U.S. patent application number 11/383080 was filed with the patent office on 2006-11-16 for breast specific genes and proteins.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Hongjun Ji, Craig A. Rosen.
Application Number | 20060257409 11/383080 |
Document ID | / |
Family ID | 26667870 |
Filed Date | 2006-11-16 |
United States Patent
Application |
20060257409 |
Kind Code |
A1 |
Ji; Hongjun ; et
al. |
November 16, 2006 |
Breast Specific Genes and Proteins
Abstract
Human breast specific gene polypeptides and DNA (RNA) encoding
such polypeptides and a procedure for producing such polypeptides
by recombinant techniques is disclosed. Also disclosed are methods
for utilizing such polynucleotides or polypeptides as a diagnostic
marker for breast cancer and as an agent to determine if breast
cancer has metastasized. Also disclosed are antibodies specific to
the breast specific gene polypeptides which may be used to target
cancer cells and be used as part of a breast cancer vaccine.
Methods of screening for antagonists for the polypeptide and
therapeutic uses thereof are also disclosed.
Inventors: |
Ji; Hongjun; (Germantown,
MD) ; Rosen; Craig A.; (Laytonsville, MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC.;INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
26667870 |
Appl. No.: |
11/383080 |
Filed: |
May 12, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10267849 |
Oct 10, 2002 |
7071309 |
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11383080 |
May 12, 2006 |
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08673284 |
Jun 28, 1996 |
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10267849 |
Oct 10, 2002 |
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60000602 |
Jun 30, 1995 |
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Current U.S.
Class: |
424/155.1 ;
435/320.1; 435/325; 435/6.14; 435/69.1; 435/7.23; 514/44R; 530/350;
530/388.8; 536/23.5 |
Current CPC
Class: |
C12Q 1/6886 20130101;
G01N 33/57415 20130101; C12Q 2539/113 20130101; G01N 2500/04
20130101; A61K 39/00 20130101; C07K 14/47 20130101; C12Q 1/6809
20130101; C12Q 2600/156 20130101; C12Q 1/6809 20130101 |
Class at
Publication: |
424/155.1 ;
536/023.5; 435/069.1; 435/320.1; 435/325; 530/350; 530/388.8;
435/006; 435/007.23; 514/044 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12Q 1/68 20060101 C12Q001/68; G01N 33/574 20060101
G01N033/574; C07H 21/04 20060101 C07H021/04; A61K 39/395 20060101
A61K039/395; C07K 14/82 20060101 C07K014/82; C07K 16/30 20060101
C07K016/30 |
Claims
1. An isolated polynucleotide comprising a member selected from the
group consisting of: (a) a polynucleotide encoding the same
polypeptide as the polynucleotide of FIG. 1 (SEQ ID NO: 1); (b) a
polynucleotide encoding the same mature polypeptide as a human gene
having a coding portion which includes DNA having at least a 90%
identity to the DNA of one of FIGS. 2-20 (SEQ ID NO:2, 4, 6, 8, 9,
11-24); (c) a polynucleotide which hybridizes to the polynucleotide
of (a) and which has at least a 70% identity thereto; and (d) a
polynucleotide encoding the same mature polypeptide as a human gene
having a coding portion which includes DNA having at least a 90%
identity to a DNA included in the deposited clone.
2. The polynucleotide of claim 1 wherein the human gene includes
DNA contained in the deposited clone.
3. The polynucleotide of claim 1 wherein the member is a
polynucleotide encoding the same polypeptide as the polynucleotide
of FIG. 1 (SEQ ID NO:1).
4. A vector containing the polynucleotide of claim 1.
5. A host cell transformed or transfected with the vector of claim
4.
6. A process for producing cells capable of expressing a
polypeptide comprising genetically engineering cells with the
vector of claim 4.
7. A process for producing a polypeptide comprising: expressing
from the host cell of claim 5 the polypeptide encoded by said
polynucleotide.
8. A polypeptide comprising a member selected from the group
consisting of: (i) a polypeptide encoded by a human gene, said
human gene having a coding portion whose DNA has at least a 90%
identity to the DNA of one of FIGS. 2-20 (SEQ ID NO:2, 4, 6, 8, 9,
11-24); (ii) a polypeptide encoded by the polynucleotide of FIG. 1
(SEQ ID NO: 1) and fragments, analogs and derivatives thereof; and
(iii) a polypeptide encoded by the human gene whose coding region
includes a DNA having at least a 90% identity to the DNA contained
in the deposited clone and fragments, analogs and derivatives of
said polypeptide.
9. The polypeptide of claim 8 wherein the polypeptide is encoded by
the polynucleotide having a sequence as set forth in FIG. 1 (SEQ ID
NO: 1).
10. An antibody against the polypeptide of claim 8.
11. A compound which inhibits activation of the polypeptide of
claim 8.
12. A method for the treatment of a patient having need to inhibit
a breast specific gene protein comprising: administering to the
patient a therapeutically effective amount of the compound of claim
11.
13. The method of claim 12 wherein the compound is a polypeptide
and the therapeutically effective amount of the compound is
administered by providing to the patient DNA encoding said
polypeptide and expressing said polypeptide in vivo.
14. A method for the treatment of a patient having need of a breast
specific gene protein comprising: administering to the patient a
therapeutically effective amount of the polypeptide of claim 8.
15. A process for diagnosing a disorder of the breast in a host
comprising: determining transcription of a human gene in a sample
derived from non-breast tissue of a host, said gene having a coding
portion which includes DNA having at least 90% identity to DNA
selected from the group consisting of the DNA of FIGS. 1-20 (SEQ ID
NO:2, 4, 6, 8, 9, 11-24), whereby said transcription indicates a
disorder of the breast in the host.
16. The process of claim 15 wherein transcription is determined by
detecting the presence of an altered level of RNA transcribed from
said human gene.
17. The process of claim 15 wherein transcription is determined by
detecting the presence of an altered level of DNA complementary to
the RNA transcribed from said human gene.
18. The process of claim 15 wherein transcription is determined by
detecting the presence of an altered level of an expression product
of said human gene.
19. A process for determining a disorder of a breast in a host
comprising: contacting the antibody of claim 10 to a fluid sample
derived from a host; determining the presence of an altered level
of a BSG gene product in said sample.
20. A process for identifying antagonists to the polypeptide of
claim 8 comprising: contacting said polypeptide with a natural
substrate and a labeled compound to be screened either
simultaneously or in either consecutive order; and determining
whether the therapeutic effectively competes with the natural
substrate in a manner sufficient to prevent binding of the protein
to its substrate.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/267,849, filed Oct. 10, 2002, which is a continuation of
U.S. application Ser. No. 08/673,284, filed Jun. 28, 1996; claims
benefit under 35 U.S.C. .sctn. 119(e) to U.S. Provisional
Application No. 60/000,602, filed Jun. 30, 1995; each of which is
hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] This invention relates to newly identified polynucleotides,
polypeptides encoded by such polynucleotides, and the use of such
polynucleotides and polypeptides for detecting disorders of the
breast, particularly the presence of breast cancer and breast
cancer metastases. The present invention further relates to
inhibiting the production and function of the polypeptides of the
present invention. The twenty breast specific genes of the present
invention are sometimes hereinafter referred to as "BSG1", "BSG2"
etc.
[0003] The mammary gland is subject to a variety of disorders that
should be readily detectable. Detection may be accomplished by
inspection which usually consists of palpation. Unfortunately, so
few periodic self-examinations are made that many breast masses are
discovered only by accidental palpation. Aspiration of suspected
cysts with a fine-gauge needle is another fairly common diagnostic
practice. Mammography or xeroradiography (soft-tissue x-ray) of the
breast of yet another. A biopsy of a lesion or suspected area is an
extreme method of diagnostic test.
[0004] There are many types of tumors and cysts which affect the
mammary gland. Fibroadenomas is the most common benign breast
tumor. As a pathological entity, it ranks third behind cystic
disease and carcinoma, respectively. These tumors are seen most
frequently in young people and are usually readily recognized
because they feel encapsulated. Fibrocystic disease, a benign
condition, is the most common disease of the female breast,
occurring in about 20% of pre-menopausal women. Lipomas of the
breast are also common and they are benign in nature. Carcinoma of
the breast is the most common malignant condition among women and
carries with it the highest fatality rate of all cancers affecting
this sex. At some during her life, one of every 15 women in the USA
will develop cancer of the breast. Its reported annual incidence is
70 per 100,000 females in the population in 1947, rising to 72.5 in
1969 for whites, and rising from 47.8 to 60.1 for blacks. The
annual mortality rate from 1930 to the present has remained fairly
constant, at approximately 23 per 100,000 female population. Breast
cancer is rare in men, but when it does occur, it usually not
recognized until late, and thus the results of treatment are poor.
In women, carcinoma of the breast is rarely seen before age 30 and
the incidence rises rapidly after menopause. For this reason,
post-menopausal breast masses should be considered cancer until
proved otherwise.
BRIEF SUMMARY OF THE INVENTION
[0005] In accordance with an aspect of the present invention, there
are provided nucleic acid probes comprising nucleic acid molecules
of sufficient length to specifically hybridize to the RNA
transcribed from the human breast specific genes of the present
invention or to DNA corresponding to such RNA.
[0006] In accordance with another aspect of the present invention
there is provided a method of and products for diagnosing breast
cancer formation and breast cancer metastases by detecting the
presence of RNA transcribed from the human breast specific genes of
the present invention or DNA corresponding to such RNA in a sample
derived from a host.
[0007] In accordance with yet another aspect of the present
invention, there is provided a method of and products for
diagnosing breast cancer formation and breast cancer metastases by
detecting an altered level of a polypeptide corresponding to the
breast specific genes of the present invention in a sample derived
from a host, whereby an elevated level of the polypeptide indicates
a breast cancer diagnosis.
[0008] In accordance with another aspect of the present invention,
there are provided isolated polynucleotides encoding human breast
specific polypeptides, including mRNAs, DNAs, cDNAs, genomic DNAs,
as well as antisense analogs and biologically active and
diagnostically or therapeutically useful fragments thereof.
[0009] In accordance with still another aspect of the present
invention there are provided human breast specific genes which
include polynucleotides as set forth in the sequence listing.
[0010] In accordance with a further aspect of the present
invention, there are provided novel polypeptides encoded by the
polynucleotides, as well as biologically active and diagnostically
or therapeutically useful fragments, analogs and derivatives
thereof.
[0011] In accordance with yet a further aspect of the present
invention, there is provided a process for producing such
polypeptides by recombinant techniques comprising culturing
recombinant prokaryotic and/or eukaryotic host cells, containing a
polynucleotide of the present invention, under conditions promoting
expression of said proteins and subsequent recovery of said
proteins.
[0012] In accordance with yet a further aspect of the present
invention, there are provided antibodies specific to such
polypeptides, which may be employed to detect breast cancer cells
or breast cancer metastasis.
[0013] In accordance with another aspect of the present invention,
there are provided processes for using one or more of the
polypeptides of the present invention to treat breast cancer and
for using the polypeptides to screen for compounds which interact
with the polypeptides, for example, compounds which inhibit or
activate the polypeptides of the present invention.
[0014] In accordance with yet another aspect of the present
invention, there is provided a screen for detecting compounds which
inhibit activation of one or more of the polynucleotides and/or
polypeptides of the present invention which may be used to
therapeutically, for example, in the treatment of breast
cancer.
[0015] In accordance with yet a further aspect of the present
invention, there are provided processes for utilizing such
polypeptides, or polynucleotides encoding such polypeptides, for in
vitro purposes related to scientific research, synthesis of DNA and
manufacture of DNA vectors.
[0016] These and other aspects of the present invention should be
apparent to those skilled in the art from the teachings herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The following drawings are illustrative of embodiments of
the invention and are not meant to limit the scope of the invention
as encompassed by the claims.
[0018] FIG. 1 (SEQ ID NO: 1) is a full length cDNA sequence of
breast specific gene 1 of the present invention.
[0019] FIG. 2 (SEQ ID NO:2) is a partial cDNA sequence and the
corresponding deduced amino acid sequence of breast specific gene 2
(HBGBP 46) of the present invention.
[0020] FIG. 3 (SEQ ID NO:4) is a partial cDNA sequence and deduced
amino acid sequence of breast specific gene 3 of the invention.
[0021] FIG. 4 (SEQ ID NO:6) is a partial cDNA sequence and the
corresponding deduced amino acid sequence of breast specific gene 4
of the present invention.
[0022] FIG. 5 (SEQ ID NO:8) is a partial cDNA sequence of breast
specific gene 5 of the present invention.
[0023] FIG. 6 (SEQ ID NO:9) is a partial cDNA and deduced amino
acid sequence of breast specific gene 6 of the present
invention.
[0024] FIG. 7 (SEQ ID NO: 11) is a partial cDNA sequence of breast
specific gene 7 of the present invention.
[0025] FIG. 8 (SEQ ID NO: 12) is a partial cDNA sequence of breast
specific gene 8 of the present invention.
[0026] FIG. 9 (SEQ ID NO: 13) is a partial cDNA sequence of breast
specific gene 9 of the present invention.
[0027] FIG. 10 (SEQ ID NO: 14) is a partial cDNA sequence of breast
specific gene 10 of the present invention.
[0028] FIG. 11 (SEQ ID NO: 15) is a partial cDNA sequence of breast
specific gene 11 of the present invention.
[0029] FIG. 12 (SEQ ID NO:16) is a partial cDNA sequence of breast
specific gene 12 of the present invention.
[0030] FIG. 13 (SEQ ID NO:17) is a partial cDNA sequence of breast
specific gene 13 of the present invention.
[0031] FIG. 14 (SEQ ID NO: 18) is a partial cDNA sequence of breast
specific gene 14 of the present invention.
[0032] FIG. 15 (SEQ ID NO: 19) is a partial cDNA sequence of breast
specific gene 15 of the present invention.
[0033] FIG. 16 (SEQ ID NO:20) is a partial cDNA sequence of breast
specific gene 16 of the present invention.
[0034] FIG. 17 (SEQ ID NO:21) is a partial cDNA sequence of breast
specific gene 17 of the present invention.
[0035] FIG. 18 (SEQ ID NO:22) is a partial cDNA sequence of breast
specific gene 18 of the present invention.
[0036] FIG. 19 (SEQ ID NO:23) is a partial cDNA sequence of breast
specific gene 19 of the present invention.
[0037] FIG. 20 (SEQ ID NO:24) is a partial cDNA sequence of breast
specific gene 20 of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The term "breast specific gene" means that such gene is
primarily expressed in tissues derived from the breast, and such
genes may be expressed in cells derived from tissues other than
from the breast. However, the expression of such genes is
significantly higher in tissues derived from the breast than from
non-breast tissues.
[0039] In accordance with one aspect of the present invention there
is provided a polynucleotide which encodes a mature polypeptide
encoded by the polynucleotide having the sequence of FIG. 1 (SEQ ID
NO: 1) and fragments, analogues and derivatives thereof.
[0040] In accordance with a further aspect of the present invention
there is provided a polynucleotide which encodes the same mature
polypeptide as a human gene having a coding portion which contains
a polynucleotide which is at least 90% identical (preferably at
least 95% identical and most preferably at least 97% or 100%
identical) to one of the polynucleotides of FIGS. 2-20 (SEQ ID
NO:2, 4, 6, 8, 9, 11-24), as well as fragments thereof.
[0041] In accordance with still another aspect of the present
invention there is provided a polynucleotide which encodes for the
same mature polypeptide as a human gene whose coding portion
includes a polynucleotide which is at least 90% identical to
(preferably at least 95% identical to and most preferably at least
97% or 100% identical) to one of the polynucleotides included in
ATCC.RTM. 97175 of Jun.2, 1995.
[0042] In accordance with yet another aspect of the present
invention, there is provided a polynucleotide probe which
hybridizes to mRNA (or the corresponding cDNA) which is transcribed
from the coding portion of a human gene which coding portion
includes a DNA sequence which is at least 90% identical to
(preferably at least 95% identical to) and most preferably at least
97% or 100% identical) to one of the polynucleotide sequences of
FIGS. 1-20 (SEQ ID NO:2, 4, 6, 8, 9, 11-24).
[0043] The present invention further relates to a mature
polypeptide encoded by a coding portion of a human gene which
coding portion includes a DNA sequence which is at least 90%
identical to (preferably at least 95% identical to and more
preferably 97% or 100% identical to) one of the polynucleotides of
FIG. 2-20 (SEQ ID NO:2, 4, 6, 8, 9, 11-24), and analogues,
derivatives and fragments thereof.
[0044] The present invention also relates to one of the mature
polypeptides encoded by the polynucleotide of FIG. 1 (SEQ ID NO: 1)
and fragments, analogues and derivatives thereof
[0045] The present invention further relates to the same mature
polypeptide encoded by a human gene whose coding portion includes
DNA which is at least 90% identical to (preferably at least 95%
identical to and more preferably at least 97% or 100% identical to)
one of the polynucleotides included in ATCC.RTM. Deposit No. 97175
deposited Jun. 2, 1995.
[0046] In accordance with an aspect of the present invention, there
are provided isolated nucleic acids (polynucleotides) which encode
for the mature polypeptides encoded by the polynucleotide of FIG. 1
(SEQ ID NO: 1) or fragments, analogues or derivatives thereof.
[0047] The polynucleotides of the present invention may be in the
form of RNA or in the form of DNA, which DNA includes cDNA, genomic
DNA, and synthetic DNA. The DNA may be double-stranded or
single-stranded, and if single stranded may be the coding strand or
non-coding (anti-sense) strand. The coding sequence which encodes
the mature polypeptide may include DNA identical to FIGS. 1-20 (SEQ
ID NO: 1-2, 4, 6, 8-9, 10-24) or that of the deposited clone or may
be a different coding sequence which coding sequence, as a result
of the redundancy or degeneracy of the genetic code, encodes the
same mature polypeptide as the coding sequence of a gene which
coding sequence includes the DNA of FIGS. 1-20 (SEQ ID NO: 1-2, 4,
6, 8-9, 10-24) or the deposited cDNA.
[0048] The polynucleotide which encodes a mature polypeptide of the
present invention may include, but is not limited to: only the
coding sequence for the mature polypeptide; the coding sequence for
the mature polypeptide and additional coding sequence such as a
leader or secretory sequence or a proprotein sequence; the coding
sequence for the mature polypeptide (and optionally additional
coding sequence) and non-coding sequence, such as introns or
non-coding sequence 5' and/or 3' of the coding sequence for the
mature polypeptide.
[0049] Thus, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only coding sequence
for the polypeptide as well as a polynucleotide which includes
additional coding and/or non-coding sequence.
[0050] The present invention further relates to variants of the
hereinabove described polynucleotides which encode fragments,
analogs and derivatives of a mature polypeptide of the present
invention. The variant of the polynucleotide may be a naturally
occurring allelic variant of the polynucleotide or a non-naturally
occurring variant of the polynucleotide.
[0051] Thus, the present invention includes polynucleotides
encoding the same mature polypeptide as hereinabove described as
well as variants of such polynucleotides which variants encode a
fragment, derivative or analog of a polypeptide of the invention.
Such nucleotide variants include deletion variants, substitution
variants and addition or insertion variants.
[0052] The polynucleotides of the invention may have a coding
sequence which is a naturally occurring allelic variant of the
human gene whose coding sequence includes DNA as shown in FIGS.
1-20 (SEQ ID NO: 1-2, 4, 6, 8-9, 10-24) or of the deposited clone.
As known in the art, an allelic variant is an alternate form of a
polynucleotide sequence which may have a substitution, deletion or
addition of one or more nucleotides, which does not substantially
alter the function of the encoded polypeptide.
[0053] The present invention also includes polynucleotides, wherein
the coding sequence for the mature polypeptide may be fused in the
same reading frame to a polynucleotide sequence which aids in
expression and secretion of a polypeptide from a host cell, for
example, a leader sequence which functions as a secretory sequence
for controlling transport of a polypeptide from the cell. The
polypeptide having a leader sequence is a preprotein and may have
the leader sequence cleaved by the host cell to form the mature
form of the polypeptide. The polynucleotides may also encode a
proprotein which is the mature protein plus additional 5' amino
acid residues. A mature protein having a prosequence is a
proprotein and is an inactive form of the protein. Once the
prosequence is cleaved an active mature protein remains.
[0054] Thus, for example, the polynucleotide of the present
invention may encode a mature protein, or a protein having a
prosequence or a protein having both a presequence and a
presequence (leader sequence).
[0055] The polynucleotides of the present invention may also have
the coding sequence fused in frame to a marker sequence which
allows for purification of the polypeptide of the present
invention. The marker sequence may be a hexa-histidine tag supplied
by a pQE-9 vector to provide for purification of the mature
polypeptide fused to the marker in the case of a bacterial host,
or, for example, the marker sequence may be a hemagglutinin (HA)
tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag
corresponds to an epitope derived from the influenza hemagglutinin
protein (Wilson, I., et al., Cell, 37:767 (1984)).
[0056] The present invention further relates to polynucleotides
which hybridize to the hereinabove-described polynucleotides if
there is at least 70%, preferably at least 90%, and more preferably
at least 95% identity between the sequences. The present invention
particularly relates to polynucleotides which hybridize under
stringent conditions to the hereinabove-described polynucleotides.
As herein used, the term "stringent conditions" means hybridization
will occur only if there is at least 95% and preferably at least
97% identity between the sequences. The polynucleotides which
hybridize to the hereinabove described polynucleotides in a
preferred embodiment encode polypeptides which retain substantially
the same biological function or activity as the mature polypeptide
of the present invention encoded by a coding sequence which
includes the DNA of FIGS. 1-20 (SEQ ID NO: 1-2, 4, 6, 8-9, 10-24)
or the deposited cDNA(s).
[0057] Alternatively, the polynucleotide may have at least 10 or 20
bases, preferably at least 30 bases, and more preferably at least
50 bases which hybridize to a polynucleotide of the present
invention and which has an identity thereto, as hereinabove
described, and which may or may not retain activity. For example,
such polynucleotides may be employed as probes for polynucleotides,
for example, for recovery of the polynucleotide or as a diagnostic
probe or as a PCR primer.
[0058] Thus, the present invention is directed to polynucleotides
having at least a 70% identity, preferably at least 90% and more
preferably at least 95% identity to a polynucleotide which encodes
the mature polypeptide encoded by a human gene which includes the
DNA of one of FIGS. 1-20 (SEQ ID NO: 1-2, 4, 6, 8-9, 10-24) as well
as fragments thereof, which fragments have at least 30 bases and
preferably at least 50 bases and to polypeptides encoded by such
polynucleotides.
[0059] The partial sequences are specific tags for messenger RNA
molecules. The complete sequence of that messenger RNA, in the form
of cDNA, is determined using the partial sequence as a probe to
identify a cDNA clone corresponding to a full-length transcript.
The partial cDNA clone can also be used as a probe to identify a
genomic clone or clones that contain the complete gene including
regulatory and promoter regions, exons, and introns.
[0060] The partial sequences of FIGS. 2-20 (SEQ ID NO:2, 4, 6, 8,
9, 11-24) may be used to identify the corresponding full length
gene from which they were derived. The partial sequences can be
nickel-translated or end-labelled with .sup.32P using
polynucleotide Idnase using labelling methods known to those with
skill in the art (Basic Methods in Molecular Biology, L. G. Davis,
M. D. Dibner, and J. F. Battey, ed., Elsevier Press, N.Y., 1986). A
lambda library prepared from human breast tissue can be directly
screened with the labelled sequences of interest or the library can
be converted en masse to pBluescript (Stratagene Cloning Systems,
La Jolla, Calif. 92037) to facilitate bacterial breasty screening.
Regarding pBluescript, see Sambrook et al., Molecular Cloning-A
Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), pg.
1.20. Both methods are well known in the art. Briefly, filters with
bacterial colonies containing the library in pBluescript or
bacterial lawns containing lambda plaques are denatured and the DNA
is fixed to the filters. The filters are hybridized with the
labelled probe using hybridization conditions described by Davis et
al., supra. The partial sequences, cloned into lambda or
pBluescript, can be used as positive controls to assess background
binding and to adjust the hybridization and washing stringencies
necessary for accurate clone identification. The resulting
autoradiograms are compared to duplicate plates of colonies or
plaques; each exposed spot corresponds to a positive breasty or
plaque. The colonies or plaques are selected, expanded and the DNA
is isolated from the colonies for further analysis and
sequencing.
[0061] Positive cDNA clones are analyzed to determine the amount of
additional sequence they contain using PCR with one primer from the
partial sequence and the other primer from the vector. Clones with
a larger vector-insert PCR product than the original partial
sequence are analyzed by restriction digestion and DNA sequencing
to determine whether they contain an insert of the same size or
similar as the mRNA size determined from Northern blot
Analysis.
[0062] Once one or more overlapping cDNA clones are identified, the
complete sequence of the clones can be determined. The preferred
method is to use exonuclease III digestion (McCombie, W. R,
Kirlcness, E., Fleming, J. T., Kerlavage, A. R., Iovannisci, D. M.,
and Martin-Gallardo, R., Methods, 3:33-40, 1991). A series of
deletion clones are generated, each of which is sequenced. The
resulting overlapping sequences are assembled into a single
contiguous sequence of high redundancy (usually three to five
overlapping sequences at each nucleotide position), resulting in a
highly accurate final sequence.
[0063] The DNA sequences (as well as the corresponding RNA
sequences) also include sequences which are or contain a DNA
sequence identical to one contained in and isolatable from
ATCC.RTM. Deposit No. 97175, deposited Jun. 2, 1995, and fragments
or portions of the isolated DNA sequences (and corresponding RNA
sequences), as well as DNA (RNA) sequences encoding the same
polypeptide. In particular, DNA (RNA) sequences encoding BSG1 (SEQ
ID NO:32) and the amino acid sequence encoded thereby (SEQ ID
NO:33) are contained in and isolatable from ATCC.RTM. Deposit No.
97175 using routine techniques known in the art. A cDNA clone,
HBGBP46, encoding BSG2 was deposited as ATCC.RTM. Deposit No.
PTA-1545 on Mar. 22, 2000, at the American Type Culture Collection,
Patent Depository, 10801 University Boulevard, Manassas, Va.
20110-2209.
[0064] The deposit(s) referred to herein will be maintained under
the terms of the Budapest Treaty on the International Recognition
of the Deposit of Micro-organisms for purposes of Patent Procedure.
These deposits are provided merely as convenience to those of skill
in the art and are not an admission that a deposit is required
under 35 U.S.C. .sctn. 112. The sequence of the polynucleotides
contained in the deposited materials, as well as the amino acid
sequence of the polypeptides encoded thereby, are incorporated
herein by reference and are controlling in the event of any
conflict with any description of sequences herein. A license may be
required to make, use or sell the deposited materials, and no such
license is hereby granted.
[0065] The present invention further relates to polynucleotides
which have at least 10 bases, preferably at least 20 bases, and may
have 30 or more bases, which polynucleotides are hybridizable to
and have at least a 70% identity to RNA (and DNA which corresponds
to such RNA) transcribed from a human gene whose coding portion
includes DNA as hereinabove described.
[0066] Thus, the polynucleotide sequences which hybridize as
described above may be used to hybridize to and detect the
expression of the human genes to which they correspond for use in
diagnostic assays as hereinafter described.
[0067] In accordance with still another aspect of the present
invention there are provided diagnostic assays for detecting
micrometastases of breast cancer in a host. While applicant does
not wish to limit the reasoning of the present invention to any
specific scientific theory, it is believed that the presence of
active transcription of a breast specific gene of the present
invention in cells of the host, other than those derived from the
breast, is indicative of breast cancer metastases. This is true
because, while the breast specific genes are found in all cells of
the body, their transcription to mRNA, cDNA and expression products
is primarily limited to the breast in non-diseased individuals.
However, if breast cancer is present, breast cancer cells migrate
from the cancer to other cells, such that these other cells are now
actively transcribing and expressing a breast specific gene at a
greater level than is normally found in non-diseased individuals,
i.e., transcription is higher than found in non-breast tissues in
healthy individuals. It is the detection of this enhanced
transcription or enhanced protein expression in cells, other than
those derived from the breast, which is indicative of metastases of
breast cancer.
[0068] In one example of such a diagnostic assay, an RNA sequence
in a sample derived from a tissue other than the breast is detected
by hybridization to a probe. The sample contains a nucleic acid or
a mixture of nucleic acids, at least one of which is suspected of
containing a human breast specific gene or fragment thereof of the
present invention which is transcribed and expressed in such
tissue. Thus, for example, in a form of an assay for determining
the presence of a specific RNA in cells, initially RNA is isolated
from the cells.
[0069] A sample may be obtained from cells derived from tissue
other than from the breast including but not limited to blood,
urine, saliva, tissue biopsy and autopsy material. The use of such
methods for detecting enhanced transcription to mRNA from a human
breast specific gene of the present invention or fragment thereof
in a sample obtained from cells derived from other than the breast
is well within the scope of those skilled in the art from the
teachings herein.
[0070] The isolation of mRNA comprises isolating total cellular RNA
by disrupting a cell and performing differential centrifugation.
Once the total RNA is isolated, mRNA is isolated by making use of
the adenine nucleotide residues known to those skilled in the art
as a poly(A) tail found on virtually every eukaryotic mRNA molecule
at the 3' end thereof. Oligonucleotides composed of only
deoxythymidine [oligo(dT)] are linked to cellulose and the oligo
(dT)-cellulose packed into small columns. When a preparation of
total cellular RNA is passed through such a column, the mRNA
molecules bind to the oligo(dT) by the poly(A)tails while the rest
of the RNA flows through the column. The bound mRNAs are then
eluted from the column and collected.
[0071] One example of detecting isolated mRNA transcribed from a
breast specific gene of the present invention comprises screening
the collected mRNAs with the gene specific oligonucleotide probes,
as hereinabove described.
[0072] It is also appreciated that such probes can be and are
preferably labeled with an analytically detectable reagent to
facilitate identification of the probe. Useful reagents include but
are not limited to radioactivity, fluorescent dyes or enzymes
capable of catalyzing the formation of a detectable product.
[0073] An example of detecting a polynucleotide complementary to
the mRNA sequence (cDNA) utilizes the polymerase chain reaction
(PCR) in conjunction with reverse transcriptase. PCR is a very
powerful method for the specific amplification of DNA or RNA
stretches (Saiki et al., Nature, 234:163-166 (1986)). One
application of this technology is in nucleic acid probe technology
to bring up nucleic acid sequences present in low copy numbers to a
detectable level. Numerous diagnostic and scientific applications
of this method have been described by H. A. Erlich (ed.) in PCR
Technology-Principles and Applications for DNA Amplification,
Stockton Press, USA, 1989, and by M. A. Inis (ed.) in PCR
Protocols, Academic Press, San Diego, USA, 1990.
[0074] RT-PCR is a combination of PCR with the reverse
transcriptase enzyme. Reverse transcriptase is an enzyme which
produces cDNA molecules from corresponding mRNA molecules. This is
important since PCR amplifies nucleic acid molecules, particularly
DNA, and this DNA may be produced from the mRNA isolated from a
sample derived from the host.
[0075] A specific example of an RT-PCR diagnostic assay involves
removing a sample from a tissue of a host. Such a sample will be
from a tissue, other than the breast, for example, blood.
Therefore, an example of such a diagnostic assay comprises whole
blood gradient isolation of nucleated cells, total RNA extraction,
RT-PCR of total RNA and agarose gel electrophoresis of PCR
products. The PCR products comprise cDNA complementary to RNA
transcribed from one or more breast specific genes of the present
invention or fragments thereof. More particularly, a blood sample
is obtained and the whole blood is combined with an equal volume of
phosphate buffered saline, centrifuged and the lymphocyte and
granulocyte layer is carefully aspirated and rediluted in phosphate
buffered saline and centrifuged again. The supernate is discarded
and the pellet containing nucleated cells is used for RNA
extraction using the RNazole B method as described by the
manufacturer (Tel-Test Inc., Friendswood, Tex).
[0076] Oligonucleotide primers and probes are prepared with high
specificity to the DNA sequences of the present invention. The
probes are at least 10 base pairs in length, preferably at least 30
base pairs in length and most preferably at least 50 base pairs in
length or more. The reverse transcriptase reaction and PCR
amplification are performed sequentially without interruption. Taq
polymerase is used during PCR and the PCR products are concentrated
and the entire sample is run on a Tris-borate-EDTA agarose gel
containing ethidium bromide.
[0077] In accordance with another aspect of the present invention,
there is provided a method of diagnosing a disorder of the breast,
for example breast cancer, by determining altered levels of the
breast specific polypeptides of the present invention in a
biological sample, derived from tissue other than from the breast.
Elevated levels of the breast specific polypeptides of the present
invention, indicates active transcription and expression of the
corresponding breast specific gene product. Assays used to detect
levels of a breast specific gene polypeptide in a sample derived
from a host are well-known to those skilled in the art and include
radioimmunoassays, competitive-binding assays, Western blot
analysis, ELISA assays and "sandwich" assays. A biological sample
may include, but is not limited to, tissue extracts, cell samples
or biological fluids, however, in accordance with the present
invention, a biological sample specifically does not include tissue
or cells of the breast.
[0078] An ELISA assay (Coligan, et al., Current Protocols in
Immunology, 1(2), Chapter 6, 1991) initially comprises preparing an
antibody specific to a breast specific polypeptide of the present
invention, preferably a monoclonal antibody. In addition, a
reporter antibody is prepared against the monoclonal antibody. To
the reporter antibody is attached a detectable reagent such as
radioactivity, fluorescence or, in this example, a horseradish
peroxidase enzyme. A sample is removed from a host and incubated on
a solid support, e.g., a polystyrene dish, that binds the proteins
in the sample. Any free protein binding sites on the dish are then
covered by incubating with a non-specific protein, such as BSA.
Next, the monoclonal antibody is incubated in the dish during which
time the monoclonal antibodies attach to the breast specific
polypeptide attached to the polystyrene dish. All unbound
monoclonal antibody is washed out with buffer. The reporter
antibody linked to horseradish peroxidase is now placed in the dish
resulting in binding of the reporter antibody to any monoclonal
antibody bound to the breast specific gene polypeptide. Unattached
reporter antibody is then washed out. Peroxidase substrates are
then added to the dish and the amount of color developed in a given
time period is a measurement of the amount of the breast specific
polypeptide present in a given volume of patient sample when
compared against a standard curve.
[0079] A competition assay may be employed where antibodies
specific to a breast specific polypeptide are attached to a solid
support. The breast specific polypeptide is then labeled and the
labeled polypeptide a sample derived from the host are passed over
the solid support and the amount of label detected, for example, by
liquid scintillation chromatography, can be correlated to a
quantity of the breast specific polypeptide in the sample.
[0080] A "sandwich" assay is similar to an ELISA assay. In a
"sandwich" assay, breast specific polypeptides are passed over a
solid support and bind to antibody attached to the solid support. A
second antibody is then bound to the breast specific polypeptide. A
third antibody which is labeled and is specific to the second
antibody, is then passed over the solid support and binds to the
second antibody and an amount can then be quantified.
[0081] In alternative methods, labeled antibodies to a breast
specific polypeptide are used. In a one-step assay, the target
molecule, if it is present, is immobilized and incubated with a
labeled antibody. The labeled antibody binds to the immobilized
target molecule. After washing to remove the unbound molecules, the
sample is assayed for the presence of the label. In a two-step
assay, immobilized target molecule is incubated with an unlabeled
antibody. The target molecule-labeled antibody complex, if present,
is then bound to a second, labeled antibody that is specific for
the unlabeled antibody. The sample is washed and assayed for the
presence of the label.
[0082] Such antibodies specific to breast specific gene proteins,
for example, anti-idiotypic antibodies, can be used to detect
breast cancer cells by being labeled and described above and
binding tightly to the breast cancer cells, and, therefore, detect
their presence.
[0083] The antibodies may also be used to target breast cancer
cells, for example, in a method of homing interaction agents which,
when contacting breast cancer cells, destroy them. This is true
since the antibodies are specific for breast specific genes which
are primarily expressed in breast cancer, and a linking of the
interaction agent to the antibody would cause the interaction agent
to be carried directly to the breast.
[0084] Antibodies of this type may also be used to do in vivo
imaging, for example, by labeling the antibodies to facilitate
scanning of the breast. One method for imaging comprises contacting
any cancer cells of the breast to be imaged with an anti-breast
specific gene protein antibody labeled with a detectable marker.
The method is performed under conditions such that the labeled
antibody binds to the breast specific gene proteins. In a specific
example, the antibodies interact with the breast, for example,
breast cancer cells, and fluoresce upon such contact such that
imaging and visibility of the breast is enhanced to allow a
determination of the diseased or non-diseased state of the
breast.
[0085] The choice of marker used to label the antibodies will vary
depending upon the application. However, the choice of marker is
readily determinable to one skilled in the art. These labeled
antibodies may be used in immunoassays as well as in histological
applications to detect the presence of the proteins. The labeled
antibodies may be polyclonal or monoclonal.
[0086] The presence of active transcription, which is greater than
that normally found, of the breast specific genes in cells other
than from the breast, by the presence of an altered level of mRNA,
cDNA or expression products is an important indication of the
presence of a breast cancer which has metastasized, since breast
cancer cells are migrating from the breast into the general
circulation. Accordingly, this phenomenon may have important
clinical implications since the method of treating a localized, as
opposed to a metastasized, tumor is entirely different.
[0087] Of the 20 breast specific genes disclosed, only breast
specific gene 1 is a full-length gene. Breast specific gene 1 is
79% identical and 83% similar to human Alzheimer disease amyloid
gene. Breast specific gene 2 is 30% identical and 48% similar to
human hydroxyindole-o-methyltransferase gene. Breast specific gene
3 is 58% identical and 62% similar to human 06-methylguanine-DNA
methyltransferase gene. Breast specific gene 4 is 34% identical and
65% similar to the mouse p120 gene. Breast specific gene 5 is 78%
identical and 89% similar to human p70 ribosomal S6 kinase alpha-II
gene. Breast specific gene 6 is 77% identical and 79% similar to
the human transcription factor NFATp gene.
[0088] As stated previously, the breast specific genes of the
present invention are putative molecular markers in the diagnosis
of breast cancer formation, and breast cancer metastases. As shown
in the following Table 1, the presence of the breast specific genes
when tested in normal breast, breast cancer, embryo and other
cancer libraries, the breast specific genes of the present
invention were found to be most prevalent in the breast cancer
library, indicating that the genes of the present invention may be
employed for detecting breast cancer, as discussed previously. The
table also indicates a putative identification, based on homology,
of BSG1 through BSG6 to known genes. TABLE-US-00001 TABLE 1 Homolog
Gene Name Norm Br Other Genes (Class) Br Ca Embryo Cancers Others
BSG1 AD Amyloid (3) 1 6 1 BSG2 Hydroxyindole- 3 1 1
o-methytransferase (2) BSG3 O-6-methylguanine- 3 1 1 DNA
methyltransferase (1) BSG4 P120 (3) 3 1 BSG5 p70 ribosomal S6 3 1
kinase alpha-II (2) BSG6 Transcription factor 2 NFATp(3) BSG7 2 1
BSG8 4 3 1 BSG8 2 BSG9 3 BSG10 3 BSG11 3 BSG12 3 3 BSG13 3 BSG14 2
BSG15 3 BSG16 1 1 1 BSG17 2 1 BSG18 2 BSG19 1 1 BSG20 2
[0089] The assays described above may also be used to test whether
bone marrow preserved before chemotherapy is contaminated with
micrometastases of a breast cancer cell. In the assay, blood cells
from the bone marrow are isolated and treated as described above,
this method allows one to determine whether preserved bone marrow
is still suitable for transplantation after chemotherapy.
[0090] The present invention further relates to mature
polypeptides, for example the BSG1 polypeptide, as well as
fragments, analogs and derivatives of such polypeptide.
[0091] The terms "fragment," "derivative" and "analog" when
referring to the polypeptides encoded by the genes of the invention
means a polypeptide which retains essentially the same biological
function or activity as such polypeptide. Thus, an analog includes
a proprotein which can be activated by cleavage of the proprotein
portion to produce an active mature polypeptide.
[0092] The polypeptides of the present invention may be recombinant
polypeptides, natural polypeptides or synthetic polypeptides,
preferably recombinant polypeptides.
[0093] The fragment, derivative or analog of the polypeptides
encoded by the genes of the invention may be (i) one in which one
or more of the amino acid residues are substituted with a conserved
or non-conserved amino acid residue (preferably a conserved amino
acid residue) and such substituted amino acid residue may or may
not be one encoded by the genetic code, or (ii) one in which one or
more of the amino acid residues includes a substituent group, or
(iii) one in which the polypeptide is fused with another compound,
such as a compound to increase the half-life of the polypeptide
(for example, polyethylene glycol), or (iv) one in which the
additional amino acids are fused to the polypeptide, such as a
leader or secretory sequence or a sequence which is employed for
purification of the mature polypeptide or a proprotein sequence.
Such fragments, derivatives and analogs are deemed to be within the
scope of those skilled in the art from the teachings herein.
[0094] The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
[0095] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide, separated
from some or all of the coexisting materials in the natural system,
is isolated. Such polynucleotides may be part of a vector.
[0096] The polypeptides of the present invention include the
polypeptides encoded by the polynucleotide of FIG. 1 (SEQ ID NO: 1)
(in particular the mature polypeptides) as well as polypeptides
which have at least 70% similarity (preferably at least a 70%
identity) to the polypeptides encoded by the polynucleotide of FIG.
1 (SEQ ID NO: 1) and preferably at least a 90% similarity
(preferably at least a 90% identity) to the polypeptides of FIGS. 8
and 9 (SEQ ID NO: 12 and 13) and more preferably at least a 95%
similarity (still more preferably at least 95% identity) to the
polypeptides encoded by the polynucleotide of (SEQ ID NO: 1) and
also include portions of such polypeptides with such portion of the
polypeptide generally containing at least 30 amino acids and more
preferably at least 50 amino acids.
[0097] As known in the art "similarity" between two polypeptides is
determined by comparing the amino acid sequence and its conserved
amino acid substitutes of one polypeptide to the sequence of a
second polypeptide.
[0098] Fragments or portions of the polypeptides of the present
invention may be employed for producing the corresponding
full-length polypeptide by peptide synthesis; therefore, the
fragments may be employed as intermediates for producing the
full-length polypeptides. Fragments or portions of the
polynucleotides of the present invention may be used to synthesize
full-length polynucleotides of the present invention.
[0099] The present invention also relates to vectors which include
polynucleotides of the present invention, host cells which are
genetically engineered with vectors of the invention and the
production of polypeptides of the invention by recombinant
techniques.
[0100] Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors of this invention
which may be, for example, a cloning vector or an expression
vector. The vector may be, for example, in the form of a plasmid, a
viral particle, a phage, etc. The engineered host cells can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying the
breast specific genes. The culture conditions, such as temperature,
pH and the like, are those previously used with the host cell
selected for expression, and will be apparent to those of
ordinarily skill in the art.
[0101] The polynucleotides of the present invention may be employed
for producing polypeptides by recombinant techniques. Thus, for
example, the polynucleotide may be included in any one of a variety
of expression vectors for expressing a polypeptide. Such vectors
include chromosomal, nonchromosomal and synthetic DNA sequences,
e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from combinations of
plasmids and phage DNA, viral DNA such as vaccinia, adenovirus,
fowl pox virus, and pseudorabies. However, any other vector may be
used as long as it is replicable and viable in the host.
[0102] The appropriate DNA sequence may be inserted into the vector
by a variety of procedures. In general, the DNA sequence is
inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Such procedures and others are deemed
to be within the scope of those skilled in the art.
[0103] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct mRNA synthesis. As representative examples of such
promoters, there may be mentioned: LTR or SV40 promoter, the E.
coli. lac or trp, the phage lambda P.sub.L promoter and other
promoters known to control expression of genes in prokaryotic or
eukaryotic cells or their viruses. The expression vector also
contains a ribosome binding site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for amplifying expression.
[0104] In addition, the expression vectors preferably contain one
or more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0105] The vector containing the appropriate DNA sequence as
hereinabove described, as well as an appropriate promoter or
control sequence, may be employed to transform an appropriate host
to permit the host to express the protein.
[0106] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptomyces,
Salmonella typhimurium; fungal cells, such as yeast; insect cells
such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO,
COS or Bowes melanoma; adenoviruses; plant cells, etc. The
selection of an appropriate host is deemed to be within the scope
of those skilled in the art from the teachings herein.
[0107] More particularly, the present invention also includes
recombinant constructs comprising one or more of the sequences as
broadly described above. The constructs comprise a vector, such as
a plasmid or viral vector, into which a sequence of the invention
has been inserted, in a forward or reverse orientation. In a
preferred aspect of this embodiment, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10,
phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a, pNH18A,
pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5
(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any
other plasmid or vector may be used as long as they are replicable
and viable in the host.
[0108] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lacI, lacZ, T3, T7,
gpt, lambda P.sub.R, P.sub.L and trp. Eukaryotic promoters include
CMV immediate early, HSV thymidine kinase, early and late SV40,
LTRs from retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art.
[0109] In a further embodiment, the present invention relates to
host cells containing the above-described constructs. The host cell
can be a higher eukaryotic cell, such as a mammalian cell, or a
lower eukaryotic cell, such as a yeast cell, or the host cell can
be a prokaryotic cell, such as a bacterial cell. Introduction of
the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)).
[0110] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Alternatively, the polypeptides of the invention can be
synthetically produced by conventional peptide synthesizers.
[0111] Proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which
is hereby incorporated by reference.
[0112] Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes is increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 10 to 300 bp that act on a
promoter to increase its transcription. Examples including the SV40
enhancer on the late side of the replication origin bp 100 to 270,
a cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus
enhancers.
[0113] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), -factor, acid phosphatase, or heat shock proteins,
among others. The heterologous structural sequence is assembled in
appropriate phase with translation initiation and termination
sequences. Optionally, the heterologous sequence can encode a
fusion protein including an N-terminal identification peptide
imparting desired characteristics, e.g., stabilization or
simplified purification of expressed recombinant product.
[0114] Useful expression vectors for bacterial use are constructed
by inserting a structural DNA sequence encoding a desired protein
together with suitable translation initiation and termination
signals in operable reading frame with a functional promoter. The
vector will comprise one or more phenotypic selectable markers and
an origin of replication to ensure maintenance of the vector and
to, if desirable, provide amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice.
[0115] As a representative but nonlimiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed.
[0116] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for
an additional period.
[0117] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification.
[0118] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents,
such methods are well know to those skilled in the art.
[0119] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell, 23:175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors
will comprise an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. DNA sequences derived from the SV40
splice, and polyadenylation sites may be used to provide the
required nontranscribed genetic elements.
[0120] The breast specific gene polypeptides can be recovered and
purified from recombinant cell cultures by methods including
ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Protein
refolding steps can be used, as necessary, in completing
configuration of the mature protein. Finally, high performance
liquid chromatography (HPLC) can be employed for final purification
steps.
[0121] The polynucleotides of the present invention may have the
coding sequence fused in frame to a marker sequence which allows
for purification of the polypeptide of the present invention. An
example of a marker sequence is a hexahistidine tag which may be
supplied by a vector, preferably a pQE-9 vector, which provides for
purification of the polypeptide fused to the marker in the case of
a bacterial host, or, for example, the marker sequence may be a
hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is
used. The HA tag corresponds to an epitope derived from the
influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767
(1984)).
[0122] The polypeptides of the present invention may be a naturally
purified product, or a product of chemical synthetic procedures, or
produced by recombinant techniques from a prokaryotic or eukaryotic
host (for example, by bacterial, yeast, higher plant, insect and
mammalian cells in culture). Depending upon the host employed in a
recombinant production procedure, the polypeptides of the present
invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial
methionine amino acid residue.
[0123] BSG1, and other breast specific genes, and the protein
product thereof may be employed for early detection of breast
cancer since they are over-expressed in the breast cancer
state.
[0124] In accordance with another aspect of the present invention
there are provided assays which may be used to screen for
therapeutics to inhibit the action of the breast specific genes or
breast specific proteins of the present invention. The present
invention discloses methods for selecting a therapeutic which forms
a complex with breast specific gene proteins with sufficient
affinity to prevent their biological action. The methods include
various assays, including competitive assays where the proteins are
immobilized to a support, and are contacted with a natural
substrate and a labeled therapeutic either simultaneously or in
either consecutive order, and determining whether the therapeutic
effectively competes with the natural substrate in a manner
sufficient to prevent binding of the protein to its substrate.
[0125] In another embodiment, the substrate is immobilized to a
support, and is contacted with both a labeled breast specific
polypeptide and a therapeutic (or unlabeled proteins and a labeled
therapeutic), and it is determined whether the amount of the breast
specific polypeptide bound to the substrate is reduced in
comparison to the assay without the therapeutic added. The breast
specific polypeptide may be labeled with antibodies.
[0126] Potential therapeutic compounds include antibodies and
anti-idiotypic antibodies as described above, or in some cases, an
oligonucleotide, which binds to the polypeptide.
[0127] Another example is an antisense construct prepared using
antisense technology, which is directed to a breast specific
polynucleotide to prevent transcription. Antisense technology can
be used to control gene expression through triple-helix formation
or antisense DNA or RNA, both of which methods are based on binding
of a polynucleotide to DNA or RNA. For example, the 5' coding
portion of the polynucleotide sequence, which encodes for the
mature polypeptides of the present invention, is used to design an
antisense RNA oligonucleotide of from about 10 to 40 base pairs in
length. A DNA oligonucleotide is designed to be complementary to a
region of the gene involved in transcription (triple helix--see Lee
et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science,
241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)),
thereby preventing transcription and the production of abreast
specific polynucleotide. The antisense RNA oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of the mRNA
molecule into the breast specific genes polypeptide
(antisense--Okano, J. Neurochem., 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988)). The oligonucleotides described
above can also be delivered to cells such that the antisense RNA or
DNA may be expressed in vivo to inhibit production of the breast
specific polypeptides.
[0128] Another example is a small molecule which binds to and
occupies the active site of the breast specific polypeptide thereby
making the active site inaccessible to substrate such that normal
biological activity is prevented. Examples of small molecules
include but are not limited to small peptides or peptide-like
molecules.
[0129] These compounds may be employed to treat breast cancer,
since they interact with the function of breast specific
polypeptides in a manner sufficient to inhibit natural function
which is necessary for the viability of breast cancer cells. This
is true since the BSGs and their protein products are primarily
expressed in breast cancer tissues and are, therefore, suspected of
being critical to the formation of this state.
[0130] The compounds may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as hereinafter
described.
[0131] The compounds of the present invention may be employed in
combination with a suitable pharmaceutical carrier. Such
compositions comprise a therapeutically effective amount of the
polypeptide, and a pharmaceutically acceptable carrier or
excipient. Such a carrier includes but is not limited to saline,
buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. The formulation should suit the mode of
administration.
[0132] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration. In addition, the pharmaceutical compositions
may be employed in conjunction with other therapeutic
compounds.
[0133] The pharmaceutical compositions may be administered in a
convenient manner such as by the oral, topical, intravenous,
intraperitoneal, intramuscular, subcutaneous, intranasal,
intra-anal or intradermal routes. The pharmaceutical compositions
are administered in an amount which is effective for treating
and/or prophylaxis of the specific indication. In general, they are
administered in an amount of at least about 10 g/kg body weight and
in most cases they will be administered in an amount not in excess
of about 8 mg/Kg body weight per day. In most cases, the dosage is
from about 10 g/kg to about 1 mg/kg body weight daily, taking into
account the routes of administration, symptoms, etc.
[0134] The breast specific genes and compounds which are
polypeptides may also be employed in accordance with the present
invention by expression of such polypeptides in vivo, which is
often referred to as "gene therapy."
[0135] Thus, for example, cells from a patient may be engineered
with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo,
with the engineered cells then being provided to a patient to be
treated with the polypeptide. Such methods are well-known in the
art. For example, cells may be engineered by procedures known in
the art by use of a retroviral particle containing RNA encoding a
polypeptide of the present invention.
[0136] Similarly, cells may be engineered in vivo for expression of
a polypeptide in vivo by, for example, procedures known in the art.
As known in the art, a producer cell for producing a retroviral
particle containing RNA encoding a polypeptide of the present
invention may be administered to a patient for engineering cells in
vivo and expression of the polypeptide in vivo. These and other
methods for administering a polypeptide of the present invention by
such method should be apparent to those skilled in the art from the
teachings of the present invention. For example, the expression
vehicle for engineering cells may be other than a retrovirus, for
example, an adenovirus which may be used to engineer cells in vivo
after combination with a suitable delivery vehicle.
[0137] Retroviruses from which the retroviral plasmid vectors
hereinabove mentioned may be derived include, but are not limited
to, Moloney Murine Leukemia Virus, spleen necrosis virus,
retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus,
avian leukosis virus, gibbon ape leukemia virus, human
immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma
Virus, and mammary tumor virus. In one embodiment, the retroviral
plasmid vector is derived from Moloney Murine Leukemia Virus.
[0138] The vector includes one or more promoters. Suitable
promoters which may be employed include, but are not limited to,
the retroviral LTR; the SV40 promoter; and the human
cytomegalovirus (CMV) promoter described in Miller, et al.,
Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter
(e.g., cellular promoters such as eukaryotic cellular promoters
including, but not limited to, the histone, pol III, and -actin
promoters). Other viral promoters which may be employed include,
but are not limited to, adenovirus promoters, thymidine kinase (TK)
promoters, and B19 parvovirus promoters. The selection of a
suitable promoter will be apparent to those skilled in the art from
the teachings contained herein.
[0139] The nucleic acid sequence encoding the polypeptide of the
present invention is under the control of a suitable promoter.
Suitable promoters which may be employed include, but are not
limited to, adenoviral promoters, such as the adenoviral major late
promoter; or heterologous promoters, such as the cytomegalovirus
(CMV) promoter; the respiratory syncytial virus (RSV) promoter;
inducible promoters, such as the MMT promoter, the metallothionein
promoter; heat shock promoters; the albumin promoter; the ApoAI
promoter; human globin promoters; viral thymidine kinase promoters,
such as the Herpes Simplex thymidine kinase promoter; retroviral
LTRs (including the modified retroviral LTRs hereinabove
described); the -actin promoter; and human growth hormone
promoters. The promoter also may be the native promoter which
controls the genes encoding the polypeptides.
[0140] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, -2, -AM, PA12, T19-14X, VT-19-17-H2,
CRE, CRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in
Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990), which is
incorporated herein by reference in its entirety. The vector may
transduce the packaging cells through any means known in the art.
Such means include, but are not limited to, electroporation, the
use of liposomes, and CaPO.sub.4 precipitation. In one alternative,
the retroviral plasmid vector may be encapsulated into a liposome,
or coupled to a lipid, and then administered to a host.
[0141] The producer cell line generates infectious retroviral
vector particles which include the nucleic acid sequence(s)
encoding the polypeptides. Such retroviral vector particles then
may be employed, to transduce eukaryotic cells, either in vitro or
in vivo. The transduced eukaryotic cells will express the nucleic
acid sequence(s) encoding the polypeptide. Eukaryotic cells which
may be transduced include, but are not limited to, embryonic stem
cells, embryonic carcinoma cells, as well as hematopoietic stem
cells, hepatocytes, fibroblasts, myoblasts, keratinocytes,
endothelial cells, and bronchial epithelial cells.
[0142] This invention is also related to the use of a breast
specific genes of the present invention as a diagnostic. For
example, some diseases result from inherited defective genes. The
breast specific genes, CSG7 and CSG10, for example, have been found
to have a reduced expression in breast cancer cells as compared to
that in normal cells. Further, the remaining breast specific genes
of the present invention are overexpressed in breast cancer.
Accordingly, a mutation in these genes allows a detection of breast
disorders, for example, breast cancer. A mutation in a breast
specific gene of the present invention at the DNA level may be
detected by a variety of techniques. Nucleic acids used for
diagnosis (genomic DNA, mRNA, etc.) may be obtained from a
patient's cells, other than from the breast, such as from blood,
urine, saliva, tissue biopsy and autopsy material. The genomic DNA
may be used directly for detection or may be amplified
enzymatically by using PCR (Saiki, et al., Nature 324:163-166
(1986)) prior to analysis. RNA or cDNA may also be used for the
same purpose. As an example, PCR primers complementary to the
nucleic acid of the instant invention can be used to identify and
analyze mutations in a breast specific polynucleotide of the
present invention. For example, deletions and insertions can be
detected by a change in size of the amplified product in comparison
to the normal genotype. Point mutations can be identified by
hybridizing amplified DNA to radiolabelled breast specific RNA or,
alternatively, radiolabelled antisense DNA sequences.
[0143] Another well-established method for screening for mutations
in particular segments of DNA after PCR amplification is
single-strand conformation polymorphism (SSCP) analysis. PCR
products are prepared for SSCP by ten cycles of reamplification to
incorporate .sup.32P-dCTP, digested with an appropriate restriction
enzyme to generate 200-300 bp fragments, and denatured by heating
to 85 C for 5 min. and then plunged into ice. Electrophoresis is
then carried out in a nondenaturing gel (5% glycerol, 5%
acrylamide) (Glavac, D. and Dean, M., Human Mutation, 2:404-414
(1993)).
[0144] Sequence differences between the reference gene and
"mutants" may be revealed by the direct DNA sequencing method. In
addition, cloned DNA segments may be used as probes to detect
specific DNA segments. The sensitivity of this method is greatly
enhanced when combined with PCR. For example, a sequencing primer
is used with double-stranded PCR product or a single-stranded
template molecule generated by a modified PCR. The sequence
determination is performed by conventional procedures with
radiolabeled nucleotides or by automatic sequencing procedures with
fluorescent-tags.
[0145] Genetic testing based on DNA sequence differences may be
achieved by detection of alteration in electrophoretic mobility of
DNA fragments and gels with or without denaturing agents. Small
sequence deletions and insertions can be visualized by
high-resolution gel electrophoresis. DNA fragments of different
sequences may be distinguished on denaturing formamide gradient
gels in which the mobilities of different DNA fragments are
retarded in the gel at different positions according to their
specific melting or partial melting temperatures (see, e.g., Myers,
et al., Science, 230:1242 (1985)). In addition, sequence
alterations, in particular small deletions, may be detected as
changes in the migration pattern of DNA.
[0146] Sequence changes at specific locations may also be revealed
by nuclease protection assays, such as Rnase and S1 protection or
the chemical cleavage method (e.g., Cotton, et al., PNAS, USA,
85:4397-4401 (1985)).
[0147] Thus, the detection of the specific DNA sequence may be
achieved by methods such as hybridization, RNase protection,
chemical cleavage, direct DNA sequencing, or the use of restriction
enzymes (e.g., Restriction Fragment Length Polymorphisms (RFLP))
and Southern blotting.
[0148] The sequences of the present invention are also valuable for
chromosome identification. The sequence is specifically targeted to
and can hybridize with a particular location on an individual human
chromosome. Moreover, there is a current need for identifying
particular sites on the chromosome. Few chromosome marketing
reagents based on actual sequence data (repeat polymorphisms) are
presently available for marking chromosomal location. The mapping
of DNAs to chromosomes according to the present invention is an
important first step in correlating those sequences with genes
associated with disease.
[0149] Briefly, sequences can be mapped to chromosomes by preparing
PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis
of the 3' untranslated region is used to rapidly select primers
that do not span more than one exon in the genomic DNA, thus
complicating the amplification process. These primers are then used
for PCR screening of somatic cell hybrids containing individual
human chromosomes. Only those hybrids containing the human gene
corresponding to the primer will yield an amplified fragment.
[0150] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular DNA to a particular chromosome. Using the
present invention with the same oligonucleotide primers,
sublocalization can be achieved with panels of fragments from
specific chromosomes or pools of large genomic clones in an
analogous manner. Other mapping strategies that can similarly be
used to map to its chromosome include in situ hybridization,
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0151] Fluorescence in situ hybridization (FISH) of a cDNA clone to
a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
cDNA as short as 50 or 60 bases. For a review of this technique,
see Verma et al., Human Chromosomes: a Manual of Basic Techniques,
Pergamon Press, New York (1988).
[0152] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man (available on
line through Johns Hopkins University Welch Medical Library). The
relationship between genes and diseases that have been mapped to
the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0153] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0154] With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a chromosomal
region associated with the disease could be one of between 50 and
500 potential causative genes. (This assumes 1 megabase mapping
resolution and one gene per 20 kb).
[0155] The polypeptides, their fragments or other derivatives, or
analogs thereof, or cells expressing them can be used as an
immunogen to produce antibodies thereto. These antibodies can be,
for example, polyclonal or monoclonal antibodies. The present
invention also includes chimeric, single chain, and humanized
antibodies, as well as Fab fragments, or the product of an Fab
expression library. Various procedures known in the art may be used
for the production of such antibodies and fragments.
[0156] Antibodies generated against the polypeptides corresponding
to a sequence of the present invention can be obtained by direct
injection of the polypeptides into an animal or by administering
the polypeptides to an animal, preferably a nonhuman. The antibody
so obtained will then bind the polypeptides itself. In this manner,
even a sequence encoding only a fragment of the polypeptides can be
used to generate antibodies binding the whole native polypeptides.
Such antibodies can then be used to isolate the polypeptide from
tissue expressing that polypeptide.
[0157] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler and
Milstein, 1975, Nature, 256:495-497), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., 1983, Immunology
Today 4:72), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
[0158] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products of this
invention. Transgenic mice may also be used to generate
antibodies.
[0159] The antibodies may also be employed to target breast cancer
cells, for example, in a method of homing interaction agents which,
when contacting breast cancer cells, destroy them. This is true
since the antibodies are specific for the breast specific
polypeptides of the present invention. A linking of the interaction
agent to the antibody would cause the interaction agent to be
carried directly to the breast.
[0160] Antibodies of this type may also be used to do in vivo
imaging, for example, by labeling the antibodies to facilitate
scanning of the pelvic area and the breast. One method for imaging
comprises contacting any cancer cells of the breast to be imaged
with an anti-breast specific protein-antibody labeled with a
detectable marker. The method is performed under conditions such
that the labeled antibody binds to the breast specific
polypeptides. In a specific example, the antibodies interact with
the breast, for example, breast cancer cells, and fluoresce upon
contact such that imaging and visibility of the breast are enhanced
to allow a determination of the diseased or non-diseased state of
the breast.
[0161] The present invention will be further described with
reference to the following examples; however, it is to be
understood that the present invention is not limited to such
examples. All parts or amounts, unless otherwise specified, are by
weight.
[0162] In order to facilitate understanding of the following
examples certain frequently occurring methods and/or terms will be
described.
[0163] "Plasmids" are designated by a lower case p preceded and/or
followed by capital letters and/or numbers. The starting plasmids
herein are either commercially available, publicly available on an
unrestricted basis, or can be constructed from available plasmids
in accord with published procedures. In addition, equivalent
plasmids to those described are known in the art and will be
apparent to the ordinarily skilled artisan.
[0164] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 .mu.g of
plasmid or DNA fragment is used with about 2 units of enzyme in
about 20 .mu.l of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 .mu.g of DNA
are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Incubation
times of about 1 hour at 37 C are ordinarily used, but may vary in
accordance with the supplier's instructions. After digestion the
reaction is electrophoresed directly on a polyacrylamide gel to
isolate the desired fragment.
[0165] Size separation of the cleaved fragments is performed using
1 percent TAE agarose gel described by Sambrook, et al., "Molecular
Cloning: A Laboratory Manual" Cold Spring Laboratory Press,
(1989).
[0166] "Oligonucleotides" refers to either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide
strands which may be chemically synthesized. Such synthetic
oligonucleotides have no 5' phosphate and thus will not ligate to
another oligonucleotide without adding a phosphate with an ATP in
the presence of a kinase. A synthetic oligonucleotide will ligate
to a fragment that has not been dephosphorylated.
[0167] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments (Maniatis,
T., et al., Id., p. 146). Unless otherwise provided, ligation may
be accomplished using known buffers and conditions with 10 units of
T4 DNA ligase ("ligase") per 0.5 .mu.g of approximately equimolar
amounts of the DNA fragments to be ligated.
[0168] Unless otherwise stated, transformation was performed as
described in the method of Graham, F. and Van der Eb, A., Virology,
52:456-457 (1973).
EXAMPLE 1
Determination of Transcription of a Breast Specific Gene
[0169] To assess the presence or absence of active transcription of
a breast specific gene RNA, approximately 6 ml of venous blood is
obtained with a standard venipuncture technique using heparinized
tubes. Whole blood is mixed with an equal volume of phosphate
buffered saline, which is then layered over 8 ml of Ficoll
(Pharmacia, Uppsala, Sweden) in a 15-ml polystyrene tube. The
gradient is centrifuged at 1800.times.g for 20 min at 5 C. The
lymphocyte and granulocyte layer (approximately 5 ml) is carefully
aspirated and rediluted up to 50 ml with phosphate-buffered saline
in a 50-ml tube, which is centrifuged again at 1800.times.g for 20
min. at 5 C. The supernatant is discarded and the pellet containing
nucleated cells is used for RNA extraction using the RNazole B
method as described by the manufacturer (Tel-Test Inc.,
Friendswood, Tex.).
[0170] To determine the quantity of mRNA, a probe is designed with
an identity to at least a portion of the mRNA sequence transcribed
from a human gene whose coding portion includes a DNA sequence of
FIGS. 1-20 (SEQ ID NO: 1-2, 4, 6, 8-9, 10-24). This probe is mixed
with the extracted RNA and the mixed DNA and RNA are precipitated
with ethanol -70 C for 15 minutes). The pellet is resuspended in
hybridization buffer and dissolved. The tubes containing the
mixture are incubated in a 72 C water bath for 10-15 mins. to
denature the DNA. The tubes are rapidly transferred to a water bath
at the desired hybridization temperature. Hybridization temperature
depends on the G+C content of the DNA. Hybridization is done for 3
hrs. 0.3 ml of nuclease-S1 buffer is added and mixed well. 50 l of
4.0 M ammonium acetate and 0.1 M EDTA is added to stop the
reaction. The mixture is extracted with phenol/chloroform and 20 g
of carrier tRNA is added and precipitation is done with an equal
volume of isopropanol. The precipitate is dissolved in 40 l of TE
(pH 7.4) and run on an alkaline agarose gel. Following
electrophoresis, the RNA is microsequenced to confirm the
nucleotide sequence. (See Favaloro, J. et al., Methods Enzymol.,
65:718 (1980) for a more detailed review).
[0171] Two oligonucleotide primers are employed to amplify the
sequence isolated by the above methods. The 5 primer is 20
nucleotides long and the 3 primer is a complimentary sequence for
the 3 end of the isolated mRNA. The primers are custom designed
according to the isolated mRNA. The reverse transcriptase reaction
and PCR amplification are performed sequentially without
interruption in a Perkin Elmer 9600 PCR machine (Emeryville,
Calif.). Four hundred ng total RNA in 20 l
diethylpyrocarbonate-treated water are placed in a 65 C water bath
for 5 min. and then quickly chilled on ice immediately prior to the
addition of PCR reagents. The 50-1 total PCR volume consisted of
2.5 units Taq polymerase (Perkin-Elmer). 2 units avian
myeloblastosis virus reverse transcriptase (Boehringer Mannheim,
Indianapolis, Ind.); 200 M each of dCTP, dATP, dGTP and dTTP
(Perkin Elmer); 18 pM each primer, 10 mM Tris-HCl; 50 mM KCl; and 2
MM MgCl.sub.2 (Perkin Elmer). PCR conditions are as follows: cycle
1 is 42 C for 15 min then 97 C for 15 s (1 cycle); cycle 2 is 95 C
for 1 min. 60 C for 1 min, and 72 C for 30 s (15 cycles); cycle 3
is 95 C for 1 min. 60 C for 1 min., and 72 C for 1 min. (10
cycles); cycle 4 is 95 C for 1 min., 60 C for 1 min., and 72 C for
2 min. (8 cycles); cycle 5 is 72 C for 15 min. (1 cycle); and the
final cycle is a 4 C hold until sample is taken out of the machine.
The 50-1 PCR products are concentrated down to 10 l with vacuum
centrifugation, and a sample is then run on a thin 1.2%
Tris-borate-EDTA agarose gel containing ethidium bromide. A band of
expected size would indicate that this gene is present in the
tissue assayed. The amount of RNA in the pellet may be quantified
in numerous ways, for example, it may be weighed.
[0172] Verification of the nucleotide sequence of the PCR products
is done by microsequencing. The PCR product is purified with a
Qiagen PCR Product Purification Kit (Qiagen, Chatsworth, Calif.) as
described by the manufacturer. One g of the PCR product undergoes
PCR sequencing by using the Taq DyeDeoxy Tenninator Cycle
sequencing kit in a Perlin-Elmer 9600 PCR machine as described by
Applied Biosystems (Foster, Calif.). The sequenced product is
purified using Centri-Sep columns (Princeton Separations, Adelphia,
N.J.) as described by the company. This product is then analyzed
with an ABI model 373A DNA sequencing system (Applied Biosystems)
integrated with a Macintosh IIci computer.
EXAMPLE 2
Bacterial Expression and Purification of the BSG Proteins and Use
for Preparing a Monoclonal Antibody
[0173] The DNA sequence encoding a polypeptide of the present
invention, for this example BSG1, ATCC # 97175, is initially
amplified using PCR oligonucleotide primers corresponding to the 5'
sequences of the protein and the vector sequences 3' to the
protein. Additional nucleotides corresponding to the DNA sequence
are added to the 5' and 3' sequences respectively. The 5'
oligonucleotide primer has the sequence 5' GCCACCATGGATGTTTTCAAG 3'
(SEQ ID NO:25) and contains an NcoI restriction enzyme site
followed by 15 nucleotides of coding sequence starting from the
initial amino acid of the processed protein. The 3' sequence 5'
GCGCAGATCTGTCTCCCCCACTCTGGGC 3' (SEQ ID NO:26) and contains a
complementary sequence to a BgII restriction enzyme site and is
followed by 18 nucleotides of the nucleic acid sequence encoding
the protein. The restriction enzyme sites correspond to the
restriction enzyme sites on a bacterial expression vector, pQE-60
(Qiagen, Inc. Chatsworth, Calif.). pQE-60 encodes antibiotic
resistance (Amp'), a bacterial origin of replication (ori), an
IPTG-regulatable promoter operator (P/O), a ribosome binding site
(RBS), a 6-His tag and restriction enzyme sites. pQE-60 is then
digested with NcoI and BgIII. The amplified sequences are ligated
into pQE-60 and inserted in frame with the sequence encoding for
the histidine tag and the RBS. The ligation mixture is then used to
transform an E. coli strain M 15/rep 4 (Qiagen) by the procedure
described in Sambrook, J. et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Laboratory Press, (1989). M15/rep4 contains
multiple copies of the plasmid pREP4, which expresses the lacI
repressor and also confers kanamycin resistance (Kan').
Transformants are identified by their ability to grow on LB plates
and ampicillin/kanamycin resistant colonies are selected. Plasmid
DNA is isolated and confirmed by restriction analysis.
[0174] Clones containing the desired constructs are grown overnight
(O/N) in liquid culture in LB media supplemented with both Amp (100
ug/ml) and Kan (25 ug/mil). The O/N culture is used to inoculate a
large culture at a ratio of 1:100 to 1:250. The cells are grown to
an optical density 600 (O.D..sup.600) of between 0.4 and 0.6. IPTG
("Isopropyl-B-D-thiogalacto pyranoside") is then added to a final
concentration of 1 mM. IPTG induces by inactivating the lacI
repressor, clearing the P/O leading to increased gene expression.
Cells are grown an extra 3 to 4 hours. Cells are then harvested by
centrifugation. The cell pellet is solubilized in the chaotropic
agent 6 Molar Guanidine HCl. After clarification, solubilized
protein is purified from this solution by chromatography on a
Nickel-Chelate column under conditions that allow for tight binding
by proteins containing the 6-His tag (Hochuli, E. et al., J.
Chromatography 411:177-184 (1984)). BSG1 protein (>90% pure) is
eluted from the column in 6 molar guanidine HCl pH 5.0 and for the
purpose of renaturation adjusted to 3 molar guanidine HCl, 100 mM
sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar
glutathione (oxidized). After incubation in this solution for 12
hours the protein is dialyzed to 10 mmolar sodium phosphate.
[0175] The protein purified in this manner may be used as an
epitope to raise monoclonal antibodies specific to such protein.
The monoclonal antibodies generated against the polypeptide the
isolated protein can be obtained by direct injection of the
polypeptides into an animal or by administering the polypeptides to
an animal. The antibodies so obtained will then bind to the protein
itself. Such antibodies can then be used to isolate the protein
from tissue expressing that polypeptide by the use of an, for
example, ELISA assay.
EXAMPLE 3
Preparation of cDNA Libraries from Breast Tissue
[0176] Total cellular RNA is prepared from tissues by the
guanidinium-phenol method as previously described (P. Chomczynski
and N. Sacchi, Anal. Biochem., 162: 156-159 (1987)) using RNAzol
(Cinna-Biotecx). An additional ethanol precipitation of the RNA is
included. Poly A mRNA is isolated from the total RNA using oligo
dT-coated latex beads (Qiagen). Two rounds of poly A selection are
performed to ensure better separation from non-polyadenylated
material when sufficient quantities of total RNA are available.
[0177] The mRNA selected on the oligo dT is used for the synthesis
of cDNA by a modification of the method of Gobbler and Hoffman
(Gobbler, U. and B. J. Hoffman, 1983, Gene, 25:263). The first
strand synthesis is performed using either Moloney murine sarcoma
virus reverse transcriptase (Stratagene) or Superscript II (RNase H
minus Moloney murine reverse transcriptase, Gibco-BRL). First
strand synthesis is primed using a primer/linker containing an Xho
I restriction site. The nucleotide mix used in the synthesis
contains methylated dCTP to prevent restriction within the cDNA
sequence. For second-strand synthesis E. coli polymerase Klenow
fragment is used and [.sup.32P]-dATP is incorporated as a tracer of
nucleotide incorporation.
[0178] Following 2nd strand synthesis, the cDNA is made blunt ended
using either T4 DNA polymerase or Klenow fragment. Eco RI adapters
are added to the cDNA and the cDNA is restricted with Xho I. The
cDNA is size fractionated over a Sephacryl S-500 column (Pharmacia)
to remove excess linkers and cDNAs under approximately 500 base
pairs.
[0179] The cDNA is cloned unidirectionally into the Eco RI-Xho I
sites of either pBluescript II phagemid or lambda Uni-zap XR
(Stratagene). In the case of cloning into pBluescript II, the
plasmids are electroporated into E. coli SURE competent cells
(Stratagene). When the cDNA is cloned into Uni-Zap XR it is
packaged using the Gigipack II packaging extract (Stratagene). The
packaged phage is used to infect SURE cells and amplified. The
pBluescript phagemid containing the cDNA inserts are excised from
the lambda Zap phage using the helper phage ExAssist (Stratagene).
The rescued phagemid is plated on SOLR E. coli cells
(Stratagene).
Preparation of Sequencing Templates
[0180] Template DNA for sequencing is prepared by 1) a boiling
method or 2) PCR amplification.
[0181] The boiling method is a modification of the method of Holmes
and Quigley (Holmes, D. S. and M. Quigley, 1981, Anal. Biochem.,
114:193). Colonies from either cDNA cloned into Bluescript II or
rescued Bluescript phagemid are grown in an enriched bacterial
media overnight. 400 .mu.l of cells are centrifuged and resuspended
in STET (0.1M NaCl, 10 mM TRIS Ph 8.0, 1.0 mM EDTA and 5% Triton
X-100) including lysozyme (80 .mu.g/ml) and RNase A (4 .mu.g/ml).
Cells are boiled for 40 seconds and centrifuged for 10 minutes. The
supernatant is removed and the DNA is precipitated with PEG/NaCl
and washed with 70% ethanol (2.times.). Templates are resuspended
in water at approximately 250 ng/.mu.l.
[0182] Preparation of templates by PCR is a modification of the
method of Rosenthal et al. (Rosenthal, et al., Nucleic Acids Res.,
1993, 21:173-174). Colonies containing cDNA cloned into pBluescript
II or rescued pBluescript phagemid are grown overnight in LB
containing ampicillin in a 96 well tissue culture plate. Two .mu.l
of the cultures are used as template in a PCR reaction (Saiki, R K,
et al., Science, 239:487-493, 1988; and Saiki, R K, et al.,
Science, 230:1350-1354, 1985) using a tricine buffer system (Ponce
and Micol., Nucleic Acids Res., 1992, 20:1992.) and 200 .mu.M
dNTPs.
[0183] The primer set chosen for amplification of the templates is
outside of primer sites chosen for sequencing of the templates. The
primers used are 5'-ATGCTTCCGGCTCGTATG-3' (SEQ ID NO:27) which is
5' of the M13 reverse sequence in pBluescript and 5'
-GGGTTTCCCAGTCACGAC-3' (SEQ ID NO:28) which is 3' of the M13
forward primer in pBluescript. Any primers which correspond to the
sequence flanking the M13 forward and reverse sequences can be
used. Perkin-Elmer 9600 thermocyclers are used for amplification of
the templates with the following cycler conditions: 5 min at 94 C
(1 cycle); (20 sec at 94 C); 20 sec at 55 C (1 min at 72 C) (30
cycles); 7 min at 72 C (1 cycle). Following amplification the PCR
templates are precipitated using PEG/NaCl and washed three times
with 70% ethanol. The templates are resuspended in water.
EXAMPLE 4
Isolation of a Selected Clone From Breast Tissue
[0184] Two approaches are used to isolate a particular clone from a
cDNA library prepared from human breast tissue.
[0185] In the first, a clone is isolated directly by screening the
library using an oligonucleotide probe. To isolate a particular
clone, a specific oligonucleotide with 30-40 nucleotides is
synthesized using an Applied Biosystems DNA synthesizer according
to one of the partial sequences described in this application. The
oligonucleotide is labeled with .sup.32P-ATP using T4
polynucleotide kinase and purified according to the standard
protocol (Maniatis et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Press, Cold Spring, N.Y., 1982). The Lambda cDNA
library is plated on 1.5% agar plate to a density of 20,000-50,000
pfu/150 mm plate. These plates are screened using Nylon membranes
according to the standard phage screening protocol (Stratagene,
1993). Specifically, the Nylon membrane with denatured and fixed
phage DNA is prehybridized in 6.times.SSC, 20 mM NaH.sub.2PO.sub.4,
0.4% SDS, 5.times. Denhardt's 500 .mu.g/ml denatured, sonicated
salmon sperm DNA; and 6.times.SSC, 0.1% SDS. After one hour of
prehybridization, the membrane is hybridized with hybridization
buffer 6.times.SSC, 20 mM NaH.sub.2PO.sub.4, 0.4% SDS, 500 .mu.g/ml
denatured, sonicated salmon sperm DNA with 1.times.10.sup.6 cpm/ml
.sup.32P-probe overnight at 42 C. The membrane is washed at 45-50 C
with washing buffer 6.times.SSC, 0.1% SDS for 20-30 minutes dried
and exposed to Kodak X-ray film overnight. Positive clones are
isolated and purified by secondary and tertiary screening. The
purified clone sequenced to verify its identity to the partial
sequence described in this application.
[0186] An alternative approach to screen the cDNA library prepared
from human breast tissue is to prepare a DNA probe corresponding to
the entire partial sequence. To prepare a probe, two
oligonucleotide primers of 17-20 nucleotides derived from both ends
of the partial sequence reported are synthesized and purified.
These two oligonucleotides are used to amplify the probe using the
cDNA library template. The DNA template is prepared from the phage
lysate of the cDNA library according to the standard phage DNA
preparation protocol (Maniatis et al.). The polymerase chain
reaction is carried out in 25 .mu.l reaction mixture with 0.5 .mu.g
of the above cDNA template. The reaction mixture is 1.5-5 mM
MgCl.sub.2, 0.01% (w/v) gelatin, 20 .mu.M each of dATP, dCTP, dGTP,
dCTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase.
Thirty five cycles of PCR (denaturation at 94 C for 1 min;
annealing at 55 C for 1 min; elongation at 72 C for 1 min) are
performed with the Perkin-Elmer Cetus automated thermal cycler. The
amplified product is analyzed by agarose gel electrophoresis and
the DNA band with expected molecular weight is excised and
purified. The PCR product is verified to be the probe by subcloning
and sequencing the DNA product. The probe is labeled with the
Multiprime DNA Labelling System (Amersham) at a specific activity
<1.times.10.sup.9 dmp/.mu.g. This probe is used to screen the
lambda cDNA library according to Stratagene's protocol.
Hybridization is carried out with 5.times.TEN 920.times.TEN:0.3M
Tris-HCl pH 8.0, 0.02M EDTA and 3MNaCl), 5.times. Denhardt's, 0.5%
sodium pyrophosphate, 0.1% SDS, 0.2 mg/ml heat denatured salmon
sperm DNA and 1.times.10.sup.6 cpm/ml of [.sup.32P]-labeled probe
at 55 C for 12 hours. The filters are washed in 0.5.times.TEN at
room temperature for 20-30 min., then at 55 C for 15 min. The
filters are dried and autoradiographed at -70 C using Kodak XAR-5
film. The positive clones are purified by secondary and tertiary
screening. The sequence of the isolated clone are verified by DNA
sequencing.
[0187] General procedures for obtaining complete sequences from
partial sequences described herein are summarized as follows;
Procedure 1
[0188] Selected human DNA from the partial sequence clone (the cDNA
clone that was sequenced to give the partial sequence) is purified
e.g., by endonuclease digestion using Eco-R1, gel electrophoresis,
and isolation of the clone by removal from low melting agarose gel.
The isolated insert DNA, is radiolabeled e.g., with .sup.32P
labels, preferably by nick translation or random primer labeling.
The labeled insert is used as a probe to screen a lambda phage cDNA
library or a plasmid cDNA library. Colonies containing clones
related to the probe cDNA are identified and purified by known
purification methods. The ends of the newly purified clones are
nucleotide sequenced to identify full length sequences. Complete
sequencing of full length clones is then performed by Exonuclease
III digestion or primer walking. Northern blots of the mRNA from
various tissues using at least part of the deposited clone from
which the partial sequence is obtained as a probe can optionally be
performed to check the size of the mRNA against that of the
purported full length cDNA.
[0189] The following procedures 2 and 3 can be used to obtain full
length genes or full length coding portions of genes where a clone
isolated from the deposited clone mixture does not contain a full
length sequence. A library derived from human breast tissue or from
the deposited clone mixture is also applicable to obtaining full
length sequences from clones obtained from sources other than the
deposited mixture by use of the partial sequences of the present
invention.
Procedure 2
RACE Protocol for Recovery of Full-Length Genes
[0190] Partial cDNA clones can be made full-length by utilizing the
rapid amplification of cDNA ends (RACE) procedure described in
Frohman, M. A., Dush, M. K. and Martin, G. R. (1988) Proc. Nafl.
Acad. Sci. USA, 85:8998-9002. A cDNA clone missing either the 5' or
3' end can be reconstructed to include the absent base pairs
extending to the translational start or stop codon, respectively.
In most cases, cDNAs are missing the start of translation therefor.
The following briefly describes a modification of this original 5'
RACE procedure. Poly A+ or total RNA is reverse transcribed with
Superscript II (Gibco/BRL) and an antisense or complementary primer
specific to the cDNA sequence. The primer is removed from the
reaction with a Microcon Concentrator (Amicon). The first-strand
cDNA is then tailed with dATP and terminal deoxynucleotide
transferase (Gibco/BRL). Thus, an anchor sequence is produced which
is needed for PCR amplification. The second strand is synthesized
from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer
Cetus), an oligo-dT primer containing three adjacent restriction
sites XhoI, SalI and ClaI) at the 5' end and a primer containing
just these restriction sites. This double-stranded cDNA is PCR
amplified for 40 cycles with the same primers as well as a nested
cDNA-specific antisense primer. The PCR products are size-separated
on an ethidium bromide-agarose gel and the region of gel containing
cDNA products the predicted size of missing protein-coding DNA is
removed. cDNA is purified from the agarose with the Magic PCR Prep
kit (Promega), restriction digested with XhoI or SalI, and ligated
to a plasmid such as pBluescript SKI (Stratagene) at ShoI and EcoRV
sites. This DNA is transformed into bacteria and the plasmid clones
sequenced to identify the correct protein-coding inserts. Correct
5' ends are confirmed by comparing this sequence with the
putatively identified homologue and overlap with the partial cDNA
clone.
[0191] Several quality-controlled kits are available for purchase.
Similar reagents and methods to those above are supplied in kit
form from Gibco/BRL. A second kit is available from Clontech which
is a modification of a related technique, SLIC (single-stranded
ligation to single-stranded cDNA) developed by Dumas et al. (Dumas,
J. B., Edwards, M., Delort, J. and Mallet, Jr., 1991, Nucleic Acids
Res., 19:5227-5232). The major differences in procedure are that
the RNA is alkaline hydrolyzed after reverse transcription and RNA
ligase is used to join a restriction site-containing anchor primer
to the first-strand cDNA. This obviates the necessity for the
dA-tailing reaction which results in a polyT stretch that is
difficult to sequence past.
[0192] An alternative to generating 5' cDNA from RNA is to use cDNA
library double-stranded DNA. An asymmetric PCR-amplified antisense
cDNA strand is synthesized with an antisense cDNA-specific primer
and a plasmid-anchored primer. These primers are removed and a
symmetric PCR reaction is performed with a nested cDNA-specific
antisense primer and the plasmid-anchored primer.
Procedure 3
RNA Ligase Protocol for Generating the 5' End Sequences to Obtain
Full Length Genes
[0193] Once a gene of interest is identified, several methods are
available for the identification of the 5' or 3' portions of the
gene which may not be present in the original deposited clone.
These methods include but are not limited to filter probing, clone
enrichment using specific probes and protocols similar and
identical to 5' and 3' RACE. While the full length gene may be
present in a library and can be identified by probing, a useful
method for generating the 5' end is to use the existing sequence
information from the original partial sequence to generate the
missing information. A method similar to 5' RACE is available for
generating the missing 5' end of a desired full-length gene. (This
method was published by Fromont-Racine et al, Nucleic Acids Res.,
21(7):1683-1684 (1993). Briefly, a specific RNA oligonucleotide is
ligated to the 5' ends of a population of RNA presumably containing
full-length gene RNA transcript and a primer set containing a
primer specific to the ligated RNA oligonucleotide. A primer
specific to a known sequence (EST) of the gene of interest is used
to PCR amplify the 5' portion of the desired full length gene which
may then be sequenced and used to generate the full length gene.
This method starts with total RNA isolated from the desired source,
poly A RNA may be used but is not a prerequisite for this
procedure. The RNA preparation may then be treated with phosphatase
if necessary to eliminate 5' phosphate groups on degraded or
damaged RNA which may interfere with the later RNA ligase step. The
phosphatase if used is then inactivated and the RNA is treated with
tobacco acid pyrophosphatase in order to remove the cap structure
present at the 5' ends of messenger RNAs. This reaction leaves a 5'
phosphate group at the 5' end of the cap-cleaved RNA which can then
be ligated to an RNA oligonucleotide using T4 RNA ligase. This
modified RNA preparation can then be used as a template for first
strand cDNA synthesis using a gene-specific oligonucleotide. The
first stand synthesis reaction can then be used as a template for
PCR amplification of the desired 5' end using a primer specific to
the ligated RNA oligonucleotide and a primer specific to the known
sequence (EST) of the gene of interest. The resultant product is
then sequenced and analyzed to confirm that the 5' end sequence
belongs to the partial sequence.
EXAMPLE 5
Cloning and Expression of BSG1 Using the Baculovirus Expression
System
[0194] The DNA sequence encoding the full length BSG1 protein, ATCC
# 97175, was amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' sequences of the gene:
[0195] The 5' primer has the sequence 5'
AAAGGATCCCCCGCCATCATGGATGTTTCAAGAAG 3' (SEQ ID NO:29) and contains
a BamHI restriction enzyme site (in bold) followed by 8 nucleotides
resembling an efficient signal for the initiation of translation in
eukaryotic cells (Kozak, M., J. Mol. Biol., 196:947-950 (1987) of
the BSG1 gene (the initiation codon for translation "ATG" is
underlined).
[0196] The 3' primer has the sequence 5'
AAATCTAGACTAGTCTCCCCCACTCTG 3' (SEQ ID NO:30) and contains the
cleavage site for the restriction endonuclease XbaI and 21
nucleotides complementary to the 3' sequence of the BSGI gene. The
amplified sequences were isolated from a 1% agarose gel using a
commercially available kit ("Geneclean," BIO 101 Inc., La Jolla,
Calif.). The fragment was then digested with the endonucleases
BamHI and Xbal and then purified again on a 1% agarose gel. This
fragment is designated F2.
[0197] The vector pA2 (modification of pVL941 vector, discussed
below) is used for the expression of the BSG1 protein using the
baculovirus expression system (for review see: Summers, M. D. and
Smith, G. E. 1987, A manual of methods for baculovirus vectors and
insect cell culture procedures, Texas Agricultural Experimental
Station Bulletin No. 1555). This expression vector contains the
strong polyhedrin promoter of the Autographa californica nuclear
polyhedrosis virus (AcMNPV) followed by the recognition sites for
the restriction endonucleases BamHI and XbaI. The polyadenylation
site of the simian virus (SV)40 is used for efficient
polyadenylation. For an easy selection of recombinant virus the
beta-galactosidase gene from E. coli is inserted in the same
orientation as the polyhedrin promoter followed by the
polyadenylation signal of the polyhedrin gene. The polyhedrin
sequences are flanked at both sides by viral sequences for the
cell-mediated homologous recombination of co-transfected wild-type
viral DNA. Many other baculovirus vectors could be used in place of
pA2 such as pRG1, pAc373, pVL941 and pAcIMI (Luckow, V. A. and
Summers, M. D., Virology, 170:31-39).
[0198] The plasmid was digested with the restriction enzymes BamHI
and XbaI and dephosphorylated using calf intestinal phosphatase by
procedures known in the art. The DNA was then isolated from a 1%
agarose gel using the commercially available kit ("Geneclean" BIO
101 Inc., La Jolla, Calif.). This vector DNA is designated V2.
[0199] Fragment F2 and the dephosphorylated plasmid pA2 were
ligated with T4 DNA ligase. E. coli HB101 cells were then
transformed and bacteria identified that contained the plasmid
(pBacBSG1) with the BSG1 gene using the enzymes BamHI and XbaI. The
sequence of the cloned fragment was confirmed by DNA
sequencing.
[0200] 5 .mu.g of the plasmid pBacBSG1 was co-transfected with 1.0
.mu.g of a commercially available linearized baculovirus
("BaculoGold.TM. baculovirus DNA", Pharmingen, San Diego, Calif.)
using the lipofection method (Felgner et al. Proc. Natl. Acad. Sci.
USA, 84:7413-7417 (1987)).
[0201] 1 .mu.g of BaculoGold.TM. virus DNA and 5 .mu.g of the
plasmid pBacBSG1 were mixed in a sterile well of a microtiter plate
containing 50 .mu.l of serum free Grace's medium (Life Technologies
Inc., Gaithersburg, Md.). Afterwards 10 .mu.l Lipofectin plus 90
.mu.l Grace's medium were added, mixed and incubated for 15 minutes
at room temperature. Then the transfection mixture was added
drop-wise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm
tissue culture plate with 1 ml Grace's medium without serum. The
plate was rocked back and forth to mix the newly added solution.
The plate was then incubated for 5 hours at 27 C. After 5 hours the
transfection solution was removed from the plate and 1 ml of
Grace's insect medium supplemented with 10% fetal calf serum was
added. The plate was put back into an incubator and cultivation
continued at 27 C for four days.
[0202] After four days the supernatant was collected and a plaque
assay performed similar as described by Summers and Smith (supra).
As a modification an agarose gel with "Blue Gal" (Life Technologies
Inc., Gaithersburg) was used which allows an easy isolation of blue
stained plaques. (A detailed description of a "plaque assay" can
also be found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
page 9-10).
[0203] Four days after the serial dilution, the virus was added to
the cells and blue stained plaques were picked with the tip of an
Eppendorf pipette. The agar containing the recombinant viruses was
then resuspended in an Eppendorf tube containing 200 .mu.l of
Grace's medium. The agar was removed by a brief centrifugation and
the supernatant containing the recombinant baculovirus was used to
infect Sf9 cells seeded in 35 mm dishes. Four days later the
supernatants of these culture dishes were harvested and then stored
at 4 C.
[0204] Sf9 cells were grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells were infected with the recombinant
baculovirus V-BSG1 at a multiplicity of infection (MOI) of 2. Six
hours later the medium was removed and replaced with SF900 II
medium minus methionine and cysteine (Life Technologies Inc.,
Gaithersburg). 42 hours later 5 .mu.Ci of .sup.35S-methionine and 5
.mu.Ci 35S cysteine (Amersham) were added. The cells were further
incubated for 16 hours before they were harvested by centrifugation
and the labelled proteins visualized by SDS-PAGE and
autoradiography.
EXAMPLE 6
Expression of Recombinant BSG1 in COS cells
[0205] The expression of plasmid, BSG1 HA is derived from a vector
pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication,
2) ampicillin resistance gene, 3) E. coli replication origin, 4)
CMV promoter followed by a polylinker region, an SV40 intron and
polyadenylation site. A DNA fragment encoding the entire precursor
and a HA tag fused in frame to its 3' end was cloned into the
polylinker region of the vector, therefore, the recombinant protein
expression is directed under the CMV promoter. The HA tag
corresponds to an epitope derived from the influenza hemagglutinin
protein as previously described (I. Wilson, H. Niman, R. Heighten,
A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37:767,
(1984)). The infusion of HA tag to the target protein allows easy
detection of the recombinant protein with an antibody that
recognizes the HA epitope.
[0206] The plasmid construction strategy is described as
follows:
[0207] The DNA sequence encoding BSG1, ATCC # 97175, was
constructed by PCR using two primers: the 5' primer
AAAGGATCCCCCGCCATCATGGATGTTCAAGAAG 3' (SEQ ID NO:29) contains a
BamHI site followed by 18 nucleotides of BSG1 coding sequence
starting from the initiation codon; the 3' sequence
AAATCTAGACTAAAGCGTAGTCTGGGACGTCGTATGGGTACTCCTGGGGTCTCCCCCACTCTGGGC
3' (SEQ ID NO:31) contains complementary sequences to an XbaI site,
translation stop codon, HA tag and the last 18 nucleotides of the
BamHI coding sequence (not including the stop codon). Therefore,
the PCR product contains an BamHI site, BSG1 coding sequence
followed by HA tag fused in frame, a translation termination stop
codon next to the HA tag, and an Xbal site. The PCR amplified DNA
fragment and the vector, pcDNAI/Amp, were digested with BamHI and
Xbal restriction enzyme and ligated. The ligation mixture was
transformed into E. coli strain SURE (available from Stratagene
Cloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif.
92037) the transformed culture was plated on ampicillin media
plates and resistant colonies were selected. Plasmid DNA was
isolated from transformants and examined by restriction analysis
for the presence of the correct fragment. For expression of the
recombinant BSG protein, COS cells were transfected with the
expression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch,
T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring
Laboratory Press, (1989)). The expression of the BSG HA protein was
detected by radiolabelling and immunoprecipitation method (E.
Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours
with .sup.35S-cysteine two days post transfection. Culture media
was then collected and cells were lysed with detergent (RIPA buffer
(150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris,
pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate
and culture media were precipitated with an HA specific monoclonal
antibody. Proteins precipitated were analyzed on 15% SDS-PAGE
gels.
[0208] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, within the scope of the appended claims, the invention
may be practiced otherwise than as particularly described.
Sequence CWU 0
0
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 33 <210>
SEQ ID NO 1 <211> LENGTH: 786 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 1
gccgctgcgg cagactcgag ccagctcaag cccgcagctc gcagggagat ccagctccgt
60 cctgcctgca gcagcmcaac cctgcacacc caccatggat gtyttcaaga
agggcttctc 120 catcgccaag gagggcgtgg tgggtgcggt ggaaaagacc
aagcaggggg tgacggaagc 180 agctgagaag accaaggagg gggtcatgta
tgtgggagcc aagaccaagg agaatgttgt 240 atgtacagag cgtgacctca
gtggccgaga agaccaagga gcaggccaac gccgtgagca 300 aggctgtggt
gagcagcgtc aacactktgg ccaccaagac cgtkgaggag gcggagaaca 360
tcgcggtcam ctccgggktg ktgcgcaagg aggayttkag gccatytkcc cccaacagga
420 gggtgaggca tcmaragara rgakwgsaag wggcmrakkr ggmscagagt
gggggagact 480 agagggctac aggccagctt ggatgacctg aagagcgctc
ctctgccttg ggacaccatc 540 ccctcctagc acaaggagtg cccgctttga
gtggacatgc ggctgtcccm acgttcctgc 600 cctcgttttc cctgggccam
ccttggcctg tccaactgtg ctgttgcaac caamcttaat 660 tgccttcctt
gggccccaac caactttttg gttctttttg amcccattta tgtttgttgt 720
gaattttttt tttaaaakga tttcaaatwa aaatttgggc ccatttttta aaaaaaaaaa
780 aaaaaa 786 <210> SEQ ID NO 2 <211> LENGTH: 549
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2 ggcacgagcc cacttccaac cccccggacc gcaggcagtg
cagatccact tcgcagcagg 60 tgactttttc agggaccccc tccccagcgc
tgagctgtac gtcctgtgcc ggatcctgca 120 tgactggcca gacgacaaag
tccacaagtt actcagcagg gtcgccgaga gctgcaagcc 180 aggggccggc
ctgctgctgg tggagacgct cctggatgag gagaagaggg tggcgcagcg 240
cgccctgatg cagtcactga acatgctggt gcagactgaa ggcaaggagc ggagcctggg
300 cgagtatcag tgcttgctgg agctgcacgg cttccaccag gtgcaggtgg
tgcacttggg 360 gggtgtcctg gatgccatct tggccaccaa agtggccccc
tgaagcccag gcagcatgtt 420 cattataggg atgtcctccc ccaggctgca
ggtggaccgc ccggtcccca agtaccatag 480 gacagtcaca taggagcgtg
tagtcgtgac tgaataaaga aagcaaaagc caaaaaaaaa 540 aaaaaaaaa 549
<210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3 Gly
Thr Ser Pro Leu Pro Thr Pro Arg Thr Ala Gly Ser Ala Asp Pro 1 5 10
15 Leu Arg Ser Arg 20 <210> SEQ ID NO 4 <211> LENGTH:
491 <212> TYPE: DNA <213> ORGANISM: Homo Sapiens
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(489) <223> OTHER INFORMATION: <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (6)..(6) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (67)..(69) <223> OTHER INFORMATION: May
be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (76)..(78) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (122)..(122)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(207)..(207) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (211)..(211) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (222)..(222) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (306)..(306) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (342)..(342)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(357)..(357) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (359)..(359) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (371)..(373) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (378)..(378) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (387)..(387)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(390)..(390) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (396)..(398) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (405)..(405) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (409)..(411) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (413)..(413)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(417)..(417) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (425)..(425) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (429)..(429) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (438)..(438) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (447)..(448)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(461)..(462) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (466)..(466) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (469)..(469) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (481)..(483) <223> OTHER
INFORMATION: May be any nucleotide <400> SEQUENCE: 4 agc ngn
ctc ttc acc atc ccg ttt tcc agc aag agt cgt tca cca gac 48 Ser Xaa
Leu Phe Thr Ile Pro Phe Ser Ser Lys Ser Arg Ser Pro Asp 1 5 10 15
agg tgt tat gga agc tgc nnn agg ttg nnn aaa ttc gga gaa gtg att 96
Arg Cys Tyr Gly Ser Cys Xaa Arg Leu Xaa Lys Phe Gly Glu Val Ile 20
25 30 tct tac cag caa tta gca gcc ctg gna ggc aac ccc aaa gcc gcg
cga 144 Ser Tyr Gln Gln Leu Ala Ala Leu Xaa Gly Asn Pro Lys Ala Ala
Arg 35 40 45 gca gtt ggg agg agc aat gag agg caa tcc tgt ccc cat
cct cat ccc 192 Ala Val Gly Arg Ser Asn Glu Arg Gln Ser Cys Pro His
Pro His Pro 50 55 60 gtg cca cag agt gtn ctt nca gca gcg gan ccg
tgg gca act tac ttc 240 Val Pro Gln Ser Val Leu Xaa Ala Ala Xaa Pro
Trp Ala Thr Tyr Phe 65 70 75 80 cgg agg act ggc cgt gaa agg aat ggc
ttc tgg ccc atg aaa ggc cac 288 Arg Arg Thr Gly Arg Glu Arg Asn Gly
Phe Trp Pro Met Lys Gly His 85 90 95 cgg ttt ggg aag cca agn ttt
gga ggg agc ttc agg ttt tgg caa ggg 336 Arg Phe Gly Lys Pro Xaa Phe
Gly Gly Ser Phe Arg Phe Trp Gln Gly 100 105 110 cct ggn ttc aag gga
gcc ggn gnt acc ttc ggg gnn ncc cgn ctt ttt 384 Pro Gly Phe Lys Gly
Ala Gly Xaa Thr Phe Gly Xaa Xaa Arg Leu Phe 115 120 125 ggn cgn aaa
ttn nnt ttt ttn cat nnn gnt ggn ttt ttt tng ccn caa 432 Gly Arg Lys
Xaa Xaa Phe Xaa His Xaa Xaa Gly Phe Phe Xaa Pro Gln 130 135 140 aaa
agn ttt aaa ttn nat tgg ttt ggg gnn ttg ngg nac cct ttt ttt 480
Lys Xaa Phe Lys Xaa Xaa Trp Phe Gly Xaa Leu Xaa Xaa Pro Phe Phe 145
150 155 160 nnn gga agt tg 491 Xaa Gly Ser <210> SEQ ID NO 5
<211> LENGTH: 163 <212> TYPE: PRT <213> ORGANISM:
Homo Sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (2)..(2) <223> OTHER
INFORMATION: The 'Xaa' at location 2 stands for Arg, Ser, Gly, a
stop codon, Trp, or Cys. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (23)..(23) <223> OTHER
INFORMATION: The 'Xaa' at location 23 stands for Lys, Asn, Arg,
Ser, Thr, Ile, Met, Glu, Asp, Gly, Ala, Val, Gln, His, Pro, Leu, a
stop codon, Tyr, Trp, Cys, or Phe. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (26)..(26) <223>
OTHER INFORMATION: The 'Xaa' at location 26 stands for Lys, Asn,
Arg, Ser, Thr, Ile, Met, Glu, Asp, Gly, Ala, Val, Gln, His, Pro,
Leu, a stop codon, Tyr, Trp, Cys, or Phe. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (41)..(41)
<223> OTHER INFORMATION: The 'Xaa' at location 41 stands for
Glu, Gly, Ala, or Val. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (71)..(71) <223> OTHER
INFORMATION: The 'Xaa' at location 71 stands for Thr, Ala, Pro, or
Ser. <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (74)..(74) <223> OTHER INFORMATION: The
'Xaa' at location 74 stands for Glu, or Asp. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(102)..(102) <223> OTHER INFORMATION: The 'Xaa' at location
102 stands for Arg, or Ser. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (120)..(120)
<223> OTHER INFORMATION: The 'Xaa' at location 120 stands for
Asp, Gly, Ala, or Val. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (124)..(124) <223> OTHER
INFORMATION: The 'Xaa' at location 124 stands for Glu, Asp, Gly,
Ala, or Val. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (125)..(125) <223> OTHER
INFORMATION: The 'Xaa' at location 125 stands for Thr, Ala, Pro, or
Ser. <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (132)..(132) <223> OTHER INFORMATION:
The 'Xaa' at location 132 stands for Leu, or Phe. <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(133)..(133) <223> OTHER INFORMATION: The 'Xaa' at location
133 stands for Asn, Ser, Thr, Ile, Asp, Gly, Ala, Val, His, Arg,
Pro, Leu, Tyr, Cys, or Phe. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (135)..(135)
<223> OTHER INFORMATION: The 'Xaa' at location 135 stands for
Leu, or Phe. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (137)..(137) <223> OTHER
INFORMATION: The 'Xaa' at location 137 stands for Lys, Asn, Arg,
Ser, Thr, Ile, Met, Glu, Asp, Gly, Ala, Val, Gln, His, Pro, Leu, a
stop codon, Tyr, Trp, Cys, or Phe. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (138)..(138)
<223> OTHER INFORMATION: The 'Xaa' at location 138 stands for
Asp, Gly, Ala, or Val. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (142)..(142) <223> OTHER
INFORMATION: The 'Xaa' at location 142 stands for a stop codon,
Trp, Ser, or Leu. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (146)..(146) <223> OTHER
INFORMATION: The 'Xaa' at location 146 stands for Arg, or Ser.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (149)..(149) <223> OTHER INFORMATION: The 'Xaa' at
location 149 stands for Leu, or Phe. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(150)..(150) <223> OTHER INFORMATION: The 'Xaa' at location
150 stands for Asn, Asp, His, or Tyr. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(154)..(154) <223> OTHER INFORMATION: The 'Xaa' at location
154 stands for Glu, Asp, Gly, Ala, or Val. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(156)..(156) <223> OTHER INFORMATION: The 'Xaa' at location
156 stands for Arg, Gly, or Trp. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (157)..(157)
<223> OTHER INFORMATION: The 'Xaa' at location 157 stands for
Asn, Asp, His, or Tyr. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (161)..(161) <223> OTHER
INFORMATION: The 'Xaa' at location 161 stands for Lys, Asn, Arg,
Ser, Thr, Ile, Met, Glu, Asp, Gly, Ala, Val, Gln, His, Pro, Leu, a
stop codon, Tyr, Trp, Cys, or Phe. <400> SEQUENCE: 5 Ser Xaa
Leu Phe Thr Ile Pro Phe Ser Ser Lys Ser Arg Ser Pro Asp 1 5 10 15
Arg Cys Tyr Gly Ser Cys Xaa Arg Leu Xaa Lys Phe Gly Glu Val Ile 20
25 30 Ser Tyr Gln Gln Leu Ala Ala Leu Xaa Gly Asn Pro Lys Ala Ala
Arg 35 40 45 Ala Val Gly Arg Ser Asn Glu Arg Gln Ser Cys Pro His
Pro His Pro 50 55 60 Val Pro Gln Ser Val Leu Xaa Ala Ala Xaa Pro
Trp Ala Thr Tyr Phe 65 70 75 80 Arg Arg Thr Gly Arg Glu Arg Asn Gly
Phe Trp Pro Met Lys Gly His 85 90 95 Arg Phe Gly Lys Pro Xaa Phe
Gly Gly Ser Phe Arg Phe Trp Gln Gly 100 105 110 Pro Gly Phe Lys Gly
Ala Gly Xaa Thr Phe Gly Xaa Xaa Arg Leu Phe 115 120 125 Gly Arg Lys
Xaa Xaa Phe Xaa His Xaa Xaa Gly Phe Phe Xaa Pro Gln 130 135 140 Lys
Xaa Phe Lys Xaa Xaa Trp Phe Gly Xaa Leu Xaa Xaa Pro Phe Phe 145 150
155 160 Xaa Gly Ser <210> SEQ ID NO 6 <211> LENGTH: 489
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (3)..(488) <223> OTHER INFORMATION: <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(58)..(58) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (214)..(214) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (291)..(293) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (298)..(298) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (325)..(325)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(339)..(339) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (357)..(359) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (368)..(368) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (402)..(404) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (416)..(416)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(426)..(428) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (432)..(432) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (434)..(434) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (438)..(438)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(441)..(441) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (455)..(455) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (478)..(478) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (481)..(481) <223> OTHER
INFORMATION: May be any nucleotide <400> SEQUENCE: 6 ga ggg
acc gcc gac cac cac cag ctg cgc tca ctg act ggc ctc atc 47 Gly Thr
Ala Asp His His Gln Leu Arg Ser Leu Thr Gly Leu Ile 1 5 10 15 cga
aac ctg tnt cgg aac gct agg aac aag gac gag atg tcc acg aag 95 Arg
Asn Leu Xaa Arg Asn Ala Arg Asn Lys Asp Glu Met Ser Thr Lys 20 25
30 gtg gtg agc cac ctg atc gag aag ctg ccg ggc agc gtg ggt gag aag
143 Val Val Ser His Leu Ile Glu Lys Leu Pro Gly Ser Val Gly Glu Lys
35 40 45 tcg ccc cca gcc gag gtg ctg gtc aac atc ata gct gtg ctc
aac aac 191 Ser Pro Pro Ala Glu Val Leu Val Asn Ile Ile Ala Val Leu
Asn Asn 50 55 60 ctg gtg gtg gcc agc ccc atc gnt gcc cga gac ctg
ctg tat ttt gac 239 Leu Val Val Ala Ser Pro Ile Xaa Ala Arg Asp Leu
Leu Tyr Phe Asp 65 70 75 gga ctc cga aag ctc atc ttc atc aag aag
aag cgg gac agc ccc gac 287 Gly Leu Arg Lys Leu Ile Phe Ile Lys Lys
Lys Arg Asp Ser Pro Asp 80 85 90 95 agt nnn aag tnc tcc cgg gca gca
tcc agc ctc ctg gnc aac ctg ttg 335 Ser Xaa Lys Xaa Ser Arg Ala Ala
Ser Ser Leu Leu Xaa Asn Leu Leu 100 105 110 gca nta caa caa gtt cca
ccg nnn ttt ccg ggn gaa ggg ttt tcg gaa 383 Ala Xaa Gln Gln Val Pro
Pro Xaa Phe Pro Gly Glu Gly Phe Ser Glu 115 120 125 gga gga ttt ctg
ggg cct nnn gtg aag ctt ttn gag gag agg nnn cgt 431 Gly Gly Phe Leu
Gly Pro Xaa Val Lys Leu Xaa Glu Glu Arg Xaa Arg 130 135 140 ngn cca
ngt nca ggg aca gat tan ttc agt ttt tgg acc cag ctt gng 479 Xaa Pro
Xaa Xaa Gly Thr Asp Xaa Phe Ser Phe Trp Thr Gln Leu Xaa 145 150 155
gng agg tat t 489 Xaa Arg Tyr 160 <210> SEQ ID NO 7
<211> LENGTH: 162 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: The 'Xaa' at location 19 stands for Tyr, Cys, Ser, or
Phe. <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (71)..(71) <223> OTHER INFORMATION: The
'Xaa' at location 71 stands for Asp, Gly, Ala, or Val. <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(97)..(97) <223> OTHER INFORMATION: The 'Xaa' at location 97
stands for Lys, Asn, Arg, Ser, Thr, Ile, Met, Glu, Asp, Gly, Ala,
Val, Gln, His, Pro, Leu, a stop codon, Tyr, Trp, Cys, or Phe.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (99)..(99) <223> OTHER INFORMATION: The 'Xaa' at
location 99 stands for Tyr, Cys, Ser, or Phe. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(108)..(108) <223> OTHER INFORMATION: The 'Xaa' at location
108 stands for Asp, Gly, Ala, or Val. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(113)..(113) <223> OTHER INFORMATION: The 'Xaa' at location
113 stands for Ile, Val, or Leu. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (119)..(119)
<223> OTHER INFORMATION: The 'Xaa' at location 119 stands for
Lys, Asn, Arg, Ser, Thr, Ile, Met, Glu, Asp, Gly, Ala, Val, Gln,
His, Pro, Leu, a stop codon, Tyr, Trp, Cys, or Phe. <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(134)..(134) <223> OTHER INFORMATION: The 'Xaa' at location
134 stands for Lys, Asn, Arg, Ser, Thr, Ile, Met, Glu, Asp, Gly,
Ala, Val, Gln, His, Pro, Leu, a stop codon, Tyr, Trp, Cys, or Phe.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (138)..(138) <223> OTHER INFORMATION: The 'Xaa' at
location 138 stands for Leu, or Phe. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(142)..(142) <223> OTHER INFORMATION: The 'Xaa' at location
142 stands for Lys, Asn, Arg, Ser, Thr, Ile, Met, Glu, Asp, Gly,
Ala, Val, Gln, His, Pro, Leu, a stop codon, Tyr, Trp, Cys, or Phe.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (144)..(144) <223> OTHER INFORMATION: The 'Xaa' at
location 144 stands for Arg, Ser, Gly, a stop codon, Trp, or Cys.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (146)..(146) <223> OTHER INFORMATION: The 'Xaa' at
location 146 stands for Ser, Gly, Arg, or Cys. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(147)..(147) <223> OTHER INFORMATION: The 'Xaa' at location
147 stands for Thr, Ala, Pro, or Ser. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(151)..(151) <223> OTHER INFORMATION: The 'Xaa' at location
151 stands for a stop codon, or Tyr. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(159)..(159) <223> OTHER INFORMATION: The 'Xaa' at location
159 stands for Glu, Gly, Ala, or Val. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(160)..(160) <223> OTHER INFORMATION: The 'Xaa' at location
160 stands for Glu, Gly, Ala, or Val. <400> SEQUENCE: 7 Gly
Thr Ala Asp His His Gln Leu Arg Ser Leu Thr Gly Leu Ile Arg 1 5 10
15 Asn Leu Xaa Arg Asn Ala Arg Asn Lys Asp Glu Met Ser Thr Lys Val
20 25 30 Val Ser His Leu Ile Glu Lys Leu Pro Gly Ser Val Gly Glu
Lys Ser 35 40 45 Pro Pro Ala Glu Val Leu Val Asn Ile Ile Ala Val
Leu Asn Asn Leu 50 55 60 Val Val Ala Ser Pro Ile Xaa Ala Arg Asp
Leu Leu Tyr Phe Asp Gly 65 70 75 80 Leu Arg Lys Leu Ile Phe Ile Lys
Lys Lys Arg Asp Ser Pro Asp Ser 85 90 95 Xaa Lys Xaa Ser Arg Ala
Ala Ser Ser Leu Leu Xaa Asn Leu Leu Ala 100 105 110 Xaa Gln Gln Val
Pro Pro Xaa Phe Pro Gly Glu Gly Phe Ser Glu Gly 115 120 125 Gly Phe
Leu Gly Pro Xaa Val Lys Leu Xaa Glu Glu Arg Xaa Arg Xaa 130 135 140
Pro Xaa Xaa Gly Thr Asp Xaa Phe Ser Phe Trp Thr Gln Leu Xaa Xaa 145
150 155 160 Arg Tyr <210> SEQ ID NO 8 <211> LENGTH: 442
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (84)..(84) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (100)..(100) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (108)..(108) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (120)..(120)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(204)..(204) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (217)..(217) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (271)..(271) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (327)..(327) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (330)..(330)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(334)..(334) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (340)..(341) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (343)..(343) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (346)..(346)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(352)..(352) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (355)..(355) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (361)..(361) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (374)..(374) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (393)..(393)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(407)..(407) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (420)..(420) <223> OTHER INFORMATION: May be any
nucleotide <400> SEQUENCE: 8 agcccagccc cagcctgccg gagcccacgg
agctacctct acctccactc ctgccaccgc 60 cgccgccctc gaccaccgcc
cctntcccca tccgtccccn ctcagggnac caagaagttn 120 caagaggggc
cgtgggcgtg ccagggcgct agggaagccg ggtgggggtg agggtagccc 180
ttgagccctg tccctgcggc tgtnaagagc agcaggnacc ctgggccagt tccagagacc
240 tgggggtgtg tttgggggtg gggtgtgagt ncgtatgaaa atgtgtgttt
gctgggggca 300 attgtgccct ggaatcatgg gcaggtnggn ccgntccggn
nanggnccgg gnttnaaatt 360 ntttccgtgg aagntttaaa gggttgaatt
tanggtaaaa aaccttnggg gaagggaagn 420 tttccaaggc aaaaaaaggt tt 442
<210> SEQ ID NO 9 <211> LENGTH: 492 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (2)..(490)
<223> OTHER INFORMATION: <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (41)..(43) <223>
OTHER INFORMATION: May be any nucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(107)..(107) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (119)..(121) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (211)..(211) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (213)..(213) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (216)..(216)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(256)..(256) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (274)..(274) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (308)..(310) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (314)..(314) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (325)..(325)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(348)..(348) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (354)..(354) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (371)..(371) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (374)..(374) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (391)..(391)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(405)..(405) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (411)..(411) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (425)..(425) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (427)..(427) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (442)..(442)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(465)..(465) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (477)..(477) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (479)..(479) <223> OTHER INFORMATION:
May be any nucleotide <400> SEQUENCE: 9 a gac tcc ccg gct ggg
tac ccc cct gtg gct ggc tct gcg nnn atc atg 49 Asp Ser Pro Ala Gly
Tyr Pro Pro Val Ala Gly Ser Ala Xaa Ile Met 1 5 10 15 gat gcc ctg
aac agc ctc gcc acg gac tcg cct tgt ggg atc ccc ccc 97 Asp Ala Leu
Asn Ser Leu Ala Thr Asp Ser Pro Cys Gly Ile Pro Pro 20 25 30 aag
atg tgg naa gac cag ccc nnn ccc ctc gcc ggt gtc tgc cgc ccc 145 Lys
Met Trp Xaa Asp Gln Pro Xaa Pro Leu Ala Gly Val Cys Arg Pro 35 40
45 atc caa ggc cgg cct gcc tcg cca cat cta ccc ggc cgt gga gtt cct
193 Ile Gln Gly Arg Pro Ala Ser Pro His Leu Pro Gly Arg Gly Val Pro
50 55 60 ggg gcc ctg cga agc agn gna gna gga gaa act cgg ctc cag
aat cca 241 Gly Ala Leu Arg Ser Xaa Xaa Xaa Gly Glu Thr Arg Leu Gln
Asn Pro 65 70 75 80 tcc tgc tgg ttc cgn cca ctt ggc cca agc cgn tgg
tgc ctg cca ttc 289 Ser Cys Trp Phe Arg Pro Leu Gly Pro Ser Arg Trp
Cys Leu Pro Phe 85 90 95 cca tct gca gca tcc cag nnn ctg nat ccc
tcc ttn cat ttg agt ggc 337 Pro Ser Ala Ala Ser Gln Xaa Leu Xaa Pro
Ser Xaa His Leu Ser Gly 100 105 110 cgt tgt tcc ant cat tna ggt ttt
aag gag ttg ngg ntt gag gtg cag 385 Arg Cys Ser Xaa His Xaa Gly Phe
Lys Glu Leu Xaa Xaa Glu Val Gln 115 120 125 cca agn caa att aac ggg
gnc cat tnt gag aaa aag gaa ncn agg gtt 433 Pro Xaa Gln Ile Asn Gly
Xaa His Xaa Glu Lys Lys Glu Xaa Arg Val 130 135 140 tta aaa ttn caa
ttg ggg caa ctt tgg ttt ant tca tgg tta ang naa 481 Leu Lys Xaa Gln
Leu Gly Gln Leu Trp Phe Xaa Ser Trp Leu Xaa Xaa 145 150 155 160 aaa
agt ttg gg 492 Lys Ser Leu <210> SEQ ID NO 10 <211>
LENGTH: 163 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (14)..(14) <223> OTHER INFORMATION: The
'Xaa' at location 14 stands for Lys, Asn, Arg, Ser, Thr, Ile, Met,
Glu, Asp, Gly, Ala, Val, Gln, His, Pro, Leu, a stop codon, Tyr,
Trp, Cys, or Phe. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (36)..(36) <223> OTHER
INFORMATION: The 'Xaa' at location 36 stands for Lys, Glu, Gln, or
a stop codon. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (40)..(40) <223> OTHER
INFORMATION: The 'Xaa' at location 40 stands for Lys, Asn, Arg,
Ser, Thr, Ile, Met, Glu, Asp, Gly, Ala, Val, Gln, His, Pro, Leu, a
stop codon, Tyr, Trp, Cys, or Phe. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (70)..(70) <223>
OTHER INFORMATION: The 'Xaa' at location 70 stands for Arg, or Ser.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (71)..(71) <223> OTHER INFORMATION: The 'Xaa' at
location 71 stands for Glu, Gly, Ala, or Val. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (72)..(72)
<223> OTHER INFORMATION: The 'Xaa' at location 72 stands for
Glu, Gly, Ala, or Val.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (103)..(103) <223> OTHER INFORMATION: The 'Xaa' at
location 103 stands for Lys, Asn, Arg, Ser, Thr, Ile, Met, Glu,
Asp, Gly, Ala, Val, Gln, His, Pro, Leu, a stop codon, Tyr, Trp,
Cys, or Phe. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (105)..(105) <223> OTHER
INFORMATION: The 'Xaa' at location 105 stands for Asn, Asp, His, or
Tyr. <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (108)..(108) <223> OTHER INFORMATION:
The 'Xaa' at location 108 stands for Leu, or Phe. <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(116)..(116) <223> OTHER INFORMATION: The 'Xaa' at location
116 stands for Asn, Ser, Thr, or Ile. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(118)..(118) <223> OTHER INFORMATION: The 'Xaa' at location
118 stands for a stop codon, Ser, or Leu. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(124)..(124) <223> OTHER INFORMATION: The 'Xaa' at location
124 stands for Arg, Gly, or Trp. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (125)..(125)
<223> OTHER INFORMATION: The 'Xaa' at location 125 stands for
Ile, Val, Leu, or Phe. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (130)..(130) <223> OTHER
INFORMATION: The 'Xaa' at location 130 stands for Arg, or Ser.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (135)..(135) <223> OTHER INFORMATION: The 'Xaa' at
location 135 stands for Asp, Gly, Ala, or Val. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(137)..(137) <223> OTHER INFORMATION: The 'Xaa' at location
137 stands for Tyr, Cys, Ser, or Phe. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(142)..(142) <223> OTHER INFORMATION: The 'Xaa' at location
142 stands for Thr, Ala, Pro, or Ser. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(147)..(147) <223> OTHER INFORMATION: The 'Xaa' at location
147 stands for Leu, or Phe. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (155)..(155)
<223> OTHER INFORMATION: The 'Xaa' at location 155 stands for
Asn, Ser, Thr, or Ile. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (159)..(159) <223> OTHER
INFORMATION: The 'Xaa' at location 159 stands for Lys, Arg, Thr, or
Met. <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (160)..(160) <223> OTHER INFORMATION:
The 'Xaa' at location 160 stands for Lys, Glu, Gln, or a stop
codon. <400> SEQUENCE: 10 Asp Ser Pro Ala Gly Tyr Pro Pro Val
Ala Gly Ser Ala Xaa Ile Met 1 5 10 15 Asp Ala Leu Asn Ser Leu Ala
Thr Asp Ser Pro Cys Gly Ile Pro Pro 20 25 30 Lys Met Trp Xaa Asp
Gln Pro Xaa Pro Leu Ala Gly Val Cys Arg Pro 35 40 45 Ile Gln Gly
Arg Pro Ala Ser Pro His Leu Pro Gly Arg Gly Val Pro 50 55 60 Gly
Ala Leu Arg Ser Xaa Xaa Xaa Gly Glu Thr Arg Leu Gln Asn Pro 65 70
75 80 Ser Cys Trp Phe Arg Pro Leu Gly Pro Ser Arg Trp Cys Leu Pro
Phe 85 90 95 Pro Ser Ala Ala Ser Gln Xaa Leu Xaa Pro Ser Xaa His
Leu Ser Gly 100 105 110 Arg Cys Ser Xaa His Xaa Gly Phe Lys Glu Leu
Xaa Xaa Glu Val Gln 115 120 125 Pro Xaa Gln Ile Asn Gly Xaa His Xaa
Glu Lys Lys Glu Xaa Arg Val 130 135 140 Leu Lys Xaa Gln Leu Gly Gln
Leu Trp Phe Xaa Ser Trp Leu Xaa Xaa 145 150 155 160 Lys Ser Leu
<210> SEQ ID NO 11 <211> LENGTH: 484 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (82)..(82)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(103)..(103) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (122)..(122) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (205)..(205) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (295)..(295) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (297)..(297)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(331)..(331) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (357)..(357) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (368)..(368) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (375)..(375) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (386)..(387)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(414)..(414) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (420)..(420) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (425)..(425) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (436)..(437) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (440)..(441)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(445)..(446) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (451)..(451) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (464)..(464) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (466)..(466) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (472)..(473)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(480)..(480) <223> OTHER INFORMATION: May be any nucleotide
<400> SEQUENCE: 11 gcagcagctg gacatcgagg gcgagctgcg
ccggctcatg gccaagcccg aggctctgaa 60 gtcactgcag gagcggcggc
gngagcagga gctgctggag cantacgtga gcaccgtgaa 120 cnaccgcagt
gtacatcgtg gactcgctgg acgaggaccg gctccgggga acaagaggag 180
gatcagatgc tgcgggacat gattnagaaa gctgggcctc cagaggaaga agttccaagt
240 ttccgtttgt tccaagatct tggtcaccaa aaagcaaaaa gcagcccctt
cccantngtt 300 agcccattag ggcccttggg ttttggcccg naaccttggg
aattccggtt tttgganttt 360 gggggggncc atggnttttg gccccnnacc
cgggaaaccc ggttttttta attngggggn 420 ccctnggttt tttggnncgn
naccnngaat ntttgggttt tttngntttg gnnaaagggn 480 tttt 484
<210> SEQ ID NO 12 <211> LENGTH: 321 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (38)..(38)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (106)..(106) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (114)..(114) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (116)..(116)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(122)..(122) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (220)..(220) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (229)..(229) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (259)..(259) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (269)..(269)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(287)..(287) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (290)..(290) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (312)..(314) <223> OTHER INFORMATION:
May be any nucleotide <400> SEQUENCE: 12 aggtcatcgg
gcagccttcc ctgtgtgcca agccagcntt cgcttctgaa aaccaaactc 60
cagccgctgc cagtcgggac ttggtcgccc ggcgctgcca gaatgntcca ctgncnaccg
120 gnccccctgc ctcggtttcc cttctgttta gtggcgacac aggcacccag
ctttggggtg 180 gtgctgacgc tcccaggggt gccaggagcc actgggacan
gggtgaggnt cccagacgtt 240 ccttcgaggt gcccagttnt ccagggagnt
tctgggccca aggcgtnttn agggatcttg 300 ttcctttaaa cnnnccaatt g 321
<210> SEQ ID NO 13 <211> LENGTH: 343 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (12)..(12)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(27)..(27) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (40)..(40) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (42)..(42) <223> OTHER INFORMATION: May
be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (45)..(45) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (75)..(76) <223>
OTHER INFORMATION: May be any nucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (97)..(97)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(99)..(99) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (101)..(101) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (171)..(171) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (208)..(208) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (210)..(210)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(219)..(219) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (224)..(224) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (236)..(236) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (254)..(254) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (267)..(267)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(269)..(269) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (273)..(273) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (275)..(276) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (289)..(289) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (296)..(298)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(302)..(302) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (304)..(305) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (312)..(312) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (315)..(315) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (318)..(318)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(332)..(332) <223> OTHER INFORMATION: May be any nucleotide
<400> SEQUENCE: 13 acgagaacag angatagagg gcatctntcc
caggtgaccn tnctnttctg tcccaggagg 60 gtgggtaatt ccctnnggga
tggggctccc acacctncnt naggtcccca ctcagaccag 120 caccagtgtc
tgcctctgag aatgttggca gctcacagag agcagggccg ncccgggatg 180
gggggcaggt actccccacc ttcctgcntn cgatcctant tctnatccag cgtccncttt
240 attaccgttt tttnactaat gcttctntng agnanngctc tttggaagna
ggagcnnnag 300 cntnntggag cntcncanga gatgtttaag gnttattaag ctt 343
<210> SEQ ID NO 14 <211> LENGTH: 480 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (16)..(16)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(60)..(60) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (124)..(124) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (155)..(155) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (204)..(204) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (246)..(246)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(265)..(265) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (269)..(269) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (320)..(320) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (331)..(331) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (377)..(377)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(385)..(385) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (395)..(396) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (398)..(398) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(402)..(402) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (413)..(413) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (431)..(431) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (435)..(435) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (439)..(441)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(453)..(454) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (462)..(462) <223> OTHER INFORMATION: May be any
nucleotide <400> SEQUENCE: 14 acatttggtg tatgtncttg
gctgaggtct acttggcttg cctgctttga tcctgagagn 60 cacccacccc
atctcacagt gaataggttg gcaggtgtgg gctggcgggt ggactacacc 120
ctgnagctcc agcctgctgc aatccgtgga agagnccatg gtgcacctgc ggctggaggt
180 ggcagctgcc ccagggaccc cagnccagcc tgttgccatg tccctctcag
cagacaattc 240 caggtnctcc tggcagaact gaagnaggnc cagaccctga
tgagctcctt gggttgagga 300 gaagggtgtt tccaggcctn tttggagccg
ncttgcccgt atggagttaa ggccttttga 360 attgtttttg gggaggnaac
ctggntttta aggtnntnaa gnccttggcc cgnattttgg 420 cttccaaatt
nccanttgnn naaatttttt ttnngaattt gnttaaggtt tgggactttt 480
<210> SEQ ID NO 15 <211> LENGTH: 481 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (57)..(57)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(187)..(187) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (223)..(224) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (244)..(244) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (248)..(248) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (251)..(251)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(272)..(272) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (294)..(294) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (297)..(297) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (299)..(299) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (312)..(312)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(328)..(328) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (332)..(332) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (336)..(336) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (340)..(340) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (354)..(354)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(362)..(362) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (367)..(367) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (381)..(381) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (383)..(383) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (385)..(385)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(396)..(396) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (404)..(405) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (407)..(407) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (409)..(410) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (412)..(412)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(415)..(415) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (428)..(429) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (431)..(431) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (437)..(437) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (442)..(442)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(459)..(460) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (463)..(464) <223> OTHER INFORMATION: May be any
nucleotide <400> SEQUENCE: 15 agcattgtgg gtaaaggcct
ggaggcagga aagtgaagga caatttcaag aaactcngtt 60 catcaatttt
catcaacacc ttcctgggcc atgcctgggt actgaggaac ccagccctga 120
atctggacat cattttccct ttcagagcat agaatgcagg gggatccagg aatgggttaa
180 caggagngaa gcttggttca aggagacctt tgcgtaccag ggnnaaggga
gttttgaact 240 ttantctnca nggcaggcag agcacgacag gnttcctacc
tggggtcaat tcanttntnc 300 tatttctctt antgttgcca tccgtagntc
cncggntaan ttggggaggt ttanggcagg 360 gngaatnggt tttgaacccc
ngngngttgg gggttngcaa ttgnngntnn gntgngaacc 420 attgaatnng
nggcttnggt gntccaggtt gggatcccnn ttnnaaaaaa aaaaaaaaat 480 t 481
<210> SEQ ID NO 16 <211> LENGTH: 484 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(47)..(47) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (76)..(76) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (162)..(162) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (310)..(310) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (320)..(320)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(323)..(323) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (357)..(357) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (361)..(361) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (369)..(369) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature
<222> LOCATION: (379)..(379) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (394)..(394) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (400)..(400)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(406)..(406) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (414)..(414) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (417)..(417) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (438)..(438) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (446)..(446)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(449)..(449) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (470)..(470) <223> OTHER INFORMATION: May be any
nucleotide <400> SEQUENCE: 16 gagacgcact ctctagcccg
gcagatgaag gcganggcgg cggccgnact tggatgagat 60 tcaccgcgag
ctgcgncctc agggatccgc acgaccccag cccgacccaa acgccgagtt 120
cgaccccgac ctgccagggg gcggcctgca ccgctgtctg gnctgcgcga ggtacttcat
180 cgattccacc aacctgaaga cccacttccg atccaaagac cacaagaaaa
ggctgaagca 240 gcttgagcgt cgagccctta cagtcaggaa gaggcggaga
gggcagcggg gttattggga 300 ttctatgtgn ccccccaggn ggnttggcag
tgcccacgga agttttccat tgaggtncct 360 nagattggnt aacttttanc
tgaaaatggg cttnaagttn caaggnaaaa gganttnccc 420 ctgggcaatt
acgaaagntt tagttnggna gggaggtttc aacccttttn cctttggttt 480 tggg 484
<210> SEQ ID NO 17 <211> LENGTH: 478 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (56)..(56)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(61)..(61) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (129)..(129) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (158)..(158) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (206)..(206) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (262)..(262)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(279)..(279) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (284)..(284) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (292)..(292) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (303)..(303) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (310)..(310)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(339)..(339) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (357)..(358) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (361)..(361) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (363)..(363) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (372)..(372)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(387)..(387) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (402)..(403) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (409)..(409) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (423)..(425) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (428)..(428)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(434)..(435) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (439)..(439) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (446)..(446) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (450)..(450) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (454)..(454)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(461)..(462) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (464)..(464) <223> OTHER INFORMATION: May be any
nucleotide <400> SEQUENCE: 17 aggcacagct gttgcgtcag
gcaaggtcac ctgcatttat ttattgagca gcagtnctgt 60 ntcaggccca
ggggccgagc ccctctccct gttcccctat ggtgtctccg aggccctctg 120
ggagggccnc acatctggga gcagcacctc agagtggnac agaaagcatt agcgtgccac
180 gagcttcacc cgacgccgag cctgtnaagg tgggctgatg gtgcccgttt
taacccagcg 240 cttcagggag gttcagaatg gnagccgaaa cccaggggnt
gttnagcatc ancttctggg 300 agnccttttn tactttttat ggactggttt
cctgggaang ggttggttgg gggaagnnca 360 ngnaggcttg gngttccttt
agggttnggg gggcctgttt tnncattcna acccaagctt 420 ttnnnttnca
tttnncttnt tttttngggn ggcnagtaaa nngntccaag gttttttt 478
<210> SEQ ID NO 18 <211> LENGTH: 480 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (85)..(85)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(90)..(90) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (94)..(94) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (274)..(275) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (285)..(285) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (292)..(292)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(300)..(300) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (309)..(309) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (321)..(321) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (323)..(324) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (330)..(330)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(332)..(332) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (342)..(342)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(347)..(347) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (349)..(350) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (356)..(356) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (360)..(360) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (373)..(374)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(376)..(376) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (390)..(390) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (392)..(392) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (402)..(402) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (408)..(408)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(411)..(411) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (413)..(413) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (430)..(430) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (433)..(434) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (442)..(442)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(449)..(449) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (470)..(470) <223> OTHER INFORMATION: May be any
nucleotide <400> SEQUENCE: 18 gggccacagc tgctcctgcg
cctgggcctt acttcctacc gagacttcct gggcaccaac 60 tggtccagct
cagctgcctg gctgngacan aagntgacca ccgactgggg tgacacgcag 120
gcctatctgg cggacccact gggggtgggg cgctgcacta gccacagccg atggacttcc
180 ttgttttcct gcgccgctcc cggcaggtgg gctgaggccc ctgggctggt
gggacgtaac 240 tggtggggca acctgagcct caggtggaac ctgnncgttg
gtttnaacct gnaggcaagn 300 ccttgtttnt tggggaattg ncnnaaaatn
gnggaccaat gnttggncnn agccanttgn 360 ccgatttttt atnntnccaa
ttgaaagttn gnattttttg gnggaaantt ngnggaagga 420 atttaacttn
ganngggggg gnacccgang ggccaaaggg gttttaaaan gaattttttg 480
<210> SEQ ID NO 19 <211> LENGTH: 285 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (85)..(85)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(186)..(186) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (190)..(190) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (210)..(212) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (214)..(214) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (218)..(218)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(221)..(221) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (226)..(226) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (230)..(230) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (234)..(234) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (238)..(238)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(250)..(250) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (252)..(252) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (265)..(265) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (271)..(271) <223> OTHER
INFORMATION: May be any nucleotide <400> SEQUENCE: 19
agccactctg agctcccacg agaaacactg cttctccagg cccggggttg ttggggagag
60 aggcagaggc agctggagcg ccgtntctct cctgctgggg acaacgtttg
ggctttgggt 120 attgactgag tggctgacag ttatctttgc aaccccaaac
tggctttggg gccaggacaa 180 ggggtnggcn tttatggtgg tccaagtttn
nntncttncc naactngggn ttgntccntg 240 actgttggan cntgttaatt
ggctntttca ntgggtttta ttttt 285 <210> SEQ ID NO 20
<211> LENGTH: 419 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (124)..(124) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (187)..(187)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(207)..(207) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (222)..(222) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (257)..(257) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (267)..(267) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (296)..(296)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(321)..(321) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (327)..(327) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (332)..(332) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (353)..(356) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (359)..(359)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(363)..(365) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (368)..(369) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (371)..(371) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (374)..(374) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (389)..(389)
<223> OTHER INFORMATION: May be any nucleotide <400>
SEQUENCE: 20 ccccgttcgc cccctgcaag cgcgcccgct tcgaggactt ctgcccggac
tcgtccccgg 60 agcgtccaac atctcaaact tgatctccat ctttggctcc
ggcttctcgg ggctggtgag 120 ccgnacagcc ggactcctcg gagcagccgc
cgccgctcaa cgggcagctg tgcgccaagc 180 aggcgtncgc cagcctcggc
gcctggnact cgagccattg tngccttcta gggacccccg 240 aggggcacag
gggaccnggg gccccgnggg ggttgggggc cagacaaaga tttggnaaag 300
gggcgagagg agggaacgag ngggggncgg gncaattggg ggtttaattt ggnnnnaang
360 ggnnnaanng nttntttttt tttaaattnt taaaaaaaaa aaaaaaaatt
ttggggggg 419 <210> SEQ ID NO 21 <211> LENGTH: 328
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(4) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (43)..(43) <223> OTHER INFORMATION: May
be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (96)..(96) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (118)..(119)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(144)..(144) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (172)..(172) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (281)..(282) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (300)..(300) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (305)..(305)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(308)..(308) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (315)..(315) <223> OTHER INFORMATION: May be any
nucleotide <400> SEQUENCE: 21 agtntcatac cgccaaccac
cgctggctgg gaggagtcgg agntgagaac ctacacagag 60 gtggtgacag
agtttgggac cgaggtggag cccgantttg ggaccaaggt ggagcccnng 120
tttgagaccc agttggagcc tgantttgag acccagctgg aacccgagtt tnaggaagag
180 gaggaggagg agaaagagga ggagatagcc actggccagg cattcccctt
cacaacagta 240 gagacctaca cagtgaactt ttggggactt tttgagatca
nngtcctacc agaccccagn 300 ccaanttnag gtttnagcag caggattt 328
<210> SEQ ID NO 22 <211> LENGTH: 524 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (24)..(24)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(94)..(94) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (140)..(140) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (146)..(146) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (266)..(266) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (271)..(271)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(288)..(288) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (292)..(292) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (295)..(295) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (298)..(298) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (301)..(301)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(304)..(304) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (306)..(306) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (311)..(311) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (315)..(315) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (323)..(323)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(327)..(327) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (331)..(331) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (337)..(337) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (359)..(360) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (365)..(365)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(377)..(377) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (381)..(381) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (387)..(387) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (402)..(403) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (406)..(406)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(411)..(413) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (434)..(435) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (454)..(454) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (461)..(461) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (464)..(464)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(466)..(466) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (476)..(477) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (499)..(501) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (514)..(514) <223> OTHER
INFORMATION: May be any nucleotide <400> SEQUENCE: 22
agggcagaag ccatggccca tgtntgcaca tccagggagg aggacagaag gcccagctca
60 catgtgagtc ctggcagaag ccatggccca tgtntgcaca tccagggagg
aggacagaag 120 gcccagctca catgtgagtn cctggncaga agccatggcc
catgtttgca catccaggga 180 ggaggacaga aaggcccagc tcacatgtga
gttcctggca gaagccatgg cccatgtttg 240 cacatccagg gaggaggaca
gaaggnccag ntcagtggcc ctggccgnca anttntantt 300 ntangnccga
ncagnagggt agntagnctt ntttggngtg tgttccaatt ccgtatttnn 360
ggttnggttt ttggggncca nttcaanaat tggtttgggg tnnttntttt nnntttaatt
420 aaagttttaa cttnnaaaaa aaaaaaaaaa tttngggggg nttngnattt
tttttnnttt 480 ttgggggggt tttaaattnn ncaagtgaaa tgtnttaaat tttg 524
<210> SEQ ID NO 23 <211> LENGTH: 490 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(72)..(72) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (202)..(202)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(210)..(210) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (218)..(218) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (254)..(254) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (270)..(270) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (280)..(280)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(298)..(298) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (309)..(309) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (311)..(311) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (317)..(317) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (324)..(324)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(326)..(326) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (330)..(330) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (345)..(345) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (350)..(350) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (356)..(356)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(375)..(375) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (380)..(380) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (384)..(384) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (391)..(391) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (428)..(428)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(442)..(442) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (446)..(446) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (449)..(450) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (453)..(453) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (456)..(456)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(458)..(458) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (469)..(469) <223> OTHER INFORMATION: May be any
nucleotide <400> SEQUENCE: 23 aggngccctc ctgagcaaga
tccacctgta cacacgcggc tgccacagcg accagagcct 60 tagccatctg
tntgtcactg aagcagagat gctcagggac ccagaggtag gccagcaact 120
gctgcgggac tctggagcgt gagaaccagc gcctggaggc tgtcctggcg tggcggcgct
180 ctgagctggt cttctggcgg angcaggcgn gcggcctngg aggcaaggct
tgaggctgtt 240 gacgggggcc aaanttgagt tccgcgcggn gggccttttn
gggaaggttt ttgaaaanga 300 gcttggagnt ntacaanatt tttngnagcn
aaaagttggc cccgnccaan cccatnggca 360 aaaacgtttg tttanaaggn
aagntttggg naaaaaggga cccggacttt gggaaatttt 420 tgttattnaa
aacctttagg gnccanagnn ttnttngngg gttttaatng gtttaaaggc 480
aattttgaaa 490 <210> SEQ ID NO 24 <211> LENGTH: 320
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (3)..(3) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (6)..(6) <223> OTHER INFORMATION: May
be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (8)..(8) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (13)..(13) <223>
OTHER INFORMATION: May be any nucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (18)..(18)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(27)..(27) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (37)..(37) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (43)..(43) <223> OTHER INFORMATION: May
be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (47)..(48) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (70)..(70) <223>
OTHER INFORMATION: May be any nucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (73)..(73)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(79)..(79) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (86)..(86) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (96)..(96) <223> OTHER INFORMATION: May
be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (97)..(97) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (107)..(107)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(110)..(114) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (122)..(122) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (124)..(124) <223> OTHER INFORMATION:
May be any nucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (137)..(137) <223> OTHER
INFORMATION: May be any nucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (204)..(204)
<223> OTHER INFORMATION: May be any nucleotide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(218)..(218) <223> OTHER INFORMATION: May be any nucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (258)..(258) <223> OTHER INFORMATION: May be any
nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (282)..(282) <223> OTHER INFORMATION:
May be any nucleotide <400> SEQUENCE: 24 cangcntntc
cangaccntg gacctcncct ggccagnctg ganaccnnca tcgtccctat 60
cagtggagtn gcnttacang gcactngcgc gtcganntac atcgccnggn nnnntatcca
120 tntnaactga tgctggngca tcaaacccgc agctgttcgc gcttatgggc
acccgggcag 180 gcatcgccag ggaagctgga gctngtggag cagcagtntc
ggctggagca gctgagtgcg 240 gcagagctgc agagcagnaa ccagggccac
tgggctgact gnctacagcg tacagagccc 300 ggttggacaa ggacctggaa 320
<210> SEQ ID NO 25 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Contains a NcoI
restriction site followed by the first 15 nucleotides of coding
sequence of BSG1 <400> SEQUENCE: 25 gccaccatgg atgttttcaa g
21 <210> SEQ ID NO 26 <211> LENGTH: 28 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Contains a complementary
sequence to a BglIII restriction site followed by 18 nucleotides of
the BSG1 sequence <400> SEQUENCE: 26 gcgcagatct gtctccccca
ctctgggc 28 <210> SEQ ID NO 27 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Primer which is 5' of
the M13 reverse sequence in pBluescript <220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide <400>
SEQUENCE: 27 atgcttccgg ctcgtatg 18 <210> SEQ ID NO 28
<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer which is 3' of the M13 forward primer in
pBluescript <400> SEQUENCE: 28 gggttttccc agtcacgac 19
<210> SEQ ID NO 29 <211> LENGTH: 36 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Contains a BamHI restriction site
followed by 8 nucleotides resembling an efficient signal for
initiation of translation in eukaryotic cells <400> SEQUENCE:
29 aaaggatccc ccgccatcat ggatgttttc aagaag 36 <210> SEQ ID NO
30 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Contains a XbaI restriction site followed by 21
nucleotides complementary to the 3' sequence of the BSG1 gene
<400> SEQUENCE: 30 aaatctagac tagtctcccc cactctg 27
<210> SEQ ID NO 31 <211> LENGTH: 66 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Contains complementary sequence to a
XbaI restriction site, translation stop codon, HA tag, and the last
18 nucleotides of the BamHI coding sequence excluding the stop
codon <400> SEQUENCE: 31 aaatctagac taaagcgtag tctgggacgt
cgtatgggta ctcctggggt ctcccccact 60 ctgggc 66 <210> SEQ ID NO
32 <211> LENGTH: 383 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (1)..(383) <223> OTHER INFORMATION:
<400> SEQUENCE: 32 atggatgtct tcaagaaggg cttctccatc
gccaaggagg gcgtggtggg tgcggtggaa 60 aagaccaagc agggggtgac
ggaagcagct gagaagacca aggagggggt catgtatgtg 120 ggagccaaga
ccaaggagaa tgttgtacag agcgtgacct cagtggccga gaagaccaag 180
gagcaggcca acgccgtgag cgaggctgtg gtgagcagcg tcaacactgt ggccaccaag
240 accgtggagg aggcggagaa catcgcggtc acctccgggg tggtgcgcaa
ggaggacttg 300 aggccatctg ccccccaaca ggagggtgag gcatccaaag
agaaagagga agtggcagag 360 gaggcccaga gtgggggaga cta 383 <210>
SEQ ID NO 33 <211> LENGTH: 127 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 33 Met Asp
Val Phe Lys Lys Gly Phe Ser Ile Ala Lys Glu Gly Val Val 1 5 10 15
Gly Ala Val Glu Lys Thr Lys Gln Gly Val Thr Glu Ala Ala Glu Lys 20
25 30 Thr Lys Glu Gly Val Met Tyr Val Gly Ala Lys Thr Lys Glu Asn
Val 35 40 45 Val Gln Ser Val Thr Ser Val Ala Glu Lys Thr Lys Glu
Gln Ala Asn 50 55 60 Ala Val Ser Glu Ala Val Val Ser Ser Val Asn
Thr Val Ala Thr Lys 65 70 75 80 Thr Val Glu Glu Ala Glu Asn Ile Ala
Val Thr Ser Gly Val Val Arg 85 90 95 Lys Glu Asp Leu Arg Pro Ser
Ala Pro Gln Gln Glu Gly Glu Ala Ser 100 105 110 Lys Glu Lys Glu Glu
Val Ala Glu Glu Ala Gln Ser Gly Gly Asp 115 120 125
* * * * *