lys
Ala Phe Thr Glu Ile Asn Arg Leu Leu Pro Tyr Phe Arg 45 50 55cag tcc
ctt tcg tgc tgt gtt tgc gga cat ttg cta caa gat cct att 246Gln Ser
Leu Ser Cys Cys Val Cys Gly His Leu Leu Gln Asp Pro Ile 60 65 70gca
ccc acc aac tcc acc tgc caa cat tat gtc tgc aaa act tgt aaa 294Ala
Pro Thr Asn Ser Thr Cys Gln His Tyr Val Cys Lys Thr Cys Lys 75 80
85ggc aag aaa atg atg atg aaa cct tcc tgt agc tgg tgc aaa gac tat
342Gly Lys Lys Met Met Met Lys Pro Ser Cys Ser Trp Cys Lys Asp
Tyr90 95 100 105gag cag ttt gag gaa aac aag cag tta agc atc cta gtg
aac tgc tac 390Glu Gln Phe Glu Glu Asn Lys Gln Leu Ser Ile Leu Val
Asn Cys Tyr 110 115 120aaa aaa cta tgc gag tat ata aca cag act aca
ctg gca cgg gat ata 438Lys Lys Leu Cys Glu Tyr Ile Thr Gln Thr Thr
Leu Ala Arg Asp Ile 125 130 135ata gaa gca gtt gac tgt tct tct gat
att ttg gct ttg ctt aat gat 486Ile Glu Ala Val Asp Cys Ser Ser Asp
Ile Leu Ala Leu Leu Asn Asp 140 145 150gga tca ttg ttt tgt gag gag
aca gaa aaa ccc tca gat tca tcc ttt 534Gly Ser Leu Phe Cys Glu Glu
Thr Glu Lys Pro Ser Asp Ser Ser Phe 155 160 165act ttg tgt ttg aca
cat tcc cct tta cct tca acc tca gaa ccc aca 582Thr Leu Cys Leu Thr
His Ser Pro Leu Pro Ser Thr Ser Glu Pro Thr170 175 180 185act gat
cct caa gct agt tta tct cca atg tct gaa agc acc ctc agc 630Thr Asp
Pro Gln Ala Ser Leu Ser Pro Met Ser Glu Ser Thr Leu Ser 190 195
200att gct att ggc agt tct gtt atc aat ggt ttg cct act tat aat ggg
678Ile Ala Ile Gly Ser Ser Val Ile Asn Gly Leu Pro Thr Tyr Asn Gly
205 210 215ctt tca ata gat aga ttt ggt ata aat att cct tca cct gaa
cat tca 726Leu Ser Ile Asp Arg Phe Gly Ile Asn Ile Pro Ser Pro Glu
His Ser 220 225 230aat acg att gac gta tgt aat act gtt gac ata aaa
act gag gat ctg 774Asn Thr Ile Asp Val Cys Asn Thr Val Asp Ile Lys
Thr Glu Asp Leu 235 240 245tct gac agc ctg cca ccc gtt tgt gac aca
gta gcc act gac tta tgt 822Ser Asp Ser Leu Pro Pro Val Cys Asp Thr
Val Ala Thr Asp Leu Cys250 255 260 265tcc aca ggc att gat atc tgc
agt ttc agt gaa gat ata aaa cct gga 870Ser Thr Gly Ile Asp Ile Cys
Ser Phe Ser Glu Asp Ile Lys Pro Gly 270 275 280gac tct ctg tta ctg
agt gtt gag gaa gta ctc cgc agc tta gaa act 918Asp Ser Leu Leu Leu
Ser Val Glu Glu Val Leu Arg Ser Leu Glu Thr 285 290 295gtt tca aat
aca gag gtc tgt tgc cct aat ttg cag ccg aac ttg gaa 966Val Ser Asn
Thr Glu Val Cys Cys Pro Asn Leu Gln Pro Asn Leu Glu 300 305 310gcc
act gta tcc aat gga cct ttt ctg cag ctt tct tcc cag tct ctt 1014Ala
Thr Val Ser Asn Gly Pro Phe Leu Gln Leu Ser Ser Gln Ser Leu 315 320
325agc cat aat gtt ttt atg tcc acc agt cct gca ctt cat ggg tta tca
1062Ser His Asn Val Phe Met Ser Thr Ser Pro Ala Leu His Gly Leu
Ser330 335 340 345tgt aca gca gca act ccg aag ata gca aaa ttg aat
aga aaa cga tcc 1110Cys Thr Ala Ala Thr Pro Lys Ile Ala Lys Leu Asn
Arg Lys Arg Ser 350 355 360aga tca gag agt gac agt gag aaa gtt cag
cca ctt cca att tct acc 1158Arg Ser Glu Ser Asp Ser Glu Lys Val Gln
Pro Leu Pro Ile Ser Thr 365 370 375att atc cga ggc cca aca ctg ggg
gca tct gct cct gtg aca gtg aaa 1206Ile Ile Arg Gly Pro Thr Leu Gly
Ala Ser Ala Pro Val Thr Val Lys 380 385 390cgg gag agc aaa att tct
ctt caa cct ata gca act gtt ccc aat gga 1254Arg Glu Ser Lys Ile Ser
Leu Gln Pro Ile Ala Thr Val Pro Asn Gly 395 400 405ggc aca aca cct
aaa atc agc aaa act gta ctt tta tct act aaa agc 1302Gly Thr Thr Pro
Lys Ile Ser Lys Thr Val Leu Leu Ser Thr Lys Ser410 415 420 425atg
aaa aag agt cat gaa cat gga tcc aag aaa tct cac tct aaa acc 1350Met
Lys Lys Ser His Glu His Gly Ser Lys Lys Ser His Ser Lys Thr 430 435
440aag cca ggt att ctt aaa aaa gac aaa gca gta aag gaa aag att cct
1398Lys Pro Gly Ile Leu Lys Lys Asp Lys Ala Val Lys Glu Lys Ile Pro
445 450 455agt cat cat ttt atg cca gga agt cct acc aag act gtg tac
aaa aaa 1446Ser His His Phe Met Pro Gly Ser Pro Thr Lys Thr Val Tyr
Lys Lys 460 465 470ccc cag gaa aag aaa ggg tgt aaa tgt ggg cgt gct
act caa aat cca 1494Pro Gln Glu Lys Lys Gly Cys Lys Cys Gly Arg Ala
Thr Gln Asn Pro 475 480 485agt gtt ctt aca tgc cga ggc caa cgc tgc
cct tgc tac tct aac cgc 1542Ser Val Leu Thr Cys Arg Gly Gln Arg Cys
Pro Cys Tyr Ser Asn Arg490 495 500 505aaa gcc tgc tta gat tgt ata
tgt cgt ggc tgc caa aac tcc tat atg 1590Lys Ala Cys Leu Asp Cys Ile
Cys Arg Gly Cys Gln Asn Ser Tyr Met 510 515 520gcc aat ggg gag aag
aag ctg gag gca ttt gcc gtg cca gaa aag gcc 1638Ala Asn Gly Glu Lys
Lys Leu Glu Ala Phe Ala Val Pro Glu Lys Ala 525 530 535ttg gag cag
acc agg ctc act ttg ggc att aac gtg act agc att gct 1686Leu Glu Gln
Thr Arg Leu Thr Leu Gly Ile Asn Val Thr Ser Ile Ala 540 545 550gtg
cgt aac gct agt acc agc acc agt gta ata aat gtc aca ggg tcc 1734Val
Arg Asn Ala Ser Thr Ser Thr Ser Val Ile Asn Val Thr Gly Ser 555 560
565cca gta acg acg ttt tta gct gcc agt aca cat gat gat aaa agt ttg
1782Pro Val Thr Thr Phe Leu Ala Ala Ser Thr His Asp Asp Lys Ser
Leu570 575 580 585gat gaa gct ata gac atg aga ttc gac tgt taa
atcagtgggt cttttaaacc 1835Asp Glu Ala Ile Asp Met Arg Phe Asp Cys *
590 595tactcctggt agggaaatag ctacagtttt acggcagcta tggttctgtt
ggtttaactt 1895gccggagctc ctgcatatag atcacttgta tcaagtgttt
tcattgctaa gttatatgtg 1955ttagtgtcgg ggaaatagtt tgcagataat
ggaggagtaa ccctacaact atatgtcctt 2015agttcttaca gaacctcata
gtttgagaac aaagctgatg caactgattt atacaaaatg 2075aactttggca
agaaaaataa cattaacctc attgtttatg gccatgcttt gtgcataatc
2135aaagtttatg attaaatgta aggaagtggt atctagtcag tccataaaga
ttgtgctaat 2195ttttttgtgg aaaagtagcc attagttcag gaaactcagt
gctgccttca gatgtcattg 2255atgtttctcc tgttggaaag ctgatgtgtc
cagctcaacc tttgtgctga catcatacca 2315tttctgatca tgaaatattg
gctactggtg tatgtagcag ttcttaaatc agcagtatta 2375tgaaaaaaaa
ttccccctca ttagaatgtt taagaaatct ttttaaaaag taaaattctg
2435tcagactaca aatgtttagc tgttactcat ttctagggaa gaaattctaa
atccctcctt 2495cactttgagc agtgttctaa ttggataaat gaaggagagt
agttttattc tgaaggtaat 2555taaatttaga ctatgtagta tgtgacagaa
tttttttaaa attataaaaa gattttattt 2615agtaattggg atttacttaa
aataattttg gaataatgct cccagacttg cccagatttg 2675tgtattgtac
ttattgccac tggccgccac tttgacttat tttctctaat agtttatttg
2735ccacagtctt tattttgaat atgctcctag ttttttttta gggtgctgtt
cattatgaag 2795gcttctttat agaggcctaa taagaatgcc tttttataaa
gcctgtgcat ttaggtaggt 2855tgaagctagg aggattttct ttagaatgct
cttttgcatg taaagcacaa agtatgtttc 2915agtttaaatg cacttcttcc
ggttaatttt tatggggaag acaagtgagt cacaaacatt 2975ctgttgaagg
gaaatctagt cagttgcttg aaagagcaca gcccaaataa aacaaggact
3035gactaggtgt aatgaaataa cctgtgattt aaaagaagag ctgcagcttt
gacagtgctt 3095atttaaagaa aaatactgct ggaaaatttc caatttctac
tacgttcacc atctctagta 3155agatctgaca tatgctgaag ttatgttttg
atttggcaca cagcatgttc aatgatggtt 3215actcgcctag tacaagacat
ggagaagaaa cctttggaca cagagcagat gacacctcct 3275tctgttttgt
agtgtatcct ggtgtcattt tctgtgaatg tggtcaggta gagttgtttt
3335tgttgttgtt gttgggcttt tttttctttt tttttttttg gtctcttttg
gtggggtggg 3395ggtgggctaa agccatagga agaaaaatgt gatgtgtcca
gtatgtacta ttttgttttt 3455gttttgcaag aagagttgaa ctatttttga
taacaagagt aaatggtgga aaatgcttct 3515tagttgtctt gtctttattt
gctttccaag atttggaatt ttatttaatt cctttaagtg 3575ttagcagtgt
cttatgaaac atgtatttac ctaacgtttg taacagtttt gtgttgaacc
3635cagatgccct gctatataaa gttgtaaatc tgttctttat tcactaatga
tcactgcaaa 3695aatgattaga aatgagattg tacacatgga tgaggatata
ttttgcaaat cgaccaaact 3755ttcctaatat tatgatctta aaattcatag
agtactttat tgcttcccaa gtttgataat 3815cttgtgggtt tttttttttt
ttgatgcatg ggaggttggc aatatagaca aagtggaaat 3875cattagtatg
tgagggcctt gattgttatg taatattgcc aatgatgaat tcaggttgtt
3935tttagcacaa gtttctcttt tttatgctgg tattctcact gccacatttt
tggaaacctg 3995tattacacct taaatctatc aataaatgat agttttctaa ttct
40394595PRTHomo sapien 4Val Gly Ser Gly Ile Ser Phe Gln Pro Gly Ala
Ile Gly Val Glu Val1 5 10 15Ser Ala Met Asn Pro Val Asn Ala Thr Ala
Leu Tyr Ile Ser Ala Ser 20 25 30Arg Leu Val Leu Asn Tyr Asp Pro Gly
Asp Pro Lys Ala Phe Thr Glu 35 40 45Ile Asn Arg Leu Leu Pro Tyr Phe
Arg Gln Ser Leu Ser Cys Cys Val 50 55 60Cys Gly His Leu Leu Gln Asp
Pro Ile Ala Pro Thr Asn Ser Thr Cys65 70 75 80Gln His Tyr Val Cys
Lys Thr Cys Lys Gly Lys Lys Met Met Met Lys 85 90 95Pro Ser Cys Ser
Trp Cys Lys Asp Tyr Glu Gln Phe Glu Glu Asn Lys 100 105 110Gln Leu
Ser Ile Leu Val Asn Cys Tyr Lys Lys Leu Cys Glu Tyr Ile 115 120
125Thr Gln Thr Thr Leu Ala Arg Asp Ile Ile Glu Ala Val Asp Cys Ser
130 135 140Ser Asp Ile Leu Ala Leu Leu Asn Asp Gly Ser Leu Phe Cys
Glu Glu145 150 155 160Thr Glu Lys Pro Ser Asp Ser Ser Phe Thr Leu
Cys Leu Thr His Ser 165 170 175Pro Leu Pro Ser Thr Ser Glu Pro Thr
Thr Asp Pro Gln Ala Ser Leu 180 185 190Ser Pro Met Ser Glu Ser Thr
Leu Ser Ile Ala Ile Gly Ser Ser Val 195 200 205Ile Asn Gly Leu Pro
Thr Tyr Asn Gly Leu Ser Ile Asp Arg Phe Gly 210 215 220Ile Asn Ile
Pro Ser Pro Glu His Ser Asn Thr Ile Asp Val Cys Asn225 230 235
240Thr Val Asp Ile Lys Thr Glu Asp Leu Ser Asp Ser Leu Pro Pro Val
245 250 255Cys Asp Thr Val Ala Thr Asp Leu Cys Ser Thr Gly Ile Asp
Ile Cys 260 265 270Ser Phe Ser Glu Asp Ile Lys Pro Gly Asp Ser Leu
Leu Leu Ser Val 275 280 285Glu Glu Val Leu Arg Ser Leu Glu Thr Val
Ser Asn Thr Glu Val Cys 290 295 300Cys Pro Asn Leu Gln Pro Asn Leu
Glu Ala Thr Val Ser Asn Gly Pro305 310 315
320Phe Leu Gln Leu Ser Ser Gln Ser Leu Ser His Asn Val Phe Met Ser
325 330 335Thr Ser Pro Ala Leu His Gly Leu Ser Cys Thr Ala Ala Thr
Pro Lys 340 345 350Ile Ala Lys Leu Asn Arg Lys Arg Ser Arg Ser Glu
Ser Asp Ser Glu 355 360 365Lys Val Gln Pro Leu Pro Ile Ser Thr Ile
Ile Arg Gly Pro Thr Leu 370 375 380Gly Ala Ser Ala Pro Val Thr Val
Lys Arg Glu Ser Lys Ile Ser Leu385 390 395 400Gln Pro Ile Ala Thr
Val Pro Asn Gly Gly Thr Thr Pro Lys Ile Ser 405 410 415Lys Thr Val
Leu Leu Ser Thr Lys Ser Met Lys Lys Ser His Glu His 420 425 430Gly
Ser Lys Lys Ser His Ser Lys Thr Lys Pro Gly Ile Leu Lys Lys 435 440
445Asp Lys Ala Val Lys Glu Lys Ile Pro Ser His His Phe Met Pro Gly
450 455 460Ser Pro Thr Lys Thr Val Tyr Lys Lys Pro Gln Glu Lys Lys
Gly Cys465 470 475 480Lys Cys Gly Arg Ala Thr Gln Asn Pro Ser Val
Leu Thr Cys Arg Gly 485 490 495Gln Arg Cys Pro Cys Tyr Ser Asn Arg
Lys Ala Cys Leu Asp Cys Ile 500 505 510Cys Arg Gly Cys Gln Asn Ser
Tyr Met Ala Asn Gly Glu Lys Lys Leu 515 520 525Glu Ala Phe Ala Val
Pro Glu Lys Ala Leu Glu Gln Thr Arg Leu Thr 530 535 540Leu Gly Ile
Asn Val Thr Ser Ile Ala Val Arg Asn Ala Ser Thr Ser545 550 555
560Thr Ser Val Ile Asn Val Thr Gly Ser Pro Val Thr Thr Phe Leu Ala
565 570 575Ala Ser Thr His Asp Asp Lys Ser Leu Asp Glu Ala Ile Asp
Met Arg 580 585 590Phe Asp Cys 59552611DNAHomo
sapienCDS(21)...(473) 5cttttttcac ctcgtctgaa atg gct gcc tcc cag
tgt ctc tgc tgc tca aaa 53 Met Ala Ala Ser Gln Cys Leu Cys Cys Ser
Lys 1 5 10ttt ctc ttc cag aga cag aac ctc gcc tgt ttc ctc aca aac
cca cac 101Phe Leu Phe Gln Arg Gln Asn Leu Ala Cys Phe Leu Thr Asn
Pro His 15 20 25tgt ggc agc ctt gtt aat gca gat ggc cat ggt gaa gtg
tgg aca gac 149Cys Gly Ser Leu Val Asn Ala Asp Gly His Gly Glu Val
Trp Thr Asp 30 35 40tgg aat aat atg tcc aag ttt ttc cag tat gga tgg
cga tgc acc act 197Trp Asn Asn Met Ser Lys Phe Phe Gln Tyr Gly Trp
Arg Cys Thr Thr 45 50 55aat gag aat acc tat tca aac cgt acc ctg atg
ggc aac tgg aac cag 245Asn Glu Asn Thr Tyr Ser Asn Arg Thr Leu Met
Gly Asn Trp Asn Gln60 65 70 75gaa aga tat gac ctg agg aat atc gtg
cag ccc aaa ccc ttg cct tcc 293Glu Arg Tyr Asp Leu Arg Asn Ile Val
Gln Pro Lys Pro Leu Pro Ser 80 85 90cag ttt gga cac tac ttt gaa aca
aca tat gat aca agc tac aac aac 341Gln Phe Gly His Tyr Phe Glu Thr
Thr Tyr Asp Thr Ser Tyr Asn Asn 95 100 105aaa atg cca ctt tca aca
cat aga ttt aag cga gag cct cac tgg ttc 389Lys Met Pro Leu Ser Thr
His Arg Phe Lys Arg Glu Pro His Trp Phe 110 115 120cca gga cat caa
cct gaa ctg gat cct ccc cga tac aaa tgc aca gaa 437Pro Gly His Gln
Pro Glu Leu Asp Pro Pro Arg Tyr Lys Cys Thr Glu 125 130 135aag tca
act tac atg aat agc tat tca aag cct taa attgggcatc 483Lys Ser Thr
Tyr Met Asn Ser Tyr Ser Lys Pro *140 145 150actcaggatg tgtataagat
cttaatattg atagtttcac atccaggttt ctaagaaatg 543ataagatact
tcacttttcc agagtgaaat gtaggaggga gcacattcta agtacagcta
603aaaatttagc tcactgtaac acagtttcac tctctgaata aataaagcaa
aaaacacagt 663aaatattctt tatccctttt tttgttgttg ttttaaccaa
gatttaaatg tcaaatttaa 723tacagcaact cagttctaca tttggggtgt
tgtagaaggg ccttaaaaag aattatttta 783ggccaggcac ggtggctcat
gcctgtaatc ccagcacttt gggaggccga ggcaggtgga 843tcacgtgagg
tcaggagttc gagaccagcc tgaccaacat ggtgaaacac tgtctctact
903aaaaacacaa aaattagctg agcatggtgg ctcacgcctg taatcccagc
tactcaggag 963gctgaggcag gggaatcgct tgaacctgag aagtggaggt
tgtggtgagc tgagatcatg 1023ccactgcact ttagcctggg tgacagagcg
agactctgtc tcaaaaaaaa aaaaaaaaaa 1083agaattattt ctctgaagtc
tacaaccact gtggtcttcc cttccttctg tcgtagcaag 1143acctcagaat
ctagcataac ttaggctagg tttggctaga tgctttctgg gtataagcca
1203gagtcgtata gtgcaacttt gctgtgacct tagtgaacat cccctcttga
ggactacaaa 1263aacaaacgta actttttaaa attattatgg agaattttac
gtaaaacaaa agtagacagg 1323ctagtctaat gaactcccat gtatcattac
ccagcatcaa ctatttatga ctaatcttac 1383ctacttctac tttgtcttat
tgaattaatt ttggagcaga tcttagaaat agaatttaat 1443ctataaaaat
cttggtgggc tgggtacggt ggctcatgcc tgtaatccca gcactttggg
1503aggctgaggt gggtggatca cctgaggtca ggagtccaag accagcctgg
ccaatgtggt 1563gaaactccat ctcttctaaa aatacaaaaa ttagctggtc
ttggtggcgg gcgcctgtaa 1623tcccagctac ttgggaggct gaggcaggag
aattgcttga acccaggaag cagaggttgc 1683agtgagctga gacggtgcca
ttgctctcca gcctgggcga caagagcgaa actccgtctc 1743aaaaaaaaaa
aagaaaagaa aaagaaaaaa aaaatcttgg tatactggct gggcacagtg
1803gctcacacct aatcccagca ctttgggagg ctgaggcagg aggatagctt
gaggctggga 1863gttcaaaacc agcctgggca acatagcaag accccatctc
taccaaaaaa aattttttta 1923aagatttcag gtatatttct caaaaagata
aggactgtca attgtctact cccccccaac 1983aaaggtcact aaggaaacct
gttgactaaa caaagctcat taaacctatt gtagtgtagc 2043aaaggagacc
atcaacttga cacagagtct tggtaatgat tcaaagggag gatgttagag
2103taaggtattt ataaggattt gagataaggg tccaactggt ttaaaatgag
tcaaaatagg 2163gaactagtag agactgagaa agggttgtga atagcttagg
tttggtaaac ttaggaaatc 2223aacagtttta attttaatat ggttaaactg
attagtattt cctatttttt tatctactgt 2283gtaagaagac cctataatat
atgggcatta ctgagagata ctgcccatat gttgtcctcg 2343taagcaagga
gatatttttt atctcccata tattaccttt caaacctttg ttactttagt
2403ttcgagatat agatccagtt tatgttgtta ctcagtagtg aggaagtttc
tttttttttt 2463ttaaatggct atcaagttgt ccccccatta gttattgaaa
agaccataat tttttcactc 2523ctattcaatg ccatttttat tgtaaataaa
ctatgtacat gtaaaaaaaa aaaaaaaaaa 2583aaaaaaaaaa aaaaaaaaaa aaaaaaaa
26116150PRTHomo sapien 6Met Ala Ala Ser Gln Cys Leu Cys Cys Ser Lys
Phe Leu Phe Gln Arg1 5 10 15Gln Asn Leu Ala Cys Phe Leu Thr Asn Pro
His Cys Gly Ser Leu Val 20 25 30Asn Ala Asp Gly His Gly Glu Val Trp
Thr Asp Trp Asn Asn Met Ser 35 40 45Lys Phe Phe Gln Tyr Gly Trp Arg
Cys Thr Thr Asn Glu Asn Thr Tyr 50 55 60Ser Asn Arg Thr Leu Met Gly
Asn Trp Asn Gln Glu Arg Tyr Asp Leu65 70 75 80Arg Asn Ile Val Gln
Pro Lys Pro Leu Pro Ser Gln Phe Gly His Tyr 85 90 95Phe Glu Thr Thr
Tyr Asp Thr Ser Tyr Asn Asn Lys Met Pro Leu Ser 100 105 110Thr His
Arg Phe Lys Arg Glu Pro His Trp Phe Pro Gly His Gln Pro 115 120
125Glu Leu Asp Pro Pro Arg Tyr Lys Cys Thr Glu Lys Ser Thr Tyr Met
130 135 140Asn Ser Tyr Ser Lys Pro145 15073296DNAHomo
sapienCDS(65)...(1735) 7cggaggaggc gaggagcgcc gggtaccggg ccgggggagc
cgcgggctct cggggaagag 60acgg atg atg aac aag ctt tac atc ggg aac
ctg agc ccc gcc gtc acc 109 Met Met Asn Lys Leu Tyr Ile Gly Asn Leu
Ser Pro Ala Val Thr 1 5 10 15gcc gac gac ctc cgg cag ctc ttt ggg
gac agg aag ctg ccc ctg gcg 157Ala Asp Asp Leu Arg Gln Leu Phe Gly
Asp Arg Lys Leu Pro Leu Ala 20 25 30gga cag gtc ctg ctg aag tcc ggc
tac gcc ttc gtg gac tac ccc gac 205Gly Gln Val Leu Leu Lys Ser Gly
Tyr Ala Phe Val Asp Tyr Pro Asp 35 40 45cag aac tgg gcc atc cgc gcc
atc gag acc ctc tcg ggt aaa gtg gaa 253Gln Asn Trp Ala Ile Arg Ala
Ile Glu Thr Leu Ser Gly Lys Val Glu 50 55 60ttg cat ggg aaa atc atg
gaa gtt gat tac tca gtc tct aaa aag cta 301Leu His Gly Lys Ile Met
Glu Val Asp Tyr Ser Val Ser Lys Lys Leu 65 70 75agg agc agg aaa att
cag att cga aac atc cct cct cac ctg cag tgg 349Arg Ser Arg Lys Ile
Gln Ile Arg Asn Ile Pro Pro His Leu Gln Trp80 85 90 95gag gtg ttg
gat gga ctt ttg gct caa tat ggg aca gtg gag aat gtg 397Glu Val Leu
Asp Gly Leu Leu Ala Gln Tyr Gly Thr Val Glu Asn Val 100 105 110gaa
caa gtc aac aca gac aca gaa acc gcc gtt gtc aac gtc aca tat 445Glu
Gln Val Asn Thr Asp Thr Glu Thr Ala Val Val Asn Val Thr Tyr 115 120
125gca aca aga gaa gaa gca aaa ata gcc atg gag aag cta agc ggg cat
493Ala Thr Arg Glu Glu Ala Lys Ile Ala Met Glu Lys Leu Ser Gly His
130 135 140cag ttt gag aac tac tcc ttc aag att tcc tac atc ccg gat
gaa gag 541Gln Phe Glu Asn Tyr Ser Phe Lys Ile Ser Tyr Ile Pro Asp
Glu Glu 145 150 155gtg agc tcc cct tcg ccc cct cag cga gcc cag cgt
ggg gac cac tct 589Val Ser Ser Pro Ser Pro Pro Gln Arg Ala Gln Arg
Gly Asp His Ser160 165 170 175tcc cgg gag caa ggc cac gcc cct ggg
ggc act tct cag gcc aga cag 637Ser Arg Glu Gln Gly His Ala Pro Gly
Gly Thr Ser Gln Ala Arg Gln 180 185 190att gat ttc ccg ctg cgg atc
ctg gtc ccc acc cag ttt gtt ggt gcc 685Ile Asp Phe Pro Leu Arg Ile
Leu Val Pro Thr Gln Phe Val Gly Ala 195 200 205atc atc gga aag gag
ggc ttg acc ata aag aac atc act aag cag acc 733Ile Ile Gly Lys Glu
Gly Leu Thr Ile Lys Asn Ile Thr Lys Gln Thr 210 215 220cag tcc cgg
gta gat atc cat aga aaa gag aac tct gga gct gca gag 781Gln Ser Arg
Val Asp Ile His Arg Lys Glu Asn Ser Gly Ala Ala Glu 225 230 235aag
cct gtc acc atc cat gcc acc cca gag ggg act tct gaa gca tgc 829Lys
Pro Val Thr Ile His Ala Thr Pro Glu Gly Thr Ser Glu Ala Cys240 245
250 255cgc atg att ctt gaa atc atg cag aaa gag gca gat gag acc aaa
cta 877Arg Met Ile Leu Glu Ile Met Gln Lys Glu Ala Asp Glu Thr Lys
Leu 260 265 270gcc gaa gag att cct ctg aaa atc ttg gca cac aat ggc
ttg gtt gga 925Ala Glu Glu Ile Pro Leu Lys Ile Leu Ala His Asn Gly
Leu Val Gly 275 280 285aga ctg att gga aaa gaa ggc aga aat ttg aag
aaa att gaa cat gaa 973Arg Leu Ile Gly Lys Glu Gly Arg Asn Leu Lys
Lys Ile Glu His Glu 290 295 300aca ggg acc aag ata aca atc tca tct
ttg cag gat ttg agc ata tac 1021Thr Gly Thr Lys Ile Thr Ile Ser Ser
Leu Gln Asp Leu Ser Ile Tyr 305 310 315aac ccg gaa aga acc atc act
gtg aag ggc aca gtt gag gcc tgt gcc 1069Asn Pro Glu Arg Thr Ile Thr
Val Lys Gly Thr Val Glu Ala Cys Ala320 325 330 335agt gct gag ata
gag att atg aag aag ctg cgt gag gcc ttt gaa aat 1117Ser Ala Glu Ile
Glu Ile Met Lys Lys Leu Arg Glu Ala Phe Glu Asn 340 345 350gat atg
ctg gct gtt aac acc cac tcc gga tac ttc tcc agc ctg tac 1165Asp Met
Leu Ala Val Asn Thr His Ser Gly Tyr Phe Ser Ser Leu Tyr 355 360
365ccc cat cac cag ttt ggc ccg ttc ccg cat cat cac tct tat cca gag
1213Pro His His Gln Phe Gly Pro Phe Pro His His His Ser Tyr Pro Glu
370 375 380cag gag att gtg aat ctc ttc atc cca acc cag gct gtg ggc
gcc atc 1261Gln Glu Ile Val Asn Leu Phe Ile Pro Thr Gln Ala Val Gly
Ala Ile 385 390 395atc ggg aag aag ggg gca cac atc aaa cag ctg gcg
aga ttc gcc gga 1309Ile Gly Lys Lys Gly Ala His Ile Lys Gln Leu Ala
Arg Phe Ala Gly400 405 410 415gcc tct atc aag att gcc cct gcg gaa
ggc cca gac gtc agc gaa agg 1357Ala Ser Ile Lys Ile Ala Pro Ala Glu
Gly Pro Asp Val Ser Glu Arg 420 425 430atg gtc atc atc acc ggg cca
ccg gaa gcc cag ttc aag gcc cag gga 1405Met Val Ile Ile Thr Gly Pro
Pro Glu Ala Gln Phe Lys Ala Gln Gly 435 440 445cgg atc ttt ggg aaa
ctg aaa gag gaa aac ttc ttt aac ccc aaa gaa 1453Arg Ile Phe Gly Lys
Leu Lys Glu Glu Asn Phe Phe Asn Pro Lys Glu 450 455 460gaa gtg aag
ctg gaa gcg cat atc aga gtg ccc tct tcc aca gct ggc 1501Glu Val Lys
Leu Glu Ala His Ile Arg Val Pro Ser Ser Thr Ala Gly 465 470 475cgg
gtg att ggc aaa ggt ggc aag acc gtg aac gaa ctg cag aac tta 1549Arg
Val Ile Gly Lys Gly Gly Lys Thr Val Asn Glu Leu Gln Asn Leu480 485
490 495acc agt gca gaa gtc atc gtg cct cgt gac caa acg cca gat gaa
aat 1597Thr Ser Ala Glu Val Ile Val Pro Arg Asp Gln Thr Pro Asp Glu
Asn 500 505 510gag gaa gtg atc gtc aga att atc ggg cac ttc ttt gct
agc cag act 1645Glu Glu Val Ile Val Arg Ile Ile Gly His Phe Phe Ala
Ser Gln Thr 515 520 525gca cag cgc aag atc agg gaa att gta caa cag
gtg aag cag cag gag 1693Ala Gln Arg Lys Ile Arg Glu Ile Val Gln Gln
Val Lys Gln Gln Glu 530 535 540cag aaa tac cct cag gga gtc gcc tca
cag cgc agc aag tga 1735Gln Lys Tyr Pro Gln Gly Val Ala Ser Gln Arg
Ser Lys * 545 550 555ggctcccaca ggcaccagca aaacaacgga tgaatgtagc
ccttccaaca cctgacagaa 1795tgagaccaaa cgcagccagc cagatcggga
gcaaaccaaa gaccatctga ggaatgagaa 1855gtctgcggag gcggccaggg
actctgccga ggccctgaga accccagggg ccgaggaggg 1915gcggggaagg
tcagccaggt ttgccagaac caccgagccc cgcctcccgc cccccagggc
1975ttctgcaggc ttcagccatc cacttcacca tccactcgga tctctcctga
actcccacga 2035cgctatccct tttagttgaa ctaacatagg tgaacgtgtt
caaagccaag caaaatgcac 2095accctttttc tgtggcaaat cgtctctgta
catgtgtgta catattagaa agggaagatg 2155ttaagatatg tggcctgtgg
gttacacagg gtgcctgcag cggtaatata ttttagaaat 2215aatatatcaa
ataactcaac taactccaat ttttaatcaa ttattaattt ttttttcttt
2275ttaaagagaa agcaggcttt tctagacttt aaagaataaa gtctttggga
ggtctcacgg 2335tgtagagagg agctttgagg ccacccgcac aaaattcacc
cagagggaaa tctcgtcgga 2395aggacactca cggcagttct ggatcacctg
tgtatgtcaa cagaagggat accgtctcct 2455tgaagaggaa actctgtcac
tcctcatgcc tgtctagctc atacacccat ttctctttgc 2515ttcacaggtt
ttaaactggt tttttgcata ctgctatata attctctgtc tctctctgtt
2575tatctctccc ctccctcccc tccccttctt ctccatctcc attcttttga
atttcctcat 2635ccctccatct caatcccgta tctacgcacc cccccccccc
aggcaaagca gtgctctgag 2695tatcacatca cacaaaagga acaaaagcga
aacacacaaa ccagcctcaa cttacacttg 2755gttactcaaa agaacaagag
tcaatggtac ttgtcctagc gttttggaag aggaaaacag 2815gaacccacca
aaccaaccaa tcaaccaaac aaagaaaaaa ttccacaatg aaagaatgta
2875ttttgtcttt ttgcattttg gtgtataagc catcaatatt cagcaaaatg
attcctttct 2935ttaaaaaaaa aaaatgtgga ggaaagtaga aatttaccaa
ggttgttggc ccagggcgtt 2995aaattcacag atttttttaa cgagaaaaac
acacagaaga agctacctca ggtgttttta 3055cctcagcacc ttgatcttgt
gtttccctta gagattttgt aaagctgata gttggagcat 3115ttttttattt
ttttaataaa aatgagttgg aaaaaaaata agatatcaac tgccagcctg
3175gagaaggtga cagtccaagt gtgcaacagc tgttctgaat tgtcttccgc
tagccaagaa 3235cctatatggc cttcttttgg acaaaccttg aaaatgttta
tttaaaaaaa aaaaaaaaaa 3295a 32968556PRTHomo sapien 8Met Met Asn Lys
Leu Tyr Ile Gly Asn Leu Ser Pro Ala Val Thr Ala 1 5 10 15Asp Asp
Leu Arg Gln Leu Phe Gly Asp Arg Lys Leu Pro Leu Ala Gly 20 25 30Gln
Val Leu Leu Lys Ser Gly Tyr Ala Phe Val Asp Tyr Pro Asp Gln 35 40
45Asn Trp Ala Ile Arg Ala Ile Glu Thr Leu Ser Gly Lys Val Glu Leu
50 55 60His Gly Lys Ile Met Glu Val Asp Tyr Ser Val Ser Lys Lys Leu
Arg65 70 75 80Ser Arg Lys Ile Gln Ile Arg Asn Ile Pro Pro His Leu
Gln Trp Glu 85 90 95Val Leu Asp Gly Leu Leu Ala Gln Tyr Gly Thr Val
Glu Asn Val Glu 100 105 110Gln Val Asn Thr Asp Thr Glu Thr Ala Val
Val Asn Val Thr Tyr Ala 115 120 125Thr Arg Glu Glu Ala Lys Ile Ala
Met Glu Lys Leu Ser Gly His Gln 130 135 140Phe Glu Asn Tyr Ser Phe
Lys Ile Ser Tyr Ile Pro Asp Glu Glu Val145 150 155 160Ser Ser Pro
Ser Pro Pro Gln Arg Ala Gln Arg Gly Asp His Ser Ser 165 170 175Arg
Glu Gln Gly His Ala Pro Gly Gly Thr Ser Gln Ala Arg Gln Ile 180 185
190Asp Phe Pro Leu Arg Ile Leu Val Pro Thr Gln Phe Val Gly Ala Ile
195 200 205Ile Gly Lys Glu Gly Leu Thr Ile Lys Asn Ile Thr Lys Gln
Thr Gln 210 215 220Ser Arg Val Asp Ile His Arg Lys Glu Asn Ser Gly
Ala Ala Glu Lys225 230 235 240Pro Val Thr Ile His Ala Thr Pro Glu
Gly Thr Ser Glu Ala Cys Arg 245
250 255Met Ile Leu Glu Ile Met Gln Lys Glu Ala Asp Glu Thr Lys Leu
Ala 260 265 270Glu Glu Ile Pro Leu Lys Ile Leu Ala His Asn Gly Leu
Val Gly Arg 275 280 285Leu Ile Gly Lys Glu Gly Arg Asn Leu Lys Lys
Ile Glu His Glu Thr 290 295 300Gly Thr Lys Ile Thr Ile Ser Ser Leu
Gln Asp Leu Ser Ile Tyr Asn305 310 315 320Pro Glu Arg Thr Ile Thr
Val Lys Gly Thr Val Glu Ala Cys Ala Ser 325 330 335Ala Glu Ile Glu
Ile Met Lys Lys Leu Arg Glu Ala Phe Glu Asn Asp 340 345 350Met Leu
Ala Val Asn Thr His Ser Gly Tyr Phe Ser Ser Leu Tyr Pro 355 360
365His His Gln Phe Gly Pro Phe Pro His His His Ser Tyr Pro Glu Gln
370 375 380Glu Ile Val Asn Leu Phe Ile Pro Thr Gln Ala Val Gly Ala
Ile Ile385 390 395 400Gly Lys Lys Gly Ala His Ile Lys Gln Leu Ala
Arg Phe Ala Gly Ala 405 410 415Ser Ile Lys Ile Ala Pro Ala Glu Gly
Pro Asp Val Ser Glu Arg Met 420 425 430Val Ile Ile Thr Gly Pro Pro
Glu Ala Gln Phe Lys Ala Gln Gly Arg 435 440 445Ile Phe Gly Lys Leu
Lys Glu Glu Asn Phe Phe Asn Pro Lys Glu Glu 450 455 460Val Lys Leu
Glu Ala His Ile Arg Val Pro Ser Ser Thr Ala Gly Arg465 470 475
480Val Ile Gly Lys Gly Gly Lys Thr Val Asn Glu Leu Gln Asn Leu Thr
485 490 495Ser Ala Glu Val Ile Val Pro Arg Asp Gln Thr Pro Asp Glu
Asn Glu 500 505 510Glu Val Ile Val Arg Ile Ile Gly His Phe Phe Ala
Ser Gln Thr Ala 515 520 525Gln Arg Lys Ile Arg Glu Ile Val Gln Gln
Val Lys Gln Gln Glu Gln 530 535 540Lys Tyr Pro Gln Gly Val Ala Ser
Gln Arg Ser Lys545 550 55593164DNAHomo sapienCDS(65)...(1603)
9cggaggaggc gaggagcgcc gggtaccggg ccgggggagc cgcgggctct cggggaagag
60acgg atg atg aac aag ctt tac atc ggg aac ctg agc ccc gcc gtc acc
109 Met Met Asn Lys Leu Tyr Ile Gly Asn Leu Ser Pro Ala Val Thr 1 5
10 15gcc gac gac ctc cgg cag ctc ttt ggg gac agg aag ctg ccc ctg
gcg 157Ala Asp Asp Leu Arg Gln Leu Phe Gly Asp Arg Lys Leu Pro Leu
Ala 20 25 30gga cag gtc ctg ctg aag tcc ggc tac gcc ttc gtg gac tac
ccc gac 205Gly Gln Val Leu Leu Lys Ser Gly Tyr Ala Phe Val Asp Tyr
Pro Asp 35 40 45cag aac tgg gcc atc cgc gcc atc gag acc ctc tcg ggt
aaa gtg gaa 253Gln Asn Trp Ala Ile Arg Ala Ile Glu Thr Leu Ser Gly
Lys Val Glu 50 55 60ttg cat ggg aaa atc atg gaa gtt gat tac tca gtc
tct aaa aag cta 301Leu His Gly Lys Ile Met Glu Val Asp Tyr Ser Val
Ser Lys Lys Leu 65 70 75agg agc agg aaa att cag att cga aac atc cct
cct cac ctg cag tgg 349Arg Ser Arg Lys Ile Gln Ile Arg Asn Ile Pro
Pro His Leu Gln Trp80 85 90 95gag gtg ttg gat gga ctt ttg gct caa
tat ggg aca gtg gag aat gtg 397Glu Val Leu Asp Gly Leu Leu Ala Gln
Tyr Gly Thr Val Glu Asn Val 100 105 110gaa caa gtc aac aca gac aca
gaa acc gcc gtt gtc aac gtc aca tat 445Glu Gln Val Asn Thr Asp Thr
Glu Thr Ala Val Val Asn Val Thr Tyr 115 120 125gca aca aga gaa gaa
gca aaa ata gcc atg gag aag cta agc ggg cat 493Ala Thr Arg Glu Glu
Ala Lys Ile Ala Met Glu Lys Leu Ser Gly His 130 135 140cag ttt gag
aac tac tcc ttc aag att tcc tac atc ccg gat gaa gag 541Gln Phe Glu
Asn Tyr Ser Phe Lys Ile Ser Tyr Ile Pro Asp Glu Glu 145 150 155ttt
gtt ggt gcc atc atc gga aag gag ggc ttg acc ata aag aac atc 589Phe
Val Gly Ala Ile Ile Gly Lys Glu Gly Leu Thr Ile Lys Asn Ile160 165
170 175act aag cag acc cag tcc cgg gta gat atc cat aga aaa gag aac
tct 637Thr Lys Gln Thr Gln Ser Arg Val Asp Ile His Arg Lys Glu Asn
Ser 180 185 190gga gct gca gag aag cct gtc acc atc cat gcc acc cca
gag ggg act 685Gly Ala Ala Glu Lys Pro Val Thr Ile His Ala Thr Pro
Glu Gly Thr 195 200 205tct gaa gca tgc cgc atg att ctt gaa atc atg
cag aaa gag gca gat 733Ser Glu Ala Cys Arg Met Ile Leu Glu Ile Met
Gln Lys Glu Ala Asp 210 215 220gag acc aaa cta gcc gaa gag att cct
ctg aaa atc ttg gca cac aat 781Glu Thr Lys Leu Ala Glu Glu Ile Pro
Leu Lys Ile Leu Ala His Asn 225 230 235ggc ttg gtt gga aga ctg att
gga aaa gaa ggc aga aat ttg aag aaa 829Gly Leu Val Gly Arg Leu Ile
Gly Lys Glu Gly Arg Asn Leu Lys Lys240 245 250 255att gaa cat gaa
aca ggg acc aag ata aca atc tca tct ttg cag gat 877Ile Glu His Glu
Thr Gly Thr Lys Ile Thr Ile Ser Ser Leu Gln Asp 260 265 270ttg agc
ata tac aac ccg gaa aga acc atc act gtg aag ggc aca gtt 925Leu Ser
Ile Tyr Asn Pro Glu Arg Thr Ile Thr Val Lys Gly Thr Val 275 280
285gag gcc tgt gcc agt gct gag ata gag att atg aag aag ctg cgt gag
973Glu Ala Cys Ala Ser Ala Glu Ile Glu Ile Met Lys Lys Leu Arg Glu
290 295 300gcc ttt gaa aat gat atg ctg gct gtt aac acc cac tcc gga
tac ttc 1021Ala Phe Glu Asn Asp Met Leu Ala Val Asn Thr His Ser Gly
Tyr Phe 305 310 315tcc agc ctg tac ccc cat cac cag ttt ggc ccg ttc
ccg cat cat cac 1069Ser Ser Leu Tyr Pro His His Gln Phe Gly Pro Phe
Pro His His His320 325 330 335tct tat cca gag cag gag att gtg aat
ctc ttc atc cca acc cag gct 1117Ser Tyr Pro Glu Gln Glu Ile Val Asn
Leu Phe Ile Pro Thr Gln Ala 340 345 350gtg ggc gcc atc atc ggg aag
aag ggg gca cac atc aaa cag ctg gcg 1165Val Gly Ala Ile Ile Gly Lys
Lys Gly Ala His Ile Lys Gln Leu Ala 355 360 365aga ttc gcc gga gcc
tct atc aag att gcc cct gcg gaa ggc cca gac 1213Arg Phe Ala Gly Ala
Ser Ile Lys Ile Ala Pro Ala Glu Gly Pro Asp 370 375 380gtc agc gaa
agg atg gtc atc atc acc ggg cca ccg gaa gcc cag ttc 1261Val Ser Glu
Arg Met Val Ile Ile Thr Gly Pro Pro Glu Ala Gln Phe 385 390 395aag
gcc cag gga cgg atc ttt ggg aaa ctg aaa gag gaa aac ttc ttt 1309Lys
Ala Gln Gly Arg Ile Phe Gly Lys Leu Lys Glu Glu Asn Phe Phe400 405
410 415aac ccc aaa gaa gaa gtg aag ctg gaa gcg cat atc aga gtg ccc
tct 1357Asn Pro Lys Glu Glu Val Lys Leu Glu Ala His Ile Arg Val Pro
Ser 420 425 430tcc aca gct ggc cgg gtg att ggc aaa ggt ggc aag acc
gtg aac gaa 1405Ser Thr Ala Gly Arg Val Ile Gly Lys Gly Gly Lys Thr
Val Asn Glu 435 440 445ctg cag aac tta acc agt gca gaa gtc atc gtg
cct cgt gac caa acg 1453Leu Gln Asn Leu Thr Ser Ala Glu Val Ile Val
Pro Arg Asp Gln Thr 450 455 460cca gat gaa aat gag gaa gtg atc gtc
aga att atc ggg cac ttc ttt 1501Pro Asp Glu Asn Glu Glu Val Ile Val
Arg Ile Ile Gly His Phe Phe 465 470 475gct agc cag act gca cag cgc
aag atc agg gaa att gta caa cag gtg 1549Ala Ser Gln Thr Ala Gln Arg
Lys Ile Arg Glu Ile Val Gln Gln Val480 485 490 495aag cag cag gag
cag aaa tac cct cag gga gtc gcc tca cag cgc agc 1597Lys Gln Gln Glu
Gln Lys Tyr Pro Gln Gly Val Ala Ser Gln Arg Ser 500 505 510aag tga
ggctcccaca ggcaccagca aaacaacgga tgaatgtagc ccttccaaca 1653Lys
*cctgacagaa tgagaccaaa cgcagccagc cagatcggga gcaaaccaaa gaccatctga
1713ggaatgagaa gtctgcggag gcggccaggg actctgccga ggccctgaga
accccagggg 1773ccgaggaggg gcggggaagg tcagccaggt ttgccagaac
caccgagccc cgcctcccgc 1833cccccagggc ttctgcaggc ttcagccatc
cacttcacca tccactcgga tctctcctga 1893actcccacga cgctatccct
tttagttgaa ctaacatagg tgaacgtgtt caaagccaag 1953caaaatgcac
accctttttc tgtggcaaat cgtctctgta catgtgtgta catattagaa
2013agggaagatg ttaagatatg tggcctgtgg gttacacagg gtgcctgcag
cggtaatata 2073ttttagaaat aatatatcaa ataactcaac taactccaat
ttttaatcaa ttattaattt 2133ttttttcttt ttaaagagaa agcaggcttt
tctagacttt aaagaataaa gtctttggga 2193ggtctcacgg tgtagagagg
agctttgagg ccacccgcac aaaattcacc cagagggaaa 2253tctcgtcgga
aggacactca cggcagttct ggatcacctg tgtatgtcaa cagaagggat
2313accgtctcct tgaagaggaa actctgtcac tcctcatgcc tgtctagctc
atacacccat 2373ttctctttgc ttcacaggtt ttaaactggt tttttgcata
ctgctatata attctctgtc 2433tctctctgtt tatctctccc ctccctcccc
tccccttctt ctccatctcc attcttttga 2493atttcctcat ccctccatct
caatcccgta tctacgcacc cccccccccc aggcaaagca 2553gtgctctgag
tatcacatca cacaaaagga acaaaagcga aacacacaaa ccagcctcaa
2613cttacacttg gttactcaaa agaacaagag tcaatggtac ttgtcctagc
gttttggaag 2673aggaaaacag gaacccacca aaccaaccaa tcaaccaaac
aaagaaaaaa ttccacaatg 2733aaagaatgta ttttgtcttt ttgcattttg
gtgtataagc catcaatatt cagcaaaatg 2793attcctttct ttaaaaaaaa
aaaatgtgga ggaaagtaga aatttaccaa ggttgttggc 2853ccagggcgtt
aaattcacag atttttttaa cgagaaaaac acacagaaga agctacctca
2913ggtgttttta cctcagcacc ttgatcttgt gtttccctta gagattttgt
aaagctgata 2973gttggagcat ttttttattt ttttaataaa aatgagttgg
aaaaaaaata agatatcaac 3033tgccagcctg gagaaggtga cagtccaagt
gtgcaacagc tgttctgaat tgtcttccgc 3093tagccaagaa cctatatggc
cttcttttgg acaaaccttg aaaatgttta tttaaaaaaa 3153aaaaaaaaaa a
316410512PRTHomo sapien 10Met Met Asn Lys Leu Tyr Ile Gly Asn Leu
Ser Pro Ala Val Thr Ala 1 5 10 15Asp Asp Leu Arg Gln Leu Phe Gly
Asp Arg Lys Leu Pro Leu Ala Gly 20 25 30Gln Val Leu Leu Lys Ser Gly
Tyr Ala Phe Val Asp Tyr Pro Asp Gln 35 40 45Asn Trp Ala Ile Arg Ala
Ile Glu Thr Leu Ser Gly Lys Val Glu Leu 50 55 60His Gly Lys Ile Met
Glu Val Asp Tyr Ser Val Ser Lys Lys Leu Arg65 70 75 80Ser Arg Lys
Ile Gln Ile Arg Asn Ile Pro Pro His Leu Gln Trp Glu 85 90 95Val Leu
Asp Gly Leu Leu Ala Gln Tyr Gly Thr Val Glu Asn Val Glu 100 105
110Gln Val Asn Thr Asp Thr Glu Thr Ala Val Val Asn Val Thr Tyr Ala
115 120 125Thr Arg Glu Glu Ala Lys Ile Ala Met Glu Lys Leu Ser Gly
His Gln 130 135 140Phe Glu Asn Tyr Ser Phe Lys Ile Ser Tyr Ile Pro
Asp Glu Glu Phe145 150 155 160Val Gly Ala Ile Ile Gly Lys Glu Gly
Leu Thr Ile Lys Asn Ile Thr 165 170 175Lys Gln Thr Gln Ser Arg Val
Asp Ile His Arg Lys Glu Asn Ser Gly 180 185 190Ala Ala Glu Lys Pro
Val Thr Ile His Ala Thr Pro Glu Gly Thr Ser 195 200 205Glu Ala Cys
Arg Met Ile Leu Glu Ile Met Gln Lys Glu Ala Asp Glu 210 215 220Thr
Lys Leu Ala Glu Glu Ile Pro Leu Lys Ile Leu Ala His Asn Gly225 230
235 240Leu Val Gly Arg Leu Ile Gly Lys Glu Gly Arg Asn Leu Lys Lys
Ile 245 250 255Glu His Glu Thr Gly Thr Lys Ile Thr Ile Ser Ser Leu
Gln Asp Leu 260 265 270Ser Ile Tyr Asn Pro Glu Arg Thr Ile Thr Val
Lys Gly Thr Val Glu 275 280 285Ala Cys Ala Ser Ala Glu Ile Glu Ile
Met Lys Lys Leu Arg Glu Ala 290 295 300Phe Glu Asn Asp Met Leu Ala
Val Asn Thr His Ser Gly Tyr Phe Ser305 310 315 320Ser Leu Tyr Pro
His His Gln Phe Gly Pro Phe Pro His His His Ser 325 330 335Tyr Pro
Glu Gln Glu Ile Val Asn Leu Phe Ile Pro Thr Gln Ala Val 340 345
350Gly Ala Ile Ile Gly Lys Lys Gly Ala His Ile Lys Gln Leu Ala Arg
355 360 365Phe Ala Gly Ala Ser Ile Lys Ile Ala Pro Ala Glu Gly Pro
Asp Val 370 375 380Ser Glu Arg Met Val Ile Ile Thr Gly Pro Pro Glu
Ala Gln Phe Lys385 390 395 400Ala Gln Gly Arg Ile Phe Gly Lys Leu
Lys Glu Glu Asn Phe Phe Asn 405 410 415Pro Lys Glu Glu Val Lys Leu
Glu Ala His Ile Arg Val Pro Ser Ser 420 425 430Thr Ala Gly Arg Val
Ile Gly Lys Gly Gly Lys Thr Val Asn Glu Leu 435 440 445Gln Asn Leu
Thr Ser Ala Glu Val Ile Val Pro Arg Asp Gln Thr Pro 450 455 460Asp
Glu Asn Glu Glu Val Ile Val Arg Ile Ile Gly His Phe Phe Ala465 470
475 480Ser Gln Thr Ala Gln Arg Lys Ile Arg Glu Ile Val Gln Gln Val
Lys 485 490 495Gln Gln Glu Gln Lys Tyr Pro Gln Gly Val Ala Ser Gln
Arg Ser Lys 500 505 510111580DNAHomo sapienCDS(220)...(1050)
11catcctgcca cccctagcct tgctggggac gtgaaccctc tccccgcgcc tgggaagcct
60tcttggcacc gggacccgga gaatccccac ggaagccagt tccaaaaggg atgaaaaggg
120ggcgtttcgg gcactgggag aagcctgtat tccagggccc ctcccagagc
aggaatctgg 180gacccaggag tgccagcctc acccacgcag atcctggcc atg aga
gct ccg cac 234 Met Arg Ala Pro His 1 5ctc cac ctc tcc gcc gcc tct
ggc gcc cgg gct ctg gcg aag ctg ctg 282Leu His Leu Ser Ala Ala Ser
Gly Ala Arg Ala Leu Ala Lys Leu Leu 10 15 20ccg ctg ctg atg gcg caa
ctc tgg gcc gca gag gcg gcg ctg ctc ccc 330Pro Leu Leu Met Ala Gln
Leu Trp Ala Ala Glu Ala Ala Leu Leu Pro 25 30 35caa aac gac acg cgc
ttg gac ccc gaa gcc tat ggc tcc ccg tgc gcg 378Gln Asn Asp Thr Arg
Leu Asp Pro Glu Ala Tyr Gly Ser Pro Cys Ala 40 45 50cgc ggc tcg cag
ccc tgg cag gtc tcg ctc ttc aac ggc ctc tcg ttc 426Arg Gly Ser Gln
Pro Trp Gln Val Ser Leu Phe Asn Gly Leu Ser Phe 55 60 65cac tgc gcg
ggt gtc ctg gtg gac cag agt tgg gtg ctg acg gcc gcg 474His Cys Ala
Gly Val Leu Val Asp Gln Ser Trp Val Leu Thr Ala Ala70 75 80 85cac
tgc gga aac aag cca ctg tgg gct cga gta ggg gat gac cac ctg 522His
Cys Gly Asn Lys Pro Leu Trp Ala Arg Val Gly Asp Asp His Leu 90 95
100ctg ctt ctt cag gga gag cag ctc cgc cgg acc act cgc tct gtt gtc
570Leu Leu Leu Gln Gly Glu Gln Leu Arg Arg Thr Thr Arg Ser Val Val
105 110 115cat ccc aag tac cac cag ggc tca ggc ccc atc ctg cca agg
cga acg 618His Pro Lys Tyr His Gln Gly Ser Gly Pro Ile Leu Pro Arg
Arg Thr 120 125 130gat gag cac gat ctc atg ttg ctg aag ctg gcc agg
ccc gta gtg ctg 666Asp Glu His Asp Leu Met Leu Leu Lys Leu Ala Arg
Pro Val Val Leu 135 140 145ggg ccc cgc gtc cgg gcc ctg cag ctt ccc
tac cgc tgt gct cag ccc 714Gly Pro Arg Val Arg Ala Leu Gln Leu Pro
Tyr Arg Cys Ala Gln Pro150 155 160 165gga gac cag tgc cag gtt gct
ggc tgg ggc acc acg gcc gcc cgg aga 762Gly Asp Gln Cys Gln Val Ala
Gly Trp Gly Thr Thr Ala Ala Arg Arg 170 175 180gtg aag tac aac aag
ggc ctg acc tgc tcc agc atc act atc ctg agc 810Val Lys Tyr Asn Lys
Gly Leu Thr Cys Ser Ser Ile Thr Ile Leu Ser 185 190 195cct aaa gag
tgt gag gtc ttc tac cct ggc gtg gtc acc aac aac atg 858Pro Lys Glu
Cys Glu Val Phe Tyr Pro Gly Val Val Thr Asn Asn Met 200 205 210ata
tgt gct gga ctg gac cgg ggc cag gac cct tgc cag agt gac tct 906Ile
Cys Ala Gly Leu Asp Arg Gly Gln Asp Pro Cys Gln Ser Asp Ser 215 220
225gga ggc ccc ctg gtc tgt gac gag acc ctc caa ggc atc ctc tcg tgg
954Gly Gly Pro Leu Val Cys Asp Glu Thr Leu Gln Gly Ile Leu Ser
Trp230 235 240 245ggt gtt tac ccc tgt ggc tct gcc cag cat cca gct
gtc tac acc cag 1002Gly Val Tyr Pro Cys Gly Ser Ala Gln His Pro Ala
Val Tyr Thr Gln 250 255 260atc tgc aaa tac atg tcc tgg atc aat aaa
gtc ata cgc tcc aac tga 1050Ile Cys Lys Tyr Met Ser Trp Ile Asn Lys
Val Ile Arg Ser Asn * 265 270 275tccagatgct acgctccagc tgatccagat
gttatgctcc tgctgatcca gatgcccaga 1110ggctccatcg tccatcctct
tcctccccag tcggctgaac tctccccttg tctgcactgt 1170tcaaacctct
gccgccctcc acacctctaa acatctcccc tctcacctca ttcccccacc
1230tatccccatt ctctgcctgt actgaagctg aaatgcagga agtggtggca
aaggtttatt 1290ccagagaagc caggaagccg gtcatcaccc agcctctgag
agcagttact ggggtcaccc
1350aacctgactt cctctgccac tccctgctgt gtgactttgg gcaagccaag
tgccctctct 1410gaacctcagt ttcctcatct gcaaaatggg aacaatgacg
tgcctacctc ttagacatgt 1470tgtgaggaga ctatgatata acatgtgtat
gtaaatcttc atggtgattg tcatgtaagg 1530cttaacacag tgggtggtga
gttctgacta aaggttacct gttgtcgtga 158012276PRTHomo sapien 12Met Arg
Ala Pro His Leu His Leu Ser Ala Ala Ser Gly Ala Arg Ala 1 5 10
15Leu Ala Lys Leu Leu Pro Leu Leu Met Ala Gln Leu Trp Ala Ala Glu
20 25 30Ala Ala Leu Leu Pro Gln Asn Asp Thr Arg Leu Asp Pro Glu Ala
Tyr 35 40 45Gly Ser Pro Cys Ala Arg Gly Ser Gln Pro Trp Gln Val Ser
Leu Phe 50 55 60Asn Gly Leu Ser Phe His Cys Ala Gly Val Leu Val Asp
Gln Ser Trp65 70 75 80Val Leu Thr Ala Ala His Cys Gly Asn Lys Pro
Leu Trp Ala Arg Val 85 90 95Gly Asp Asp His Leu Leu Leu Leu Gln Gly
Glu Gln Leu Arg Arg Thr 100 105 110Thr Arg Ser Val Val His Pro Lys
Tyr His Gln Gly Ser Gly Pro Ile 115 120 125Leu Pro Arg Arg Thr Asp
Glu His Asp Leu Met Leu Leu Lys Leu Ala 130 135 140Arg Pro Val Val
Leu Gly Pro Arg Val Arg Ala Leu Gln Leu Pro Tyr145 150 155 160Arg
Cys Ala Gln Pro Gly Asp Gln Cys Gln Val Ala Gly Trp Gly Thr 165 170
175Thr Ala Ala Arg Arg Val Lys Tyr Asn Lys Gly Leu Thr Cys Ser Ser
180 185 190Ile Thr Ile Leu Ser Pro Lys Glu Cys Glu Val Phe Tyr Pro
Gly Val 195 200 205Val Thr Asn Asn Met Ile Cys Ala Gly Leu Asp Arg
Gly Gln Asp Pro 210 215 220Cys Gln Ser Asp Ser Gly Gly Pro Leu Val
Cys Asp Glu Thr Leu Gln225 230 235 240Gly Ile Leu Ser Trp Gly Val
Tyr Pro Cys Gly Ser Ala Gln His Pro 245 250 255Ala Val Tyr Thr Gln
Ile Cys Lys Tyr Met Ser Trp Ile Asn Lys Val 260 265 270Ile Arg Ser
Asn 275131512DNAHomo sapienCDS(246)...(980) 13aggcggacaa aacccgattg
ttcctgggcc ctttccccat cgcgcctggg cctgctcccc 60agcccggggc aggggcgggg
gccagtgtgg tgacacacgc tgtagctgtc tccccggctg 120gctggctcgc
tctctcctgg ggacacagag gtcggcaggc agcacacaga gggacctacg
180ggcagctgtt ccttcccccg actcaagaat ccccggaggc ccggaggcct
gcagcaggag 240cggcc atg aag aag ctg atg gtg gtg ctg agt ctg att gct
gca gcc tgg 290 Met Lys Lys Leu Met Val Val Leu Ser Leu Ile Ala Ala
Ala Trp 1 5 10 15gca gag gag cag aat aag ttg gtg cat ggc gga ccc
tgc gac aag aca 338Ala Glu Glu Gln Asn Lys Leu Val His Gly Gly Pro
Cys Asp Lys Thr 20 25 30tct cac ccc tac caa gct gcc ctc tac acc tcg
ggc cac ttg ctc tgt 386Ser His Pro Tyr Gln Ala Ala Leu Tyr Thr Ser
Gly His Leu Leu Cys 35 40 45ggt ggg gtc ctt atc cat cca ctg tgg gtc
ctc aca gct gcc cac tgc 434Gly Gly Val Leu Ile His Pro Leu Trp Val
Leu Thr Ala Ala His Cys 50 55 60aaa aaa ccg aat ctt cag gtc ttc ctg
ggg aag cat aac ctt cgg caa 482Lys Lys Pro Asn Leu Gln Val Phe Leu
Gly Lys His Asn Leu Arg Gln 65 70 75agg gag agt tcc cag gag cag agt
tct gtt gtc cgg gct gtg atc cac 530Arg Glu Ser Ser Gln Glu Gln Ser
Ser Val Val Arg Ala Val Ile His80 85 90 95cct gac tat gat gcc gcc
agc cat gac cag gac atc atg ctg ttg cgc 578Pro Asp Tyr Asp Ala Ala
Ser His Asp Gln Asp Ile Met Leu Leu Arg 100 105 110ctg gca cgc cca
gcc aaa ctc tct gaa ctc atc cag ccc ctt ccc ctg 626Leu Ala Arg Pro
Ala Lys Leu Ser Glu Leu Ile Gln Pro Leu Pro Leu 115 120 125gag agg
gac tgc tca gcc aac acc acc agc tgc cac atc ctg ggc tgg 674Glu Arg
Asp Cys Ser Ala Asn Thr Thr Ser Cys His Ile Leu Gly Trp 130 135
140ggc aag aca gca gat ggt gat ttc cct gac acc atc cag tgt gca tac
722Gly Lys Thr Ala Asp Gly Asp Phe Pro Asp Thr Ile Gln Cys Ala Tyr
145 150 155atc cac ctg gtg tcc cgt gag gag tgt gag cat gcc tac cct
ggc cag 770Ile His Leu Val Ser Arg Glu Glu Cys Glu His Ala Tyr Pro
Gly Gln160 165 170 175atc acc cag aac atg ttg tgt gct ggg gat gag
aag tac ggg aag gat 818Ile Thr Gln Asn Met Leu Cys Ala Gly Asp Glu
Lys Tyr Gly Lys Asp 180 185 190tcc tgc cag ggt gat tct ggg ggt ccg
ctg gta tgt gga gac cac ctc 866Ser Cys Gln Gly Asp Ser Gly Gly Pro
Leu Val Cys Gly Asp His Leu 195 200 205cga ggc ctt gtg tca tgg ggt
aac atc ccc tgt gga tca aag gag aag 914Arg Gly Leu Val Ser Trp Gly
Asn Ile Pro Cys Gly Ser Lys Glu Lys 210 215 220cca gga gtc tac acc
aac gtc tgc aga tac acg aac tgg atc caa aaa 962Pro Gly Val Tyr Thr
Asn Val Cys Arg Tyr Thr Asn Trp Ile Gln Lys 225 230 235acc att cag
gcc aag tga ccctgacatg tgacatctac ctcccgacct 1010Thr Ile Gln Ala
Lys *240accaccccac tggctggttc cagaacgtct ctcacctaga ccttgcctcc
cctcctctcc 1070tgcccagctc tgaccctgat gcttaataaa cgcagcgacg
tgagggtcct gattctccct 1130ggttttaccc cagctccatc cttgcatcac
tggggaggac gtgatgagtg aggacttggg 1190tcctcggtct tacccccacc
actaagagaa tacaggaaaa tcccttctag gcatctcctc 1250tccccaaccc
ttccacacgt ttgatttctt cctgcagagg cccagccacg tgtctggaat
1310cccagctccg ctgcttactg tcggtgtccc cttgggatgt acctttcttc
actgcagatt 1370tctcacctgt aagatgaaga taaggatgat acagtctcca
taaggcagtg gctgttggaa 1430agatttaagg tttcacacct atgacataca
tggaatagca cctgggccac catgcactca 1490ataaagaatg aattttatta tg
151214244PRTHomo sapien 14Met Lys Lys Leu Met Val Val Leu Ser Leu
Ile Ala Ala Ala Trp Ala1 5 10 15Glu Glu Gln Asn Lys Leu Val His Gly
Gly Pro Cys Asp Lys Thr Ser 20 25 30His Pro Tyr Gln Ala Ala Leu Tyr
Thr Ser Gly His Leu Leu Cys Gly 35 40 45Gly Val Leu Ile His Pro Leu
Trp Val Leu Thr Ala Ala His Cys Lys 50 55 60Lys Pro Asn Leu Gln Val
Phe Leu Gly Lys His Asn Leu Arg Gln Arg65 70 75 80Glu Ser Ser Gln
Glu Gln Ser Ser Val Val Arg Ala Val Ile His Pro 85 90 95Asp Tyr Asp
Ala Ala Ser His Asp Gln Asp Ile Met Leu Leu Arg Leu 100 105 110Ala
Arg Pro Ala Lys Leu Ser Glu Leu Ile Gln Pro Leu Pro Leu Glu 115 120
125Arg Asp Cys Ser Ala Asn Thr Thr Ser Cys His Ile Leu Gly Trp Gly
130 135 140Lys Thr Ala Asp Gly Asp Phe Pro Asp Thr Ile Gln Cys Ala
Tyr Ile145 150 155 160His Leu Val Ser Arg Glu Glu Cys Glu His Ala
Tyr Pro Gly Gln Ile 165 170 175Thr Gln Asn Met Leu Cys Ala Gly Asp
Glu Lys Tyr Gly Lys Asp Ser 180 185 190Cys Gln Gly Asp Ser Gly Gly
Pro Leu Val Cys Gly Asp His Leu Arg 195 200 205Gly Leu Val Ser Trp
Gly Asn Ile Pro Cys Gly Ser Lys Glu Lys Pro 210 215 220Gly Val Tyr
Thr Asn Val Cys Arg Tyr Thr Asn Trp Ile Gln Lys Thr225 230 235
240Ile Gln Ala Lys152892DNAHomo sapienCDS(88)...(618) 15gcggcggcgg
cggcggcggc aggagcccgg gaggcggagg cgggaggcgg cggcggcgcg 60cggagacgca
gcagcggcag cggcagc atg tcg gcc ggc gga gcg tca gtc ccg 114 Met Ser
Ala Gly Gly Ala Ser Val Pro 1 5ccg ccc ccg aac ccc gcc gtg tcc ttc
ccg ccg ccc cgg gtc acc ctg 162Pro Pro Pro Asn Pro Ala Val Ser Phe
Pro Pro Pro Arg Val Thr Leu10 15 20 25ccc gcc ggc ccc gac atc ctg
cgg acc tac tcg ggc gcc ttc gtc tgc 210Pro Ala Gly Pro Asp Ile Leu
Arg Thr Tyr Ser Gly Ala Phe Val Cys 30 35 40ctg gag att ctg ttc ggg
ggt ctt gtc tgg att ttg gtt gcc tcc tcc 258Leu Glu Ile Leu Phe Gly
Gly Leu Val Trp Ile Leu Val Ala Ser Ser 45 50 55aat gtt cct cta cct
cta cta caa gga tgg gtc atg ttt gtg tcc gtg 306Asn Val Pro Leu Pro
Leu Leu Gln Gly Trp Val Met Phe Val Ser Val 60 65 70aca gcg ttt ttc
ttt tcg ctc ctc ttt ctg ggc atg ttc ctc tct ggc 354Thr Ala Phe Phe
Phe Ser Leu Leu Phe Leu Gly Met Phe Leu Ser Gly 75 80 85atg gtg gct
caa att gat gct aac tgg aac ttc ctg gat ttt gcc tac 402Met Val Ala
Gln Ile Asp Ala Asn Trp Asn Phe Leu Asp Phe Ala Tyr90 95 100 105cat
ttt aca gta ttt gtc ttc tat ttt gga gcc ttt tta ttg gaa gca 450His
Phe Thr Val Phe Val Phe Tyr Phe Gly Ala Phe Leu Leu Glu Ala 110 115
120gca gcc aca tcc ctg cat gat ttg cat tgc aat aca acc ata acc ggg
498Ala Ala Thr Ser Leu His Asp Leu His Cys Asn Thr Thr Ile Thr Gly
125 130 135cag cca ctc ctg agt gat aac cag tat aac ata aac gta gca
gcc tca 546Gln Pro Leu Leu Ser Asp Asn Gln Tyr Asn Ile Asn Val Ala
Ala Ser 140 145 150att ttt gcc ttt atg acg aca gct tgt tat ggt tgc
agt ttg ggt ctg 594Ile Phe Ala Phe Met Thr Thr Ala Cys Tyr Gly Cys
Ser Leu Gly Leu 155 160 165gct tta cga aga tgg cga ccg taa
cactccttag aaactggcag tcgtatgtta 648Ala Leu Arg Arg Trp Arg Pro
*170 175gtttcacttg tctactttat atgtctgatc aatttggata ccattttgtc
cagatgcaaa 708aacattccaa aagtaatgtg tttagtagag agagactcta
agctcaagtt ctggtttatt 768tcatggatgg aatgttaatt ttattatgat
attaaagaaa tggcctttta ttttacatct 828ctcccctttt tccctttccc
cctttatttt cctccttttc tttctgaaag tttcctttta 888tgtccataaa
atacaaatat attgttcata aaaaattagt atcccttttg tttggttgct
948gagtcacctg aaccttaatt ttaattggta attacagccc ctaaaaaaaa
cacatttcaa 1008ataggcttcc cactaaactc tatattttag tgtaaaccag
gaattggcac acttttttta 1068gaatgggcca gatggtaaat atttatgctt
cacggtccat acagtctctg tcacaactat 1128tcagttctgc tagtatagcg
tgaaagcagc tatacacaat acagaaatga atgagtgtgg 1188ttatgttcta
ataaaactta tttataaaaa caaggggagg ctgggtttag cctgtgggcc
1248atagtttgtc aaccactggt gtaaaacctt agttatatat gatctgcatt
ttcttgaact 1308gatcattgaa aacttataaa cctaacagaa aagccacata
atatttagtg tcattatgca 1368ataatcacat tgcctttgtg ttaatagtca
aatacttacc tttggagaat acttaccttt 1428ggaggaatgt ataaaatttc
tcaggcagag tcctggatat aggaaaaagt aatttatgaa 1488gtaaacttca
gttgcttaat caaactaatg atagtctaac aactgagcaa gatcctcatc
1548tgagagtgct taaaatggga tccccagaga ccattaacca atactggaac
tggtatctag 1608ctactgatgt cttactttga gtttatttat gcttcagaat
acagttgttt gccctgtgca 1668tgaatatacc catatttgtg tgtggatatg
tgaagctttt ccaaatagag ctctcagaag 1728aattaagttt ttacttctaa
ttattttgca ttactttgag ttaaatttga atagagtatt 1788aaatataaag
ttgtagattc ttatgtgttt ttgtattagc ccagacatct gtaatgtttt
1848tgcactggtg acagacaaaa tctgttttaa aatcatatcc agcacaaaaa
ctatttctgg 1908ctgaatagca cagaaaagta ttttaaccta cctgtagaga
tcctcgtcat ggaaaggtgc 1968caaactgttt tgaatggaag gacaagtaag
agtgaggcca cagttcccac cacacgaggg 2028cttttgtatt gttctacttt
ttcagccctt tactttctgg ctgaagcatc cccttggagt 2088gccatgtata
agttgggcta ttagagttca tggaacatag aacaaccatg aatgagtggc
2148atgatccgtg cttaatgatc aagtgttact tatctaataa tcctctagaa
agaaccctgt 2208tagatcttgg tttgtgataa aaatataaag acagaagaca
tgaggaaaaa caaaaggttt 2268gaggaaatca ggcatatgac tttatactta
acatcagatc ttttctataa tatcctacta 2328ctttggtttt cctagctcca
taccacacac ctaaacctgt attatgaatt acatattaca 2388aagtcataaa
tgtgccatat ggatatacag tacattctag ttggaatcgt ttactctgct
2448agaatttagg tgtgagattt tttgtttccc aggtatagca ggcttatgtt
tggtggcatt 2508aaattggttt ctttaaaatg ctttggtggc acttttgtaa
acagattgct tctagattgt 2568tacaaaccaa gcctaagaca catctgtgaa
tacttagatt tgtagcttaa tcacattcta 2628gacttgtgag ttgaatgaca
aagcagttga acaaaaatta tggcatttaa gaatttaaca 2688tgtcttagct
gtaaaaatga gaaagtgttg gttggtttta aaatctggta actccatgat
2748gaaaagaaat ttattttata cgtgttatgt ctctaataaa gtattcattt
gataaaaaaa 2808aaaaaaaaaa gggcggccgc tctagaggat ccaagcttac
gtacgcgtgc atgcgacgtc 2868atagctcttc tatagtgtca ccta
289216176PRTHomo sapien 16Met Ser Ala Gly Gly Ala Ser Val Pro Pro
Pro Pro Asn Pro Ala Val1 5 10 15Ser Phe Pro Pro Pro Arg Val Thr Leu
Pro Ala Gly Pro Asp Ile Leu 20 25 30Arg Thr Tyr Ser Gly Ala Phe Val
Cys Leu Glu Ile Leu Phe Gly Gly 35 40 45Leu Val Trp Ile Leu Val Ala
Ser Ser Asn Val Pro Leu Pro Leu Leu 50 55 60Gln Gly Trp Val Met Phe
Val Ser Val Thr Ala Phe Phe Phe Ser Leu65 70 75 80Leu Phe Leu Gly
Met Phe Leu Ser Gly Met Val Ala Gln Ile Asp Ala 85 90 95Asn Trp Asn
Phe Leu Asp Phe Ala Tyr His Phe Thr Val Phe Val Phe 100 105 110Tyr
Phe Gly Ala Phe Leu Leu Glu Ala Ala Ala Thr Ser Leu His Asp 115 120
125Leu His Cys Asn Thr Thr Ile Thr Gly Gln Pro Leu Leu Ser Asp Asn
130 135 140Gln Tyr Asn Ile Asn Val Ala Ala Ser Ile Phe Ala Phe Met
Thr Thr145 150 155 160Ala Cys Tyr Gly Cys Ser Leu Gly Leu Ala Leu
Arg Arg Trp Arg Pro 165 170 175174458DNAHomo sapienCDS(21)...(404)
17gtcgacccac gcgtccggcg atg cct cgc tgg ctt ctg ctt tca ttg acc ttt
53 Met Pro Arg Trp Leu Leu Leu Ser Leu Thr Phe 1 5 10gcg ggt ctg
ttc ccg ctg cgg cgc cgg cag ctg ctt ggt agt tgc ggg 101Ala Gly Leu
Phe Pro Leu Arg Arg Arg Gln Leu Leu Gly Ser Cys Gly 15 20 25ggg cgt
gag ggc ggt ggc cca gac caa ccg gct ggc agc cca gct ccg 149Gly Arg
Glu Gly Gly Gly Pro Asp Gln Pro Ala Gly Ser Pro Ala Pro 30 35 40ctc
cgc ccg ccc ctg cct cgg acc ctg cgc ctg agg aag tat cga ggc 197Leu
Arg Pro Pro Leu Pro Arg Thr Leu Arg Leu Arg Lys Tyr Arg Gly 45 50
55aac cct ctg cca ccc gaa gtt cgt ggg tcg ctc cca gag ggc gcg ccc
245Asn Pro Leu Pro Pro Glu Val Arg Gly Ser Leu Pro Glu Gly Ala
Pro60 65 70 75tgg agc cga gcg ccc ttg ggc ggc cat ctg gag gcc agg
tgc ggg ccg 293Trp Ser Arg Ala Pro Leu Gly Gly His Leu Glu Ala Arg
Cys Gly Pro 80 85 90cga acc cgc gag gag cgc gcg gcg ggc gcg gcg gcg
acg gca gga gga 341Arg Thr Arg Glu Glu Arg Ala Ala Gly Ala Ala Ala
Thr Ala Gly Gly 95 100 105ggg gcc ggg agc ccg ggc gcc gcc gaa gga
cgc ccc gtc ctc cac atg 389Gly Ala Gly Ser Pro Gly Ala Ala Glu Gly
Arg Pro Val Leu His Met 110 115 120ctg cca ctt ggc tga gccgggcgcc
ggcgagaagg cggcgccgct gccctggcag 444Leu Pro Leu Gly * 125ctggactgca
ctttgccccc gcccggcctc agctgccgcc cgcccagacg ccagcaagcc
504cccctcccac gacagggctg ctccgggagc ttcggagacc cgccccgggc
ctgagcgcag 564gctgcctccg ggaccccacg gctgtccgga cgtgccatgg
gcgcgcagct gccgggcaac 624gtgttgtgta agtgaacatc tgggaggtaa
acactacacg tgaagagtgg tgaaagggaa 684cattgattac tgaagtgccc
tggagaggga aagcactggt caacatcaca tggacaaatt 744tcattgtttt
ctaaagatgg cctggaagta gtctttgcca ctgcttcctc cacaaacagc
804tcttcataac atgggctgca tgaaatcaaa gcaaactttc ccatttccta
ccatatatga 864aggtgagaag cagcatgaga gtgaagaacc ctttatgcca
gaagagagat gtctacctag 924gatggcttct ccagttaatg tcaaagagga
agtgaaggaa cctccaggga ccaatattgt 984gatcttggaa tatgcacacc
gcctgtctca ggatatcttg tgtgatgcct tgcagcaatg 1044ggcatgcaat
aacatcaagt accatgacat tccatacatt gagagtgagg ggccttgagg
1104ctgtaggatg acaacacttt gactgtggag gtgctagttt gaataaatgt
gacaaaagca 1164aaaactggtg tgaaaaagta caaataacta tctggattta
aaaatgtgtc tacgataatg 1224tcactattat aagaacaact aggatgaaat
gcattttaag tacttctatg ttaacagcaa 1284tttctgttta gtcttagatt
ttagtcatct gaagggctga acagaggtcc tgtgacaccc 1344aataatcagc
tgaatgtcac agcacttctt cctaagtaat ggcatcacca aagaaaatgc
1404taaggaataa aaactgcccc aaattccaat ggttgaagtt tatcctttaa
aataacaatt 1464tttgtttata cccaaaaaaa gtccagatat gaaaagggct
tttctaaaat ttcttggcga 1524gggaatggca ctcaaatcat agtgattaac
agtaagtctt gtttgtttgt caaggatctc 1584tacttcttga cacaaatgaa
ccctgtcttt aataagataa gatatttatt tttgtagatg 1644agaagtgtaa
ctaccacctt ggacctcagg gccctaacta attacagctg ttactggacg
1704actcagactt tgtgcctaaa gccatcttag agataacagt ttatagaagc
catgacatta 1764gtgtttattg cattgaatta agcccagtga tataactata
caagaaaaca agtatgggta 1824ccttttacaa agagcaatcc aataaatctt
aaaaataaca gaaacttagt ctgcaaggta 1884gaaagtttca gttttaattc
tgtattaagc tttactatct cagaggtaca gagggctgga 1944atatgggcat
ttatttccag ttttttcttg actagtaagg cggtcaccat taaaatagac
2004cagatgataa tgcatgaaga tttacagttg tattgcaaaa cggaaaagat
aaaactgtcc 2064tttgaggaga gtactcgttt tctgggtttt tgttattttt
tagtggtaac acaagcctat 2124agggcattta tagccaccta ttatactgtt
tccataagcc tggctacctt ttagggaagc
2184tattttttct ctttcatttt tactgtcaca gcacatacac acacaccttt
ttgttttaaa 2244ggattaagta ctgtttgaag atcagtggta acagaaaatt
tgggagggag aagaagaaat 2304taagacatga cttgttagaa aattaagact
tcagtttcta gaattatctt ttcatcaaga 2364tttggtagac attgagttta
aatggaaagg aaattattta agcctgtgta tgttagatcc 2424acaatacacc
attggtattg aaatataaag gttaaaaaaa aggcttatga cctctttaat
2484gagataaata tgtatttgtc ttgtaagcag gcagaaaatc tacctctaat
tttaacacta 2544atactttgaa acccacaatc aaatagagtg aattctccaa
gttacataag caaggaaaac 2604attatttgaa atatgccatg ttttcgttgc
ctttggacac ctcatcattc aactctaatt 2664ttaccgagtc ccgggatttg
tactgtccca ttgtacttgc aatctacaat ttatataata 2724gaaaaacaac
caaacccatt catacaagga tctgaagtta taaggttaag ggcagaaagt
2784ttcccataag tataaaacat ttccaggtca tgaagagtag tttaggttga
gtgacaaaag 2844cctaggtgtg gttgtttttc attcattttg catctcacac
caagacattt ttgctgcaag 2904gtcatctgct gcttaaaatg tacaattagg
tatataaaat aagtacaatg gtgaaaacac 2964aaagccaggt aaagcagcat
gccccactaa atttttcagt atacataggg acagacaagt 3024gagttttggt
tgtatctaaa tattttaatt tcaggttcct tctgtgccct gggccactat
3084ttcccagggg tgtgacagag atgcctgcca gatccatatc aactagaagt
ctgatttctg 3144ttgctgccct tcctcagcaa ctatggcagt atacttttat
caccaagcac cactcccttg 3204tccctgaatc acattttaat agagtacaat
atcttctgta caatatttct gaaacactta 3264tgtctgaaat atatgctgta
ttgtatgtta acccatgaca tatatgaact acaaggcttg 3324cataatcagt
gagctagtgg ataaatcaag acaggagcaa atgggagaaa gatgaataaa
3384caaatgaaaa aagatgaata aatgaataag agagatgaat aaacaaattt
acattacatg 3444tgatagttat catggtatgg ccttcatgac aagatggatg
agaatatcac tgataggata 3504ttagccttct ttcatatctt tatattgaaa
tatgggcttt acttcaattt gaaggtcttt 3564catgaacaat aaaagagagt
agaaggactg tctgagaagg caggagacat ataaaacaga 3624tgactgaaag
actgactagc tcctggaaag ggaaacattt ggaacatcca gagtaaggca
3684aatgggcttc taccagcaca acaaagagcc tccaggtggc aacatggaag
caggttatca 3744gagaaaataa atgtgcaaat tccttattta caatgactca
cttaacccca caaacatgtt 3804tcactgctgc cttccccagt tgtcgcttat
gtactgttgt tacctttcag ttacatgcct 3864ttgatcctaa aattctctac
ttttgttgcc ttatcagttc tttgcaatct gcctgtggtt 3924atcagcactt
aaagcacaat tttgaagggg aaaaaaatga taatcacctt agtcccaaag
3984aaataatttg tcaaactgcc ttattagtat taaaaacaga cacactgaat
gaagtagcat 4044gatacgcata tatcctactc agtatcattg gccttttatc
aaatggggaa actatacttt 4104tgtattacat agttttagaa atcgaaagtt
agagactctt tataagtaat gtcaaggaac 4164agtaatttaa aaacaaagtt
ctaacaaata tattgtttgc ttaatcacaa tgccctcaac 4224ttgtatttga
ataactaaat aggacatgtc ttccttggag ctgtgggcat tagttcagaa
4284gcactacctg catcttaatt ttcaaaactt aagttttatt agcaaatcct
cttctctgta 4344agacttagct atgaagtggt atattttttc caaatatttt
tctgaaaaca tttgttgttg 4404taactgcaca ataaaagtcc agttgcaatt
aaaaaaaaaa aaaaaaaaaa aaaa 445818127PRTHomo sapien 18Met Pro Arg
Trp Leu Leu Leu Ser Leu Thr Phe Ala Gly Leu Phe Pro1 5 10 15Leu Arg
Arg Arg Gln Leu Leu Gly Ser Cys Gly Gly Arg Glu Gly Gly 20 25 30Gly
Pro Asp Gln Pro Ala Gly Ser Pro Ala Pro Leu Arg Pro Pro Leu 35 40
45Pro Arg Thr Leu Arg Leu Arg Lys Tyr Arg Gly Asn Pro Leu Pro Pro
50 55 60Glu Val Arg Gly Ser Leu Pro Glu Gly Ala Pro Trp Ser Arg Ala
Pro65 70 75 80Leu Gly Gly His Leu Glu Ala Arg Cys Gly Pro Arg Thr
Arg Glu Glu 85 90 95Arg Ala Ala Gly Ala Ala Ala Thr Ala Gly Gly Gly
Ala Gly Ser Pro 100 105 110Gly Ala Ala Glu Gly Arg Pro Val Leu His
Met Leu Pro Leu Gly 115 120 125191127DNAHomo sapienCDS(48)...(851)
19accaaatcaa ccataggtcc aagaacaatt gtctctggac ggcagct atg cga ctc
56 Met Arg Leu 1acc gtg ctg tgt gct gtg tgc ctg ctg cct ggc agc ctg
gcc ctg ccg 104Thr Val Leu Cys Ala Val Cys Leu Leu Pro Gly Ser Leu
Ala Leu Pro 5 10 15ctg cct cag gag gcg gga ggc atg agt gag cta cag
tgg gaa cag gct 152Leu Pro Gln Glu Ala Gly Gly Met Ser Glu Leu Gln
Trp Glu Gln Ala20 25 30 35cag gac tat ctc aag aga ttt tat ctc tat
gac tca gaa aca aaa aat 200Gln Asp Tyr Leu Lys Arg Phe Tyr Leu Tyr
Asp Ser Glu Thr Lys Asn 40 45 50gcc aac agt tta gaa gcc aaa ctc aag
gag atg caa aaa ttc ttt ggc 248Ala Asn Ser Leu Glu Ala Lys Leu Lys
Glu Met Gln Lys Phe Phe Gly 55 60 65cta cct ata act gga atg tta aac
tcc cgc gtc ata gaa ata atg cag 296Leu Pro Ile Thr Gly Met Leu Asn
Ser Arg Val Ile Glu Ile Met Gln 70 75 80aag ccc aga tgt gga gtg cca
gat gtt gca gaa tac tca cta ttt cca 344Lys Pro Arg Cys Gly Val Pro
Asp Val Ala Glu Tyr Ser Leu Phe Pro 85 90 95aat agc cca aaa tgg act
tcc aaa gtg gtc acc tac agg atc gta tca 392Asn Ser Pro Lys Trp Thr
Ser Lys Val Val Thr Tyr Arg Ile Val Ser100 105 110 115tat act cga
gac tta ccg cat att aca gtg gat cga tta gtg tca aag 440Tyr Thr Arg
Asp Leu Pro His Ile Thr Val Asp Arg Leu Val Ser Lys 120 125 130gct
tta aac atg tgg ggc aaa gag atc ccc ctg cat ttc agg aaa gtt 488Ala
Leu Asn Met Trp Gly Lys Glu Ile Pro Leu His Phe Arg Lys Val 135 140
145gta tgg gga act gct gac atc atg att ggc ttt gcg cga gga gct cat
536Val Trp Gly Thr Ala Asp Ile Met Ile Gly Phe Ala Arg Gly Ala His
150 155 160ggg gac tcc tac cca ttt gat ggg cca gga aac acg ctg gct
cat gcc 584Gly Asp Ser Tyr Pro Phe Asp Gly Pro Gly Asn Thr Leu Ala
His Ala 165 170 175ttt gcg cct ggg aca ggt ctc gga gga gat gct cac
ttc gat gag gat 632Phe Ala Pro Gly Thr Gly Leu Gly Gly Asp Ala His
Phe Asp Glu Asp180 185 190 195gaa cgc tgg acg gat ggt agc agt cta
ggg att aac ttc ctg tat gct 680Glu Arg Trp Thr Asp Gly Ser Ser Leu
Gly Ile Asn Phe Leu Tyr Ala 200 205 210gca act cat gaa ctt ggc cat
tct ttg ggt atg gga cat tcc tct gat 728Ala Thr His Glu Leu Gly His
Ser Leu Gly Met Gly His Ser Ser Asp 215 220 225cct aat gca gtg atg
tat cca acc tat gga aat gga gat ccc caa aat 776Pro Asn Ala Val Met
Tyr Pro Thr Tyr Gly Asn Gly Asp Pro Gln Asn 230 235 240ttt aaa ctt
tcc cag gat gat att aaa ggc att cag aaa cta tat gga 824Phe Lys Leu
Ser Gln Asp Asp Ile Lys Gly Ile Gln Lys Leu Tyr Gly 245 250 255aag
aga agt aat tca aga aag aaa tag aaacttcagg cagaacatcc 871Lys Arg
Ser Asn Ser Arg Lys Lys *260 265attcattcat tcattggatt gtatatcatt
gttgcacaat cagaattgat aagcactgtt 931cctccactcc atttagcaat
tatgtcaccc ttttttattg cagttggttt ttgaatgtct 991ttcactcctt
ttattggtta aactccttta tggtgtgact gtgtcttatt ccatctatga
1051gctttgtcag tgcgcgtaga tgtcaataaa tgttacatac acaaataaat
aaaatgttta 1111ttccatggta aattta 112720267PRTHomo sapien 20Met Arg
Leu Thr Val Leu Cys Ala Val Cys Leu Leu Pro Gly Ser Leu1 5 10 15Ala
Leu Pro Leu Pro Gln Glu Ala Gly Gly Met Ser Glu Leu Gln Trp 20 25
30Glu Gln Ala Gln Asp Tyr Leu Lys Arg Phe Tyr Leu Tyr Asp Ser Glu
35 40 45Thr Lys Asn Ala Asn Ser Leu Glu Ala Lys Leu Lys Glu Met Gln
Lys 50 55 60Phe Phe Gly Leu Pro Ile Thr Gly Met Leu Asn Ser Arg Val
Ile Glu65 70 75 80Ile Met Gln Lys Pro Arg Cys Gly Val Pro Asp Val
Ala Glu Tyr Ser 85 90 95Leu Phe Pro Asn Ser Pro Lys Trp Thr Ser Lys
Val Val Thr Tyr Arg 100 105 110Ile Val Ser Tyr Thr Arg Asp Leu Pro
His Ile Thr Val Asp Arg Leu 115 120 125Val Ser Lys Ala Leu Asn Met
Trp Gly Lys Glu Ile Pro Leu His Phe 130 135 140Arg Lys Val Val Trp
Gly Thr Ala Asp Ile Met Ile Gly Phe Ala Arg145 150 155 160Gly Ala
His Gly Asp Ser Tyr Pro Phe Asp Gly Pro Gly Asn Thr Leu 165 170
175Ala His Ala Phe Ala Pro Gly Thr Gly Leu Gly Gly Asp Ala His Phe
180 185 190Asp Glu Asp Glu Arg Trp Thr Asp Gly Ser Ser Leu Gly Ile
Asn Phe 195 200 205Leu Tyr Ala Ala Thr His Glu Leu Gly His Ser Leu
Gly Met Gly His 210 215 220Ser Ser Asp Pro Asn Ala Val Met Tyr Pro
Thr Tyr Gly Asn Gly Asp225 230 235 240Pro Gln Asn Phe Lys Leu Ser
Gln Asp Asp Ile Lys Gly Ile Gln Lys 245 250 255Leu Tyr Gly Lys Arg
Ser Asn Ser Arg Lys Lys 260 265215093DNAHomo
sapienCDS(136)...(1935) 21aggtgacagc tggagggagg agcgggggtg
gagccggggg aagggtgggg aggggatggg 60ctggagctcc gggcagtgtg cgaggcgcac
gcacaggagc ctgcactctg cgtcccgcac 120cccagcagcc gcgcc atg agc cgg
agt ctc ttg ctc tgg ttc ttg ctg ttc 171 Met Ser Arg Ser Leu Leu Leu
Trp Phe Leu Leu Phe 1 5 10ctg ctc ctg ctc ccg ccg ctc ccc gtc ctg
ctc gcg gac cca ggg gcg 219Leu Leu Leu Leu Pro Pro Leu Pro Val Leu
Leu Ala Asp Pro Gly Ala 15 20 25ccc acg cca gtg aat ccc tgt tgt tac
tat cca tgc cag cac cag ggc 267Pro Thr Pro Val Asn Pro Cys Cys Tyr
Tyr Pro Cys Gln His Gln Gly 30 35 40atc tgt gtc cgc ttc ggc ctt gac
cgc tac cag tgt gac tgc acc cgc 315Ile Cys Val Arg Phe Gly Leu Asp
Arg Tyr Gln Cys Asp Cys Thr Arg45 50 55 60acg ggc tat tcc ggc ccc
aac tgc acc atc cct ggc ctg tgg acc tgg 363Thr Gly Tyr Ser Gly Pro
Asn Cys Thr Ile Pro Gly Leu Trp Thr Trp 65 70 75ctc cgg aat tca ctg
cgg ccc agc ccc tct ttc acc cac ttc ctg ctc 411Leu Arg Asn Ser Leu
Arg Pro Ser Pro Ser Phe Thr His Phe Leu Leu 80 85 90act cac ggg cgc
tgg ttc tgg gag ttt gtc aat gcc acc ttc atc cga 459Thr His Gly Arg
Trp Phe Trp Glu Phe Val Asn Ala Thr Phe Ile Arg 95 100 105gag atg
ctc atg cgc ctg gta ctc aca gtg cgc tcc aac ctt atc ccc 507Glu Met
Leu Met Arg Leu Val Leu Thr Val Arg Ser Asn Leu Ile Pro 110 115
120agt ccc ccc acc tac aac tca gca cat gac tac atc agc tgg gag tct
555Ser Pro Pro Thr Tyr Asn Ser Ala His Asp Tyr Ile Ser Trp Glu
Ser125 130 135 140ttc tcc aac gtg agc tat tac act cgt att ctg ccc
tct gtg cct aaa 603Phe Ser Asn Val Ser Tyr Tyr Thr Arg Ile Leu Pro
Ser Val Pro Lys 145 150 155gat tgc ccc aca ccc atg gga acc aaa ggg
aag aag cag ttg cca gat 651Asp Cys Pro Thr Pro Met Gly Thr Lys Gly
Lys Lys Gln Leu Pro Asp 160 165 170gcc cag ctc ctg gcc cgc cgc ttc
ctg ctc agg agg aag ttc ata cct 699Ala Gln Leu Leu Ala Arg Arg Phe
Leu Leu Arg Arg Lys Phe Ile Pro 175 180 185gac ccc caa ggc acc aac
ctc atg ttt gcc ttc ttt gca caa cac ttc 747Asp Pro Gln Gly Thr Asn
Leu Met Phe Ala Phe Phe Ala Gln His Phe 190 195 200acc cac cag ttc
ttc aaa act tct ggc aag atg ggt cct ggc ttc acc 795Thr His Gln Phe
Phe Lys Thr Ser Gly Lys Met Gly Pro Gly Phe Thr205 210 215 220aag
gcc ttg ggc cat ggg gta gac ctc ggc cac att tat gga gac aat 843Lys
Ala Leu Gly His Gly Val Asp Leu Gly His Ile Tyr Gly Asp Asn 225 230
235ctg gag cgt cag tat caa ctg cgg ctc ttt aag gat ggg aaa ctc aag
891Leu Glu Arg Gln Tyr Gln Leu Arg Leu Phe Lys Asp Gly Lys Leu Lys
240 245 250tac cag gtg ctg gat gga gaa atg tac ccg ccc tcg gta gaa
gag gcg 939Tyr Gln Val Leu Asp Gly Glu Met Tyr Pro Pro Ser Val Glu
Glu Ala 255 260 265cct gtg ttg atg cac tac ccc cga ggc atc ccg ccc
cag agc cag atg 987Pro Val Leu Met His Tyr Pro Arg Gly Ile Pro Pro
Gln Ser Gln Met 270 275 280gct gtg ggc cag gag gtg ttt ggg ctg ctt
cct ggg ctc atg ctg tat 1035Ala Val Gly Gln Glu Val Phe Gly Leu Leu
Pro Gly Leu Met Leu Tyr285 290 295 300gcc acg ctc tgg cta cgt gag
cac aac cgt gtg tgt gac ctg ctg aag 1083Ala Thr Leu Trp Leu Arg Glu
His Asn Arg Val Cys Asp Leu Leu Lys 305 310 315gct gag cac ccc acc
tgg ggc gat gag cag ctt ttc cag acg acc cgc 1131Ala Glu His Pro Thr
Trp Gly Asp Glu Gln Leu Phe Gln Thr Thr Arg 320 325 330ctc atc ctc
ata ggg gag acc atc aag att gtc atc gag gag tac gtg 1179Leu Ile Leu
Ile Gly Glu Thr Ile Lys Ile Val Ile Glu Glu Tyr Val 335 340 345cag
cag ctg agt ggc tat ttc ctg cag ctg aaa ttt gac cca gag ctg 1227Gln
Gln Leu Ser Gly Tyr Phe Leu Gln Leu Lys Phe Asp Pro Glu Leu 350 355
360ctg ttc ggt gtc cag ttc caa tac cgc aac cgc att gcc atg gag ttc
1275Leu Phe Gly Val Gln Phe Gln Tyr Arg Asn Arg Ile Ala Met Glu
Phe365 370 375 380aac cat ctc tac cac tgg cac ccc ctc atg cct gac
tcc ttc aag gtg 1323Asn His Leu Tyr His Trp His Pro Leu Met Pro Asp
Ser Phe Lys Val 385 390 395ggc tcc cag gag tac agc tac gag cag ttc
ttg ttc aac acc tcc atg 1371Gly Ser Gln Glu Tyr Ser Tyr Glu Gln Phe
Leu Phe Asn Thr Ser Met 400 405 410ttg gtg gac tat ggg gtt gag gcc
ctg gtg gat gcc ttc tct cgc cag 1419Leu Val Asp Tyr Gly Val Glu Ala
Leu Val Asp Ala Phe Ser Arg Gln 415 420 425att gct ggc cgg atc ggt
ggg ggc agg aac atg gac cac cac atc ctg 1467Ile Ala Gly Arg Ile Gly
Gly Gly Arg Asn Met Asp His His Ile Leu 430 435 440cat gtg gct gtg
gat gtc atc agg gag tct cgg gag atg cgg ctg cag 1515His Val Ala Val
Asp Val Ile Arg Glu Ser Arg Glu Met Arg Leu Gln445 450 455 460ccc
ttc aat gag tac cgc aag agg ttt ggc atg aaa ccc tac acc tcc 1563Pro
Phe Asn Glu Tyr Arg Lys Arg Phe Gly Met Lys Pro Tyr Thr Ser 465 470
475ttc cag gag ctc gta gga gag aag gag atg gca gca gag ttg gag gaa
1611Phe Gln Glu Leu Val Gly Glu Lys Glu Met Ala Ala Glu Leu Glu Glu
480 485 490ttg tat gga gac att gat gcg ttg gag ttc tac cct gga ctg
ctt ctt 1659Leu Tyr Gly Asp Ile Asp Ala Leu Glu Phe Tyr Pro Gly Leu
Leu Leu 495 500 505gaa aag tgc cat cca aac tct atc ttt ggg gag agt
atg ata gag att 1707Glu Lys Cys His Pro Asn Ser Ile Phe Gly Glu Ser
Met Ile Glu Ile 510 515 520ggg gct ccc ttt tcc ctc aag ggt ctc cta
ggg aat ccc atc tgt tct 1755Gly Ala Pro Phe Ser Leu Lys Gly Leu Leu
Gly Asn Pro Ile Cys Ser525 530 535 540ccg gag tac tgg aag ccg agc
aca ttt ggc ggc gag gtg ggc ttt aac 1803Pro Glu Tyr Trp Lys Pro Ser
Thr Phe Gly Gly Glu Val Gly Phe Asn 545 550 555att gtc aag acg gcc
aca ctg aag aag ctg gtc tgc ctc aac acc aag 1851Ile Val Lys Thr Ala
Thr Leu Lys Lys Leu Val Cys Leu Asn Thr Lys 560 565 570acc tgt ccc
tac gtt tcc ttc cgt gtg ccg gat gcc agt cag gat gat 1899Thr Cys Pro
Tyr Val Ser Phe Arg Val Pro Asp Ala Ser Gln Asp Asp 575 580 585ggg
cct gct gtg gag cga cca tcc aca gag ctc tga ggggcaggaa 1945Gly Pro
Ala Val Glu Arg Pro Ser Thr Glu Leu * 590 595agcagcattc tggaggggag
agctttgtgc ttgtcattcc agagtgctga ggccagggct 2005gatggtctta
aatgctcatt ttctggtttg gcatggtgag tgttggggtt gacatttaga
2065actttaagtc tcacccatta tctggaatat tgtgattctg tttattcttc
cagaatgctg 2125aactccttgt tagcccttca gattgttagg agtggttctc
atttggtctg ccagaatact 2185gggttcttag ttgacaacct agaatgtcag
atttctggtt gatttgtaac acagtcattc 2245taggatgtgg agctactgat
gaaatctgct agaaagttag ggggttctta ttttgcattc 2305cagaatcttg
actttctgat tggtgattca aagtgttgtg ttcctggctg atgatccaga
2365acagtggctc gtatcccaaa tctgtcagca tctggctgtc tagaatgtgg
atttgattca 2425ttttcctgtt cagtgagata tcatagagac ggagatccta
aggtccaaca agaatgcatt 2485ccctgaatct gtgcctgcac tgagagggca
aggaagtggg gtgttcttct tgggaccccc 2545actaagaccc tggtctgagg
atgtagagag aacaggtggg ctgtattcac gccattggtt 2605ggaagctacc
agagctctat ccccatccag gtcttgactc atggcagctg tttctcatga
2665agctaataaa attcgctttc taaagttacc tgttatatat ctcttttggt
cccatcctct 2725aaagcagagg caacactgga acatggctag cctttcttgt
agccatggct gggcgtgcta 2785gaggttgcag catgagactt tctgctggga
tccttgggcc catcactgta tagacatgct 2845accactggta cttcctttct
ccctgcgggc caggcactgc ccttttcagg aagctctctt 2905aaaataccca
ttgccccaga cctggaagat ataacattca gttcccacca tctgattaaa
2965acaacttcct cccttacaga gcatacaaca gagggggcac ccggggagga
gagcacatac 3025tgtgttccaa tttcacgctt ttaattctca tttgttctca
caccaacagt gtgaagtgcg 3085tggtataatc tccatttcaa aaccaaggaa
gcagcctcag agtggtcgag tgacacacct 3145cacgcaggct gagtccagag
cttgtgctcc tcttgattcc tggtttgact cagttccagg 3205cctgatcttg
cctgtctggc tcagggtcaa agacagaatg gtggagtgta gcctccacct
3265gatattcagg ctactcattc agtcccaaat atgtattttc ctaagtgttt
actatgtgcc 3325agttcctgta acaggtgtgg ggacacagca gtgagtaatc
aatacagaca aggttctgcc 3385cttatggagc tcacactcca gtggcagaca
aacagaccat aaataaggaa acgatgaaat 3445aagatatata caaggtgagt
gtgacttccc ttctaacccc ctctgctctg tcctccccta 3505ttgcgctctc
aagaccagag acccaacagc agtgatctca gggcagacag ccctccactc
3565cagctctgag acccttttct caggacctct gtaggcagca gagagagagg
acagaggggt 3625aagatgaggg gttgagggaa ggttcttcat gatccacact
ttgggcttag tatttctcag 3685gaagagctat ggcccagaaa caacagggga
aactagagtt cggtctgaca gtccttgggg 3745ttaagtctcc tgtcttatgg
tccagaaact cctgtttctc cttagttggc tggaaactgc 3805tcccatcatt
ccttctggcc tctgctgaat gcagggaatg caatccttcc ctgctcttgc
3865agttgctctg acgtagaaag atccttcggg tgctggaagt ctccatgaag
agcttgtgtc 3925ctgtcctttc ttgcagattc tatttcccct cttctgctaa
tacctcttac tttgcttgag 3985aatcctctcc tttcttatta atttcagtct
tggtggttct atcaggggtg cattctggcc 4045aaggggtggg cctgtgaatc
aatcctgggc aatcagacac cctctcctta aaaactggcc 4105cgtggagact
gagatcactg actctgactc atccccacag ctggctctga caagatggtc
4165catttgttcc tgcttccgag atccccaggg cagcctggat ccctgccctt
ctcaagactt 4225tagcttttcc ttccatccgg tggcctattc caggaattcc
tcttttgctt aaatcagttg 4285gagtttgtgt ctgttgcttg taatcaagcc
tttatggctg ctgggctgag tgacacaagc 4345actttaatgg cctggaggga
cttttaatca gtgaagatgc aatcagacaa gtgttttgga 4405aagagcaccc
tcgagaaggg tggatgacag ggcagagcag gaaggacagg aagctggcag
4465aacggaggag gctgcagccg tggtccaacc aggagctgat ggcagctggg
gctaggggaa 4525gggctttgag ggtggaagga tgggatgggt tccagaggta
ttcctctctt aaatgcaagt 4585gcctagatta ggtagacttt gcttagtatt
gacaactgca catgaaagtt ttgcaaaggg 4645aaacaggcta aatgcaccaa
gaaagcttct tcagagtgaa gaatcttaat gcttgtaatt 4705taaacatttg
ttcctggagt tttgatttgg tggatgtgat ggttggtttt atttgtcagt
4765ttggttgggc tatagcacac agttatttaa tcaaacagta atctaggtgt
ggctgtgaag 4825gtattttgta gatgtgatta acatctacaa tcagttgact
ttaagtgaaa gagattactt 4885aaataatttg ggtgagctgc acctgattag
ttgaaaggcc tcaagaacaa acactgcagt 4945ttcctggaaa agaagaaact
ttgcctcaag actatagcca tcgactcctg cctgagtttc 5005cagcctgcta
gtctgcccta tggatttgaa gtttgccaac cccaacaatt gtgtgaatta
5065atttctaaaa ataaagctat atacagcc 509322599PRTHomo sapien 22Met
Ser Arg Ser Leu Leu Leu Trp Phe Leu Leu Phe Leu Leu Leu Leu1 5 10
15Pro Pro Leu Pro Val Leu Leu Ala Asp Pro Gly Ala Pro Thr Pro Val
20 25 30Asn Pro Cys Cys Tyr Tyr Pro Cys Gln His Gln Gly Ile Cys Val
Arg 35 40 45Phe Gly Leu Asp Arg Tyr Gln Cys Asp Cys Thr Arg Thr Gly
Tyr Ser 50 55 60Gly Pro Asn Cys Thr Ile Pro Gly Leu Trp Thr Trp Leu
Arg Asn Ser65 70 75 80Leu Arg Pro Ser Pro Ser Phe Thr His Phe Leu
Leu Thr His Gly Arg 85 90 95Trp Phe Trp Glu Phe Val Asn Ala Thr Phe
Ile Arg Glu Met Leu Met 100 105 110Arg Leu Val Leu Thr Val Arg Ser
Asn Leu Ile Pro Ser Pro Pro Thr 115 120 125Tyr Asn Ser Ala His Asp
Tyr Ile Ser Trp Glu Ser Phe Ser Asn Val 130 135 140Ser Tyr Tyr Thr
Arg Ile Leu Pro Ser Val Pro Lys Asp Cys Pro Thr145 150 155 160Pro
Met Gly Thr Lys Gly Lys Lys Gln Leu Pro Asp Ala Gln Leu Leu 165 170
175Ala Arg Arg Phe Leu Leu Arg Arg Lys Phe Ile Pro Asp Pro Gln Gly
180 185 190Thr Asn Leu Met Phe Ala Phe Phe Ala Gln His Phe Thr His
Gln Phe 195 200 205Phe Lys Thr Ser Gly Lys Met Gly Pro Gly Phe Thr
Lys Ala Leu Gly 210 215 220His Gly Val Asp Leu Gly His Ile Tyr Gly
Asp Asn Leu Glu Arg Gln225 230 235 240Tyr Gln Leu Arg Leu Phe Lys
Asp Gly Lys Leu Lys Tyr Gln Val Leu 245 250 255Asp Gly Glu Met Tyr
Pro Pro Ser Val Glu Glu Ala Pro Val Leu Met 260 265 270His Tyr Pro
Arg Gly Ile Pro Pro Gln Ser Gln Met Ala Val Gly Gln 275 280 285Glu
Val Phe Gly Leu Leu Pro Gly Leu Met Leu Tyr Ala Thr Leu Trp 290 295
300Leu Arg Glu His Asn Arg Val Cys Asp Leu Leu Lys Ala Glu His
Pro305 310 315 320Thr Trp Gly Asp Glu Gln Leu Phe Gln Thr Thr Arg
Leu Ile Leu Ile 325 330 335Gly Glu Thr Ile Lys Ile Val Ile Glu Glu
Tyr Val Gln Gln Leu Ser 340 345 350Gly Tyr Phe Leu Gln Leu Lys Phe
Asp Pro Glu Leu Leu Phe Gly Val 355 360 365Gln Phe Gln Tyr Arg Asn
Arg Ile Ala Met Glu Phe Asn His Leu Tyr 370 375 380His Trp His Pro
Leu Met Pro Asp Ser Phe Lys Val Gly Ser Gln Glu385 390 395 400Tyr
Ser Tyr Glu Gln Phe Leu Phe Asn Thr Ser Met Leu Val Asp Tyr 405 410
415Gly Val Glu Ala Leu Val Asp Ala Phe Ser Arg Gln Ile Ala Gly Arg
420 425 430Ile Gly Gly Gly Arg Asn Met Asp His His Ile Leu His Val
Ala Val 435 440 445Asp Val Ile Arg Glu Ser Arg Glu Met Arg Leu Gln
Pro Phe Asn Glu 450 455 460Tyr Arg Lys Arg Phe Gly Met Lys Pro Tyr
Thr Ser Phe Gln Glu Leu465 470 475 480Val Gly Glu Lys Glu Met Ala
Ala Glu Leu Glu Glu Leu Tyr Gly Asp 485 490 495Ile Asp Ala Leu Glu
Phe Tyr Pro Gly Leu Leu Leu Glu Lys Cys His 500 505 510Pro Asn Ser
Ile Phe Gly Glu Ser Met Ile Glu Ile Gly Ala Pro Phe 515 520 525Ser
Leu Lys Gly Leu Leu Gly Asn Pro Ile Cys Ser Pro Glu Tyr Trp 530 535
540Lys Pro Ser Thr Phe Gly Gly Glu Val Gly Phe Asn Ile Val Lys
Thr545 550 555 560Ala Thr Leu Lys Lys Leu Val Cys Leu Asn Thr Lys
Thr Cys Pro Tyr 565 570 575Val Ser Phe Arg Val Pro Asp Ala Ser Gln
Asp Asp Gly Pro Ala Val 580 585 590Glu Arg Pro Ser Thr Glu Leu
595234982DNAHomo sapienCDS(136)...(1824) 23aggtgacagc tggagggagg
agcgggggtg gagccggggg aagggtgggg aggggatggg 60ctggagctcc gggcagtgtg
cgaggcgcac gcacaggagc ctgcactctg cgtcccgcac 120cccagcagcc gcgcc atg
agc cgg agt ctc ttg ctc tgg ttc ttg ctg ttc 171 Met Ser Arg Ser Leu
Leu Leu Trp Phe Leu Leu Phe 1 5 10ctg ctc ctg ctc ccg ccg ctc ccc
gtc ctg ctc gcg gac cca ggg gcg 219Leu Leu Leu Leu Pro Pro Leu Pro
Val Leu Leu Ala Asp Pro Gly Ala 15 20 25ccc acg cca gtg aat ccc tgt
tgt tac tat cca tgc cag cac cag ggc 267Pro Thr Pro Val Asn Pro Cys
Cys Tyr Tyr Pro Cys Gln His Gln Gly 30 35 40atc tgt gtc cgc ttc ggc
ctt gac cgc tac cag tgt gac tgc acc cgc 315Ile Cys Val Arg Phe Gly
Leu Asp Arg Tyr Gln Cys Asp Cys Thr Arg45 50 55 60acg ggc tat tcc
ggc ccc aac tgc acc atc cct ggc ctg tgg acc tgg 363Thr Gly Tyr Ser
Gly Pro Asn Cys Thr Ile Pro Gly Leu Trp Thr Trp 65 70 75ctc cgg aat
tca ctg cgg ccc agc ccc tct ttc acc cac ttc ctg ctc 411Leu Arg Asn
Ser Leu Arg Pro Ser Pro Ser Phe Thr His Phe Leu Leu 80 85 90act cac
ggg cgc tgg ttc tgg gag ttt gtc aat gcc acc ttc atc cga 459Thr His
Gly Arg Trp Phe Trp Glu Phe Val Asn Ala Thr Phe Ile Arg 95 100
105gag atg ctc atg cgc ctg gta ctc aca gtg cgc tcc aac ctt atc ccc
507Glu Met Leu Met Arg Leu Val Leu Thr Val Arg Ser Asn Leu Ile Pro
110 115 120agt ccc ccc acc tac aac tca gca cat gac tac atc agc tgg
gag tct 555Ser Pro Pro Thr Tyr Asn Ser Ala His Asp Tyr Ile Ser Trp
Glu Ser125 130 135 140ttc tcc aac gtg agc tat tac act cgt att ctg
ccc tct gtg cct aaa 603Phe Ser Asn Val Ser Tyr Tyr Thr Arg Ile Leu
Pro Ser Val Pro Lys 145 150 155gat tgc ccc aca ccc atg gga acc aaa
ggg aag aag cag ttg cca gat 651Asp Cys Pro Thr Pro Met Gly Thr Lys
Gly Lys Lys Gln Leu Pro Asp 160 165 170gcc cag ctc ctg gcc cgc cgc
ttc ctg ctc agg agg aag ttc ata cct 699Ala Gln Leu Leu Ala Arg Arg
Phe Leu Leu Arg Arg Lys Phe Ile Pro 175 180 185gac ccc caa ggc acc
aac ctc atg ttt gcc ttc ttt gca caa cac ttc 747Asp Pro Gln Gly Thr
Asn Leu Met Phe Ala Phe Phe Ala Gln His Phe 190 195 200acc cac cag
ttc ttc aaa act tct ggc aag atg ggt cct ggc ttc acc 795Thr His Gln
Phe Phe Lys Thr Ser Gly Lys Met Gly Pro Gly Phe Thr205 210 215
220aag gcc ttg ggc cat ggg gta gac ctc ggc cac att tat gga gac aat
843Lys Ala Leu Gly His Gly Val Asp Leu Gly His Ile Tyr Gly Asp Asn
225 230 235ctg gag cgt cag tat caa ctg cgg ctc ttt aag gat ggg aaa
ctc aag 891Leu Glu Arg Gln Tyr Gln Leu Arg Leu Phe Lys Asp Gly Lys
Leu Lys 240 245 250tac cag gtg ctg gat gga gaa atg tac ccg ccc tcg
gta gaa gag gcg 939Tyr Gln Val Leu Asp Gly Glu Met Tyr Pro Pro Ser
Val Glu Glu Ala 255 260 265cct gtg ttg atg cac tac ccc cga ggc atc
ccg ccc cag agc cag atg 987Pro Val Leu Met His Tyr Pro Arg Gly Ile
Pro Pro Gln Ser Gln Met 270 275 280gct gtg ggc cag gag gtg ttt ggg
ctg ctt cct ggg ctc atg ctg tat 1035Ala Val Gly Gln Glu Val Phe Gly
Leu Leu Pro Gly Leu Met Leu Tyr285 290 295 300gcc acg ctc tgg cta
cgt gag cac aac cgt gtg tgt gac ctg ctg aag 1083Ala Thr Leu Trp Leu
Arg Glu His Asn Arg Val Cys Asp Leu Leu Lys 305 310 315gct gag cac
ccc acc tgg ggc gat gag cag ctt ttc cag acg acc cgc 1131Ala Glu His
Pro Thr Trp Gly Asp Glu Gln Leu Phe Gln Thr Thr Arg 320 325 330ctc
atc ctc ata ggg gag acc atc aag att gtc atc gag gag tac gtg 1179Leu
Ile Leu Ile Gly Glu Thr Ile Lys Ile Val Ile Glu Glu Tyr Val 335 340
345cag cag ctg agt ggc tat ttc ctg cag ctg aaa ttt gac cca gag ctg
1227Gln Gln Leu Ser Gly Tyr Phe Leu Gln Leu Lys Phe Asp Pro Glu Leu
350 355 360ctg ttc ggt gtc cag ttc caa tac cgc aac cgc att gcc atg
gag ttc 1275Leu Phe Gly Val Gln Phe Gln Tyr Arg Asn Arg Ile Ala Met
Glu Phe365 370 375 380aac cat ctc tac cac tgg cac ccc ctc atg cct
gac tcc ttc aag atc 1323Asn His Leu Tyr His Trp His Pro Leu Met Pro
Asp Ser Phe Lys Ile 385 390 395ggt ggg ggc agg aac atg gac cac cac
atc ctg cat gtg gct gtg gat 1371Gly Gly Gly Arg Asn Met Asp His His
Ile Leu His Val Ala Val Asp 400 405 410gtc atc agg gag tct cgg gag
atg cgg ctg cag ccc ttc aat gag tac 1419Val Ile Arg Glu Ser Arg Glu
Met Arg Leu Gln Pro Phe Asn Glu Tyr 415 420 425cgc aag agg ttt ggc
atg aaa ccc tac acc tcc ttc cag gag ctc gta 1467Arg Lys Arg Phe Gly
Met Lys Pro Tyr Thr Ser Phe Gln Glu Leu Val 430 435 440gga gag aag
gag atg gca gca gag ttg gag gaa ttg tat gga gac att 1515Gly Glu Lys
Glu Met Ala Ala Glu Leu Glu Glu Leu Tyr Gly Asp Ile445 450 455
460gat gcg ttg gag ttc tac cct gga ctg ctt ctt gaa aag tgc cat cca
1563Asp Ala Leu Glu Phe Tyr Pro Gly Leu Leu Leu Glu Lys Cys His Pro
465 470 475aac tct atc ttt ggg gag agt atg ata gag att ggg gct ccc
ttt tcc 1611Asn Ser Ile Phe Gly Glu Ser Met Ile Glu Ile Gly Ala Pro
Phe Ser 480 485 490ctc aag ggt ctc cta ggg aat ccc atc tgt tct ccg
gag tac tgg aag 1659Leu Lys Gly Leu Leu Gly Asn Pro Ile Cys Ser Pro
Glu Tyr Trp Lys 495 500 505ccg agc aca ttt ggc ggc gag gtg ggc ttt
aac att gtc aag acg gcc 1707Pro Ser Thr Phe Gly Gly Glu Val Gly Phe
Asn Ile Val Lys Thr Ala 510 515 520aca ctg aag aag ctg gtc tgc ctc
aac acc aag acc tgt ccc tac gtt 1755Thr Leu Lys Lys Leu Val Cys Leu
Asn Thr Lys Thr Cys Pro Tyr Val525 530 535 540tcc ttc cgt gtg ccg
gat gcc agt cag gat gat ggg cct gct gtg gag 1803Ser Phe Arg Val Pro
Asp Ala Ser Gln Asp Asp Gly Pro Ala Val Glu 545 550 555cga cca tcc
aca gag ctc tga ggggcaggaa agcagcattc tggaggggag 1854Arg Pro Ser
Thr Glu Leu * 560agctttgtgc ttgtcattcc agagtgctga ggccagggct
gatggtctta aatgctcatt 1914ttctggtttg gcatggtgag tgttggggtt
gacatttaga actttaagtc tcacccatta 1974tctggaatat tgtgattctg
tttattcttc cagaatgctg aactccttgt tagcccttca 2034gattgttagg
agtggttctc atttggtctg ccagaatact gggttcttag ttgacaacct
2094agaatgtcag atttctggtt gatttgtaac acagtcattc taggatgtgg
agctactgat 2154gaaatctgct agaaagttag ggggttctta ttttgcattc
cagaatcttg actttctgat 2214tggtgattca aagtgttgtg ttcctggctg
atgatccaga acagtggctc gtatcccaaa 2274tctgtcagca tctggctgtc
tagaatgtgg atttgattca ttttcctgtt cagtgagata 2334tcatagagac
ggagatccta aggtccaaca agaatgcatt ccctgaatct gtgcctgcac
2394tgagagggca aggaagtggg gtgttcttct tgggaccccc actaagaccc
tggtctgagg 2454atgtagagag aacaggtggg ctgtattcac gccattggtt
ggaagctacc agagctctat 2514ccccatccag gtcttgactc atggcagctg
tttctcatga agctaataaa attcgctttc 2574taaagttacc tgttatatat
ctcttttggt cccatcctct aaagcagagg caacactgga 2634acatggctag
cctttcttgt agccatggct gggcgtgcta gaggttgcag catgagactt
2694tctgctggga tccttgggcc catcactgta tagacatgct accactggta
cttcctttct 2754ccctgcgggc caggcactgc ccttttcagg aagctctctt
aaaataccca ttgccccaga 2814cctggaagat ataacattca gttcccacca
tctgattaaa acaacttcct cccttacaga 2874gcatacaaca gagggggcac
ccggggagga gagcacatac tgtgttccaa tttcacgctt 2934ttaattctca
tttgttctca caccaacagt gtgaagtgcg tggtataatc tccatttcaa
2994aaccaaggaa gcagcctcag agtggtcgag tgacacacct cacgcaggct
gagtccagag 3054cttgtgctcc tcttgattcc tggtttgact cagttccagg
cctgatcttg cctgtctggc 3114tcagggtcaa agacagaatg gtggagtgta
gcctccacct gatattcagg ctactcattc 3174agtcccaaat atgtattttc
ctaagtgttt actatgtgcc agttcctgta acaggtgtgg 3234ggacacagca
gtgagtaatc aatacagaca aggttctgcc cttatggagc tcacactcca
3294gtggcagaca aacagaccat aaataaggaa acgatgaaat aagatatata
caaggtgagt 3354gtgacttccc ttctaacccc ctctgctctg tcctccccta
ttgcgctctc aagaccagag 3414acccaacagc agtgatctca gggcagacag
ccctccactc cagctctgag acccttttct 3474caggacctct gtaggcagca
gagagagagg acagaggggt aagatgaggg gttgagggaa 3534ggttcttcat
gatccacact ttgggcttag tatttctcag gaagagctat ggcccagaaa
3594caacagggga aactagagtt cggtctgaca gtccttgggg ttaagtctcc
tgtcttatgg 3654tccagaaact cctgtttctc cttagttggc tggaaactgc
tcccatcatt ccttctggcc 3714tctgctgaat gcagggaatg caatccttcc
ctgctcttgc agttgctctg acgtagaaag 3774atccttcggg tgctggaagt
ctccatgaag agcttgtgtc ctgtcctttc ttgcagattc 3834tatttcccct
cttctgctaa tacctcttac tttgcttgag aatcctctcc tttcttatta
3894atttcagtct tggtggttct atcaggggtg cattctggcc aaggggtggg
cctgtgaatc 3954aatcctgggc aatcagacac cctctcctta aaaactggcc
cgtggagact gagatcactg 4014actctgactc atccccacag ctggctctga
caagatggtc catttgttcc tgcttccgag 4074atccccaggg cagcctggat
ccctgccctt ctcaagactt tagcttttcc ttccatccgg 4134tggcctattc
caggaattcc tcttttgctt aaatcagttg gagtttgtgt ctgttgcttg
4194taatcaagcc tttatggctg ctgggctgag tgacacaagc actttaatgg
cctggaggga 4254cttttaatca gtgaagatgc aatcagacaa gtgttttgga
aagagcaccc tcgagaaggg 4314tggatgacag ggcagagcag gaaggacagg
aagctggcag aacggaggag gctgcagccg 4374tggtccaacc aggagctgat
ggcagctggg gctaggggaa gggctttgag ggtggaagga 4434tgggatgggt
tccagaggta ttcctctctt aaatgcaagt gcctagatta ggtagacttt
4494gcttagtatt gacaactgca catgaaagtt ttgcaaaggg aaacaggcta
aatgcaccaa 4554gaaagcttct tcagagtgaa gaatcttaat gcttgtaatt
taaacatttg ttcctggagt 4614tttgatttgg tggatgtgat ggttggtttt
atttgtcagt ttggttgggc tatagcacac 4674agttatttaa tcaaacagta
atctaggtgt ggctgtgaag gtattttgta gatgtgatta 4734acatctacaa
tcagttgact ttaagtgaaa gagattactt aaataatttg ggtgagctgc
4794acctgattag ttgaaaggcc tcaagaacaa acactgcagt ttcctggaaa
agaagaaact 4854ttgcctcaag actatagcca tcgactcctg cctgagtttc
cagcctgcta gtctgcccta 4914tggatttgaa gtttgccaac cccaacaatt
gtgtgaatta atttctaaaa ataaagctat 4974atacagcc 498224562PRTHomo
sapien 24Met Ser Arg Ser Leu Leu Leu Trp Phe Leu Leu Phe Leu Leu
Leu Leu1 5 10 15Pro Pro Leu Pro Val Leu Leu Ala Asp Pro Gly Ala Pro
Thr Pro Val 20 25 30Asn Pro Cys Cys Tyr Tyr Pro Cys Gln His Gln Gly
Ile Cys Val Arg 35 40 45Phe Gly Leu Asp Arg Tyr Gln Cys Asp Cys Thr
Arg Thr Gly Tyr Ser 50 55 60Gly Pro Asn Cys Thr Ile Pro Gly Leu Trp
Thr Trp Leu Arg Asn Ser65 70 75 80Leu Arg Pro Ser Pro Ser Phe Thr
His Phe Leu
Leu Thr His Gly Arg 85 90 95Trp Phe Trp Glu Phe Val Asn Ala Thr Phe
Ile Arg Glu Met Leu Met 100 105 110Arg Leu Val Leu Thr Val Arg Ser
Asn Leu Ile Pro Ser Pro Pro Thr 115 120 125Tyr Asn Ser Ala His Asp
Tyr Ile Ser Trp Glu Ser Phe Ser Asn Val 130 135 140Ser Tyr Tyr Thr
Arg Ile Leu Pro Ser Val Pro Lys Asp Cys Pro Thr145 150 155 160Pro
Met Gly Thr Lys Gly Lys Lys Gln Leu Pro Asp Ala Gln Leu Leu 165 170
175Ala Arg Arg Phe Leu Leu Arg Arg Lys Phe Ile Pro Asp Pro Gln Gly
180 185 190Thr Asn Leu Met Phe Ala Phe Phe Ala Gln His Phe Thr His
Gln Phe 195 200 205Phe Lys Thr Ser Gly Lys Met Gly Pro Gly Phe Thr
Lys Ala Leu Gly 210 215 220His Gly Val Asp Leu Gly His Ile Tyr Gly
Asp Asn Leu Glu Arg Gln225 230 235 240Tyr Gln Leu Arg Leu Phe Lys
Asp Gly Lys Leu Lys Tyr Gln Val Leu 245 250 255Asp Gly Glu Met Tyr
Pro Pro Ser Val Glu Glu Ala Pro Val Leu Met 260 265 270His Tyr Pro
Arg Gly Ile Pro Pro Gln Ser Gln Met Ala Val Gly Gln 275 280 285Glu
Val Phe Gly Leu Leu Pro Gly Leu Met Leu Tyr Ala Thr Leu Trp 290 295
300Leu Arg Glu His Asn Arg Val Cys Asp Leu Leu Lys Ala Glu His
Pro305 310 315 320Thr Trp Gly Asp Glu Gln Leu Phe Gln Thr Thr Arg
Leu Ile Leu Ile 325 330 335Gly Glu Thr Ile Lys Ile Val Ile Glu Glu
Tyr Val Gln Gln Leu Ser 340 345 350Gly Tyr Phe Leu Gln Leu Lys Phe
Asp Pro Glu Leu Leu Phe Gly Val 355 360 365Gln Phe Gln Tyr Arg Asn
Arg Ile Ala Met Glu Phe Asn His Leu Tyr 370 375 380His Trp His Pro
Leu Met Pro Asp Ser Phe Lys Ile Gly Gly Gly Arg385 390 395 400Asn
Met Asp His His Ile Leu His Val Ala Val Asp Val Ile Arg Glu 405 410
415Ser Arg Glu Met Arg Leu Gln Pro Phe Asn Glu Tyr Arg Lys Arg Phe
420 425 430Gly Met Lys Pro Tyr Thr Ser Phe Gln Glu Leu Val Gly Glu
Lys Glu 435 440 445Met Ala Ala Glu Leu Glu Glu Leu Tyr Gly Asp Ile
Asp Ala Leu Glu 450 455 460Phe Tyr Pro Gly Leu Leu Leu Glu Lys Cys
His Pro Asn Ser Ile Phe465 470 475 480Gly Glu Ser Met Ile Glu Ile
Gly Ala Pro Phe Ser Leu Lys Gly Leu 485 490 495Leu Gly Asn Pro Ile
Cys Ser Pro Glu Tyr Trp Lys Pro Ser Thr Phe 500 505 510Gly Gly Glu
Val Gly Phe Asn Ile Val Lys Thr Ala Thr Leu Lys Lys 515 520 525Leu
Val Cys Leu Asn Thr Lys Thr Cys Pro Tyr Val Ser Phe Arg Val 530 535
540Pro Asp Ala Ser Gln Asp Asp Gly Pro Ala Val Glu Arg Pro Ser
Thr545 550 555 560Glu Leu25607DNAHomo sapienCDS(114)...(398)
25ggactgttga agacaggtct ccacacacag ctccagcagc cacatttgca accttggcca
60tctgtccaga acctgctccc acctcaggcc caggccaacc gtgcactgct gca atg
116 Met 1ggc tct gag ctg gag acg gcg atg gag acc ctc atc aac gtg
ttc cac 164Gly Ser Glu Leu Glu Thr Ala Met Glu Thr Leu Ile Asn Val
Phe His 5 10 15gcc cac tcg ggc aaa gag ggg gac aag tac aag ctg agc
aag aag gag 212Ala His Ser Gly Lys Glu Gly Asp Lys Tyr Lys Leu Ser
Lys Lys Glu 20 25 30ctg aaa gag ctg ctg cag acg gag ctc tct ggc ttc
ctg gat gcc cag 260Leu Lys Glu Leu Leu Gln Thr Glu Leu Ser Gly Phe
Leu Asp Ala Gln 35 40 45aag gat gtg gat gct gtg gac aag gtg atg aag
gag cta gac gag aat 308Lys Asp Val Asp Ala Val Asp Lys Val Met Lys
Glu Leu Asp Glu Asn50 55 60 65gga gac ggg gag gtg gac ttc cag gag
tat gtg gtg ctt gtg gct gct 356Gly Asp Gly Glu Val Asp Phe Gln Glu
Tyr Val Val Leu Val Ala Ala 70 75 80ctc aca gtg gcc tgt aac aat ttc
ttc tgg gag aac agt tga 398Leu Thr Val Ala Cys Asn Asn Phe Phe Trp
Glu Asn Ser * 85 90gcagacagcc acattgggca gcgcccttcc tctccaccct
cccagacctg cctcttcccc 458ctgcttccac ctcaccccac ttatccctct
ccataacccc acccttgccc accccacccc 518cacccccacc aagggcgcaa
gagtagcggt ccaagcctgc aactcatctt tcattaaagg 578cttctctctc
accagcaaaa aaaaaaaaa 6072694PRTHomo sapien 26Met Gly Ser Glu Leu
Glu Thr Ala Met Glu Thr Leu Ile Asn Val Phe 1 5 10 15His Ala His
Ser Gly Lys Glu Gly Asp Lys Tyr Lys Leu Ser Lys Lys 20 25 30Glu Leu
Lys Glu Leu Leu Gln Thr Glu Leu Ser Gly Phe Leu Asp Ala 35 40 45Gln
Lys Asp Val Asp Ala Val Asp Lys Val Met Lys Glu Leu Asp Glu 50 55
60Asn Gly Asp Gly Glu Val Asp Phe Gln Glu Tyr Val Val Leu Val Ala65
70 75 80Ala Leu Thr Val Ala Cys Asn Asn Phe Phe Trp Glu Asn Ser 85
9027517DNAHomo sapienCDS(65)...(352) 27cctccacagc aacttccttg
atccctgcca cgcacgactg aacacagaca gcagccgcct 60cgcc atg aag ctg ctg
atg gtc ctc atg ctg gcg gcc ctc ctc ctg cac 109 Met Lys Leu Leu Met
Val Leu Met Leu Ala Ala Leu Leu Leu His 1 5 10 15tgc tat gca gat
tct ggc tgc aaa ctc ctg gag gac atg gtt gaa aag 157Cys Tyr Ala Asp
Ser Gly Cys Lys Leu Leu Glu Asp Met Val Glu Lys 20 25 30acc atc aat
tcc gac ata tct ata cct gaa tac aaa gag ctt ctt caa 205Thr Ile Asn
Ser Asp Ile Ser Ile Pro Glu Tyr Lys Glu Leu Leu Gln 35 40 45gag ttc
ata gac agt gat gcc gct gca gag gct atg ggg aaa ttc aag 253Glu Phe
Ile Asp Ser Asp Ala Ala Ala Glu Ala Met Gly Lys Phe Lys 50 55 60cag
tgt ttc ctc aac cag tca cat aga act ctg aaa aac ttt gga ctg 301Gln
Cys Phe Leu Asn Gln Ser His Arg Thr Leu Lys Asn Phe Gly Leu 65 70
75atg atg cat aca gtg tac gac agc att tgg tgt aat atg aag agt aat
349Met Met His Thr Val Tyr Asp Ser Ile Trp Cys Asn Met Lys Ser
Asn80 85 90 95taa ctttacccaa ggcgtttggc tcagagggct acagactatg
gccagaactc 402*atctgttgat tgctagaaac cacttttctt tcttgtgttg
tctttttatg tggaaactgc 462tagacaactg ttgaaacctc aaattcattt
ccatttcaat aactaactgc aaatc 5172895PRTHomo sapien 28Met Lys Leu Leu
Met Val Leu Met Leu Ala Ala Leu Leu Leu His Cys 1 5 10 15Tyr Ala
Asp Ser Gly Cys Lys Leu Leu Glu Asp Met Val Glu Lys Thr 20 25 30Ile
Asn Ser Asp Ile Ser Ile Pro Glu Tyr Lys Glu Leu Leu Gln Glu 35 40
45Phe Ile Asp Ser Asp Ala Ala Ala Glu Ala Met Gly Lys Phe Lys Gln
50 55 60Cys Phe Leu Asn Gln Ser His Arg Thr Leu Lys Asn Phe Gly Leu
Met65 70 75 80Met His Thr Val Tyr Asp Ser Ile Trp Cys Asn Met Lys
Ser Asn 85 90 95292192DNAHomo sapienCDS(101)...(2044) 29agtctggccc
tggacaaccc cagcaaagcc gccctcagcc agcccagaag cactgggcct 60tggccacagc
aacacccact gagcacgctg ggagctgagt atg gcg tcc ctg gtc 115 Met Ala
Ser Leu Val 1 5tcg ctg gag ctg ggg ctg ctt ctg gct gtg ctg gtg gtg
acg gcg acg 163Ser Leu Glu Leu Gly Leu Leu Leu Ala Val Leu Val Val
Thr Ala Thr 10 15 20gcg tcc ccg cct gct ggt ctg ctg agc ctg ctc acc
tct ggc cag ggc 211Ala Ser Pro Pro Ala Gly Leu Leu Ser Leu Leu Thr
Ser Gly Gln Gly 25 30 35gct ctg gat caa gag gct ctg ggc ggc ctg tta
aat acg ctg gcg gac 259Ala Leu Asp Gln Glu Ala Leu Gly Gly Leu Leu
Asn Thr Leu Ala Asp 40 45 50cgt gtg cac tgc acc aac ggg ccg tgt gga
aag tgc ctg tct gtg gag 307Arg Val His Cys Thr Asn Gly Pro Cys Gly
Lys Cys Leu Ser Val Glu 55 60 65gac gcc ctg ggc ctg ggc gag cct gag
ggg tca ggg ctg ccc ccg ggc 355Asp Ala Leu Gly Leu Gly Glu Pro Glu
Gly Ser Gly Leu Pro Pro Gly70 75 80 85ccg gtc ctg gag gcc agg tac
gtc gcc cgc ctc agt gcc gcc gcc gtc 403Pro Val Leu Glu Ala Arg Tyr
Val Ala Arg Leu Ser Ala Ala Ala Val 90 95 100ctg tac ctc agc aac
ccc gag ggc acc tgt gag gac act cgg gct ggc 451Leu Tyr Leu Ser Asn
Pro Glu Gly Thr Cys Glu Asp Thr Arg Ala Gly 105 110 115ctc tgg gcc
tct cat gca gac cac ctc ctg gcc ctg ctc gag agc ccc 499Leu Trp Ala
Ser His Ala Asp His Leu Leu Ala Leu Leu Glu Ser Pro 120 125 130aag
gcc ctg acc ccg ggc ctg agc tgg ctg ctg cag agg atg cag gcc 547Lys
Ala Leu Thr Pro Gly Leu Ser Trp Leu Leu Gln Arg Met Gln Ala 135 140
145cgg gct gcc ggc cag acc ccc aag acg gcc tgc gta gat atc cct cag
595Arg Ala Ala Gly Gln Thr Pro Lys Thr Ala Cys Val Asp Ile Pro
Gln150 155 160 165ctg ctg gag gag gcg gtg ggg gcg ggg gct ccg ggc
agt gct ggc ggc 643Leu Leu Glu Glu Ala Val Gly Ala Gly Ala Pro Gly
Ser Ala Gly Gly 170 175 180gtc ctg gct gcc ctg ctg gac cat gtc agg
agc ggg tct tgc ttc cac 691Val Leu Ala Ala Leu Leu Asp His Val Arg
Ser Gly Ser Cys Phe His 185 190 195gcc ttg ccg agc cct cag tac ttc
gtg gac ttt gtg ttc cag cag cac 739Ala Leu Pro Ser Pro Gln Tyr Phe
Val Asp Phe Val Phe Gln Gln His 200 205 210agc agc gag gtc cct atg
acg ctg gcc gag ctg tca gcc ttg atg cag 787Ser Ser Glu Val Pro Met
Thr Leu Ala Glu Leu Ser Ala Leu Met Gln 215 220 225cgc ctg ggg gtg
ggc agg gag gcc cac agt gac cac agt cat cgg cac 835Arg Leu Gly Val
Gly Arg Glu Ala His Ser Asp His Ser His Arg His230 235 240 245agg
gga gcc agc agc cgg gac cct gtg ccc ctc atc agc tcc agc aac 883Arg
Gly Ala Ser Ser Arg Asp Pro Val Pro Leu Ile Ser Ser Ser Asn 250 255
260agc tcc agt gtg tgg gac acg gta tgc ctg agt gcc agg gac gtg atg
931Ser Ser Ser Val Trp Asp Thr Val Cys Leu Ser Ala Arg Asp Val Met
265 270 275gct gca tat gga ctg tcg gaa cag gct ggg gtg acc ccg gag
gcc tgg 979Ala Ala Tyr Gly Leu Ser Glu Gln Ala Gly Val Thr Pro Glu
Ala Trp 280 285 290gcc caa ctg agc cct gcc ctg ctc caa cag cag ctg
agt gga gcc tgc 1027Ala Gln Leu Ser Pro Ala Leu Leu Gln Gln Gln Leu
Ser Gly Ala Cys 295 300 305acc tcc cag tcc agg ccc ccc gtc cag gac
cag ctc agc cag tca gag 1075Thr Ser Gln Ser Arg Pro Pro Val Gln Asp
Gln Leu Ser Gln Ser Glu310 315 320 325agg tat ctg tac ggc tcc ctg
gcc acg ctg ctc atc tgc ctc tgc gcg 1123Arg Tyr Leu Tyr Gly Ser Leu
Ala Thr Leu Leu Ile Cys Leu Cys Ala 330 335 340gtc ttt ggc ctc ctg
ctg ctg acc tgc act ggc tgc agg ggg gtc gcc 1171Val Phe Gly Leu Leu
Leu Leu Thr Cys Thr Gly Cys Arg Gly Val Ala 345 350 355cac tac atc
ctg cag acc ttc ctg agc ctg gca gtg ggt gca ctc act 1219His Tyr Ile
Leu Gln Thr Phe Leu Ser Leu Ala Val Gly Ala Leu Thr 360 365 370ggg
gac gct gtc ctg cat ctg acg ccc aag gtg ctg ggg ctg cat aca 1267Gly
Asp Ala Val Leu His Leu Thr Pro Lys Val Leu Gly Leu His Thr 375 380
385cac agc gaa gag ggc ctc agc cca cag ccc acc tgg cgc ctc ctg gct
1315His Ser Glu Glu Gly Leu Ser Pro Gln Pro Thr Trp Arg Leu Leu
Ala390 395 400 405atg ctg gcc ggg ctc tac gcc ttc ttc ctg ttt gag
aac ctc ttc aat 1363Met Leu Ala Gly Leu Tyr Ala Phe Phe Leu Phe Glu
Asn Leu Phe Asn 410 415 420ctc ctg ctg ccc agg gac ccg gag gac ctg
gag gac ggg ccc tgc ggc 1411Leu Leu Leu Pro Arg Asp Pro Glu Asp Leu
Glu Asp Gly Pro Cys Gly 425 430 435cac agc agc cat agc cac ggg ggc
cac agc cac ggt gtg tcc ctg cag 1459His Ser Ser His Ser His Gly Gly
His Ser His Gly Val Ser Leu Gln 440 445 450ctg gca ccc agc gag ctc
cgg cag ccc aag ccc ccc cac gag ggc tcc 1507Leu Ala Pro Ser Glu Leu
Arg Gln Pro Lys Pro Pro His Glu Gly Ser 455 460 465cgc gca gac ctg
gtg gcg gag gag agc ccg gag ctg ctg aac cct gag 1555Arg Ala Asp Leu
Val Ala Glu Glu Ser Pro Glu Leu Leu Asn Pro Glu470 475 480 485ccc
agg aga ctg agc cca gag ttg agg cta ctg ccc tat atg atc act 1603Pro
Arg Arg Leu Ser Pro Glu Leu Arg Leu Leu Pro Tyr Met Ile Thr 490 495
500ctg ggc gac gcc gtg cac aac ttc gcc gac ggg ctg gcc gtg ggc gcc
1651Leu Gly Asp Ala Val His Asn Phe Ala Asp Gly Leu Ala Val Gly Ala
505 510 515gcc ttc gcg tcc tcc tgg aag acc ggg ctg gcc acc tcg ctg
gcc gtg 1699Ala Phe Ala Ser Ser Trp Lys Thr Gly Leu Ala Thr Ser Leu
Ala Val 520 525 530ttc tgc cac gag ttg cca cac gag ctg ggg gac ttc
gcc gcc ttg ctg 1747Phe Cys His Glu Leu Pro His Glu Leu Gly Asp Phe
Ala Ala Leu Leu 535 540 545cac gcg ggg ctg tcc gtg cgc caa gca ctg
ctg ctg aac ctg gcc tcc 1795His Ala Gly Leu Ser Val Arg Gln Ala Leu
Leu Leu Asn Leu Ala Ser550 555 560 565gcg ctc acg gcc ttc gct ggt
ctc tac gtg gca ctc gcg gtt gga gtc 1843Ala Leu Thr Ala Phe Ala Gly
Leu Tyr Val Ala Leu Ala Val Gly Val 570 575 580agc gag gag agc gag
gcc tgg atc ctg gca gtg gcc acc ggc ctg ttc 1891Ser Glu Glu Ser Glu
Ala Trp Ile Leu Ala Val Ala Thr Gly Leu Phe 585 590 595ctc tac gta
gca ctc tgc gac atg ctc ccg gcg atg ttg aaa gta cgg 1939Leu Tyr Val
Ala Leu Cys Asp Met Leu Pro Ala Met Leu Lys Val Arg 600 605 610gac
ccg cgg ccc tgg ctc ctc ttc ctg ctg cac aac gtg ggc ctg ctg 1987Asp
Pro Arg Pro Trp Leu Leu Phe Leu Leu His Asn Val Gly Leu Leu 615 620
625ggc ggc tgg acc gtc ctg ctg ctg ctg tcc ctg tac gag gat gac atc
2035Gly Gly Trp Thr Val Leu Leu Leu Leu Ser Leu Tyr Glu Asp Asp
Ile630 635 640 645acc ttc tga taccctgccc tagtccccca cctttgactt
aagatcccac 2084Thr Phe *acctcacaaa cctacagccc agaaaccaga agcccctata
gaggccccag tcccaactcc 2144agtaaagaca ctcttgtcct tggaaaaaaa
aaaaaaaaaa aaaaaaaa 219230647PRTHomo sapien 30Met Ala Ser Leu Val
Ser Leu Glu Leu Gly Leu Leu Leu Ala Val Leu1 5 10 15Val Val Thr Ala
Thr Ala Ser Pro Pro Ala Gly Leu Leu Ser Leu Leu 20 25 30Thr Ser Gly
Gln Gly Ala Leu Asp Gln Glu Ala Leu Gly Gly Leu Leu 35 40 45Asn Thr
Leu Ala Asp Arg Val His Cys Thr Asn Gly Pro Cys Gly Lys 50 55 60Cys
Leu Ser Val Glu Asp Ala Leu Gly Leu Gly Glu Pro Glu Gly Ser65 70 75
80Gly Leu Pro Pro Gly Pro Val Leu Glu Ala Arg Tyr Val Ala Arg Leu
85 90 95Ser Ala Ala Ala Val Leu Tyr Leu Ser Asn Pro Glu Gly Thr Cys
Glu 100 105 110Asp Thr Arg Ala Gly Leu Trp Ala Ser His Ala Asp His
Leu Leu Ala 115 120 125Leu Leu Glu Ser Pro Lys Ala Leu Thr Pro Gly
Leu Ser Trp Leu Leu 130 135 140Gln Arg Met Gln Ala Arg Ala Ala Gly
Gln Thr Pro Lys Thr Ala Cys145 150 155 160Val Asp Ile Pro Gln Leu
Leu Glu Glu Ala Val Gly Ala Gly Ala Pro 165 170 175Gly Ser Ala Gly
Gly Val Leu Ala Ala Leu Leu Asp His Val Arg Ser 180 185 190Gly Ser
Cys Phe His Ala Leu Pro Ser Pro Gln Tyr Phe Val Asp Phe 195 200
205Val Phe Gln Gln His Ser Ser Glu Val Pro Met Thr Leu Ala Glu Leu
210 215 220Ser Ala Leu Met Gln Arg Leu Gly Val Gly Arg Glu Ala His
Ser Asp225 230 235 240His Ser His Arg His Arg Gly Ala Ser Ser Arg
Asp Pro Val Pro Leu 245 250 255Ile Ser Ser Ser Asn Ser Ser Ser Val
Trp Asp Thr Val Cys Leu Ser 260 265
270Ala Arg Asp Val Met Ala Ala Tyr Gly Leu Ser Glu Gln Ala Gly Val
275 280 285Thr Pro Glu Ala Trp Ala Gln Leu Ser Pro Ala Leu Leu Gln
Gln Gln 290 295 300Leu Ser Gly Ala Cys Thr Ser Gln Ser Arg Pro Pro
Val Gln Asp Gln305 310 315 320Leu Ser Gln Ser Glu Arg Tyr Leu Tyr
Gly Ser Leu Ala Thr Leu Leu 325 330 335Ile Cys Leu Cys Ala Val Phe
Gly Leu Leu Leu Leu Thr Cys Thr Gly 340 345 350Cys Arg Gly Val Ala
His Tyr Ile Leu Gln Thr Phe Leu Ser Leu Ala 355 360 365Val Gly Ala
Leu Thr Gly Asp Ala Val Leu His Leu Thr Pro Lys Val 370 375 380Leu
Gly Leu His Thr His Ser Glu Glu Gly Leu Ser Pro Gln Pro Thr385 390
395 400Trp Arg Leu Leu Ala Met Leu Ala Gly Leu Tyr Ala Phe Phe Leu
Phe 405 410 415Glu Asn Leu Phe Asn Leu Leu Leu Pro Arg Asp Pro Glu
Asp Leu Glu 420 425 430Asp Gly Pro Cys Gly His Ser Ser His Ser His
Gly Gly His Ser His 435 440 445Gly Val Ser Leu Gln Leu Ala Pro Ser
Glu Leu Arg Gln Pro Lys Pro 450 455 460Pro His Glu Gly Ser Arg Ala
Asp Leu Val Ala Glu Glu Ser Pro Glu465 470 475 480Leu Leu Asn Pro
Glu Pro Arg Arg Leu Ser Pro Glu Leu Arg Leu Leu 485 490 495Pro Tyr
Met Ile Thr Leu Gly Asp Ala Val His Asn Phe Ala Asp Gly 500 505
510Leu Ala Val Gly Ala Ala Phe Ala Ser Ser Trp Lys Thr Gly Leu Ala
515 520 525Thr Ser Leu Ala Val Phe Cys His Glu Leu Pro His Glu Leu
Gly Asp 530 535 540Phe Ala Ala Leu Leu His Ala Gly Leu Ser Val Arg
Gln Ala Leu Leu545 550 555 560Leu Asn Leu Ala Ser Ala Leu Thr Ala
Phe Ala Gly Leu Tyr Val Ala 565 570 575Leu Ala Val Gly Val Ser Glu
Glu Ser Glu Ala Trp Ile Leu Ala Val 580 585 590Ala Thr Gly Leu Phe
Leu Tyr Val Ala Leu Cys Asp Met Leu Pro Ala 595 600 605Met Leu Lys
Val Arg Asp Pro Arg Pro Trp Leu Leu Phe Leu Leu His 610 615 620Asn
Val Gly Leu Leu Gly Gly Trp Thr Val Leu Leu Leu Leu Ser Leu625 630
635 640Tyr Glu Asp Asp Ile Thr Phe 645311910DNAHomo
sapienCDS(101)...(1762) 31agtctggccc tggacaaccc cagcaaagcc
gccctcagcc agcccagaag cactgggcct 60tggccacagc aacacccact gagcacgctg
ggagctgagt atg gcg tcc ctg gtc 115 Met Ala Ser Leu Val 1 5tcg ctg
gag ctg gga ctg ctt ctg gct gtg ctg gtg gtg acg gcg acg 163Ser Leu
Glu Leu Gly Leu Leu Leu Ala Val Leu Val Val Thr Ala Thr 10 15 20gcg
tcc ccg cct gct ggt ctg ctg agc ctg ctc acc tct ggc cag ggc 211Ala
Ser Pro Pro Ala Gly Leu Leu Ser Leu Leu Thr Ser Gly Gln Gly 25 30
35gct ctg gat caa gag gct ctg ggc ggc ctg tta aat acg ctg gcg gac
259Ala Leu Asp Gln Glu Ala Leu Gly Gly Leu Leu Asn Thr Leu Ala Asp
40 45 50cgt gtg cac tgc gcc aac ggg ccg tgt gga aag gcc tgc gta gat
atc 307Arg Val His Cys Ala Asn Gly Pro Cys Gly Lys Ala Cys Val Asp
Ile 55 60 65cct cag ctg ctg gag gag gcg gtg ggg gcg ggg gct ccg ggc
agt gct 355Pro Gln Leu Leu Glu Glu Ala Val Gly Ala Gly Ala Pro Gly
Ser Ala70 75 80 85ggc ggc gtc ctg gct gcc ctg ctg gac cat gtc agg
agc ggg tct tgc 403Gly Gly Val Leu Ala Ala Leu Leu Asp His Val Arg
Ser Gly Ser Cys 90 95 100ttc cac gcc ttg ccg agc cct cag tac ttc
gtg gac ttt gtg ttc cag 451Phe His Ala Leu Pro Ser Pro Gln Tyr Phe
Val Asp Phe Val Phe Gln 105 110 115cag cac agc agc gag gtc cct atg
acg ctg gcc gag ctg tca gcc ttg 499Gln His Ser Ser Glu Val Pro Met
Thr Leu Ala Glu Leu Ser Ala Leu 120 125 130atg cag cgc ctg ggg gtg
ggc agg gag gcc cac agt gac cac agt cat 547Met Gln Arg Leu Gly Val
Gly Arg Glu Ala His Ser Asp His Ser His 135 140 145cgg cac agg gga
gcc agc agc cgg gac cct gtg ccc ctc atc agc tcc 595Arg His Arg Gly
Ala Ser Ser Arg Asp Pro Val Pro Leu Ile Ser Ser150 155 160 165agc
aac agc tcc agt gtg tgg gac acg gta tgc ctg agt gcc agg gac 643Ser
Asn Ser Ser Ser Val Trp Asp Thr Val Cys Leu Ser Ala Arg Asp 170 175
180gtg atg gct gca tat gga ctg tcg gaa cag gct ggg gtg acc ccg gag
691Val Met Ala Ala Tyr Gly Leu Ser Glu Gln Ala Gly Val Thr Pro Glu
185 190 195gcc tgg gcc caa ctg agc cct gcc ctg ctc caa cag cag ctg
agt gga 739Ala Trp Ala Gln Leu Ser Pro Ala Leu Leu Gln Gln Gln Leu
Ser Gly 200 205 210gcc tgc acc tcc cag tcc agg ccc ccc gtc cag gac
cag ctc agc cag 787Ala Cys Thr Ser Gln Ser Arg Pro Pro Val Gln Asp
Gln Leu Ser Gln 215 220 225tca gag agg tat ctg tac ggc tcc ctg gcc
acg ctg ctc atc tgc ctc 835Ser Glu Arg Tyr Leu Tyr Gly Ser Leu Ala
Thr Leu Leu Ile Cys Leu230 235 240 245tgc gcg gtc ttt ggc ctc ctg
ctg ctg acc tgc act ggc tgc agg ggg 883Cys Ala Val Phe Gly Leu Leu
Leu Leu Thr Cys Thr Gly Cys Arg Gly 250 255 260gtc acc cac tac atc
ctg cag acc ttc ctg agc ctg gca gtg ggt gca 931Val Thr His Tyr Ile
Leu Gln Thr Phe Leu Ser Leu Ala Val Gly Ala 265 270 275gtc act ggg
gac gct gtc ctg cat ctg acg ccc aag gtg ctg ggg ctg 979Val Thr Gly
Asp Ala Val Leu His Leu Thr Pro Lys Val Leu Gly Leu 280 285 290cat
aca cac agc gaa gag ggc ctc agc cca cag ccc acc tgg cgc ctc 1027His
Thr His Ser Glu Glu Gly Leu Ser Pro Gln Pro Thr Trp Arg Leu 295 300
305ctg gct atg ctg gcc ggg ctc tac gcc ttc ttc ctg ttt gag aac ctc
1075Leu Ala Met Leu Ala Gly Leu Tyr Ala Phe Phe Leu Phe Glu Asn
Leu310 315 320 325ttc aat ctc ctg ctg ccc agg gac ccg gag gac ctg
gag gac ggg ccc 1123Phe Asn Leu Leu Leu Pro Arg Asp Pro Glu Asp Leu
Glu Asp Gly Pro 330 335 340tgc ggc cac agc agc cat agc cac ggg ggc
cac agc cac ggt gtg tcc 1171Cys Gly His Ser Ser His Ser His Gly Gly
His Ser His Gly Val Ser 345 350 355ctg cag ctg gca ccc agc gag ctc
cgg cag ccc aag ccc ccc cac gag 1219Leu Gln Leu Ala Pro Ser Glu Leu
Arg Gln Pro Lys Pro Pro His Glu 360 365 370ggc tcc cgc gca gac ctg
gtg gcg gag gag agc ccg gag ctg ctg aac 1267Gly Ser Arg Ala Asp Leu
Val Ala Glu Glu Ser Pro Glu Leu Leu Asn 375 380 385cct gag ccc agg
aga ctg agc cca gag ttg agg cta ctg ccc tat atg 1315Pro Glu Pro Arg
Arg Leu Ser Pro Glu Leu Arg Leu Leu Pro Tyr Met390 395 400 405atc
act ctg ggc gac gcc gtg cac aac ttc gcc gac ggg ctg gcc gtg 1363Ile
Thr Leu Gly Asp Ala Val His Asn Phe Ala Asp Gly Leu Ala Val 410 415
420ggc gcc gcc ttc gcg tcc tcc tgg aag acc ggg ctg gcc acc tcg ctg
1411Gly Ala Ala Phe Ala Ser Ser Trp Lys Thr Gly Leu Ala Thr Ser Leu
425 430 435gcc gtg ttc tgc cac gag ttg cca cac gag ctg ggg gac ttc
gcc gcc 1459Ala Val Phe Cys His Glu Leu Pro His Glu Leu Gly Asp Phe
Ala Ala 440 445 450ttg ctg cac gcg ggg ctg tcc gtg cgc caa gca ctg
ctg ctg aac ctg 1507Leu Leu His Ala Gly Leu Ser Val Arg Gln Ala Leu
Leu Leu Asn Leu 455 460 465gcc tcc gcg ctc acg gcc ttc gct ggt ctc
tac gtg gca ctc gcg gtt 1555Ala Ser Ala Leu Thr Ala Phe Ala Gly Leu
Tyr Val Ala Leu Ala Val470 475 480 485gga gtc agc gag gag agc gag
gcc tgg atc ctg gca gtg gcc acc ggc 1603Gly Val Ser Glu Glu Ser Glu
Ala Trp Ile Leu Ala Val Ala Thr Gly 490 495 500ctg ttc ctc tac gta
gca ctc tgc gac atg ctc ccg gcg atg ttg aaa 1651Leu Phe Leu Tyr Val
Ala Leu Cys Asp Met Leu Pro Ala Met Leu Lys 505 510 515gta cgg gac
ccg cgg ccc tgg ctc ctc ttc ctg ctg cac aac gtg ggc 1699Val Arg Asp
Pro Arg Pro Trp Leu Leu Phe Leu Leu His Asn Val Gly 520 525 530ctg
ctg ggc ggc tgg acc gtc ctg ctg ctg ctg tcc ctg tac gag gat 1747Leu
Leu Gly Gly Trp Thr Val Leu Leu Leu Leu Ser Leu Tyr Glu Asp 535 540
545gac atc acc ttc tga taccctgccc tagtccccca cctttgactt aagatcccac
1802Asp Ile Thr Phe *550acctcacaaa cctacagccc agaaaccaga agcccctata
gaggccccag tcccaactcc 1862agtaaagaca ctcttgtcct tggaaaaaaa
aaaaaaaaaa aaaaaaaa 191032553PRTHomo sapien 32Met Ala Ser Leu Val
Ser Leu Glu Leu Gly Leu Leu Leu Ala Val Leu1 5 10 15Val Val Thr Ala
Thr Ala Ser Pro Pro Ala Gly Leu Leu Ser Leu Leu 20 25 30Thr Ser Gly
Gln Gly Ala Leu Asp Gln Glu Ala Leu Gly Gly Leu Leu 35 40 45Asn Thr
Leu Ala Asp Arg Val His Cys Ala Asn Gly Pro Cys Gly Lys 50 55 60Ala
Cys Val Asp Ile Pro Gln Leu Leu Glu Glu Ala Val Gly Ala Gly65 70 75
80Ala Pro Gly Ser Ala Gly Gly Val Leu Ala Ala Leu Leu Asp His Val
85 90 95Arg Ser Gly Ser Cys Phe His Ala Leu Pro Ser Pro Gln Tyr Phe
Val 100 105 110Asp Phe Val Phe Gln Gln His Ser Ser Glu Val Pro Met
Thr Leu Ala 115 120 125Glu Leu Ser Ala Leu Met Gln Arg Leu Gly Val
Gly Arg Glu Ala His 130 135 140Ser Asp His Ser His Arg His Arg Gly
Ala Ser Ser Arg Asp Pro Val145 150 155 160Pro Leu Ile Ser Ser Ser
Asn Ser Ser Ser Val Trp Asp Thr Val Cys 165 170 175Leu Ser Ala Arg
Asp Val Met Ala Ala Tyr Gly Leu Ser Glu Gln Ala 180 185 190Gly Val
Thr Pro Glu Ala Trp Ala Gln Leu Ser Pro Ala Leu Leu Gln 195 200
205Gln Gln Leu Ser Gly Ala Cys Thr Ser Gln Ser Arg Pro Pro Val Gln
210 215 220Asp Gln Leu Ser Gln Ser Glu Arg Tyr Leu Tyr Gly Ser Leu
Ala Thr225 230 235 240Leu Leu Ile Cys Leu Cys Ala Val Phe Gly Leu
Leu Leu Leu Thr Cys 245 250 255Thr Gly Cys Arg Gly Val Thr His Tyr
Ile Leu Gln Thr Phe Leu Ser 260 265 270Leu Ala Val Gly Ala Val Thr
Gly Asp Ala Val Leu His Leu Thr Pro 275 280 285Lys Val Leu Gly Leu
His Thr His Ser Glu Glu Gly Leu Ser Pro Gln 290 295 300Pro Thr Trp
Arg Leu Leu Ala Met Leu Ala Gly Leu Tyr Ala Phe Phe305 310 315
320Leu Phe Glu Asn Leu Phe Asn Leu Leu Leu Pro Arg Asp Pro Glu Asp
325 330 335Leu Glu Asp Gly Pro Cys Gly His Ser Ser His Ser His Gly
Gly His 340 345 350Ser His Gly Val Ser Leu Gln Leu Ala Pro Ser Glu
Leu Arg Gln Pro 355 360 365Lys Pro Pro His Glu Gly Ser Arg Ala Asp
Leu Val Ala Glu Glu Ser 370 375 380Pro Glu Leu Leu Asn Pro Glu Pro
Arg Arg Leu Ser Pro Glu Leu Arg385 390 395 400Leu Leu Pro Tyr Met
Ile Thr Leu Gly Asp Ala Val His Asn Phe Ala 405 410 415Asp Gly Leu
Ala Val Gly Ala Ala Phe Ala Ser Ser Trp Lys Thr Gly 420 425 430Leu
Ala Thr Ser Leu Ala Val Phe Cys His Glu Leu Pro His Glu Leu 435 440
445Gly Asp Phe Ala Ala Leu Leu His Ala Gly Leu Ser Val Arg Gln Ala
450 455 460Leu Leu Leu Asn Leu Ala Ser Ala Leu Thr Ala Phe Ala Gly
Leu Tyr465 470 475 480Val Ala Leu Ala Val Gly Val Ser Glu Glu Ser
Glu Ala Trp Ile Leu 485 490 495Ala Val Ala Thr Gly Leu Phe Leu Tyr
Val Ala Leu Cys Asp Met Leu 500 505 510Pro Ala Met Leu Lys Val Arg
Asp Pro Arg Pro Trp Leu Leu Phe Leu 515 520 525Leu His Asn Val Gly
Leu Leu Gly Gly Trp Thr Val Leu Leu Leu Leu 530 535 540Ser Leu Tyr
Glu Asp Asp Ile Thr Phe545 55033598DNAHomo sapienCDS(23)...(421)
33cagagtcact cctgccttca cc atg aag tcc agc ggc ctc ttc ccc ttc ctg
52 Met Lys Ser Ser Gly Leu Phe Pro Phe Leu 1 5 10gtg ctg ctt gcc
ctg gga act ctg gca cct tgg gct gtg gaa ggc tct 100Val Leu Leu Ala
Leu Gly Thr Leu Ala Pro Trp Ala Val Glu Gly Ser 15 20 25gga aag tcc
ttc aaa gct gga gtc tgt cct cct aag aaa tct gcc cag 148Gly Lys Ser
Phe Lys Ala Gly Val Cys Pro Pro Lys Lys Ser Ala Gln 30 35 40tgc ctt
aga tac aag aaa cct gag tgc cag agt gac tgg cag tgt cca 196Cys Leu
Arg Tyr Lys Lys Pro Glu Cys Gln Ser Asp Trp Gln Cys Pro 45 50 55ggg
aag aag aga tgt tgt cct gac act tgt ggc atc aaa tgc ctg gat 244Gly
Lys Lys Arg Cys Cys Pro Asp Thr Cys Gly Ile Lys Cys Leu Asp 60 65
70cct gtt gac acc cca aac cca aca agg agg aag cct ggg aag tgc cca
292Pro Val Asp Thr Pro Asn Pro Thr Arg Arg Lys Pro Gly Lys Cys
Pro75 80 85 90gtg act tat ggc caa tgt ttg atg ctt aac ccc ccc aat
ttc tgt gag 340Val Thr Tyr Gly Gln Cys Leu Met Leu Asn Pro Pro Asn
Phe Cys Glu 95 100 105atg gat ggc cag tgc aag cgt gac ttg aag tgt
tgc atg ggc atg tgt 388Met Asp Gly Gln Cys Lys Arg Asp Leu Lys Cys
Cys Met Gly Met Cys 110 115 120ggg aaa tcc tgc gtt tcc cct gtg aaa
gct tga ttcctgccat atggaggagg 441Gly Lys Ser Cys Val Ser Pro Val
Lys Ala * 125 130ctctggagtc ctgctctgtg tggtccaggt cctttccacc
ctgagacttg gctccaccac 501tgatatcctc ctttggggaa aggcttggca
cacagcaggc tttcaagaag tgccagttga 561tcaatgaata aataaacgag
cctatttctc tttgcac 59834132PRTHomo sapien 34Met Lys Ser Ser Gly Leu
Phe Pro Phe Leu Val Leu Leu Ala Leu Gly 1 5 10 15Thr Leu Ala Pro
Trp Ala Val Glu Gly Ser Gly Lys Ser Phe Lys Ala 20 25 30Gly Val Cys
Pro Pro Lys Lys Ser Ala Gln Cys Leu Arg Tyr Lys Lys 35 40 45Pro Glu
Cys Gln Ser Asp Trp Gln Cys Pro Gly Lys Lys Arg Cys Cys 50 55 60Pro
Asp Thr Cys Gly Ile Lys Cys Leu Asp Pro Val Asp Thr Pro Asn65 70 75
80Pro Thr Arg Arg Lys Pro Gly Lys Cys Pro Val Thr Tyr Gly Gln Cys
85 90 95Leu Met Leu Asn Pro Pro Asn Phe Cys Glu Met Asp Gly Gln Cys
Lys 100 105 110Arg Asp Leu Lys Cys Cys Met Gly Met Cys Gly Lys Ser
Cys Val Ser 115 120 125Pro Val Lys Ala 130353079DNAHomo
sapienCDS(25)...(2448) 35ggcacaaagt tgggggccgc gaag atg agg ctg tcc
ccg gcg ccc ctg aag 51 Met Arg Leu Ser Pro Ala Pro Leu Lys 1 5ctg
agc cgg act ccg gca ctg ctg gcc ctg gcg ctg ccc ctg gcc gcg 99Leu
Ser Arg Thr Pro Ala Leu Leu Ala Leu Ala Leu Pro Leu Ala Ala10 15 20
25gcg ctg gcc ttc tcc gac gag acc ctg gac aaa gtg ccc aag tca gag
147Ala Leu Ala Phe Ser Asp Glu Thr Leu Asp Lys Val Pro Lys Ser Glu
30 35 40ggc tac tgt agc cgt atc ctg cgc gcc cag ggc acg cgg cgc gag
ggc 195Gly Tyr Cys Ser Arg Ile Leu Arg Ala Gln Gly Thr Arg Arg Glu
Gly 45 50 55tac acc gag ttc agc ctc cgc gtg gag ggc gac ccc gac ttc
tac aag 243Tyr Thr Glu Phe Ser Leu Arg Val Glu Gly Asp Pro Asp Phe
Tyr Lys 60 65 70ccg gga acc agc tac cgc gta aca ctt tca gct gct cct
ccc tcc tac 291Pro Gly Thr Ser Tyr Arg Val Thr Leu Ser Ala Ala Pro
Pro Ser Tyr 75 80 85ttc aga gga ttc aca tta att gcc ctc aga gag aac
aga gag ggt gat 339Phe Arg Gly Phe Thr Leu Ile Ala Leu Arg Glu Asn
Arg Glu Gly Asp90 95 100 105aag gaa gaa gac cat gct ggg acc ttc cag
atc ata gac gaa gaa gaa 387Lys Glu Glu Asp His Ala Gly Thr Phe Gln
Ile Ile Asp Glu Glu Glu
110 115 120act cag ttt atg agc aat tgc cct gtt gca gtc act gaa agc
act cca 435Thr Gln Phe Met Ser Asn Cys Pro Val Ala Val Thr Glu Ser
Thr Pro 125 130 135cgg agg agg acc cgg atc cag gtg ttt tgg ata gca
cca cca gcg gga 483Arg Arg Arg Thr Arg Ile Gln Val Phe Trp Ile Ala
Pro Pro Ala Gly 140 145 150aca ggc tgc gtg att ctg aag gcc agc atc
gta caa aaa cgc att att 531Thr Gly Cys Val Ile Leu Lys Ala Ser Ile
Val Gln Lys Arg Ile Ile 155 160 165tat ttt caa gat gag ggc tct ctg
acc aag aaa ctt tgt gaa caa gat 579Tyr Phe Gln Asp Glu Gly Ser Leu
Thr Lys Lys Leu Cys Glu Gln Asp170 175 180 185tcc aca ttt gat ggg
gtg act gac aaa ccc atc tta gac tgc tgt gcc 627Ser Thr Phe Asp Gly
Val Thr Asp Lys Pro Ile Leu Asp Cys Cys Ala 190 195 200tgc gga act
gcc aag tac aga ctc aca ttt tat ggg aat tgg tcc gag 675Cys Gly Thr
Ala Lys Tyr Arg Leu Thr Phe Tyr Gly Asn Trp Ser Glu 205 210 215aag
aca cac cca aag gat tac cct cgt cgg gcc aac cac tgg tct gcg 723Lys
Thr His Pro Lys Asp Tyr Pro Arg Arg Ala Asn His Trp Ser Ala 220 225
230atc atc gga gga tcc cac tcc aag aat tat gta ctg tgg gaa tat gga
771Ile Ile Gly Gly Ser His Ser Lys Asn Tyr Val Leu Trp Glu Tyr Gly
235 240 245gga tat gcc agc gaa ggc gtc aaa caa gtt gca gaa ttg ggc
tca ccc 819Gly Tyr Ala Ser Glu Gly Val Lys Gln Val Ala Glu Leu Gly
Ser Pro250 255 260 265gtg aaa atg gag gaa gaa att cga caa cag agt
gat gag gtc ctc acc 867Val Lys Met Glu Glu Glu Ile Arg Gln Gln Ser
Asp Glu Val Leu Thr 270 275 280gtc atc aaa gcc aaa gcc caa tgg cca
gcc tgg cag cct ctc aac gtg 915Val Ile Lys Ala Lys Ala Gln Trp Pro
Ala Trp Gln Pro Leu Asn Val 285 290 295aga gca gca cct tca gct gaa
ttt tcc gtg gac aga acg cgc cat tta 963Arg Ala Ala Pro Ser Ala Glu
Phe Ser Val Asp Arg Thr Arg His Leu 300 305 310atg tcc ttc ctg acc
atg atg ggc cct agt ccc gac tgg aac gta ggc 1011Met Ser Phe Leu Thr
Met Met Gly Pro Ser Pro Asp Trp Asn Val Gly 315 320 325tta tct gca
gaa gat ctg tgc acc aag gaa tgt ggc tgg gtc cag aag 1059Leu Ser Ala
Glu Asp Leu Cys Thr Lys Glu Cys Gly Trp Val Gln Lys330 335 340
345gtg gtg caa gac ctg att ccc tgg gac gct ggc acc gac agc ggg gtg
1107Val Val Gln Asp Leu Ile Pro Trp Asp Ala Gly Thr Asp Ser Gly Val
350 355 360acc tat gag tca ccc aac aaa ccc acc att ccc cag gag aaa
atc cgg 1155Thr Tyr Glu Ser Pro Asn Lys Pro Thr Ile Pro Gln Glu Lys
Ile Arg 365 370 375ccc ctg acc agc ctg gac cat cct cag agt cct ttc
tat gac cca gag 1203Pro Leu Thr Ser Leu Asp His Pro Gln Ser Pro Phe
Tyr Asp Pro Glu 380 385 390ggt ggg tcc atc act caa gta gcc aga gtt
gtc atc gag aga atc gca 1251Gly Gly Ser Ile Thr Gln Val Ala Arg Val
Val Ile Glu Arg Ile Ala 395 400 405cgg aag ggt gaa caa tgc aat att
gta cct gac aat gtc gat gat att 1299Arg Lys Gly Glu Gln Cys Asn Ile
Val Pro Asp Asn Val Asp Asp Ile410 415 420 425gta gct gac ctg gct
cca gaa gag aaa gat gaa gat gac acc cct gaa 1347Val Ala Asp Leu Ala
Pro Glu Glu Lys Asp Glu Asp Asp Thr Pro Glu 430 435 440acc tgc atc
tac tcc aac tgg tcc cca tgg tcc gcc tgc agc tcc tcc 1395Thr Cys Ile
Tyr Ser Asn Trp Ser Pro Trp Ser Ala Cys Ser Ser Ser 445 450 455acc
tgt gac aaa ggc aag agg atg cga cag cgc atg ctg aaa gca cag 1443Thr
Cys Asp Lys Gly Lys Arg Met Arg Gln Arg Met Leu Lys Ala Gln 460 465
470ctg gac ctc agc gtc ccc tgc cct gac acc cag gac ttc cag ccc tgc
1491Leu Asp Leu Ser Val Pro Cys Pro Asp Thr Gln Asp Phe Gln Pro Cys
475 480 485atg ggc cct ggc tgc agt gac gaa gac ggc tcc acc tgc acc
atg tcc 1539Met Gly Pro Gly Cys Ser Asp Glu Asp Gly Ser Thr Cys Thr
Met Ser490 495 500 505gag tgg atc acc tgg tcg ccc tgc agc atc tcc
tgc ggc atg ggc atg 1587Glu Trp Ile Thr Trp Ser Pro Cys Ser Ile Ser
Cys Gly Met Gly Met 510 515 520agg tcc cgg gag agg tat gtg aag cag
ttc ccg gag gac ggc tcc gtg 1635Arg Ser Arg Glu Arg Tyr Val Lys Gln
Phe Pro Glu Asp Gly Ser Val 525 530 535tgc acg ctg ccc act gag gaa
atg gag aag tgc acg gtc aac gag gag 1683Cys Thr Leu Pro Thr Glu Glu
Met Glu Lys Cys Thr Val Asn Glu Glu 540 545 550tgc tct ccc agc agc
tgc ctg atg acc gag tgg ggc gag tgg gac gag 1731Cys Ser Pro Ser Ser
Cys Leu Met Thr Glu Trp Gly Glu Trp Asp Glu 555 560 565tgc agc gcc
acc tgc ggc atg ggc atg aag aag cgg cac cgc atg atc 1779Cys Ser Ala
Thr Cys Gly Met Gly Met Lys Lys Arg His Arg Met Ile570 575 580
585aag atg aac ccc gca gat ggc tcc atg tgc aaa gcc gag aca tca cag
1827Lys Met Asn Pro Ala Asp Gly Ser Met Cys Lys Ala Glu Thr Ser Gln
590 595 600gca gag aag tgc atg atg cca gag tgc cac acc atc cca tgc
ttg ctg 1875Ala Glu Lys Cys Met Met Pro Glu Cys His Thr Ile Pro Cys
Leu Leu 605 610 615tcc cca tgg tcc gag tgg agt gac tgc agc gtg acc
tgc ggg aag ggc 1923Ser Pro Trp Ser Glu Trp Ser Asp Cys Ser Val Thr
Cys Gly Lys Gly 620 625 630atg cga acc cga cag cgg atg ctc aag tct
ctg gca gaa ctt gga gac 1971Met Arg Thr Arg Gln Arg Met Leu Lys Ser
Leu Ala Glu Leu Gly Asp 635 640 645tgc aat gag gat ctg gag cag gtg
gag aag tgc atg ctc cct gaa tgc 2019Cys Asn Glu Asp Leu Glu Gln Val
Glu Lys Cys Met Leu Pro Glu Cys650 655 660 665ccc att gac tgt gag
ctc acc gag tgg tcc cag tgg tcg gaa tgt aac 2067Pro Ile Asp Cys Glu
Leu Thr Glu Trp Ser Gln Trp Ser Glu Cys Asn 670 675 680aag tca tgt
ggg aaa ggc cac gtg att cga acc cgg atg atc caa atg 2115Lys Ser Cys
Gly Lys Gly His Val Ile Arg Thr Arg Met Ile Gln Met 685 690 695gag
cct cag ttt gga ggt gca ccc tgc cca gag act gtg cag cga aaa 2163Glu
Pro Gln Phe Gly Gly Ala Pro Cys Pro Glu Thr Val Gln Arg Lys 700 705
710aag tgc cgc atc cga aaa tgc ctt cga aat cca tcc atc caa aag cca
2211Lys Cys Arg Ile Arg Lys Cys Leu Arg Asn Pro Ser Ile Gln Lys Pro
715 720 725cgc tgg agg gag gcc cga gag agc cgg cgg agt gag cag ctg
aag gaa 2259Arg Trp Arg Glu Ala Arg Glu Ser Arg Arg Ser Glu Gln Leu
Lys Glu730 735 740 745gag tct gaa ggg gag cag ttc cca ggt tgt agg
atg cgc cca tgg acg 2307Glu Ser Glu Gly Glu Gln Phe Pro Gly Cys Arg
Met Arg Pro Trp Thr 750 755 760gcc tgg tca gaa tgc acc aaa ctg tgc
gga ggt gga att cag gaa cgt 2355Ala Trp Ser Glu Cys Thr Lys Leu Cys
Gly Gly Gly Ile Gln Glu Arg 765 770 775tac atg act gta aag aag aga
ttc aaa agc tcc cag ttt acc agc tgc 2403Tyr Met Thr Val Lys Lys Arg
Phe Lys Ser Ser Gln Phe Thr Ser Cys 780 785 790aaa gac aag aag gag
atc aga gca tgc aat gtt cat cct tgt tag 2448Lys Asp Lys Lys Glu Ile
Arg Ala Cys Asn Val His Pro Cys * 795 800 805caagggtacg agttccccag
ggctgcactc tagattccag agtcaccaat ggctggatta 2508tttgcttgtt
taagacaatt taaattgtgt acgctagttt tcatttttgc agtgtggttc
2568gcccagtagt cttgtggatg ccagagacat cctttctgaa tacttcttga
tgggtacagg 2628ctgagtgggg cgccctcacc tccagccagc ctcttcctgc
agaggagtag tgtcagccac 2688cttgtactaa gctgaaacat gtccctctgg
agcttccacc tggccaggga ggacggagac 2748tttgacctac tccacatgga
gaggcaacca tgtctggaag tgactatgcc tgagtcccag 2808ggtgcggcag
gtaggaaaca ttcacagatg aagacagcag attccccaca ttctcatctt
2868tggcctgttc aatgaaacca ttgtttgccc atctcttctt agtggaactt
taggtctctt 2928ttcaagtctc ctcagtcatc aatagttcct ggggaaaaac
agagctggta gacttgaaga 2988ggagcattga tgttgggtgg cttttgttct
ttcactgaga aattcggaat acatttgtct 3048cacccctgat attggttcct
gatgccccag c 307936807PRTHomo sapien 36Met Arg Leu Ser Pro Ala Pro
Leu Lys Leu Ser Arg Thr Pro Ala Leu1 5 10 15Leu Ala Leu Ala Leu Pro
Leu Ala Ala Ala Leu Ala Phe Ser Asp Glu 20 25 30Thr Leu Asp Lys Val
Pro Lys Ser Glu Gly Tyr Cys Ser Arg Ile Leu 35 40 45Arg Ala Gln Gly
Thr Arg Arg Glu Gly Tyr Thr Glu Phe Ser Leu Arg 50 55 60Val Glu Gly
Asp Pro Asp Phe Tyr Lys Pro Gly Thr Ser Tyr Arg Val65 70 75 80Thr
Leu Ser Ala Ala Pro Pro Ser Tyr Phe Arg Gly Phe Thr Leu Ile 85 90
95Ala Leu Arg Glu Asn Arg Glu Gly Asp Lys Glu Glu Asp His Ala Gly
100 105 110Thr Phe Gln Ile Ile Asp Glu Glu Glu Thr Gln Phe Met Ser
Asn Cys 115 120 125Pro Val Ala Val Thr Glu Ser Thr Pro Arg Arg Arg
Thr Arg Ile Gln 130 135 140Val Phe Trp Ile Ala Pro Pro Ala Gly Thr
Gly Cys Val Ile Leu Lys145 150 155 160Ala Ser Ile Val Gln Lys Arg
Ile Ile Tyr Phe Gln Asp Glu Gly Ser 165 170 175Leu Thr Lys Lys Leu
Cys Glu Gln Asp Ser Thr Phe Asp Gly Val Thr 180 185 190Asp Lys Pro
Ile Leu Asp Cys Cys Ala Cys Gly Thr Ala Lys Tyr Arg 195 200 205Leu
Thr Phe Tyr Gly Asn Trp Ser Glu Lys Thr His Pro Lys Asp Tyr 210 215
220Pro Arg Arg Ala Asn His Trp Ser Ala Ile Ile Gly Gly Ser His
Ser225 230 235 240Lys Asn Tyr Val Leu Trp Glu Tyr Gly Gly Tyr Ala
Ser Glu Gly Val 245 250 255Lys Gln Val Ala Glu Leu Gly Ser Pro Val
Lys Met Glu Glu Glu Ile 260 265 270Arg Gln Gln Ser Asp Glu Val Leu
Thr Val Ile Lys Ala Lys Ala Gln 275 280 285Trp Pro Ala Trp Gln Pro
Leu Asn Val Arg Ala Ala Pro Ser Ala Glu 290 295 300Phe Ser Val Asp
Arg Thr Arg His Leu Met Ser Phe Leu Thr Met Met305 310 315 320Gly
Pro Ser Pro Asp Trp Asn Val Gly Leu Ser Ala Glu Asp Leu Cys 325 330
335Thr Lys Glu Cys Gly Trp Val Gln Lys Val Val Gln Asp Leu Ile Pro
340 345 350Trp Asp Ala Gly Thr Asp Ser Gly Val Thr Tyr Glu Ser Pro
Asn Lys 355 360 365Pro Thr Ile Pro Gln Glu Lys Ile Arg Pro Leu Thr
Ser Leu Asp His 370 375 380Pro Gln Ser Pro Phe Tyr Asp Pro Glu Gly
Gly Ser Ile Thr Gln Val385 390 395 400Ala Arg Val Val Ile Glu Arg
Ile Ala Arg Lys Gly Glu Gln Cys Asn 405 410 415Ile Val Pro Asp Asn
Val Asp Asp Ile Val Ala Asp Leu Ala Pro Glu 420 425 430Glu Lys Asp
Glu Asp Asp Thr Pro Glu Thr Cys Ile Tyr Ser Asn Trp 435 440 445Ser
Pro Trp Ser Ala Cys Ser Ser Ser Thr Cys Asp Lys Gly Lys Arg 450 455
460Met Arg Gln Arg Met Leu Lys Ala Gln Leu Asp Leu Ser Val Pro
Cys465 470 475 480Pro Asp Thr Gln Asp Phe Gln Pro Cys Met Gly Pro
Gly Cys Ser Asp 485 490 495Glu Asp Gly Ser Thr Cys Thr Met Ser Glu
Trp Ile Thr Trp Ser Pro 500 505 510Cys Ser Ile Ser Cys Gly Met Gly
Met Arg Ser Arg Glu Arg Tyr Val 515 520 525Lys Gln Phe Pro Glu Asp
Gly Ser Val Cys Thr Leu Pro Thr Glu Glu 530 535 540Met Glu Lys Cys
Thr Val Asn Glu Glu Cys Ser Pro Ser Ser Cys Leu545 550 555 560Met
Thr Glu Trp Gly Glu Trp Asp Glu Cys Ser Ala Thr Cys Gly Met 565 570
575Gly Met Lys Lys Arg His Arg Met Ile Lys Met Asn Pro Ala Asp Gly
580 585 590Ser Met Cys Lys Ala Glu Thr Ser Gln Ala Glu Lys Cys Met
Met Pro 595 600 605Glu Cys His Thr Ile Pro Cys Leu Leu Ser Pro Trp
Ser Glu Trp Ser 610 615 620Asp Cys Ser Val Thr Cys Gly Lys Gly Met
Arg Thr Arg Gln Arg Met625 630 635 640Leu Lys Ser Leu Ala Glu Leu
Gly Asp Cys Asn Glu Asp Leu Glu Gln 645 650 655Val Glu Lys Cys Met
Leu Pro Glu Cys Pro Ile Asp Cys Glu Leu Thr 660 665 670Glu Trp Ser
Gln Trp Ser Glu Cys Asn Lys Ser Cys Gly Lys Gly His 675 680 685Val
Ile Arg Thr Arg Met Ile Gln Met Glu Pro Gln Phe Gly Gly Ala 690 695
700Pro Cys Pro Glu Thr Val Gln Arg Lys Lys Cys Arg Ile Arg Lys
Cys705 710 715 720Leu Arg Asn Pro Ser Ile Gln Lys Pro Arg Trp Arg
Glu Ala Arg Glu 725 730 735Ser Arg Arg Ser Glu Gln Leu Lys Glu Glu
Ser Glu Gly Glu Gln Phe 740 745 750Pro Gly Cys Arg Met Arg Pro Trp
Thr Ala Trp Ser Glu Cys Thr Lys 755 760 765Leu Cys Gly Gly Gly Ile
Gln Glu Arg Tyr Met Thr Val Lys Lys Arg 770 775 780Phe Lys Ser Ser
Gln Phe Thr Ser Cys Lys Asp Lys Lys Glu Ile Arg785 790 795 800Ala
Cys Asn Val His Pro Cys 805372805DNAHomo sapienCDS(616)...(1587)
37cgggtctgat agtccctacc tgtcaggact ggtgttagga tgagataatg tttgtgaact
60gtaaacatat ataaacgtgt gctactgtga gaactggaac aaagaagaga gggagtgaga
120gaaatcaagg gagggctggg gctgggaaag aacgaaaagg gagtcgcgta
tagaggagag 180gcgacagtcg cgagccacac tttgcaatga aactctttag
actttctgcc gggagagcgg 240cccagacgcg ccaggtctgt agcaggaggc
cgcgagggcg ggtccccaga agcctacagg 300tgagtatcgg ttctcccctt
cccggctttc ggtccggagg aggcgggagc agcttccctg 360ttctgatcct
atcgcgggcg gcgcagggcc ggcttggcct tccgtgggac ggggaggggg
420gcgggatgtg tcacccaaat accagtgggg acggtcggtg gtggaaccag
ccgggcaggt 480cgggtagagt ataagagccg gagggagcgg ccggggcgca
gacgcctgca gaccatccca 540gacgccggag cccgagcccc gacgagtccc
cgcgcctcat ccgcccgcgt ccggtccgcg 600ttcctccgcc ccacc atg gct cgg
ggc ccc ggc ctc gcg ccg cca ccg ctg 651 Met Ala Arg Gly Pro Gly Leu
Ala Pro Pro Pro Leu 1 5 10cgg ctg ccg ctg ctg ctg ctg gtg ctg gcg
gcg gtg acc ggc cac acg 699Arg Leu Pro Leu Leu Leu Leu Val Leu Ala
Ala Val Thr Gly His Thr 15 20 25gcc gcg cag gac aac tgc acg tgt ccc
acc aac aag atg acc gtg tgc 747Ala Ala Gln Asp Asn Cys Thr Cys Pro
Thr Asn Lys Met Thr Val Cys 30 35 40agc ccc gac ggc ccc ggc ggc cgc
tgc cag tgc cgc gcg ctg ggc tcg 795Ser Pro Asp Gly Pro Gly Gly Arg
Cys Gln Cys Arg Ala Leu Gly Ser45 50 55 60ggc atg gcg gtc gac tgc
tcc acg ctg acc tcc aag tgt ctg ctg ctc 843Gly Met Ala Val Asp Cys
Ser Thr Leu Thr Ser Lys Cys Leu Leu Leu 65 70 75aag gcg cgc atg agc
gcc ccc aag aac gcc cgc acg ctg gtg cgg ccg 891Lys Ala Arg Met Ser
Ala Pro Lys Asn Ala Arg Thr Leu Val Arg Pro 80 85 90agt gag cac gcg
ctc gtg gac aac gat ggc ctc tac gac ccc gac tgc 939Ser Glu His Ala
Leu Val Asp Asn Asp Gly Leu Tyr Asp Pro Asp Cys 95 100 105gac ccc
gag ggc cgc ttc aag gcg cgc cag tgc aac cag acg tcg gtg 987Asp Pro
Glu Gly Arg Phe Lys Ala Arg Gln Cys Asn Gln Thr Ser Val 110 115
120tgc tgg tgc gtg aac tcg gtg ggc gtg cgc cgc acg gac aag ggc gac
1035Cys Trp Cys Val Asn Ser Val Gly Val Arg Arg Thr Asp Lys Gly
Asp125 130 135 140ctg agc cta cgc tgc gat gag ctg gtg cgc acc cac
cac atc ctc att 1083Leu Ser Leu Arg Cys Asp Glu Leu Val Arg Thr His
His Ile Leu Ile 145 150 155gac ctg cgc cac cgc ccc acc gcc ggc gcc
ttc aac cac tca gac ctg 1131Asp Leu Arg His Arg Pro Thr Ala Gly Ala
Phe Asn His Ser Asp Leu 160 165 170gac gcc gag ctg agg cgg ctc ttc
cgc gag cgc tat cgg ctg cac ccc 1179Asp Ala Glu Leu Arg Arg Leu Phe
Arg Glu Arg Tyr Arg Leu His Pro 175 180 185aag ttc gtg gcg gcc gtg
cac tac gag cag ccc acc atc cag atc gag 1227Lys Phe Val Ala Ala Val
His Tyr Glu Gln Pro Thr Ile Gln Ile Glu 190 195 200ctg cgg cag aac
acg tct cag aag gcc gcc ggt gaa gtg gat atc ggc
1275Leu Arg Gln Asn Thr Ser Gln Lys Ala Ala Gly Glu Val Asp Ile
Gly205 210 215 220gat gcc gcc tac tac ttc gag agg gac atc aag ggc
gag tct cta ttc 1323Asp Ala Ala Tyr Tyr Phe Glu Arg Asp Ile Lys Gly
Glu Ser Leu Phe 225 230 235cag ggc cgc ggc ggc ctg gac ttg cgc gtg
cgc gga gaa ccc ctg cag 1371Gln Gly Arg Gly Gly Leu Asp Leu Arg Val
Arg Gly Glu Pro Leu Gln 240 245 250gtg gag cgc acg ctc atc tat tac
ctg gac gag att ccc ccg aag ttc 1419Val Glu Arg Thr Leu Ile Tyr Tyr
Leu Asp Glu Ile Pro Pro Lys Phe 255 260 265tcc atg aag cgc ctc acc
gcc ggc ctc atc gcc gtc atc gtg gtg gtc 1467Ser Met Lys Arg Leu Thr
Ala Gly Leu Ile Ala Val Ile Val Val Val 270 275 280gtg gtg gcc ctc
gtc gcc ggc atg gcc gtc ctg gtg atc acc aac cgg 1515Val Val Ala Leu
Val Ala Gly Met Ala Val Leu Val Ile Thr Asn Arg285 290 295 300aga
aag tcg ggg aag tac aag aag gtg gag atc aag gaa ctg ggg gag 1563Arg
Lys Ser Gly Lys Tyr Lys Lys Val Glu Ile Lys Glu Leu Gly Glu 305 310
315ttg aga aag gaa ccg agc ttg tag gtacccggcg gggcagggga tggggtgggg
1617Leu Arg Lys Glu Pro Ser Leu * 320taccggattt cggtatcgtc
ccagacccaa gtgagtcacg cttcctgatt cctcggcgca 1677aaggagacgt
ttatcctttc aaattcctgc cttccccctc ccttttgcgc acacaccagg
1737tttaatagat cctggcctca gggtctcctt tctttctcac ttctgtcttg
agggaagcat 1797ttctaaaatg tatccccttt cggtccaaca acaggaaacc
tgactggggc agtgaaggaa 1857gggatggcac agcgttatgt gtaaaaaaca
agtatctgta tgacaacccg ggatcgtttg 1917caagtaactg aatccattgc
gacattgtga aggcttaaat gagtttagat gggaaatagc 1977gttgttatcg
ccttgggttt aaattatttg atgagttcca cttgtatcat ggcctacccg
2037aggagaagag gagtttgtta actgggccta tgtagtagcc tcatttacca
tcgtttgtat 2097tactgaccac atatgcttgt cactgggaaa gaagcctgtt
tcagctgcct gaacgcagtt 2157tggatgtctt tgaggacaga cattgcccgg
aaactcagtc tatttattct tcagcttgcc 2217cttactgcca ctgatattgg
taatgttctt ttttgtaaaa tgtttgtaca tatgttgtct 2277ttgataatgt
tgctgtaatt ttttaaaata aaacacgaat ttaataaaat atgggaaagg
2337cacaaaccag aagtcggcat ttgtgaaaag tccctccaga tttctatcac
tttggtctct 2397aatttcccaa gacttgtatt ttttttttat ttcaaattat
aacacttttt tttcccccag 2457aagtgggtgt ttcatgttgc tactctggtg
tgtcccaaga tatcctaact ggccagtgta 2517aatgctattc tttctaaata
agattatttg gaaacttcct tcaaactgca ggagggcgag 2577ctctgagggc
acgagaagct aaaactagct gcttttgatg aaaaagagtg ccagtctttg
2637gtcatctcta aacaaggctt atcaccaatg gagacagaaa actctagttc
aagagctgta 2697cctcctttga atcccagccc tactcgaaat aagtggtact
atttccattt agcctttgag 2757caaatcactt aactcaaagg cgttgtggct
ctaagattaa acgacttt 280538323PRTHomo sapien 38Met Ala Arg Gly Pro
Gly Leu Ala Pro Pro Pro Leu Arg Leu Pro Leu1 5 10 15Leu Leu Leu Val
Leu Ala Ala Val Thr Gly His Thr Ala Ala Gln Asp 20 25 30Asn Cys Thr
Cys Pro Thr Asn Lys Met Thr Val Cys Ser Pro Asp Gly 35 40 45Pro Gly
Gly Arg Cys Gln Cys Arg Ala Leu Gly Ser Gly Met Ala Val 50 55 60Asp
Cys Ser Thr Leu Thr Ser Lys Cys Leu Leu Leu Lys Ala Arg Met65 70 75
80Ser Ala Pro Lys Asn Ala Arg Thr Leu Val Arg Pro Ser Glu His Ala
85 90 95Leu Val Asp Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Pro Glu
Gly 100 105 110Arg Phe Lys Ala Arg Gln Cys Asn Gln Thr Ser Val Cys
Trp Cys Val 115 120 125Asn Ser Val Gly Val Arg Arg Thr Asp Lys Gly
Asp Leu Ser Leu Arg 130 135 140Cys Asp Glu Leu Val Arg Thr His His
Ile Leu Ile Asp Leu Arg His145 150 155 160Arg Pro Thr Ala Gly Ala
Phe Asn His Ser Asp Leu Asp Ala Glu Leu 165 170 175Arg Arg Leu Phe
Arg Glu Arg Tyr Arg Leu His Pro Lys Phe Val Ala 180 185 190Ala Val
His Tyr Glu Gln Pro Thr Ile Gln Ile Glu Leu Arg Gln Asn 195 200
205Thr Ser Gln Lys Ala Ala Gly Glu Val Asp Ile Gly Asp Ala Ala Tyr
210 215 220Tyr Phe Glu Arg Asp Ile Lys Gly Glu Ser Leu Phe Gln Gly
Arg Gly225 230 235 240Gly Leu Asp Leu Arg Val Arg Gly Glu Pro Leu
Gln Val Glu Arg Thr 245 250 255Leu Ile Tyr Tyr Leu Asp Glu Ile Pro
Pro Lys Phe Ser Met Lys Arg 260 265 270Leu Thr Ala Gly Leu Ile Ala
Val Ile Val Val Val Val Val Ala Leu 275 280 285Val Ala Gly Met Ala
Val Leu Val Ile Thr Asn Arg Arg Lys Ser Gly 290 295 300Lys Tyr Lys
Lys Val Glu Ile Lys Glu Leu Gly Glu Leu Arg Lys Glu305 310 315
320Pro Ser Leu39590DNAHomo sapienCDS(28)...(402) 39acctgcaccc
cgcccgggca tagcacc atg cct gct tgt cgc cta ggc ccg cta 54 Met Pro
Ala Cys Arg Leu Gly Pro Leu 1 5gcc gcc gcc ctc ctc ctc agc ctg ctg
ctg ttc ggc ttc acc cta gtc 102Ala Ala Ala Leu Leu Leu Ser Leu Leu
Leu Phe Gly Phe Thr Leu Val10 15 20 25tca ggc aca gga gca gag aag
act ggc gtg tgc ccc gag ctc cag gct 150Ser Gly Thr Gly Ala Glu Lys
Thr Gly Val Cys Pro Glu Leu Gln Ala 30 35 40gac cag aac tgc acg caa
gag tgc gtc tcg gac agc gaa tgc gcc gac 198Asp Gln Asn Cys Thr Gln
Glu Cys Val Ser Asp Ser Glu Cys Ala Asp 45 50 55aac ctc aag tgc tgc
agc gcg ggc tgt gcc acc ttc tgc tct ctg ccc 246Asn Leu Lys Cys Cys
Ser Ala Gly Cys Ala Thr Phe Cys Ser Leu Pro 60 65 70aat gat aag gag
ggt tcc tgc ccc cag gtg aac att aac ttt ccc cag 294Asn Asp Lys Glu
Gly Ser Cys Pro Gln Val Asn Ile Asn Phe Pro Gln 75 80 85ctc ggc ctc
tgt cgg gac cag tgc cag gtg gac agc cag tgt cct ggc 342Leu Gly Leu
Cys Arg Asp Gln Cys Gln Val Asp Ser Gln Cys Pro Gly90 95 100 105cag
atg aaa tgc tgc cgc aat ggc tgt ggg aag gtg tcc tgt gtc act 390Gln
Met Lys Cys Cys Arg Asn Gly Cys Gly Lys Val Ser Cys Val Thr 110 115
120ccc aat ttc tga gctccagcca ccaccaggct gagcagtgag gagagaaagt
442Pro Asn Phe *ttctgcctgg ccctgcatct ggttccagcc cacctgccct
cccctttttc gggactctgt 502attccctctt gggctgacca cagcttctcc
ctttcccaac caataaagta accactttca 562gcaaaaaaaa aaaaaaaaaa aaaaaaaa
59040124PRTHomo sapien 40Met Pro Ala Cys Arg Leu Gly Pro Leu Ala
Ala Ala Leu Leu Leu Ser1 5 10 15Leu Leu Leu Phe Gly Phe Thr Leu Val
Ser Gly Thr Gly Ala Glu Lys 20 25 30Thr Gly Val Cys Pro Glu Leu Gln
Ala Asp Gln Asn Cys Thr Gln Glu 35 40 45Cys Val Ser Asp Ser Glu Cys
Ala Asp Asn Leu Lys Cys Cys Ser Ala 50 55 60Gly Cys Ala Thr Phe Cys
Ser Leu Pro Asn Asp Lys Glu Gly Ser Cys65 70 75 80Pro Gln Val Asn
Ile Asn Phe Pro Gln Leu Gly Leu Cys Arg Asp Gln 85 90 95Cys Gln Val
Asp Ser Gln Cys Pro Gly Gln Met Lys Cys Cys Arg Asn 100 105 110Gly
Cys Gly Lys Val Ser Cys Val Thr Pro Asn Phe 115 12041450DNAHomo
sapienCDS(67)...(288) 41cccaagatgg actcaggcag gcagctctgc tgtatgtgaa
gcccagtgag gggcagtggg 60ggggcc atg ctg cag gta caa gtt aat ctc cct
gta tcg cct ctg ccc 108 Met Leu Gln Val Gln Val Asn Leu Pro Val Ser
Pro Leu Pro 1 5 10act tac cct tac tcc ttt ttc tac cca gat aag gag
ggt tcc tgc ccc 156Thr Tyr Pro Tyr Ser Phe Phe Tyr Pro Asp Lys Glu
Gly Ser Cys Pro15 20 25 30cag gtg aac att aac ttt ccc cag ctc ggc
ctc tgt cgg gac cag tgc 204Gln Val Asn Ile Asn Phe Pro Gln Leu Gly
Leu Cys Arg Asp Gln Cys 35 40 45cag gtg gac agc cag tgt cct ggc cag
atg aaa tgc tgc cgc aat ggc 252Gln Val Asp Ser Gln Cys Pro Gly Gln
Met Lys Cys Cys Arg Asn Gly 50 55 60tgt ggg aag gtg tcc tgt gtc act
ccc aat ttc tga ggtccagcca 298Cys Gly Lys Val Ser Cys Val Thr Pro
Asn Phe * 65 70ccaccaggct gagcagtgag gagagaaagt ttctgcctgg
ccctgcatct ggttccagcc 358cacctgccct cccctttttc gggactctgt
attccctctt gggctgacca cagcttctcc 418ctttcccaac caataaagta
accactttca gc 4504273PRTHomo sapien 42Met Leu Gln Val Gln Val Asn
Leu Pro Val Ser Pro Leu Pro Thr Tyr 1 5 10 15Pro Tyr Ser Phe Phe
Tyr Pro Asp Lys Glu Gly Ser Cys Pro Gln Val 20 25 30Asn Ile Asn Phe
Pro Gln Leu Gly Leu Cys Arg Asp Gln Cys Gln Val 35 40 45Asp Ser Gln
Cys Pro Gly Gln Met Lys Cys Cys Arg Asn Gly Cys Gly 50 55 60Lys Val
Ser Cys Val Thr Pro Asn Phe65 7043705DNAHomo sapienCDS(339)...(626)
43ctgccgcccg cccagacgcc agcaagcccc cctcccacga cagggctgct ccgggagctt
60cggagacccg ccccgggcct gagcgcaggc tgcctccggg accccacggc tgtccggacg
120tgccatgggc gcgcagctgc cgggcaacgt gttgtgtaag tgaacatctg
ggaggtaaac 180actacacgtg aagagtggtg aaagggaaca ttgattactg
aagtgccctg gagagggaaa 240gcactggtca acatcacatg gacaaatttc
attgttttct aaagatggcc tggaagtagt 300ctttgccact gcttcctcca
caaacagctc ttcataac atg ggc tgc atg aaa tca 356 Met Gly Cys Met Lys
Ser 1 5aag caa act ttc cca ttt cct acc ata tat gaa ggt gag aag cag
cat 404Lys Gln Thr Phe Pro Phe Pro Thr Ile Tyr Glu Gly Glu Lys Gln
His 10 15 20gag agt gaa gaa ccc ttt atg cca gaa gag aga tgt cta cct
agg atg 452Glu Ser Glu Glu Pro Phe Met Pro Glu Glu Arg Cys Leu Pro
Arg Met 25 30 35gct tct cca gtt aat gtc aaa gag gaa gtg aag gaa cct
cca ggg acc 500Ala Ser Pro Val Asn Val Lys Glu Glu Val Lys Glu Pro
Pro Gly Thr 40 45 50aat att gtg atc ttg gaa tat gca cac cgc ctg tct
cag gat atc ttg 548Asn Ile Val Ile Leu Glu Tyr Ala His Arg Leu Ser
Gln Asp Ile Leu55 60 65 70tgt gat gcc ttg cag caa tgg gca tgc aat
aac atc aag tac cat gac 596Cys Asp Ala Leu Gln Gln Trp Ala Cys Asn
Asn Ile Lys Tyr His Asp 75 80 85att cca tac att gag agt gag ggg cct
tga ggctgtagga tgacaacact 646Ile Pro Tyr Ile Glu Ser Glu Gly Pro *
90 95ttgactgtgg aggtgctagt ttgaataaat gtgacaaaag caaaaaaaaa
aaaaaaaaa 7054495PRTHomo sapien 44Met Gly Cys Met Lys Ser Lys Gln
Thr Phe Pro Phe Pro Thr Ile Tyr1 5 10 15Glu Gly Glu Lys Gln His Glu
Ser Glu Glu Pro Phe Met Pro Glu Glu 20 25 30Arg Cys Leu Pro Arg Met
Ala Ser Pro Val Asn Val Lys Glu Glu Val 35 40 45Lys Glu Pro Pro Gly
Thr Asn Ile Val Ile Leu Glu Tyr Ala His Arg 50 55 60Leu Ser Gln Asp
Ile Leu Cys Asp Ala Leu Gln Gln Trp Ala Cys Asn65 70 75 80Asn Ile
Lys Tyr His Asp Ile Pro Tyr Ile Glu Ser Glu Gly Pro 85 90 95
* * * * *