U.S. patent application number 11/812518 was filed with the patent office on 2008-01-17 for novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of prostate cancer.
This patent application is currently assigned to COMPUGEN LTD.. Invention is credited to Pinchas Akiva, Michal Avalon-Soffer, Dvir Dahary, Zurit Levine, Sarah Pollock, Shirley Sameah-Greenwald, Osnat Sella-Tavor, Rotern Sorek, Amir Toporik.
Application Number | 20080014590 11/812518 |
Document ID | / |
Family ID | 35431336 |
Filed Date | 2008-01-17 |
United States Patent
Application |
20080014590 |
Kind Code |
A1 |
Dahary; Dvir ; et
al. |
January 17, 2008 |
Novel nucleotide and amino acid sequences, and assays and methods
of use thereof for diagnosis of prostate cancer
Abstract
Novel markers for prostate cancer that are both sensitive and
accurate. Furthermore, these markers are able to distinguish
between prostate cancer and benign prostate hyperplasia ("BPH").
These markers are overexpressed in prostate cancer specifically, as
opposed to normal prostate tissue and/or BPH. The measurement of
these markers, alone or in combination, in patient samples provides
information that the diagnostician can correlate with a probable
diagnosis of prostate cancer. The markers of the present invention,
alone or in combination, show a high degree of differential
detection between prostate cancer and non-cancerous states.
Inventors: |
Dahary; Dvir; (Tel-Aviv,
IL) ; Pollock; Sarah; (Tel-Aviv, IL) ; Levine;
Zurit; (Herzlia, IL) ; Sorek; Rotern;
(Rechovot, IL) ; Avalon-Soffer; Michal;
(Ramat-HaSharon, IL) ; Akiva; Pinchas; (Ramat-Gan,
IL) ; Toporik; Amir; (Azur, IL) ; Sella-Tavor;
Osnat; (Kfar Kish, IL) ; Sameah-Greenwald;
Shirley; (Kfar-Saba, IL) |
Correspondence
Address: |
STAAS & HALSEY LLP
SUITE 700
1201 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
COMPUGEN LTD.
Tel Aviv
IL
|
Family ID: |
35431336 |
Appl. No.: |
11/812518 |
Filed: |
June 19, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11043806 |
Jan 27, 2005 |
|
|
|
11812518 |
Jun 19, 2007 |
|
|
|
60620916 |
Oct 22, 2004 |
|
|
|
60628123 |
Nov 17, 2004 |
|
|
|
60621131 |
Oct 25, 2004 |
|
|
|
60628101 |
Nov 17, 2004 |
|
|
|
60620874 |
Oct 22, 2004 |
|
|
|
60628134 |
Nov 17, 2004 |
|
|
|
60620853 |
Oct 22, 2004 |
|
|
|
60628112 |
Nov 17, 2004 |
|
|
|
60628145 |
Nov 17, 2004 |
|
|
|
60620656 |
Oct 22, 2004 |
|
|
|
60628251 |
Nov 17, 2004 |
|
|
|
60628178 |
Nov 17, 2004 |
|
|
|
60628231 |
Nov 17, 2004 |
|
|
|
60620918 |
Oct 22, 2004 |
|
|
|
60628156 |
Nov 17, 2004 |
|
|
|
60620677 |
Oct 22, 2004 |
|
|
|
60628167 |
Nov 17, 2004 |
|
|
|
60539129 |
Jan 27, 2004 |
|
|
|
60539128 |
Jan 27, 2004 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/7.1; 530/324; 530/326; 530/328; 530/350; 530/387.9; 536/23.5;
536/24.33 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C07K 14/82 20130101; C07K 16/3069 20130101; G01N 33/57434 20130101;
C07K 14/705 20130101; C07K 14/47 20130101; C12Q 2600/112 20130101;
C12Q 1/6886 20130101 |
Class at
Publication: |
435/006 ;
435/007.1; 530/324; 530/326; 530/328; 530/350; 530/387.9;
536/023.5; 536/024.33 |
International
Class: |
G01N 33/574 20060101
G01N033/574; C07H 21/04 20060101 C07H021/04; C07K 14/82 20060101
C07K014/82; C07K 16/30 20060101 C07K016/30; C12Q 1/68 20060101
C12Q001/68 |
Claims
1. An isolated polynucleotide comprising the polynucleotide
sequence set forth in a member selected from the group consisting
of 5, 6, 7, 8, 9, 10, 34, 35, 36, 37, 38, 39, 13, 14, 435, 436,
437-450 and 451.
2. An isolated polynucleotide, comprising the polynucleotide
sequence set forth in a member selected from the group consisting
of 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 222, 223,
224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,
237, 136, 137, 138, 139, 140, 141, 142, 422, 423, 424-433 and
434.
3. An isolated polypeptide comprising the polypeptide sequence set
forth in a member selected from the group consisting of 331, 332,
333, 334, 335, 359, 360, 361, 362, 363, 338, 339, 452, 453-462 and
463.
4. An isolated polypeptide, comprising the polypeptide selected
from the group consisting of: a polypeptide comprising a first
amino acid sequence being at least about 95% homologous to amino
acids 1-63 of SEQ ID NO:334, and a second amino acid sequence being
at least about 95% homologous to amino acids 64-84 of SEQ ID
NO:334, wherein said first and second amino acid sequences are
contiguous and in a sequential order; a polypeptide comprising a
first amino acid sequence being at least 95% homologous to amino
acids 1-163 of SEQ ID NO. 359, a bridging amino acid H
corresponding to amino acid 164 of SEQ ID NO. 359, a second amino
acid sequence being at least 95% homologous to amino acids 165-445
of SEQ ID NO. 359, and a third amino acid sequence being at least
about 95% homologous to amino acids 446-496 of SEQ ID NO. 359,
wherein said first amino acid sequence, bridging amino acid, second
amino acid sequence and third amino acid sequence are contiguous
and in a sequential order; a polypeptide comprising a first amino
acid sequence being at least about 95% homologous to amino acids
1-163 of SEQ ID NO:360, a bridging amino acid H corresponding to
amino acid 164 of SEQ ID NO. 360, a second amino acid sequence
being at least about 95% homologous to amino acids 165-358 of SEQ
ID NO. 360, and a third amino acid sequence being at least about
95% homologous to amino acids 359-382 of SEQ ID NO. 360, wherein
said first amino acid sequence, bridging amino acid, second amino
acid sequence and third amino acid sequence are contiguous and in a
sequential order; a polypeptide comprising a first amino acid
sequence being at least about 95% homologous to amino acids 1-163
of SEQ ID NO. 361, a bridging amino acid H corresponding to amino
acid 164 of SEQ ID NO. 361, a second amino acid sequence being at
least about 95% homologous to amino acids 165-359 of SEQ ID NO.
361, and a third amino acid sequence being at least about 95%
homologous to amino acids 360-370 of SEQ ID NO. 361, wherein said
first amino acid sequence, bridging amino acid, second amino acid
sequence and third amino acid sequence are contiguous and in a
sequential order; a polypeptide comprising a first amino acid
sequence being at least about 95% homologous to amino acids 1-163
of SEQ ID NO. 362, a bridging amino acid H corresponding to amino
acid 164 of SEQ ID NO. 362, a second amino acid sequence being at
least about 95% homologous to amino acids 165-286 of SEQ ID NO.
362, and a third amino acid sequence being at least about 95%
homologous to amino acids 287-301 of SEQ ID NO. 362, wherein said
first amino acid sequence, bridging amino acid, second amino acid
sequence and third amino acid sequence are contiguous and in a
sequential order; a polypeptide comprising a first amino acid
sequence being at least about 95% homologous to amino acids 1-96 of
SEQ ID NO. 363, a second amino acid sequence being at least about
95% homologous to amino acids 97-147 of SEQ ID NO. 363, a bridging
amino acid H corresponding to amino acid 148 of SEQ ID NO. 363, a
third amino acid sequence being at least about 95% homologous to
amino acids 149-343 of SEQ ID NO. 363, and a fourth amino acid
sequence being at least about 95% homologous to amino acids 344-354
of SEQ ID NO. 363, wherein said first amino acid sequence, second
amino acid sequence, bridging amino acid, third amino acid sequence
and fourth amino acid sequence are contiguous and in a sequential
order; a polypeptide comprising a first amino acid sequence being
at least about 95% homologous to amino acids 1-27 of SEQ ID NO.
339, and a second amino acid sequence being at least about 95%
homologous to amino acids 28-41 of SEQ ID NO. 339, wherein said
first amino acid sequence and second amino acid sequence are
contiguous and in a sequential order; a polypeptide comprising a
first amino acid sequence being at least about 95% homologous to
amino acids 1-110 of SEQ ID NO. 332, and a second amino acid
sequence being at least about 95% homologous to amino acids 111-222
of SEQ ID NO. 332, wherein said first and second amino acid
sequences are contiguous and in a sequential order; a polypeptide
comprising a first amino acid sequence being at least about 95%
homologous to amino acids 1-5 of SEQ ID NO. 333, a second amino
acid sequence being at least about 95% homologous to amino acids
6-64 of SEQ ID NO. 333, and a third amino acid sequence being at
least about 95% homologous to amino acids 65-93 of SEQ ID NO. 333,
wherein said first, second and third amino acid sequences are
contiguous and in a sequential order; a polypeptide comprising a
first amino acid sequence being at least about 95% homologous to
amino acids 1-63 of SEQ ID NO. 334, and a second amino acid
sequence being at least about 95% homologous to amino acids 64-84
of SEQ ID NO. 334, wherein said first and second amino acid
sequences are contiguous and in a sequential order; and a
polypeptide comprising a first amino acid sequence being at least
about 95% homologous to amino acids 1-63 of SEQ ID NO. 335, and a
second amino acid sequence being at least about 95% homologous to a
polypeptide sequence corresponding to amino acids 64-90 of SEQ ID
NO. 335, wherein said first and second amino acid sequences are
contiguous and in a sequential order.
5. The isolated polypeptide of claim 4, comprising a member
selected from the group consisting of a polypeptide comprising an
amino acid sequence being at least about 95% about homologous to
SEQ ID NO: 538; a polypeptide comprising an amino acid sequence
being at least about 95% about homologous to amino acids 446-496 in
SEQ ID NO. 359; a polypeptide comprising an amino acid sequence
being at least about 95% about homologous to amino acids 359-382 in
SEQ ID NO. 360; a polypeptide comprising an amino acid sequence
being at least about 95% homologous to amino acids 360-370 in SEQ
ID NO. 361; a polypeptide comprising an amino acid sequence being
at least about 95% homologous to amino acids 287-301 in SEQ ID NO.
362; a polypeptide comprising an amino acid sequence being at least
about 95% homologous to the sequence in SEQ ID NO. 363; a
polypeptide comprising an amino acid sequence being at least about
95% homologous to amino acids 28-41 in SEQ ID NO. 339; a
polypeptide comprising an amino acid sequence being at least about
95% homologous to amino acids 84-222 in SEQ ID NO. 332; a
polypeptide comprising an amino acid sequence being at least about
95% homologous to amino acids 65-93 in SEQ ID NO. 333; a
polypeptide comprising an amino acid sequence being at least about
95% homologous to amino acids 64-84 in SEQ ID NO. 334; a
polypeptide comprising an amino acid sequence being at least about
95% homologous to amino acids 64-90 in SEQ ID NO. 335; and a
polypeptide comprising an amino acid sequence being at least about
95% homologous to amino acids 1-110 of SEQ ID NO. 332.
6. An isolated peptide, the peptide having a length "n", wherein n
is selected from the group consisting of 10 amino acids in length,
20 amino acids in length, 30 amino acids in length, 40 amino acids
in length and 50 amino acids in length, wherein at least two amino
acids comprise KR, having a structure as follows: a sequence
starting from any of amino acid numbers 96-x to 96 of SEQ ID
NO:363; and ending at any of amino acid numbers 97+((n-2)-x) of SEQ
ID NO:363, in which x varies from 0 to n-2.
7. An isolated peptide comprising the amino acid sequence set forth
in a 50 amino acid long segment of SEQ ID NO:363 anywhere from
amino acid position 48 to amino acid position 145, and including
amino acids at positions 96 and 97.
8. An isolated peptide comprising the amino acid sequence set forth
in a 40 amino acid long segment of SEQ ID NO:363 anywhere from
amino acid position 58 to amino acid position 135, and including
amino acids at positions 96 and 97.
9. An isolated peptide comprising the amino acid sequence set forth
in a 30 amino acid long segment of SEQ ID NO:363 anywhere from
amino acid position 68 to amino acid position 125, and including
amino acids at positions 96 and 97.
10. An isolated peptide comprising the amino acid sequence set
forth in a 20 amino acid long segment of SEQ ID NO:363 anywhere
from amino acid position 78 to amino acid position 115, and
including amino acids at positions 96 and 97.
11. An isolated peptide comprising the amino acid sequence set
forth in a member selected from the group consisting of SEQ ID NOs
464, 465-471 and 472.
12. An isolated primer pair, comprising the pair of nucleic acid
sequences selected from the group consisting of: SEQ NOs 490 and
491; 41.6 and 417; 419 and 420.
13. An antibody to specifically bind to the amino acid sequence of
claim 3.
14. An antibody to specifically bind to the amino acid sequence of
claim 4.
15. An antibody to specifically bind to the amino acid sequence of
claim 5.
16. A kit for detecting prostate cancer, comprising at least one
primer pair of claim 12.
17. A kit for detecting prostate cancer, comprising the antibody of
claim 13.
18. The kit of claim 17, wherein said kit further comprises at
least one ELISA reagent or at least one Western blot reagent.
19. A kit for detecting prostate cancer, comprising the antibody of
claim 14.
20. The kit of claim 19, wherein said kit further comprises at
least one ELISA reagent or at least one Western blot reagent.
21. A kit for detecting prostate cancer, comprising the antibody of
claim 15.
22. The kit of claim 21, wherein said kit further comprises at
least one ELISA reagent or at least one Western blot reagent.
23. A method for detecting prostate cancer, comprising detecting
overexpression of the polynucleotide of claim 1 in a sample from a
patient.
24. The method of claim 23, wherein said detecting overexpression
comprises performing nucleic acid amplification.
25. A method for detecting prostate cancer, comprising detecting
overexpression of the polynucleotide of claim 2 in a sample from a
patient.
26. The method of claim 25, wherein said detecting overexpression
comprises performing nucleic acid amplification.
27. A method for detecting prostate cancer, comprising detecting
overexpression of the polypeptide of claim 3 in a sample from a
patient.
28. The method of claim 27, wherein said detecting comprises
detecting binding of the antibody of claim 13 to the polypeptide of
claim 3 in a sample from a patient.
29. A method for detecting prostate cancer, comprising detecting
overexpression of the polypeptide of claim 4 in a sample from a
patient.
30. The method of claim 29, wherein said detecting comprises
detecting binding of the antibody of claim 14 to the polypeptide of
claim 4 in a sample from a patient.
31. A method for detecting prostate cancer, comprising detecting
overexpression of the polypeptide of claim 5 in a sample from a
patient.
32. The method of claim 31, wherein said detecting comprises
detecting binding of the antibody of claim 15 to the polypeptide of
claim 5 in a sample from a patient.
33. A biomarker to detect prostate cancer, comprising an amino acid
sequence of claim 3, marked with a label.
34. A biomarker to detect prostate cancer, comprising an amino acid
sequence of claim 4, marked with a label.
35. A biomarker to detect prostate cancer, comprising an amino acid
sequence of claim 5, marked with a label.
36. A method to screen for prostate cancer, comprising detecting
prostate cancer cells with the biomarker of claim 33.
37. A method to screen for prostate cancer, comprising detecting
prostate cancer cells with the biomarker of claim 34.
38. A method to screen for prostate cancer, comprising detecting
prostate cancer cells with the biomarker of claim 35.
39. A method to diagnose prostate cancer, comprising detecting
prostate cancer cells with the biomarker of claim 33.
40. A method to diagnose prostate cancer, comprising detecting
prostate cancer cells with the biomarker of claim 34.
41. A method to diagnose prostate cancer, comprising detecting
prostate cancer cells with the biomarker of claim 35.
42. A method for monitoring disease progression, treatment efficacy
or relapse of prostate cancer, comprising detecting prostate cancer
cells with the biomarker of claim 33.
43. A method for monitoring disease progression, treatment efficacy
or relapse of prostate cancer, comprising detecting prostate cancer
cells with the biomarker of claim 34.
44. A method for monitoring disease progression, treatment efficacy
or relapse of prostate cancer, comprising detecting prostate cancer
cells with the biomarker of claim 35.
45. A method of selecting a therapy for prostate cancer, comprising
detecting prostate cancer cells with the biomarker of claim 33 and
selecting a therapy according to said detection.
46. A method of selecting a therapy for prostate cancer, comprising
detecting prostate cancer cells with the biomarker of claim 34 and
selecting a therapy according to said detection.
47. A method of selecting a therapy for prostate cancer, comprising
detecting prostate cancer cells with the biomarker of claim 35 and
selecting a therapy according to said detection.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application is related to Novel Nucleotide and Amino
Acid Sequences, and Assays and Methods of use thereof for Diagnosis
of Prostate Cancer, and claims priority to the below U.S.
provisional and non-provisional applications, each of which is
incorporated herein by reference in its entirety:
[0002] Application No. 60/620,916 filed Oct. 22, 2004--Differential
Expression of Markers in Colon Cancer
[0003] Application No. 60/628,123 filed Nov. 17, 2004--Differential
Expression of Markers in Colon Cancer II
[0004] Application No. 60/621,131 filed Oct. 25, 2004--Diagnostic
Markers for Colon Cancer, and Assays and Methods of use
thereof.
[0005] Application No. 60/628,101 filed Nov. 17, 2004--Differential
Expression of Markers in Breast Cancer II
[0006] Application No. 60/620,874 filed Oct. 22, 2004--Differential
Expression of Markers in Ovarian Cancer
[0007] Application No. 60/628,134 filed Nov. 17, 2004--Differential
Expression of Markers in Ovarian Cancer II
[0008] Application No. 60/620,853 filed Oct. 22,
2004-28814--Differential Expression of Markers in Lung Cancer
[0009] Application No. 60/628,112 filed Nov. 17, 2004--Differential
Expression of Markers in Lung Cancer II
[0010] Application No. 60/628,145 filed Nov. 17, 2004--Differential
Expression of Markers in Pancreatic Cancer II
[0011] Application No. 60/620,656 filed Oct. 22, 2004--Differential
Expression of Markers in Prostate Cancer
[0012] Application No. 60/628,251 filed Nov. 17, 2004--Differential
Expression of Markers in Prostate Cancer II
[0013] Application No. 60/628,178 filed Nov. 17, 2004--Differential
Expression of Markers in Brain Cancer II
[0014] Application No. 60/628,231 filed Nov. 17, 2004--Novel
Diagnostic Serum Markers, and Assays and Methods of use
thereof.
[0015] Application No. 60/620,918 filed Oct. 22, 2004--Diagnostic
Markers for Renal Cancer, and Assays and Methods of Use
thereof.
[0016] Application No. 60/628,156 filed Nov. 17, 2004--Diagnostic
Markers for Renal Cancer, and Assays and Methods of Use thereof
II
[0017] Application No. 60/620,677 filed Oct. 22, 2004--Differential
Expression of Markers in Bladder Cancer I
[0018] Application No. 60/628,167 filed Nov. 17, 2004--Differential
Expression of Markers in Bladder Cancer II
[0019] Application No. - - - NA - - - filed Nov. 17, 2004--Novel
Diagnostic Markers, and Assays and Methods of Use thereof.
[0020] Application No. 60/539,129 filed Jan. 27, 2004--Methods and
Systems for Annotating Biomolecular Sequences
[0021] Application No. 60/539,128 filed Jan. 27, 2004--Evolutionary
Conserved Spliced Sequences and Methods and Systems for Identifying
thereof.
[0022] application Ser. No. 11/043,806 filed Jan. 27, 2005--NOVEL
NUCLEOTIDE AND AMINO ACID SEQUENCES, AND ASSAYS AND METHODS OF USE
THEREOF FOR DIAGNOSIS OF PROSTATE CANCER
FIELD OF THE INVENTION
[0023] The present invention is related to novel nucleotide and
protein sequences that are diagnostic markers for prostate cancer,
and assays and methods of use thereof.
BACKGROUND OF THE INVENTION
[0024] Prostate cancer is the most commonly diagnosed malignancy
and the second most frequent cause of cancer-related deaths in the
western male population. Prostate cancer therapies are most
effective in the earlier stages of the disease, before metastasis
has occurred. Treatment is expected to be even more effective
before significant local growth of the cancerous tissue has taken
place. Therefore, efforts to control the disease (i.e., to decrease
prostate cancer mortality) have focused on increasing detection of
the cancer while it is still locally confined and potentially
curable, through diagnostic assays that are suitable for early
detection of prostate cancer. Unfortunately, such detection also
has significant drawbacks, because diagnostic assays that use
currently available prostate cancer markers lead to high numbers of
false positive diagnoses, and/or are not sufficiently sensitive
(potentially leading to high numbers of false negative
diagnoses).
[0025] Measurements of serum concentrations of prostatic marker
enzymes have recognized value in the clinical detection, diagnosis
and management of prostate cancer. The two most widely used
prostatic marker enzymes are prostatic acid phosphatase (PAP) and
prostate-specific antigen (PSA). Normally, both enzymes are
secreted from the prostatic epithelial cells into the seminal
fluid, but in patients with prostatic disease they leak into the
circulation, where they can be detected by means of immunological
assays (Armbruster, Clin. Che. 39:181-95 (1993)).
[0026] Prostatic acid phosphatase, one of the earliest serum
markers for prostate, has an as yet undetermined function and is
one of the most predominant protein components in human prostatic
secretions. The use of PAP as a marker for prostatic tumors is
complicated by the reported structural similarities between the
prostate-specific acid phosphatase and the lysosomal acid
phosphatase occurring in all tissues. Furthermore, there is a
tendency towards lower PAP mRNA and protein levels in prostate
cancer in comparison with benign prostatic hyperplasia (BPH). In
recent years, PAP measurements were superseded by serum PSA
measurements in the clinical management of prostate cancer.
[0027] Prostate-specific antigen (PSA) was identified by several
groups as a prostate-specific protein from the seminal fluid, and
was subsequently determined to be an antigen from prostate cancer
tissue. PSA is produced exclusively by the columnar epithelial
cells of the prostate and periuretural glands. Normal prostate
epithelium and benign hyperplastic tissue actually produce more PSA
mRNA and protein than does prostate cancer tissue. Furthermore, it
was shown that loss of differentiation of prostatic carcinomas is
associated with a decrease in the level of intraprostatic PSA.
[0028] Prostate-specific membrane antigen (PSM) was originally
identified using an antibody developed by immunizing mice with the
membrane fraction of LNCaP human prostatic adenocarcinoma cells.
Like PAP and PSA, PSM can be detected in normal prostate, BPH and
prostate cancer and is absent from most other tissues. However, the
usefulness of PSM as marker for prostatic cancer has not been fully
established.
[0029] Other markers have recently been considered. For example,
PCA3 DD3 is a new marker from DiagnoCure, which has been described
as being useful in a urine-based test (PCA3 itself is described in
PCT Application Nos. WO 98/45420 and WO 2000/123550). This marker
is apparently only expressed in prostate cancer, and therefore may
be used to distinguish between BPH and prostate cancer. However, as
described in greater detail below, the sensitivity and accuracy of
this marker may be improved when used in combination with one or
more additional markers.
[0030] Therefore, PSA is recognized as the best available marker
for prostate cancer, being useful for screening selected
populations of patients with symptoms indicative of prostate cancer
and for monitoring patients after therapy, especially after
surgical prostatectomy. However, PSA has significant drawbacks in
terms of false positive measurements, since it cannot distinguish
prostate cancer from BPH. It may also lead to false negative
measurements, since de-differentiation of prostate cancerous tissue
(which may occur with some types of prostate cancers) also leads to
decreased expression of this marker. New markers are currently
being developed to overcome this problem, but these markers have
their own drawbacks. Clearly, new markers are required.
SUMMARY OF THE INVENTION
[0031] The background art does not teach or suggest markers for
prostate cancer that are sufficiently sensitive and/or accurate,
alone or in combination.
[0032] The present invention overcomes these deficiencies of the
background art by providing novel markers for prostate cancer that
are both sensitive and accurate. Furthermore, at least some of
these markers are able to distinguish between prostate cancer and
benign prostate hyperplasia ("BPH"). These markers are
differentially expressed, and preferably overexpressed in prostate
cancer specifically, as opposed to normal prostate tissue and/or
BPH. The measurement of these markers, alone or in combination, in
patient samples (biological samples) provides information that the
diagnostician can correlate with a probable diagnosis of prostate
cancer. The markers of the present invention, alone or in
combination, show a high degree of differential detection between
prostate cancer and non-cancerous states.
[0033] According to preferred embodiments of the present invention,
examples of suitable biological samples include but are not limited
to blood, serum, plasma, blood cells, urine, sputum, saliva, stool,
spinal fluid or CSF, lymph fluid, the external secretions of the
skin, respiratory, intestinal, and genitourinary tracts, tears,
milk, neuronal tissue, prostate tissue or mucous and any human
organ or tissue, or any sample obtained by lavage (for example of
the bronchial system), and also samples of in vivo cell culture
constituents. In a preferred embodiment, the biological sample
comprises prostate tissue and/or other tissues of the male
genitalia, or reproductive or urinary tracts, and/or a serum
(and/or any blood) sample and/or a urine sample and/or a semen
sample and/or any other tissue or liquid sample. The sample can
optionally be diluted with a suitable eluant before contacting the
sample to an antibody and/or performing any other diagnostic
assay.
[0034] Information given in the text with regard to cellular
localization was determined according to four different software
programs: (i) tmhmm (from Center for Biological Sequence Analysis,
Technical University of Denmark DTU, www "dot" cbs "dot" dtu "dot"
dk/services/TMHMM/TMHMM2.0b.guide.php) or (ii) tmpred (from EMBnet,
maintained by the ISREC Bionformatics group and the LICR
Information Technology Office, Ludwig Institute for Cancer
Research, Swiss Institute of Bioinformatics, www "dot" ch "dot"
embnet "dot" org/software/TMPRED_form "dot" html) for transmembrane
region prediction; (iii) signalp_hmm or (iv) signalp_nn (both from
Center for Biological Sequence Analysis, Technical University of
Denmark DTU, www "dot" cbs "dot" dtu "dot"
dk/services/SignalP/background/prediction.php) for signal peptide
prediction. The terms "signalp_hrm" and "signalp_nn" refer to two
modes of operation for the program SignalP: hmm refers to Hidden
Markov Model, while nn refers to neural networks. Localization was
also determined through manual inspection of known protein
localization and/or gene structure, and the use of heuristics by
the individual inventor. In some cases for the manual inspection of
cellular localization prediction inventors used the ProLoc
computational platform [Einat Hazkani-Covo, Erez Levanon, Galit
Rotman, Dan Graur and Amit Novik; (2004) "Evolution of
multicellularity in metazoa: comparative analysis of the
subcellular localization of proteins in Saccharomyces, Drosophila
and Caenorhabditis." Cell Biology International 2004;
28(3):171-8.], which predicts protein localization based on various
parameters including, protein domains (e.g., prediction of
trans-membranous regions and localization thereof within the
protein), pI, protein length, amino acid composition, homology to
pre-annotated proteins, recognition of sequence patterns which
direct the protein to a certain organelle (such as, nuclear
localization signal, NLS, mitochondria localization signal), signal
peptide and anchor modeling and using unique domains from Pfam that
are specific to a single compartment.
[0035] Information is given in the text with regard to SNPs (single
nucleotide polymorphisms). A description of the abbreviations is as
follows. "T->C", for example, means that the SNP results in a
change at the position given in the table from T to C. Similarly,
"M->Q", for example, means that the SNP has caused a change in
the corresponding amino acid sequence, from methionine (M) to
glutamine (Q). If, in place of a letter at the right hand side for
the nucleotide sequence SNP, there is a space, it indicates that a
frameshift has occurred. A frameshift may also be indicated with a
hyphen (-). A stop codon is indicated with an asterisk at the right
hand side (*). As part of the description of an SNP, a comment may
be found in parentheses after the above description of the SNP
itself. This comment may include an FTId, which is an identifier to
a SwissProt entry that was created with the indicated SNP. An FTId
is a unique and stable feature identifier, which allows
construction of links directly from position-specific annotation in
the feature table to specialized protein-related databases. The
FTId is always the last component of a feature in the description
field, as follows: FTId=XXX_number, in which XXX is the 3-letter
code for the specific feature key, separated by an underscore from
a 6-digit number. In the table of the amino acid mutations of the
wild type proteins of the selected splice variants of the
invention, the header of the first column is "SNP position(s) on
amino acid sequence", representing a position of a known mutation
on amino acid sequence.
[0036] SNPs may optionally be used as diagnostic markers according
to the present invention, alone or in combination with one or more
other SNPs and/or any other diagnostic marker. Preferred
embodiments of the present invention comprise such SNPs, including
but not limited to novel SNPs on the known (WT or wild type)
protein sequences given below, as well as novel nucleic acid and/or
amino acid sequences formed through such SNPs, and/or any SNP on a
variant amino acid and/or nucleic acid sequence described
herein.
[0037] Information given in the text with regard to the Homology to
the known proteins was determined by Smith-Waterman version 5.1.2
using special (non default) parameters as follows:
[0038] model=sw.model
[0039] GAPEXT=0
[0040] GAPOP=100.0 [0041] MATRIX=blosum 100
[0042] Information is given with regard to overexpression of a
cluster in cancer based on ESTs. A key to the p values with regard
to the analysis of such overexpression is as follows: [0043]
library-based statistics: P-value without including the level of
expression in cell-lines (P1) [0044] library based statistics:
P-value including the level of expression in cell-lines (P2) [0045]
EST clone statistics: P-value without including the level of
expression in cell-lines (SP1) [0046] EST clone statistics:
predicted overexpression ratio without including the level of
expression in cell-lines (R3) [0047] EST clone statistics: P-value
including the level of expression in cell-lines (SP2) [0048] EST
clone statistics: predicted overexpression ratio including the
level of expression in cell-lines (R4)
[0049] Library-based statistics refer to statistics over an entire
library, while EST clone statistics refer to expression only for
ESTs from a particular tissue or cancer.
[0050] Information is given with regard to overexpression of a
cluster in cancer based on microarrays. As a microarray reference,
in the specific segment paragraphs, the unabbreviated tissue name
was used as the reference to the type of chip for which expression
was measured.
[0051] There are two types of microarray results: those from
microarrays prepared according to a design by the present
inventors, for which the microarray fabrication procedure is
described in detail in Materials and Experimental Procedures
section herein; and those results from microarrays using Affymetrix
technology. As a microarray reference, in the specific segment
paragraphs, the unabbreviated tissue name was used as the reference
to the type of chip for which expression was measured. For
microarrays prepared according to a design by the present
inventors, the probe name begins with the name of the cluster
(gene), followed by an identifying number. Oligonucleotide
microarray results taken from Affymetrix data were from chips
available from Affymetrix Inc, Santa Clara, Calif., USA (see for
example data regarding the Human Genome U133 (HG-U133) Set at www
"dot" affymetrix "dot" com/products/arrays/specific/hgu133.affx;
GeneChip Human Genome U133A 2.0 Array at www "dot" affymetrix "dot"
com/products/arrays/specific/hgu133av2.affx; and Human Genome U133
Plus 2.0 Array at www "dot" affymetrix "dot"
com/products/arrays/specific/hgu133plus.affx). The probe names
follow the Affymetrix naming convention. The data is available from
NCBI Gene Expression Omnibus (see
www.ncbi.nlm.nih.gov/projects/geo/ and Edgar et al, Nucleic Acids
Research, 2002, Vol. 30, No. 1 207-210). The dataset (including
results) is available from www "dot" ncbi "dot" nlm "dot" nih "dot"
gov/geo/query/acc.cgi?acc=GSE1133 for the Series GSE1133 database
(published on March 2004); a reference to these results is as
follows: Su et al (Proc Natl Acad Sci USA. 2004 Apr. 20;
101(16):6062-7. Epub 2004 Apr. 9). A list of probes designed
according to the present inventors is given below. TABLE-US-00001
>HSSTROL3_0_0_12518
ATGAGAGTAACCTCACCCGTGCACTAGTTTACAGAGCATTCACTGCCCCA
>HSSTROL3_0_0_12517
CAGAGATGAGAGCCTGGAGCATTGCAGATGCCAGGGACTTCACAAATGAA
[0052] The following list of abbreviations for tissues was used in
the TAA histograms. The term "TAA" stands for "Tumor Associated
Antigen", and the TAA histograms, given in the text, represent the
cancerous tissue expression pattern as predicted by the biomarkers
selection engine, as described in detail in examples 1-5 below:
[0053] "BONE" for "bone"; [0054] "COL" for "colon"; [0055] "EPI"
for "epithelial"; [0056] "GEN" for "general"; [0057] "LIVER" for
"liver"; [0058] "LUN" for "lung"; [0059] "LYMPH" for "lymph nodes";
[0060] "MARROW" for "bone marrow"; [0061] "OVA" for "ovary"; [0062]
"PANCREAS" for "pancreas"; [0063] "PRO" for "prostate"; [0064]
"STOMACH" for "stomach"; [0065] "TCELL" for "T cells"; [0066]
"THYROID" for "Thyroid"; [0067] "MAM" for "breast"; [0068] "BRAIN"
for "brain"; [0069] "UTERUS" for "uterus"; [0070] "SKIN" for
"skin"; [0071] "KIDNEY" for "kidney"; [0072] "MUSCLE" for "muscle";
[0073] "ADREN" for "adrenal"; [0074] "HEAD" for "head and neck";
[0075] "BLADDER" for "bladder";
[0076] It should be noted that the terms "segment", "seg" and
"node" are used interchangeably in reference to nucleic acid
sequences of the present invention; they refer to portions of
nucleic acid sequences that were shown to have one or more
properties as described below. They are also the building blocks
that were used to construct complete nucleic acid sequences as
described in greater detail below. Optionally and preferably, they
are examples of oligonucleotides which are embodiments of the
present invention, for example as amplicons, hybridization units
and/or from which primers and/or complementary oligonucleotides may
optionally be derived, and/or for any other use.
[0077] As used herein the phrase "prostate cancer" refers to
cancers of the prostate tissue and/or other tissues of the male
genitalia, or reproductive or urinary tracts.
[0078] The term "marker" in the context of the present invention
refers to a nucleic acid fragment, a peptide, or a polypeptide,
which is differentially present in a sample taken from subjects
(patients) having prostate cancer as compared to a comparable
sample taken from subjects who do not have prostate cancer.
[0079] The phrase "differentially present" refers to differences in
the quantity of a marker present in a sample taken from patients
having prostate cancer as compared to a comparable sample taken
from patients who do not have prostate cancer. For example, a
nucleic acid fragment may optionally be differentially present
between the two samples if the amount of the nucleic acid fragment
in one sample is significantly different from the amount of the
nucleic acid fragment in the other sample, for example as measured
by hybridization and/or NAT-based assays. A polypeptide is
differentially present between the two samples if the amount of the
polypeptide in one sample is significantly different from the
amount of the polypeptide in the other sample. It should be noted
that if the marker is detectable in one sample and not detectable
in the other, then such a marker can be considered to be
differentially present.
[0080] As used herein the phrase "diagnostic" means identifying the
presence or nature of a pathologic condition. Diagnostic methods
differ in their sensitivity and specificity. The "sensitivity" of a
diagnostic assay is the percentage of diseased individuals who test
positive (percent of "true positives"). Diseased individuals not
detected by the assay are "false negatives." Subjects who are not
diseased and who test negative in the assay are termed "true
negatives." The "specificity" of a diagnostic assay is 1 minus the
false positive rate, where the "false positive" rate is defined as
the proportion of those without the disease who test positive.
While a particular diagnostic method may not provide a definitive
diagnosis of a condition, it suffices if the method provides a
positive indication that aids in diagnosis.
[0081] As used herein the phrase "diagnosing" refers to classifying
a disease or a symptom, determining a severity of the disease,
monitoring disease progression, forecasting an outcome of a disease
and/or prospects of recovery. The term "detecting" may also
optionally encompass any of the above.
[0082] Diagnosis of a disease according to the present invention
can be affected by determining a level of a polynucleotide or a
polypeptide of the present invention in a biological sample
obtained from the subject, wherein the level determined can be
correlated with predisposition to, or presence or absence of the
disease. It should be noted that a "biological sample obtained from
the subject" may also optionally comprise a sample that has not
been physically removed from the subject, as described in greater
detail below.
[0083] As used herein, the term "level" refers to expression levels
of RNA and/or protein or to DNA copy number of a marker of the
present invention.
[0084] Typically the level of the marker in a biological sample
obtained from the subject is different (i.e., increased or
decreased) from the level of the same variant in a similar sample
obtained from a healthy individual (examples of biological samples
are described herein).
[0085] Numerous well known tissue or fluid collection methods can
be utilized to collect the biological sample from the subject in
order to determine the level of DNA, RNA and/or polypeptide of the
variant of interest in the subject.
[0086] Examples include, but are not limited to, fine needle
biopsy, needle biopsy, core needle biopsy and surgical biopsy
(e.g., brain biopsy), and lavage. Regardless of the procedure
employed, once a biopsy/sample is obtained the level of the variant
can be determined and a diagnosis can thus be made.
[0087] Determining the level of the same variant in normal tissues
of the same origin is preferably effected along-side to detect an
elevated expression and/or amplification and/or a decreased
expression, of the variant as opposed to the normal tissues.
[0088] A "test amount" of a marker refers to an amount of a marker
in a subject's sample that is consistent with a diagnosis of
prostate cancer. A test amount can be either in absolute amount
(e.g., microgram/ml) or a relative amount (e.g., relative intensity
of signals).
[0089] A "control amount" of a marker can be any amount or a range
of amounts to be compared against a test amount of a marker. For
example, a control amount of a marker can be the amount of a marker
in a patient with prostate cancer or a person without prostate
cancer. A control amount can be either in absolute amount (e.g.,
microgram/ml) or a relative amount (e.g., relative intensity of
signals).
[0090] "Detect" refers to identifying the presence, absence or
amount of the object to be detected.
[0091] A "label" includes any moiety or item detectable by
spectroscopic, photo chemical, biochemical, immunochemical, or
chemical means. For example, useful labels include .sup.32P,
.sup.35S, fluorescent dyes, electron-dense reagents, enzymes (e.g.,
as commonly used in an ELISA), biotin-streptavadin, dioxigenin,
haptens and proteins for which antisera or monoclonal antibodies
are available, or nucleic acid molecules with a sequence
complementary to a target. The label often generates a measurable
signal, such as a radioactive, chromogenic, or fluorescent signal,
that can be used to quantify the amount of bound label in a sample.
The label can be incorporated in or attached to a primer or probe
either covalently, or through ionic, van der Waals or hydrogen
bonds, e.g., incorporation of radioactive nucleotides, or
biotinylated nucleotides that are recognized by streptavadin. The
label may be directly or indirectly detectable. Indirect detection
can involve the binding of a second label to the first label,
directly or indirectly. For example, the label can be the ligand of
a binding partner, such as biotin, which is a binding partner for
streptavadin, or a nucleotide sequence, which is the binding
partner for a complementary sequence, to which it can specifically
hybridize. The binding partner may itself be directly detectable,
for example, an antibody may be itself labeled with a fluorescent
molecule. The binding partner also may be indirectly detectable,
for example, a nucleic acid having a complementary nucleotide
sequence can be a part of a branched DNA molecule that is in turn
detectable through hybridization with other labeled nucleic acid
molecules (see, e.g., P. D. Fahrlander and A. Klausner,
Bio/Technology 6:1165 (1988)). Quantitation of the signal is
achieved by, e.g., scintillation counting, densitometry, or flow
cytometry.
[0092] Exemplary detectable labels, optionally and preferably for
use with immunoassays, include but are not limited to magnetic
beads, fluorescent dyes, radiolabels, enzymes (e.g., horse radish
peroxide, alkaline phosphatase and others commonly used in an
ELISA), and calorimetric labels such as colloidal gold or colored
glass or plastic beads. Alternatively, the marker in the sample can
be detected using an indirect assay, wherein, for example, a
second, labeled antibody is used to detect bound marker-specific
antibody, and/or in a competition or inhibition assay wherein, for
example, a monoclonal antibody which binds to a distinct epitope of
the marker are incubated simultaneously with the mixture.
[0093] "Immunoassay" is an assay that uses an antibody to
specifically bind an antigen. The immunoassay is characterized by
the use of specific binding properties of a particular antibody to
isolate, target, and/or quantify the antigen.
[0094] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide (or other epitope), refers
to a binding reaction that is determinative of the presence of the
protein in a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein at least two
times greater than the background (non-specific signal) and do not
substantially bind in a significant amount to other proteins
present in the sample. Specific binding to an antibody under such
conditions may require an antibody that is selected for its
specificity for a particular protein. For example, polyclonal
antibodies raised to seminal basic protein from specific species
such as rat, mouse, or human can be selected to obtain only those
polyclonal antibodies that are specifically immunoreactive with
seminal basic protein and not with other proteins, except for
polymorphic variants and alleles of seminal basic protein. This
selection may be achieved by subtracting out antibodies that
cross-react with seminal basic protein molecules from other
species. A variety of immunoassay formats may be used to select
antibodies specifically immunoreactive with a particular protein.
For example, solid-phase ELISA immunoassays are routinely used to
select antibodies specifically immunoreactive with a protein (see,
e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988),
for a description of immunoassay formats and conditions that can be
used to determine specific immunoreactivity). Typically a specific
or selective reaction will be at least twice background signal or
noise and more typically more than 10 to 100 times background.
[0095] According to preferred embodiments of the present invention,
there is provided an isolated polynucleotide comprising the
sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7,
8, 9 and 10.
[0096] According to preferred embodiments of the present invention,
there is provided an isolated polynucleotide comprising the segment
selected from the group consisting of SEQ ID NOs: 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, 111, 112, 113, 114 and 115.
[0097] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide comprising SEQ ID NOs:
331, 332, 333, 334 and 335.
[0098] According to preferred embodiments of the present invention,
there is provided an isolated polynucleotide comprising the
sequence selected from the group consisting of SEQ ID NOs: 34, 35,
36, 37, 38 and 39.
[0099] According to preferred embodiments of the present invention,
there is provided an isolated polynucleotide comprising a segment
SEQ ID NOs: 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,
233, 234, 235, 236 and 237.
[0100] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide comprising SEQ ID NOs:
359, 360, 361, 362 and 363.
[0101] According to preferred embodiments of the present invention,
there is provided an isolated polynucleotide comprising the
sequence selected from the group consisting of SEQ ID NOs: 13 and
14.
[0102] According to preferred embodiments of the present invention,
there is provided an isolated polynucleotide comprising the node
selected from the group consisting of SEQ ID NOs: 136, 137, 138,
139, 140, 141 and 142.
[0103] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide comprising SEQ ID NOs:
338 and 339.
[0104] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide of SEQ ID NO.
359, comprising a first amino acid sequence being at least 90%
homologous to corresponding to amino acids 1-163 of SEQ ID NOs.
391, which also corresponds to amino acids 1-163 of SEQ ID NOs.
359, a bridging amino acid H corresponding to amino acid 164 of SEQ
ID NOs. 359, a second amino acid sequence being at least 90%
homologous to corresponding to amino acids 165-445 of SEQ ID NOs.
391, which also corresponds to amino acids 165-445 of SEQ ID NO.
359, and a third amino acid sequence being at least 70%, optionally
at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having
the sequence corresponding to amino acids 446-496 of SEQ ID NO.
359, wherein said first amino acid sequence, bridging amino acid,
second amino acid sequence and third amino acid sequence are
contiguous and in a sequential order.
[0105] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 359, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 446-496 in SEQ ID NO. 359.
[0106] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide encoding for SEQ
ID NOs.360, comprising a first amino acid sequence being at least
90% homologous to corresponding to amino acids 1-163 of SEQ ID NOs.
391, which also corresponds to amino acids 1-163 of SEQ ID NO:360,
a bridging amino acid H corresponding to amino acid 164 of SEQ ID
NO. 360, a second amino acid sequence being at least 90% homologous
to corresponding to amino acids 165-358 of SEQ ID NOs. 391, which
also corresponds to amino acids 165-358 of SEQ ID NO. 360, and a
third amino acid sequence being at least 70%, optionally at least
80%, preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide having the
sequence corresponding to amino acids 359-382 of SEQ ID NO. 360,
wherein said first amino acid sequence, bridging amino acid, second
amino acid sequence and third amino acid sequence are contiguous
and in a sequential order.
[0107] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 360, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 359-382 in SEQ ID NO. 360.
[0108] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide of SEQ ID NO.
361, comprising a first amino acid sequence being at least 90%
homologous to corresponding to amino acids 1-163 of SEQ ID NOs.
391, which also corresponds to amino acids 1-163 of SEQ ID NO. 361,
a bridging amino acid H corresponding to amino acid 164 of SEQ ID
NO. 361, a second amino acid sequence being at least 90% homologous
to corresponding to amino acids 165-359 of SEQ ID NOs. 391, which
also corresponds to amino acids 165-359 of SEQ ID NO. 361, and a
third amino acid sequence being at least 70%, optionally at least
80%, preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide having the
sequence corresponding to amino acids 360-370 of SEQ ID NO. 361,
wherein said first amino acid sequence, bridging amino acid, second
amino acid sequence and third amino acid sequence are contiguous
and in a sequential order.
[0109] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 361, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 360-370 in SEQ ID NO. 361.
[0110] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide of SEQ ID NO.
362, comprising a first amino acid sequence being at least 90%
homologous to corresponding to amino acids 1-163 of SEQ ID NOs. SEQ
ID NOs. 391, which also corresponds to amino acids 1-163 of SEQ ID
NO:362, a bridging amino acid H corresponding to amino acid 164 of
SEQ ID NO:362, a second amino acid sequence being at least 90%
homologous to corresponding to amino acids 165-286 of SEQ ID NOs.
391, which also corresponds to amino acids 165-286 of SEQ ID
NO:362, and a third amino acid sequence being at least 70%,
optionally at least 80%, preferably at least 85%, more preferably
at least 90% and most preferably at least 95% homologous to a
polypeptide having the sequence corresponding to amino acids
287-301 of SEQ ID NO:362, wherein said first amino acid sequence,
bridging amino acid, second amino acid sequence and third amino
acid sequence are contiguous and in a sequential order.
[0111] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO:362, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 287-301 in SEQ ID NO. 362.
[0112] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide of SEQ ID NO.
363, comprising a first amino acid sequence being at least 90%
homologous to corresponding to amino acids 1-96 of SEQ ID NOs. 391,
which also corresponds to amino acids 1-96 of SEQ ID NO. 363, a
second amino acid sequence being at least 90% homologous to
corresponding to amino acids 113-163 of SEQ ID NOs. 391, which also
corresponds to amino acids 97-147 of SEQ ID NO:363, a bridging
amino acid H corresponding to amino acid 148 of SEQ ID NO. 363, a
third amino acid sequence being at least 90% homologous to
corresponding to amino acids 165-359 of SEQ ID NOs. 391, which also
corresponds to amino acids 149-343 of SEQ ID NO:363, and a fourth
amino acid sequence being at least 70%, optionally at least 80%,
preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide sequence
corresponding to amino acids 344-354 of SEQ ID NO. 363, wherein
said first amino acid sequence, second amino acid sequence,
bridging amino acid, third amino acid sequence and fourth amino
acid sequence are contiguous and in a sequential order.
[0113] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide of an edge
portion of SEQ ID NO:363, comprising a polypeptide having a length
"n", wherein n is at least about 10 amino acids in length,
optionally at least about 20 amino acids in length, preferably at
least about 30 amino acids in length, more preferably at least
about 40 amino acids in length and most preferably at least about
50 amino acids in length, wherein at least two amino acids comprise
KR, having a structure as follows: a sequence starting from any of
amino acid numbers 96-x to 96; and ending at any of amino acid
numbers 97+((n-2)-x), in which x varies from 0 to n-2.
[0114] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 363, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to the sequence in SEQ ID NO. 363.
[0115] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide of SEQ ID NO.
339, comprising a first amino acid sequence being at least 90%
homologous to corresponding to amino acids 1-27 of SEQ ID NO. 387,
which also corresponds to amino acids 1-27 of SEQ ID NO. 339, and a
second amino acid sequence being at least 70%, optionally at least
80%, preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide having the
sequence corresponding to amino acids 28-41 of SEQ ID NO. 339,
wherein said first amino acid sequence and second amino acid
sequence are contiguous and in a sequential order.
[0116] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 339, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 28-41 in SEQ ID NO. 339.
[0117] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide encoding for SEQ
ID NO. 332, comprising a first amino acid sequence being at least
70%, optionally at least 80%, preferably at least 85%, more
preferably at least 90% and most preferably at least 95% homologous
to a polypeptide sequence corresponding to amino acids 1-110 of SEQ
ID NO. 332, and a second amino acid sequence being at least 90%
homologous to TQ corresponding to amino acids 1-112 of Q8IXM0,
which also corresponds to amino acids 111-222 of SEQ ID NO. 332,
wherein said first and second amino acid sequences are contiguous
and in a sequential order.
[0118] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a head of
SEQ ID NO. 332, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 1-110 of SEQ ID NO. 332.
[0119] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide of SEQ ID NO.
332, comprising a first amino acid sequence being at least 90%
homologous to amino acids 1-83 of Q96AC2, which also corresponds to
amino acids 1-83 of SEQ ID NO. 332, and a second amino acid
sequence being at least 70%, optionally at least 80%, preferably at
least 85%, more preferably at least 90% and most preferably at
least 95% homologous to a polypeptide sequence corresponding to
amino acids 84-222 of SEQ ID NO. 332, wherein said first and second
amino acid sequences are contiguous and in a sequential order.
[0120] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 332, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 84-222 in SEQ ID NO. 332.
[0121] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide of SEQ ID NO.
332, comprising a first amino acid sequence being at least 90%
homologous to amino acids 1-83 of Q8N2G4, which also corresponds to
amino acids 1-83 of SEQ ID NO. 332, and a second amino acid
sequence being at least 70%, optionally at least 80%, preferably at
least 85%, more preferably at least 90% and most preferably at
least 95% homologous to a polypeptide sequence corresponding to
amino acids 84-222 of SEQ ID NO. 332, wherein said first and second
amino acid sequences are contiguous and in a sequential order.
[0122] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 332, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 84-222 in SEQ ID NO. 332.
[0123] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide encoding for SEQ
ID NO. 332, comprising a first amino acid sequence being at least
90% homologous to amino acids 24-106 of BAC85518, which also
corresponds to amino acids 1-83 of SEQ ID NO. 332, and a second
amino acid sequence being at least 70%, optionally at least 80%,
preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide sequence
corresponding to amino acids 84-222 of SEQ ID NO. 332, wherein said
first and second amino acid sequences are contiguous and in a
sequential order.
[0124] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 332, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 84-222 in SEQ ID NO. 332.
[0125] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide encoding for SEQ
ID NO. 333, comprising a first amino acid sequence being at least
90% homologous to amino acids 1-64 of Q96AC2, which also
corresponds to amino acids 1-64 of SEQ ID NO. 333, and a second
amino acid sequence being at least 70%, optionally at least 80%,
preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide sequence
corresponding to amino acids 65-93 of SEQ ID NO. 333, wherein said
first and second amino acid sequences are contiguous and in a
sequential order.
[0126] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 333, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 65-93 in SEQ ID NO. 333.
[0127] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide encoding for SEQ
ID NO. 333, comprising a first amino acid sequence being at least
90% homologous to amino acids 1-64 of Q8N2G4, which also
corresponds to amino acids 1-64 of SEQ ID NO. 333, and a second
amino acid sequence being at least 70%, optionally at least 80%,
preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide sequence
corresponding to amino acids 65-93 of SEQ ID NO. 333, wherein said
first and second amino acid sequences are contiguous and in a
sequential order.
[0128] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 333, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 65-93 in SEQ ID NO. 333.
[0129] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide encoding for SEQ
ID NO. 333, comprising a first amino acid sequence being at least
70%, optionally at least 80%, preferably at least 85%, more
preferably at least 90% and most preferably at least 95% homologous
to a polypeptide sequence corresponding to amino acids 1-5 of SEQ
ID NO. 333, second amino acid sequence being at least 90%
homologous to amino acids 22-80 of BAC85273, which also corresponds
to amino acids 6-64 of SEQ ID NO. 333, and a third amino acid
sequence being at least 70%, optionally at least 80%, preferably at
least 85%, more preferably at least 90% and most preferably at
least 95% homologous to a polypeptide sequence corresponding to
amino acids 65-93 of SEQ ID NO. 333, wherein said first, second and
third amino acid sequences are contiguous and in a sequential
order.
[0130] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a head of
SEQ ID NO. 333, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 1-5 of SEQ ID NO. 333.
[0131] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 333, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 65-93 in SEQ ID NO. 333.
[0132] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide encoding for SEQ
ID NO. 333, comprising a first amino acid sequence being at least
90% homologous to amino acids 24-87 of BAC85518, which also
corresponds to amino acids 1-64 of SEQ ID NO. 333, and a second
amino acid sequence being at least 70%, optionally at least 80%,
preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide sequence
corresponding to amino acids 65-93 of SEQ ID NO. 333, wherein said
first and second amino acid sequences are contiguous and in a
sequential order.
[0133] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 333, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 65-93 in SEQ ID NO. 333.
[0134] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide encoding for SEQ
ID NO. 334, comprising a first amino acid sequence being at least
90% homologous to amino acids 1-63 of Q96AC2, which also
corresponds to amino acids 1-63 of SEQ ID NO. 334, and a second
amino acid sequence being at least 70%, optionally at least 80%,
preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide sequence
corresponding to amino acids 64-84 of SEQ ID NO. 334, wherein said
first and second amino acid sequences are contiguous and in a
sequential order.
[0135] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 334, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 64-84 in SEQ ID NO. 334.
[0136] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide encoding for SEQ
ID NO. 335, comprising a first amino acid sequence being at least
90% homologous to amino acids 1-63 of SEQ ID NOs. Q96AC2, which
also corresponds to amino acids 1-63 of SEQ ID NO. 335, and a
second amino acid sequence being at least 70%, optionally at least
80%, preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide sequence
corresponding to amino acids 64-90 of SEQ ID NO. 335, wherein said
first and second amino acid sequences are contiguous and in a
sequential order.
[0137] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 335, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 64-90 in SEQ ID NO. 335.
[0138] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide encoding for SEQ
ID NO. 335, comprising a first amino acid sequence being at least
90% homologous to amino acids 1-63 of Q8N2G4, which also
corresponds to amino acids 1-63 of SEQ ID NO. 335, and a second
amino acid sequence being at least 70%, optionally at least 80%,
preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide sequence
corresponding to amino acids 64-90 of SEQ ID NO. 335, wherein said
first and second amino acid sequences are contiguous and in a
sequential order.
[0139] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 335, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 64-90 in SEQ ID NO. 335.
[0140] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide encoding for SEQ
ID NO. 335, comprising a first amino acid sequence being at least
70%, optionally at least 80%, preferably at least 85%, more
preferably at least 90% and most preferably at least 95% homologous
to a polypeptide sequence corresponding to amino acids 1-5 of SEQ
ID NO. 335, second amino acid sequence being at least 90%
homologous to amino acids 22-79 of BAC85273, which also corresponds
to amino acids 6-63 of SEQ ID NO. 335, and a third amino acid
sequence being at least 70%, optionally at least 80%, preferably at
least 85%, more preferably at least 90% and most preferably at
least 95% homologous to a polypeptide sequence corresponding to
amino acids 64-90 of SEQ ID NO. 335, wherein said first, second and
third amino acid sequences are contiguous and in a sequential
order.
[0141] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a head of
SEQ ID NO. 335, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 1-5 of SEQ ID NO. 335.
[0142] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 335, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to the amino acids 64-90 in SEQ ID NO. 335.
[0143] According to preferred embodiments of the present invention,
there is provided an isolated chimeric polypeptide encoding for SEQ
ID NO. 335, comprising a first amino acid sequence being at least
90% homologous to amino acids 24-86 of BAC85518, which also
corresponds to amino acids 1-63 of SEQ ID NO. 335, and a second
amino acid sequence being at least 70%, optionally at least 80%,
preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide sequence
corresponding to amino acids 64-90 of SEQ ID NO. 335, wherein said
first and second amino acid sequences are contiguous and in a
sequential order.
[0144] According to preferred embodiments of the present invention,
there is provided an isolated polypeptide encoding for a tail of
SEQ ID NO. 335, comprising a polypeptide being at least 70%,
optionally at least about 80%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% homologous to amino acids 64-90 in SEQ ID NO. 335.
[0145] According to preferred embodiments of the present invention,
there is provided an antibody from cluster R11723, HUTMTREFAC,
HSSTROL3, capable of specifically binding to an epitope of an amino
acid sequence.
[0146] Optionally the amino acid sequence corresponds to a bridge,
edge portion, tail, head or insertion.
[0147] Optionally the antibody is capable of differentiating
between a splice variant having said epitope and a corresponding
known protein.
[0148] According to preferred embodiments of the present invention,
there is provided a kit for detecting prostate cancer, comprising a
kit from cluster R11723, HUMTREFAC, HSSTROL3 for detecting
overexpression of a splice variant.
[0149] Optionally the kit comprises a NAT-based technology.
[0150] Optionally the kit further comprises at least one primer
pair capable of selectively hybridizing to a nucleic acid
sequence.
[0151] Optionally the kit further comprises at least one
oligonucleotide capable of selectively hybridizing to a nucleic
acid sequence.
[0152] Optionally the kit comprises an antibody.
[0153] Optionally the kit further comprises at least one reagent
for performing an ELISA or a Western blot.
[0154] According to preferred embodiments of the present invention,
there is provided a method for detecting prostate cancer,
comprising detecting overexpression of a splice variant from
cluster R11723, HUMTREFAC, and HSSTROL3.
[0155] Optionally detecting overexpression is performed with a
NAT-based technology.
[0156] Optionally detecting overexpression is performed with an
immunoassay.
[0157] Optionally the immunoassay comprises an antibody.
[0158] According to preferred embodiments of the present invention,
there is provided a biomarker capable of detecting prostate cancer,
comprising nucleic acid sequences or a fragment thereof, or amino
acid sequences or a fragment thereof from cluster R11723, HUMTREFAC
and HSSTROL3.
[0159] According to preferred embodiments of the present invention,
there is provided a method for screening for prostate cancer,
comprising detecting prostate cancer cells with a biomarker or an
antibody or a method or assay from cluster R11723, HUMTREFAC and
HSSTROL3.
[0160] According to preferred embodiments of the present invention,
there is provided a method for diagnosing prostate cancer,
comprising detecting prostate cancer cells with a biomarker or an
antibody or a method or assay from cluster R11723, HUMTREFAC and
HSSTROL3.
[0161] According to preferred embodiments of the present invention,
there is provided a method for monitoring disease progression,
treatment efficacy, relapse of prostate cancer, comprising
detecting prostate cancer cells with a biomarker or an antibody or
a method or assay from cluster R11723, HUMTREFAC and HSSTROL3.
[0162] According to preferred embodiments of the present invention,
there is provided a method of selecting a therapy for prostate
cancer, comprising detecting prostate cancer cells with a biomarker
or an antibody or a method or assay from cluster R11723, HUMTREFAC
and HSSTROL3.
[0163] According to preferred embodiments of the present invention,
preferably any of the above nucleic acid and/or amino acid
sequences further comprises any sequence having at least about 70%,
preferably at least about 80%, more preferably at least about 90%,
most preferably at least about 95% homology thereto.
[0164] All nucleic acid sequences and/or amino acid sequences shown
herein as embodiments of the present invention relate to their
isolated form, as isolated polynucleotides (including for all
transcripts), oligonucleotides (including for all segments,
amplicons and primers), peptides (including for all tails, bridges,
insertions or heads, optionally including other antibody epitopes
as described herein) and/or polypeptides (including for all
proteins). It should be noted that oligonucleotide and
polynucleotide, or peptide and polypeptide, may optionally be used
interchangeably.
[0165] Unless otherwise noted, all experimental data relates to
variants of the present invention, named according to the segment
being tested (as expression was tested through RT-PCR as
described).
[0166] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs. The following
references provide one of skill with a general definition of many
of the terms used in this invention: Singleton et al., Dictionary
of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge
Dictionary of Science and Technology (Walker ed., 1988); The
Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer
Verlag (1991); and Hale & Marham, The Harper Collins Dictionary
of Biology (1991). All of these are hereby incorporated by
reference as if fully set forth herein. As used herein, the
following terms have the meanings ascribed to them unless specified
otherwise.
BRIEF DESCRIPTION OF DRAWINGS
[0167] FIG. 1 is a schematic description of the cancer biomarker
selection engine.
[0168] FIG. 2 is a schematic illustration, depicting grouping of
transcripts of a given cluster based on presence or absence of
unique sequence regions.
[0169] FIG. 3 is a schematic summary of quantitative real-time PCR
analysis.
[0170] FIG. 4 is a schematic presentation of the oligonucleotide
based microarray fabrication.
[0171] FIG. 5 is a schematic summary of the oligonucleotide based
microarray experimental flow.
[0172] FIG. 6 is a histogram showing Cancer and cell-line vs.
normal tissue expression for Cluster R11723, demonstrating
overexpression in epithelial malignant tumors, a mixture of
malignant tumors from different tissues and kidney malignant
tumors.
[0173] FIG. 7 is a histogram showing over expression of the R11723
transcripts which are detectable by amplicon as depicted in
sequence name R11723 seg13 (SEQ ID NO:492) in cancerous prostate
samples relative to the normal samples.
[0174] FIG. 8 is a histogram showing expression of R11723
transcripts, which are detectable by amplicon as depicted in
sequence name R11723seg13 (SEQ ID NO:492), in different normal
tissues.
[0175] FIG. 9A are histograms showing over expression of the R11723
transcripts, which are detectable by amplicon as depicted in
sequence name R11723 junc11-18 (SEQ ID NO:495) in cancerous
prostate samples relative to the normal samples (FIG. 9A) or
expression in normal tissues (FIG. 9B).
[0176] FIG. 10 is a histogram showing Cancer and cell-line vs.
normal tissue expression for Cluster HUMTREFAC, demonstrating
overexpression in a mixture of malignant tumors from different
tissues, breast malignant tumors, pancreas carcinoma and prostate
cancer.
[0177] FIG. 11 is a histogram showing Cancer and cell-line vs.
normal tissue expression for Cluster HSSTROL3, demonstrating
overexpression in transitional cell carcinoma, epithelial malignant
tumors, a mixture of malignant tumors from different tissues and
pancreas carcinoma.
[0178] FIG. 12 is a histogram showing the over expression of the
Stromelysin-3 precursor (SEQ ID NO:391) transcripts, which are
detectable by amplicon as depicted in sequence name HSSTROL3 seg24
(SEQ ID NO:499), in cancerous Prostate samples relative to the
normal samples.
[0179] FIG. 13 is a histogram demonstrating the expression of
Stromelysin-3 transcripts which are detectable by amplicon as
depicted in sequence name HSSTROL3 seg24 (SEQ ID NO:499) in
different normal tissues.
[0180] FIGS. 14 and 15 are histograms showing over expression of
the Thrombospondin 1 (THBS1) transcripts in cancerous and benign
(BPH) prostate samples relative to the normal samples.
[0181] FIG. 16 is a histogram showing the relative expression of
Thrombospondin 1 (THBS1) variants in normal, benign and tumor
derived prostate samples as determined by oligonucleotide-based
micro-array experiments with SEQ ID NOs: 477, 478, 479, 480, 481,
482.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0182] The present invention is of novel markers for prostate
cancer that are both sensitive and accurate. Biomolecular sequences
(amino acid and/or nucleic acid sequences) uncovered using the
methodology of the present invention and described herein can be
efficiently utilized as tissue or pathological markers and/or as
drugs or drug targets for treating or preventing a disease.
[0183] These markers are specifically released to the bloodstream
under conditions of prostate cancer and/or other prostate
pathology, and/or are otherwise expressed at a much higher level
and/or specifically expressed in prostate cancer tissue or cells.
The measurement of these markers, alone or in combination, in
patient samples provides information that the diagnostician can
correlate with a probable diagnosis of prostate cancer and/or
pathology.
[0184] The present invention therefore also relates to diagnostic
assays for prostate cancer and/or prostate pathology, and methods
of use of such markers for detection of prostate cancer and/or
prostate pathology, optionally and preferably in a sample taken
from a subject (patient), which is more preferably some type of
blood sample.
[0185] The markers of the present invention, alone or in
combination, can be used for prognosis, prediction, screening,
early diagnosis, staging, therapy selection and treatment
monitoring of prostate cancer. For example, optionally and
preferably, these markers may be used for staging prostate cancer
and/or monitoring the progression of the disease. Furthermore, the
markers of the present invention, alone or in combination, can be
used for detection of the source of metastasis found in anatomical
places other then prostate. Also, one or more of the markers may
optionally be used in combination with one or more other prostate
cancer markers (other than those described herein).
[0186] Biomolecular sequences (amino acid and/or nucleic acid
sequences) uncovered using the methodology of the present invention
and described herein can be efficiently utilized as tissue or
pathological markers and/or as drugs or drug targets for treating
or preventing a disease.
[0187] These markers are specifically released to the bloodstream
under conditions of prostate cancer (or one of the above indicative
conditions), and/or are otherwise expressed at a much higher level
and/or specifically expressed in prostate cancer tissue or cells,
and/or tissue or cells under one of the above indicative
conditions. The measurement of these markers, alone or in
combination, in patient samples provides information that the
diagnostician can correlate with a probable diagnosis of prostate
cancer and/or a condition that it is indicative of a higher risk
for prostate cancer.
[0188] The present invention therefore also relates to diagnostic
assays for prostate cancer and/or an indicative condition, and
methods of use of such markers for detection of prostate cancer
and/or an indicative condition, optionally and preferably in a
sample taken from a subject (patient), which is more preferably
some type of blood sample.
[0189] According to a preferred embodiment of the present
invention, use of the marker optionally and preferably permits a
non-cancerous prostate disease state to be distinguished from
prostate cancer and/or an indicative condition. A non limiting
example of a non-cancerous prostate disease state includes BPH.
According to another preferred embodiment of the present invention,
use of the marker optionally and preferably permits an indicative
condition to be distinguished from prostate cancer.
[0190] In another embodiment, the present invention relates to
bridges, tails, heads and/or insertions, and/or analogs, homologs
and derivatives of such peptides. Such bridges, tails, heads and/or
insertions are described in greater detail below with regard to the
Examples.
[0191] As used herein a "tail" refers to a peptide sequence at the
end of an amino acid sequence that is unique to a splice variant
according to the present invention. Therefore, a splice variant
having such a tail may optionally be considered as a chimera, in
that at least a first portion of the splice variant is typically
highly homologous (often 100% identical) to a portion of the
corresponding known protein, while at least a second portion of the
variant comprises the tail.
[0192] As used herein a "head" refers to a peptide sequence at the
beginning of an amino acid sequence that is unique to a splice
variant according to the present invention. Therefore, a splice
variant having such a head may optionally be considered as a
chimera, in that at least a first portion of the splice variant
comprises the head, while at least a second portion is typically
highly homologous (often 100% identical) to a portion of the
corresponding known protein.
[0193] As used herein "an edge portion" refers to a connection
between two portions of a splice variant according to the present
invention that were not joined in the wild type or known protein.
An edge may optionally arise due to a join between the above "known
protein" portion of a variant and the tail, for example, and/or may
occur if an internal portion of the wild type sequence is no longer
present, such that two portions of the sequence are now contiguous
in the splice variant that were not contiguous in the known
protein. A "bridge" may optionally be an edge portion as described
above, but may also include a join between a head and a "known
protein" portion of a variant, or a join between a tail and a
"known protein" portion of a variant, or a join between an
insertion and a "known protein" portion of a variant.
[0194] Optionally and preferably, a bridge between a tail or a head
or a unique insertion, and a "known protein" portion of a variant,
comprises at least about 10 amino acids, more preferably at least
about 20 amino acids, most preferably at least about 30 amino
acids, and even more preferably at least about 40 amino acids, in
which at least one amino acid is from the tail/head/insertion and
at least one amino acid is from the "known protein" portion of a
variant. Also optionally, the bridge may comprise any number of
amino acids from about 10 to about 40 amino acids (for example, 10,
11, 12, 13 . . . 37, 38, 39, 40 amino acids in length, or any
number in between).
[0195] It should be noted that a bridge cannot be extended beyond
the length of the sequence in either direction, and it should be
assumed that every bridge description is to be read in such manner
that the bridge length does not extend beyond the sequence
itself.
[0196] Furthermore, bridges are described with regard to a sliding
window in certain contexts below. For example, certain descriptions
of the bridges feature the following format: a bridge between two
edges (in which a portion of the known protein is not present in
the variant) may optionally be described as follows: a bridge
portion of CONTIG-NAME_P1 (representing the name of the protein),
comprising a polypeptide having a length "n", wherein n is at least
about 10 amino acids in length, optionally at least about 20 amino
acids in length, preferably at least about 30 amino acids in
length, more preferably at least about 40 amino acids in length and
most preferably at least about 50 amino acids in length, wherein at
least two amino acids comprise XX (2 amino acids in the center of
the bridge, one from each end of the edge), having a structure as
follows (numbering according to the sequence of CONTIG-NAME_P1): a
sequence starting from any of amino acid numbers 49-x to 49 (for
example); and ending at any of amino acid numbers 50+((n-2)-x) (for
example), in which x varies from 0 to n-2. In this example, it
should also be read as including bridges in which n is any number
of amino acids between 10-50 amino acids in length. Furthermore,
the bridge polypeptide cannot extend beyond the sequence, so it
should be read such that 49-x (for example) is not less than 1, nor
50+((n-2)-x) (for example) greater than the total sequence
length.
[0197] In another embodiment, this invention provides antibodies
specifically recognizing the splice variants and polypeptide
fragments thereof of this invention. Preferably such antibodies
differentially recognize splice variants of the present invention
but do not recognize a corresponding known protein (such known
proteins are discussed with regard to their splice variants in the
Examples below).
[0198] In another embodiment, this invention provides an isolated
nucleic acid molecule encoding for a splice variant according to
the present invention, having a nucleotide sequence as set forth in
any one of the sequences listed herein, or a sequence complementary
thereto. In another embodiment, this invention provides an isolated
nucleic acid molecule, having a nucleotide sequence as set forth in
any one of the sequences listed herein, or a sequence complementary
thereto. In another embodiment, this invention provides an
oligonucleotide of at least about 12 nucleotides, specifically
hybridizable with the nucleic acid molecules of this invention. In
another embodiment, this invention provides vectors, cells,
liposomes and compositions comprising the isolated nucleic acids of
this invention.
[0199] In another embodiment, this invention provides a method for
detecting a splice variant according to the present invention in a
biological sample, comprising: contacting a biological sample with
an antibody specifically recognizing a splice variant according to
the present invention under conditions whereby the antibody
specifically interacts with the splice variant in the biological
sample but do not recognize known corresponding proteins (wherein
the known protein is discussed with regard to its splice variant(s)
in the Examples below), and detecting said interaction; wherein the
presence of an interaction correlates with the presence of a splice
variant in the biological sample.
[0200] In another embodiment, this invention provides a method for
detecting a splice variant nucleic acid sequences in a biological
sample, comprising: hybridizing the isolated nucleic acid molecules
or oligonucleotide fragments of at least about a minimum length to
a nucleic acid material of a biological sample and detecting a
hybridization complex; wherein the presence of a hybridization
complex correlates with the presence of a splice variant nucleic
acid sequence in the biological sample.
[0201] According to the present invention, the splice variants
described herein are non-limiting examples of markers for
diagnosing prostate cancer and/or prostate pathology. Each splice
variant marker of the present invention can be used alone or in
combination, for various uses, including but not limited to,
prognosis, prediction, screening, early diagnosis, determination of
progression, therapy selection and treatment monitoring of prostate
cancer and/or prostate pathology.
[0202] According to optional but preferred embodiments of the
present invention, any marker according to the present invention
may optionally be used alone or combination. Such a combination may
optionally comprise a plurality of markers described herein,
optionally including any subcombination of markers, and/or a
combination featuring at least one other marker, for example a
known marker. Furthermore, such a combination may optionally and
preferably be used as described above with regard to determining a
ratio between a quantitative or semi-quantitative measurement of
any marker described herein to any other marker described herein,
and/or any other known marker, and/or any other marker. With regard
to such a ratio between any marker described herein (or a
combination thereof) and a known marker, more preferably the known
marker comprises the "known protein" as described in greater detail
below with regard to each cluster or gene.
[0203] According to other preferred embodiments of the present
invention, a splice variant protein or a fragment thereof, or a
splice variant nucleic acid sequence or a fragment thereof, may be
featured as a biomarker for detecting prostate cancer and/or
prostate pathology, such that a biomarker may optionally comprise
any of the above.
[0204] According to still other preferred embodiments, the present
invention optionally and preferably encompasses any amino acid
sequence or fragment thereof encoded by a nucleic acid sequence
corresponding to a splice variant protein as described herein. Any
oligopeptide or peptide relating to such an amino acid sequence or
fragment thereof may optionally also (additionally or
alternatively) be used as a biomarker, including but not limited to
the unique amino acid sequences of these proteins that are depicted
as tails, heads, insertions, edges or bridges. The present
invention also optionally encompasses antibodies capable of
recognizing, and/or being elicited by, such oligopeptides or
peptides.
[0205] The present invention also optionally and preferably
encompasses any nucleic acid sequence or fragment thereof, or amino
acid sequence or fragment thereof, corresponding to a splice
variant of the present invention as described above, optionally for
any application.
[0206] Non-limiting examples of methods or assays are described
below.
[0207] The present invention also relates to kits based upon such
diagnostic methods or assays.
Nucleic Acid Sequences and Oligonucleotides
[0208] Various embodiments of the present invention encompass
nucleic acid sequences described hereinabove; fragments thereof,
sequences hybridizable therewith, sequences homologous thereto,
sequences encoding similar polypeptides with different codon usage,
altered sequences characterized by mutations, such as deletion,
insertion or substitution of one or more nucleotides, either
naturally occurring or artificially induced, either randomly or in
a targeted fashion.
[0209] The present invention encompasses nucleic acid sequences
described herein; fragments thereof, sequences hybridizable
therewith, sequences homologous thereto [e.g., at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 95% or more say 100% identical
to the nucleic acid sequences set forth below], sequences encoding
similar polypeptides with different codon usage, altered sequences
characterized by mutations, such as deletion, insertion or
substitution of one or more nucleotides, either naturally occurring
or man induced, either randomly or in a targeted fashion. The
present invention also encompasses homologous nucleic acid
sequences (i.e., which form a part of a polynucleotide sequence of
the present invention) which include sequence regions unique to the
polynucleotides of the present invention.
[0210] In cases where the polynucleotide sequences of the present
invention encode previously unidentified polypeptides, the present
invention also encompasses novel polypeptides or portions thereof,
which are encoded by the isolated polynucleotide and respective
nucleic acid fragments thereof described hereinabove.
[0211] A "nucleic acid fragment" or an "oligonucleotide" or a
"polynucleotide" are used herein interchangeably to refer to a
polymer of nucleic acids. A polynucleotide sequence of the present
invention refers to a single or double stranded nucleic acid
sequences which is isolated and provided in the form of an RNA
sequence, a complementary polynucleotide sequence (cDNA), a genomic
polynucleotide sequence and/or a composite polynucleotide sequences
(e.g., a combination of the above).
[0212] As used herein the phrase "complementary polynucleotide
sequence" refers to a sequence, which results from reverse
transcription of messenger RNA using a reverse transcriptase or any
other RNA dependent DNA polymerase. Such a sequence can be
subsequently amplified in vivo or in vitro using a DNA dependent
DNA polymerase.
[0213] As used herein the phrase "genomic polynucleotide sequence"
refers to a sequence derived (isolated) from a chromosome and thus
it represents a contiguous portion of a chromosome.
[0214] As used herein the phrase "composite polynucleotide
sequence" refers to a sequence, which is composed of genomic and
cDNA sequences. A composite sequence can include some exonal
sequences required to encode the polypeptide of the present
invention, as well as some intronic sequences interposing
therebetween. The intronic sequences can be of any source,
including of other genes, and typically will include conserved
splicing signal sequences. Such intronic sequences may further
include cis acting expression regulatory elements.
[0215] Preferred embodiments of the present invention encompass
oligonucleotide probes.
[0216] An example of an oligonucleotide probe which can be utilized
by the present invention is a single stranded polynucleotide which
includes a sequence complementary to the unique sequence region of
any variant according to the present invention, including but not
limited to a nucleotide sequence coding for an amino sequence of a
bridge, tail, head and/or insertion according to the present
invention, and/or the equivalent portions of any nucleotide
sequence given herein (including but not limited to a nucleotide
sequence of a node, segment or amplicon described herein).
[0217] Alternatively, an oligonucleotide probe of the present
invention can be designed to hybridize with a nucleic acid sequence
encompassed by any of the above nucleic acid sequences,
particularly the portions specified above, including but not
limited to a nucleotide sequence coding for an amino sequence of a
bridge, tail, head and/or insertion according to the present
invention, and/or the equivalent portions of any nucleotide
sequence given herein (including but not limited to a nucleotide
sequence of a node, segment or amplicon described herein).
[0218] Oligonucleotides designed according to the teachings of the
present invention can be generated according to any oligonucleotide
synthesis method known in the art such as enzymatic synthesis or
solid phase synthesis. Equipment and reagents for executing
solid-phase synthesis are commercially available from, for example,
Applied Biosystems. Any other means for such synthesis may also be
employed; the actual synthesis of the oligonucleotides is well
within the capabilities of one skilled in the art and can be
accomplished via established methodologies as detailed in, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988) and "Oligonucleotide Synthesis" Gait, M. J.,
ed. (1984) utilizing solid phase chemistry, e.g. cyanoethyl
phosphoramidite followed by deprotection, desalting and
purification by for example, an automated trityl-on method or
HPLC.
[0219] Oligonucleotides used according to this aspect of the
present invention are those having a length selected from a range
of about 10 to about 200 bases preferably about 15 to about 150
bases, more preferably about 20 to about 100 bases, most preferably
about 20 to about 50 bases. Preferably, the oligonucleotide of the
present invention features at least 17, at least 18, at least 19,
at least 20, at least 22, at least 25, at least 30 or at least 40,
bases specifically hybridizable with the biomarkers of the present
invention.
[0220] The oligonucleotides of the present invention may comprise
heterocylic nucleosides consisting of purines and the pyrimidines
bases, bonded in a 3' to 5' phosphodiester linkage.
[0221] Preferably used oligonucleotides are those modified at one
or more of the backbone, internucleoside linkages or bases, as is
broadly described hereinunder.
[0222] Specific examples of preferred oligonucleotides useful
according to this aspect of the present invention include
oligonucleotides containing modified backbones or non-natural
internucleoside linkages. Oligonucleotides having modified
backbones include those that retain a phosphorus atom in the
backbone, as disclosed in U.S. Pat. Nos. 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;
5,563,253; 5,571,799; 5,587,361; and 5,625,050.
[0223] Preferred modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free acid forms can also be
used.
[0224] Alternatively, modified oligonucleotide backbones that do
not include a phosphorus atom therein have backbones that are
formed by short chain alkyl or cycloalkyl internucleoside linkages,
mixed heteroatom and alkyl or cycloalkyl internucleoside linkages,
or one or more short chain heteroatomic or heterocyclic
internucleoside linkages. These include those having morpholino
linkages (formed in part from the sugar portion of a nucleoside);
siloxane backbones; sulfide, sulfoxide and sulfone backbones;
formacetyl and thioformacetyl backbones; methylene formacetyl and
thioformacetyl backbones; alkene containing backbones; sulfamate
backbones; methyleneimino and methylenehydrazino backbones;
sulfonate and sulfonamide backbones; amide backbones; and others
having mixed N, O, S and CH.sub.2 component parts, as disclosed in
U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134;
5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;
5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086;
5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704;
5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.
[0225] Other oligonucleotides which can be used according to the
present invention, are those modified in both sugar and the
internucleoside linkage, i.e., the backbone, of the nucleotide
units are replaced with novel groups. The base units are maintained
for complementation with the appropriate polynucleotide target. An
example for such an oligonucleotide mimetic, includes peptide
nucleic acid (PNA). United States patents that teach the
preparation of PNA compounds include, but are not limited to, U.S.
Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is
herein incorporated by reference. Other backbone modifications,
which can be used in the present invention are disclosed in U.S.
Pat. No. 6,303,374.
[0226] Oligonucleotides of the present invention may also include
base modifications or substitutions. As used herein, "unmodified"
or "natural" bases include the purine bases adenine (A) and guanine
(G), and the pyrimidine bases thymine (T), cytosine (C) and uracil
(U). Modified bases include but are not limited to other synthetic
and natural bases such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine
and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,
8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted
adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,
7-deazaguanine and 7-deazaadenine and 3-deazaguanine and
3-deazaadenine. Further bases particularly useful for increasing
the binding affinity of the oligomeric compounds of the invention
include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6
and O-6 substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2.degree. C. and are presently preferred base
substitutions, even more particularly when combined with
2'-O-methoxyethyl sugar modifications.
[0227] Another modification of the oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates, which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. Such
moieties include but are not limited to lipid moieties such as a
cholesterol moiety, cholic acid, a thioether, e.g.,
hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,
dodecandiol or undecyl residues, a phospholipid, e.g.,
di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid, a palmityl
moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol
moiety, as disclosed in U.S. Pat. No. 6,303,374.
[0228] It is not necessary for all positions in a given
oligonucleotide molecule to be uniformly modified, and in fact more
than one of the aforementioned modifications may be incorporated in
a single compound or even at a single nucleoside within an
oligonucleotide.
[0229] It will be appreciated that oligonucleotides of the present
invention may include further modifications for more efficient use
as diagnostic agents and/or to increase bioavailability,
therapeutic efficacy and reduce cytotoxicity.
[0230] To enable cellular expression of the polynucleotides of the
present invention, a nucleic acid construct according to the
present invention may be used, which includes at least a coding
region of one of the above nucleic acid sequences, and further
includes at least one cis acting regulatory element. As used
herein, the phrase "cis acting regulatory element" refers to a
polynucleotide sequence, preferably a promoter, which binds a trans
acting regulator and regulates the transcription of a coding
sequence located downstream thereto.
[0231] Any suitable promoter sequence can be used by the nucleic
acid construct of the present invention.
[0232] Preferably, the promoter utilized by the nucleic acid
construct of the present invention is active in the specific cell
population transformed. Examples of cell type-specific and/or
tissue-specific promoters include promoters such as albumin that is
liver specific, lymphoid specific promoters [Calame et al., (1988)
Adv. Immunol. 43:235-275]; in particular promoters of T-cell
receptors [Winoto et al., (1989) EMBO J. 8:729-733] and
immunoglobulins; [Banerji et al. (1983) Cell 33729-740],
neuron-specific promoters such as the neurofilament promoter [Byrne
et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477],
pancreas-specific promoters [Edlunch et al. (1985) Science
230:912-916] or mammary gland-specific promoters such as the milk
whey promoter (U.S. Pat. No. 4,873,316 and European Application
Publication No. 264,166). The nucleic acid construct of the present
invention can further include an enhancer, which can be adjacent or
distant to the promoter sequence and can function in up regulating
the transcription therefrom.
[0233] The nucleic acid construct of the present invention
preferably further includes an appropriate selectable marker and/or
an origin of replication. Preferably, the nucleic acid construct
utilized is a shuttle vector, which can propagate both in E. coli
(wherein the construct comprises an appropriate selectable marker
and origin of replication) and be compatible for propagation in
cells, or integration in a gene and a tissue of choice. The
construct according to the present invention can be, for example, a
plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an
artificial chromosome.
[0234] Examples of suitable constructs include, but are not limited
to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay,
pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available
from Invitrogen Co. (www "dot" invitrogen "dot" com). Examples of
retroviral vector and packaging systems are those sold by Clontech,
San Diego, Calif., including Retro-X vectors pLNCX and pLXSN, which
permit cloning into multiple cloning sites and the transgene is
transcribed from CMV promoter. Vectors derived from Mo-MuLV are
also included such as pBabe, where the transgene will be
transcribed from the 5'LTR promoter.
[0235] Currently preferred in vivo nucleic acid transfer techniques
include transfection with viral or non-viral constructs, such as
adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated
virus (AAV) and lipid-based systems. Useful lipids for
lipid-mediated transfer of the gene are, for example, DOTMA, DOPE,
and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65
(1996)]. The most preferred constructs for use in gene therapy are
viruses, most preferably adenoviruses, AAV, lentiviruses, or
retroviruses. A viral construct such as a retroviral construct
includes at least one transcriptional promoter/enhancer or
locus-defining element(s), or other elements that control gene
expression by other means such as alternate splicing, nuclear RNA
export, or post-translational modification of messenger. Such
vector constructs also include a packaging signal, long terminal
repeats (LTRs) or portions thereof, and positive and negative
strand primer binding sites appropriate to the virus used, unless
it is already present in the viral construct. In addition, such a
construct typically includes a signal sequence for secretion of the
peptide from a host cell in which it is placed. Preferably the
signal sequence for this purpose is a mammalian signal sequence or
the signal sequence of the polypeptide variants of the present
invention. Optionally, the construct may also include a signal that
directs polyadenylation, as well as one or more restriction sites
and a translation termination sequence. By way of example, such
constructs will typically include a 5' LTR, a tRNA binding site, a
packaging signal, an origin of second-strand DNA synthesis, and a
3' LTR or a portion thereof. Other vectors can be used that are
non-viral, such as cationic lipids, polylysine, and dendrimers.
Hybridization Assays
[0236] Detection of a nucleic acid of interest in a biological
sample may optionally be effected by hybridization-based assays
using an oligonucleotide probe (non-limiting examples of probes
according to the present invention were previously described).
[0237] Traditional hybridization assays include PCR, RT-PCR,
Real-time PCR, RNase protection, in-situ hybridization, primer
extension, Southern blots (DNA detection), dot or slot blots (DNA,
RNA), and Northern blots (RNA detection) (NAT type assays are
described in greater detail below). More recently, PNAs have been
described (Nielsen et al. 1999, Current Opin. Biotechnol.
10:71-75). Other detection methods include kits containing probes
on a dipstick setup and the like.
[0238] Hybridization based assays which allow the detection of a
variant of interest (i.e., DNA or RNA) in a biological sample rely
on the use of oligonucleotides which can be 10, 15, 20, or to 100
nucleotides long preferably from 10 to 50, more preferably from 40
to 50 nucleotides long.
[0239] Thus, the isolated polynucleotides (oligonucleotides) of the
present invention are preferably hybridizable with any of the
herein described nucleic acid sequences under moderate to stringent
hybridization conditions.
[0240] Moderate to stringent hybridization conditions are
characterized by a hybridization solution such as containing 10%
dextrane sulfate, 1 M NaCl, 1% SDS and 5.times.10.sup.6 cpm
.sup.32P labeled probe, at 65.degree. C., with a final wash
solution of 0.2.times.SSC and 0.1% SDS and final wash at 65.degree.
C. and whereas moderate hybridization is effected using a
hybridization solution containing 10% dextrane sulfate, 1 M NaCl,
1% SDS and 5.times.10.sup.6 cpm .sup.32P labeled probe, at
65.degree. C., with a final wash solution of 1.times.SSC and 0.1%
SDS and final wash at 50.degree. C.
[0241] More generally, hybridization of short nucleic acids (below
200 bp in length, e.g. 17-40 bp in length) can be effected using
the following exemplary hybridization protocols which can be
modified according to the desired stringency; (i) hybridization
solution of 6.times.SSC and 1% SDS or 3 M TMACI, 0.01 M sodium
phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 .mu.g/ml
denatured salmon sperm DNA and 0.1% nonfat dried milk,
hybridization temperature of 1-1.5.degree. C. below the T.sub.m,
final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8),
1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5.degree. C. below the T.sub.m;
(ii) hybridization solution of 6.times.SSC and 0.1% SDS or 3 M
TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5%
SDS, 100 .mu.g/ml denatured salmon sperm DNA and 0.1% nonfat dried
milk, hybridization temperature of 2-2.5.degree. C. below the
T.sub.m, final wash solution of 3 M TMACI, 0.01 M sodium phosphate
(pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5.degree. C. below
the T.sub.m, final wash solution of 6.times.SSC, and final wash at
22.degree. C.; (iii) hybridization solution of 6.times.SSC and 1%
SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH
7.6), 0.5% SDS, 100 .mu.g/ml denatured salmon sperm DNA and 0.1%
nonfat dried milk, hybridization temperature.
[0242] The detection of hybrid duplexes can be carried out by a
number of methods. Typically, hybridization duplexes are separated
from unhybridized nucleic acids and the labels bound to the
duplexes are then detected. Such labels refer to radioactive,
fluorescent, biological or enzymatic tags or labels of standard use
in the art. A label can be conjugated to either the oligonucleotide
probes or the nucleic acids derived from the biological sample.
[0243] Probes can be labeled according to numerous well known
methods. Non-limiting examples of radioactive labels include 3H,
14C, 32P, and 35S, Non-limiting examples of detectable markers
include ligands, fluorophores, chemiluminescent agents, enzymes,
and antibodies. Other detectable markers for use with probes, which
can enable an increase in sensitivity of the method of the
invention, include biotin and radio-nucleotides. It will become
evident to the person of ordinary skill that the choice of a
particular label dictates the manner in which it is bound to the
probe.
[0244] For example, oligonucleotides of the present invention can
be labeled subsequent to synthesis, by incorporating biotinylated
dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a
psoralen derivative of biotin to RNAs), followed by addition of
labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin)
or the equivalent. Alternatively, when fluorescently-labeled
oligonucleotide probes are used, fluorescein, lissamine,
phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5,
Cy5, Cy5.5, Cy7, FluorX (Amersham) and others [e.g., Kricka et al.
(1992), Academic Press San Diego, Calif] can be attached to the
oligonucleotides.
[0245] Those skilled in the art will appreciate that wash steps may
be employed to wash away excess target DNA or probe as well as
unbound conjugate. Further, standard heterogeneous assay formats
are suitable for detecting the hybrids using the labels present on
the oligonucleotide primers and probes.
[0246] It will be appreciated that a variety of controls may be
usefully employed to improve accuracy of hybridization assays. For
instance, samples may be hybridized to an irrelevant probe and
treated with RNAse A prior to hybridization, to assess false
hybridization.
[0247] Although the present invention is not specifically dependent
on the use of a label for the detection of a particular nucleic
acid sequence, such a label might be beneficial, by increasing the
sensitivity of the detection. Furthermore, it enables automation.
Probes can be labeled according to numerous well known methods.
[0248] As commonly known, radioactive nucleotides can be
incorporated into probes of the invention by several methods.
Non-limiting examples of radioactive labels include .sup.3H,
.sup.14C, .sup.32P, and .sup.35S Those skilled in the art will
appreciate that wash steps may be employed to wash away excess
target DNA or probe as well as unbound conjugate. Further, standard
heterogeneous assay formats are suitable for detecting the hybrids
using the labels present on the oligonucleotide primers and
probes.
[0249] It will be appreciated that a variety of controls may be
usefully employed to improve accuracy of hybridization assays.
[0250] Probes of the invention can be utilized with naturally
occurring sugar-phosphate backbones as well as modified backbones
including phosphorothioates, dithionates, alkyl phosphonates and
a-nucleotides and the like. Probes of the invention can be
constructed of either ribonucleic acid (RNA) or deoxyribonucleic
acid (DNA), and preferably of DNA.
NAT Assays
[0251] Detection of a nucleic acid of interest in a biological
sample may also optionally be effected by NAT-based assays, which
involve nucleic acid amplification technology, such as PCR for
example (or variations thereof such as real-time PCR for
example).
[0252] As used herein, a "primer" defines an oligonucleotide which
is capable of annealing to (hybridizing with) a target sequence,
thereby creating a double stranded region which can serve as an
initiation point for DNA synthesis under suitable conditions.
[0253] Amplification of a selected, or target, nucleic acid
sequence may be carried out by a number of suitable methods. See
generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14 Numerous
amplification techniques have been described and can be readily
adapted to suit particular needs of a person of ordinary skill.
Non-limiting examples of amplification techniques include
polymerase chain reaction (PCR), ligase chain reaction (LCR),
strand displacement amplification (SDA), transcription-based
amplification, the q3 replicase system and NASBA (Kwoh et al.,
1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al.,
1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol.
Biol., 28:253-260; and Sambrook et al., 1989, supra).
[0254] The terminology "amplification pair" (or "primer pair")
refers herein to a pair of oligonucleotides (oligos) of the present
invention, which are selected to be used together in amplifying a
selected nucleic acid sequence by one of a number of types of
amplification processes, preferably a polymerase chain reaction.
Other types of amplification processes include ligase chain
reaction, strand displacement amplification, or nucleic acid
sequence-based amplification, as explained in greater detail below.
As commonly known in the art, the oligos are designed to bind to a
complementary sequence under selected conditions.
[0255] In one particular embodiment, amplification of a nucleic
acid sample from a patient is amplified under conditions which
favor the amplification of the most abundant differentially
expressed nucleic acid. In one preferred embodiment, RT-PCR is
carried out on an mRNA sample from a patient under conditions which
favor the amplification of the most abundant mRNA. In another
preferred embodiment, the amplification of the differentially
expressed nucleic acids is carried out simultaneously. It will be
realized by a person skilled in the art that such methods could be
adapted for the detection of differentially expressed proteins
instead of differentially expressed nucleic acid sequences.
[0256] The nucleic acid (i.e. DNA or RNA) for practicing the
present invention may be obtained according to well known
methods.
[0257] Oligonucleotide primers of the present invention may be of
any suitable length, depending on the particular assay format and
the particular needs and targeted genomes employed. Optionally, the
oligonucleotide primers are at least 12 nucleotides in length,
preferably between 15 and 24 molecules, and they may be adapted to
be especially suited to a chosen nucleic acid amplification system.
As commonly known in the art, the oligonucleotide primers can be
designed by taking into consideration the melting point of
hybridization thereof with its targeted sequence (Sambrook et al.,
1989, Molecular Cloning--A Laboratory Manual, 2nd Edition, CSH
Laboratories; Ausubel et al., 1989, in Current Protocols in
Molecular Biology, John Wiley & Sons Inc., N.Y.).
[0258] It will be appreciated that antisense oligonucleotides may
be employed to quantify expression of a splice isoform of interest.
Such detection is effected at the pre-mRNA level. Essentially the
ability to quantitate transcription from a splice site of interest
can be effected based on splice site accessibility.
Oligonucleotides may compete with splicing factors for the splice
site sequences. Thus, low activity of the antisense oligonucleotide
is indicative of splicing activity.
[0259] The polymerase chain reaction and other nucleic acid
amplification reactions are well known in the art (various
non-limiting examples of these reactions are described in greater
detail below). The pair of oligonucleotides according to this
aspect of the present invention are preferably selected to have
compatible melting temperatures (Tm), e.g., melting temperatures
which differ by less than that 7.degree. C., preferably less than
5.degree. C., more preferably less than 4.degree. C., most
preferably less than 3.degree. C., ideally between 3.degree. C. and
0.degree. C.
[0260] Polymerase Chain Reaction (PCR): The polymerase chain
reaction (PCR), as described in U.S. Pat. Nos. 4,683,195 and
4,683,202 to Mullis and Mullis et al., is a method of increasing
the concentration of a segment of target sequence in a mixture of
genomic DNA without cloning or purification. This technology
provides one approach to the problems of low target sequence
concentration. PCR can be used to directly increase the
concentration of the target to an easily detectable level. This
process for amplifying the target sequence involves the
introduction of a molar excess of two oligonucleotide primers which
are complementary to their respective strands of the
double-stranded target sequence to the DNA mixture containing the
desired target sequence. The mixture is denatured and then allowed
to hybridize. Following hybridization, the primers are extended
with polymerase so as to form complementary strands. The steps of
denaturation, hybridization (annealing), and polymerase extension
(elongation) can be repeated as often as needed, in order to obtain
relatively high concentrations of a segment of the desired target
sequence.
[0261] The length of the segment of the desired target sequence is
determined by the relative positions of the primers with respect to
each other, and, therefore, this length is a controllable
parameter. Because the desired segments of the target sequence
become the dominant sequences (in terms of concentration) in the
mixture, they are said to be "PCR-amplified."
[0262] Ligase Chain Reaction (LCR or LAR): The ligase chain
reaction [LCR; sometimes referred to as "Ligase Amplification
Reaction" (LAR)] has developed into a well-recognized alternative
method of amplifying nucleic acids. In LCR, four oligonucleotides,
two adjacent oligonucleotides which uniquely hybridize to one
strand of target DNA, and a complementary set of adjacent
oligonucleotides, which hybridize to the opposite strand are mixed
and DNA ligase is added to the mixture. Provided that there is
complete complementarity at the junction, ligase will covalently
link each set of hybridized molecules. Importantly, in LCR, two
probes are ligated together only when they base-pair with sequences
in the target sample, without gaps or mismatches. Repeated cycles
of denaturation, and ligation amplify a short segment of DNA. LCR
has also been used in combination with PCR to achieve enhanced
detection of single-base changes: see for example Segev, PCT
Publication No. W09001069 A1 (1990). However, because the four
oligonucleotides used in this assay can pair to form two short
ligatable fragments, there is the potential for the generation of
target-independent background signal. The use of LCR for mutant
screening is limited to the examination of specific nucleic acid
positions.
[0263] Self-Sustained Synthetic Reaction (3SR/NASBA): The
self-sustained sequence replication reaction (3SR) is a
transcription-based in vitro amplification system that can
exponentially amplify RNA sequences at a uniform temperature. The
amplified RNA can then be utilized for mutation detection. In this
method, an oligonucleotide primer is used to add a phage RNA
polymerase promoter to the 5' end of the sequence of interest. In a
cocktail of enzymes and substrates that includes a second primer,
reverse transcriptase, RNase H, RNA polymerase and ribo- and
deoxyribonucleoside triphosphates, the target sequence undergoes
repeated rounds of transcription, cDNA synthesis and second-strand
synthesis to amplify the area of interest. The use of 3SR to detect
mutations is kinetically limited to screening small segments of DNA
(e.g., 200-300 base pairs).
[0264] Q-Beta (Q.beta.) Replicase: In this method, a probe which
recognizes the sequence of interest is attached to the replicatable
RNA template for Q.beta. replicase. A previously identified major
problem with false positives resulting from the replication of
unhybridized probes has been addressed through use of a
sequence-specific ligation step. However, available thermostable
DNA ligases are not effective on this RNA substrate, so the
ligation must be performed by T4 DNA ligase at low temperatures (37
degrees C.). This prevents the use of high temperature as a means
of achieving specificity as in the LCR, the ligation event can be
used to detect a mutation at the junction site, but not
elsewhere.
[0265] A successful diagnostic method must be very specific. A
straight-forward method of controlling the specificity of nucleic
acid hybridization is by controlling the temperature of the
reaction. While the 3SR/NASBA, and Q.beta. systems are all able to
generate a large quantity of signal, one or more of the enzymes
involved in each cannot be used at high temperature (i.e., >55
degrees C.). Therefore the reaction temperatures cannot be raised
to prevent non-specific hybridization of the probes. If probes are
shortened in order to make them melt more easily at low
temperatures, the likelihood of having more than one perfect match
in a complex genome increases. For these reasons, PCR and LCR
currently dominate the research field in detection
technologies.
[0266] The basis of the amplification procedure in the PCR and LCR
is the fact that the products of one cycle become usable templates
in all subsequent cycles, consequently doubling the population with
each cycle. The final yield of any such doubling system can be
expressed as: (1+X).sup.n=y, where "X" is the mean efficiency
(percent copied in each cycle), "n" is the number of cycles, and
"y" is the overall efficiency, or yield of the reaction. If every
copy of a target DNA is utilized as a template in every cycle of a
polymerase chain reaction, then the mean efficiency is 100%. If 20
cycles of PCR are performed, then the yield will be 2.sup.20, or
1,048,576 copies of the starting material. If the reaction
conditions reduce the mean efficiency to 85%, then the yield in
those 20 cycles will be only 1.85.sup.20, or 220,513 copies of the
starting material. In other words, a PCR running at 85% efficiency
will yield only 21% as much final product, compared to a reaction
running at 100% efficiency. A reaction that is reduced to 50% mean
efficiency will yield less than 1% of the possible product.
[0267] In practice, routine polymerase chain reactions rarely
achieve the theoretical maximum yield, and PCRs are usually run for
more than 20 cycles to compensate for the lower yield. At 50% mean
efficiency, it would take 34 cycles to achieve the million-fold
amplification theoretically possible in 20, and at lower
efficiencies, the number of cycles required becomes prohibitive. In
addition, any background products that amplify with a better mean
efficiency than the intended target will become the dominant
products.
[0268] Also, many variables can influence the mean efficiency of
PCR, including target DNA length and secondary structure, primer
length and design, primer and dNTP concentrations, and buffer
composition, to name but a few. Contamination of the reaction with
exogenous DNA (e.g., DNA spilled onto lab surfaces) or
cross-contamination is also a major consideration. Reaction
conditions must be carefully optimized for each different primer
pair and target sequence, and the process can take days, even for
an experienced investigator. The laboriousness of this process,
including numerous technical considerations and other factors,
presents a significant drawback to using PCR in the clinical
setting. Indeed, PCR has yet to penetrate the clinical market in a
significant way. The same concerns arise with LCR, as LCR must also
be optimized to use different oligonucleotide sequences for each
target sequence. In addition, both methods require expensive
equipment, capable of precise temperature cycling.
[0269] Many applications of nucleic acid detection technologies,
such as in studies of allelic variation, involve not only detection
of a specific sequence in a complex background, but also the
discrimination between sequences with few, or single, nucleotide
differences. One method of the detection of allele-specific
variants by PCR is based upon the fact that it is difficult for Taq
polymerase to synthesize a DNA strand when there is a mismatch
between the template strand and the 3' end of the primer. An
allele-specific variant may be detected by the use of a primer that
is perfectly matched with only one of the possible alleles; the
mismatch to the other allele acts to prevent the extension of the
primer, thereby preventing the amplification of that sequence. This
method has a substantial limitation in that the base composition of
the mismatch influences the ability to prevent extension across the
mismatch, and certain mismatches do not prevent extension or have
only a minimal effect.
[0270] A similar 3'-mismatch strategy is used with greater effect
to prevent ligation in the LCR. Any mismatch effectively blocks the
action of the thermostable ligase, but LCR still has the drawback
of target-independent background ligation products initiating the
amplification. Moreover, the combination of PCR with subsequent LCR
to identify the nucleotides at individual positions is also a
clearly cumbersome proposition for the clinical laboratory.
[0271] The direct detection method according to various preferred
embodiments of the present invention may be, for example a cycling
probe reaction (CPR) or a branched DNA analysis.
[0272] When a sufficient amount of a nucleic acid to be detected is
available, there are advantages to detecting that sequence
directly, instead of making more copies of that target, (e.g., as
in PCR and LCR). Most notably, a method that does not amplify the
signal exponentially is more amenable to quantitative analysis.
Even if the signal is enhanced by attaching multiple dyes to a
single oligonucleotide, the correlation between the final signal
intensity and amount of target is direct. Such a system has an
additional advantage that the products of the reaction will not
themselves promote further reaction, so contamination of lab
surfaces by the products is not as much of a concern. Recently
devised techniques have sought to eliminate the use of
radioactivity and/or improve the sensitivity in automatable
formats. Two examples are the "Cycling Probe Reaction" (CPR), and
"Branched DNA" (bDNA).
[0273] Cycling probe reaction (CPR): The cycling probe reaction
(CPR), uses a long chimeric oligonucleotide in which a central
portion is made of RNA while the two termini are made of DNA.
Hybridization of the probe to a target DNA and exposure to a
thermostable RNase H causes the RNA portion to be digested. This
destabilizes the remaining DNA portions of the duplex, releasing
the remainder of the probe from the target DNA and allowing another
probe molecule to repeat the process. The signal, in the form of
cleaved probe molecules, accumulates at a linear rate. While the
repeating process increases the signal, the RNA portion of the
oligonucleotide is vulnerable to RNases that may carried through
sample preparation.
[0274] Branched DNA: Branched DNA (bDNA), involves oligonucleotides
with branched structures that allow each individual oligonucleotide
to carry 35 to 40 labels (e.g., alkaline phosphatase enzymes).
While this enhances the signal from a hybridization event, signal
from non-specific binding is similarly increased.
[0275] The detection of at least one sequence change according to
various preferred embodiments of the present invention may be
accomplished by, for example restriction fragment length
polymorphism (RFLP analysis), allele specific oligonucleotide (ASO)
analysis, Denaturing/Temperature Gradient Gel Electrophoresis
(DGGE/TGGE), Single-Strand Conformation Polymorphism (SSCP)
analysis or Dideoxy fingerprinting (ddF).
[0276] The demand for tests which allow the detection of specific
nucleic acid sequences and sequence changes is growing rapidly in
clinical diagnostics. As nucleic acid sequence data for genes from
humans and pathogenic organisms accumulates, the demand for fast,
cost-effective, and easy-to-use tests for as yet mutations within
specific sequences is rapidly increasing.
[0277] A handful of methods have been devised to scan nucleic acid
segments for mutations. One option is to determine the entire gene
sequence of each test sample (e.g., a bacterial isolate). For
sequences under approximately 600 nucleotides, this may be
accomplished using amplified material (e.g., PCR reaction
products). This avoids the time and expense associated with cloning
the segment of interest. However, specialized equipment and highly
trained personnel are required, and the method is too labor-intense
and expensive to be practical and effective in the clinical
setting.
[0278] In view of the difficulties associated with sequencing, a
given segment of nucleic acid may be characterized on several other
levels. At the lowest resolution, the size of the molecule can be
determined by electrophoresis by comparison to a known standard run
on the same gel. A more detailed picture of the molecule may be
achieved by cleavage with combinations of restriction enzymes prior
to electrophoresis, to allow construction of an ordered map. The
presence of specific sequences within the fragment can be detected
by hybridization of a labeled probe, or the precise nucleotide
sequence can be determined by partial chemical degradation or by
primer extension in the presence of chain-terminating nucleotide
analogs.
[0279] Restriction fragment length polymorphism (RFLP): For
detection of single-base differences between like sequences, the
requirements of the analysis are often at the highest level of
resolution. For cases in which the position of the nucleotide in
question is known in advance, several methods have been developed
for examining single base changes without direct sequencing. For
example, if a mutation of interest happens to fall within a
restriction recognition sequence, a change in the pattern of
digestion can be used as a diagnostic tool (e.g., restriction
fragment length polymorphism [RFLP] analysis).
[0280] Single point mutations have been also detected by the
creation or destruction of RFLPs. Mutations are detected and
localized by the presence and size of the RNA fragments generated
by cleavage at the mismatches. Single nucleotide mismatches in DNA
heteroduplexes are also recognized and cleaved by some chemicals,
providing an alternative strategy to detect single base
substitutions, generically named the "Mismatch Chemical Cleavage"
(MCC). However, this method requires the use of osmium tetroxide
and piperidine, two highly noxious chemicals which are not suited
for use in a clinical laboratory.
[0281] RFLP analysis suffers from low sensitivity and requires a
large amount of sample. When RFLP analysis is used for the
detection of point mutations, it is, by its nature, limited to the
detection of only those single base changes which fall within a
restriction sequence of a known restriction endonuclease. Moreover,
the majority of the available enzymes have 4 to 6 base-pair
recognition sequences, and cleave too frequently for many
large-scale DNA manipulations. Thus, it is applicable only in a
small fraction of cases, as most mutations do not fall within such
sites.
[0282] A handful of rare-cutting restriction enzymes with 8
base-pair specificities have been isolated and these are widely
used in genetic mapping, but these enzymes are few in number, are
limited to the recognition of G+C-rich sequences, and cleave at
sites that tend to be highly clustered. Recently, endonucleases
encoded by group I introns have been discovered that might have
greater than 12 base-pair specificity, but again, these are few in
number.
[0283] Allele specific oligonucleotide (ASO): If the change is not
in a recognition sequence, then allele-specific oligonucleotides
(ASOs), can be designed to hybridize in proximity to the mutated
nucleotide, such that a primer extension or ligation event can
bused as the indicator of a match or a mis-match. Hybridization
with radioactively labeled allelic specific oligonucleotides (ASO)
also has been applied to the detection of specific point mutations.
The method is based on the differences in the melting temperature
of short DNA fragments differing by a single nucleotide. Stringent
hybridization and washing conditions can differentiate between
mutant and wild-type alleles. The ASO approach applied to PCR
products also has been extensively utilized by various researchers
to detect and characterize point mutations in ras genes and gsp/gip
oncogenes. Because of the presence of various nucleotide changes in
multiple positions, the ASO method requires the use of many
oligonucleotides to cover all possible oncogenic mutations.
[0284] With either of the techniques described above (i.e., RFLP
and ASO), the precise location of the suspected mutation must be
known in advance of the test. That is to say, they are inapplicable
when one needs to detect the presence of a mutation within a gene
or sequence of interest.
[0285] Denaturing/Temperature Gradient Gel Electrophoresis
(DGGE/TGGE): Two other methods rely on detecting changes in
electrophoretic mobility in response to minor sequence changes. One
of these methods, termed "Denaturing Gradient Gel Electrophoresis"
(DGGE) is based on the observation that slightly different
sequences will display different patterns of local melting when
electrophoretically resolved on a gradient gel. In this manner,
variants can be distinguished, as differences in melting properties
of homoduplexes versus heteroduplexes differing in a single
nucleotide can detect the presence of mutations in the target
sequences because of the corresponding changes in their
electrophoretic mobilities. The fragments to be analyzed, usually
PCR products, are "clamped" at one end by a long stretch of G-C
base pairs (30-80) to allow complete denaturation of the sequence
of interest without complete dissociation of the strands. The
attachment of a GC "clamp" to the DNA fragments increases the
fraction of mutations that can be recognized by DGGE. Attaching a
GC clamp to one primer is critical to ensure that the amplified
sequence has a low dissociation temperature. Modifications of the
technique have been developed, using temperature gradients, and the
method can be also applied to RNA:RNA duplexes.
[0286] Limitations on the utility of DGGE include the requirement
that the denaturing conditions must be optimized for each type of
DNA to be tested. Furthermore, the method requires specialized
equipment to prepare the gels and maintain the needed high
temperatures during electrophoresis. The expense associated with
the synthesis of the clamping tail on one oligonucleotide for each
sequence to be tested is also a major consideration. In addition,
long running times are required for DGGE. The long running time of
DGGE was shortened in a modification of DGGE called constant
denaturant gel electrophoresis (CDGE). CDGE requires that gels be
performed under different denaturant conditions in order to reach
high efficiency for the detection of mutations.
[0287] A technique analogous to DGGE, termed temperature gradient
gel electrophoresis (TGGE), uses a thermal gradient rather than a
chemical denaturant gradient. TGGE requires the use of specialized
equipment which can generate a temperature gradient perpendicularly
oriented relative to the electrical field. TGGE can detect
mutations in relatively small fragments of DNA therefore scanning
of large gene segments requires the use of multiple PCR products
prior to running the gel.
[0288] Single-Strand Conformation Polymorphism (SSCP): Another
common method, called "Single-Strand Conformation Polymorphism"
(SSCP) was developed by Hayashi, Sekya and colleagues and is based
on the observation that single strands of nucleic acid can take on
characteristic conformations in non-denaturing conditions, and
these conformations influence electrophoretic mobility. The
complementary strands assume sufficiently different structures that
one strand may be resolved from the other. Changes in sequences
within the fragment will also change the conformation, consequently
altering the mobility and allowing this to be used as an assay for
sequence variations.
[0289] The SSCP process involves denaturing a DNA segment (e.g., a
PCR product) that is labeled on both strands, followed by slow
electrophoretic separation on a non-denaturing polyacrylamide gel,
so that intra-molecular interactions can form and not be disturbed
during the run. This technique is extremely sensitive to variations
in gel composition and temperature. A serious limitation of this
method is the relative difficulty encountered in comparing data
generated in different laboratories, under apparently similar
conditions.
[0290] Dideoxy fingerprinting (ddF): The dideoxy fingerprinting
(ddF) is another technique developed to scan genes for the presence
of mutations. The ddF technique combines components of Sanger
dideoxy sequencing with SSCP. A dideoxy sequencing reaction is
performed using one dideoxy terminator and then the reaction
products are electrophoresed on nondenaturing polyacrylamide gels
to detect alterations in mobility of the termination segments as in
SSCP analysis. While ddF is an improvement over SSCP in terms of
increased sensitivity, ddF requires the use of expensive
dideoxynucleotides and this technique is still limited to the
analysis of fragments of the size suitable for SSCP (i.e.,
fragments of 200-300 bases for optimal detection of mutations).
[0291] In addition to the above limitations, all of these methods
are limited as to the size of the nucleic acid fragment that can be
analyzed. For the direct sequencing approach, sequences of greater
than 600 base pairs require cloning, with the consequent delays and
expense of either deletion sub-cloning or primer walking, in order
to cover the entire fragment. SSCP and DGGE have even more severe
size limitations. Because of reduced sensitivity to sequence
changes, these methods are not considered suitable for larger
fragments. Although SSCP is reportedly able to detect 90% of
single-base substitutions within a 200 base-pair fragment, the
detection drops to less than 50% for 400 base pair fragments.
Similarly, the sensitivity of DGGE decreases as the length of the
fragment reaches 500 base-pairs. The ddF technique, as a
combination of direct sequencing and SSCP, is also limited by the
relatively small size of the DNA that can be screened.
[0292] According to a presently preferred embodiment of the present
invention the step of searching for any of the nucleic acid
sequences described here, in tumor cells or in cells derived from a
cancer patient is effected by any suitable technique, including,
but not limited to, nucleic acid sequencing, polymerase chain
reaction, ligase chain reaction, self-sustained synthetic reaction,
Q.beta.-Replicase, cycling probe reaction, branched DNA,
restriction fragment length polymorphism analysis, mismatch
chemical cleavage, heteroduplex analysis, allele-specific
oligonucleotides, denaturing gradient gel electrophoresis, constant
denaturant gel electrophoresis, temperature gradient gel
electrophoresis and dideoxy fingerprinting.
[0293] Detection may also optionally be performed with a chip or
other such device. The nucleic acid sample which includes the
candidate region to be analyzed is preferably isolated, amplified
and labeled with a reporter group. This reporter group can be a
fluorescent group such as phycoerythrin. The labeled nucleic acid
is then incubated with the probes immobilized on the chip using a
fluidics station. describe the fabrication of fluidics devices and
particularly microcapillary devices, in silicon and glass
substrates.
[0294] Once the reaction is completed, the chip is inserted into a
scanner and patterns of hybridization are detected. The
hybridization data is collected, as a signal emitted from the
reporter groups already incorporated into the nucleic acid, which
is now bound to the probes attached to the chip. Since the sequence
and position of each probe immobilized on the chip is known, the
identity of the nucleic acid hybridized to a given probe can be
determined.
[0295] It will be appreciated that when utilized along with
automated equipment, the above described detection methods can be
used to screen multiple samples for a disease and/or pathological
condition both rapidly and easily.
Amino Acid Sequences and Peptides
[0296] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an analog or mimetic of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers. Polypeptides can be modified, e.g., by the
addition of carbohydrate residues to form glycoproteins. The terms
"polypeptide," "peptide" and "protein" include glycoproteins, as
well as non-glycoproteins.
[0297] Polypeptide products can be biochemically synthesized such
as by employing standard solid phase techniques. Such methods
include but are not limited to exclusive solid phase synthesis,
partial solid phase synthesis methods, fragment condensation,
classical solution synthesis. These methods are preferably used
when the peptide is relatively short (i.e., 10 kDa) and/or when it
cannot be produced by recombinant techniques (i.e., not encoded by
a nucleic acid sequence) and therefore involves different
chemistry.
[0298] Solid phase polypeptide synthesis procedures are well known
in the art and further described by John Morrow Stewart and Janis
Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce
Chemical Company, 1984).
[0299] Synthetic polypeptides can optionally be purified by
preparative high performance liquid chromatography [Creighton T.
(1983) Proteins, structures and molecular principles. WH Freeman
and Co. N.Y.], after which their composition can be confirmed via
amino acid sequencing.
[0300] In cases where large amounts of a polypeptide are desired,
it can be generated using recombinant techniques such as described
by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier
et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984)
Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311,
Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984)
Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol.
6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant
Molecular Biology, Academic Press, NY, Section VIII, pp
421-463.
[0301] The present invention also encompasses polypeptides encoded
by the polynucleotide sequences of the present invention, as well
as polypeptides according to the amino acid sequences described
herein. The present invention also encompasses homologues of these
polypeptides, such homologues can be at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 95% or more say 100% homologous to the amino
acid sequences set forth below, as can be determined using BlastP
software of the National Center of Biotechnology Information (NCBI)
using default parameters, optionally and preferably including the
following: filtering on (this option filters repetitive or
low-complexity sequences from the query using the Seg (protein)
program), scoring matrix is BLOSUM62 for proteins, word size is 3,
E value is 10, gap costs are 11, 1 (initialization and extension),
and number of alignments shown is 50. Optionally and preferably,
nucleic acid sequence identity/homology is determined with BlastN
software of the National Center of Biotechnology Information (NCBI)
using default parameters, which preferably include using the DUST
filter program, and also preferably include having an E value of
10, filtering low complexity sequences and a word size of 11.
Finally, the present invention also encompasses fragments of the
above described polypeptides and polypeptides having mutations,
such as deletions, insertions or substitutions of one or more amino
acids, either naturally occurring or artificially induced, either
randomly or in a targeted fashion.
[0302] It will be appreciated that peptides identified according
the present invention may be degradation products, synthetic
peptides or recombinant peptides as well as peptidomimetics,
typically, synthetic peptides and peptoids and semipeptoids which
are peptide analogs, which may have, for example, modifications
rendering the peptides more stable while in a body or more capable
of penetrating into cells. Such modifications include, but are not
limited to N terminus modification, C terminus modification,
peptide bond modification, including, but not limited to, CH2-NH,
CH2-S, CH2-S.dbd.O, O.dbd.C--NH, CH2-O, CH2-CH2, S.dbd.C--NH,
CH.dbd.CH or CF.dbd.CH, backbone modifications, and residue
modification. Methods for preparing peptidomimetic compounds are
well known in the art and are specified. Further details in this
respect are provided hereinunder.
[0303] Peptide bonds (--CO--NH--) within the peptide may be
substituted, for example, by N-methylated bonds (--N(CH3)-CO--),
ester bonds (--C(R)H--C--O--O--C(R)--N--), ketomethylen bonds
(--CO--CH2-), .alpha.-aza bonds (--NH--N(R)--CO--), wherein R is
any alkyl, e.g., methyl, carba bonds (--CH2-NH--), hydroxyethylene
bonds (--CH(OH)--CH.sub.2--), thioamide bonds (--CS--NH--),
olefinic double bonds (--CH.dbd.CH--), retro amide bonds
(--NH--CO--), peptide derivatives (--N(R)--CH.sub.2--CO--), wherein
R is the "normal" side chain, naturally presented on the carbon
atom.
[0304] These modifications can occur at any of the bonds along the
peptide chain and even at several (2-3) at the same time.
[0305] Natural aromatic amino acids, Trp, Tyr and Phe, may be
substituted for synthetic non-natural acid such as Phenylglycine,
TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe,
halogenated derivatives of Phe or o-methyl-Tyr.
[0306] In addition to the above, the peptides of the present
invention may also include one or more modified amino acids or one
or more non-amino acid monomers (e.g. fatty acids, complex
carbohydrates etc).
[0307] As used herein in the specification and in the claims
section below the term "amino acid" or "amino acids" is understood
to include the 20 naturally occurring amino acids; those amino
acids often modified post-translationally in vivo, including, for
example, hydroxyproline, phosphoserine and phosphothreonine; and
other unusual amino acids including, but not limited to,
2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,
nor-leucine and ornithine. Furthermore, the term "amino acid"
includes both D- and L-amino acids.
[0308] Table 1 non-conventional or modified amino acids which can
be used with the present invention. TABLE-US-00002 TABLE 1
Non-conventional amino acid Code Non-conventional amino acid Code
.alpha.-aminobutyric acid Abu L-N-methylalanine Nmala
.alpha.-amino-.alpha.-methylbutyrate Mgabu L-N-methylarginine Nmarg
aminocyclopropane- Cpro L-N-methylasparagine Nmasn Carboxylate
L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib
L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine
Nmgin Carboxylate L-N-methylglutamic acid Nmglu Cyclohexylalanine
Chexa L-N-methylhistidine Nmhis Cyclopentylalanine Cpen
L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp
L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine
Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid
Dglu L-N-methylornithine Nmorn D-histidine Dhis
L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline
Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys
L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan
Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine
Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine
Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine
Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine
Dtyr .alpha.-methyl-aminoisobutyrate Maib D-valine Dval
.alpha.-methyl-.gamma.-aminobutyrate Mgabu D-.alpha.-methylalanine
Dmala .alpha.-methylcyclohexylalanine Mchexa
D-.alpha.-methylarginine Dmarg .alpha.-methylcyclopentylalanine
Mcpen D-.alpha.-methylasparagine Dmasn
.alpha.-methyl-.alpha.-napthylalanine Manap
D-.alpha.-methylaspartate Dmasp .alpha.-methylpenicillamine Mpen
D-.alpha.-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
D-.alpha.-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
D-.alpha.-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
D-.alpha.-methylisoleucine Dmile N-amino-.alpha.-methylbutyrate
Nmaabu D-.alpha.-methylleucine Dmleu .alpha.-napthylalanine Anap
D-.alpha.-methyllysine Dmlys N-benzylglycine Nphe
D-.alpha.-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln
D-.alpha.-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-.alpha.-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu
D-.alpha.-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-.alpha.-methylserine Dmser N-cyclobutylglycine Ncbut
D-.alpha.-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-.alpha.-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-.alpha.-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-.alpha.-methylvaline Dmval N-cyclododeclglycine Ncdod
D-.alpha.-methylalnine Dnmala N-cyclooctylglycine Ncoct
D-.alpha.-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-.alpha.-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-.alpha.-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-.alpha.-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe
D-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-.gamma.-aminobutyrate Nmgabu N-
Nmchexa D-N-methylmethionine Dnmmet methylcyclohexylalanine
D-N-methylornithine Dnmom N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nva
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
Penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t-butylglycine
Mtbug L-.alpha.-methylcysteine Mcys L-methylethylglycine Metg
L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomo Mhphe
phenylalanine L-.alpha.-methylisoleucine Mile
N-(2-methylthioethyl)glycine Nmet D-N-methylglutamine Dnmgln
N-(3-guanidinopropyl)glycine Narg D-N-methylglutamate Dnmglu
N-(1-hydroxyethyl)glycine Nthr D-N-methylhistidine Dnmhis
N-(hydroxyethyl)glycine Nser D-N-methylisoleucine Dnmile
N-(imidazolylethyl)glycine Nhis D-N-methylleucine Dnmleu
N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine Dnmlys
N-methyl-.gamma.-aminobutyrate Nmgabu N- Nmchexa
D-N-methylmethionine Dnmmet methylcyclohexylalanine
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
Penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t-butylglycine
Mtbug L-.alpha.-methylcysteine Mcys L-methylethylglycine Metg
L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomophenylalanine
Mhphe L-.alpha.-methylisoleucine Mile N-(2-methylthioethyl)glycine
Nmet L-.alpha.-methylleucine Mleu L-.alpha.-methyllysine Mlys
L-.alpha.-methylmethionine Mmet L-.alpha.-methylnorleucine Mnle
L-.alpha.-methylnorvaline Mnva L-.alpha.-methylornithine Morn
L-.alpha.-methylphenylalanine Mphe L-.alpha.-methylproline Mpro
L-.alpha.-methylserine Mser L-.alpha.-methylthreonine Mthr
L-.alpha.-methylvaline Mtrp L-.alpha.-methyltyrosine Mtyr
L-.alpha.-methylleucine Mval L-N- Nmhphe Nnbbm
methylhomophenylalanine N-(N-(2,2-diphenylethyl)
N-(N-(3,3-diphenylpropyl) carbamylmethyl-glycine Nnbhm
Carbamylmethyl(1)glycine Nnbhe 1-carboxy-1-(2,2-diphenyl Nmbc
ethylamino)cyclopropane
[0309] Since the peptides of the present invention are preferably
utilized in diagnostics which require the peptides to be in soluble
form, the peptides of the present invention preferably include one
or more non-natural or natural polar amino acids, including but not
limited to serine and threonine which are capable of increasing
peptide solubility due to their hydroxyl-containing side chain.
[0310] The peptides of the present invention are preferably
utilized in a linear form, although it will be appreciated that in
cases where cyclicization does not severely interfere with peptide
characteristics, cyclic forms of the peptide can also be
utilized.
[0311] The peptides of present invention can be biochemically
synthesized such as by using standard solid phase techniques. These
methods include exclusive solid phase synthesis well known in the
art, partial solid phase synthesis methods, fragment condensation,
classical solution synthesis. These methods are preferably used
when the peptide is relatively short (i.e., 10 kDa) and/or when it
cannot be produced by recombinant techniques (i.e., not encoded by
a nucleic acid sequence) and therefore involves different
chemistry.
[0312] Synthetic peptides can be purified by preparative high
performance liquid chromatography and the composition of which can
be confirmed via amino acid sequencing.
[0313] In cases where large amounts of the peptides of the present
invention are desired, the peptides of the present invention can be
generated using recombinant techniques such as described by Bitter
et al., (1987) Methods in Enzymol. 153:516-544, Studier et al.
(1990)
[0314] Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature
310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et
al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science
224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and
Weissbach & Weissbach, 1988, Methods for Plant Molecular
Biology, Academic Press, NY, Section VIII, pp 421-463 and also as
described above.
Antibodies
[0315] "Antibody" refers to a polypeptide ligand that is preferably
substantially encoded by an immunoglobulin gene or immunoglobulin
genes, or fragments thereof, which specifically binds and
recognizes an epitope (e.g., an antigen). The recognized
immunoglobulin genes include the kappa and lambda light chain
constant region genes, the alpha, gamma, delta, epsilon and mu
heavy chain constant region genes, and the myriad-immunoglobulin
variable region genes. Antibodies exist, e.g., as intact
immunoglobulins or as a number of well characterized fragments
produced by digestion with various peptidases. This includes, e.g.,
Fab' and F(ab)'.sub.2 fragments. The term "antibody," as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies or those synthesized de novo using
recombinant DNA methodologies. It also includes polyclonal
antibodies, monoclonal antibodies, chimeric antibodies, humanized
antibodies, or single chain antibodies. "Fc" portion of an antibody
refers to that portion of an immunoglobulin heavy chain that
comprises one or more heavy chain constant region domains, CH1, CH2
and CH3, but does not include the heavy chain variable region.
[0316] The functional fragments of antibodies, such as Fab,
F(ab')2, and Fv that are capable of binding to macrophages, are
described as follows: (1) Fab, the fragment which contains a
monovalent antigen-binding fragment of an antibody molecule, can be
produced by digestion of whole antibody with the enzyme papain to
yield an intact light chain and a portion of one heavy chain; (2)
Fab', the fragment of an antibody molecule that can be obtained by
treating whole antibody with pepsin, followed by reduction, to
yield an intact light chain and a portion of the heavy chain; two
Fab' fragments are obtained per antibody molecule; (3)
(Fab').sub.2, the fragment of the antibody that can be obtained by
treating whole antibody with the enzyme pepsin without subsequent
reduction; F(ab')2 is a dimer of two Fab' fragments held together
by two disulfide bonds; (4) Fv, defined as a genetically engineered
fragment containing the variable region of the light chain and the
variable region of the heavy chain expressed as two chains; and (5)
Single chain antibody ("SCA"), a genetically engineered molecule
containing the variable region of the light chain and the variable
region of the heavy chain, linked by a suitable polypeptide linker
as a genetically fused single chain molecule.
[0317] Methods of producing polyclonal and monoclonal antibodies as
well as fragments thereof are well known in the art (See for
example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1988, incorporated herein by
reference).
[0318] Antibody fragments according to the present invention can be
prepared by proteolytic hydrolysis of the antibody or by expression
in E. coli or mammalian cells (e.g. Chinese hamster ovary cell
culture or other protein expression systems) of DNA encoding the
fragment. Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. For example,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
This fragment can be further cleaved using a thiol reducing agent,
and optionally a blocking group for the sulfhydryl groups resulting
from cleavage of disulfide linkages, to produce 3.5S Fab'
monovalent fragments. Alternatively, an enzymatic cleavage using
pepsin produces two monovalent Fab' fragments and an Fc fragment
directly. These methods are described, for example, by Goldenberg,
U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained
therein, which patents are hereby incorporated by reference in
their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126
(1959)]. Other methods of cleaving antibodies, such as separation
of heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0319] Fv fragments comprise an association of VH and VL chains.
This association may be noncovalent, as described in Inbar et al.
[Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the
variable chains can be linked by an intermolecular disulfide bond
or cross-linked by chemicals such as glutaraldehyde. Preferably,
the Fv fragments comprise VH and VL chains connected by a peptide
linker. These single-chain antigen binding proteins (sFv) are
prepared by constructing a structural gene comprising DNA sequences
encoding the VH and VL domains connected by an oligonucleotide. The
structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
sFvs are described, for example, by [Whitlow and Filpula, Methods
2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et
al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778,
which is hereby incorporated by reference in its entirety.
[0320] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick and Fry [Methods, 2: 106-10
(1991)].
[0321] Humanized forms of non-human (e.g., murine) antibodies are
chimeric molecules of immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab') or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues from a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)].
[0322] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as import
residues, which are typically taken from an import variable domain.
Humanization can be essentially performed following the method of
Winter and co-workers [Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
[0323] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introduction of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10: 779-783 (1992);
Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368
812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51
(1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg
and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).
[0324] Preferably, the antibody of this aspect of the present
invention specifically binds at least one epitope of the
polypeptide variants of the present invention. As used herein, the
term "epitope" refers to any antigenic determinant on an antigen to
which the paratope of an antibody binds.
[0325] Epitopic determinants usually consist of chemically active
surface groupings of molecules such as amino acids or carbohydrate
side chains and usually have specific three dimensional structural
characteristics, as well as specific charge characteristics.
[0326] Optionally, a unique epitope may be created in a variant due
to a change in one or more post-translational modifications,
including but not limited to glycosylation and/or phosphorylation,
as described below. Such a change may also cause a new epitope to
be created, for example through removal of glycosylation at a
particular site.
[0327] An epitope according to the present invention may also
optionally comprise part or all of a unique sequence portion of a
variant according to the present invention in combination with at
least one other portion of the variant which is not contiguous to
the unique sequence portion in the linear polypeptide itself, yet
which are able to form an epitope in combination. One or more
unique sequence portions may optionally combine with one or more
other non-contiguous portions of the variant (including a portion
which may have high homology to a portion of the known protein) to
form an epitope.
Immunoassays
[0328] In another embodiment of the present invention, an
immunoassay can be used to qualitatively or quantitatively detect
and analyze markers in a sample. This method comprises: providing
an antibody that specifically binds to a marker; contacting a
sample with the antibody; and detecting the presence of a complex
of the antibody bound to the marker in the sample.
[0329] To prepare an antibody that specifically binds to a marker,
purified protein markers can be used. Antibodies that specifically
bind to a protein marker can be prepared using any suitable methods
known in the art.
[0330] After the antibody is provided, a marker can be detected
and/or quantified using any of a number of well recognized
immunological binding assays. Useful assays include, for example,
an enzyme immune assay (EIA) such as enzyme-linked immunosorbent
assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or
a slot blot assay see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;
4,517,288; and 4,837,168). Generally, a sample obtained from a
subject can be contacted with the antibody that specifically binds
the marker.
[0331] Optionally, the antibody can be fixed to a solid support to
facilitate washing and subsequent isolation of the complex, prior
to contacting the antibody with a sample. Examples of solid
supports include but are not limited to glass or plastic in the
form of, e.g., a microtiter plate, a stick, a bead, or a microbead.
Antibodies can also be attached to a solid support.
[0332] After incubating the sample with antibodies, the mixture is
washed and the antibody-marker complex formed can be detected. This
can be accomplished by incubating the washed mixture with a
detection reagent. Alternatively, the marker in the sample can be
detected using an indirect assay, wherein, for example, a second,
labeled antibody is used to detect bound marker-specific antibody,
and/or in a competition or inhibition assay wherein, for example, a
monoclonal antibody which binds to a distinct epitope of the marker
are incubated simultaneously with the mixture.
[0333] Throughout the assays, incubation and/or washing steps may
be required after each combination of reagents. Incubation steps
can vary from about 5 seconds to several hours, preferably from
about 5 minutes to about 24 hours. However, the incubation time
will depend upon the assay format, marker, volume of solution,
concentrations and the like. Usually the assays will be carried out
at ambient temperature, although they can be conducted over a range
of temperatures, such as 10.degree. C. to 40.degree. C.
[0334] The immunoassay can be used to determine a test amount of a
marker in a sample from a subject. First, a test amount of a marker
in a sample can be detected using the immunoassay methods described
above. If a marker is present in the sample, it will form an
antibody-marker complex with an antibody that specifically binds
the marker under suitable incubation conditions described above.
The amount of an antibody-marker complex can optionally be
determined by comparing to a standard. As noted above, the test
amount of marker need not be measured in absolute units, as long as
the unit of measurement can be compared to a control amount and/or
signal.
[0335] Preferably used are antibodies which specifically interact
with the polypeptides of the present invention and not with wild
type proteins or other isoforms thereof, for example. Such
antibodies are directed, for example, to the unique sequence
portions of the polypeptide variants of the present invention,
including but not limited to bridges, heads, tails and insertions
described in greater detail below. Preferred embodiments of
antibodies according to the present invention are described in
greater detail with regard to the section entitled
"Antibodies".
[0336] Radio-immunoassay (RJA): In one version, this method
involves precipitation of the desired substrate and in the methods
detailed hereinbelow, with a specific antibody and radiolabelled
antibody binding protein (e.g., protein A labeled with I.sup.125)
immobilized on a precipitable carrier such as agarose beads. The
number of counts in the precipitated pellet is proportional to the
amount of substrate.
[0337] In an alternate version of the RIA, a labeled substrate and
an unlabelled antibody binding protein are employed. A sample
containing an unknown amount of substrate is added in varying
amounts. The decrease in precipitated counts from the labeled
substrate is proportional to the amount of substrate in the added
sample.
[0338] Enzyme linked immunosorbent assay (ELISA): This method
involves fixation of a sample (e.g., fixed cells or a proteinaceous
solution) containing a protein substrate to a surface such as a
well of a microtiter plate. A substrate specific antibody coupled
to an enzyme is applied and allowed to bind to the substrate.
Presence of the antibody is then detected and quantitated by a
colorimetric reaction employing the enzyme coupled to the antibody.
Enzymes commonly employed in this method include horseradish
peroxidase and alkaline phosphatase. If well calibrated and within
the linear range of response, the amount of substrate present in
the sample is proportional to the amount of color produced. A
substrate standard is generally employed to improve quantitative
accuracy.
[0339] Western blot: This method involves separation of a substrate
from other protein by means of an acrylamide gel followed by
transfer of the substrate to a membrane (e.g., nylon or PVDF).
Presence of the substrate is then detected by antibodies specific
to the substrate, which are in turn detected by antibody binding
reagents. Antibody binding reagents may be, for example, protein A,
or other antibodies. Antibody binding reagents may be radiolabelled
or enzyme linked as described hereinabove. Detection may be by
autoradiography, colorimetric reaction or chemiluminescence. This
method allows both quantitation of an amount of substrate and
determination of its identity by a relative position on the
membrane which is indicative of a migration distance in the
acrylamide gel during electrophoresis.
[0340] Immunohistochemical analysis: This method involves detection
of a substrate in situ in fixed cells by substrate specific
antibodies. The substrate specific antibodies may be enzyme linked
or linked to fluorophores. Detection is by microscopy and
subjective evaluation. If enzyme linked antibodies are employed, a
colorimetric reaction may be required.
[0341] Fluorescence activated cell sorting (FACS): This method
involves detection of a substrate in situ in cells by substrate
specific antibodies. The substrate specific antibodies are linked
to fluorophores. Detection is by means of a cell sorting machine
which reads the wavelength of light emitted from each cell as it
passes through a light beam. This method may employ two or more
antibodies simultaneously.
Radio-Imaging Methods
[0342] These methods include but are not limited to, positron
emission tomography (PET) single photon emission computed
tomography (SPECT). Both of these techniques are non-invasive, and
can be used to detect and/or measure a wide variety of tissue
events and/or functions, such as detecting cancerous cells for
example. Unlike PET, SPECT can optionally be used with two labels
simultaneously. SPECT has some other advantages as well, for
example with regard to cost and the types of labels that can be
used. For example, U.S. Pat. No. 6,696,686 describes the use of
SPECT for detection of breast cancer, and is hereby incorporated by
reference as if fully set forth herein.
Display Libraries
[0343] According to still another aspect of the present invention
there is provided a display library comprising a plurality of
display vehicles (such as phages, viruses or bacteria) each
displaying at least 6, at least 7, at least 8, at least 9, at least
10, 10-15, 12-17, 15-20, 15-30 or 20-50 consecutive amino acids
derived from the polypeptide sequences of the present
invention.
[0344] Methods of constructing such display libraries are well
known in the art. Such methods are described in, for example, Young
A C, et al., "The three-dimensional structures of a polysaccharide
binding antibody to Cryptococcus neoformans and its complex with a
peptide from a phage display library: implications for the
identification of peptide mimotopes" J Mol Biol 1997 Dec. 12;
274(4):622-34; Giebel L B et al. "Screening of cyclic peptide phage
libraries identifies ligands that bind streptavidin with high
affinities" Biochemistry 1995 Nov. 28; 34(47):15430-5; Davies E L
et al., "Selection of specific phage-display antibodies using
libraries derived from chicken immunoglobulin genes" J Immunol
Methods 1995 Oct. 12; 186(1):125-35; Jones C R T al. "Current
trends in molecular recognition and bioseparation" J Chromatogr A
1995 Jul. 14; 707(1):3-22; Deng S J et al. "Basis for selection of
improved carbohydrate-binding single-chain antibodies from
synthetic gene libraries" Proc Natl Acad Sci USA 1995 May 23;
92(11):4992-6; and Deng S J et al. "Selection of antibody
single-chain variable fragments with improved carbohydrate binding
by phage display" J Biol Chem 1994 Apr. 1; 269(13):9533-8, which
are incorporated herein by reference.
[0345] The following sections relate to Candidate Marker
Examples.
Candidate Marker Examples Section
[0346] This Section relates to Examples of sequences according to
the present invention, including illustrative methods of selection
thereof.
[0347] Description of the Methodology Undertaken to Uncover the
Biomolecular Sequences of the Present Invention
[0348] Human ESTs and cDNAs were obtained from GenBank versions 136
(Jun. 15, 2003
ftp.ncbi.nih.gov/genbank/release.notes/gb136.release.notes); NCBI
genome assembly of April 2003; RefSeq sequences from June 2003;
Genbank version 139 (December 2003); Human Genome from NCBI (Build
34) (from October 2003); and RefSeq sequences from December 2003;
and the LifeSeq library from Incyte Corporation (Wilmington, Del.,
USA; ESTs only). With regard to GenBank sequences, the human EST
sequences from the EST (GBEST) section and the human mRNA sequences
from the primate (GBPRI) section were used; also the human
nucleotide RefSeq mRNA sequences were used (see for example www
"dot" ncbi "dot" nlm "dot" nih "dot" gov/Genbank/GenbankOverview
"dot" html and for a reference to the EST section, see www "dot"
ncbi "dot" nlm "dot" nih "dot" gov/dbEST/; a general reference to
dbEST, the EST database in GenBank, may be found in Boguski et al,
Nat. Genet. 1993 August; 4(4):332-3; all of which are hereby
incorporated by reference as if fully set forth herein).
[0349] Novel splice variants were predicted using the LEADS
clustering and assembly system as described in Sorek, R., Ast, G.
& Graur, D. Alu-containing exons are alternatively spliced.
Genome Res 12, 1060-7 (2002); U.S. Pat. No. 6,625,545; and U.S.
patent application Ser. No. 10/426,002, published as US20040101876
on May 27, 2004; all of which are hereby incorporated by reference
as if fully set forth herein. Briefly, the software cleans the
expressed sequences from repeats, vectors and immunoglobulins. It
then aligns the expressed sequences to the genome taking
alternatively splicing into account and clusters overlapping
expressed sequences into "clusters" that represent genes or partial
genes.
[0350] These were annotated using the GeneCarta (Compugen,
Tel-Aviv, Israel) platform. The GeneCarta platform includes a rich
pool of annotations, sequence information (particularly of spliced
sequences), chromosomal information, alignments, and additional
information such as SNPs, gene ontology terms, expression profiles,
functional analyses, detailed domain structures, known and
predicted proteins and detailed homology reports.
[0351] A brief explanation is provided with regard to the method of
selecting the candidates. However, it should noted that this
explanation is provided for descriptive purposes only, and is not
intended to be limiting in any way. The potential markers were
identified by a computational process that was designed to find
genes and/or their splice variants that are over-expressed in tumor
tissues, by using databases of expressed sequences. Various
parameters related to the information in the EST libraries,
determined according to a manual classification process, were used
to assist in locating genes and/or splice variants thereof that are
over-expressed in cancerous tissues. The detailed description of
the selection method is presented in Example 1 below. The cancer
biomarkers selection engine and the following wet validation stages
are schematically summarized in FIG. 1.
Example 1
Identification of Differentially Expressed Gene Products
Algorithm
[0352] In order to distinguish between differentially expressed
gene products and constitutively expressed genes (i.e., house
keeping genes) an algorithm based on an analysis of frequencies was
configured. A specific algorithm for identification of transcripts
over expressed in cancer is described hereinbelow.
[0353] Dry Analysis
[0354] Library Annotation--Est Libraries are Manually Classified
According to:
[0355] (i) Tissue origin
[0356] (ii) Biological source--Examples of frequently used
biological sources for construction of EST libraries include cancer
cell-lines; normal tissues; cancer tissues; fetal tissues; and
others such as normal cell lines and pools of normal cell-lines,
cancer cell-lines and combinations thereof. A specific description
of abbreviations used below with regard to these tissues/cell lines
etc is given above.
[0357] (iii) Protocol of library construction--various methods are
known in the art for library construction including normalized
library construction; non-normalized library construction;
subtracted libraries; ORESTES and others. It will be appreciated
that at times the protocol of library construction is not indicated
in GenBank and/or other library annotaion.
[0358] The following rules are followed:
[0359] EST libraries originating from identical biological samples
are considered as a single library.
[0360] EST libraries which included above-average levels of
contamination, such as DNA contamination for example, were
eliminated. The presence of such contamination was determined as
follows. For each library, the number of unspliced ESTs that are
not fully contained within other spliced sequences was counted. If
the percentage of such sequences (as compared to all other
sequences) was at least 4 standard deviations above the average for
all libraries being analyzed, this library was tagged as being
contaminated and was eliminated from further consideration in the
below analysis (see also Sorek, R. & Safer, H. M. A novel
algorithm for computational identification of contaminated EST
libraries. Nucleic Acids Res 31, 1067-74 (2003) for further
details).
[0361] Clusters (genes) having at least five sequences including at
least two sequences from the tissue of interest were analyzed.
Splice variants were identified by using the LEADS software package
as described above.
Example 2
Identification of Genes Over Expressed in Cancer
[0362] Two different scoring algorithms were developed.
[0363] Libraries score--candidate sequences which are supported by
a number of cancer libraries, are more likely to serve as specific
and effective diagnostic markers.
[0364] The basic algorithm--for each cluster the number of cancer
and normal libraries contributing sequences to the cluster was
counted. Fisher exact test was used to check if cancer libraries
are significantly over-represented in the cluster as compared to
the total number of cancer and normal libraries.
[0365] Library counting: Small libraries (e.g., less than 1000
sequences) were excluded from consideration unless they participate
in the cluster. For this reason, the total number of libraries is
actually adjusted for each cluster.
[0366] Clones no. score--Generally, when the number of ESTs is much
higher in the cancer libraries relative to the normal libraries it
might indicate actual over-expression.
[0367] The algorithm--
[0368] Clone counting: For counting EST clones each library
protocol class was given a weight based on our belief of how much
the protocol reflects actual expression levels:
[0369] (i) non-normalized: 1
[0370] (ii) normalized: 0.2
[0371] (iii) all other classes: 0.1
[0372] Clones number score--The total weighted number of EST clones
from cancer libraries was compared to the EST clones from normal
libraries. To avoid cases where one library contributes to the
majority of the score, the contribution of the library that gives
most clones for a given cluster was limited to 2 clones.
[0373] The score was computed as c + 1 C / n + 1 N ##EQU1##
[0374] Where:
[0375] c--weighted number of "cancer" clones in the cluster.
[0376] C--weighted number of clones in all "cancer" libraries.
[0377] n--weighted number of "normal" clones in the cluster.
[0378] N--weighted number of clones in all "normal" libraries.
[0379] Clones number score significance--Fisher exact test was used
to check if EST clones from cancer libraries are significantly
over-represented in the cluster as compared to the total number of
EST clones from cancer and normal libraries.
[0380] Two search approaches were used to find either general
cancer-specific candidates or tumor specific candidates. [0381]
Libraries/sequences originating from tumor tissues are counted as
well as libraries originating from cancer cell-lines ("normal"
cell-lines were ignored). [0382] Only libraries/sequences
originating from tumor tissues are counted
Example 3
Identification of Tissue Specific Genes
[0383] For detection of tissue specific clusters, tissue
libraries/sequences were compared to the total number of
libraries/sequences in cluster. Similar statistical tools to those
described in above were employed to identify tissue specific genes.
Tissue abbreviations are the same as for cancerous tissues, but are
indicated with the header "normal tissue".
[0384] The algorithm--for each tested tissue T and for each tested
cluster the following were examined:
[0385] 1. Each cluster includes at least 2 libraries from the
tissue T. At least 3 clones (weighed--as described above) from
tissue T in the cluster; and
[0386] 2. Clones from the tissue T are at least 40% from all the
clones participating in the tested cluster
[0387] Fisher exact test P-values were computed both for library
and weighted clone counts to check that the counts are
statistically significant.
Example 4
Identification of Splice Variants Over Expressed in Cancer of
Clusters which are not Over Expressed in Cancer
[0388] Cancer-Specific Splice Variants Containing a Unique Region
were Identified.
[0389] Identification of unique sequence regions in splice
variants
[0390] A Region is defined as a group of adjacent exons that always
appear or do not appear together in each splice variant.
[0391] A "segment" (sometimes referred also as "seg" or "node") is
defined as the shortest contiguous transcribed region without known
splicing inside.
[0392] Only reliable ESTs were considered for region and segment
analysis. An EST was defined as unreliable if:
[0393] (i) Unspliced;
[0394] (ii) Not covered by RNA;
[0395] (iii) Not covered by spliced ESTs; and
[0396] (iv) Alignment to the genome ends in proximity of long
poly-A stretch or starts in proximity of long poly-T stretch.
[0397] Only reliable regions were selected for further scoring.
Unique sequence regions were considered reliable if:
[0398] (i) Aligned to the genome; and
[0399] (ii) Regions supported by more than 2 ESTs.
[0400] The algorithm
[0401] Each unique sequence region divides the set of transcripts
into 2 groups:
[0402] (i) Transcripts containing this region (group TA).
[0403] (ii) Transcripts not containing this region (group TB).
[0404] The set of EST clones of every cluster is divided into 3
groups:
[0405] (i) Supporting (originating from) transcripts of group TA
(S1).
[0406] (ii) Supporting transcripts of group TB (S2).
[0407] (iii) Supporting transcripts from both groups (S3).
[0408] Library and clones number scores described above were given
to S1 group.
[0409] Fisher Exact Test P-values were used to check if:
[0410] S1 is significantly enriched by cancer EST clones compared
to S2; and
[0411] S1 is significantly enriched by cancer EST clones compared
to cluster background (S1+S2+S3).
[0412] Identification of unique sequence regions and division of
the group of transcripts accordingly is illustrated in FIG. 2. Each
of these unique sequence regions corresponds to a segment, also
termed herein a "node".
[0413] Region 1: common to all transcripts, thus it is not
considered; Region 2: specific to Transcript 1: T.sub.--1 unique
regions (2+6) against T.sub.--2+3 unique regions (3+4); Region 3:
specific to Transcripts 2+3: T.sub.--2+3 unique regions (3+4)
against Ti unique regions (2+6); Region 4: specific to Transcript
3: T.sub.--3 unique regions (4) against Tl+2 unique regions
(2+5+6); Region 5: specific to Transcript 1+2: T.sub.--1+2 unique
regions (2+5+6) against T3 unique regions (4); Region 6: specific
to Transcript 1: same as region 2.
Example 5
[0414] Identification of cancer specific splice variants of genes
over expressed in cancer
[0415] A search for EST supported (no mRNA) regions for genes
of:
[0416] (i) known cancer markers
[0417] (ii) Genes shown to be over-expressed in cancer in published
micro-array experiments.
[0418] Reliable EST supported-regions were defined as supported by
minimum of one of the following:
[0419] (i) 3 spliced ESTs; or
[0420] (ii) 2 spliced ESTs from 2 libraries;
[0421] (iii) 10 unspliced ESTs from 2 libraries, or
[0422] (iv) 3 libraries.
Actual Marker Examples
[0423] The following examples relate to specific actual marker
examples.
EXPERIMENTAL EXAMPLES SECTION
[0424] This Section relates to Examples describing experiments
involving these sequences, and illustrative, non-limiting examples
of methods, assays and uses thereof. The materials and experimental
procedures are explained first, as all experiments used them as a
basis for the work that was performed.
[0425] The markers of the present invention were tested with regard
to their expression in various cancerous and non-cancerous tissue
samples. A description of the samples used in the panel is provided
in Table 2 below. A description of the samples used in the normal
tissue panel is provided in Table 3 below. Tests were then
performed as described in the "Materials and Experimental
Procedures" section below. TABLE-US-00003 TABLE 2 Tissue samples in
testing panel Lot No. Pathology Sex/Age Source 66-A-Adeno G1 GS-4
160202 Adenocarcinoma Gleason score 4 M/64 ABS 73-A-Adeno G1 GS-4
16026T2 Acinar Adenocarcinoma Gleason score 4(2 + 2) M/77 ABS
68-A-Adeno G1 GS-5 160172 Adenocarcinoma Gleason score 5 M/66 ABS
56-Am-Adeno G1 GS-5 36467 Adenocarcinoma, Gleason score 5(3 + 2);
stage 2 M/72 Ambion 58-Am-Adeno G1 GS-5 37192 Adenocarcinoma,
Gleason score 5; stage 2 M/52 Ambion 65-A-Adeno G2 GS-5 160022
Adenocarcinoma Gleason score 5 M/66 ABS 69-A-Adeno GS-5 160182
Acinar Adenocarcinoma Gleason score 5 M/58 ABS 55-Am-Adeno GS-5
36464 Adenocarcinoma, Gleason score 5; stage 1 M/53 Ambion
64-A-Adeno G2 GS-6 160092 Acinar Adenocarcinoma Gleason score 6
M/71 ABS 70-A-Adeno G2 GS-6 160192 Adenocarcinoma Gleason score 6
M/53 ABS 18-A-Adeno GS-6 5610020069T Adenocarcinoma, Gleason score
6 (3 + 3) M ABS 67-A-Adeno GS-6 160142 Acinar Adenocarcinoma
Gleason score 6 M/62 ABS 25-A-Adeno GS-7 5605020052T
Adenocarcinoma, Gleason score 7 (4 + 3) M ABS 26-A-Adeno GS-7
5609020067T Adenocarcinoma, Gleason score 7 (4 + 3) M ABS
72-A-Adeno GS-7 160122 Acinar Adenocarcinoma Gleason score 7 M/66
ABS 71-A-Adeno GS-7 160242 Acinar Adenocarcinoma Gleason score 7
M/70 ABS 57-Am-Adeno GS-7 26442 Adenocarcinoma, Gleason score 7
M/62 Ambion 32-A-Adeno GS-9 5604020042T Adenocarcinoma, Gleason
score 9 (5 + 4) M ABS 54-B-Adeno G3 A610031 Adenocarcinoma Biochair
33-A-BPH 5607020058 BPH M ABS 34-A-BPH 5607020059 BPH M ABS
35-A-BPH 5607020060 BPH M ABS 43-B-PBH A609267 BPH M/66 Biochair
44-B-PBH A609268 BPH M/72 Biochair 45-B-PBH A609269 BPH M/69
Biochair 46-B-PBH A609270 BPH M/65 Biochair 47-B-PBH A609271 BPH
M/71 Biochair 40-A-N M26 5609020067N Normal Matched M ABS 41-A-N
M32 5604020042N Normal Matched M ABS 48-B-N A609257 Normal PM M/24
Biochair 49-B-N A609256 Normal PM M/36 Biochair 50-B-N A609255
Normal PM M/26 Biochair 51-B-N A609258 Normal PM M/27 Biochair
52-B-N A609254 Normal PM M/29 Biochair 53-Cl-N 1070317 Normal -
Pool of 47 M&F Clontech 42-Am-N 061P04A Normal (IC BLEED) M/47
ambion 59-Am-N 25955 Normal PM (Head trauma) M/62 Ambion 60-Am-N
33605 Normal PM (Myocardial infraction) M/69 Ambion 61-Am-N 34077
Normal PM (Alzheimer's) M/71 Ambion 62-Am-N 31316 Normal (Renal
failure) M/79 Ambion 63-Am-N 30991 Normal (Gall Bladder cancer)
M/78 Ambion
[0426] TABLE-US-00004 TABLE 3 Tissue samples in normal panel: Lot
no. Source Tissue Pathology Sex/Age 1-Am-Colon (C71) 071P10B Ambion
Colon PM F/43 2-B-Colon (C69) A411078 Biochain Colon PM-Pool of 10
M&F 3-Cl-Colon (C70) 1110101 Clontech Colon PM-Pool of 3
M&F 4-Am-Small Intestine 091P0201A Ambion Small Intestine PM
M/75 5-B-Small Intestine A501158 Biochain Small Intestine PM M/63
6-B-Rectum A605138 Biochain Rectum PM M/25 7-B-Rectum A610297
Biochain Rectum PM M/24 8-B-Rectum A610298 Biochain Rectum PM M/27
9-Am-Stomach 110P04A Ambion Stomach PM M/16 10-B-Stomach A501159
Biochain Stomach PM M/24 11-B-Esophagus A603814 Biochain Esophagus
PM M/26 12-B-Esophagus A603813 Biochain Esophagus PM M/41
13-Am-Pancreas 071P25C Ambion Pancreas PM M/25 14-CG-Pancreas
CG-255-2 Ichilov Pancreas PM M/75 15-B-Lung A409363 Biochain Lung
PM F/26 16-Am-Lung (L93) 111P0103A Ambion Lung PM F/61 17-B-Lung
(L92) A503204 Biochain Lung PM M/28 18-Am-Ovary (O47) 061P43A
Ambion Ovary PM F/16 19-B-Ovary (O48) A504087 Biochain Ovary PM
F/51 20-B-Ovary (O46) A504086 Biochain Ovary PM F/41 21-Am-Cervix
101P0101A Ambion Cervix PM F/40 22-B-Cervix A408211 Biochain Cervix
PM F/36 23-B-Cervix A504089 Biochain Cervix PM-Pool of 5 M&F
24-B-Uterus A411074 Biochain Uterus PM-Pool of 10 M&F
25-B-Uterus A409248 Biochain Uterus PM F/43 26-B-Uterus A504090
Biochain Uterus PM-Pool of 5 M&F 27-B-Bladder A501157 Biochain
Bladder PM M/29 28-Am-Bladder 071P02C Ambion Bladder PM M/20
29-B-Bladder A504088 Biochain Bladder PM-Pool of 5 M&F
30-Am-Placenta 021P33A Ambion Placenta PB F/33 31-B-Placenta
A410165 Biochain Placenta PB F/26 32-B-Placenta A411073 Biochain
Placenta PB-Pool of 5 M&F 33-B-Breast (B59) A607155 Biochain
Breast PM F/36 34-Am-Breast (B63) 26486 Ambion Breast PM F/43
35-Am-Breast (B64) 23036 Ambion Breast PM F/57 36-Cl-Prostate (P53)
1070317 Clontech Prostate PB-Pool of 47 M&F 37-Am-Prostate
(P42) 061P04A Ambion Prostate PM M/47 38-Am-Prostate (P59) 25955
Ambion Prostate PM M/62 39-Am-Testis 111P0104A Ambion Testis PM
M/25 40-B-Testis A411147 Biochain Testis PM M/74 41-Cl-Testis
1110320 Clontech Testis PB-Pool of 45 M&F 42-CG-Adrenal
CG-184-10 Ichilov Adrenal PM F/81 43-B-Adrenal A610374 Biochain
Adrenal PM F/83 44-B-Heart A411077 Biochain Heart PB-Pool of 5
M&F 45-CG-Heart CG-255-9 Ichilov Heart PM M/75 46-CG-Heart
CG-227-1 Ichilov Heart PM F/36 47-Am-Liver 081P0101A Ambion Liver
PM M/64 48-CG-Liver CG-93-3 Ichilov Liver PM F/19 49-CG-Liver
CG-124-4 Ichilov Liver PM F/34 50-Cl-BM 1110932 Clontech Bone
Marrow PM-Pool of 8 M&F 51-CGEN-Blood WBC#5 CGEN Blood M
52-CGEN-Blood WBC#4 CGEN Blood M 53-CGEN-Blood WBC#3 CGEN Blood M
54-CG-Spleen CG-267 Ichilov Spleen PM F/25 55-CG-Spleen 111P0106B
Ambion Spleen PM M/25 56-CG-Spleen A409246 Biochain Spleen PM F/12
56-CG-Thymus CG-98-7 Ichilov Thymus PM F/28 58-Am-Thymus 101P0101A
Ambion Thymus PM M/14 59-B-Thymus A409278 Biochain Thymus PM M/28
60-B-Thyroid A610287 Biochain Thyroid PM M/27 61-B-Thyroid A610286
Biochain Thyroid PM M/24 62-CG-Thyroid CG-119-2 Ichilov Thyroid PM
F/66 63-Cl-Salivary Gland 1070319 Clontech Salivary Gland PM-Pool
of 24 M&F 64-Am-Kidney 111P0101B Ambion Kidney PM-Pool of 14
M&F 65-Cl-Kidney 1110970 Clontech Kidney PM-Pool of 14 M&F
66-B-Kidney A411080 Biochain Kidney PM-Pool of 5 M&F
67-CG-Cerebellum CG-183-5 Ichilov Cerebellum PM M/74
68-CG-Cerebellum CG-212-5 Ichilov Cerebellum PM M/54 69-B-Brain
A411322 Biochain Brain PM M/28 70-Cl-Brain 1120022 Clontech Brain
PM-Pool of 2 M&F 71-B-Brain A411079 Biochain Brain PM-Pool of 2
M&F 72-CG-Brain CG-151-1 Ichilov Brain PM F/86 73-Am-Skeletal
Muscle 101P013A Ambion Skeletal Muscle PM F/28 74-Cl-Skeletal
Muscle 1061038 Clontech Skeletal Muscle PM-Pool of 2 M&F
[0427] Materials and Experimental Procedures
[0428] RNA preparation --RNA was obtained from Clontech (Franklin
Lakes, N.J. USA 07417, www"dot" clontech"dot" com), BioChain Inst.
Inc. (Hayward, Calif. 94545 USA www.biochain.com), ABS (Wilmington,
Del. 19801, USA, www"dot" absbioreagents"dot" com) or Ambion
(Austin, Tex. 78744 USA, www"dot" ambion"dot" com). Alternatively,
RNA was generated from tissue samples using TR1--Reagent (Molecular
Research Center), according to Manufacturer's instructions. Tissue
and RNA samples were obtained from patients or from postmortem.
Total RNA samples were treated with DNaseI (Ambion) and purified
using RNeasy columns (Qiagen).
[0429] RT PCR--Purified RNA (1 .mu.g) was mixed with 150 ng Random
Hexamer primers (Invitrogen) and 500 .mu.M dNTP in a total volume
of 15.6 .mu.l. The mixture was incubated for 5 min at 65.degree. C.
and then quickly chilled on ice. Thereafter, 5 .mu.l of 5.times.
SuperscriptII first strand buffer (Invitrogen), 2.4 .mu.l 0.1M DTT
and 40 units RNasin (Promega) were added, and the mixture was
incubated for 10 min at 25.degree. C., followed by further
incubation at 42.degree. C. for 2 min. Then, 1 .mu.l (200 units) of
SuperscriptII (Invitrogen) was added and the reaction (final volume
of 25 .mu.l) was incubated for 50 min at 42.degree. C. and then
inactivated at 70.degree. C. for 15 min. The resulting cDNA was
diluted 1:20 in TE buffer (10 mM Tris pH=8, 1 mM EDTA pH=8).
[0430] Real-Time RT-PCR analysis--cDNA (5 .mu.l), prepared as
described above, was used as a template in Real-Time PCR reactions
using the SYBR Green I assay (PE Applied Biosystem) with specific
primers and UNG Enzyme (Eurogentech or ABI or Roche). The
amplification was
[0431] effected as follows: 50.degree. C. for 2 min, 95.degree. C.
for 10 min, and then 40 cycles of 95.degree. C. for 15 sec,
followed by 60.degree. C. for 1 min. Detection was performed by
using the PE Applied Biosystem SDS 7000. The cycle in which the
reactions achieved a threshold level (Ct) of fluorescence was
registered and was used to calculate the relative transcript
quantity in the RT reactions. The relative quantity was calculated
using the equation Q=efficiency .sup.-Ct. The efficiency of the PCR
reaction was calculated from a standard curve, created by using
serial dilutions of several reverse transcription (RT) reactions.
To minimize inherent differences in the RT reaction, the resulting
relative quantities were normalized to the geometric mean of the
relative quantities of several housekeeping (HSKP) genes. Schematic
summary of quantitative real-time PCR analysis is presented in FIG.
3. As shown, the x-axis shows the cycle number. The CT=Threshold
Cycle point, which is the cycle that the amplification curve
crosses the fluorescence threshold that was set in the experiment.
This point is a calculated cycle number in which PCR product signal
is above the background level (passive dye ROX) and still in the
Geometric/Exponential phase (as shown, once the level of
fluorescence crosses the measurement threshold, it has a
geometrically increasing phase, during which measurements are most
accurate, followed by a linear phase and a plateau phase; for
quantitative measurements, the latter two phases do not provide
accurate measurements). The y-axis shows the normalized reporter
fluorescence. It should be noted that this type of analysis
provides relative quantification.
[0432] The sequences of the housekeeping genes measured in all the
examples below on prostate panel were as follows: TABLE-US-00005
SDHA (GenBank Accession No. NM_004168 (SEQ ID NO: 508)) SDHA
Forward primer (SEQ ID NO: 405): TGGGAACAAGAGGGCATCTG SDHA Reverse
primer (SEQ ID NO: 406): CCACCACTGCATCAAATTCATG SDHA-amplicon (SEQ
ID NO: 407): TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGT
ATCCAGTAGTGGATCATGAATTTGATGCAGTGGTGG PBGD (GenBank Accession No.
BC019323 (SEQ ID NO: 509)), PBGD Forward primer (SEQ ID NO: 402):
TGAGAGTGATTCGCGTGGG PBGD Reverse primer (SEQ ID NO: 403):
CCAGGGTACGAGGCTTTCAAT PBGD-amplicon (SEQ ID NO: 404):
TGAGAGTGATTCGCGTGGGTACCCGCAAGAGCCAGCTTGCTCGCATACAG
ACGGACAGTGTGGTGGCAACATTGAAAGCCTCGTACCCTGG HPRT1 (GenBank Accession
No. NM_000194 (SEQ ID NO: 510)), HPRT1 Forward primer (SEQ ID NO:
399): TGACACTGGCAAAACAATGCA HPRT1 Reverse primer (SEQ ID NO: 400):
GGTCCTTTTCACCAGCAAGCT HPRT1-amplicon (SEQ ID NO: 401):
TGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATA
ATCCAAAGATGGTCAAGGTCGCAAGCTTGCTGGTGAAAAGGACC RPL19 (GenBank
Accession No. NM_000981 (SEQ ID NO: 511) RPL19Forward primer (SEQ
ID NO: 408): TGGCAAGAAGAAGGTCTGGTTAG RPL19Reverse primer (SEQ ID
NO: 409): TGATCAGCCCATCTTTGATGAG RPL19-amplicon (SEQ ID NO: 410):
TGGCAAGAAGAAGGTCTGGTTAGACCCCAATGAGACCAATGAAATCGCCA
ATGCCAACTCCCGTCAGCAGATCCGGAAGCTCATCAAAGATGGGCTGATC A
[0433] The sequences of the housekeeping genes measured in all the
examples on normal tissue samples panel were as follows:
TABLE-US-00006 RPL19 (GenBank Accession No. NM_000981 (SEQ ID NO:
511)), RPL19 Forward primer (SEQ ID NO: 408):
TGGCAAGAAGAAGGTCTGGTTAG RPL19 Reverse primer (SEQ ID NO: 409):
TGATCAGCCCATCTTTGATGAG RPL19 -amplicon (SEQ ID NO: 410):
TGGCAAGAAGAAGGTCTGGTTAGACCCCAATGAGACCAATGAAATCGCCA
ATGCCAACTCCCGTCAGCAGATCCGGAAGCTCATCAAAGATGGGCTGATC A TATA box
(GenBank Accession No. NM_003194 (SEQ ID NO: 512)), TATA box
Forward primer (SEQ ID NO: 513): CGGTTTGCTGCGGTAATCAT TATA box
Reverse primer (SEQ ID NO: 514): TTTCTTGCTGCCAGTCTGGAC TATA box
-amplicon (SEQ ID NO: 515):
CGGTTTGCTGCGGTAATCATGAGGATAAGAGAGCCACGAACCACGGCACT
GATTTTCAGTTCTGGGAAAATGGTGTGCACAGGAGCCAAGAGTGAAGAAC
AGTCCAGACTGGCAGCAAGAAA UBC (GenBank Accession No. BC000449 (SEQ ID
NO: 516)) UBC Forward primer (SEQ ID NO: 517): ATTTGGGTCGCGGTTCTTG
UBC Reverse primer (SEQ ID NO: 518): TGCCTTGACATTCTCGATGGT UBC
-amplicon (SEQ ID NO: 519):
ATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGACAA
TGCAGATCTTCGTGAAGACTCTGACTGGTAAGACCATCACCCTCGAGG
TTGAGCCCAGTGACACCATCGAGAATGTCAAGGCA SDHA (GenBank Accession No.
NM_004168 (SEQ ID NO: 508)) SDHA Forward primer (SEQ ID NO: 405):
TGGGAACAAGAGGGCATCTG SDHA Reverse primer (SEQ ID NO: 406):
CCACCACTGCATCAAATTCATG SDHA-amplicon (SEQ ID NO: 407):
TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGT
ATCCAGTAGTGGATCATGAATTTGATGCAGTGGTGG
[0434] Oligonucleotide-Based Micro-Array Experiment Protocol--
Microarray Fabrication
[0435] Microarrays (chips) were printed by pin deposition using the
MicroGrid II MGII 600 robot from BioRobtics Limited (Cambridge,
UK). 50-mer oligonucleotides target sequences were designed by
Compugen Ltd (Tel-Aviv, Ill.) as described by A. Shoshan et al,
"Optical technologies and informatics", Proceedings of SPIE. Vol
4266, pp. 86-95 (2001). The designed oligonucleotides were
synthesized and purified by desalting with the Sigma-Genosys system
(The Woodlands, Tex., US) and all of the oligonucleotides were
joined to a C6 amino-modified linker at the 5' end, or being
attached directly to CodeLink slides (Cat #25-6700-01. Amersham
Bioscience, Piscataway, N.J., US). The 50-mer oligonucleotides,
forming the target sequences, were first suspended in Ultra-pure
DDW (Cat # 01-866-1A Kibbutz Beit-Haemek, Israel) to a
concentration of 50 .mu.M. Before printing the slides, the
oligonucleotides were resuspended in 300 mM sodium phosphate (pH
8.5) to final concentration of 150 mM and printed at 35-40%
relative humidity at 21.degree. C.
[0436] Each slide contained a total of 9792 features in 32
subarrays. Of these features, 4224 features were sequences of
interest according to the present invention and negative controls
that were printed in duplicate. An additional 288 features (96
target sequences printed in triplicate) contained housekeeping
genes from Human Evaluation Library2, Compugen Ltd, Israel. Another
384 features are E. coli spikes 1-6, which are oligos to E-Coli
genes which are commercially available in the Array Control product
(Array control-sense oligo spots, Ambion Inc. Austin, Tex. Cat
#1781, Lot #112K06).
Post-Coupling Processing of Printed Slides
[0437] After the spotting of the oligonucleotides to the glass
(CodeLink) slides, the slides were incubated for 24 hours in a
sealed saturated NaCl humidification chamber (relative humidity
70-75%).
[0438] Slides were treated for blocking of the residual reactive
groups by incubating them in blocking solution at 50.degree. C. for
15 minutes (10 ml/slide of buffer containing 0.1M Tris, 50 mM
ethanolamine, 0.1% SDS). The slides were then rinsed twice with
Ultra-pure DDW (double distilled water). The slides were then
washed with wash solution (10 ml/slide. 4.times.SSC, 0.1% SDS)) at
50.degree. C. for 30 minutes on the shaker. The slides were then
rinsed twice with Ultra-pure DDW, followed by drying by
centrifugation for 3 minutes at 800 rpm.
[0439] Next, in order to assist in automatic operation of the
hybridization protocol, the slides were treated with Ventana
Discovery hybridization station barcode adhesives. The printed
slides were loaded on a Bio-Optica (Milan, Italy) hematology
staining device and were incubated for 10 minutes in 50 ml of
3-Aminopropyl Triethoxysilane (Sigma A3648 lot #122K589). Excess
fluid was dried and slides were then incubated for three hours in
20 mm/Hg in a dark vacuum desiccator (Pelco 2251, Ted Pella, Inc.
Redding Calif.).
[0440] The following protocol was then followed with the Genisphere
900-RP (random primer), with mini elute columns on the Ventana
Discovery HybStation.TM., to perform the microarray experiments.
Briefly, the protocol was performed as described with regard to the
instructions and information provided with the device itself. The
protocol included cDNA synthesis and labeling. cDNA concentration
was measured with the TBS-380 (Turner Biosystems. Sunnyvale,
Calif.) PicoFlour, which is used with the OliGreen ssDNA
Quantitation reagent and kit.
[0441] Hybridization was performed with the Ventana Hybridization
device, according to the provided protocols (Discovery
Hybridization Station Tuscon Ariz.).
[0442] The slides were then scanned with GenePix 4000B dual laser
scanner from Axon Instruments Inc, and analyzed by GenePix Pro 5.0
software.
[0443] Schematic summary of the oligonucleotide based microarray
fabrication and the experimental flow is presented in FIGS. 4 and
5.
[0444] Briefly, as shown in FIG. 4, DNA oligonucleotides at 25 uM
were deposited (printed) onto Amersham `CodeLink` glass slides
generating a well defined `spot`. These slides are covered with a
long-chain, hydrophilic polymer chemistry that creates an active
3-D surface that covalently binds the DNA oligonucleotides 5'-end
via the C6-amine modification. This binding ensures that the full
length of the DNA oligonucleotides is available for hybridization
to the cDNA and also allows lower background, high sensitivity and
reproducibility.
[0445] FIG. 5 shows a schematic method for performing the
microarray experiments. It should be noted that stages on the
left-hand or right-hand side may optionally be performed in any
order, including in parallel, until stage 4 (hybridization).
Briefly, on the left-hand side, the target oligonucleotides are
being spotted on a glass microscope slide (although optionally
other materials could be used) to form a spotted slide (stage 1).
On the right hand side, control sample RNA and cancer sample RNA
are Cy3 and Cy5 labeled, respectively (stage 2), to form labeled
probes. It should be noted that the control and cancer samples come
from corresponding tissues (for example, normal prostate tissue and
cancerous prostate tissue). Furthermore, the tissue from which the
RNA was taken is indicated below in the specific examples of data
for particular clusters, with regard to overexpression of an
oligonucleotide from a "chip" (microarray), as for example
"prostate" for chips in which prostate cancerous tissue and normal
tissue were tested as described above. In stage 3, the probes are
mixed. In stage 4, hybridization is performed to form a processed
slide. In stage 5, the slide is washed and scanned to form an image
file, followed by data analysis in stage 6.
Description for Cluster R11723
[0446] Cluster R11723 features 6 transcript(s) and 26 segment(s) of
interest, the names for which are given in Tables 4 and 5,
respectively, the sequences themselves are given at the end of the
application. The selected protein variants are given in table 6.
TABLE-US-00007 TABLE 4 Transcripts of interest Transcript Name
Sequence ID No. R11723_PEA_1_T15 5 R11723_PEA_1_T17 6
R11723_PEA_1_T19 7 R11723_PEA_1_T20 8 R11723_PEA_1_T5 9
R11723_PEA_1_T6 10
[0447] TABLE-US-00008 TABLE 5 Segments of interest Segment Name
Sequence ID No. R11723_PEA_1_node_13 90 R11723_PEA_1_node_16 91
R11723_PEA_1_node_19 92 R11723_PEA_1_node_2 93 R11723_PEA_1_node_22
94 R11723_PEA_1_node_31 95 R11723_PEA_1_node_10 96
R11723_PEA_1_node_11 97 R11723_PEA_1_node_15 98
R11723_PEA_1_node_18 99 R11723_PEA_1_node_20 100
R11723_PEA_1_node_21 101 R11723_PEA_1_node_23 102
R11723_PEA_1_node_24 103 R11723_PEA_1_node_25 104
R11723_PEA_1_node_26 105 R11723_PEA_1_node_27 106
R11723_PEA_1_node_28 107 R11723_PEA_1_node_29 108
R11723_PEA_1_node_3 109 R11723_PEA_1_node_30 110
R11723_PEA_1_node_4 111 R11723_PEA_1_node_5 112 R11723_PEA_1_node_6
113 R11723_PEA_1_node_7 114 R11723_PEA_1_node_8 115
[0448] TABLE-US-00009 TABLE 6 Proteins of interest Protein Name
Sequence ID No. R11723_PEA_1_P2 331 R11723_PEA_1_6 332
R11723_PEA_1_P7 333 R11723_PEA_1_P13 334 R11723_PEA_1_P10 335
[0449] Cluster R11723 can be used as a diagnostic marker according
to overexpression of transcripts of this cluster in cancer.
Expression of such transcripts in normal tissues is also given
according to the previously described methods. The term "number" in
the right hand column of the table and the numbers on the y-axis of
FIG. 6 refer to weighted expression of ESTs in each category, as
"parts per million" (ratio of the expression of ESTs for a
particular cluster to the expression of all ESTs in that category,
according to parts per million).
[0450] Overall, the following results were obtained as shown with
regard to the histograms in FIG. 6 and Table 7. This cluster is
overexpressed (at least at a minimum level) in the following
pathological conditions: epithelial malignant tumors, a mixture of
malignant tumors from different tissues and kidney malignant
tumors. TABLE-US-00010 TABLE 7 Normal tissue distribution Name of
Tissue Number Adrenal 0 Brain 30 Epithelial 3 General 17 head and
neck 0 Kidney 0 Lung 0 Breast 0 Ovary 0 Pancreas 10 Skin 0 Uterus
0
[0451] TABLE-US-00011 TABLE 8 P values and ratios for expression in
cancerous tissue Name of Tissue P1 P2 SP1 R3 SP2 R4 Adrenal 4.2e-01
4.6e-01 4.6e-01 2.2 5.3e-01 1.9 Brain 2.2e-01 2.0e-01 1.2e-02 2.8
5.0e-02 2.0 Epithelial 3.0e-05 6.3e-05 1.8e-05 6.3 3.4e-06 6.4
General 7.2e-03 4.0e-02 1.3e-04 2.1 1.1e-03 1.7 head and neck 1
5.0e-01 1 1.0 7.5e-01 1.3 Kidney 1.5e-01 2.4e-01 4.4e-03 5.4
2.8e-02 3.6 Lung 1.2e-01 1.6e-01 1 1.6 1 1.3 Breast 5.9e-01 4.4e-01
1 1.1 6.8e-01 1.5 Ovary 1.6e-02 1.3e-02 1.0e-01 3.8 7.0e-02 3.5
Pancreas 5.5e-01 2.0e-01 3.9e-01 1.9 1.4e-01 2.7 Skin 1 4.4e-01 1
1.0 1.9e-02 2.1 Uterus 1.5e-02 5.4e-02 1.9e-01 3.1 1.4e-01 2.5
[0452] As noted above, cluster R11723 features 6 transcript(s),
which were listed in Table 4 above. A description of each variant
protein according to the present invention is now provided.
[0453] Variant protein R11723_PEA.sub.--1_P2 (SEQ ID NO:331)
according to the present invention has an amino acid sequence as
given at the end of the application; it is encoded by transcript(s)
R11723_PEA.sub.--1_T6 (SEQ ID NO:10). The location of the variant
protein was determined according to results from a number of
different software programs and analyses, including analyses from
SignalP and other specialized programs. The variant protein is
believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted
because both signal-peptide prediction programs predict that this
protein has a signal peptide, and neither trans-membrane region
prediction program predicts that this protein has a trans-membrane
region.
[0454] Variant protein R11723_PEA.sub.--1_P2 (SEQ ID NO:331) also
has the following non-silent SNPs (Single Nucleotide Polymorphisms)
as listed in Table 9, (given according to their position(s) on the
amino acid sequence, with the alternative amino acid(s) listed; the
last column indicates whether the SNP is known or not; the presence
of known SNPs in variant protein R11723_PEA.sub.--1_P2 (SEQ ID
NO:331) sequence provides support for the deduced sequence of this
variant protein according to the present invention). TABLE-US-00012
TABLE 9 Amino acid mutations SNP position(s) on amino acid
Alternative sequence amino acid(s) Previously known SNP? 107 H
-> P Yes 70 G -> No 70 G -> C No
[0455] Variant protein R11723_PEA.sub.--1_P2 (SEQ ID NO:331) is
encoded by the following transcript(s): R11723_PEA.sub.--1_T6 (SEQ
ID NO:10), for which the sequence(s) is/are given at the end of the
application. The coding portion of transcript R11723_PEA.sub.--1_T6
(SEQ ID NO:10) is shown in bold; this coding portion starts at
position 1716 and ends at position 2051. The transcript also has
the following SNPs as listed in Table 10 (given according to their
position on the nucleotide sequence, with the alternative nucleic
acid listed; the last column indicates whether the SNP is known or
not; the presence of known SNPs in variant protein
R11723_PEA.sub.--1_P2 (SEQ ID NO:331) sequence provides support for
the deduced sequence of this variant protein according to the
present invention). TABLE-US-00013 TABLE 10 Nucleic acid SNPs SNP
position on nucleotide Previously sequence Alternative nucleic acid
known SNP? 1231 C -> T Yes 1278 G -> C Yes 1923 G -> No
1923 G -> T No 2035 A -> C Yes 2048 A -> C No 2057 A ->
G Yes
[0456] Variant protein R11723_PEA.sub.--1_P6 (SEQ ID NO:332)
according to the present invention has an amino acid sequence as
given at the end of the application; it is encoded by transcript(s)
R11723_PEA.sub.--1_T15 (SEQ ID NO:5). One or more alignments to one
or more previously published protein sequences are given at the end
of the application. A brief description of the relationship of the
variant protein according to the present invention to each such
aligned protein is as follows:
[0457] Comparison report between R11723_PEA.sub.--1_P6 (SEQ ID
NO:332) and Q8IXM0 (SEQ ID NO:485):
[0458] 1. An isolated chimeric polypeptide encoding for
R11723_PEA.sub.--1_P6 (SEQ ID NO:332), comprising a first amino
acid sequence being at least 70%, optionally at least 80%,
preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide having the
sequence MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSAGIMYRKSCASSAACLIASAGSPCRGLAPGREEQRALHKAGAVGGGVR (SEQ ID NO:
534) corresponding to amino acids 1-110 of R11723_PEA.sub.--1_P6
(SEQ ID NO:332), and a second amino acid sequence being at least
90% homologous to
MYAQALLVVGVLQRQAAAQHLHEHPPKLLRGHRVQERVDDRAEVEKRLREGEEDHV
RPEVGPRPVVLGFGRSHDPPNLVGHPAYGQCHNNQPWADTSRRERQRKEKHSMRTQ
corresponding to amino acids 1-112 of Q8IXM0, which also
corresponds to amino acids 111-222 of R11723_PEA.sub.--1_P6 (SEQ ID
NO:332), wherein said first and second amino acid sequences are
contiguous and in a sequential order.
[0459] 2. An isolated polypeptide encoding for a head of
R11723_PEA.sub.--1_P6 (SEQ ID NO:332), comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence
TABLE-US-00014 (SEQ ID NO: 534)
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV
QDMCQKEVMEQSAGIMYRKSCASSAACLIASAGSPCRGLAPGREEQRALH KAGAVGGGVR of
(SEQ ID NO: 332) R11723_PEA_1_P6.
[0460] Comparison report between R11723_PEA.sub.--1_P6 (SEQ ID
NO:332) and Q96AC2 (SEQ ID NO:486):
[0461] 1. An isolated chimeric polypeptide encoding for
R11723_PEA.sub.--1_P6 (SEQ ID NO:332), comprising a first amino
acid sequence being at least 90% homologous to
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSAGIMYRKSCASSAACLIASAG corresponding to amino acids 1-83 of
Q96AC2, which also corresponds to amino acids 1-83 of
R11723_PEA.sub.--1_P6 (SEQ ID NO:332), and a second amino acid
sequence being at least 70%, optionally at least 80%, preferably at
least 85%, more preferably at least 90% and most preferably at
least 95% homologous to a polypeptide having the sequence
SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQ
CHNNQPWADTSRRERQRKEKHSMRTQ (SEQ ID NO: 535) corresponding to amino
acids 84-222 of R11723_PEA.sub.--1_P6 (SEQ ID NO:332), wherein said
first and second amino acid sequences are contiguous and in a
sequential order.
[0462] 2. An isolated polypeptide encoding for a tail of
R11723_PEA.sub.--1_P6 (SEQ ID NO:332) comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence
TABLE-US-00015 (SEQ ID NO: 535)
SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHE
HPPKLLRGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSH
DPPNLVGHPAYGQCHNNQPWADTSRRERQRKEKHSMRTQ (SEQ ID NO: 332) in
R11723_PEA_1_P6.
[0463] Comparison report between R11723_PEA.sub.--1_P6 (SEQ ID
NO:332) and Q8N2G4 (SEQ ID NO:487):
[0464] 1. An isolated chimeric polypeptide encoding for
R11723_PEA.sub.--1_P6 (SEQ ID NO:332), comprising a first amino
acid sequence being at least 90% homologous to
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSAGIMYRKSCASSAACLIASAG corresponding to amino acids 1-83 of
Q8N2G4, which also corresponds to amino acids 1-83 of
R11723_PEA.sub.--1_P6 (SEQ ID.NO:332), and a second amino acid
sequence being at least 70%, optionally at least 80%, preferably at
least 85%, more preferably at least 90% and most preferably at
least 95% homologous to a polypeptide having the sequence
SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQ
CHNNQPWADTSRRERQRKEKHSMRTQ (SEQ ID NO: 535) corresponding to amino
acids 84-222 of R11723_PEA.sub.--1_P6 (SEQ ID NO:332), wherein said
first and second amino acid sequences are contiguous and in a
sequential order.
[0465] 2. An isolated polypeptide encoding for a tail of
R11723_PEA.sub.--1_P6 (SEQ ID NO:332) comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence
TABLE-US-00016 (SEQ ID NO: 535)
SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHE
HPPKLLRGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSH
DPPNLVGHPAYGQCHNNQPWADTSRRERQRKEKHSMRTQ (SEQ ID NO: 332) in
R11723_PEA_1_P6
[0466] Comparison report between R11723_PEA.sub.--1_P6 (SEQ ID
NO:332) and BAC85518 (SEQ ID NO:488):
[0467] 1. An isolated chimeric polypeptide encoding for
R11723_PEA.sub.--1_P6 (SEQ ID NO:332), comprising a first amino
acid sequence being at least 90% homologous to
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV
MEQSAGIMYRKSCASSAACLIASAG corresponding to amino acids 24-106 of
BAC85518, which also corresponds to amino acids 1-83 of
R11723_PEA.sub.--1_P6 (SEQ ID NO:332), and a second amino acid
sequence being at least 70%, optionally at least 80%, preferably at
least 85%, more preferably at least 90% and most preferably at
least 95% homologous to a polypeptide having the sequence
SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHEHPPKLL
RGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQ
CHNNQPWADTSRRERQRKEKHSMRTQ (SEQ ID NO: 535) corresponding to amino
acids 84-222 of R11723_PEA.sub.--1_P6 (SEQ ID NO:332), wherein said
first and second amino acid sequences are contiguous and in a
sequential order.
[0468] 2. An isolated polypeptide encoding for a tail of
R11723_PEA.sub.--1_P6 (SEQ ID NO:332), comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence
TABLE-US-00017 (SEQ ID NO: 535)
SPCRGLAPGREEQRALHKAGAVGGGVRMYAQALLVVGVLQRQAAAQHLHE
HPPKLLRGHRVQERVDDRAEVEKRLREGEEDHVRPEVGPRPVVLGFGRSH
DPPNLVGHPAYGQCHNNQPWADTSRRERQRKEKHSMRTQ (SEQ ID NO: 332) in
R11723_PEA_1_P6.
[0469] The location of the variant protein was determined according
to results from a number of different software programs and
analyses, including analyses from SignalP and other specialized
programs. The variant protein is believed to be located as follows
with regard to the cell: secreted. The protein localization is
believed to be secreted because both signal-peptide prediction
programs predict that this protein has a signal peptide, and
neither trans-membrane region prediction program predicts that this
protein has a trans-membrane region.
[0470] Variant protein R11723_PEA.sub.--1_P6 (SEQ ID NO:332) also
has the following non-silent SNPs (Single Nucleotide Polymorphisms)
as listed in Table 11, (given according to their position(s) on the
amino acid sequence, with the alternative amino acid(s) listed; the
last column indicates whether the SNP is known or not; the presence
of known SNPs in variant protein R11723_PEA.sub.--1_P6 (SEQ ID
NO:332) sequence provides support for the deduced sequence of this
variant protein according to the present invention). TABLE-US-00018
TABLE 11 Amino acid mutations SNP position(s) on amino acid
Alternative sequence amino acid(s) Previously known SNP? 180 G
-> No 180 G -> C No 217 H -> P Yes
[0471] Variant protein R11723_PEA.sub.--1_P6 (SEQ ID NO:332) is
encoded by the following transcript(s): R11723_PEA.sub.--1_T15 (SEQ
ID NO:5), for which the sequence(s) is/are given at the end of the
application. The coding portion of transcript
R11723_PEA.sub.--1_T15 (SEQ ID NO:5) is shown in bold; this coding
portion starts at position 434 and ends at position 1099. The
transcript also has the following SNPs as listed in Table 12 (given
according to their position on the nucleotide sequence, with the
alternative nucleic acid listed; the last column indicates whether
the SNP is known or not; the presence of known SNPs in variant
protein R11723_PEA.sub.--1_P6 (SEQ ID NO:332) sequence provides
support for the deduced sequence of this variant protein according
to the present invention). TABLE-US-00019 TABLE 12 Nucleic acid
SNPs SNP position on nucleotide Previously sequence Alternative
nucleic acid known SNP? 971 G -> No 971 G -> T No 1083 A
-> C Yes 1096 A -> C No 1105 A -> G Yes
[0472] Variant protein R11723_PEA_l_P7 (SEQ ID NO:333) according to
the present invention has an amino acid sequence as given at the
end of the application; it is encoded by transcript(s)
R11723_PEA.sub.--1_T17 (SEQ ID NO:6). One or more alignments to one
or more previously published protein sequences are given at the end
of the application. A brief description of the relationship of the
variant protein according to the present invention to each such
aligned protein is as follows:
[0473] Comparison report between R11723_PEA.sub.--1_P7 (SEQ ID
NO:333) and Q96AC2 (SEQ ID NO:486):
[0474] 1. An isolated chimeric polypeptide encoding for
R11723_PEA.sub.--1P7 (SEQ ID NO:333), comprising a first amino acid
sequence being at least 90% homologous to
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV MEQSAG
corresponding to amino acids 1-64 of Q96AC2 (SEQ ID NO:486), which
also corresponds to amino acids 1-64 of R11723_PEA.sub.--1_P7 (SEQ
ID NO:333), and a second amino acid sequence being at least 70%,
optionally at least 80%, preferably at least 85%, more preferably
at least 90% and most preferably at least 95% homologous to a
polypeptide having the sequence SHCVTRLECSGTISAHCNLCLPGSNDHPT (SEQ
ID NO: 536) corresponding to amino acids 65-93 of
R11723_PEA.sub.--1_P7 (SEQ ID NO:333), wherein said first and
second amino acid sequences are contiguous and in a sequential
order.
[0475] 2. An isolated polypeptide encoding for a tail of
R11723_PEA.sub.--1_P7 (SEQ ID NO:333), comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence
SHCVTRLECSGTISAHCNLCLPGSNDHPT (SEQ ID NO: 536) in
R11723_PEA.sub.--1_P7 (SEQ ID NO:333).
[0476] Comparison report between R11723_PEA.sub.--1_P7 (SEQ ID
NO:333) and Q8N2G4 (SEQ ID NO:487):
[0477] 1. An isolated chimeric polypeptide encoding for
R11723_PEA.sub.--1_P7 (SEQ ID NO:333), comprising a first amino
acid sequence being at least 90% homologous to
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV MEQSAG
corresponding to amino acids 1-64 of Q8N2G4 (SEQ ID NO:487), which
also corresponds to amino acids 1-64 of R11723_PEA.sub.--1P7 (SEQ
ID NO:333), and a second amino acid sequence being at least 70%,
optionally at least 80%, preferably at least 85%, more preferably
at least 90% and most preferably at least 95% homologous to a
polypeptide having the sequence SHCVTRLECSGTISAHCNLCLPGSNDHPT (SEQ
ID NO: 536) corresponding to amino acids 65-93 of
R11723_PEA.sub.--1_P7 (SEQ ID NO:333), wherein said first and
second amino acid sequences are contiguous and in a sequential
order.
[0478] 2. An isolated polypeptide encoding for a tail of
R11723_PEA.sub.--1_P7 (SEQ ID NO:333), comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence
SHCVTRLECSGTISAHCNLCLPGSNDHPT (SEQ ID NO: 536) in
R11723_PEA.sub.--1_P7 (SEQ ID NO:333).
[0479] Comparison report between R11723_PEA.sub.--1_P7 (SEQ ID
NO:333) and BAC85273 (SEQ ID NO:489):
[0480] 1. An isolated chimeric polypeptide encoding for
R11723_PEA.sub.--1_P7 (SEQ ID NO:333), comprising a first amino
acid sequence being at least 70%, optionally at least 80%,
preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide having the
sequence MWVLG (SEQ ID NO: 537) corresponding to amino acids 1-5 of
R11723_PEA.sub.--1_P7 (SEQ ID NO:333), second amino acid sequence
being at least 90% homologous to
IAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEVMEQSAG
corresponding to amino acids 22-80 of BAC85273, which also
corresponds to amino acids 6-64 of R11723_PEA.sub.--1_P7 (SEQ ID
NO:333), and a third amino acid sequence being at least 70%,
optionally at least 80%, preferably at least 85%, more preferably
at least 90% and most preferably at least 95% homologous to a
polypeptide having the sequence SHCVTRLECSGTISAHCNLCLPGSNDHPT (SEQ
ID NO: 536) corresponding to amino acids 65-93 of
R11723_PEA.sub.--1_P7 (SEQ ID NO:333), wherein said first, second
and third amino acid sequences are contiguous and in a sequential
order.
[0481] 2. An isolated polypeptide encoding for a head of
R11723_PEA.sub.--1_P7 (SEQ ID NO:333), comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence MWVLG (SEQ
ID NO: 537) of R 1723_PEA.sub.--1_P7 (SEQ ID NO:333).
[0482] 3. An isolated polypeptide encoding for a tail of
R11723_PEA.sub.--1P7 (SEQ ID NO:333), comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence
SHCVTRLECSGTISAHCNLCLPGSNDHPT (SEQ ID NO: 536) in
R11723_PEA.sub.--1_P7 (SEQ ID NO:333).
[0483] Comparison report between R11723_PEA.sub.--1_P7 (SEQ ID
NO:333) and BAC85518 (SEQ ID NO:489):
[0484] 1. An isolated chimeric polypeptide encoding for
R11723_PEA.sub.--1_P7 (SEQ ID NO:333), comprising a first amino
acid sequence being at least 90% homologous to
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV MEQSAG
corresponding to amino acids 24-87 of BAC85518, which also
corresponds to amino acids 1-64 of R11723_PEA.sub.--1_P7 (SEQ ID
NO:333), and a second amino acid sequence being at least 70%,
optionally at least 80%, preferably at least 85%, more preferably
at least 90% and most preferably at least 95% homologous to a
polypeptide having the sequence SHCVTRLECSGTISAHCNLCLPGSNDHPT (SEQ
ID NO: 536) corresponding to amino acids 65-93 of
R11723_PEA.sub.--1_P7 (SEQ ID NO:333), wherein said first and
second amino acid sequences are contiguous and in a sequential
order.
[0485] 2. An isolated polypeptide encoding for a tail of
R11723_PEA.sub.--1_P7 (SEQ ID NO:333), comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence
SHCVTRLECSGTISAHCNLCLPGSNDHPT (SEQ ID NO: 536) in
R11723_PEA.sub.--1_P7 (SEQ ID NO:333).
[0486] The location of the variant protein was determined according
to results from a number of different software programs and
analyses, including analyses from SignalP and other specialized
programs. The variant protein is believed to be located as follows
with regard to the cell: secreted. The protein localization is
believed to be secreted because both signal-peptide prediction
programs predict that this protein has a signal peptide, and
neither trans-membrane region prediction program predicts that this
protein has a trans-membrane region.
[0487] Variant protein R11723_PEA.sub.--1_P7 (SEQ ID NO:333) also
has the following non-silent SNPs (Single Nucleotide Polymorphisms)
as listed in Table 13, (given according to their position(s) on the
amino acid sequence, with the alternative amino acid(s) listed; the
last column indicates whether the SNP is known or not; the presence
of known SNPs in variant protein R11723_PEA.sub.--1_P7 (SEQ ID
NO:333) sequence provides support for the deduced sequence of this
variant protein according to the present invention). TABLE-US-00020
TABLE 13 Amino acid mutations SNP position(s) on amino acid
Alternative sequence amino acid(s) Previously known SNP? 67 C ->
S Yes
[0488] Variant protein R11723_PEA.sub.--1_P7 (SEQ ID NO:333) is
encoded by the following transcript(s): R11723_PEA.sub.--1_T17 (SEQ
ID NO:6), for which the sequence(s) is/are given at the end of the
application. The coding portion of transcript
R11723_PEA.sub.--1_T17 (SEQ ID NO:6) is shown in bold; this coding
portion starts at position 434 and ends at position 712. The
transcript also has the following SNPs as listed in Table 14 (given
according to their position on the nucleotide sequence, with the
alternative nucleic acid listed; the last column indicates whether
the SNP is known or not; the presence of known SNPs in variant
protein R11723_PEA.sub.--1_P7 (SEQ ID NO:333) sequence provides
support for the deduced sequence of this variant protein according
to the present invention). TABLE-US-00021 TABLE 14 Nucleic acid
SNPs SNP position on nucleotide Alternative sequence nucleic acid
Previously known SNP? 625 G -> T Yes 633 G -> C Yes 1303 C
-> T Yes
[0489] Variant protein R11723_PEA.sub.--1_P13 (SEQ ID NO:334)
according to the present invention has an amino acid sequence as
given at the end of the application; it is encoded by transcript(s)
R11723_PEA.sub.--1_T19 (SEQ ID NO:7) and R11723_PEA.sub.--1_T5 (SEQ
ID NO:9). One or more alignments to one or more previously
published protein sequences are given at the end of the
application. A brief description of the relationship of the variant
protein according to the present invention to each such aligned
protein is as follows:
[0490] Comparison report between R11723_PEA.sub.--1_P13 (SEQ ID
NO:334) and Q96AC2:
[0491] 1. An isolated chimeric polypeptide encoding for
R11723_PEA_-1P13 (SEQ ID NO:334) comprising a first amino acid
sequence being at least 90% homologous to
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV MEQSA
corresponding to amino acids 1-63 of Q96AC2, which also corresponds
to amino acids 1-63 of R11723_PEA.sub.--1_P13 (SEQ ID NO:334), and
a second amino acid sequence being at least 70%, optionally at
least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having
the sequence DTKRTNTLLFEMRHFAKQLTT (SEQ ID NO: 538) corresponding
to amino acids 64-84 of R11723_PEA.sub.--1_P13 (SEQ ID NO:334),
wherein said first and second amino acid sequences are contiguous
and in a sequential order.
[0492] 2. An isolated polypeptide encoding for a tail of
R11723_PEA.sub.--1_P13 (SEQ ID NO:334), comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence
DTKRTNTLLFEMRHFAKQLTT (SEQ ID NO: 538) in R11723_PEA.sub.--1_P13
(SEQ ID NO:334).
[0493] The location of the variant protein was determined according
to results from a number of different software programs and
analyses, including analyses from SignalP and other specialized
programs. The variant protein is believed to be located as follows
with regard to the cell: secreted. The protein localization is
believed to be secreted because both signal-peptide prediction
programs predict that this protein has a signal peptide, and
neither trans-membrane region prediction program predicts that this
protein has a trans-membrane region.
[0494] Variant protein R11723_PEA.sub.--1_P13 (SEQ ID NO:334) is
encoded by the following transcript(s): R11723_PEA.sub.--1_T19 (SEQ
ID NO:7), for which the sequence(s) is/are given at the end of the
application. The coding portion of transcript
R11723_PEA.sub.--1_T19 (SEQ ID NO:7) is shown in bold; this coding
portion starts at position 434 and ends at position 685. The
transcript also has the following SNPs as listed in Table 15 (given
according to their position on the nucleotide sequence, with the
alternative nucleic acid listed; the last column indicates whether
the SNP is known or not; the presence of known SNPs in variant
protein R11723_PEA.sub.--1_P13 (SEQ ID NO:334) sequence provides
support for the deduced sequence of this variant protein according
to the present invention). TABLE-US-00022 TABLE 15 Nucleic acid
SNPs SNP position on nucleotide Alternative sequence nucleic acid
Previously known SNP? 778 G -> T Yes 786 G -> C Yes 1456 C
-> T Yes
[0495] Variant protein R11723_PEA.sub.--1_P10 (SEQ ID NO:335)
according to the present invention has an amino acid sequence as
given at the end of the application; it is encoded by transcript(s)
R11723_PEA.sub.--1_T20 (SEQ ID NO:8). One or more alignments to one
or more previously published protein sequences are given at the end
of the application. A brief description of the relationship of the
variant protein according to the present invention to each such
aligned protein is as follows:
[0496] Comparison report between R11723_PEA.sub.--1_P10 (SEQ ID
NO:335) and Q96AC2:
[0497] 1. An isolated chimeric polypeptide encoding for
R11723_PEA.sub.--1P10 (SEQ ID NO:335), comprising a first amino
acid sequence being at least 90% homologous to
MWVLGLAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV MEQSA
corresponding to amino acids 1-63 of Q96AC2, which also corresponds
to amino acids 1-63 of R11723_PEA_lP10 (SEQ ID NO:335), and a
second amino acid sequence being at least 70%, optionally at least
80%, preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide having the
sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK (SEQ ID NO: 539) corresponding
to amino acids 64-90 of R11723_PEA.sub.--1_P10 (SEQ ID NO:335),
wherein said first and second amino acid sequences are contiguous
and in a sequential order.
[0498] 2. An isolated polypeptide encoding for a tail of
R11723_PEA.sub.--1_P10 (SEQ ID NO:335), comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence
DRVSLCHEAGVQWNNFSTLQPLPPRLK (SEQ ID NO: 539) in
R11723_PEA.sub.--1_P10 (SEQ ID NO:335).
[0499] Comparison report between R11723_PEA.sub.--1_P10 (SEQ ID
NO:335) and Q8N2G4 (SEQ ID NO:487):
[0500] 1. An isolated chimeric polypeptide encoding for
R11723_PEA.sub.--1_P10 (SEQ ID NO:335) comprising a first amino
acid sequence being at least 90% homologous to
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV MEQSA
corresponding to amino acids 1-63 of Q8N2G4, which also corresponds
to amino acids 1-63 of R11723_PEA.sub.--1P10 (SEQ ID NO:335), and a
second amino acid sequence being at least 70%, optionally at least
80%, preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide having the
sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK (SEQ ID NO: 539) corresponding
to amino acids 64-90 of R11723_PEA.sub.--1_P10 (SEQ ID NO:335),
wherein said first and second amino acid sequences are contiguous
and in a sequential order.
[0501] 2. An isolated polypeptide encoding for a tail of
R11723_PEA.sub.--1_P10 (SEQ ID NO:335), comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence
DRVSLCHEAGVQWNNFSTLQPLPPRLK (SEQ ID NO: 539) in
R11723_PEA.sub.--1_P10 (SEQ ID NO:335).
[0502] Comparison report between R11723_PEA.sub.--1_P10 (SEQ ID
NO:335) and BAC85273:
[0503] 1. An isolated chimeric polypeptide encoding for
R11723_PEA.sub.--1_P10 (SEQ ID NO:335), comprising a first amino
acid sequence being at least 70%, optionally at least 80%,
preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide having the
sequence MWVLG (SEQ ID NO: 537) corresponding to amino acids 1-5 of
R11723_PEA.sub.--1 P10 (SEQ ID NO:335), second amino acid sequence
being at least 90% homologous to
IAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEVMEQSA
corresponding to amino acids 22-79 of BAC85273, which also
corresponds to amino acids 6-63 of R11723_PEA.sub.--1P10 (SEQ ID
NO:335), and a third amino acid sequence being at least 70%,
optionally at least 80%, preferably at least 85%, more preferably
at least 90% and most preferably at least 95% homologous to a
polypeptide having the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK (SEQ ID
NO: 539) corresponding to amino acids 64-90 of
R11723_PEA.sub.--1_P10 (SEQ ID NO:335), wherein said first, second
and third amino acid sequences are contiguous and in a sequential
order.
[0504] 2. An isolated polypeptide encoding for a head of
R11723_PEA.sub.--1_P10 (SEQ ID NO:335), comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence MWVLG (SEQ
ID NO: 537) of R11723_PEA.sub.--1_P10 (SEQ ID NO:335).
[0505] 3. An isolated polypeptide encoding for a tail of
R11723_PEA.sub.--1_P10 (SEQ ID NO:335), comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence
DRVSLCHEAGVQWNNFSTLQPLPPRLK (SEQ ID NO: 539) in
R11723_PEA.sub.--1_P10 (SEQ ID NO:335).
[0506] Comparison report between R11723_PEA.sub.--1_P10 (SEQ ID
NO:335) and BAC85518:
[0507] 1. An isolated chimeric polypeptide encoding for
R11723_PEA.sub.--1P10 (SEQ ID NO:335) comprising a first amino acid
sequence being at least 90% homologous to
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEV MEQSA
corresponding to amino acids 24-86 of BAC85518, which also
corresponds to amino acids 1-63 of R11723_PEA.sub.--1P10 (SEQ ID
NO:335), and a second amino acid sequence being at least 70%,
optionally at least 80%, preferably at least 85%, more preferably
at least 90% and most preferably at least 95% homologous to a
polypeptide having the sequence DRVSLCHEAGVQWNNFSTLQPLPPRLK (SEQ ID
NO: 539) corresponding to amino acids 64-90 of
R11723_PEA.sub.--1_P10 (SEQ ID NO:335), wherein said first and
second amino acid sequences are contiguous and in a sequential
order.
[0508] 2. An isolated polypeptide encoding for a tail of
R11723_PEA.sub.--1_P10 (SEQ ID NO:335), comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence
DRVSLCHEAGVQWNNFSTLQPLPPRLK (SEQ ID NO: 539) in
R11723_PEA.sub.--1_P10 (SEQ ID NO:335).
[0509] The location of the variant protein was determined according
to results from a number of different software programs and
analyses, including analyses from SignalP and other specialized
programs. The variant protein is believed to be located as follows
with regard to the cell: secreted. The protein localization is
believed to be secreted because both signal-peptide prediction
programs predict that this protein has a signal peptide, and
neither trans-membrane region prediction program predicts that this
protein has a trans-membrane region.
[0510] Variant protein R11723_PEA.sub.--1_P10 (SEQ ID NO:335) also
has the following non-silent SNPs (Single Nucleotide Polymorphisms)
as listed in Table 16, (given according to their position(s) on the
amino acid sequence, with the alternative amino acid(s) listed; the
last column indicates whether the SNP is known or not; the presence
of known SNPs in variant protein R11723_PEA.sub.--1_P10 (SEQ ID
NO:335) sequence provides support for the deduced sequence of this
variant protein according to the present invention). TABLE-US-00023
TABLE 16 Amino acid mutations SNP position(s) on amino acid
Alternative sequence amino acid(s) Previously known SNP? 66 V ->
F Yes
[0511] Variant protein R11723_PEA.sub.--1_P10 (SEQ ID NO:335) is
encoded by the following transcript(s): R11723_PEA.sub.--1_T20 (SEQ
ID NO:8), for which the sequence(s) is/are given at the end of the
application. The coding portion of transcript
R11723_PEA.sub.--1_T20 (SEQ ID NO:8) is shown in bold; this coding
portion starts at position 434 and ends at position 703. The
transcript also has the following SNPs as listed in Table 17 (given
according to their position on the nucleotide sequence, with the
alternative nucleic acid listed; the last column indicates whether
the SNP is known or not; the presence of known SNPs in variant
protein R11723_PEA.sub.--1_P10 (SEQ ID NO:335) sequence provides
support for the deduced sequence of this variant protein according
to the present invention). TABLE-US-00024 TABLE 17 Nucleic acid
SNPs SNP position on nucleotide Alternative sequence nucleic acid
Previously known SNP? 629 G -> T Yes 637 G -> C Yes 1307 C
-> T Yes
[0512] As noted above, cluster R11723 features 26 segment(s), which
were listed in Table 49 above and for which the sequence(s) are
given at the end of the application. These segment(s) are portions
of nucleic acid sequence(s) which are described herein separately
because they are of particular interest. A description of each
segment according to the present invention is now provided.
[0513] Segment cluster R11723_PEA.sub.--1_node.sub.--13 (SEQ ID
NO:90) according to the present invention is supported by 5
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1IT19 (SEQ ID NO:7),
R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1_T6 (SEQ
ID NO:10). Table 18 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00025 TABLE
18 Segment location on transcripts Segment Segment Transcript name
starting position ending position R11723_PEA_1_T19 (SEQ 624 776 ID
NO: 7) R11723_PEA_1_T5 (SEQ ID 624 776 NO: 9) R11723_PEA_1_T6 (SEQ
ID 658 810 NO: 10)
[0514] Segment cluster R11723_PEA.sub.--1_node.sub.--16 (SEQ ID
NO:91) according to the present invention is supported by 3
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1IT17 (SEQ ID NO:6),
R11723_PEA.sub.--1_T19 (SEQ ID NO:7) and R11723_PEA.sub.--1_T20
(SEQ ID NO:8). Table 19 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00026 TABLE
19 Segment location on transcripts Segment Segment Transcript name
starting position ending position R11723_PEA_1_T17 (SEQ 624 1367 ID
NO: 6) R11723_PEA_1_T19 (SEQ 777 1520 ID NO: 7) R11723_PEA_1_T20
(SEQ 628 1371 ID NO: 8)
[0515] Segment cluster R11723_PEA.sub.--1_node.sub.--19 (SEQ ID
NO:92) according to the present invention is supported by 45
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and
R11723_PEA.sub.--1_T6 (SEQ ID NO:10). Table 20 below describes the
starting and ending position of this segment on each transcript.
TABLE-US-00027 TABLE 20 Segment location on transcripts Segment
Segment Transcript name starting position ending position
R11723_PEA_1_T5 (SEQ ID 835 1008 NO: 9) R11723_PEA_1_T6 (SEQ ID 869
1042 NO: 10)
[0516] Segment cluster R11723_PEA.sub.--1_node.sub.--2 (SEQ ID
NO:93) according to the present invention is supported by 29
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723_PEA.sub.--1_T17 (SEQ ID NO:6), R11723_PEA.sub.--1_T19 (SEQ
ID NO:7) R11723_PEA.sub.--1_T20 (SEQ ID NO:8),
R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1_T6 (SEQ
ID NO:10). Table 21 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00028 TABLE
21 Segment location on transcripts Segment Segment Transcript name
starting position ending position R11723_PEA_1_T15 (SEQ 1 309 ID
NO: 5) R11723_PEA_1_T17 (SEQ 1 309 ID NO: 6) R11723_PEA_1_T19 (SEQ
1 309 ID NO: 7) R11723_PEA_1_T20 (SEQ 1 309 ID NO: 8)
R11723_PEA_1_T5 (SEQ ID 1 309 NO: 9) R11723_PEA_1_T6 (SEQ ID 1 309
NO: 10)
[0517] Segment cluster R11723_PEA.sub.--1_node.sub.--22 (SEQ ID
NO:94) according to the present invention is supported by 65
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1T5 (SEQ ID NO:9) and
R11723_PEA.sub.--1_T6 (SEQ ID NO: 10). Table 22 below describes the
starting and ending position of this segment on each transcript.
TABLE-US-00029 TABLE 22 Segment location on transcripts Segment
Segment Transcript name starting position ending position
R11723_PEA_1_T5 (SEQ ID 1083 1569 NO: 9) R11723_PEA_1_T6 (SEQ ID
1117 1603 NO: 10)
[0518] Segment cluster R11723_PEA.sub.--1_node.sub.--31 (SEQ ID
NO:95) according to the present invention is supported by 70
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1IT6 (SEQ
ID NO:10). Table 23 below describes the starting and ending
position of this segment on each transcript (it should be noted
that these transcripts show alternative polyadenylation).
TABLE-US-00030 TABLE 23 Segment location on transcripts Segment
starting Segment ending Transcript name position position
R11723_PEA_1_T15 (SEQ 1060 1295 ID NO: 5) R11723_PEA_1_T5 (SEQ ID
1978 2213 NO: 9) R11723_PEA_1_T6 (SEQ ID 2012 2247 NO: 10)
[0519] According to an optional embodiment of the present
invention, short segments related to the above cluster are also
provided. These segments are up to about 120 bp in length, and so
are included in a separate description.
[0520] Segment cluster R11723_PEA.sub.--1_node.sub.--10 (SEQ ID
NO:96) according to the present invention is supported by 38
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1T15 (SEQ ID NO:5),
R11723_PEA.sub.--1_T17 (SEQ ID NO:6), R11723_PEA.sub.--1_T19 (SEQ
ID NO:7), R11723_PEA.sub.--1_T20 (SEQ ID NO:8),
R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and
[0521] R11723PEA.sub.--1_T6 (SEQ ID NO:10). Table 24 below
describes the starting and ending position of this segment on each
transcript. TABLE-US-00031 TABLE 24 Segment location on transcripts
Segment starting Segment ending Transcript name position position
R11723_PEA_1_T15 (SEQ 486 529 ID NO: 5) R11723_PEA_1_T17 (SEQ 486
529 ID NO: 6) R11723_PEA_1_T19 (SEQ 486 529 ID NO: 7)
R11723_PEA_1_T20 (SEQ 486 529 ID NO: 8) R11723_PEA_1_T5 (SEQ ID 486
529 NO: 9) R11723_PEA_1_T6 (SEQ ID 520 563 NO: 10)
[0522] Segment cluster R11723_PEA.sub.--1_node.sub.--11 (SEQ ID
NO:97) according to the present invention is supported by 42
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723_PEA.sub.--1_T17 (SEQ ID NO:6), R11723_PEA.sub.--1_T19 (SEQ
ID NO:7), R11723_PEA.sub.--1_T20 (SEQ ID NO:8),
R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1_T6 (SEQ
ID NO:10). Table 25 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00032 TABLE
25 Segment location on transcripts Segment starting Segment ending
Transcript name position position R11723_PEA_1_T15 (SEQ 530 623 ID
NO: 5) R11723_PEA_1_T17 (SEQ 530 623 ID NO: 6) R11723_PEA_1_T19
(SEQ 530 623 ID NO: 7) R11723_PEA_1_T20 (SEQ 530 623 ID NO: 8)
R11723_PEA_1_T5 (SEQ ID 530 623 NO: 9) R11723_PEA_1_T6 (SEQ ID 564
657 NO: 10)
[0523] Segment cluster R11723_PEA.sub.--1_node.sub.--15 (SEQ ID
NO:98) according to the present invention can be found in the
following transcript(s): R11723_PEA.sub.--1_T20 (SEQ ID NO:8).
Table 26 below describes the starting and ending position of this
segment on each transcript. TABLE-US-00033 TABLE 26 Segment
location on transcripts Segment starting Segment ending Transcript
name position position R11723_PEA_1_T20 (SEQ 624 627 ID NO: 8)
[0524] Segment cluster R11723_PEA.sub.--1_node.sub.--18 (SEQ ID
NO:99) according to the present invention is supported by 40
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723-PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1_T6 (SEQ
ID NO:10). Table 27 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00034 TABLE
27 Segment location on transcripts Segment starting Segment ending
Transcript name position position R11723_PEA_1_T15 (SEQ 624 681 ID
NO: 5) R11723_PEA_1_T5 (SEQ ID 777 834 NO: 9) R11723_PEA_1_T6 (SEQ
ID 811 868 NO: 10)
[0525] Segment cluster R11723_PEA.sub.--1_node.sub.--20 (SEQ ID
NO:100) according to the present invention can be found in the
following transcript(s): R11723_PEA.sub.--1_T5 (SEQ ID NO:9)
and
[0526] R11723_PEA.sub.--1T6 (SEQ ID NO:10). Table 28 below
describes the starting and ending position of this segment on each
transcript. TABLE-US-00035 TABLE 28 Segment location on transcripts
Segment starting Segment ending Transcript name position position
R11723_PEA_1_T5 (SEQ ID 1009 1019 NO: 9) R11723_PEA_1_T6 (SEQ ID
1043 1053 NO: 10)
[0527] Segment cluster R11723_PEA.sub.--1_node.sub.--21 (SEQ ID
NO:101) according to the present invention is supported by 36
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1T5 (SEQ ID NO:9) and
R11723_PEA.sub.--1_T6 (SEQ ID NO:10). Table 29 below describes the
starting and ending position of this segment on each transcript.
TABLE-US-00036 TABLE 29 Segment location on transcripts Segment
starting Segment ending Transcript name position position
R11723_PEA_1_T5 (SEQ ID 1020 1082 NO: 9) R11723_PEA_1_T6 (SEQ ID
1054 1116 NO: 10)
[0528] Segment cluster R11723_PEA.sub.--1_node.sub.--23 (SEQ ID NO:
102) according to the present invention is supported by 39
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and
R11723_PEA.sub.--1T6 (SEQ ID NO:10). Table 30 below describes the
starting and ending position of this segment on each transcript.
TABLE-US-00037 TABLE 30 Segment location on transcripts Segment
starting Segment ending Transcript name position position
R11723_PEA_1_T5 (SEQ ID 1570 1599 NO: 9) R11723_PEA_1_T6 (SEQ ID
1604 1633 NO: 10)
[0529] Segment cluster R11723_PEA.sub.--1_node.sub.--24 (SEQ ID
NO:103) according to the present invention is supported by 51
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1IT6 (SEQ
ID NO:10). Table 31 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00038 TABLE
31 Segment location on transcripts Segment starting Segment ending
Transcript name position position R11723_PEA_1_T15 (SEQ 682 765 ID
NO: 5) R11723_PEA_1_T5 (SEQ ID 1600 1683 NO: 9) R11723_PEA_1_T6
(SEQ ID 1634 1717 NO: 10)
[0530] Segment cluster R11723_PEA.sub.--1_node.sub.--25 (SEQ ID NO:
104) according to the present invention is supported by 54
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1_T6 (SEQ
ID NO:10). Table 32 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00039 TABLE
32 Segment location on transcripts Segment starting Segment ending
Transcript name position position R11723_PEA_1_T15 (SEQ 766 791 ID
NO: 5) R11723_PEA_1_T5 (SEQ ID 1684 1709 NO: 9) R11723_PEA_1_T6
(SEQ ID 1718 1743 NO: 10)
[0531] Segment cluster R11723_PEA.sub.--1_node.sub.--26 (SEQ ID
NO:105) according to the present invention is supported by 62
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1_T6 (SEQ
ID NO:10). Table 33 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00040 TABLE
33 Segment location on transcripts Segment starting Segment ending
Transcript name position position R11723_PEA_1_T15 (SEQ 792 904 ID
NO: 5) R11723_PEA_1_T5 (SEQ ID 1710 1822 NO: 9) R11723_PEA_1_T6
(SEQ ID 1744 1856 NO: 10)
[0532] Segment cluster R11723_PEA.sub.--1_node 27 (SEQ ID NO: 106)
according to the present invention is supported by 67 libraries.
The number of libraries was determined as previously described.
This segment can be found in the following transcript(s):
R11723_PEA.sub.--1_T15 (SEQ ID NO:5), R11723_PEA.sub.--1_T5 (SEQ ID
NO:9) and R11723_PEA.sub.--1IT6 (SEQ ID NO:10) Table 34 below
describes the starting and ending position of this segment on each
transcript. TABLE-US-00041 TABLE 34 Segment location on transcripts
Segment starting Segment ending Transcript name position position
R11723_PEA_1_T15 (SEQ 905 986 ID NO: 5) R11723_PEA_1_T5 (SEQ ID
1823 1904 NO: 9) R11723_PEA_1_T6 (SEQ ID 1857 1938 NO: 10)
[0533] Segment cluster R11723_PEA.sub.--1_node.sub.--28 (SEQ ID
NO:107) according to the present invention can be found in the
following transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1IT6 (SEQ
ID NO:10). Table 35 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00042 TABLE
35 Segment location on transcripts Segment starting Segment ending
Transcript name position position R11723_PEA_1_T15 (SEQ 987 1010 ID
NO: 5) R11723_PEA_1_T5 (SEQ ID 1905 1928 NO: 9) R11723_PEA_1_T6
(SEQ ID 1939 1962 NO: 10)
[0534] Segment cluster R11723_PEA.sub.--1_node.sub.--29 (SEQ ID
NO:108) according to the present invention is supported by 69
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723-PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1_T6 (SEQ
ID NO:10). Table 36 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00043 TABLE
36 Segment location on transcripts Segment starting Segment ending
Transcript name position position R11723_PEA_1_T15 (SEQ 1011 1038
ID NO: 5) R11723_PEA_1_T5 (SEQ ID 1929 1956 NO: 9) R11723_PEA_1_T6
(SEQ ID 1963 1990 NO: 10)
[0535] Segment cluster R11723_PEA.sub.--1_node.sub.--3 (SEQ ID
NO:109) according to the present invention can be found in the
following transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723_PEA.sub.--1_T17 (SEQ ID NO:6), R11723_PEA.sub.--1_T19 (SEQ
ID NO:7), R11723_PEA.sub.--1_T20 (SEQ ID NO:8),
R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1_T6 (SEQ
ID NO:10). Table 37 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00044 TABLE
37 Segment location on transcripts Segment starting Segment ending
Transcript name position position R11723_PEA_1_T15 (SEQ 310 319 ID
NO: 5) R11723_PEA_1_T17 (SEQ 310 319 ID NO: 6) R11723_PEA_1_T19
(SEQ 310 319 ID NO: 7) R11723_PEA_1_T20 (SEQ 310 319 ID NO: 8)
R11723_PEA_1_T5 (SEQ ID 310 319 NO: 9) R11723_PEA_1_T6 (SEQ ID 310
319 NO: 10)
[0536] Segment cluster R11723_PEA.sub.--1_node.sub.--30 (SEQ ID NO:
110) according to the present invention can be found in the
following transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1T6 (SEQ
ID NO:10). Table 38 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00045 TABLE
38 Segment location on transcripts Segment starting Segment ending
Transcript name position position R11723_PEA_1_T15 (SEQ 1039 1059
ID NO: 5) R11723_PEA_1_T5 (SEQ ID 1957 1977 NO: 9) R11723_PEA_1_T6
(SEQ ID 1991 2011 NO: 10)
[0537] Segment cluster R11723_PEA.sub.--1_node.sub.--4 (SEQ ID
NO:111) according to the present invention is supported by 25
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723_PEA.sub.--1_T17 (SEQ ID NO:6), R11723_PEA.sub.--1_T19 (SEQ
ID NO:7) R11723_PEA.sub.--1_T20 (SEQ ID NO:8),
R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1_T6 (SEQ
ID NO:10). Table 39 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00046 TABLE
39 Segment location on transcripts Segment starting Segment ending
Transcript name position position R11723_PEA_1_T15 (SEQ 320 371 ID
NO: 5) R11723_PEA_1_T17 (SEQ 320 371 ID NO: 6) R11723_PEA_1_T19
(SEQ 320 371 ID NO: 7) R11723_PEA_1_T20 (SEQ 320 371 ID NO: 8)
R11723_PEA_1_T5 (SEQ ID 320 371 NO: 9) R11723_PEA_1_T6 (SEQ ID 320
371 NO: 10)
[0538] Segment cluster R11723_PEA.sub.--1_node.sub.--5 (SEQ ID
NO:112) according to the present invention is supported by 26
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723_PEA.sub.--1_T17 (SEQ ID NO:6), R11723_PEA.sub.--1T19 (SEQ ID
NO:7), R11723_PEA.sub.--1_T20 (SEQ ID NO:8), R11723_PEA.sub.--1_T5
(SEQ ID NO:9) and R11723PEA.sub.--1T6 (SEQ ID NO:10). Table 40
below describes the starting and ending position of this segment on
each transcript. TABLE-US-00047 TABLE 40 Segment location on
transcripts Segment starting Segment ending Transcript name
position position R11723_PEA_1_T15 (SEQ 372 414 ID NO: 5)
R11723_PEA_1_T17 (SEQ 372 414 ID NO: 6) R11723_PEA_1_T19 (SEQ 372
414 ID NO: 7) R11723_PEA_1_T20 (SEQ 372 414 ID NO: 8)
R11723_PEA_1_T5 (SEQ ID 372 414 NO: 9) R11723_PEA_1_T6 (SEQ ID 372
414 NO: 10)
[0539] Segment cluster R11723_PEA.sub.--1_node.sub.--6 (SEQ ID
NO:113) according to the present invention is supported by 27
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723_PEA.sub.--1_T17 (SEQ ID NO:6), R11723_PEA.sub.--1_T19 (SEQ
ID NO:7), R11723_PEA.sub.--1_T20 (SEQ ID NO:8),
R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1_T6 (SEQ
ID NO:10). Table 41 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00048 TABLE
41 Segment location on transcripts Segment starting Segment ending
Transcript name position position R11723_PEA_1_T15 (SEQ 415 446 ID
NO: 5) R11723_PEA_1_T17 (SEQ 415 446 ID NO: 6) R11723_PEA_1_T19
(SEQ 415 446 ID NO: 7) R11723_PEA_1_T20 (SEQ 415 446 ID NO: 8)
R11723_PEA_1_T5 (SEQ ID 415 446 NO: 9) R11723_PEA_1_T6 (SEQ ID 415
446 NO: 10)
[0540] Segment cluster R11723_PEA.sub.--1_node.sub.--7 (SEQ ID
NO:114) according to the present invention is supported by 29
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1_T15 (SEQ ID NO:5),
R11723_PEA.sub.--1_T17 (SEQ ID NO:6), R11723_PEA.sub.--1_T19 (SEQ
ID NO:7), R11723_PEA.sub.--1_T20 (SEQ ID NO:8),
R11723_PEA.sub.--1_T5 (SEQ ID NO:9) and R11723_PEA.sub.--1_T6 (SEQ
ID NO:10). Table 42 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00049 TABLE
42 Segment location on transcripts Segment starting Segment ending
Transcript name position position R11723_PEA_1_T15 (SEQ 447 485 ID
NO: 5) R11723_PEA_1_T17 (SEQ 447 485 ID NO: 6) R11723_PEA_1_T19
(SEQ 447 485 ID NO: 7) R11723_PEA_1_T20 (SEQ 447 485 ID NO: 8)
R11723_PEA_1_T5 (SEQ ID 447 485 NO: 9) R11723_PEA_1_T6 (SEQ ID 447
485 NO: 10)
[0541] Segment cluster R11723_PEA.sub.--1_node.sub.--8 (SEQ ID
NO:115) according to the present invention is supported by 2
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): R11723_PEA.sub.--1T6 (SEQ ID NO: 10). Table 43 below
describes the starting and ending position of this segment on each
transcript. TABLE-US-00050 TABLE 43 Segment location on transcripts
Segment Segment ending Transcript name starting position position
R11723_PEA_1_T6 (SEQ ID 486 519 NO: 10)
[0542] It should be noted that the variants of this cluster are
variants of the hypothetical protein PSEC0181 (referred to herein
as "PSEC"). Furthermore, use of the known protein (WT protein) for
detection of ovarian cancer, alone or in combination with one or
more variants of this cluster and/or of any other cluster and/or of
any known marker, also comprises an embodiment of the present
invention. It should be noted that the nucleotide transcript
sequence of known protein (PSEC, also referred to herein as the
"wild type" or WT protein) features at least one SNP that appears
to affect the coding region, in addition to certain silent SNPs.
This SNP does not have an effect on the R11723_PEA.sub.--1_T5 (SEQ
ID NO:9) splice variant sequence): "G->" resulting in a missing
nucleotide (affects amino acids from position 91 onwards). The
missing nucleotide creates a frame shift, resulting in a new
protein. This SNP was not previously identified and is supported by
5 ESTs out of .about.70 ESTs in this exon. TABLE-US-00051 Variant
protein alignment to the previously known protein: Sequence name:
/tmp/gp6eQTLWqk/mFtjupUzhb: Q8IXM0 Sequence documentation:
Alignment of: R11723_PEA_1_P6 (SEQ ID NO: 332) .times. Q8IXM0 . .
Alignment segment 1/1: Quality: 1128.00 Escore: 0 Matching length:
112 Total length: 112 Matching Percent 100.00 Matching Percent
100.00 Similarity: Identity: Total Percent 100.00 Total Percent
100.00 Similarity: Identity: Gaps: 0 Alignment: . . . . . 111
MYAQALLVVGVLQRQAAAQHLHEHPPKLLRGHRVQERVDDRAEVEKRLRE 160
|||||||||||||||||||||||||||||||||||||||||||||||||| 1
MYAQALLVVGVLQRQAAAQHLHEHPPKLLRGHRVQERVDDRAEVEKRLRE 50 . . . . . 161
GEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQCHNNQPWADTSRRE 210
|||||||||||||||||||||||||||||||||||||||||||||||||| 51
GEEDHVRPEVGPRPVVLGFGRSHDPPNLVGHPAYGQCHNNQPWADTSRRE 100 . 211
RQRKEKHSMRTQ 222 |||||||||||| 101 RQRKEKHSMRTQ 112 Sequence name:
/tmp/gp6eQTLWqk/mFtjUpUzhb: Q96AC2 Sequence documentation:
Alignment of: R11723_PEA_1_P6 (SEQ ID NO: 332) .times. Q96AC2 . .
Alignment segment 1/1: Quality: 835.00 Escore: 0 Matching length:
83 Total length: 83 Matching Percent 100.00 Matching Percent 100.00
Similarity: Identity: Total Percent 100.00 Total Percent 100.00
Similarity: Identity: Gaps: 0 Alignment: . . . . . 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50 . . . 51
QDMCQKEVMEQSAGIMYRKSCASSAACLIASAG 83
||||||||||||||||||||||||||||||||| 51
QDMCQKEVMEQSAGIMYRKSCASSAACLIASAG 83 Sequence name:
/tmp/gp6eQTLWqk/mFtjUpUzhb: Q8N2G4 Sequence documentation:
Alignment of: R11723_PEA_1_P6 (SEQ ID NO: 332) .times. Q8N2G4 . .
Alignment segment 1/1: Quality: 835.00 Escore: 0 Matching length:
83 Total length: 83 Matching Percent 100.00 Matching Percent 100.00
Similarity: Identity: Total Percent 100.00 Total Percent 100.00
Similarity: Identity: Gaps: 0 Alignment: . . . . . 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50 . . . 51
QDMCQKEVMEQSAGIMYRKSCASSAACLIASAG 83
||||||||||||||||||||||||||||||||| 51
QDMCQKEVMEQSAGIMYRKSCASSAACLIASAG 83 Sequence name:
/tmp/gp6eQTLWqk/mFtjUpUzhb: BAC85518 Sequence documentation:
Alignment of: R11723_PEA_1_P6 (SEQ ID NO: 332) .times. BAC85518 . .
Alignment segment 1/1: Quality: 835.00 Escore: 0 Matching length:
83 Total length: 83 Matching Percent 100.00 Matching Percent 100.00
Similarity: Identity: Total Percent 100.00 Total Percent 100.00
Similarity: Identity: Gaps: 0 Alignment: . . . . . 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 24
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 73 . . . 51
QDMCQKEVMEQSAGIMYRKSCASSAACLIASAG 83
||||||||||||||||||||||||||||||||| 74
QDMCQKEVMEQSAGIMYRKSCASSAACLIASAG 106 Sequence name:
/tmp/VXjdFlzdBX/bexTxTh0Th: Q96AC2 Sequence documentation:
Alignment of: R11723_PEA_1_P7 (SEQ ID NO: 333) .times. Q96AC2 . .
Alignment segment 1/1: Quality: 654.00 Escore: 0 Matching length:
64 Total length: 64 Matching Percent 100.00 Matching Percent 100.00
Similarity: Identity: Total Percent 100.00 Total Percent 100.00
Similarity: Identity: Gaps: 0 Alignment: . . . . . 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50 . 51
QDMCQKEVMEQSAG 64 |||||||||||||| 51 QDMCQKEVMEQSAG 64 Sequence
name: /tmp/VXjdFlzdBX/bexTxTh0Th: Q8N2G4 Sequence documentation:
Alignment of: R11723_PEA_1_P7 (SEQ ID NO: 333) .times. Q8N2G4 . .
Alignment segment 1/1: Quality: 654.00 Escore: 0 Matching length:
64 Total length: 64 Matching Percent 100.00 Matching Percent 100.00
Similarity: Identity: Total Percent 100.00 Total Percent 100.00
Similarity: Identity: Gaps: 0 Alignment: . . . . . 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50 . 51
QDMCQKEVMEQSAG 64 |||||||||||||| 51 QDMCQKEVMEQSAG 64 Sequence
name: /tmp/VXjdFlzdBX/bexTxm0Th: BAC85273 Sequence documentation:
Alignment of: R11723_PEA_1_P7 (SEQ ID NO: 333) .times. BAC85273 . .
Alignment segment 1/1: Quality: 600.00 Escore: 0 Matching length:
59 Total length: 59 Matching Percent 100.00 Matching Percent 100.00
Similarity: Identity: Total Percent 100.00 Total Percent 100.00
Similarity: Identity: Gaps: 0 Alignment: . . . . . 6
IAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQ 55
|||||||||||||||||||||||||||||||||||||||||||||||||| 22
IAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQ 71 . 56
KEVMEQSAG 64 |||||||||
72 KEVMEQSAG 80 Sequence name: /tmp/VXjdFlzdBX/bexTxTh0Th: BACS5518
Sequence documentation: Alignment of: R11723_PEA_1_P7 (SEQ ID NO:
333) .times. BAC85518 . . Alignment segment 1/1: Quality: 654.00
Escore: 0 Matching length: 64 Total length: 64 Matching Percent
100.00 Matching Percent 100.00 Similarity: Identity: Total Percent
100.00 Total Percent 100.00 Similarity: Identity: Gaps: 0
Alignment: . . . . . 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 24
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 73 . 51
QDMCQKEVMEQSAG 64 |||||||||||||| 74 QDMCQKEVMEQSAG 87 Sequence
name: /tmp/OLMSexEmIh/pc7Z7Xm1YR: Q96AC2 Sequence documentation:
Alignment of: R11723_PEA_1_P10 (SEQ ID NO: 335) .times. Q96AC2 . .
Alignment segment 1/1: Quality: 645.00 Escore: 0 Matching length:
63 Total length: 63 Matching Percent 100.00 Matching Percent 100.00
Similarity: Identity: Total Percent 100.00 Total Percent 100.00
Similarity: Identity: Gaps: 0 Alignment: . . . . . 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50 . 51
QDMCQKEVMEQSA 63 ||||||||||||| 51 QDMCQKEVMEQSA 63 Sequence name:
/tmp/OLMSexEmIh/pc7Z7Xm1YR: Q8N2G4 Sequence documentation:
Alignment of: R11723_PEA_1_P10 (SEQ ID NO: 335) .times. Q8N2G4 . .
Alignment segment 1/1: Quality: 645.00 Escore: 0 Matching length:
63 Total length: 63 Matching Percent 100.00 Matching Percent 100.00
Similarity: Identity: Total Percent 100.00 Total Percent 100.00
Similarity: Identity: Gaps: 0 Alignment: . . . . . 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50 . 51
QDMCQKEVMEQSA 63 ||||||||||||| 51 QDMCQKEVMEQSA 63 Sequence name:
/tmp/OLMSexEmIh/pc7Z7Xm1YR: BAC85273 Sequence documentation:
Alignment of: R11723_PEA_1_P10 (SEQ ID NO: 335) .times. BAC85273 .
. Alignment segment 1/1: Quality: 591.00 Escore: 0 Matching length:
58 Total length: 58 Matching Percent 100.00 Matching Percent 100.00
Similarity: Identity: Total Percent 100.00 Total Percent 100.00
Similarity: Identity: Gaps: 0 Alignment: . . . . . 6
IAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQ 55
|||||||||||||||||||||||||||||||||||||||||||||||||| 22
IAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQ 71 56 KEVMEQSA
63 |||||||| 72 KEVMEQSA 79 Sequence name:
/tmp/OLMSexEmIh/pc7Z7Xm1YR: BAC85518 Sequence documentation:
Alignment of: R11723_PEA_1_P10 (SEQ ID NO: 335) .times. BAC85518 .
. Alignment segment 1/1: Quality: 645.00 Escore: 0 Matching length:
63 Total length: 63 Matching Percent 100.00 Matching Percent 100.00
Similarity: Identity: Total Percent 100.00 Total Percent 100.00
Similarity: Identity: Gaps: 0 Alignment: . . . . . 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 24
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 73 . 51
QDMCQKEVMEQSA 63 ||||||||||||| 74 QDMCQKEVMEQSA 86 Alignment of:
R11723_PEA_1_P13 (SEQ ID NO: 334) .times. Q96AC2 . . Alignment
segment 1/1: Quality: 645.00 Escore: 0 Matching length: 63 Total
length: 63 Matching Percent 100.00 Matching Percent 100.00
Similarity: Identity: Total Percent 100.00 Total Percent 100.00
Similarity: Identity: Gaps: 0 Alignment: . . . . . 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 1
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV 50 . 51
QDMCQKEVMEQSA 63 ||||||||||||| 51 QDMCQKEVMEQSA 63
Expression of R11723 Transcripts which are Detectable by Amplicon
as Depicted in Sequence Name R11723 seg13 in Normal and Cancerous
Prostate Tissues
[0543] Expression of transcripts detectable by or according to
seg13, R11732seg13 (SEQ ID NO:492) amplicon (s) and R11732seg13F
(SEQ ID NO:490) and R11732seg13R (SEQ ID NO:491) primers was
measured by real time PCR. In parallel the expression of four
housekeeping genes--PBGD (GenBank Accession No. BC019323 (SEQ ID
NO:509); amplicon--PBGD--amplicon, HPRT1 (GenBank Accession No.
NM.sub.--000194 (SEQ ID NO:510); amplicon--HPRT1-amplicon (SEQ ID
NO:401), SDHA (GenBank Accession No. NM.sub.--004168 (SEQ ID
NO:508); amplicon--SDHA-, RPL19 (GenBank Accession No.
NM.sub.--000981 (SEQ ID NO:511); RPL19 amplicon (SEQ ID NO:410) was
measured similarly. For each RT sample, the expression of the above
amplicon was normalized to the geometric mean of the quantities of
the housekeeping genes. The normalized quantity of each RT sample
was then divided by the median of the quantities of the normal
post-mortem (PM) samples (Sample Nos. 42, 48-53, 59-63, Table 1
above, "Tissue samples in testing panel"), to obtain a value of
fold up-regulation for each sample relative to median of the normal
PM samples.
[0544] FIG. 7 is a histogram showing over expression of the
above-indicated transcripts in cancerous prostate samples relative
to the normal samples. Values represent the average of duplicate
experiments. Error bars indicate the minimal and maximal values
obtained).
[0545] As is evident from FIG. 7, the expression of transcripts
detectable by the above amplicon in cancer samples was higher than
in the non-cancerous samples (Sample Nos. 42, 48-53, 59-63, Table 1
above, "Tissue samples in testing panel"). Notably an
over-expression of at least 5 fold was found in 4 out of 19
adenocarcinoma samples Statistical analysis was applied to verify
the significance of these results, as described below.
[0546] The P value for the difference in the expression levels of
transcripts detectable by the above amplicon (s) in prostate cancer
samples versus the normal tissue samples was determined by T test
as 7.57E-02.
[0547] The above values demonstrate statistical significance of the
results. Primer pairs are also optionally and preferably
encompassed within the present invention; for example, for the
above experiment, the following primer pair was used as a
non-limiting illustrative example only of a suitable primer pair:
R11732seg13F forward primer (SEQ ID NO:490); and R11732seg13R
reverse primer (SEQ ID NO:491).
[0548] The present invention also preferably encompasses any
amplicon obtained through the use of any suitable primer pair; for
example, for the above experiment, the following amplicon was
obtained as a non-limiting illustrative example only of a suitable
amplicon:R11732seg13 (SEQ ID NO:492) TABLE-US-00052 R11732seg13F
(SEQ ID NO: 490)- ACACTAAAAGAACAAACACCTTGCTC R11732seg13R (SEQ ID
NO: 491)- TCCTCAGAAGGCACATGAAAGA R11732seg13 (SEQ ID NO: 492) (SEQ
ID NO: 492) - ACACTAAAAGAACAAACACCTTGCTCTTCGAGATGAGACATTTTGCCAAG
CAGTTGACCACTTAGTTCTCAAGAAGCAACTATCTCTTTCATGTGCCTTC TGAGGA
Expression of R11723 Transcripts which are Detectable by Amplicon
as Depicted in Sequence Name R11723seg13 (SEQ ID NO:492) in
Different Normal Tissues
[0549] Expression of R11723 transcripts detectable by or according
to R11723seg13 (SEQ ID NO:492) amplicon and R11723seg13F (SEQ ID
NO:490), R11723seg13R (SEQ ID NO:491) was measured by real time
PCR. In parallel the expression of four housekeeping genes: RPL19
(GenBank Accession No. NM.sub.--000981 (SEQ ID NO:511); RPL19
amplicon (SEQ ID NO:410), TATA box (GenBank Accession No.
NM.sub.--003194 (SEQ ID NO:512); TATA amplicon (SEQ ID NO:515),
Ubiquitin (GenBank Accession No. BC000449 (SEQ ID NO:516);
amplicon--Ubiquitin-amplicon (SEQ ID NO:519)) and SDHA (GenBank
Accession No. NM.sub.--004168 (SEQ ID NO:508);
amplicon--SDHA-amplicon was measured similarly. For each RT sample,
the expression of the above amplicon was normalized to the
geometric mean of the quantities of the housekeeping genes. The
normalized quantity of each RT sample was then divided by the
median of the quantities of the ovary samples (Sample Nos. 18-20
Table 2 "Tissue samples in normal panel", above), to obtain a value
of relative expression of each sample relative to median of the
ovary samples. Primers and amplicon are as above.
[0550] The results are presented in FIG. 8, demonstrating the
expression of R11723 transcripts which are detectable by amplicon
as depicted in sequence name R11723seg13 (SEQ ID NO:492) in
different normal tissues.
Expression of R11723 Transcripts, which are Detectable by Amplicon
as Depicted in Sequence Name R11723junc11-18 (SEQ ID NO:495) in
Normal and Cancerous Prostate Tissues.
[0551] Expression of transcripts detectable by or according to
junc11-18 R11732junc11-18 amplicon (SEQ ID NO:495) and
R11732junc11-18F (SEQ ID NO:493) and R11732junc11-18R (SEQ ID
NO:494) primers was measured by real time PCR (this junction is
found in the known protein sequence or "wild type" (WT) sequence,
also termed herein the PSEC sequence). In parallel the expression
of four housekeeping genes--PBGD (GenBank Accession No. BC019323
(SEQ ID NO:509); amplicon--PBGD-amplicon (SEQ ID NO:404)), HPRT1
(GenBank Accession No. NM.sub.--000194 (SEQ ID NO:510);
amplicon--HPRT1-amplicon (SEQ ID NO:401)), SDHA (GenBank Accession
No. NM.sub.--004168 (SEQ ID NO:508); amplicon--SDHA-amplicon, and
RPL19 (GenBank Accession No. NM.sub.--000981 (SEQ ID NO:511); RPL19
amplicon (SEQ ID NO:410) was measured similarly. For each RT
sample, the expression of the above amplicon was normalized to the
geometric mean of the quantities of the housekeeping genes. The
normalized quantity of each RT sample was then divided by the
median of the quantities of the normal post-mortem (PM) samples
(Sample Nos. 42, 48-53, 59-63 Table 1, above "Tissue samples in
prostate cancer testing panel"), to obtain a value of fold
up-regulation for each sample relative to median of the normal PM
samples.
[0552] FIG. 9A is a histogram showing over expression of the
above-indicated transcripts in cancerous prostate samples relative
to the normal samples.
[0553] As is evident from FIG. 9A, the expression of transcripts
detectable by the above amplicon in a few cancer samples was higher
than in the non-cancerous samples (Sample Nos. 42, 48-53, 59-63,
Table 1, above: "Tissue samples in prostate cancer testing panel").
Notably an over-expression of at least 5 fold was found in 2 out of
19 adenocarcinoma samples
[0554] Primer pairs are also optionally and preferably encompassed
within the present invention; for example, for the above
experiment, the following primer pair was used as a non-limiting
illustrative example only of a suitable primer pair:
R11732junc11-18F forward primer (SEQ ID NO:493); and R11732
junc11-18R reverse primer (SEQ ID NO:494).
[0555] The present invention also preferably encompasses any
amplicon obtained through the use of any suitable primer pair; for
example, for the above experiment, the following amplicon was
obtained as a non-limiting illustrative example only of a suitable
amplicon: R11732 junc11-18 (SEQ ID NO:495) TABLE-US-00053
R11723junc11-18F (SEQ ID NO: 493)- AGTGATGGAGCAAAGTGCCG R11723
junc11-18R (SEQ ID NO: 494)- CAGCAGCTGATGCAAACTGAG R11723 junc11-18
(SEQ ID NO: 495)-
AGTGATGGAGCAAAGTGCCGGGATCATGTACCGCAAGTCCTGTGCATCAT
CAGCGGCCTGTCTCATCGCCTCTGCCGGGTACCAGTCCTTCTGCTCCCCA
GGGAAACTGAACTCAGTTTGCATCAGCTGCTG
Expression of R11723 Transcripts, which were Detected by Amplicon
as Depicted in the Sequence Name R11723 junc11-18 (SEQ ID NO:495)
in Different Normal Tissues.
[0556] Expression of R11723 transcripts detectable by or according
to R1723seg13 amplicon (SEQ ID NO:495) and R11723junc11-18F (SEQ ID
NO:493), R11723junc11-18R (SEQ ID NO:494) was measured by real time
PCR. In parallel the expression of four housekeeping genes RPL19
(GenBank Accession No. NM.sub.--000981 (SEQ ID NO:511); RPL19
amplicon (SEQ ID NO:410), TATA box (GenBank Accession No.
NM.sub.--003194 (SEQ ID NO:512); TATA amplicon (SEQ ID NO:515), UBC
(GenBank Accession No. BC000449 (SEQ ID NO:516);
amplicon--Ubiquitin-amplicon (SEQ ID NO:519) and SDHA (GenBank
Accession No. NM.sub.--004168 (SEQ ID NO:508);
amplicon--SDHA-amplicon (SEQ ID NO:407) was measured similarly. For
each RT sample, the expression of the above amplicon was normalized
to the geometric mean of the quantities of the housekeeping genes.
The normalized quantity of each RT sample was then divided by the
median of the quantities of the ovary samples (Sample Nos. 18-20,
Table 2, "Tissue samples in normal panel", above), to obtain a
value of relative expression of each sample relative to median of
the ovary samples. Results are shown in FIG. 9B; primers and
amplicon are as above.
[0557] The expression of variant transcripts relating to the R11723
cluster (also known as PSEC) was found to be similar to that of the
WT (known or wild type) protein; however in some cancers,
expression of one or more variant transcripts was found to be
higher (R11723_T5 for example in certain tissues).
DESCRIPTION FOR CLUSTER HUMTREFAC
[0558] Cluster HUMTREFAC features 2 transcript(s) and 7 segment(s)
of interest, the names for which are given in Tables 44 and 45,
respectively, the sequences themselves are given at the end of the
application. The selected protein variants are given in table 46.
TABLE-US-00054 TABLE 44 Transcripts of interest Transcript Name
Sequence ID No. HUMTREFAC_PEA_2_T4 13 HUMTREFAC_PEA_2_T5 14
[0559] TABLE-US-00055 TABLE 45 Segments of interest Segment Name
Sequence ID No. HUMTREFAC_PEA_2_node_0 136 HUMTREFAC_PEA_2_node_9
137 HUMTREFAC_PEA_2_node_2 138 HUMTREFAC_PEA_2_node_3 139
HUMTREFAC_PEA_2_node_4 140 HUMTREFAC_PEA_2_node_5 141
HUMTREFAC_PEA_2_node_8 142
[0560] TABLE-US-00056 TABLE 46 Proteins of interest Sequence
Protein Name ID No. Corresponding Transcript(s) HUMTREFAC_PEA_2_P7
338 HUMTREFAC_PEA_2_T5 (SEQ ID NO: 14) HUMTREFAC_PEA_2_P8 339
HUMTREFAC_PEA_2_T4 (SEQ ID NO: 13)
[0561] These sequences are variants of the known protein Trefoil
factor 3 precursor (SEQ ID NO:387) (SwissProt accession identifier
TFF3--HUMAN (SEQ ID NO 387); known also according to the synonyms
Intestinal trefoil factor; hP1.B), SEQ ID NO: 387, referred to
herein as the previously known protein.
[0562] Protein Trefoil factor 3 precursor (SEQ ID NO:387) is known
or believed to have the following function(s): May have a role in
promoting cell migration (motogen). The sequence for protein
Trefoil factor 3 precursor (SEQ ID NO:387) is given at the end of
the application, as "Trefoil factor 3 precursor (SEQ ID NO:387)
amino acid sequence". Known polymorphisms for this sequence are as
shown in Table 47. TABLE-US-00057 TABLE 47 Amino acid mutations for
Known Protein SNP position(s) on amino acid sequence Comment 74-76
QEA -> TRKT
[0563] Protein Trefoil factor 3 precursor (SEQ ID NO:387)
localization is believed to be Secreted.
[0564] The following GO Annotation(s) apply to the previously known
protein. The following annotation(s) were found: defense response;
digestion, which are annotation(s) related to Biological Process;
and extracellular, which are annotation(s) related to Cellular
Component.
[0565] The GO assignment relies on information from one or more of
the SwissProt/TremBl Protein knowledgebase, available from
<http://www.expasy.ch/sprot/>; or Locuslink, available from
<http://www.ncbi.nlm.nih.gov/projects/LocusLink/>.
[0566] Cluster HUMTREFAC can be used as a diagnostic marker
according to overexpression of transcripts of this cluster in
cancer. Expression of such transcripts in normal tissues is also
given according to the previously described methods. The term
"number" in the left hand column of the table and the numbers on
the y-axis of FIG. 10 refer to weighted expression of ESTs in each
category, as "parts per million" (ratio of the expression of ESTs
for a particular cluster to the expression of all ESTs in that
category, according to parts per million).
[0567] Overall, the following results were obtained as shown with
regard to the histograms in FIG. 10 and Table 48. This cluster is
overexpressed (at least at a minimum level) in the following
pathological conditions: a mixture of malignant tumors from
different tissues, breast malignant tumors, pancreas carcinoma and
prostate cancer. TABLE-US-00058 TABLE 48 Normal tissue distribution
Name of Tissue Number Adrenal 40 Colon 797 Epithelial 95 General 39
Liver 0 Lung 57 Lymph nodes 3 Breast 0 Muscle 3 Pancreas 2 Prostate
16 Stomach 0 Thyroid 257 Uterus 54
[0568] TABLE-US-00059 TABLE 49 P values and ratios for expression
in cancerous tissue Name of Tissue P1 P2 SP1 R3 SP2 R4 Adrenal
6.4e-01 6.9.sup.e-01 7.1e-01 1.1 7.8e-01 0.9 Colon 4.6e-01
5.7.sup.e-01 9.7e-01 0.5 1 0.4 Epithelial 2.4e-02 3.4.sup.e-01
9.5e-10 2.0 5.3e-02 1.1 General 2.5e-04 3.9e-02 1.4e-28 3.6 1.9e-10
1.9 Liver 1 6.8e-01 1 1.0 6.9e-01 1.4 Lung 4.8e-01 7.6.sup.e-01
2.2e-03 1.0 1.6e-01 0.5 Lymph nodes 5.1e-01 8.0.sup.e-01 2.3e-02
5.0 1.9e-01 2.1 Breast 7.6e-02 1.2.sup.e-01 3.1e-06 12.0 1.1e-03
6.5 Muscle 9.2e-01 4.8e-01 1 0.8 3.9e-01 2.1 Pancreas 1.2e-01
2.4.sup.e-01 5.7e-03 6.5 2.1e-02 4.6 Prostate 1.5e-01 2.7e-01
9.9e-10 8.1 3.1e-07 5.7 Stomach 3.0e-01 1.3.sup.e-01 5.0e-01 2.0
6.7e-02 2.8 Thyroid 6.4e-01 6.4.sup.e-01 9.6e-01 0.5 9.6e-01 0.5
Uterus 4.1e-01 7.3.sup.e-01 7.5e-02 1.3 4.0e-01 0.8
[0569] As noted above, cluster HUMTREFAC features 2 transcript(s),
which were listed in Table 44 above. These transcript(s) encode for
protein(s) which are variant(s) of protein Trefoil factor 3
precursor (SEQ ID NO:387). A description of each variant protein
according to the present invention is now provided.
[0570] Variant protein HUMTREFAC_PEA.sub.--2_P7 (SEQ ID NO:338)
according to the present invention has an amino acid sequence as
given at the end of the application; it is encoded by transcript(s)
HUMTREFAC_PEA.sub.--2_T5 (SEQ ID NO:14). The location of the
variant protein was determined according to results from a number
of different software programs and analyses, including analyses
from SignalP and other specialized programs. The variant protein is
believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted
because both signal-peptide prediction programs predict that this
protein has a signal peptide, and neither trans-membrane region
prediction program predicts that this protein has a trans-membrane
region.
[0571] Variant protein HUMTREFAC_PEA.sub.--2_P7 (SEQ ID NO:338)
also has the following non-silent SNPs (Single Nucleotide
Polymorphisms) as listed in Table 50, (given according to their
position(s) on the amino acid sequence, with the alternative amino
acid(s) listed; the last column indicates whether the SNP is known
or not; the presence of known SNPs in variant protein
HUMTREFAC_PEA.sub.--2 P7 (SEQ ID NO:338) sequence provides support
for the deduced sequence of this variant protein according to the
present invention). TABLE-US-00060 TABLE 50 Amino acid mutations
SNP position(s) on amino acid Previously sequence Alternative amino
acid(s) known SNP? 5 A -> S No 5 A -> T No 14 A -> V Yes
43 L -> M No 60 P -> S Yes 123 S -> * Yes
[0572] Variant protein HUMTREFAC_PEA.sub.--2_P7 (SEQ ID NO:338) is
encoded by the following transcript(s): HUMTREFAC_PEA.sub.--2_T5
(SEQ ID NO:14), for which the sequence(s) is/are given at the end
of the application. The coding portion of transcript
HUMTREFAC_PEA.sub.--2_T5 (SEQ ID NO:14) is shown in bold; this
coding portion starts at position 278 and ends at position 688. The
transcript also has the following SNPs as listed in Table 51 (given
according to their position on the nucleotide sequence, with the
alternative nucleic acid listed; the last column indicates whether
the SNP is known or not; the presence of known SNPs in variant
protein HUMTREFAC_PEA.sub.--2_P7 (SEQ ID NO:338) sequence provides
support for the deduced sequence of this variant protein according
to the present invention). TABLE-US-00061 TABLE 51 Nucleic acid
SNPs SNP position on nucleotide Previously sequence Alternative
nucleic acid known SNP? 233 A -> G Yes 290 G -> A No 290 G
-> T No 318 C -> T Yes 404 C -> A No 404 C -> T No 455
C -> T Yes 645 C -> A Yes 685 C -> T No
[0573] Variant protein HUMTREFAC_PEA.sub.--2_P8 (SEQ ID NO:339)
according to the present invention has an amino acid sequence as
given at the end of the application; it is encoded by transcript(s)
HUMTREFAC_PEA-2-T4 (SEQ ID NO: 13). An alignment is given to the
known protein (Trefoil factor 3 precursor (SEQ ID NO:387)) at the
end of the application. One or more alignments to one or more
previously published protein sequences are given at the end of the
application. A brief description of the relationship of the variant
protein according to the present invention to each such aligned
protein is as follows:
[0574] Comparison report between HUMTREFAC_PEA.sub.--2_P8 (SEQ ID
NO:339) and
[0575] TFF3--HUMAN:
[0576] 1. An isolated chimeric polypeptide encoding for
HUMTREFAC_PEA.sub.--2_P8 (SEQ ID NO:339), comprising a first amino
acid sequence being at least 90% homologous to
MAARALCMLGLVLALLSSSSAEEYVGL corresponding to amino acids 1-27 of
TFF3--HUMAN, which also corresponds to amino acids 1-27 of
HUMTREFAC_PEA.sub.--2_P8 (SEQ ID NO:339), and a second amino acid
sequence being at least 70%, optionally at least 80%, preferably at
least 85%, more preferably at least 90% and most preferably at
least 95% homologous to a polypeptide having the sequence
WKVHLPKGEGFSSG (SEQ ID NO: 543) corresponding to amino acids 28-41
of HUMTREFAC_PEA.sub.--2_P8 (SEQ ID NO:339) wherein said first
amino acid sequence and second amino acid sequence are contiguous
and in a sequential order.
[0577] 2. An isolated polypeptide encoding for a tail of
HUMTREFAC_PEA.sub.--2_P8 (SEQ ID NO:339), comprising a polypeptide
being at least 70%, optionally at least about 80%, preferably at
least about 85%, more preferably at least about 90% and most
preferably at least about 95% homologous to the sequence
WKVHLPKGEGFSSG (SEQ ID NO: 543) in HUMTREFAC_PEA.sub.--2_P8 (SEQ ID
NO:339).
[0578] The location of the variant protein was determined according
to results from a number of different software programs and
analyses, including analyses from SignalP and other specialized
programs. The variant protein is believed to be located as follows
with regard to the cell: secreted. The protein localization is
believed to be secreted because both signal-peptide prediction
programs predict that this protein has a signal peptide, and
neither trans-membrane region prediction program predicts that this
protein has a trans-membrane region.
[0579] Variant protein HUMTREFAC_PEA.sub.--2_P8 (SEQ ID NO:339)
also has the following non-silent SNPs (Single Nucleotide
Polymorphisms) as listed in Table 52, (given according to their
position(s) on the amino acid sequence, with the alternative amino
acid(s) listed; the last column indicates whether the SNP is known
or not; the presence of known SNPs in variant protein
HUMTREFAC_PEA.sub.--2-P8 (SEQ ID NO:339) sequence provides support
for the deduced sequence of this variant protein according to the
present invention). TABLE-US-00062 TABLE 52 Amino acid mutations
SNP position(s) on amino acid Previously sequence Alternative amino
acid(s) known SNP? 5 A -> S No 5 A -> T No 14 A -> V
Yes
[0580] Variant protein HUMTREFAC_PEA.sub.--2_P8 (SEQ ID NO:339) is
encoded by the following transcript(s): HUMTREFAC_PEA.sub.--2_T4
(SEQ ID NO:13), for which the sequence(s) is/are given at the end
of the application. The coding portion of transcript HUMTREFAC_PEA
2_T4 (SEQ ID NO:13) is shown in bold; this coding portion starts at
position 278 and ends at position 400. The transcript also has the
following SNPs as listed in Table 53 (given according to their
position on the nucleotide sequence, with the alternative nucleic
acid listed; the last column indicates whether the SNP is known or
not; the presence of known SNPs in variant protein
HUMTREFAC_PEA.sub.--2_P8 (SEQ ID NO:339) sequence provides support
for the deduced sequence of this variant protein according to the
present invention). TABLE-US-00063 TABLE 53 Nucleic acid SNPs SNP
position on nucleotide Previously sequence Alternative nucleic acid
known SNP? 233 A -> G Yes 290 G -> A No 290 G -> T No 318
C -> T Yes 515 C -> A No 515 C -> T No 566 C -> T Yes
756 C -> A Yes 796 C -> T No 1265 A -> C No 1266 A -> T
No
[0581] As noted above, cluster HUMTREFAC features 7 segment(s),
which were listed in Table 121 above and for which the sequence(s)
are given at the end of the application. These segment(s) are
portions of nucleic acid sequence(s) which are described herein
separately because they are of particular interest. A description
of each segment according to the present invention is now
provided.
[0582] Segment cluster HUMTREFAC_PEA.sub.--2_node.sub.--0 (SEQ ID
NO:136) according to the present invention is supported by 188
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): HUMTREFAC_PEA.sub.--2_T4 (SEQ ID NO:13) and
HUMTREFAC_PEA.sub.--2_T5 (SEQ ID NO: 14). Table 54 below describes
the starting and ending position of this segment on each
transcript. TABLE-US-00064 TABLE 54 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HUMTREFAC_PEA_2_T4 (SEQ 1 359 ID NO: 13) HUMTREFAC_PEA_2_T5 (SEQ 1
359 ID NO: 14)
[0583] Segment cluster HUMTREFAC_PEA.sub.--2_node.sub.--9 (SEQ ID
NO:137) according to the present invention is supported by 150
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): HUMTREFAC_PEA.sub.--2_T4 (SEQ ID NO: 13) and
HUMTREFAC_PEA.sub.--2_T5 (SEQ ID NO: 14). Table 55 below describes
the starting and ending position of this segment on each
transcript. TABLE-US-00065 TABLE 55 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HUMTREFAC_PEA_2_T4 (SEQ 681 1266 ID NO: 13) HUMTREFAC_PEA_2_T5 (SEQ
570 747 ID NO: 14)
[0584] According to an optional embodiment of the present
invention, short segments related to the above cluster are also
provided. These segments are up to about 120 bp in length, and so
are included in a separate description.
[0585] Segment cluster HUMTREFAC_PEA.sub.--2_node.sub.--2 (SEQ ID
NO: 138) according to the present invention is supported by 4
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): HUMTREFAC_PEA.sub.--2_T4 (SEQ ID NO: 13). Table 56
below describes the starting and ending position of this segment on
each transcript. TABLE-US-00066 TABLE 56 Segment location on
transcripts Segment Segment Transcript name starting position
ending position HUMTREFAC_PEA_2_T4 (SEQ 360 470 ID NO: 13)
[0586] Segment cluster HUMTREFAC_PEA.sub.--2_node.sub.--3 (SEQ ID
NO:139) according to the present invention is supported by 10
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): HUMTREFAC_PEA.sub.--2_T4 (SEQ ID NO: 13) and
HUMTREFAC_PEA.sub.--2_T5 (SEQ ID NO: 14). Table 57 below describes
the starting and ending position of this segment on each
transcript. TABLE-US-00067 TABLE 57 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HUMTREFAC_PEA_2_T4 (SEQ 471 514 ID NO: 13) HUMTREFAC_PEA_2_T5 (SEQ
360 403 ID NO: 14)
[0587] Segment cluster HUMTREFAC_PEA.sub.--2_node.sub.--4 (SEQ ID
NO:140) according to the present invention is supported by 197
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): HUMTREFAC_PEA.sub.--2_T4 (SEQ ID NO:13) and
HUMTREFAC_PEA.sub.--2_T5 (SEQ ID NO:14). Table 58 below describes
the starting and ending position of this segment on each
transcript. TABLE-US-00068 TABLE 58 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HUMTREFAC_PEA_2_T4 (SEQ 515 611 ID NO: 13) HUMTREFAC_PEA_2_T5 (SEQ
404 500 ID NO: 14)
[0588] Segment cluster HUMTREFAC_PEA.sub.--2_node.sub.--5 (SEQ ID
NO:141) according to the present invention is supported by 187
libraries. The number of libraries was determined as previously
described. This segment can be found in the following
transcript(s): HUMTREFAC_PEA.sub.--2_T4 (SEQ ID NO: 13) and
HUMTREFAC_PEA.sub.--2_T5 (SEQ ID NO: 14). Table 59 below describes
the starting and ending position of this segment on each
transcript. TABLE-US-00069 TABLE 59 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HUMTREFAC_PEA_2_T4 (SEQ 612 661 ID NO: 13) HUMTREFAC_PEA_2_T5 (SEQ
501 550 ID NO: 14)
[0589] Segment cluster HUMTREFAC_PEA.sub.--2_node.sub.--8 (SEQ ID
NO:142) according to the present invention can be found in the
following transcript(s): HUMTREFAC_PEA.sub.--2_T4 (SEQ ID NO:13)
and HUMTREFAC_PEA.sub.--2_T5 (SEQ ID NO:14). Table 60 below
describes the starting and ending position of this segment on each
transcript. TABLE-US-00070 TABLE 60 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HUMTREFAC_PEA_2_T4 (SEQ 662 680 ID NO: 13) HUMTREFAC_PEA_2_T5 (SEQ
551 569 ID NO: 14)
[0590] Variant protein alignment to the previously known protein:
TABLE-US-00071 Sequence name: TFF3_HUMAN Sequence documentation:
Alignment of: HUMTREFAC_PEA_2_P8 (SEQ ID NO: 339) .times.
TFF3_HUMAN . . Alignment segment 1/1: Quality: 246.00 Escore: 0
Matching length: 27 Total length: 27 Matching Percent 100.00
Matching Percent 100.00 Similarity: Identity: Total Percent 100.00
Total Percent 100.00 Similarity: Identity: Gaps: 0 Alignment: . . 1
MAARALCMLGLVLALLSSSSAEEYVGL 27 ||||||||||||||||||||||||||| 1
MAARALCMLGLVLALLSSSSAEEYVGL 27
[0591] Description for Cluster HSSTROL3
[0592] Cluster HSSTROL3 features 6 transcript(s) and 16 segment(s)
of interest, the names for which are given in Tables 61 and 62,
respectively, the sequences themselves are given at the end of the
application. The selected protein variants are given in table 63.
TABLE-US-00072 TABLE 61 Transcripts of interest Transcript Name
Sequence ID No. HSSTROL3_T5 34 HSSTROL3_T8 35 HSSTROL3_T9 36
HSSTROL3_T10 37 HSSTROL3_T11 38 HSSTROL3_T12 39
[0593] TABLE-US-00073 TABLE 62 Segments of interest Segment Name
Sequence ID No. HSSTROL3_node_6 222 HSSTROL3_node_10 223
HSSTROL3_node_13 224 HSSTROL3_node_15 225 HSSTROL3_node_19 226
HSSTROL3_node_21 227 HSSTROL3_node_24 228 HSSTROL3_node_25 229
HSSTROL3_node_26 230 HSSTROL3_node_28 231 HSSTROL3_node_29 232
HSSTROL3_node_11 233 HSSTROL3_node_17 234 HSSTROL3_node_18 235
HSSTROL3_node_20 236 HSSTROL3_node_27 237
[0594] TABLE-US-00074 TABLE 63 Proteins of interest Protein Name
Sequence ID No. Corresponding Transcript(s) HSSTROL3_P4 359
HSSTROL3_T5 (SEQ ID NO: 34) HSSTROL3_P5 360 HSSTROL3_T8 (SEQ ID NO:
35); HSSTROL3_T9 (SEQ ID NO: 36) HSSTROL3_P7 361 HSSTROL3_T10 (SEQ
ID NO: 37) HSSTROL3_P8 362 HSSTROL3_T11 (SEQ ID NO: 38) HSSTROL3_P9
363 HSSTROL3_T12 (SEQ ID NO: 39)
[0595] These sequences are variants of the known protein
Stromelysin-3 precursor (SEQ ID NO:391) (SwissProt accession
identifier MM11_HUMAN (SEQ ID NO: 391); known also according to the
synonyms EC 3.4.24.-; Matrix metalloproteinase-11; MMP-11; ST3;
SL-3), SEQ ID NO: 391) referred to herein as the previously known
protein.
[0596] Protein Stromelysin-3 precursor (SEQ ID NO:391) is known or
believed to have the following function(s): May play an important
role in the progression of epithelial malignancies. The sequence
for protein Stromelysin-3 precursor (SEQ ID NO:391) is given at the
end of the application, as "Stromelysin-3 precursor (SEQ ID NO:391)
amino acid sequence".
[0597] The following GO Annotation(s) apply to the previously known
protein. The following annotation(s) were found: proteolysis and
peptidolysis; developmental processes; morphogenesis, which are
annotation(s) related to Biological Process; stromelysin 3; calcium
binding; zinc binding; hydrolase, which are annotation(s) related
to Molecular Function; and extracellular matrix, which are
annotation(s) related to Cellular Component.
[0598] The GO assignment relies on information from one or more of
the SwissProt/TremBl Protein knowledgebase, available from
<http://www.expasy.ch/sprot/>; or Locuslink, available from
<http://www.ncbi.nlm.nih.gov/projects/LocusLink/>.
[0599] Cluster HSSTROL3 can be used as a diagnostic marker
according to overexpression of transcripts of this cluster in
cancer. Expression of such transcripts in normal tissues is also
given according to the previously described methods. The term
"number" in the left hand column of the table and the numbers on
the y-axis of FIG. 11 refer to weighted expression of ESTs in each
category, as "parts per million" (ratio of the expression of ESTs
for a particular cluster to the expression of all ESTs in that
category, according to parts per million).
[0600] Overall, the following results were obtained as shown with
regard to the histograms in FIG. 11 and Table 64. This cluster is
overexpressed (at least at a minimum level) in the following
pathological conditions: transitional cell carcinoma, epithelial
malignant tumors, a mixture of malignant tumors from different
tissues and pancreas carcinoma. TABLE-US-00075 TABLE 64 Normal
tissue distribution Name of Tissue Number Adrenal 0 Bladder 0 Brain
1 Colon 63 Epithelial 33 General 13 head and neck 101 Kidney 0 Lung
11 Breast 8 Ovary 14 Pancreas 0 Prostate 2 Skin 99 Thyroid 0 Uterus
181
[0601] TABLE-US-00076 TABLE 65 P values and ratios for expression
in cancerous tissue Name of Tissue P1 P2 SP1 R3 SP2 R4 Adrenal 1
4.6e-01 1 1.0 5.3e-01 1.9 Bladder 2.7e-01 3.4e-01 3.3e-03 4.9
2.1e-02 3.3 Brain 3.5e-01 2.6e-01 1 1.7 3.3e-01 2.8 Colon 7.7e-02
1.5e-01 3.1e-01 1.4 5.2e-01 1.0 Epithelial 1.2e-04 1.2e-02 1.3e-06
2.7 4.6e-02 1.4 General 5.4e-09 3.1e-05 1.8e-16 5.0 3.1e-07 2.6
head and neck 4.6e-01 4.3e-01 1 0.6 9.4e-01 0.7 Kidney 2.5e-01
3.5e-01 1.1e-01 4.0 2.4e-01 2.8 Lung 1.8e-01 4.5e-01 1.9e-01 2.7
5.1e-01 1.4 Breast 2.0e-01 3.4e-01 7.3e-02 3.3 2.5e-01 2.0 Ovary
2.6e-01 3.2e-01 2.2e-02 2.0 7.0e-02 1.6 Pancreas 9.5e-02 1.8e-01
1.8e-04 7.8 1.6e-03 5.5 Prostate 8.2e-01 7.8e-01 4.5e-01 1.8
5.6e-01 1.5 Skin 5.2e-01 5.8e-01 7.1e-01 0.8 1 0.3 Thyroid 2.9e-01
2.9e-01 1 1.1 1 1.1 Uterus 4.2e-01 8.0e-01 7.5e-01 0.6 9.9e-01
0.4
[0602] As noted above, cluster HSSTROL3 features 6 transcript(s),
which were listed in Table 65 above. These transcript(s) encode for
protein(s) which are variant(s) of protein Stromelysin-3 precursor
(SEQ ID NO:391). A description of each variant protein according to
the present invention is now provided.
[0603] Variant protein HSSTROL3_P4 (SEQ ID NO:359) according to the
present invention has an amino acid sequence as given at the end of
the application; it is encoded by transcript(s) HSSTROL3_T5 (SEQ ID
NO:34). An alignment is given to the known protein (Stromelysin-3
precursor (SEQ ID NO:391)) at the end of the application. One or
more alignments to one or more previously published protein
sequences are given at the end of the application. A brief
description of the relationship of the variant protein according to
the present invention to each such aligned protein is as
follows:
[0604] Comparison report between HSSTROL3_P4 (SEQ ID NO:359) and
MM11_HUMAN:
[0605] 1. An isolated chimeric polypeptide encoding for HSSTROL3_P4
(SEQ ID NO:359) comprising a first amino acid sequence being at
least 90% homologous to
MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQPWHAALPSS
PAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVLSGGRWEKTDLTYRILRFP
WQLVQEQVRQTMAEALKVWSDVTPLTFTEVHEGRADIMIDFARYW corresponding to
amino acids 1-163 of MM11_HUMAN, which also corresponds to amino
acids 1-163 of HSSTROL3_P4 (SEQ ID NO:359), a bridging amino acid H
corresponding to amino acid 164 of HSSTROL3_P4 (SEQ ID NO:359), a
second amino acid sequence being at least 90% homologous to
GDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGHVLG
LQHTTAAKALMSAFYTFRYPLSLSPDDCRGVQHLYGQPWPTVTSRTPALGPQAGIDTN
EIAPLEPDAPPDACEASFDAVSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGL
PSPVDAAFEDAQGHIWFFQGAQYWVYDGEKPVLGPAPLTELGLVRFPVHAALVWGPE
KNKIYFFRGRDYWRFHPSTRRVDSPVPRRATDWRGVPSEIDAAFQDADG corresponding to
amino acids 165-445 of MM11_HUMAN, which also corresponds to amino
acids 165-445 of HSSTROL3_P4 (SEQ ID NO:359), and a third amino
acid sequence being at least 70%, optionally at least 80%,
preferably at least 85%, more preferably at least 90% and most
preferably at least 95% homologous to a polypeptide having the
sequence ALGVRQLVGGGHSSRFSHLVVAGLPHACHRKSGSSSQVLCPEPSALLSVAG (SEQ
ID NO: 564) corresponding to amino acids 446-496 of HSSTROL3_P4
(SEQ ID NO:359), wherein said first amino acid sequence, bridging
amino acid, second amino acid sequence and third amino acid
sequence are contiguous and in a sequential order.
[0606] 2. An isolated polypeptide encoding for a tail of
HSSTROL3_P4 (SEQ ID NO:359), comprising a polypeptide being at
least 70%, optionally at least about 80%, preferably at least about
85%, more preferably at least about 90% and most preferably at
least about 95% homologous to the sequence
ALGVRQLVGGGHSSRFSHLVVAGLPHACHRKSGSSSQVLCPEPSALLSVAG in (SEQ ID NO:
564) HSSTROL3_P4 (SEQ ID NO:359).
[0607] The location of the variant protein was determined according
to results from a number of different software programs and
analyses, including analyses from SignalP and other specialized
programs. The variant protein is believed to be located as follows
with regard to the cell: secreted. The protein localization is
believed to be secreted because both signal-peptide prediction
programs predict that this protein has a signal peptide, and
neither trans-membrane region prediction program predicts that this
protein has a trans-membrane region.
[0608] Variant protein HSSTROL3_P4 (SEQ ID NO:359) also has the
following non-silent SNPs (Single Nucleotide Polymorphisms) as
listed in Table 66, (given according to their position(s) on the
amino acid sequence, with the alternative amino acid(s) listed; the
last column indicates whether the SNP is known or not; the presence
of known SNPs in variant protein HSSTROL3_P4 (SEQ ID NO:359)
sequence provides support for the deduced sequence of this variant
protein according to the present invention). TABLE-US-00077 TABLE
66 Amino acid mutations SNP position(s) on Alternative Previously
amino acid sequence amino acid(s) known SNP? 38 V -> A Yes 104 R
-> P Yes 214 A -> No 323 Q -> H Yes
[0609] Variant protein HSSTROL3_P4 (SEQ ID NO:359) is encoded by
the following transcript(s): HSSTROL3_T5 (SEQ ID NO:34), for which
the sequence(s) is/are given at the end of the application. The
coding portion of transcript HSSTROL3_T5 (SEQ ID NO:34) is shown in
bold; this coding portion starts at position 24 and ends at
position 1511. The transcript also has the following SNPs as listed
in Table 67 (given according to their position on the nucleotide
sequence, with the alternative nucleic acid listed; the last column
indicates whether the SNP is known or not; the presence of known
SNPs in variant protein HSSTROL3_P4 (SEQ ID NO:359) sequence
provides support for the deduced sequence of this variant protein
according to the present invention). TABLE-US-00078 TABLE 67
Nucleic acid SNPs SNP position on Alternative Previously nucleotide
sequence nucleic acid known SNP? 136 T -> C Yes 334 G -> C
Yes 663 G -> No 699 -> T No 992 G -> C Yes 1528 A -> G
Yes 1710 A -> G Yes 2251 A -> G Yes 2392 C -> No 2444 C
-> A Yes 2470 A -> T Yes 2687 -> G No 2696 -> G No 2710
C -> No 2729 -> A No 2755 T -> C No 2813 A -> No 2813 A
-> C No 2963 A -> No 2963 A -> C No 2993 T -> C Yes
3140 -> T No
[0610] Variant protein HSSTROL3_P5 (SEQ ID NO:360) according to the
present invention has an amino acid sequence as given at the end of
the application; it is encoded by transcript(s) HSSTROL3_T8 (SEQ ID
NO:35) and HSSTROL3_T9 (SEQ ID NO:36). An alignment is given to the
known protein (Stromelysin-3 precursor (SEQ ID NO:391) at the end
of the application. One or more alignments to one or more
previously published protein sequences are given at the end of the
application. A brief description of the relationship of the variant
protein according to the present invention to each such aligned
protein is as follows:
[0611] Comparison report between HSSTROL3_P5 (SEQ ID NO:360) and
MM11_HUMAN:
[0612] 1. An isolated chimeric polypeptide encoding for HSSTROL3_P5
(SEQ ID NO:360), comprising a first amino acid sequence being at
least 90% homologous to
MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQPWHAALPSS
PAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVLSGGRWEKTDLTYRILRFP
WQLVQEQVRQTMAEALKVWSDVTPLTFTEVHEGRADIMIDFARYW corresponding to
amino acids 1-163 of MM11_HUMAN, which also corresponds to amino
acids 1-163 of HSSTROL3_P5 (SEQ ID NO:360), a bridging amino acid H
corresponding to amino acid 164 of HSSTROL3_P5 (SEQ ID NO:360), a
second amino acid sequence being at least 90% homologous to
GDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGHVLG
LQHTTAAKALMSAFYTFRYPLSLSPDDCRGVQHLYGQPWPTVTSRTPALGPQAGIDTN
EIAPLEPDAPPDACEASFDAVSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGL
PSPVDAAFEDAQGHIWFFQ corresponding to amino acids 165-358 of
MM11_HUMAN, which also corresponds to amino acids 165-358 of
HSSTROL3_P5 (SEQ ID NO:360), and a third amino acid sequence being
at least 70%, optionally at least 80%, preferably at least 85%,
more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence
ELGFPSSTGRDESLEHCRCQGLHK (SEQ ID NO: 565) corresponding to amino
acids 359-382 of HSSTROL3_P5 (SEQ ID NO:360), wherein said first
amino acid sequence, bridging amino acid, second amino acid
sequence and third amino acid sequence are contiguous and in a
sequential order.
[0613] 2. An isolated polypeptide encoding for a tail of
HSSTROL3_P5 (SEQ ID NO:360), comprising a polypeptide being at
least 70%, optionally at least about 80%, preferably at least about
85%, more preferably at least about 90% and most preferably at
least about 95% homologous to the sequence ELGFPSSTGRDESLEHCRCQGLHK
(SEQ ID NO: 565) in HSSTROL3_P5 (SEQ ID NO:360).
[0614] The location of the variant protein was determined according
to results from a number of different software programs and
analyses, including analyses from SignalP and other specialized
programs. The variant protein is believed to be located as follows
with regard to the cell: secreted. The protein localization is
believed to be secreted because both signal-peptide prediction
programs predict that this protein has a signal peptide, and
neither trans-membrane region prediction program predicts that this
protein has a trans-membrane region.
[0615] Variant protein HSSTROL3_P5 (SEQ ID NO:360) also has the
following non-silent SNPs (Single Nucleotide Polymorphisms) as
listed in Table 68, (given according to their position(s) on the
amino acid sequence, with the alternative amino acid(s) listed; the
last column indicates whether the SNP is known or not; the presence
of known SNPs in variant protein HSSTROL3_P5 (SEQ ID NO:360)
sequence provides support for the deduced sequence of this variant
protein according to the present invention). TABLE-US-00079 TABLE
68 Amino acid mutations SNP position(s) on Alternative Previously
amino acid sequence amino acid(s) known SNP? 38 V -> A Yes 104 R
-> P Yes 214 A -> No 323 Q -> H Yes
[0616] Variant protein HSSTROL3_P5 (SEQ ID NO:360) is encoded by
the following transcript(s): HSSTROL3_T8 (SEQ ID NO:35) and
HSSTROL3_T9 (SEQ ID NO:36), for which the sequence(s) is/are given
at the end of the application.
[0617] The coding portion of transcript HSSTROL3_T8 (SEQ ID NO:35)
is shown in bold; this coding portion starts at position 24 and
ends at position 1169. The transcript also has the following SNPs
as listed in Table 69 (given according to their position on the
nucleotide sequence, with the alternative nucleic acid listed; the
last column indicates whether the SNP is known or not; the presence
of known SNPs in variant protein HSSTROL3_P5 (SEQ ID NO:360)
sequence provides support for the deduced sequence of this variant
protein according to the present invention). TABLE-US-00080 TABLE
69 Nucleic acid SNPs SNP position on Alternative Previously
nucleotide sequence nucleic acid known SNP? 136 T -> C Yes 334 G
-> C Yes 663 G -> No 699 -> T No 992 G -> C Yes 1903 C
-> No 1955 C -> A Yes 1981 A -> T Yes 2198 -> G No 2207
-> G No 2221 C -> No 2240 -> A No 2266 T -> C No 2324 A
-> No 2324 A -> C No 2474 A -> No 2474 A -> C No 2504 T
-> C Yes 2651 -> T No
[0618] The coding portion of transcript HSSTROL3_T9 (SEQ ID NO:36)
is shown in bold; this coding portion starts at position 24 and
ends at position 1169. The transcript also has the following SNPs
as listed in Table 70 (given according to their position on the
nucleotide sequence, with the alternative nucleic acid listed; the
last column indicates whether the SNP is known or not; the presence
of known SNPs in variant protein HSSTROL3_P5 (SEQ ID NO:360)
sequence provides support for the deduced sequence of this variant
protein according to the present invention). TABLE-US-00081 TABLE
70 Nucleic acid SNPs SNP position on Alternative Previously
nucleotide sequence nucleic acid known SNP? 136 T -> C Yes 334 G
-> C Yes 663 G -> No 699 -> T No 992 G -> C Yes 1666 A
-> G Yes 1848 A -> G Yes 2389 A -> G Yes 2530 C -> No
2582 C -> A Yes 2608 A -> T Yes 2825 -> G No 2834 -> G
No 2848 C -> No 2867 -> A No 2893 T -> C No 2951 A ->
No 2951 A -> C No 3101 A -> No 3101 A -> C No 3131 T ->
C Yes 3278 -> T No
[0619] Variant protein HSSTROL3_P7 (SEQ ID NO:361) according to the
present invention has an amino acid sequence as given at the end of
the application; it is encoded by transcript(s) HSSTROL3_T10 (SEQ
ID NO:37). An alignment is given to the known protein
(Stromelysin-3 precursor (SEQ ID NO:391)) at the end of the
application. One or more alignments to one or more previously
published protein sequences are given at the end of the
application. A brief description of the relationship of the variant
protein according to the present invention to each such aligned
protein is as follows:
[0620] Comparison report between HSSTROL3_P7 (SEQ ID NO:361) and
MM11_HUMAN:
[0621] 1. An isolated chimeric polypeptide encoding for HSSTROL3_P7
(SEQ ID NO:361) comprising a first amino acid sequence being at
least 90% homologous to
MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQPWHAALPSS
PAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVLSGGRWEKTDLTYRILRFP
WQLVQEQVRQTMAEALKVWSDVTPLTFTEVHEGRADIMIDFARYW corresponding to
amino acids 1-163 of MM11_HUMAN, which also corresponds to amino
acids 1-163 of HSSTROL3_P7 (SEQ ID NO:361), a bridging amino acid H
corresponding to amino acid 164 of HSSTROL3_P7 (SEQ ID NO:361), a
second amino acid sequence being at least 90% homologous to
GDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGHVLG
LQHTTAAKALMSAFYTFRYPLSLSPDDCRGVQHLYGQPWPTVTSRTPALGPQAGIDTN
EIAPLEPDAPPDACEASFDAVSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGL
PSPVDAAFEDAQGHIWFFQG corresponding to amino acids 165-359 of
MM11_HUMAN, which also corresponds to amino acids 165-359 of
HSSTROL3_P7 (SEQ ID NO:361), and a third amino acid sequence being
at least 70%, optionally at least 80%, preferably at least 85%,
more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence TTGVSTPAPGV (SEQ ID
NO: 566) corresponding to amino acids 360-370 of HSSTROL3_P7 (SEQ
ID NO:361), wherein said first amino acid sequence, bridging amino
acid, second amino acid sequence and third amino acid sequence are
contiguous and in a sequential order.
[0622] 2. An isolated polypeptide encoding for a tail of
HSSTROL3_P7 (SEQ ID NO:361) comprising a polypeptide being at least
70%, optionally at least about 80%, preferably at least about 85%,
more preferably at least about 90% and most preferably at least
about 95% homologous to the sequence TTGVSTPAPGV (SEQ ID NO: 566)
in HSSTROL3_P7 (SEQ ID NO:361).
[0623] The location of the variant protein was determined according
to results from a number of different software programs and
analyses, including analyses from SignalP and other specialized
programs. The variant protein is believed to be located as follows
with regard to the cell: secreted. The protein localization is
believed to be secreted because both signal-peptide prediction
programs predict that this protein has a signal peptide, and
neither trans-membrane region prediction program predicts that this
protein has a trans-membrane region.
[0624] Variant protein HSSTROL3_P7 (SEQ ID NO:361) also has the
following non-silent SNPs (Single Nucleotide Polymorphisms) as
listed in Table 71, (given according to their position(s) on the
amino acid sequence, with the alternative amino acid(s) listed; the
last column indicates whether the SNP is known or not; the presence
of known SNPs in variant protein HSSTROL3_P7 (SEQ ID NO:361)
sequence provides support for the deduced sequence of this variant
protein according to the present invention). TABLE-US-00082 TABLE
71 Amino acid mutations SNP position(s) on Alternative Previously
amino acid sequence amino acid(s) known SNP? 38 V -> A Yes 104 R
-> P Yes 214 A -> No 323 Q -> H Yes
[0625] Variant protein HSSTROL3_P7 (SEQ ID NO:361) is encoded by
the following transcript(s): HSSTROL3_T10 (SEQ ID NO:37), for which
the sequence(s) is/are given at the end of the application. The
coding portion of transcript HSSTROL3_T10 (SEQ ID NO:37) is shown
in bold; this coding portion starts at position 24 and ends at
position 1133. The transcript also has the following SNPs as listed
in Table 72 (given according to their position on the nucleotide
sequence, with the alternative nucleic acid listed; the last column
indicates whether the SNP is known or not; the presence of known
SNPs in variant protein HSSTROL3_P7 (SEQ ID NO:361) sequence
provides support for the deduced sequence of this variant protein
according to the present invention). TABLE-US-00083 TABLE 72
Nucleic acid SNPs SNP position on Alternative Previously nucleotide
sequence nucleic acid known SNP? 136 T -> C Yes 334 G -> C
Yes 663 G -> No 699 -> T No 992 G -> C Yes 1386 A -> G
Yes 1568 A -> G Yes 2109 A -> G Yes 2250 C -> No 2302 C
-> A Yes 2328 A -> T Yes 2545 -> G No 2554 -> G No 2568
C -> No 2587 -> A No 2613 T -> C No 2671 A -> No 2671 A
-> C No 2821 A -> No 2821 A -> C No 2851 T -> C Yes
2998 -> T No
[0626] Variant protein HSSTROL3_P8 (SEQ ID NO:362) according to the
present invention has an amino acid sequence as given at the end of
the application; it is encoded by transcript(s) HSSTROL3_T11 (SEQ
ID NO:38). An alignment is given to the known protein
(Stromelysin-3 precursor (SEQ ID NO:391)) at the end of the
application. One or more alignments to one or more previously
published protein sequences are given at the end of the
application. A brief description of the relationship of the variant
protein according to the present invention to each such aligned
protein is as follows:
[0627] Comparison report between HSSTROL3_P8 (SEQ ID NO:362) and
MM11_HUMAN:
[0628] 1. An isolated chimeric polypeptide encoding for HSSTROL3_P8
(SEQ ID NO:362) comprising a first amino acid sequence being at
least 90% homologous to
MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQPWHAALPSS
PAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVLSGGRWEKTDLTYRILRFP
WQLVQEQVRQTMAEALKVWSDVTPLTFTEVHEGRADIMIDFARYW corresponding to
amino acids 1-163 of MM 11_HUMAN, which also corresponds to amino
acids 1-163 of HSSTROL3_P8 (SEQ ID NO:362), a bridging amino acid H
corresponding to amino acid 164 of HSSTROL3_P8 (SEQ ID NO:362), a
second amino acid sequence being at least 90% homologous to
GDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGHVLG
LQHTTAAKALMSAFYTFRYPLSLSPDDCRGVQHLYGQPWPTVTSRTPALGPQAGIDTN EIAPLE
corresponding to amino acids 165-286 of MM11_HUMAN, which also
corresponds to amino acids 165-286 of HSSTROL3_P8 (SEQ ID NO:362),
and a third amino acid sequence being at least 70%, optionally at
least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having
the sequence VRPCLPVPLLLCWPL (SEQ ID NO: 567) corresponding to
amino acids 287-301 of HSSTROL3_P8 (SEQ ID NO:362), wherein said
first amino acid sequence, bridging amino acid, second amino acid
sequence and third amino acid sequence are contiguous and in a
sequential order.
[0629] 2. An isolated polypeptide encoding for a tail of
HSSTROL3_P8 (SEQ ID NO:362), comprising a polypeptide being at
least 70%, optionally at least about 80%, preferably at least about
85%, more preferably at least about 90% and most preferably at
least about 95% homologous to the sequence VRPCLPVPLLLCWPL (SEQ ID
NO: 567) in HSSTROL3_P8 (SEQ ID NO:362).
[0630] The location of the variant protein was determined according
to results from a number of different software programs and
analyses, including analyses from SignalP and other specialized
programs. The variant protein is believed to be located as follows
with regard to the cell: secreted. The protein localization is
believed to be secreted because both signal-peptide prediction
programs predict that this protein has a signal peptide, and
neither trans-membrane region prediction program predicts that this
protein has a trans-membrane region.
[0631] Variant protein HSSTROL3_P8 (SEQ ID NO:362) also has the
following non-silent SNPs (Single Nucleotide Polymorphisms) as
listed in Table 73, (given according to their position(s) on the
amino acid sequence, with the alternative amino acid(s) listed; the
last column indicates whether the SNP is known or not; the presence
of known SNPs in variant protein HSSTROL3_P8 (SEQ ID NO:362)
sequence provides support for the deduced sequence of this variant
protein according to the present invention). TABLE-US-00084 TABLE
73 Amino acid mutations SNP position(s) on Alternative Previously
amino acid sequence amino acid(s) known SNP? 38 V -> A Yes 104 R
-> P Yes 214 A -> No
[0632] Variant protein HSSTROL3_P8 (SEQ ID NO:362) is encoded by
the following transcript(s): HSSTROL3_T11 (SEQ ID NO:38), for which
the sequence(s) is/are given at the end of the application. The
coding portion of transcript HSSTROL3_T11 (SEQ ID NO:38) is shown
in bold; this coding portion starts at position 24 and ends at
position 926. The transcript also has the following SNPs as listed
in Table 74 (given according to their position on the nucleotide
sequence, with the alternative nucleic acid listed; the last column
indicates whether the SNP is known or not; the presence of known
SNPs in variant protein HSSTROL3_P8 (SEQ ID NO:362) sequence
provides support for the deduced sequence of this variant protein
according to the present invention). TABLE-US-00085 TABLE 74
Nucleic acid SNPs SNP position on Alternative Previously nucleotide
sequence nucleic acid known SNP? 136 T -> C Yes 334 G -> C
Yes 663 G -> No 699 -> T No 935 G -> A Yes 948 G -> A
Yes 1084 G -> C Yes 1557 C -> No 1609 C -> A Yes 1635 A
-> T Yes 1852 -> G No 1861 -> G No 1875 C -> No 1894
-> A No 1920 T -> C No 1978 A -> No 1978 A -> C No 2128
A -> No 2128 A -> C No 2158 T -> C Yes 2305 -> T No
[0633] Variant protein HSSTROL3_P9 (SEQ ID NO:363) according to the
present invention has an amino acid sequence as given at the end of
the application; it is encoded by transcript(s) HSSTROL3_T12 (SEQ
ID NO:39). An alignment is given to the known protein
(Stromelysin-3 precursor (SEQ ID NO:391) at the end of the
application. One or more alignments to one or more previously
published protein sequences are given at the end of the
application. A brief description of the relationship of the variant
protein according to the present invention to each such aligned
protein is as follows:
[0634] Comparison report between HSSTROL3_P9 (SEQ ID NO:363) and
MM11_HUMAN:
[0635] 1. An isolated chimeric polypeptide encoding for HSSTROL3_P9
(SEQ ID NO:363) comprising a first amino acid sequence being at
least 90% homologous to
MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQPWHAALPSS
PAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQK corresponding to amino acids
1-96 of MM11_HUMAN, which also corresponds to amino acids 1-96 of
HSSTROL3_P9 (SEQ ID NO:363), a second amino acid sequence being at
least 90% homologous to
RILRFPWQLVQEQVRQTMAEALKVWSDVTPLTFTEVHEGRADIMIDFARYW corresponding
to amino acids 113-163 of MM11_HUMAN, which also corresponds to
amino acids 97-147 of HSSTROL3_P9 (SEQ ID NO:363), a bridging amino
acid H corresponding to amino acid 148 of HSSTROL3_P9 (SEQ ID
NO:363), a third amino acid sequence being at least 90% homologous
to GDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWTIGDDQGTDLLQVAAHEFGHVLG
LQHTTAAKALMSAFYTFRYPLSLSPDDCRGVQHLYGQPWPTVTSRTPALGPQAGIDTN
EIAPLEPDAPPDACEASFDAVSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGL
PSPVDAAFEDAQGHIWFFQG corresponding to amino acids 165-359 of
MM11_HUMAN, which also corresponds to amino acids 149-343 of
HSSTROL3_P9 (SEQ ID NO:363), and a fourth amino acid sequence being
at least 70%, optionally at least 80%, preferably at least 85%,
more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence TTGVSTPAPGV (SEQ ID
NO: 566) corresponding to amino acids 344-354 of HSSTROL3_P9 (SEQ
ID NO:363), wherein said first amino acid sequence, second amino
acid sequence, bridging amino acid, third amino acid sequence and
fourth amino acid sequence are contiguous and in a sequential
order.
[0636] 2. An isolated chimeric polypeptide encoding for an edge
portion of HSSTROL3_P9 (SEQ ID NO:363), comprising a polypeptide
having a length "n", wherein n is at least about 10 amino acids in
length, optionally at least about 20 amino acids in length,
preferably at least about 30 amino acids in length, more preferably
at least about 40 amino acids in length and most preferably at
least about 50 amino acids in length, wherein at least two amino
acids comprise KR, having a structure as follows: a sequence
starting from any of amino acid numbers 96-x to 96; and ending at
any of amino acid numbers 97+((n-2)-x), in which x varies from 0 to
n-2.
[0637] 3. An isolated polypeptide encoding for a tail of
HSSTROL3_P9 (SEQ ID NO:363) comprising a polypeptide being at least
70%, optionally at least about 80%, preferably at least about 85%,
more preferably at least about 90% and most preferably at least
about 95% homologous to the sequence TTGVSTPAPGV (SEQ ID NO: 566)
in HSSTROL3_P9 (SEQ ID NO:363).
[0638] The location of the variant protein was determined according
to results from a number of different software programs and
analyses, including analyses from SignalP and other specialized
programs. The variant protein is believed to be located as follows
with regard to the cell: secreted. The protein localization is
believed to be secreted because both signal-peptide prediction
programs predict that this protein has a signal peptide, and
neither trans-membrane region prediction program predicts that this
protein has a trans-membrane region.
[0639] Variant protein HSSTROL3_P9 (SEQ ID NO:363) also has the
following non-silent SNPs (Single Nucleotide Polymorphisms) as
listed in Table 75, (given according to their position(s) on the
amino acid sequence, with the alternative amino acid(s) listed; the
last column indicates whether the SNP is known or not; the presence
of known SNPs in variant protein HSSTROL3_P9 (SEQ ID NO:363)
sequence provides support for the deduced sequence of this variant
protein according to the present invention). TABLE-US-00086 TABLE
75 Amino acid mutations SNP position(s) on amino acid Previously
sequence Alternative amino acid(s) known SNP? 38 V -> A Yes 198
A -> No 307 Q -> H Yes
[0640] Variant protein HSSTROL3_P9 (SEQ ID NO:363) is encoded by
the following transcript(s): HSSTROL3_T12 (SEQ ID NO:39), for which
the sequence(s) is/are given at the end of the application. The
coding portion of transcript HSSTROL3_T12 (SEQ ID NO:39) is shown
in bold; this coding portion starts at position 24 and ends at
position 1085. The transcript also has the following SNPs as listed
in Table 76 (given according to their position on the nucleotide
sequence, with the alternative nucleic acid listed; the last column
indicates whether the SNP is known or not; the presence of known
SNPs in variant protein HSSTROL3_P9 (SEQ ID NO:363) sequence
provides support for the deduced sequence of this variant protein
according to the present invention). TABLE-US-00087 TABLE 76
Nucleic acid SNPs SNP position on nucleotide Previously sequence
Alternative nucleic acid known SNP? 136 T -> C Yes 615 G ->
No 651 -> T No 944 G -> C Yes 1275 C -> No 1327 C -> A
Yes 1353 A -> T Yes 1570 -> G No 1579 -> G No 1593 C ->
No 1612 -> A No 1638 T -> C No 1696 A -> No 1696 A -> C
No 1846 A -> No 1846 A -> C No 1876 T -> C Yes 2023 ->
T No
[0641] As noted above, cluster HSSTROL3 features 16 segment(s),
which were listed in Table 63 above and for which the sequence(s)
are given at the end of the application. These segment(s) are
portions of nucleic acid sequence(s) which are described herein
separately because they are of particular interest. A description
of each segment according to the present invention is now
provided.
[0642] Segment cluster HSSTROL3_node.sub.--6 (SEQ ID NO:222)
according to the present invention is supported by 14 libraries.
The number of libraries was determined as previously described.
This segment can be found in the following transcript(s):
HSSTROL3_T5 (SEQ ID NO:34), HSSTROL3_T8 (SEQ ID NO:35), HSSTROL3_T9
(SEQ ID NO:36), HSSTROL3_T10 (SEQ ID NO:37), HSSTROL3_T11 (SEQ ID
NO:38) and HSSTROL3_T12 (SEQ ID NO:39). Table 77 below describes
the starting and ending position of this segment on each
transcript. TABLE-US-00088 TABLE 77 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HSSTROL3_T5 (SEQ ID NO: 34) 1 131 HSSTROL3_T8 (SEQ ID NO: 35) 1 131
HSSTROL3_T9 (SEQ ID NO: 36) 1 131 HSSTROL3_T10 (SEQ ID NO: 37) 1
131 HSSTROL3_T11 (SEQ ID NO: 38) 1 131 HSSTROL3_T12 (SEQ ID NO: 39)
1 131
[0643] Segment cluster HSSTROL3_node.sub.--10 (SEQ ID NO:223)
according to the present invention is supported by 21 libraries.
The number of libraries was determined as previously described.
This segment can be found in the following transcript(s):
HSSTROL3_T5 (SEQ ID NO:34), HSSTROL3_T8 (SEQ ID NO:35), HSSTROL3_T9
(SEQ ID NO:36), HSSTROL3_T10 (SEQ ID NO:37), HSSTROL3_T11 (SEQ ID
NO:38) and HSSTROL3_T12 (SEQ ID NO:39). Table 78 below describes
the starting and ending position of this segment on each
transcript. TABLE-US-00089 TABLE 78 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HSSTROL3_T5 (SEQ ID NO: 34) 132 313 HSSTROL3_T8 (SEQ ID NO: 35) 132
313 HSSTROL3_T9 (SEQ ID NO: 36) 132 313 HSSTROL3_T10 (SEQ ID NO:
37) 132 313 HSSTROL3_T11 (SEQ ID NO: 38) 132 313 HSSTROL3_T12 (SEQ
ID NO: 39) 132 313
[0644] Segment cluster HSSTROL3_node.sub.--13 (SEQ ID NO:224)
according to the present invention is supported by 36 libraries.
The number of libraries was determined as previously described.
This segment can be found in the following transcript(s):
HSSTROL3_T5 (SEQ ID NO:34), HSSTROL3_T8 (SEQ ID NO:35), HSSTROL3_T9
(SEQ ID NO:36) HSSTROL3_T10 (SEQ ID NO:37), HSSTROL3_T11 (SEQ ID
NO:38) and HSSTROL3_T12 (SEQ ID NO:39). Table 79 below describes
the starting and ending position of this segment on each
transcript. TABLE-US-00090 TABLE 79 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HSSTROL3_T5 (SEQ ID NO: 34) 362 505 HSSTROL3_T8 (SEQ ID NO: 35) 362
505 HSSTROL3_T9 (SEQ ID NO: 36) 362 505 HSSTROL3_T10 (SEQ ID NO:
37) 362 505 HSSTROL3_T11 (SEQ ID NO: 38) 362 505 HSSTROL3_T12 (SEQ
ID NO: 39) 314 457
[0645] Segment cluster HSSTROL3_node.sub.--15 (SEQ ID NO:225)
according to the present invention is supported by 47 libraries.
The number of libraries was determined as previously described.
This segment can be found in the following transcript(s):
HSSTROL3_T5 (SEQ ID NO:34), HSSTROL3_T8 (SEQ ID NO:35), HSSTROL3_T9
(SEQ ID NO:36), HSSTROL3_T10 (SEQ ID NO:37), HSSTROL3_T11 (SEQ ID
NO:38) and HSSTROL3_T12 (SEQ ID NO:39). Table 80 below describes
the starting and ending position of this segment on each
transcript. TABLE-US-00091 TABLE 80 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HSSTROL3_T5 (SEQ ID NO: 34) 506 639 HSSTROL3_T8 (SEQ ID NO: 35) 506
639 HSSTROL3_T9 (SEQ ID NO: 36) 506 639 HSSTROL3_T10 (SEQ ID NO:
37) 506 639 HSSTROL3_T11 (SEQ ID NO: 38) 506 639 HSSTROL3_T12 (SEQ
ID NO: 39) 458 591
[0646] Segment cluster HSSTROL3_node.sub.--19 (SEQ ID NO:226)
according to the present invention is supported by 63 libraries.
The number of libraries was determined as previously described.
This segment can be found in the following transcript(s):
HSSTROL3_T5 (SEQ ID NO:34), HSSTROL3_T8 (SEQ ID NO:35), HSSTROL3_T9
(SEQ ID NO:36), HSSTROL3_T10 (SEQ ID NO:37), HSSTROL3_T11 (SEQ ID
NO:38) and HSSTROL3_T12 (SEQ ID NO:39). Table 81 below describes
the starting and ending position of this segment on each
transcript. TABLE-US-00092 TABLE 81 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HSSTROL3_T5 (SEQ ID NO: 34) 699 881 HSSTROL3_T8 (SEQ ID NO: 35) 699
881 HSSTROL3_T9 (SEQ ID NO: 36) 699 881 HSSTROL3_T10 (SEQ ID NO:
37) 699 881 HSSTROL3_T11 (SEQ ID NO: 38) 699 881 HSSTROL3_T12 (SEQ
ID NO: 39) 651 833
[0647] Segment cluster HSSTROL3_node.sub.--21 (SEQ ID NO:227)
according to the present invention is supported by 61 libraries.
The number of libraries was determined as previously described.
This segment can be found in the following transcript(s):
HSSTROL3_T5 (SEQ ID NO:34), HSSTROL3_T8 (SEQ ID NO:35), HSSTROL3_T9
(SEQ ID NO:36), HSSTROL3_T10 (SEQ ID NO:37), HSSTROL3_T11 (SEQ ID
NO:38) and HSSTROL3_T12 (SEQ ID NO:39). Table 82 below describes
the starting and ending position of this segment on each
transcript. TABLE-US-00093 TABLE 82 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HSSTROL3_T5 (SEQ ID NO: 34) 882 1098 HSSTROL3_T8 (SEQ ID NO: 35)
882 1098 HSSTROL3_T9 (SEQ ID NO: 36) 882 1098 HSSTROL3_T10 (SEQ ID
NO: 37) 882 1098 HSSTROL3_T11 (SEQ ID NO: 38) 974 1190 HSSTROL3_T12
(SEQ ID NO: 39) 834 1050
[0648] Segment cluster HSSTROL3_node.sub.--24 (SEQ ID NO:228)
according to the present invention is supported by 7 libraries. The
number of libraries was determined as previously described. This
segment can be found in the following transcript(s): HSSTROL3_T8
(SEQ ID NO:35) and HSSTROL3_T9 (SEQ ID NO:36). Table 83 below
describes the starting and ending position of this segment on each
transcript. TABLE-US-00094 TABLE 83 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HSSTROL3_T8 (SEQ ID NO: 35) 1099 1236 HSSTROL3_T9 (SEQ ID NO: 36)
1099 1236
[0649] Segment cluster HSSTROL3_node.sub.--25 (SEQ ID NO:229)
according to the present invention is supported by 13 libraries.
The number of libraries was determined as previously described.
This segment can be found in the following transcript(s):
HSSTROL3_T8 (SEQ ID NO:35). Table 84 below describes the starting
and ending position of this segment on each transcript.
TABLE-US-00095 TABLE 84 Segment location on transcripts Segment
Segment Transcript name starting position ending position
HSSTROL3_T8 (SEQ ID NO: 35) 1237 1536
[0650] Segment cluster HSSTROL3_node.sub.--26 (SEQ ID NO:230)
according to the present invention is supported by 55 libraries.
The number of libraries was determined as previously described.
This segment can be found in the following transcript(s):
HSSTROL3_T5 (SEQ ID NO:34), HSSTROL3_T8 (SEQ ID NO:35), HSSTROL3_T9
(SEQ ID NO:36) and HSSTROL3_T111 (SEQ ID NO:38). Table 85 below
describes the starting and ending position of this segment on each
transcript. TABLE-US-00096 TABLE 85 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HSSTROL3_T5 (SEQ ID NO: 34) 1099 1240 HSSTROL3_T8 (SEQ ID NO: 35)
1537 1678 HSSTROL3_T9 (SEQ ID NO: 36) 1237 1378 HSSTROL3_T11 (SEQ
ID NO: 38) 1191 1332
[0651] Segment cluster HSSTROL3_node.sub.--28 (SEQ ID NO:231)
according to the present invention is supported by 10 libraries.
The number of libraries was determined as previously described.
This segment can be found in the following transcript(s):
HSSTROL3_T5 (SEQ ID NO:34), HSSTROL3_T9 (SEQ ID NO:36) and
HSSTROL3_T10 (SEQ ID NO:37). Table 86 below describes the starting
and ending position of this segment on each transcript.
TABLE-US-00097 TABLE 86 Segment location on transcripts Segment
Segment Transcript name starting position ending position
HSSTROL3_T5 (SEQ ID NO: 34) 1357 2283 HSSTROL3_T9 (SEQ ID NO: 36)
1495 2421 HSSTROL3_T10 (SEQ ID NO: 37) 1215 2141
[0652] Segment cluster HSSTROL3_node.sub.--29 (SEQ ID NO:232)
according to the present invention is supported by 109 libraries.
The number of libraries was determined as previously described.
This segment can be found in the following transcript(s):
HSSTROL3_T5 (SEQ ID NO:34), HSSTROL3_T8 (SEQ ID NO:35), HSSTROL3_T9
(SEQ ID NO:36), HSSTROL3_T10 (SEQ ID NO:37), HSSTROL3_T11 (SEQ ID
NO:38) and HSSTROL3_T12 (SEQ ID NO:39). Table 87 below describes
the starting and ending position of this segment on each
transcript. TABLE-US-00098 TABLE 87 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HSSTROL3_T5 (SEQ ID NO: 34) 2284 3194 HSSTROL3_T8 (SEQ ID NO: 35)
1795 2705 HSSTROL3_T9 (SEQ ID NO: 36) 2422 3332 HSSTROL3_T10 (SEQ
ID NO: 37) 2142 3052 HSSTROL3_T11 (SEQ ID NO: 38) 1449 2359
HSSTROL3_T12 (SEQ ID NO: 39) 1167 2077
[0653] According to an optional embodiment of the present
invention, short segments related to the above cluster are also
provided. These segments are up to about 120 bp in length, and so
are included in a separate description.
[0654] Segment cluster HSSTROL3_node.sub.--11 (SEQ ID NO:233)
according to the present invention is supported by 25 libraries.
The number of libraries was determined as previously described.
This segment can be found in the following transcript(s):
HSSTROL3_T5 (SEQ ID NO:34), HSSTROL3_T8 (SEQ ID NO:35), HSSTROL3_T9
(SEQ ID NO:36), HSSTROL3_T10 (SEQ ID NO:37) and HSSTROL3_T11 (SEQ
ID NO:38). Table 88 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00099 TABLE
88 Segment location on transcripts Segment Segment Transcript name
starting position ending position HSSTROL3_T5 (SEQ ID NO: 34) 314
361 HSSTROL3_T8 (SEQ ID NO: 35) 314 361 HSSTROL3_T9 (SEQ ID NO: 36)
314 361 HSSTROL3_T10 (SEQ ID NO: 37) 314 361 HSSTROL3_T11 (SEQ ID
NO: 38) 314 361
[0655] Segment cluster HSSTROL3_node.sub.--17 (SEQ ID NO:234)
according to the present invention is supported by 45 libraries.
The number of libraries was determined as previously described.
This segment can be found in the following transcript(s):
HSSTROL3_T5 (SEQ ID NO:34), HSSTROL3_T8 (SEQ ID NO:35), HSSTROL3_T9
(SEQ ID NO:36), HSSTROL3_T10 (SEQ ID NO:37), HSSTROL3_T11 (SEQ ID
NO:38) and HSSTROL3_T12 (SEQ ID NO:39). Table 89 below describes
the starting and ending position of this segment on each
transcript. TABLE-US-00100 TABLE 89 Segment location on transcripts
Segment Segment Transcript name starting position ending position
HSSTROL3_T5 (SEQ ID NO: 34) 640 680 HSSTROL3_T8 (SEQ ID NO: 35) 640
680 HSSTROL3_T9 (SEQ ID NO: 36) 640 680 HSSTROL3_T10 (SEQ ID NO:
37) 640 680 HSSTROL3_T11 (SEQ ID NO: 38) 640 680 HSSTROL3_T12 (SEQ
ID NO: 39) 592 632
[0656] Segment cluster HSSTROL3_node.sub.--18 (SEQ ID NO:235)
according to the present invention can be found in the following
transcript(s): HSSTROL3_T5 (SEQ ID NO:34), HSSTROL3_T8 (SEQ ID
NO:35), HSSTROL3_T9 (SEQ ID NO:36), HSSTROL3_T10 (SEQ ID NO:37),
HSSTROL3_T11 (SEQ ID NO:38) and HSSTROL3_T12 (SEQ ID NO:39). Table
90 below describes the starting and ending position of this segment
on each transcript. TABLE-US-00101 TABLE 90 Segment location on
transcripts Segment Segment Transcript name starting position
ending position HSSTROL3_T5 (SEQ ID NO: 34) 681 698 HSSTROL3_T8
(SEQ ID NO: 35) 681 698 HSSTROL3_T9 (SEQ ID NO: 36) 681 698
HSSTROL3_T10 (SEQ ID NO: 37) 681 698 HSSTROL3_T11 (SEQ ID NO: 38)
681 698 HSSTROL3_T12 (SEQ ID NO: 39) 633 650
[0657] Segment cluster HSSTROL3_node.sub.--20 (SEQ ID NO:236)
according to the present invention is supported by 1 libraries. The
number of libraries was determined as previously described. This
segment can be found in the following transcript(s): HSSTROL3_T11
(SEQ ID NO:38). Table 91 below describes the starting and ending
position of this segment on each transcript. TABLE-US-00102 TABLE
91 Segment location on transcripts Segment Segment Transcript name
starting position ending position HSSTROL3_T11 (SEQ ID NO: 38) 882
973
[0658] Segment cluster HSSTROL3_node.sub.--27 (SEQ ID NO:237)
according to the present invention is supported by 50 libraries.
The number of libraries was determined as previously described.
This segment can be found in the following transcript(s):
HSSTROL3_T5 (SEQ ID NO:34), HSSTROL3_T8 (SEQ ID NO:35), HSSTROL3_T9
(SEQ ID NO:36), HSSTROL3_T10 (SEQ ID NO:37), HSSTROL3_T11 (SEQ ID
NO:38) and HSSTROL3_T12 (SEQ ID NO:39). Table 92 below describes
the starting and ending position of this segment on each
transcript. TABLE-US-00103 TABLE 92 Segment location on transcripts
Segment Segment Transcript name Starting position ending position
HSSTROL3_T5 (SEQ ID NO:34) 1241 1356 HSSTROL3_T8 (SEQ ID NO:35)
1679 1794 HSSTROL3_T9 (SEQ ID NO:36) 1379 1494 HSSTROL3_T10 (SEQ ID
NO:37) 1099 1214 HSSTROL3_T11 (SEQ ID NO:38) 1333 1448 HSSTROL3_T12
(SEQ ID NO:39) 1051 1166
[0659] TABLE-US-00104 Variant protein alignment to the previously
known protein: Sequence name: MM11_HUMAN Sequence documentation:
Alignment of: HSSTROL3_P4 (SEQ ID NO: 359) .times. MM11_HUMAN . .
Alignment segment 1/1: Quality: 4444.00 Escore: 0 Matching length:
445 Total length: 445 Matching Percent 99.78 Matching Percent 99.78
Similarity: Identity: Total Percent 99.78 Total Percent 99.78
Similarity: Identity: Gaps: 0 Alignment: . . . . . 1
MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQP 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 1
MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQP 50 . . . . . 51
WHAALPSSPAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVL 100
|||||||||||||||||||||||||||||||||||||||||||||||||| 51
WHAALPSSPAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVL 100 . . . . .
101 SGGRWEKTDLTYRILRFPWQLVQEQVRQTMAEALKVWSDVTPLTFTEVHE 150
|||||||||||||||||||||||||||||||||||||||||||||||||| 101
SGGRWEKTDLTYRILRFPWQLVQEQVRQTMAEALKVWSDVTPLTFTEVHE 150 . . . . .
151 GRADIMIDFARYWHGDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWT 200
|||||||||||||||||||||||||||||||||||||||||||||||||| 151
GRADIMIDFARYWDGDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWT 200 . . . . .
201 IGDDQGTDLLQVAAHEFGHVLGLQHTTAAKALMSAFYTFRYPLSLSPDDC 250
|||||||||||||||||||||||||||||||||||||||||||||||||| 201
IGDDQGTDLLQVAAHEFGHVLGLQHTTAAKALMSAFYTFRYPLSLSPDDC 250 . . . . .
251 RGVQHLYGQPWPTVTSRTPALGPQAGIDTNEIAPLEPDAPPDACEASFDA 300
|||||||||||||||||||||||||||||||||||||||||||||||||| 251
RGVQHLYGQPWPTVTSRTPALGPQAGIDTNEIAPLEPDAPPDACEASFDA 300 . . . . .
301 VSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGLPSPVDAAFEDA 350
|||||||||||||||||||||||||||||||||||||||||||||||||| 301
VSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGLPSPVDAAFEDA 350 . . . . .
351 QGHIWFFQGAQYWVYDGEKPVLGPAPLTELGLVRFPVHAALVWGPEKNKI 400
|||||||||||||||||||||||||||||||||||||||||||||||||| 351
QGHIWFFQGAQYWVYDGEKPVLGPAPLTELGLVRFPVHAALVWGPEKNKI 400 . . . . 401
YFFRGRDYWRFHPSTRRVDSPVPRRATDWRGVPSEIDAAFQDADG 445
||||||||||||||||||||||||||||||||||||||||||||| 401
YFFRGRDYWRFHPSTRRVDSPVPRRATDWRGVPSEIDAAFQDADG 445 Sequence name:
MM11_HUMAN Sequence documentation: Alignment of: HSSTROL3_P5 (SEQ
ID NO: 360) .times. MM11_HUMAN . . Alignment segment 1/1: Quality:
3566.00 Escore: 0 Matching length: 358 Total length: 358 Matching
Percent 99.72 Matching Percent 99.72 Similarity: Identity: Total
Percent 99.72 Total Percent 99.72 Similarity: Identity: Gaps: 0
Alignment: . . . . . 1
MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQP 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 1
MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQP 50 . . . . . 51
WHAALPSSPAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVL 100
|||||||||||||||||||||||||||||||||||||||||||||||||| 51
WHAALPSSPAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVL 100 . . . . .
101 SGGRWEKTDLTYRILRFPWQLVQEQVRQTMAEALKVWSDVTPLTFTEVHE 150
|||||||||||||||||||||||||||||||||||||||||||||||||| 101
SGGRWEKTDLTYRILRFPWQLVQEQVRQTMAEALKVWSDVTPLTFTEVHE 150 . . . . .
151 GRADIMIDFARYWHGDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWT 200
|||||||||||||||||||||||||||||||||||||||||||||||||| 151
GRADIMIDFARYWDGDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWT 200 . . . . .
201 IGDDQGTDLLQVAAHEFGHVLGLQHTTAAKALMSAFYTFRYPLSLSPDDC 250
|||||||||||||||||||||||||||||||||||||||||||||||||| 201
IGDDQGTDLLQVAAHEFGHVLGLQHTTAAKALMSAFYTFRYPLSLSPDDC 250 . . . . .
251 RGVQHLYGQPWPTVTSRTPALGPQAGIDTNEIAPLEPDAPPDACEASFDA 300
|||||||||||||||||||||||||||||||||||||||||||||||||| 251
RGVQHLYGQPWPTVTSRTPALGPQAGIDTNEIAPLEPDAPPDACEASFDA 300 . . . . .
301 VSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGLPSPVDAAFEDA 350
|||||||||||||||||||||||||||||||||||||||||||||||||| 301
VSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGLPSPVDAAFEDA 350 351 QGHIWFFQ
358 |||||||| 351 QGHIWFFQ 358 Sequence name: MM11_HUMAN Sequence
documentation: Alignment of: HSSTROL3_P7 (SEQ ID NO: 361) .times.
MM11_HUMAN . . Alignment segment 1/1: Quality: 3575.00 Escore: 0
Matching length: 359 Total length: 359 Matching Percent 99.72
Matching Percent 99.72 Similarity: Identity: Total Percent 99.72
Total Percent 99.72 Similarity: Identity: Gaps: 0 Alignment: . . .
. . 1 MAPAAWLRSAAAPALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQP 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 1
MAPAAWLRSAAAPALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQP 50 . . . . . 51
WHAALPSSPAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVL 100
|||||||||||||||||||||||||||||||||||||||||||||||||| 51
WHAALPSSPAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVL 100 . . . . .
101 SGGRWEKTDLTYRILRFPWQLVQEQVRQTMAEALKVWSDVTPLTFTEVHE 150
|||||||||||||||||||||||||||||||||||||||||||||||||| 101
SGGRWEKTDLTYRILRFPWQLVQEQVRQTMAEALKVWSDVTPLTFTEVHE 150 . . . . .
151 GRADIMIDFARYWHGDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWT 200
|||||||||||||||||||||||||||||||||||||||||||||||||| 151
GRADIMIDFARYWDGDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWT 200 . . . . .
201 IGDDQGTDLLQVAAHEFGHVLGLQHTTAAKALMSAFYTFRYPLSLSPDDC 250
|||||||||||||||||||||||||||||||||||||||||||||||||| 201
IGDDQGTDLLQVAAHEFGHVLGLQHTTAAKALMSAFYTFRYPLSLSPDDC 250 . . . . .
251 RGVQHLYGQPWPTVTSRTPALGPQAGIDTNEIAPLEPDAPPDACEASFDA 300
|||||||||||||||||||||||||||||||||||||||||||||||||| 251
RGVQHLYGQPWPTVTSRTPALGPQAGIDTNEIAPLEPDAPPDACEASFDA 300 . . . . .
301 VSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGLPSPVDAAFEDA 350
|||||||||||||||||||||||||||||||||||||||||||||||||| 301
VSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGLPSPVDAAFEDA 350 351
QGHIWFFQG 359 ||||||||| 351 QGHIWFFQG 359 Sequence name: MM11_HUMAN
Sequence documentation: Alignment of: HSSTROL3_P8 (SEQ ID NO: 362)
.times. MM11_HUMAN . . Alignment segment 1/1: Quality: 2838.00
Escore: 0 Matching length: 286 Total length: 286 Matching Percent
99.65 Matching Percent 99.65 Similarity: Identity: Total Percent
99.65 Total Percent 99.65 Similarity: Identity: Gaps: 0 Alignment:
. . . . . 1 MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQP 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 1
MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQP 50 . . . . . 51
WHAALPSSPAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVL 100
|||||||||||||||||||||||||||||||||||||||||||||||||| 51
WHAALPSSPAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVL 100 . . . . .
101 SGGRWEKTDLTYRILRFPWQLVQEQVRQTMAEALKVWSDVTPLTFTEVHE 150
|||||||||||||||||||||||||||||||||||||||||||||||||| 101
SGGRWEKTDLTYRILRFPWQLVQEQVRQTMAEALKVWSDVTPLTFTEVHE 150 . . . . .
151 GPADIMIDFARYWHGDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWT 200
|||||||||||||||||||||||||||||||||||||||||||||||||| 151
GRADIMIDFARYWDGDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWT 200 . . . . .
201 IGDDQGTDLLQVAAHEFGHVLGLQHTTAAKALMSAFYTFRYPLSLSPDDC 250
|||||||||||||||||||||||||||||||||||||||||||||||||| 201
IGDDQGTDLLQVAAHEFGHVLGLQHTTAAKALMSAFYTFRYPLSLSPDDC 250 . . . 251
RGVQHLYGQPWPTVTSRTPALGPQAGIDTNEIAPLE 286
|||||||||||||||||||||||||||||||||||| 251
RGVQHLYGQPWPTVTSRTPALGPQAGIDTNEIAPLE 286 Sequence name: MM11_HUMAN
Sequence documentation:
Alignment of: HSSTROL3_P9 (SEQ ID NO: 363) .times. MM11_HUMAN . .
Alignment segment 1/1: Quality: 3316.00 Escore: 0 Matching length:
343 Total length: 359 Matching Percent 99.71 Matching Percent 99.71
Similarity: Identity: Total Percent 95.26 Total Percent 95.26
Similarity: Identity: Gaps: 1 Alignment: . . . . . 1
MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQP 50
|||||||||||||||||||||||||||||||||||||||||||||||||| 1
MAPAAWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQP 50 . . . . . 51
WHAALPSSPAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQK.... 96
|||||||||||||||||||||||||||||||||||||||||||||| 51
WHAALPSSPAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVL 100 . . . . . 97
............RILRFPWQLVQEQVRQTMAEALKVWSDVTPLTFTEVHE 134
|||||||||||||||||||||||||||||||||||||| 101
SGGRWEKTDLTYRILRFPWQLVQEQVRQTMAEALKVWSDVTPLTFTEVHE 150 . . . . .
135 GRADIMIDFARYWHGDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWT 184
||||||||||||| |||||||||||||||||||||||||||||||||||| 151
GRADIMIDFARYWDGDDLPFDGPGGILAHAFFPKTHREGDVHFDYDETWT 200 . . . . .
185 IGDDQGTDLLQVAAHEFGHVLGLQHTTAAKALMSAFYTFRYPLSLSPDDC 234
|||||||||||||||||||||||||||||||||||||||||||||||||| 201
IGDDQGTDLLQVAAHEFGHVLGLQHTTAAKALMSAFYTFRYPLSLSPDDC 250 . . . . .
235 RGVQHLYGQPWPTVTSRTPALGPQAGIDTNEIAPLEPDAPPDACEASFDA 284
|||||||||||||||||||||||||||||||||||||||||||||||||| 251
RGVQHLYGQPWPTVTSRTPALGPQAGIDTNEIAPLEPDAPPDACEASFDA 300 . . . . .
285 VSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGLPSPVDAAFEDA 334
|||||||||||||||||||||||||||||||||||||||||||||||||| 301
VSTIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGLPSPVDAAFEDA 350 335
QGHIWFFQG 343 ||||||||| 351 QGHIWFFQG 359
Expression of Stromelysin-3 precursor (SEQ ID NO:391) (EC 3.4.24.-)
(Matrix metalloproteinase-11) (MMP-11) (ST3) (SL-3HSSTROL3)
transcripts which are detectable by amplicon as depicted in
sequence name HSSTROL3 seg24 (SEQ ID NO:499) in normal and
cancerous Prostate tissues
[0660] Expression of Stromelysin-3 precursor (SEQ ID NO:391) (EC
3.4.24.-) (Matrix metalloproteinase-11) (MMP-11) (ST3) (SL-3)
transcripts detectable by or according to seg24, HSSTROL3 seg24
(SEQ ID NO:499) amplicon(s) and HSSTROL3 seg24F (SEQ ID NO:497) and
HSSTROL3 seg24R (SEQ ID NO:498) primers was measured by real time
PCR. In parallel the expression of four housekeeping genes --PBGD
(GenBank Accession No. BC019323 (SEQ ID NO:509);
amplicon--PBGD-amplicon (SEQ ID NO:404), HPRT1 (GenBank Accession
No. NM.sub.--000194 (SEQ ID NO:510); amplicon--HPRT1-amplicon (SEQ
ID NO:401), SDHA (GenBank Accession No. NM.sub.--004168 (SEQ ID
NO:508); amplicon--SDHA-amplicon (SEQ ID NO:407), and RPL19
(GenBank Accession No. NM.sub.--000981 (SEQ ID NO:511); RPL19
amplicon (SEQ ID NO:410) was measured similarly. For each RT
sample, the expression of the above amplicon was normalized to the
geometric mean of the quantities of the housekeeping genes. The
normalized quantity of each RT sample was then divided by the
median of the quantities of the normal post-mortem (PM) samples
(Sample Nos. 42, 48-53, 59-63, Table 1, "Tissue samples in testing
panel", above), to obtain a value of fold up-regulation for each
sample relative to median of the normal PM samples.
[0661] FIG. 12 is a histogram showing over expression of the
above-indicated Stromelysin-3 precursor (SEQ ID NO:391) transcripts
in cancerous Prostate samples relative to the normal samples.
Values represent the average of duplicate experiments. Error bars
indicate the minimal and maximal values obtained.)
[0662] As is evident from FIG. 12, the expression of Stromelysin-3
precursor (SEQ ID NO:391) transcripts detectable by the above
amplicon(s) in cancer samples was higher than in several
non-cancerous samples (Sample Nos. 42, 48-53, 59-63, Table 1,
"Tissue samples in testing panel"). Notably an over-expression of
at least 3 fold was found in 4 out of 19 adenocarcinoma
samples.
[0663] Statistical analysis was applied to verify the significance
of these results, as described below.
[0664] The P value for the difference in the expression levels of
Stromelysin-3 precursor (SEQ ID NO:391) transcripts detectable by
the above amplicon(s) in Prostate cancer samples versus the normal
tissue samples was determined by T test as 2.34E-03.
[0665] The above value demonstrate statistical significance of the
results.
[0666] Primer pairs are also optionally and preferably encompassed
within the present invention; for example, for the above
experiment, the following primer pair was used as a non-limiting
illustrative example only of a suitable primer pair: HSSTROL3
seg24F forward primer (SEQ ID NO:497); and HSSTROL3 seg24R reverse
primer (SEQ ID NO:498).
[0667] The present invention also preferably encompasses any
amplicon obtained through the use of any suitable primer pair; for
example, for the above experiment, the following amplicon was
obtained as a non-limiting illustrative example only of a suitable
amplicon: HSSTROL seg24 (SEQ ID NO:499). TABLE-US-00105 HSSTROL
seg24 Forward primer (SEQ ID NO: 497): ATTTCCATCCTCAACTGGCAGA
HSSTROL seg24 Reverse primer (SEQ ID NO: 498): TGCCCTGGAACCCACG
HSSTROL seg24 Amplicon (SEQ ID NO: 499):
ATTTCCATCCTCAACTGGCAGAGATGAGAGCCTGGAGCATTGCAGATGCC
AGGGACTTCACAAATGAAGGCACAGCATGGGAAACCTGCGTGGGTTCCAG GGCA
Expression of Stromelysin-3 precursor (SEQ ID NO:391) transcripts
which are detectable by amplicon as depicted in sequence name
HSSTROL3 seg24 (SEQ ID NO:499) in different normal tissues
[0668] Expression of Stromelysin-3 precursor (SEQ ID NO:391)
transcripts detectable by or according to HSSTROL3 seg24 (SEQ ID
NO:499) amplicon(s) and HSSTROL3 seg24F (SEQ ID NO:497) and
HSSTROL3 seg24R (SEQ ID NO:498) was measured by real time PCR. In
parallel the expression of four housekeeping genes Ubiquitin
(GenBank Accession No. BC000449 (SEQ ID NO:516);
amplicon--Ubiquitin-amplicon (SEQ ID NO:519) and SDHA (GenBank
Accession No. NM.sub.--004168 (SEQ ID NO:508);
amplicon--SDHA-amplicon (SEQ ID NO:407), RPL19 (GenBank Accession
No. NM.sub.--000981 (SEQ ID NO:511); RPL19 amplicon (SEQ ID
NO:410)), TATA box (GenBank Accession No. NM.sub.--003194 (SEQ ID
NO:512); TATA amplicon (SEQ ID NO:515)) was measured similarly. For
each RT sample, the expression of the above amplicon was normalized
to the geometric mean of the quantities of the housekeeping genes.
The normalized quantity of each RT sample was then divided by the
median of the quantities of the lung samples (Sample Nos. 15-17,
Table 2 "Tissue samples in normal panel", above), to obtain a value
of relative expression of each sample relative to median of the
lung samples.
[0669] The results are shown in FIG. 13, demonstrating the
expression of Stromelysin-3 transcripts which are detectable by
amplicon as depicted in sequence name HSSTROL3 seg24 (SEQ ID
NO:499) in different normal tissues.
Expression of Thrombospondin 1 (THBS1) Transcripts which are
Detectable by SEQ ID NO:421 in Normal, Benign and Cancerous
Prostate Tissues
[0670] Expression of Thrombospondin 1 (THBS1) transcripts
detectable by SEQ ID NO:421, segment 24 (e.g., variants no. 10,11
and 30; SEQ ID NOs: 441, 442, and 451) was measured by real time
PCR, according to the exemplary marker HUMTHROM-segment 24 (SEQ ID
NO:425). In parallel the expression of four housekeeping
genes--PBGD (GenBank Accession No. BC019323 (SEQ ID NO:509);
amplicon--SEQ ID NO:404), HPRT1 (GenBank Accession No.
NM.sub.--000194 (SEQ ID NO:510); amplicon--SEQ ID NO:401), RPL19
(GenBank Accession No. NM.sub.--000981 (SEQ ID NO:511);
amplicon--SEQ ID NO:410) and SDHA (GenBank Accession No.
NM.sub.--004168 (SEQ ID NO:508); amplicon--SEQ ID NO:407), was
measured similarly. For each RT sample, the expression of SEQ ID
NO:421 was normalized to the geometric mean of the quantities of
the housekeeping genes. The normalized quantity of each RT sample
was then divided by the median of the quantities of the normal
post-mortem (PM) samples (Sample Nos. 42, 48-53, 59-63, Table 2,
above), to obtain a value of fold up-regulation for each sample
relative to median of the normal PM samples.
[0671] FIG. 14 is a histogram showing over expression of the
above-indicated Thrombospondin 1 (THBS1) transcripts in cancerous
and benign (BPH) prostate samples relative to the normal samples.
The number and percentage of cancer samples that exhibit at least 3
fold over-expression, out of the total number of samples tested is
indicated in the bottom.
[0672] As is evident from FIG. 14, the expression of Thrombospondin
1 (THBS1) transcripts detectable by SEQ ID NO:421 in cancer samples
was significantly higher than in the normal PM samples (Sample Nos.
42, 48-53, 59-63, Table 2). Notably an over-expression of at least
3 fold was found in 10 out of 19 adenocarcinoma samples. Over
expression of at least 3 fold was observed also in 7 out of the 8
BPH samples, and in the 2 matched normal samples. Since matched
samples are histologically non-cancerous tissue that surrounds the
tumor, such samples could have been contaminated with cancer or
pre-cancer cells.
[0673] Statistical analysis was applied to verify the significance
of these results, as described below.
[0674] The P value for the difference in the expression levels of
Thrombospondin 1 (THBS1) transcripts detectable by SEQ ID NO:421 in
prostate cancer samples versus the normal prostate samples was
determined by T test as 9.92E-03.
[0675] Threshold of 3 fold overexpression was found to
differentiate between cancer and normal samples with P value of
2.08E-03 as checked by exact fisher test.
[0676] The above value demonstrates statistical significance of the
results.
[0677] According to the present invention, HUMTHROM is a
non-limiting example of a marker for diagnosing prostate cancer.
The HUMTHROM marker of the present invention, can be used alone or
in combination, for prognosis, prediction, screening, early
diagnosis, therapy selection and treatment monitoring of prostate
cancer. Although optionally any method may be used to detect
overexpression and/or differential expression of this marker,
preferably a NAT-based technology is used. Therefore, optionally
and preferably, any nucleic acid molecule capable of selectively
hybridizing to HUMTHROM as previously defined is also encompassed
within the present invention. Primer pairs are also optionally and
preferably encompassed within the present invention; for example,
for the above experiment, the following primer pair was used as a
non-limiting illustrative example only of a suitable primer pair:
HUMTHROM-seg24-forward (SEQ ID NO:419): CTGCAGGCTCAGCAACTTCTT; and
HUMTHROM-seg24-reverse (SEQ ID NO:420): TTTCAAATCCCTCCCTTGTCA.
[0678] The present invention also preferably encompasses any
amplicon obtained through the use of any suitable primer pair; for
example, for the above experiment, the following amplicon was
obtained as a non-limiting illustrative example only of a suitable
amplicon:Amplicon from seg 24: (SEQ ID NO:421) TABLE-US-00106
CTGCAGGCTCAGCAACTTCTTTTAATGAAAAACAAACTCACCCTC
TTCCCCAGCATTCTTTCCATGTGTCAGAGAAGCAGAGGTTTCTTGAACGG
GCTTAGGAGAGTCTATGACAAGGGAGGGATTTGAAA.
[0679] According to other preferred embodiments of the present
invention, HUMTHROM or a fragment thereof comprises a biomarker for
detecting prostate cancer. Optionally and more preferably, the
fragment of HUMTHROM comprises HUMTHROM-seg24 (SEQ ID NO:425). Also
optionally and more preferably, any suitable method may be used for
detecting a fragment such as HUMTHROM-seg24 for example. Most
preferably, NAT-based technology used, such as any nucleic acid
molecule capable of specifically hybridizing with the fragment.
Optionally and most preferably, a primer pair is used for obtaining
the fragment.
[0680] Optionally the HUMTHROM transcript could (additionally or
alternatively) comprise any one or more of the following sequences:
SEQ ID NOs: 435-440; 443-445; 447-450.
[0681] According to still other preferred embodiments, the present
invention optionally and preferably encompasses any amino acid
sequence or fragment thereof encoded by a nucleic acid sequence
corresponding to HUMTHROM as described above or below. Any
oligopeptide or peptide relating to such an amino acid sequence or
fragment thereof may optionally also (additionally or
alternatively) be used as a biomarker. The present invention also
optionally encompasses antibodies capable of recognizing, and/or
being elicited by, such an oligopeptide or peptide.
[0682] Also, optionally and preferably HUMTHROM could be detected
by detection of an amino acid sequence according to any of SEQ ID
NOs 452-463, for which the unique regions relating to the splice
variants are given separately and additionally in SEQ ID NOs
464-472. The present invention also encompasses these amino acid
sequences as a biomarker for detecting prostate cancer.
[0683] The present invention also optionally and preferably
encompasses any nucleic acid sequence or fragment thereof, or amino
acid sequence or fragment thereof, corresponding to HUMTHROM as
described above or below, optionally for any application.
Expression of Thrombospondin 1 (THBS1) Transcripts which are
Detectable by SEQ ID NO:418 in Normal, Benign and Cancerous
Prostate Tissues
[0684] Expression of Thrombospondin 1 (THBS1) transcripts
detectable by SEQ ID NO:418, segment 19 (e.g., variant no. 18; SEQ
ID NO: 446) was measured by real time PCR, according to the
exemplary, illustrative marker HUMTHROM-segment 19 (SEQ ID NO:
423). In parallel the expression of four housekeeping genes--PBGD
(GenBank Accession No. BC019323 (SEQ ID NO:509); amplicon--SEQ ID
NO:404), HPRT1 (GenBank Accession No. NM.sub.--000194 (SEQ ID
NO:510); amplicon--SEQ ID NO:401), RPL19 (GenBank Accession No.
NM.sub.--000981 (SEQ ID NO:511); amplicon--SEQ ID NO:410) and SDHA
(GenBank Accession No. NM.sub.--004168 (SEQ ID NO:508);
amplicon--SEQ ID NO:407), was measured similarly. For each RT
sample, the expression of SEQ ID NO:418 was normalized to the
geometric mean of the quantities of the housekeeping genes. The
normalized quantity of each RT sample was then divided by the
median of the quantities of the normal post-mortem (PM) samples
(Sample Nos. 42, 48-53, 59-63, Table 2, above), to obtain a value
of fold up-regulation for each sample relative to median of the
normal PM samples.
[0685] FIG. 15 is a histogram showing over expression of the
above-indicated Thrombospondin 1 (THBS1) transcripts in cancerous
and benign (BPH) prostate samples relative to the normal samples.
The number and percentage of cancer samples that exhibit at least 3
fold over-expression, out of the total number of samples tested is
indicated in the bottom.
[0686] As is evident from FIG. 15, the expression of Thrombospondin
1 (THBS1) transcripts detectable by SEQ ID NO: 418 in cancer
samples was significantly higher than in the normal PM samples
(Sample Nos. 42, 48-53, 59-63, Table 2). Notably an over-expression
of at least 3 fold was found in 17 out of 19 adenocarcinoma
samples. Over expression of at least 3 fold was observed also in 6
out of the 8 BPH samples, and in the 2 matched normal samples.
Since matched samples are histologically non-cancerous tissue that
surrounds the tumor, such samples could have been contaminated with
cancer or pre-cancer cells. These samples were purchased
commercially with the matching non-cancerous tissue samples, as
described above.
[0687] Statistical analysis was applied to verify the significance
of these results, as described below.
[0688] The P value for the difference in the expression levels of
Thrombospondin 1 (THBS1) transcripts detectable by SEQ ID NO: 418
in prostate cancer samples versus the normal prostate samples was
determined by T test as 1.17E-04.
[0689] Threshold of 3 fold overexpression was found to
differentiate between cancer and normal samples with P value of
1.00E-05 as checked by exact fisher test.
[0690] The P value for the difference between the expression levels
of Thrombospondin 1 (THBS1) transcripts detectable by SEQ ID NO:
418 in the prostate cancer samples versus the BPH and normal
prostate samples was determined by T test as 7.36E-02. Threshold of
3 fold overexpression was found to differentiate between cancer
sample and BPH and normal sample with P value of 5.42E-04 as
checked by exact fisher test.
[0691] All the above values demonstrate statistical significance of
the results.
[0692] According to the present invention, HUMTHROM is a
non-limiting example of a marker for diagnosing prostate cancer.
Although optionally any method may be used to detect overexpression
and/or differential expression of this marker, preferably a
NAT-based technology is used. Therefore, optionally and preferably,
any nucleic acid molecule capable of selectively hybridizing to
HUMTHROM as previously defined is also encompassed within the
present invention. Primer pairs are also optionally and preferably
encompassed within the present invention; for example, for the
above experiment, the following primer pair was used as a
non-limiting illustrative example only of a suitable primer
pair:HUMTHROM-seg19-forward (SEQ ID NO:416): AAAGCATCCGATTACCCCACT
and HUMTHROM-segl9-reverse (SEQ ID NO:417):
CCGGCACAAAGTTGCAGTTA.
[0693] The present invention also preferably encompasses any
amplicon obtained through the use of any suitable primer pair; for
example, for the above experiment, the following amplicon was
obtained as a non-limiting illustrative example only of a suitable
amplicon:Amplicon from seg 19: TABLE-US-00107
Kits and Diagnostic Assays and Methods
[0694] The markers described with regard to any of Examples 1-6
above can be used alone, in combination with other markers
described above, and/or with other entirely different markers
(including but not limited to DD3, PSA or prostate specific
membrane antigen) to aid in the diagnosis of prostate cancer,
benign prostate hyperplasia or a negative diagnosis. These markers
can be used in combination with other markers for a number of uses,
including but not limited to, prognosis, prediction, screening,
early diagnosis, therapy selection and treatment monitoring of
prostate cancer, and also optionally including staging of the
disease. Used together, they tend to provide more information for
the diagnostician, increasing the percentage of true positive and
true negative diagnoses and decreasing the percentage of false
positive or false negative diagnoses, than a single marker
alone.
[0695] Assays and methods according to the present invention, as
described above, include but are not limited to, immunoassays,
hybridization assays and NAT-based assays. The combination of the
markers of the present invention with other markers described
above, and/or with other entirely different markers to aid in the
diagnosis of prostate cancer could be carried out as a mix of
NAT-based assays, immunoassays and hybridization assays. According
to preferred embodiments of the present invention, the assays are
NAT-based assays, as described for example with regard to the
Examples above.
[0696] In yet another aspect, the present invention provides kits
for aiding a diagnosis of prostate cancer, wherein the kits can be
used to detect the markers of the present invention. For example,
the kits can be used to detect any one or combination of markers
described above, which markers are differentially present in
samples of a prostate cancer patient, BPH and normal patients. The
kits of the invention have many applications. For example, the kits
can be used to differentiate if a subject has prostate cancer, BPH
or has a negative diagnosis, thus aiding a prostate cancer
diagnosis. In another example, the kits can be used to identify
compounds that modulate expression of the markers in in vitro
prostate cells or in vivo animal models for prostate cancer.
[0697] In one embodiment, a kit comprises: (a) a substrate
comprising an adsorbent thereon, wherein the adsorbent is suitable
for binding a marker, and (b) a washing solution or instructions
for making a washing solution, wherein the combination of the
adsorbent and the washing solution allows detection of the marker
as previously described.
[0698] Optionally, the kit can further comprise instructions for
suitable operational parameters in the form of a label or a
separate insert. For example, the kit may have standard
instructions informing a consumer/kit user how to wash the probe
after a sample of seminal plasma or other tissue sample is
contacted on the probe.
[0699] In another embodiment, a kit comprises (a) an antibody that
specifically binds to a marker; and (b) a detection reagent. Such
kits can be prepared from the materials described above.
[0700] In either embodiment, the kit may optionally further
comprise a standard or control information, and/or a control amount
of material, so that the test sample can be compared with the
control information standard and/or control amount to determine if
the test amount of a marker detected in a sample is a diagnostic
amount consistent with a diagnosis of prostate cancer.
[0701] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0702] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents and patent applications mentioned in this
specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20080014590A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20080014590A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References