U.S. patent application number 11/538566 was filed with the patent office on 2007-03-01 for compositions and methods for the diagnosis and treatment of tumor.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Gretchen D. Frantz, Kenneth J. Hillan, Heidi Phillips, Paul Polakis, Susan D. Spencer, P. Mickey Williams, Thomas D. Wu, Zemin Zhang.
Application Number | 20070048218 11/538566 |
Document ID | / |
Family ID | 27569669 |
Filed Date | 2007-03-01 |
United States Patent
Application |
20070048218 |
Kind Code |
A1 |
Frantz; Gretchen D. ; et
al. |
March 1, 2007 |
Compositions and Methods for the Diagnosis and Treatment of
Tumor
Abstract
The present invention is directed to compositions of matter
useful for the diagnosis and treatment of tumor in mammals and to
methods of using those compositions of matter for the same.
Inventors: |
Frantz; Gretchen D.; (San
Francisco, CA) ; Hillan; Kenneth J.; (San Francisco,
CA) ; Phillips; Heidi; (San Carlos, CA) ;
Polakis; Paul; (Burlingame, CA) ; Spencer; Susan
D.; (Tiburon, CA) ; Williams; P. Mickey; (Half
Moon Bay, CA) ; Wu; Thomas D.; (San Francisco,
CA) ; Zhang; Zemin; (Foster City, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
27569669 |
Appl. No.: |
11/538566 |
Filed: |
October 4, 2006 |
Related U.S. Patent Documents
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Application
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Patent Number |
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10827972 |
Apr 20, 2004 |
6898863 |
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11538566 |
Oct 4, 2006 |
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10241220 |
Sep 11, 2002 |
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11538566 |
Oct 4, 2006 |
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60323268 |
Sep 18, 2001 |
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60339227 |
Oct 19, 2001 |
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60336827 |
Nov 7, 2001 |
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60331906 |
Nov 20, 2001 |
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60345444 |
Jan 2, 2002 |
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60369724 |
Apr 3, 2002 |
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60404809 |
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Current U.S.
Class: |
424/1.49 ;
424/155.1; 424/178.1; 530/388.8; 530/391.1 |
Current CPC
Class: |
A61K 47/6809 20170801;
A61K 47/6843 20170801; A61P 35/02 20180101; A61P 35/04 20180101;
C07K 16/30 20130101; A61P 35/00 20180101; C07K 14/705 20130101;
G01N 33/574 20130101; A61K 47/6851 20170801; A61K 47/6803 20170801;
C07K 14/4748 20130101; C07K 16/18 20130101; C07K 14/71 20130101;
A61P 43/00 20180101; A61K 2039/505 20130101 |
Class at
Publication: |
424/001.49 ;
424/155.1; 424/178.1; 530/391.1; 530/388.8 |
International
Class: |
A61K 51/00 20060101
A61K051/00; A61K 39/395 20060101 A61K039/395; C07K 16/46 20070101
C07K016/46; C07K 16/30 20070101 C07K016/30 |
Claims
1. An isolated antibody that binds to a polypeptide having at least
80% amino acid sequence identity to: (a) the polypeptide shown in
any one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112,
114, 116, 118 or 120); (b) the polypeptide shown in any one of
FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116,
118 or 120), lacking its associated signal peptide; (c) an
extracellular domain of the polypeptide shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120), with its associated signal peptide; (d) an extracellular
domain of the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide; (e) a polypeptide encoded by the
nucleotide sequence shown in any one of FIGS. 1-56, 113, 115, 117
or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or (f) a
polypeptide encoded by the full-length coding region of the
nucleotide sequence shown in any one of FIGS. 1-56, 113, 115, 117
or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
2. An isolated antibody that binds to a polypeptide having: (a) the
amino acid sequence shown in any one of FIGS. 57-112, 114, 116, 118
or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120); (b) the amino
acid sequences shown in any one of FIGS. 57-112, 114, 116, 118 or
120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its
associated signal peptide sequence; (c) an amino acid sequence of
an extracellular domain of the polypeptide shown in any one of
FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116,
118 or 120), with its associated signal peptide sequence; (d) an
amino acid sequence of an extracellular domain of the polypeptide
shown in any one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID
NOS:57-112, 114, 116, 118 or 120), lacking its associated signal
peptide sequence; (e) an amino acid sequence encoded by the
nucleotide sequence shown in any one of FIGS. 1-56, 113, 115, 117
or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or (f) an amino
acid sequence encoded by the full-length coding region of the
nucleotide sequence shown in any one of FIGS. 1-56, 113, 115, 117
or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
3. The antibody of claim 1 which is a monoclonal antibody.
4. The antibody of claim 1 which is an antibody fragment.
5. The antibody of claim 1 which is a chimeric or a humanized
antibody.
6. The antibody of claim 1 which is conjugated to a growth
inhibitory agent.
7. The antibody of claim 1 which is conjugated to a cytotoxic
agent.
8. The antibody of claim 7, wherein the cytotoxic agent is selected
from the group consisting of toxins, antibiotics, radioactive
isotopes and nucleolytic enzymes.
9. The antibody of claim 7, wherein the cytotoxic agent is a
toxin.
10. The antibody of claim 9, wherein the toxin is selected from the
group consisting of maytansinoid and calicheamicin.
11. The antibody of claim 9, wherein the toxin is a
maytansinoid.
12. The antibody of claim 1 which is produced in bacteria.
13. The antibody of claim 1 which is produced in CHO cells.
14. The antibody of claim 1 which induces death of a cell to which
it binds.
15. The antibody of claim 1 which is detectably labeled.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to compositions of matter
useful for the diagnosis and treatment of tumor in mammals and to
methods of using those compositions of matter for the same.
BACKGROUND OF THE INVENTION
[0002] Malignant tumors (cancers) are the second leading cause of
death in the United States, after heart disease (Boring et al., CA
Cancel J. Clin. 43:7 (1993)). Cancer is characterized by the
increase in the number of abnormal, or neoplastic, cells derived
from a normal tissue which proliferate to form a tumor mass, the
invasion of adjacent tissues by these neoplastic tumor cells, and
the generation of malignant cells which eventually spread via the
blood or lymphatic system to regional lymph nodes and to distant
sites via a process called metastasis. In a cancerous state, a cell
proliferates under conditions in which normal cells would not grow.
Cancer manifests itself in a wide variety of forms, characterized
by different degrees of invasiveness and aggressiveness.
[0003] In attempts to discover effective cellular targets for
cancer diagnosis and therapy, researchers have sought to identify
transmembrane or otherwise membrane-associated polypeptides that
are specifically expressed on the surface of one or more particular
type(s) of cancer cell as compared to on one or more normal
non-cancerous cell(s). Often, such membrane-associated polypeptides
are more abundantly expressed on the surface of the cancer cells as
compared to on the surface of the non-cancerous cells. The
identification of such tumor-associated cell surface antigen
polypeptides has given rise to the ability to specifically target
cancer cells for destruction via antibody-based therapies. In this
regard, it is noted that antibody-based therapy has proved very
effective in the treatment of certain cancers. For example,
HERCEPTIN.RTM. and RITUXAN.RTM. (both from Genentech Inc., South
San Francisco, Calif.) are antibodies that have been used
successfully to treat breast cancer and non-Hodgkin's lymphoma,
respectively. More specifically, HERCEPTIN.RTM. is a recombinant
DNA-derived humanized monoclonal antibody that selectively binds to
the extracellular domain of the human epidermal growth factor
receptor 2 (HER2) proto-oncogene. HER2 protein overexpression is
observed in 25-30% of primary breast cancers. RITUXAN.RTM. is a
genetically engineered chimeric murine/human monoclonal antibody
directed against the CD20 antigen found on the surface of normal
and malignant B lymphocytes. Both these antibodies are
recombinantly produced in CHO cells.
[0004] In other attempts to discover effective cellular targets for
cancer diagnosis and therapy, researchers have sought to identify
(1) non-membrane-associated polypeptides that are specifically
produced by one or more particular type(s) of cancer cell(s) as
compared to by one or more particular type(s) of non-cancerous
normal cell(s), (2) polypeptides that are produced by cancer cells
at an expression level that is significantly higher than that of
one or more normal non-cancerous cell(s), or (3) polypeptides whose
expression is specifically limited to only a single (or very
limited number of different) tissue type(s) in both the cancerous
and non-cancerous state (e.g., normal prostate and prostate tumor
tissue). Such polypeptides may remain intracellularly located or
may be secreted by the cancer cell. Moreover, such polypeptides may
be expressed not by the cancer cell itself, but rather by cells
which produce and/or secrete polypeptides having a potentiating or
growth-enhancing effect on cancer cells. Such secreted polypeptides
are often proteins that provide cancer cells with a growth
advantage over normal cells and include such things as, for
example, angiogenic factors, cellular adhesion factors, growth
factors, and the like. Identification of antagonists of such
non-membrane associated polypeptides would be expected to serve as
effective therapeutic agents for the treatment of such cancers.
Furthermore, identification of the expression pattern of such
polypeptides would be useful for the diagnosis of particular
cancers in mammals.
[0005] Despite the above identified advances in mammalian cancer
therapy, there is a great need for additional diagnostic and
therapeutic agents capable of detecting the presence of tumor in a
mammal and for effectively inhibiting neoplastic cell growth,
respectively. Accordingly, it is an objective of the present
invention to identify:
[0006] (1) cell membrane-associated polypeptides that are more
abundantly expressed on one or more type(s) of cancer cell(s) as
compared to on normal cells or on other different cancer cells, (2)
non-membrane-associated polypeptides that are specifically produced
by one or more particular type(s) of cancer cell(s) (or by other
cells that produce polypeptides having a potentiating effect on the
growth of cancer cells) as compared to by one or more particular
type(s) of non-cancerous normal cell(s), (3)
non-membrane-associated polypeptides that are produced by cancer
cells at an expression level that is significantly higher than that
of one or more normal non-cancerous cell(s), or (4) polypeptides
whose expression is specifically limited to only a single (or very
limited number of different) tissue type(s) in both a cancerous and
non-cancerous state (e.g., normal prostate and prostate tumor
tissue), and to use those polypeptides, and their encoding nucleic
acids, to produce compositions of matter useful in the therapeutic
treatment and diagnostic detection of cancer in mammals. It is also
an objective of the present invention to identify cell
membrane-associated, secreted or intracellular polypeptides whose
expression is limited to a single or very limited number of
tissues, and to use those polypeptides, and their encoding nucleic
acids, to produce compositions of matter useful in the therapeutic
treatment and diagnostic detection of cancer in mammals.
SUMMARY OF THE INVENTION
A. Embodiments
[0007] In the present specification, Applicants describe for the
first time the identification of various cellular polypeptides (and
their encoding nucleic acids or fragments thereof) which are
expressed to a greater degree on the surface of or by one or more
types of cancer cell(s) as compared to on the surface of or by one
or more types of normal non-cancer cells. Alternatively, such
polypeptides are expressed by cells which produce and/or secrete
polypeptides having a potentiating or growth-enhancing effect on
cancer cells. Again alternatively, such polypeptides may not be
overexpressed by tumor cells as compared to normal cells of the
same tissue type, but rather may be specifically expressed by both
tumor cells and normal cells of only a single or very limited
number of tissue types (preferably tissues which are not essential
for life, e.g., prostate, etc.). All of the above polypeptides are
herein referred to as Tumor-associated Antigenic Target
polypeptides ("TAT" polypeptides) and are expected to serve as
effective targets for cancer therapy and diagnosis in mammals.
[0008] Accordingly, in one embodiment of the present invention, the
invention provides an isolated nucleic acid molecule having a
nucleotide sequence that encodes a tumor-associated antigenic
target polypeptide or fragment thereof (a "TAT" polypeptide).
[0009] In certain aspects, the isolated nucleic acid molecule
comprises a nucleotide sequence having at least about 80% nucleic
acid sequence identity, alternatively at least about 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA
molecule encoding a full-length TAT polypeptide having an amino
acid sequence as disclosed herein, a TAT polypeptide amino acid
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a transmembrane TAT polypeptide, with or
without the signal peptide, as disclosed herein or any other
specifically defined fragment of a full-length TAT polypeptide
amino acid sequence as disclosed herein, or (b) the complement of
the DNA molecule of (a).
[0010] In other aspects, the isolated nucleic acid molecule
comprises a nucleotide sequence having at least about 80% nucleic
acid sequence identity, alternatively at least about 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA
molecule comprising the coding sequence of a full-length TAT
polypeptide cDNA as disclosed herein, the coding sequence of a TAT
polypeptide lacking the signal peptide as disclosed herein, the
coding sequence of an extracellular domain of a transmembrane TAT
polypeptide, with or without the signal peptide, as disclosed
herein or the coding sequence of any other specifically defined
fragment of the full-length TAT polypeptide amino acid sequence as
disclosed herein, or (b) the complement of the DNA molecule of
(a).
[0011] In further aspects, the invention concerns an isolated
nucleic acid molecule comprising a nucleotide sequence having at
least about 80% nucleic acid sequence identity, alternatively at
least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid
sequence identity, to (a) a DNA molecule that encodes the same
mature polypeptide encoded by the full-length coding region of any
of the human protein cDNAs deposited with the ATCC as disclosed
herein, or (b) the complement of the DNA molecule of (a).
[0012] Another aspect of the invention provides an isolated nucleic
acid molecule comprising a nucleotide sequence encoding a TAT
polypeptide which is either transmembrane domain-deleted or
transmembrane domain-inactivated, or is complementary to such
encoding nucleotide sequence, wherein the transmembrane domain(s)
of such polypeptide(s) are disclosed herein. Therefore, soluble
extracellular domains of the herein described TAT polypeptides are
contemplated.
[0013] In other aspects, the present invention is directed to
isolated nucleic acid molecules which hybridize to (a) a nucleotide
sequence encoding a TAT polypeptide having a full-length amino acid
sequence as disclosed herein, a TAT polypeptide amino acid sequence
lacking the signal peptide as disclosed herein, an extracellular
domain of a transmembrane TAT polypeptide, with or without the
signal peptide, as disclosed herein or any other specifically
defined fragment of a full-length TAT polypeptide amino acid
sequence as disclosed herein, or (b) the complement of the
nucleotide sequence of (a). In this regard, an embodiment of the
present invention is directed to fragments of a full-length TAT
polypeptide coding sequence, or the complement thereof, as
disclosed herein, that may find use as, for example, hybridization
probes useful as, for example, diagnostic probes, antisense
oligonucleotide probes, or for encoding fragments of a full-length
TAT polypeptide that may optionally encode a polypeptide comprising
a binding site for an anti-TAT polypeptide antibody, a TAT binding
oligopeptide or other small organic molecule that binds to a TAT
polypeptide. Such nucleic acid fragments are usually at least about
5 nucleotides in length, alternatively at least about 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,
165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250,
260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,
390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,
520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,
650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,
780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900,
910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in
length, wherein in this context the term "about" means the
referenced nucleotide sequence length plus or minus 10% of that
referenced length. It is noted that novel fragments of a TAT
polypeptide-encoding nucleotide sequence may be determined in a
routine manner by aligning the TAT polypeptide-encoding nucleotide
sequence with other known nucleotide sequences using any of a
number of well known sequence alignment programs and determining
which TAT polypeptide-encoding nucleotide sequence fragment(s) are
novel. All of such novel fragments of TAT polypeptide-encoding
nucleotide sequences are contemplated herein. Also contemplated are
the TAT polypeptide fragments encoded by these nucleotide molecule
fragments, preferably those TAT polypeptide fragments that comprise
a binding site for an anti-TAT antibody, a TAT binding oligopeptide
or other small organic molecule that binds to a TAT
polypeptide.
[0014] In another embodiment, the invention provides isolated TAT
polypeptides encoded by any of the isolated nucleic acid sequences
hereinabove identified.
[0015] In a certain aspect, the invention concerns an isolated TAT
polypeptide, comprising an amino acid sequence having at least
about 80% amino acid sequence identity, alternatively at least
about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence
identity, to a TAT polypeptide having a full-length amino acid
sequence as disclosed herein, a TAT polypeptide amino acid sequence
lacking the signal peptide as disclosed herein, an extracellular
domain of a transmembrane TAT polypeptide protein, with or without
the signal peptide, as disclosed herein, an amino acid sequence
encoded by any of the nucleic acid sequences disclosed herein or
any other specifically defined fragment of a full-length TAT
polypeptide amino acid sequence as disclosed herein.
[0016] In a further aspect, the invention concerns an isolated TAT
polypeptide comprising an amino acid sequence having at least about
80% amino acid sequence identity, alternatively at least about 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to an
amino acid sequence encoded by any of the human protein cDNAs
deposited with the ATCC as disclosed herein.
[0017] In a specific aspect, the invention provides an isolated TAT
polypeptide without the N-terminal signal sequence and/or without
the initiating methionine and is encoded by a nucleotide sequence
that encodes such an amino acid sequence as hereinbefore described.
Processes for producing the same are also herein described, wherein
those processes comprise culturing a host cell comprising a vector
which comprises the appropriate encoding nucleic acid molecule
under conditions suitable for expression of the TAT polypeptide and
recovering the TAT polypeptide from the cell culture.
[0018] Another aspect of the invention provides an isolated TAT
polypeptide which is either transmembrane domain-deleted or
transmembrane domain-inactivated. Processes for producing the same
are also herein described, wherein those processes comprise
culturing a host cell comprising a vector which comprises the
appropriate encoding nucleic acid molecule under conditions
suitable for expression of the TAT polypeptide and recovering the
TAT polypeptide from the cell culture.
[0019] In other embodiments of the present invention, the invention
provides vectors comprising DNA encoding any of the herein
described polypeptides. Host cells comprising any such vector are
also provided. By way of example, the host cells may be CHO cells,
E. coli cells, or yeast cells. A process for producing any of the
herein described polypeptides is further provided and comprises
culturing host cells under conditions suitable for expression of
the desired polypeptide and recovering the desired polypeptide from
the cell culture.
[0020] In other embodiments, the invention provides isolated
chimeric polypeptides comprising any of the herein described TAT
polypeptides fused to a heterologous (non-TAT) polypeptide. Example
of such chimeric molecules comprise any of the herein described TAT
polypeptides fused to a heterologous polypeptide such as, for
example, an epitope tag sequence or a Fc region of an
immunoglobulin.
[0021] In another embodiment, the invention provides an antibody
which binds, preferably specifically, to any of the above or below
described polypeptides. Optionally, the antibody is a monoclonal
antibody, antibody fragment, chimeric antibody, humanized antibody,
single-chain antibody or antibody that competitively inhibits the
binding of an anti-TAT polypeptide antibody to its respective
antigenic epitope. Antibodies of the present invention may
optionally be conjugated to a growth inhibitory agent or cytotoxic
agent such as a toxin, including, for example, a maytansinoid or
calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic
enzyme, or the like. The antibodies of the present invention may
optionally be produced in CHO cells or bacterial cells and
preferably induce death of a cell to which they bind. For
diagnostic purposes, the antibodies of the present invention may be
detectably labeled, attached to a solid support, or the like.
[0022] In other embodiments of the present invention, the invention
provides vectors comprising DNA encoding any of the herein
described antibodies. Host cell comprising any such vector are also
provided. By way of example, the host cells may be CHO cells, E.
coli cells, or yeast cells. A process for producing any of the
herein described antibodies is further provided and comprises
culturing host cells under conditions suitable for expression of
the desired antibody and recovering the desired antibody from the
cell culture.
[0023] In another embodiment, the invention provides oligopeptides
("TAT binding oligopeptides") which bind, preferably specifically,
to any of the above or below described TAT polypeptides.
Optionally, the TAT binding oligopeptides of the present invention
may be conjugated to a growth inhibitory agent or cytotoxic agent
such as a toxin, including, for example, a maytansinoid or
calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic
enzyme, or the like. The TAT binding oligopeptides of the present
invention may optionally be produced in CHO cells or bacterial
cells and preferably induce death of a cell to which they bind. For
diagnostic purposes, the TAT binding oligopeptides of the present
invention may be detectably labeled, attached to a solid support,
or the like.
[0024] In other embodiments of the present invention, the invention
provides vectors comprising DNA encoding any of the herein
described TAT binding oligopeptides. Host cell comprising any such
vector are also provided. By way of example, the host cells may be
CHO cells, E. coli cells, or yeast cells. A process for producing
any of the herein described TAT binding oligopeptides is further
provided and comprises culturing host cells under conditions
suitable for expression of the desired oligopeptide and recovering
the desired oligopeptide from the cell culture.
[0025] In another embodiment, the invention provides small organic
molecules ("TAT binding organic molecules") which bind, preferably
specifically, to any of the above or below described TAT
polypeptides. Optionally, the TAT binding organic molecules of the
present invention may be conjugated to a growth inhibitory agent or
cytotoxic agent such as a toxin, including, for example, a
maytansinoid or calicheamicin, an antibiotic, a radioactive
isotope, a nucleolytic enzyme, or the like. The TAT binding organic
molecules of the present invention preferably induce death of a
cell to which they bind. For diagnostic purposes, the TAT binding
organic molecules of the present invention may be detectably
labeled, attached to a solid support, or the like.
[0026] In a still further embodiment, the invention concerns a
composition of matter comprising a TAT polypeptide as described
herein, a chimeric TAT polypeptide as described herein, an anti-TAT
antibody as described herein, a TAT binding oligopeptide as
described herein, or a TAT binding organic molecule as described
herein, in combination with a carrier. Optionally, the carrier is a
pharmaceutically acceptable carrier.
[0027] In yet another embodiment, the invention concerns an article
of manufacture comprising a container and a composition of matter
contained within the container, wherein the composition of matter
may comprise a TAT polypeptide as described herein, a chimeric TAT
polypeptide as described herein, an anti-TAT antibody as described
herein, a TAT binding oligopeptide as described herein, or a TAT
binding organic molecule as described herein. The article may
further optionally comprise a label affixed to the container, or a
package insert included with the container, that refers to the use
of the composition of matter for the therapeutic treatment or
diagnostic detection of a tumor.
[0028] Another embodiment of the present invention is directed to
the use of a TAT polypeptide as described herein, a chimeric TAT
polypeptide as described herein, an anti-TAT polypeptide antibody
as described herein, a TAT binding oligopeptide as described
herein, or a TAT binding organic molecule as described herein, for
the preparation of a medicament useful in the treatment of a
condition which is responsive to the TAT polypeptide, chimeric TAT
polypeptide, anti-TAT polypeptide antibody, TAT binding
oligopeptide, or TAT binding organic molecule.
B. Additional Embodiments
[0029] Another embodiment of the present invention is directed to a
method for inhibiting the growth of a cell that expresses a TAT
polypeptide, wherein the method comprises contacting the cell with
an antibody, an oligopeptide or a small organic molecule that binds
to the TAT polypeptide, and wherein the binding of the antibody,
oligopeptide or organic molecule to the TAT polypeptide causes
inhibition of the growth of the cell expressing the TAT
polypeptide. In preferred embodiments, the cell is a cancer cell
and binding of the antibody, oligopeptide or organic molecule to
the TAT polypeptide causes death of the cell expressing the TAT
polypeptide. Optionally, the antibody is a monoclonal antibody,
antibody fragment, chimeric antibody, humanized antibody, or
single-chain antibody. Antibodies, TAT binding oligopeptides and
TAT binding organic molecules employed in the methods of the
present invention may optionally be conjugated to a growth
inhibitory agent or cytotoxic agent such as a toxin, including, for
example, a maytansinoid or calicheamicin, an antibiotic, a
radioactive isotope, a nucleolytic enzyme, or the like. The
antibodies and TAT binding oligopeptides employed in the methods of
the present invention may optionally be produced in CHO cells or
bacterial cells.
[0030] Yet another embodiment of the present invention is directed
to a method of therapeutically treating a mammal having a cancerous
tumor comprising cells that express a TAT polypeptide, wherein the
method comprises administering to the mammal a therapeutically
effective amount of an antibody, an oligopeptide or a small organic
molecule that binds to the TAT polypeptide, thereby resulting in
the effective therapeutic treatment of the tumor. Optionally, the
antibody is a monoclonal antibody, antibody fragment, chimeric
antibody, humanized antibody, or single-chain antibody. Antibodies,
TAT binding oligopeptides and TAT binding organic molecules
employed in the methods of the present invention may optionally be
conjugated to a growth inhibitory agent or cytotoxic agent such as
a toxin, including, for example, a maytansinoid or calicheamicin,
an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the
like. The antibodies and oligopeptides employed in the methods of
the present invention may optionally be produced in CHO cells or
bacterial cells.
[0031] Yet another embodiment of the present invention is directed
to a method of determining the presence of a TAT polypeptide in a
sample suspected of containing the TAT polypeptide, wherein the
method comprises exposing the sample to an antibody, oligopeptide
or small organic molecule that binds to the TAT polypeptide and
determining binding of the antibody, oligopeptide or organic
molecule to the TAT polypeptide in the sample, wherein the presence
of such binding is indicative of the presence of the TAT
polypeptide in the sample. Optionally, the sample may contain cells
(which may be cancer cells) suspected of expressing the TAT
polypeptide. The antibody, TAT binding oligopeptide or TAT binding
organic molecule employed in the method may optionally be
detectably labeled, attached to a solid support, or the like.
[0032] A further embodiment of the present invention is directed to
a method of diagnosing the presence of a tumor in a mammal, wherein
the method comprises detecting the level of expression of a gene
encoding a TAT polypeptide (a) in a test sample of tissue cells
obtained from said mammal, and (b) in a control sample of known
normal non-cancerous cells of the same tissue origin or type,
wherein a higher level of expression of the TAT polypeptide in the
test sample, as compared to the control sample, is indicative of
the presence of tumor in the mammal from which the test sample was
obtained.
[0033] Another embodiment of the present invention is directed to a
method of diagnosing the presence of a tumor in a mammal, wherein
the method comprises (a) contacting a test sample comprising tissue
cells obtained from the mammal with an antibody, oligopeptide or
small organic molecule that binds to a TAT polypeptide and (b)
detecting the formation of a complex between the antibody,
oligopeptide or small organic molecule and the TAT polypeptide in
the test sample, wherein the formation of a complex is indicative
of the presence of a tumor in the mammal. Optionally, the antibody,
TAT binding oligopeptide or TAT binding organic molecule employed
is detectably labeled, attached to a solid support, or the like,
and/or the test sample of tissue cells is obtained from an
individual suspected of having a cancerous tumor.
[0034] Yet another embodiment of the present invention is directed
to a method for treating or preventing a cell proliferative
disorder associated with altered, preferably increased, expression
or activity of a TAT polypeptide, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of a TAT polypeptide. Preferably, the cell
proliferative disorder is cancer and the antagonist of the TAT
polypeptide is an anti-TAT polypeptide antibody, TAT binding
oligopeptide, TAT binding organic molecule or antisense
oligonucleotide. Effective treatment or prevention of the cell
proliferative disorder may be a result of direct killing or growth
inhibition of cells that express a TAT polypeptide or by
antagonizing the cell growth potentiating activity of a TAT
polypeptide.
[0035] Yet another embodiment of the present invention is directed
to a method of binding an antibody, oligopeptide or small organic
molecule to a cell that expresses a TAT polypeptide, wherein the
method comprises contacting a cell that expresses a TAT polypeptide
with said antibody, oligopeptide or small organic molecule under
conditions which are suitable for binding of the antibody,
oligopeptide or small organic molecule to said TAT polypeptide and
allowing binding therebetween.
[0036] Other embodiments of the present invention are directed to
the use of (a) a TAT polypeptide, (b) a nucleic acid encoding a TAT
polypeptide or a vector or host cell comprising that nucleic acid,
(c) an anti-TAT polypeptide antibody, (d) a TAT-binding
oligopeptide, or (e) a TAT-binding small organic molecule in the
preparation of a medicament useful for (i) the therapeutic
treatment or diagnostic detection of a cancer or tumor, or (ii) the
therapeutic treatment or prevention of a cell proliferative
disorder.
[0037] Another embodiment of the present invention is directed to a
method for inhibiting the growth of a cancer cell, wherein the
growth of said cancer cell is at least in part dependent upon the
growth potentiating effect(s) of a TAT polypeptide (wherein the TAT
polypeptide may be expressed either by the cancer cell itself or a
cell that produces polypeptide(s) that have a growth potentiating
effect on cancer cells), wherein the method comprises contacting
the TAT polypeptide with an antibody, an oligopeptide or a small
organic molecule that binds to the TAT polypeptide, thereby
antagonizing the growth-potentiating activity of the TAT
polypeptide and, in turn, inhi biting the growth of the cancer
cell. Preferably the growth of the cancer cell is completely
inhibited. Even more preferably, binding of the antibody,
oligopeptide or small organic molecule to the TAT polypeptide
induces the death of the cancer cell. Optionally, the antibody is a
monoclonal antibody, antibody fragment, chimeric antibody,
humanized antibody, or single-chain antibody. Antibodies, TAT
binding oligopeptides and TAT binding organic molecules employed in
the methods of the present invention may optionally be conjugated
to a growth inhibitory agent or cytotoxic agent such as a toxin,
including, for example, a maytansinoid or calicheamicin, an
antibiotic, a radioactive isotope, a nucleolytic enzyme, or the
like. The antibodies and TAT binding oligopeptides employed in the
methods of the present invention may optionally be produced in CHO
cells or bacterial cells.
[0038] Yet another embodiment of the present invention is directed
to a method of therapeutically treating a tumor in a mammal,
wherein the growth of said tumor is at least in part dependent upon
the growth potentiating effect(s) of a TAT polypeptide, wherein the
method comprises administering to the mammal a therapeutically
effective amount of an antibody, an oligopeptide or a small organic
molecule that binds to the TAT polypeptide, thereby antagonizing
the growth potentiating activity of said TAT polypeptide and
resulting in the effective therapeutic treatment of the tumor.
Optionally, the antibody is a monoclonal antibody, antibody
fragment, chimeric antibody, humanized antibody, or single-chain
antibody. Antibodies, TAT binding oligopeptides and TAT binding
organic molecules employed in the methods of the present invention
may optionally be conjugated to a growth inhibitory agent or
cytotoxic agent such as a toxin, including, for example, a
maytansinoid or calicheamicin, an antibiotic, a radioactive
isotope, a nucleolytic enzyme, or the like. The antibodies and
oligopeptides employed in the methods of the present invention may
optionally be produced in CHO cells or bacterial cells.
C. Further Additional Embodiments
[0039] In yet further embodiments, the invention is directed to the
following set of potential claims for this application:
[0040] 1. Isolated nucleic acid having a nucleotide sequence that
has at least 80% nucleic acid sequence identity to:
[0041] (a) a DNA molecule encoding the amino acid sequence shown in
any one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112,
114, 116, 118 or 120);
[0042] (b) a DNA molecule encoding the amino acid sequence shown in
any one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112,
114, 116, 118 or 120), lacking its associated signal peptide;
[0043] (c) a DNA molecule encoding an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated
signal peptide;
[0044] (d) a DNA molecule encoding an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated
signal peptide;
[0045] (e) the nucleotide sequence shown in any one of FIGS. 1-56,
113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119);
[0046] (f) the full-length coding region of the nucleotide sequence
shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID
NOS:1-56, 113, 115, 117 or 119); or
[0047] (g) the complement of (a), (b), (c), (d), (e) or (f).
[0048] 2. Isolated nucleic acid having:
[0049] (a) a nucleotide sequence that encodes the amino acid
sequence shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0050] (b) a nucleotide sequence that encodes the amino acid
sequence shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated
signal peptide;
[0051] (c) a nucleotide sequence that encodes an extracellular
domain of the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its
associated signal peptide;
[0052] (d) a nucleotide sequence that encodes an extracellular
domain of the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide;
[0053] (e) the nucleotide sequence shown in any one of FIGS. 1-56,
113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119);
[0054] (f) the full-length coding region of the nucleotide sequence
shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID
NOS:1-56, 113, 115, 117 or 119); or
[0055] (g) the complement of (a), (b), (c), (d), (e) or (f).
[0056] 3. Isolated nucleic acid that hybridizes to:
[0057] (a) a nucleic acid that encodes the amino acid sequence
shown in any one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID
NOS:57-112, 114, 116, 118 or 120);
[0058] (b) a nucleic acid that encodes the amino acid sequence
shown in any one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID
NOS:57-112, 114, 116, 118 or 120), lacking its associated signal
peptide;
[0059] (c) a nucleic acid that encodes an extracellular domain of
the polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or
120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated
signal peptide;
[0060] (d) a nucleic acid that encodes an extracellular domain of
the polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or
120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its
associated signal peptide;
[0061] (e) the nucleotide sequence shown in any one of FIGS. 1-56,
113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119);
[0062] (f) the full-length coding region of the nucleotide sequence
shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID
NOS:1-56, 113, 115, 117 or 119); or
[0063] (g) the complement of (a), (b), (c), (d), (e) or (f).
[0064] 4. The nucleic acid of Claim 3, wherein the hybridization
occurs under stringent conditions.
[0065] 5. The nucleic acid of Claim 3 which is at least about 5
nucleotides in length.
[0066] 6. An expression vector comprising the nucleic acid of Claim
1, 2 or 3.
[0067] 7. The expression vector of Claim 6, wherein said nucleic
acid is operably linked to control sequences recognized by a host
cell transformed with the vector.
[0068] 8. A host cell comprising the expression vector of Claim
7.
[0069] 9. The host cell of Claim 8 which is a CHO cell, an E. coli
cell or a yeast cell.
[0070] 10. A process for producing a polypeptide comprising
culturing the host cell of Claim 8 under conditions suitable for
expression of said polypeptide and recovering said polypeptide from
the cell culture.
[0071] 11. An isolated polypeptide having at least 80% amino acid
sequence identity to:
[0072] (a) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0073] (b) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide;
[0074] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), with its associated signal peptide;
[0075] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), lacking its associated signal peptide;
[0076] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56,
113, 115, 117 or 119); or
[0077] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-56, 113,
115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0078] 12. An isolated polypeptide having:
[0079] (a) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120);
[0080] (b) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120), lacking its associated signal peptide sequence;
[0081] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated
signal peptide sequence;
[0082] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated
signal peptide sequence;
[0083] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ
ID NOS:1-56, 113, 115, 117 or 119); or
[0084] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-56,
113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0085] 13. A chimeric polypeptide comprising the polypeptide of
Claim 11 or 12 fused to a heterologous polypeptide.
[0086] 14. The chimeric polypeptide of Claim 13, wherein said
heterologous polypeptide is an epitope tag sequence or an Fc region
of an immunoglobulin.
[0087] 15. An isolated antibody that binds to a polypeptide having
at least 80% amino acid sequence identity to:
[0088] (a) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0089] (b) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide;
[0090] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), with its associated signal peptide;
[0091] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), lacking its associated signal peptide;
[0092] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56,
113, 115, 117 or 119); or
[0093] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-56, 113,
115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0094] 16. An isolated antibody that binds to a polypeptide
having:
[0095] (a) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120);
[0096] (b) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120), lacking its associated signal peptide sequence;
[0097] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated
signal peptide sequence;
[0098] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated
signal peptide sequence;
[0099] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ
ID NOS:1-56, 113, 115, 117 or 119); or
[0100] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-56,
113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0101] 17. The antibody of Claim 15 or 16 which is a monoclonal
antibody.
[0102] 18. The antibody of Claim 15 or 16 which is an antibody
fragment.
[0103] 19. The antibody of Claim 15 or 16 which is a chimeric or a
humanized antibody.
[0104] 20. The antibody of Claim 15 or 16 which is conjugated to a
growth inhibitory agent.
[0105] 21. The antibody of Claim 15 or 16 which is conjugated to a
cytotoxic agent.
[0106] 22. The antibody of Claim 21, wherein the cytotoxic agent is
selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0107] 23. The antibody of Claim 21, wherein the cytotoxic agent is
a toxin.
[0108] 24. The antibody of Claim 23, wherein the toxin is selected
from the group consisting of maytansinoid and calicheamicin.
[0109] 25. The antibody of Claim 23, wherein the toxin is a
maytansinoid.
[0110] 26. The antibody of Claim 15 or 16 which is produced in
bacteria.
[0111] 27. The antibody of Claim 15 or 16 which is produced in CHO
cells.
[0112] 28. The antibody of Claim 15 or 16 which induces death of a
cell to which it binds.
[0113] 29. The antibody of Claim 15 or 16 which is detectably
labeled.
[0114] 30. An isolated nucleic acid having a nucleotide sequence
that encodes the antibody of Claim 15 or 16.
[0115] 31. An expression vector comprising the nucleic acid of
Claim 30 operably linked to control sequences recognized by a host
cell transformed with the vector.
[0116] 32. A host cell comprising the expression vector of Claim
31.
[0117] 33. The host cell of Claim 32 which is a CHO cell, an E.
coli cell or a yeast cell.
[0118] 34. A process for producing an antibody comprising culturing
the host cell of Claim 32 under conditions suitable for expression
of said antibody and recovering said antibody from the cell
culture.
[0119] 35. An isolated oligopeptide that binds to a polypeptide
having at least 80% amino acid sequence identity to:
[0120] (a) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0121] (b) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide;
[0122] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), with its associated signal peptide;
[0123] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), lacking its associated signal peptide;
[0124] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56,
113, 115, 117 or 119); or
[0125] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-56, 113,
115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0126] 36. An isolated oligopeptide that binds to a polypeptide
having:
[0127] (a) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120);
[0128] (b) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120), lacking its associated signal peptide sequence;
[0129] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated
signal peptide sequence;
[0130] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated
signal peptide sequence;
[0131] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ
ID NOS:1-56, 113, 115, 117 or 119); or
[0132] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-56,
113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 19).
[0133] 37. The oligopeptide of Claim 35 or 36 which is conjugated
to a growth inhibitory agent.
[0134] 38. The oligopeptide of Claim 35 or 36 which is conjugated
to a cytotoxic agent.
[0135] 39. The oligopeptide of Claim 38, wherein the cytotoxic
agent is selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0136] 40. The oligopeptide of Claim 38, wherein the cytotoxic
agent is a toxin.
[0137] 41. The oligopeptide of Claim 40, wherein the toxin is
selected from the group consisting of maytansinoid and
calicheamicin.
[0138] 42. The oligopeptide of Claim 40, wherein the toxin is a
maytansinoid.
[0139] 43. The oligopeptide of Claim 35 or 36 which induces death
of a cell to which it binds.
[0140] 44. The oligopeptide of Claim 35 or 36 which is detectably
labeled.
[0141] 45. A TAT binding organic molecule that binds to a
polypeptide having at least 80% amino acid sequence identity
to:
[0142] (a) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0143] (b) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide;
[0144] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), with its associated signal peptide;
[0145] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), lacking its associated signal peptide;
[0146] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56,
113, 115, 117 or 119); or
[0147] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-56, 113,
115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0148] 46. The organic molecule of Claim 45 that binds to a
polypeptide having:
[0149] (a) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120);
[0150] (b) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120), lacking its associated signal peptide sequence;
[0151] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated
signal peptide sequence;
[0152] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated
signal peptide sequence;
[0153] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ
ID NOS:1-56, 113, 115, 117 or 119); or
[0154] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-56,
113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0155] 47. The organic molecule of Claim 45 or 46 which is
conjugated to a growth inhibitory agent.
[0156] 48. The organic molecule of Claim 45 or 46 which is
conjugated to a cytotoxic agent.
[0157] 49. The organic molecule of Claim 48, wherein the cytotoxic
agent is selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0158] 50. The organic molecule of Claim 48, wherein the cytotoxic
agent is a toxin.
[0159] 51. The organic molecule of Claim 50, wherein the toxin is
selected from the group consisting of maytansinoid and
calicheamicin.
[0160] 52. The organic molecule of Claim 50, wherein the toxin is a
maytansinoid.
[0161] 53. The organic molecule of Claim 45 or 46 which induces
death of a cell to which it binds.
[0162] 54. The organic molecule of Claim 45 or 46 which is
detectably labeled.
[0163] 55. A composition of matter comprising:
[0164] (a) the polypeptide of Claim 11;
[0165] (b) the polypeptide of Claim 12;
[0166] (c) the chimeric polypeptide of Claim 13;
[0167] (d) the antibody of Claim 15;
[0168] (e) the antibody of Claim 16;
[0169] (f) the oligopeptide of Claim 35;
[0170] (g) the oligopeptide of Claim 36;
[0171] (h) the TAT binding organic molecule of Claim 45; or
[0172] (i) the TAT binding organic molecule of Claim 46; in
combination with a carrier.
[0173] 56. The composition of matter of Claim 55, wherein said
carrier is a pharmaceutically acceptable carrier.
[0174] 57. An article of manufacture comprising:
[0175] (a) a container; and
[0176] (b) the composition of matter of Claim 55 contained within
said container.
[0177] 58. The article of manufacture of Claim 57 further
comprising a label affixed to said container, or a package insert
included with said container, referring to the use of said
composition of matter for the therapeutic treatment of or the
diagnostic detection of a cancer.
[0178] 59. A method of inhibiting the growth of a cell that
expresses a protein having at least 80% amino acid sequence
identity to:
[0179] (a) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0180] (b) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide;
[0181] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), with its associated signal peptide;
[0182] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), lacking its associated signal peptide;
[0183] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56,
113, 115, 117 or 119); or
[0184] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-56, 113,
115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said
method comprising contacting said cell with an antibody,
oligopeptide or organic molecule that binds to said protein, the
binding of said antibody, oligopeptide or organic molecule to said
protein thereby causing an inhibition of growth of said cell.
[0185] 60. The method of claim 59, wherein said antibody is a
monoclonal antibody.
[0186] 61. The method of Claim 59, wherein said antibody is an
antibody fragment.
[0187] 62. The method of Claim 59, wherein said antibody is a
chimeric or a humanized antibody.
[0188] 63. The method of Claim 59, wherein said antibody,
oligopeptide or organic molecule is conjugated to a growth
inhibitory agent.
[0189] 64. The method of Claim 59, wherein said antibody,
oligopeptide or organic molecule is conjugated to a cytotoxic
agent.
[0190] 65. The method of Claim 64, wherein said cytotoxic agent is
selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0191] 66. The method of Claim 64, wherein the cytotoxic agent is a
toxin.
[0192] 67. The method of Claim 66, wherein the toxin is selected
from the group consisting of maytansinoid and calicheamicin.
[0193] 68. The method of Claim 66, wherein the toxin is a
maytansinoid.
[0194] 69. The method of Claim 59, wherein said antibody is
produced in bacteria.
[0195] 70. The method of Claim 59, wherein said antibody is
produced in CHO cells.
[0196] 71. The method of Claim 59, wherein said cell is a cancer
cell.
[0197] 72. The method of Claim 71, wherein said cancer cell is
further exposed to radiation treatment or a chemotherapeutic
agent.
[0198] 73. The method of Claim 71, wherein said cancer cell is
selected from the group consisting of a breast cancer cell, a
colorectal cancer cell, a lung cancer cell, an ovarian cancer cell,
a central nervous system cancer cell, a liver cancer cell, a
bladder cancer cell, a pancreatic cancer cell, a cervical cancer
cell, a melanoma cell and a leukemia cell.
[0199] 74. The method of Claim 71, wherein said protein is more
abundantly expressed by said cancer cell as compared to a normal
cell of the same tissue origin.
[0200] 75. The method of Claim 59 which causes the death of said
cell.
[0201] 76. The method of Claim 59, wherein said protein has:
[0202] (a) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120);
[0203] (b) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120), lacking its associated signal peptide sequence;
[0204] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated
signal peptide sequence;
[0205] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated
signal peptide sequence;
[0206] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ
ID NOS:1-56, 113, 115, 117 or 119); or
[0207] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-56,
113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0208] 77. A method of therapeutically treating a mammal having a
cancerous tumor comprising cells that express a protein having at
least 80% amino acid sequence identity to:
[0209] (a) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0210] (b) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide;
[0211] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), with its associated signal peptide;
[0212] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS: 57-112, 114,
116, 118 or 120), lacking its associated signal peptide;
[0213] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56,
113, 115, 117 or 119); or
[0214] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-56, 113,
115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said
method comprising administering to said mammal a therapeutically
effective amount of an antibody, oligopeptide or organic molecule
that binds to said protein, thereby effectively treating said
mammal.
[0215] 78. The method of Claim 77, wherein said antibody is a
monoclonal antibody.
[0216] 79. The method of Claim 77, wherein said antibody is an
antibody fragment.
[0217] 80. The method of Claim 77, wherein said antibody is a
chimeric or a humanized antibody.
[0218] 81. The method of Claim 77, wherein said antibody,
oligopeptide or organic molecule is conjugated to a growth
inhibitory agent.
[0219] 82. The method of Claim 77, wherein said antibody,
oligopeptide or organic molecule is conjugated to a cytotoxic
agent.
[0220] 83. The method of Claim 82, wherein said cytotoxic agent is
selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0221] 84. The method of Claim 82, wherein the cytotoxic agent is a
toxin.
[0222] 85. The method of Claim 84, wherein the toxin is selected
from the group consisting of maytansinoid and calicheamicin.
[0223] 86. The method of Claim 84, wherein the toxin is a
maytansinoid.
[0224] 87. The method of Claim 77, wherein said antibody is
produced in bacteria.
[0225] 88. The method of Claim 77, wherein said antibody is
produced in CHO cells.
[0226] 89. The method of Claim 77, wherein said tumor is further
exposed to radiation treatment or a chemotherapeutic agent.
[0227] 90. The method of Claim 77, wherein said tumor is a breast
tumor, a colorectal tumor, a lung tumor, an ovarian tumor, a
central nervous system tumor, a liver tumor, a bladder tumor, a
pancreatic tumor, or a cervical tumor.
[0228] 91. The method of Claim 77, wherein said protein is more
abundantly expressed by the cancerous cells of said tumor as
compared to a normal cell of the same tissue origin.
[0229] 92. The method of Claim 77, wherein said protein has:
[0230] (a) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120);
[0231] (b) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120), lacking its associated signal peptide sequence;
[0232] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS: 57-112, 114, 116, 118 or 120), with its associated
signal peptide sequence;
[0233] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated
signal peptide sequence;
[0234] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ
ID NOS:1-56, 113, 115, 117 or 119); or
[0235] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-56,
113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0236] 93. A method of determining the presence of a protein in a
sample suspected of containing said protein, wherein said protein
has at least 80% amino acid sequence identity to:
[0237] (a) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0238] (b) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide;
[0239] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), with its associated signal peptide;
[0240] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), lacking its associated signal peptide;
[0241] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56,
113, 115, 117 or 119); or
[0242] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-56, 113,
115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said
method comprising exposing said sample to an antibody, oligopeptide
or organic molecule that binds to said protein and determining
binding of said antibody, oligopeptide or organic molecule to said
protein in said sample, wherein binding of the antibody,
oligopeptide or organic molecule to said protein is indicative of
the presence of said protein in said sample.
[0243] 94. The method of Claim 93, wherein said sample comprises a
cell suspected of expressing said protein.
[0244] 95. The method of Claim 94, wherein said cell is a cancer
cell.
[0245] 96. The method of Claim 93, wherein said antibody,
oligopeptide or organic molecule is detectably labeled.
[0246] 97. The method of Claim 93, wherein said protein has:
[0247] (a) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120);
[0248] (b) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120), lacking its associated signal peptide sequence;
[0249] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS: 57-112, 114, 116, 118 or 120), with its associated
signal peptide sequence;
[0250] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated
signal peptide sequence;
[0251] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ
ID NOS:1-56, 113, 115, 117 or 119); or
[0252] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-56,
113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0253] 98. A method of diagnosing the presence of a tumor in a
mammal, said method comprising determining the level of expression
of a gene encoding a protein having at least 80% amino acid
sequence identity to:
[0254] (a) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0255] (b) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide;
[0256] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), with its associated signal peptide;
[0257] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), lacking its associated signal peptide;
[0258] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56,
113, 115, 117 or 119); or
[0259] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-56, 113,
115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), in a test
sample of tissue cells obtained from said mammal and in a control
sample of known normal cells of the same tissue origin, wherein a
higher level of expression of said protein in the test sample, as
compared to the control sample, is indicative of the presence of
tumor in the mammal from which the test sample was obtained.
[0260] 99. The method of Claim 98, wherein the step of determining
the level of expression of a gene encoding said protein comprises
employing an oligonucleotide in an in situ hybridization or RT-PCR
analysis.
[0261] 100. The method of Claim 98, wherein the step determining
the level of expression of a gene encoding said protein comprises
employing an antibody in an immunohistochemistry or Western blot
analysis.
[0262] 101. The method of Claim 98, wherein said protein has:
[0263] (a) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120);
[0264] (b) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120), lacking its associated signal peptide sequence;
[0265] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated
signal peptide sequence;
[0266] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated
signal peptide sequence;
[0267] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ
ID NOS:1-56, 113, 115, 117 or 119); or
[0268] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-56,
113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0269] 102. A method of diagnosing the presence of a tumor in a
mammal, said method comprising contacting a test sample of tissue
cells obtained from said mammal with an antibody, oligopeptide or
organic molecule that binds to a protein having at least 80% amino
acid sequence identity to:
[0270] (a) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0271] (b) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide;
[0272] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), with its associated signal peptide;
[0273] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), lacking its associated signal peptide;
[0274] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56,
113, 115, 117 or 119); or
[0275] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-56, 113,
115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), and
detecting the formation of a complex between said antibody,
oligopeptide or organic molecule and said protein in the test
sample, wherein the formation of a complex is indicative of the
presence of a tumor in said mammal.
[0276] 103. The method of Claim 102, wherein said antibody,
oligopeptide or organic molecule is detectably labeled.
[0277] 104. The method of Claim 102, wherein said test sample of
tissue cells is obtained from an individual suspected of having a
cancerous tumor.
[0278] 105. The method of Claim 102, wherein said protein has:
[0279] (a) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120);
[0280] (b) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120), lacking its associated signal peptide sequence;
[0281] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS: 57-112, 114, 116, 118 or 120), with its associated
signal peptide sequence;
[0282] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated
signal peptide sequence;
[0283] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ
ID NOS:1-56, 113, 115, 117 or 119); or
[0284] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-56,
113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0285] 106. A method for treating or preventing a cell
proliferative disorder associated with increased expression or
activity of a protein having at least 80% amino acid sequence
identity to:
[0286] (a) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0287] (b) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide;
[0288] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), with its associated signal peptide; [0289] (d) an
extracellular domain of the polypeptide shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120), lacking its associated signal peptide;
[0290] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56,
113, 115, 117 or 19); or
[0291] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-56, 113,
115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said
method comprising administering to a subject in need of such
treatment an effective amount of an antagonist of said protein,
thereby effectively treating or preventing said cell proliferative
disorder.
[0292] 107. The method of Claim 106, wherein said cell
proliferative disorder is cancer.
[0293] 108. The method of Claim 106, wherein said antagonist is an
anti-TAT polypeptide antibody, TAT binding oligopeptide, TAT
binding organic molecule or antisense oligonucleotide.
[0294] 109. A method of binding an antibody, oligopeptide or
organic molecule to a cell that expresses a protein having at least
80% amino acid sequence identity to:
[0295] (a) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0296] (b) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide;
[0297] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), with its associated signal peptide;
[0298] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), lacking its associated signal peptide;
[0299] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56,
113, 115, 117 or 119); or
[0300] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-56, 113,
115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said
method comprising contacting said cell with an antibody,
oligopeptide or organic molecule that binds to said protein and
allowing the binding of the antibody, oligopeptide or organic
molecule to said protein to occur, thereby binding said antibody,
oligopeptide or organic molecule to said cell.
[0301] 110. The method of Claim 109, wherein said antibody is a
monoclonal antibody.
[0302] 111. The method of Claim 109, wherein said antibody is an
antibody fragment.
[0303] 112. The method of Claim 109, wherein said antibody is a
chimeric or a humanized antibody.
[0304] 113. The method of Claim 109, wherein said antibody,
oligopeptide or organic molecule is conjugated to a growth
inhibitory agent.
[0305] 114. The method of Claim 109, wherein said antibody,
oligopeptide or organic molecule is conjugated to a cytotoxic
agent.
[0306] 115. The method of Claim 114, wherein said cytotoxic agent
is selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0307] 116. The method of Claim 114, wherein the cytotoxic agent is
a toxin.
[0308] 117. The method of Claim 116, wherein the toxin is selected
from the group consisting of maytansinoid and calicheamicin.
[0309] 118. The method of Claim 116, wherein the toxin is a
maytansinoid.
[0310] 119. The method of Claim 109, wherein said antibody is
produced in bacteria.
[0311] 120. The method of Claim 109, wherein said antibody is
produced in CHO cells.
[0312] 121. The method of Claim 109, wherein said cell is a cancer
cell.
[0313] 122. The method of Claim 121, wherein said cancer cell is
further exposed to radiation treatment or a chemotherapeutic
agent.
[0314] 123. The method of Claim 121, wherein said cancer cell is
selected from the group consisting of a breast cancer cell, a
colorectal cancer cell, a lung cancer cell, an ovarian cancer cell,
a central nervous system cancer cell, a liver cancer cell, a
bladder cancer cell, a pancreatic cancer cell, a cervical cancer
cell, a melanoma cell and a leukemia cell.
[0315] 124. The method of Claim 123, wherein said protein is more
abundantly expressed by said cancer cell as compared to a normal
cell of the same tissue origin.
[0316] 125. The method of Claim 109 which causes the death of said
cell.
[0317] 126. Use of a nucleic acid as claimed in any of Claims 1 to
5 or 30 in the preparation of a medicament for the therapeutic
treatment or diagnostic detection of a cancer.
[0318] 127. Use of a nucleic acid as claimed in any of Claims 1 to
5 or 30 in the preparation of a medicament for treating a
tumor.
[0319] 128. Use of a nucleic acid as claimed in any of Claims 1 to
5 or 30 in the preparation of a medicament for treatment or
prevention of a cell proliferative disorder.
[0320] 129. Use of an expression vector as claimed in any of Claims
6, 7 or 31 in the preparation of a medicament for the therapeutic
treatment or diagnostic detection of a cancer.
[0321] 130. Use of an expression vector as claimed in any of Claims
6, 7 or 31 in the preparation of medicament for treating a
tumor.
[0322] 131. Use of an expression vector as claimed in any of Claims
6, 7 or 31 in the preparation of a medicament for treatment or
prevention of a cell proliferative disorder.
[0323] 132. Use of a host cell as claimed in any of Claims 8, 9,
32, or 33 in the preparation of a medicament for the therapeutic
treatment or diagnostic detection of a cancer.
[0324] 133. Use of a host cell as claimed in any of Claims 8, 9, 32
or 33 in the preparation of a medicament for treating a tumor.
[0325] 134. Use of a host cell as claimed in any of Claims 8, 9, 32
or 33 in the preparation of a medicament for treatment or
prevention of a cell proliferative disorder.
[0326] 135. Use of a polypeptide as claimed in any of Claims 11 to
14 in the preparation of a medicament for the therapeutic treatment
or diagnostic detection of a cancer.
[0327] 136. Use of a polypeptide as claimed in any of Claims 11 to
14 in the preparation of a medicament for treating a tumor.
[0328] 137. Use of a polypeptide as claimed in any of Claims 11 to
14 in the preparation of a medicament for treatment or prevention
of a cell proliferative disorder.
[0329] 138. Use of an antibody as claimed in any of Claims 15 to 29
in the preparation of a medicament for the therapeutic treatment or
diagnostic detection of a cancer.
[0330] 139. Use of an antibody as claimed in any of Claims 15 to 29
in the preparation of a medicament for treating a tumor.
[0331] 140. Use of an antibody as claimed in any of Claims 15 to 29
in the preparation of a medicament for treatment or prevention of a
cell proliferative disorder.
[0332] 141. Use of an oligopeptide as claimed in any of Claims 35
to 44 in the preparation of a medicament for the therapeutic
treatment or diagnostic detection of a cancer.
[0333] 142. Use of an oligopeptide as claimed in any of Claims 35
to 44 in the preparation of a medicament for treating a tumor.
[0334] 143. Use of an oligopeptide as claimed in any of Claims 35
to 44 in the preparation of a medicament for treatment or
prevention of a cell proliferative disorder.
[0335] 144. Use of a TAT binding organic molecule as claimed in any
of Claims 45 to 54 in the preparation of a medicament for the
therapeutic treatment or diagnostic detection of a cancer.
[0336] 145. Use of a TAT binding organic molecule as claimed in any
of Claims 45 to 54 in the preparation of a medicament for treating
a tumor.
[0337] 146. Use of a TAT binding organic molecule as claimed in any
of Claims 45 to 54 in the preparation of a medicament for treatment
or prevention of a cell proliferative disorder.
[0338] 147. Use of a composition of matter as claimed in any of
Claims 55 or 56 in the preparation of a medicament for the
therapeutic treatment or diagnostic detection of a cancer.
[0339] 148. Use of a composition of matter as claimed in any of
Claims 55 or 56 in the preparation of a medicament for treating a
tumor.
[0340] 149. Use of a composition of matter as claimed in any of
Claims 55 or 56 in the preparation of a medicament for treatment or
prevention of a cell proliferative disorder.
[0341] 150. Use of an article of manufacture as claimed in any of
Claims 57 or 58 in the preparation of a medicament for the
therapeutic treatment or diagnostic detection of a cancer.
[0342] 151. Use of an article of manufacture as claimed in any of
Claims 57 or 58 in the preparation of a medicament for treating a
tumor.
[0343] 152. Use of an article of manufacture as claimed in any of
Claims 57 or 58 in the preparation of a medicament for treatment or
prevention of a cell proliferative disorder.
[0344] 153. A method for inhibiting the growth of a cell, wherein
the growth of said cell is at least in part dependent upon a growth
potentiating effect of a protein having at least 80% amino acid
sequence identity to:
[0345] (a) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0346] (b) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide;
[0347] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), with its associated signal peptide;
[0348] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), lacking its associated signal peptide;
[0349] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56,
113, 115, 117 or 119); or
[0350] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-56, 113,
115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said
method comprising contacting said protein with an antibody,
oligopeptide or organic molecule that binds to said protein, there
by inhibiting the growth of said cell.
[0351] 154. The method of Claim 153, wherein said cell is a cancer
cell.
[0352] 155. The method of Claim 153, wherein said protein is
expressed by said cell.
[0353] 156. The method of Claim 153, wherein the binding of said
antibody, oligopeptide or organic molecule to said protein
antagonizes a cell growth-potentiating activity of said
protein.
[0354] 157. The method of Claim 153, wherein the binding of said
antibody, oligopeptide or organic molecule to said protein induces
the death of said cell.
[0355] 158. The method of Claim 153, wherein said antibody is a
monoclonal antibody.
[0356] 159. The method of Claim 153, wherein said antibody is an
antibody fragment.
[0357] 160. The method of Claim 153, wherein said antibody is a
chimeric or a humanized antibody.
[0358] 161. The method of Claim 153, wherein said antibody,
oligopeptide or organic molecule is conjugated to a growth
inhibitory agent.
[0359] 162. The method of Claim 153, wherein said antibody,
oligopeptide or organic molecule is conjugated to a cytotoxic
agent.
[0360] 163. The method of Claim 162, wherein said cytotoxic agent
is selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0361] 164. The method of Claim 162, wherein the cytotoxic agent is
a toxin.
[0362] 165. The method of Claim 164, wherein the toxin is selected
from the group consisting of maytansinoid and calicheamicin.
[0363] 166. The method of Claim 164, wherein the toxin is a
maytansinoid.
[0364] 167. The method of Claim 153, wherein said antibody is
produced in bacteria.
[0365] 168. The method of Claim 153, wherein said antibody is
produced in CHO cells.
[0366] 169. The method of Claim 153, wherein said protein has:
[0367] (a) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120);
[0368] (b) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120), lacking its associated signal peptide sequence;
[0369] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated
signal peptide sequence;
[0370] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated
signal peptide sequence;
[0371] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ
ID NOS:1-56, 113, 115, 117 or 119); or
[0372] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-56,
113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0373] 170. A method of therapeutically treating a tumor in a
mammal, wherein the growth of said tumor is at least in part
dependent upon a growth potentiating effect of a protein having at
least 80% amino acid sequence identity to:
[0374] (a) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0375] (b) the polypeptide shown in any one of FIGS. 57-112, 114,
116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking
its associated signal peptide;
[0376] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), with its associated signal peptide;
[0377] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114,
116, 118 or 120), lacking its associated signal peptide;
[0378] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56,
113, 115, 117 or 119); or
[0379] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-56, 113,
115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said
method comprising contacting said protein with an antibody,
oligopeptide or organic molecule that binds to said protein,
thereby effectively treating said tumor.
[0380] 171. The method of Claim 170, wherein said protein is
expressed by cells of said tumor.
[0381] 172. The method of Claim 170, wherein the binding of said
antibody, oligopeptide or organic molecule to said protein
antagonizes a cell growth-potentiating activity of said
protein.
[0382] 173. The method of Claim 170, wherein said antibody is a
monoclonal antibody.
[0383] 174. The method of Claim 170, wherein said antibody is an
antibody fragment.
[0384] 175. The method of Claim 170, wherein said antibody is a
chimeric or a humanized antibody.
[0385] 176. The method of Claim 170, wherein said antibody,
oligopeptide or organic molecule is conjugated to a growth
inhibitory agent.
[0386] 177. The method of Claim 170, wherein said antibody,
oligopeptide or organic molecule is conjugated to a cytotoxic
agent.
[0387] 178. The method of Claim 177, wherein said cytotoxic agent
is selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0388] 179. The method of Claim 177, wherein the cytotoxic agent is
a toxin.
[0389] 180. The method of Claim 179, wherein the toxin is selected
from the group consisting of maytansinoid and calicheamicin.
[0390] 181. The method of Claim 179, wherein the toxin is a
maytansinoid.
[0391] 182. The method of Claim 170, wherein said antibody is
produced in bacteria.
[0392] 183. The method of Claim 170, wherein said antibody is
produced in CHO cells.
[0393] 184. The method of Claim 170, wherein said protein has:
[0394] (a) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120);
[0395] (b) the amino acid sequence shown in any one of FIGS.
57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or
120), lacking its associated signal peptide sequence;
[0396] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated
signal peptide sequence;
[0397] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 57-112, 114, 116, 118 or 120
(SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated
signal peptide sequence;
[0398] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-56, 113, 115, 117 or 119 (SEQ
ID NOS:1-56, 113, 115, 117 or 119); or
[0399] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-56,
113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0400] Yet further embodiments of the present invention will be
evident to the skilled artisan upon a reading of the present
specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0401] FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a TAT207
cDNA, wherein SEQ ID NO:1 is a clone designated herein as
"DNA67962".
[0402] FIG. 2 shows a nucleotide sequence (SEQ ID NO:2) of a TAT177
cDNA, wherein SEQ ID NO:2 is a clone designated herein as
"DNA77507".
[0403] FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a TAT235
cDNA, wherein SEQ ID NO:3 is a clone designated herein as
"DNA87993".
[0404] FIG. 4 shows a nucleotide sequence (SEQ ID NO:4) of a TAT234
cDNA, wherein SEQ ID NO:4 is a clone designated herein as
"DNA92980".
[0405] FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a TAT239
cDNA, wherein SEQ ID NO:5 is a clone designated herein as
"DNA96792".
[0406] FIG. 6 shows a nucleotide sequence (SEQ ID NO:6) of a TAT193
cDNA, wherein SEQ ID NO:6 is a clone designated herein as
"DNA96964".
[0407] FIG. 7 shows a nucleotide sequence (SEQ ID NO:7) of a TAT233
cDNA, wherein SEQ ID NO:7 is a clone designated herein as
"DNA105792".
[0408] FIG. 8 shows a nucleotide sequence (SEQ ID NO:8) of a TAT226
cDNA, wherein SEQ ID NO:8 is a clone designated herein as
"DNA119474".
[0409] FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a TAT199
cDNA, wherein SEQ ID NO:9 is a clone designated herein as
"DNA142915".
[0410] FIGS. 10A-B show a nucleotide sequence (SEQ ID NO:10) of a
TAT204 cDNA, wherein SEQ ID NO:10 is a clone designated herein as
"DNA150491".
[0411] FIGS. 11A-B show a nucleotide sequence (SEQ ID NO:11) of a
TAT248 cDNA, wherein SEQ ID NO:11 is a clone designated herein as
"DNA280351".
[0412] FIG. 12 shows a nucleotide sequence (SEQ ID NO:12) of a
TAT232 cDNA, wherein SEQ ID NO:12 is a clone designated herein as
"DNA150648".
[0413] FIG. 13 shows a nucleotide sequence (SEQ ID NO:13) of a
TAT219 cDNA, wherein SEQ ID NO:13 is a clone designated herein as
"DNA172500".
[0414] FIG. 14 shows a nucleotide sequence (SEQ ID NO:14) of a
TAT224 cDNA, wherein SEQ ID NO:14 is a clone designated herein as
"DNA179651".
[0415] FIG. 15 shows a nucleotide sequence (SEQ ID NO:15) of a
TAT237 cDNA, wherein SEQ ID NO:15 is a clone designated herein as
"DNA207698".
[0416] FIG. 16 shows a nucleotide sequence (SEQ ID NO:16) of a
TAT178 cDNA, wherein SEQ ID NO:16 is a clone designated herein as
"DNA208551".
[0417] FIGS. 17A-B show a nucleotide sequence (SEQ ID NO:17) of a
TAT198 cDNA, wherein SEQ ID NO:17 is a clone designated herein as
"DNA210159".
[0418] FIGS. 18A-B show a nucleotide sequence (SEQ ID NO:18) of a
TAT194 cDNA, wherein SEQ ID NO:18 is a clone designated herein as
"DNA225706".
[0419] FIGS. 19A-B show a nucleotide sequence (SEQ ID NO:19) of a
TAT223 cDNA, wherein SEQ ID NO:19 is a clone designated herein as
"DNA225793".
[0420] FIG. 20 shows a nucleotide sequence (SEQ ID NO:20) of a
TAT196 cDNA, wherein SEQ ID NO:20 is a clone designated herein as
"DNA225796".
[0421] FIG. 21 shows a nucleotide sequence (SEQ ID NO:21) of a
TAT236 cDNA, wherein SEQ ID NO:21 is a clone designated herein as
"DNA225886".
[0422] FIG. 22 shows a nucleotide sequence (SEQ ID NO:22) of a
TAT195 cDNA, wherein SEQ ID NO:22 is a clone designated herein as
"DNA225943".
[0423] FIG. 23 shows a nucleotide sequence (SEQ ID NO:23) of a
TAT203 cDNA, wherein SEQ ID NO:23 is a clone designated herein as
"DNA226283".
[0424] FIGS. 24A-B show a nucleotide sequence (SEQ ID NO:24) of a
TAT200 cDNA, wherein SEQ ID NO:24 is a clone designated herein as
"DNA226589".
[0425] FIGS. 25A-B show a nucleotide sequence (SEQ ID NO:25) of a
TAT205 cDNA, wherein SEQ ID NO:25 is a clone designated herein as
"DNA226622".
[0426] FIGS. 26A-B show a nucleotide sequence (SEQ ID NO:26) of a
TAT 85 cDNA, wherein SEQ ID NO:26 is a clone designated herein as
"DNA226717".
[0427] FIGS. 27A-B show a nucleotide sequence (SEQ ID NO:27) of a
TAT225 cDNA, wherein SEQ ID NO:27 is a clone designated herein as
"DNA227162".
[0428] FIG. 28 shows a nucleotide sequence (SEQ ID NO:28) of a
TAT247 cDNA, wherein SEQ ID NO:28 is a clone designated herein as
"DNA277804".
[0429] FIG. 29 shows a nucleotide sequence (SEQ ID NO:29) of a
TAT197 cDNA, wherein SEQ ID NO:29 is a clone designated herein as
"DNA227545".
[0430] FIG. 30 shows a nucleotide sequence (SEQ ID NO:30) of a
TAT175 cDNA, wherein SEQ ID NO:30 is a clone designated herein as
"DNA227611".
[0431] FIG. 31 shows a nucleotide sequence (SEQ ID NO:31) of a
TAT208 cDNA, wherein SEQ ID NO:31 is a clone designated herein as
"DNA261021".
[0432] FIG. 32 shows a nucleotide sequence (SEQ ID NO:32) of a
TAT174 cDNA, wherein SEQ ID NO:32 is a clone designated herein as
"DNA233034".
[0433] FIG. 33 shows a nucleotide sequence (SEQ ID NO:33) of a
TAT214 cDNA, wherein SEQ ID NO:33 is a clone designated herein as
"DNA266920".
[0434] FIG. 34 shows a nucleotide sequence (SEQ ID NO:34) of a
TAT220 cDNA, wherein SEQ ID NO:34 is a clone designated herein as
"DNA266921".
[0435] FIG. 35 shows a nucleotide sequence (SEQ ID NO:35) of a
TAT221 cDNA, wherein SEQ ID NO:35 is a clone designated herein as
"DNA266922".
[0436] FIG. 36 shows a nucleotide sequence (SEQ ID NO:36) of a
TAT201 cDNA, wherein SEQ ID NO:36 is a clone designated herein as
"DNA234441".
[0437] FIGS. 37A-B show a nucleotide sequence (SEQ ID NO:37) of a
TAT179 cDNA, wherein SEQ ID NO:37 is a clone designated herein as
"DNA234834".
[0438] FIG. 38 shows a nucleotide sequence (SEQ ID NO:38) of a
TAT216 cDNA, wherein SEQ ID NO:38 is a clone designated herein as
"DNA247587".
[0439] FIG. 39 shows a nucleotide sequence (SEQ ID NO:39) of a
TAT218 cDNA, wherein SEQ ID NO:39 is a clone designated herein as
"DNA255987".
[0440] FIG. 40 shows a nucleotide sequence (SEQ ID NO:40) of a
TAT206 cDNA, wherein SEQ ID NO:40 is a clone designated herein as
"DNA5604 1".
[0441] FIGS. 41A-B show a nucleotide sequence (SEQ ID NO:41) of a
TAT374 cDNA, wherein SEQ ID NO:41 is a clone designated herein as
"DNA257845".
[0442] FIG. 42 shows a nucleotide sequence (SEQ ID NO:42) of a
TAT209 cDNA, wherein SEQ ID NO:42 is a clone designated herein as
"DNA260655".
[0443] FIG. 43 shows a nucleotide sequence (SEQ ID NO:43) of a
TAT192 cDNA, wherein SEQ ID NO:43 is a clone designated herein as
"DNA260945".
[0444] FIG. 44 shows a nucleotide sequence (SEQ ID NO:44) of a
TAT180 cDNA, wherein SEQ ID NO:44 is a clone designated herein as
"DNA247476".
[0445] FIG. 45 shows a nucleotide sequence (SEQ ID NO:45) of a
TAT375 cDNA, wherein SEQ ID NO:45 is a clone designated herein as
"DNA260990".
[0446] FIG. 46 shows a nucleotide sequence (SEQ ID NO:46) of a
TAT181 cDNA, wherein SEQ ID NO:46 is a clone designated herein as
"DNA261001".
[0447] FIG. 47 shows a nucleotide sequence (SEQ ID NO:47) of a
TAT176 cDNA, wherein SEQ ID NO:47 is a clone designated herein as
"DNA261013".
[0448] FIG. 48 shows a nucleotide sequence (SEQ ID NO:48) of a
TAT184 cDNA, wherein SEQ ID NO:48 is a clone designated herein as
"DNA262144".
[0449] FIG. 49 shows a nucleotide sequence (SEQ ID NO:49) of a
TAT182 cDNA, wherein SEQ ID NO:49 is a clone designated herein as
"DNA266928".
[0450] FIGS. 50A-B show a nucleotide sequence (SEQ ID NO:50) of a
TAT213 cDNA, wherein SEQ ID NO:50 is a clone designated herein as
"DNA267342".
[0451] FIGS. 51A-C show a nucleotide sequence (SEQ ID NO:51) of a
TAT217 cDNA, wherein SEQ ID NO:51 is a clone designated herein as
"DNA267626".
[0452] FIG. 52 shows a nucleotide sequence (SEQ ID NO:52) of a
TAT222 cDNA, wherein SEQ ID NO:52 is a clone designated herein as
"DNA268035".
[0453] FIG. 53 shows a nucleotide sequence (SEQ ID NO:53) of a
TAT202 cDNA, wherein SEQ ID NO:53 is a clone designated herein as
"DNA268334".
[0454] FIG. 54 shows a nucleotide sequence (SEQ ID NO:54) of a
TAT215 cDNA, wherein SEQ ID NO:54 is a clone designated herein as
"DNA269238".
[0455] FIG. 55 shows a nucleotide sequence (SEQ ID NO:55) of a
TAT238 cDNA, wherein SEQ ID NO:55 is a clone designated herein as
"DNA272578".
[0456] FIG. 56 shows a nucleotide sequence (SEQ ID NO:56) of a
TAT212 cDNA, wherein SEQ ID NO:56 is a clone designated herein as
"DNA277797".
[0457] FIG. 57 shows the amino acid sequence (SEQ ID NO:57) derived
from the coding sequence of SEQ ID NO:1 shown in FIG. 1.
[0458] FIG. 58 shows the amino acid sequence (SEQ ID NO:58) derived
from the coding sequence of SEQ ID NO:2 shown in FIG. 2.
[0459] FIG. 59 shows the amino acid sequence (SEQ ID NO:59) derived
from the coding sequence of SEQ ID NO: 3 shown in FIG. 3.
[0460] FIG. 60 shows the amino acid sequence (SEQ ID NO:60) derived
from the coding sequence of SEQ ID NO:4 shown in FIG. 4.
[0461] FIG. 61 shows the amino acid sequence (SEQ ID NO:61) derived
from the coding sequence of SEQ ID NO:5 shown in FIG. 5.
[0462] FIG. 62 shows the amino acid sequence (SEQ ID NO:62) derived
from the coding sequence of SEQ ID NO:6 shown in FIG. 6.
[0463] FIG. 63 shows the amino acid sequence (SEQ ID NO:63) derived
from the coding sequence of SEQ ID NO:7 shown in FIG. 7.
[0464] FIG. 64 shows the amino acid sequence (SEQ ID NO: 64)
derived from the coding sequence of SEQ ID NO:8 shown in FIG.
8.
[0465] FIG. 65 shows the amino acid sequence (SEQ ID NO:65) derived
from the coding sequence of SEQ ID NO:9 shown in FIG. 9.
[0466] FIG. 66 shows the amino acid sequence (SEQ ID NO:66) derived
from the coding sequence of SEQ ID NO:10 shown in FIGS. 10A-B.
[0467] FIG. 67 shows the amino acid sequence (SEQ ID NO:67) derived
from the coding sequence of SEQ ID NO:11 shown in FIGS. 11A-B.
[0468] FIG. 68 shows the amino acid sequence (SEQ ID NO:68) derived
from the coding sequence of SEQ ID NO:12 shown in FIG. 12.
[0469] FIG. 69 shows the amino acid sequence (SEQ ID NO:69) derived
from the coding sequence of SEQ ID NO:13 shown in FIG. 13.
[0470] FIG. 70 shows the amino acid sequence (SEQ ID NO:70) derived
from the coding sequence of SEQ ID NO:14 shown in FIG. 14.
[0471] FIG. 71 shows the amino acid sequence (SEQ ID NO:71) derived
from the coding sequence of SEQ ID NO:15 shown in FIG. 15.
[0472] FIG. 72 shows the amino acid sequence (SEQ ID NO:72) derived
from the coding sequence of SEQ ID NO:16 shown in FIG. 16.
[0473] FIG. 73 shows the amino acid sequence (SEQ ID NO:73) derived
from the coding sequence of SEQ ID NO:17 shown in FIGS. 17A-B.
[0474] FIG. 74 shows the amino acid sequence (SEQ ID NO:74) derived
from the coding sequence of SEQ ID NO:18 shown in FIGS. 18A-B.
[0475] FIG. 75 shows the amino acid sequence (SEQ ID NO:75) derived
from the coding sequence of SEQ ID NO:19 shown in FIGS. 19A-B.
[0476] FIG. 76 shows the amino acid sequence (SEQ ID NO:76) derived
from the coding sequence of SEQ ID NO:20 shown in FIG. 20.
[0477] FIG. 77 shows the amino acid sequence (SEQ ID NO:77) derived
from the coding sequence of SEQ ID NO:21 shown in FIG. 21.
[0478] FIG. 78 shows the amino acid sequence (SEQ ID NO:78) derived
from the coding sequence of SEQ ID NO:22 shown in FIG. 22.
[0479] FIG. 79 shows the amino acid sequence (SEQ ID NO:79) derived
from the coding sequence of SEQ ID NO:23 shown in FIG. 23.
[0480] FIG. 80 shows the amino acid sequence (SEQ ID NO:80) derived
from the coding sequence of SEQ ID NO:24 shown in FIGS. 24A-B.
[0481] FIG. 81 shows the amino acid sequence (SEQ ID NO:81) derived
from the coding sequence of SEQ ID NO:25 shown in FIGS. 25A-B.
[0482] FIG. 82 shows the amino acid sequence (SEQ ID NO:82) derived
from the coding sequence of SEQ ID NO:26 shown in FIGS. 26A-B.
[0483] FIG. 83 shows the amino acid sequence (SEQ ID NO:83) derived
from the coding sequence of SEQ ID NO:27 shown in FIGS. 27A-B.
[0484] FIG. 84 shows the amino acid sequence (SEQ ID NO:84) derived
from the coding sequence of SEQ ID NO:28 shown in FIG. 28.
[0485] FIG. 85 shows the amino acid sequence (SEQ ID NO:85) derived
from the coding sequence of SEQ ID NO:29 shown in FIG. 29.
[0486] FIG. 86 shows the amino acid sequence (SEQ ID NO:86) derived
from the coding sequence of SEQ ID NO:30 shown in FIG. 30.
[0487] FIG. 87 shows the amino acid sequence (SEQ ID NO: 87)
derived from the coding sequence of SEQ ID NO:31 shown in FIG.
31.
[0488] FIG. 88 shows the amino acid sequence (SEQ ID NO:88) derived
from the coding sequence of SEQ ID NO:32 shown in FIG. 32.
[0489] FIG. 89 shows the amino acid sequence (SEQ ID NO: 89)
derived from the coding sequence of SEQ ID NO:33 shown in FIG.
33.
[0490] FIG. 90 shows the amino acid sequence (SEQ ID NO:90) derived
from the coding sequence of SEQ ID NO:34 shown in FIG. 34.
[0491] FIG. 91 shows the amino acid sequence (SEQ ID NO:91) derived
from the coding sequence of SEQ ID NO:35 shown in FIG. 35.
[0492] FIG. 92 shows the amino acid sequence (SEQ ID NO:92) derived
from the coding sequence of SEQ ID NO:36 shown in FIG. 36.
[0493] FIG. 93 shows the amino acid sequence (SEQ ID NO:93) derived
from the coding sequence of SEQ ID NO:37 shown in FIGS. 37A-B.
[0494] FIG. 94 shows the amino acid sequence (SEQ ID NO:94) derived
from the coding sequence of SEQ ID NO:38 shown in FIG. 38.
[0495] FIG. 95 shows the amino acid sequence (SEQ ID NO:95) derived
from the coding sequence of SEQ ID NO:39 shown in FIG. 39.
[0496] FIG. 96 shows the amino acid sequence (SEQ ID NO:96) derived
from the coding sequence of SEQ ID NO:40 shown in FIG. 40.
[0497] FIG. 97 shows the amino acid sequence (SEQ ID NO:97) derived
from the coding sequence of SEQ ID NO:41 shown in FIGS. 41A-B.
[0498] FIG. 98 shows the amino acid sequence (SEQ ID NO:98) derived
from the coding sequence of SEQ ID NO:42 shown in FIG. 42.
[0499] FIG. 99 shows the amino acid sequence (SEQ ID NO:99) derived
from the coding sequence of SEQ ID NO:43 shown in FIG. 43.
[0500] FIG. 100 shows the amino acid sequence (SEQ ID NO:100)
derived from the coding sequence of SEQ ID NO:44 shown in FIG.
44.
[0501] FIG. 101 shows the amino acid sequence (SEQ ID NO:101)
derived from the coding sequence of SEQ ID NO:45 shown in FIG.
45.
[0502] FIG. 102 shows the amino acid sequence (SEQ ID NO:102)
derived from the coding sequence of SEQ ID NO:46 shown in FIG.
46.
[0503] FIG. 103 shows the amino acid sequence (SEQ ID NO:103)
derived from the coding sequence of SEQ ID NO:47 shown in FIG.
47.
[0504] FIG. 104 shows the amino acid sequence (SEQ ID NO:104)
derived from the coding sequence of SEQ ID NO:48 shown in FIG.
48.
[0505] FIG. 105 shows the amino acid sequence (SEQ ID NO:105)
derived from the coding sequence of SEQ ID NO:49 shown in FIG.
49.
[0506] FIG. 106 shows the amino acid sequence (SEQ ID NO:106)
derived from the coding sequence of SEQ ID NO:50 shown in FIGS.
50A-B.
[0507] FIGS. 107A-B show the amino acid sequence (SEQ ID NO:107)
derived from the coding sequence of SEQ ID NO:51 shown in FIGS.
51A-C.
[0508] FIG. 108 shows the amino acid sequence (SEQ ID NO:108)
derived from the coding sequence of SEQ ID NO:52 shown in FIG.
52.
[0509] FIG. 109 shows the amino acid sequence (SEQ ID NO:109)
derived from the coding sequence of SEQ ID NO:53 shown in FIG.
53.
[0510] FIG. 110 shows the amino acid sequence (SEQ ID NO:110)
derived from the coding sequence of SEQ ID NO:54 shown in FIG.
54.
[0511] FIG. 111 shows the amino acid sequence (SEQ ID NO:111)
derived from the coding sequence of SEQ ID NO:55 shown in FIG.
55.
[0512] FIG. 112 shows the amino acid sequence (SEQ ID NO:112)
derived from the coding sequence of SEQ ID NO:56 shown in FIG.
56.
[0513] FIG. 113 shows a nucleotide sequence (SEQ ID NO:113) of a
TAT376 cDNA, wherein SEQ ID NO:113 is a clone designated herein as
"DNA304853".
[0514] FIG. 114 shows the amino acid sequence (SEQ ID NO:114)
derived from the coding sequence of SEQ ID NO:113 shown in FIG.
113.
[0515] FIG. 115 shows a nucleotide sequence (SEQ ID NO:115) of a
TAT377 cDNA, wherein SEQ ID NO:115 is a clone designated herein as
"DNA304854".
[0516] FIG. 116 shows the amino acid sequence (SEQ ID NO:116)
derived from the coding sequence of SEQ ID NO:115 shown in FIG.
115.
[0517] FIG. 117 shows a nucleotide sequence (SEQ ID NO:117) of a
TAT378 cDNA, wherein SEQ ID NO:117 is a clone designated herein as
"DNA304855".
[0518] FIG. 118 shows the amino acid sequence (SEQ ID NO:118)
derived from the coding sequence of SEQ ID NO:117 shown in FIG.
117.
[0519] FIGS. 119A-B show a nucleotide sequence (SEQ ID NO:119) of a
TAT379 cDNA, wherein SEQ ID NO:119 is a clone designated herein as
"DNA287971".
[0520] FIG. 120 shows the amino acid sequence (SEQ ID NO:120)
derived from the coding sequence of SEQ ID NO:119 shown in FIGS.
119A-B.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0521] The terms "TAT polypeptide" and "TAT" as used herein and
when immediately followed by a numerical designation, refer to
various polypeptides, wherein the complete designation (i.e.,
TAT/number) refers to specific polypeptide sequences as described
herein. The terms "TAT/number polypeptide" and "TAT/number" wherein
the term "number" is provided as an actual numerical designation as
used herein encompass native sequence polypeptides, polypeptide
variants and fragments of native sequence polypeptides and
polypeptide variants (which are further defined herein). The TAT
polypeptides described herein may be isolated from a variety of
sources, such as from human tissue types or from another source, or
prepared by recombinant or synthetic methods. The term "TAT
polypeptide" refers to each individual TAT/number polypeptide
disclosed herein. All disclosures in this specification which refer
to the "TAT polypeptide" refer to each of the polypeptides
individually as well as jointly. For example, descriptions of the
preparation of, purification of, derivation of, formation of
antibodies to or against, formation of TAT binding oligopeptides to
or against, formation of TAT binding organic molecules to or
against, administration of, compositions containing, treatment of a
disease with, etc., pertain to each polypeptide of the invention
individually. The term "TAT polypeptide" also includes variants of
the TAT/number polypeptides disclosed herein.
[0522] A "native sequence TAT polypeptide" comprises a polypeptide
having the same amino acid sequence as the corresponding TAT
polypeptide derived from nature. Such native sequence TAT
polypeptides can be isolated from nature or can be produced by
recombinant or synthetic means. The term "native sequence TAT
polypeptide" specifically encompasses naturally-occurring truncated
or secreted forms of the specific TAT polypeptide (e.g., an
extracellular domain sequence), naturally-occurring variant forms
(e.g., alternatively spliced forms) and naturally-occurring allelic
variants of the polypeptide. In certain embodiments of the
invention, the native sequence TAT polypeptides disclosed herein
are mature or full-length native sequence polypeptides comprising
the full-length amino acids sequences shown in the accompanying
figures. Start and stop codons (if indicated) are shown in bold
font and underlined in the figures. Nucleic acid residues indicated
as "N" in the accompanying figures are any nucleic acid residue.
However, while the TAT polypeptides disclosed in the accompanying
figures are shown to begin with methionine residues designated
herein as amino acid position 1 in the figures, it is conceivable
and possible that other methionine residues located either upstream
or downstream from the amino acid position 1 in the figures may be
employed as the starting amino acid residue for the TAT
polypeptides.
[0523] The TAT polypeptide "extracellular domain" or "ECD" refers
to a form of the TAT polypeptide which is essentially free of the
transmembrane and cytoplasmic domains. Ordinarily, a TAT
polypeptide ECD will have less than 1% of such transmembrane and/or
cytoplasmic domains and preferably, will have less than 0.5% of
such domains. It will be understood that any transmembrane domains
identified for the TAT polypeptides of the present invention are
identified pursuant to criteria routinely employed in the art for
identifying that type of hydrophobic domain. The exact boundaries
of a transmembrane domain may vary but most likely by no more than
about 5 amino acids at either end of the domain as initially
identified herein. Optionally, therefore, an extracellular domain
of a TAT polypeptide may contain from about 5 or fewer amino acids
on either side of the transmembrane domain/extracellular domain
boundary as identified in the Examples or specification and such
polypeptides, with or without the associated signal peptide, and
nucleic acid encoding them, are contemplated by the present
invention.
[0524] The approximate location of the "signal peptides" of the
various TAT polypeptides disclosed herein may be shown in the
present specification and/or the accompanying figures. It is noted,
however, that the C-terminal boundary of a signal peptide may vary,
but most likely by no more than about 5 amino acids on either side
of the signal peptide C-terminal boundary as initially identified
herein, wherein the C-terminal boundary of the signal peptide may
be identified pursuant to criteria routinely employed in the art
for identifying that type of amino acid sequence element (e.g.,
Nielsen et al., Prot. Eng. 10: 1-6 (1997) and von Heinje et al.,
Nucl. Acids. Res. 14:4683-4690 (1986)). Moreover, it is also
recognized that, in some cases, cleavage of a signal sequence from
a secreted polypeptide is not entirely uniform, resulting in more
than one secreted species. These mature polypeptides, where the
signal peptide is cleaved within no more than about 5 amino acids
on either side of the C-terminal boundary of the signal peptide as
identified herein, and the polynucleotides encoding them, are
contemplated by the present invention.
[0525] "TAT polypeptide variant" means a TAT polypeptide,
preferably an active TAT polypeptide, as defined herein having at
least about 80% amino acid sequence identity with a full-length
native sequence TAT polypeptide sequence as disclosed herein, a TAT
polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain of a TAT polypeptide, with or
without the signal peptide, as disclosed herein or any other
fragment of a full-length TAT polypeptide sequence as disclosed
herein (such as those encoded by a nucleic acid that represents
only a portion of the complete coding sequence for a full-length
TAT polypeptide). Such TAT polypeptide variants include, for
instance, TAT polypeptides wherein one or more amino acid residues
are added, or deleted, at the N- or C-terminus of the full-length
native amino acid sequence. Ordinarily, a TAT polypeptide variant
will have at least about 80% amino acid sequence identity,
alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino
acid sequence identity, to a full-length native sequence TAT
polypeptide sequence as disclosed herein, a TAT polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a TAT polypeptide, with or without the
signal peptide, as disclosed herein or any other specifically
defined fragment of a full-length TAT polypeptide sequence as
disclosed herein. Ordinarily, TAT variant polypeptides are at least
about 10 amino acids in length, alternatively at least about 20,
30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,
310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,
440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,
570, 580, 590, 600 amino acids in length, or more. Optionally, TAT
variant polypeptides will have no more than one conservative amino
acid substitution as compared to the native TAT polypeptide
sequence, alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10
conservative amino acid substitution as compared to the native TAT
polypeptide sequence.
[0526] "Percent (%) amino acid sequence identity" with respect to
the TAT polypeptide sequences identified herein is defined as the
percentage of amino acid residues in a candidate sequence that are
identical with the amino acid residues in the specific TAT
polypeptide sequence, after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
identity, and not considering any conservative substitutions as
part of the sequence identity. Alignment for purposes of
determining percent amino acid sequence identity can be achieved in
various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
ALIGN or Megalign (DNASTAR) software. Those skilled in the art can
determine appropriate parameters for measuring alignment, including
any algorithms needed to achieve maximal alignment over the full
length of the sequences being compared. For purposes herein,
however, % amino acid sequence identity values are generated using
the sequence comparison computer program ALIGN-2, wherein the
complete source code for the ALIGN-2 program is provided in Table 1
below. The ALIGN-2 sequence comparison computer program was
authored by Genentech, Inc. and the source code shown in Table 1
below has been filed with user documentation in the U.S. Copyright
Office, Washington D.C., 20559, where it is registered under U.S.
Copyright Registration No. TXU510087. The ALIGN-2 program is
publicly available through Genentech, Inc., South San Francisco,
Calif. or may be compiled from the source code provided in Table 1
below. The ALIGN-2 program should be compiled for use on a UNIX
operating system, preferably digital UNIX V4.0D. All sequence
comparison parameters are set by the ALIGN-2 program and do not
vary.
[0527] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows: 100 times the fraction X/Y where X is
the number of amino acid residues scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of A and B, and where Y is the total number of amino acid residues
in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the length of amino acid sequence B, the
% amino acid sequence identity of A to B will not equal the % amino
acid sequence identity of B to A. As examples of % amino acid
sequence identity calculations using this method, Tables 2 and 3
demonstrate how to calculate the % amino acid sequence identity of
the amino acid sequence designated "Comparison Protein" to the
amino acid sequence designated "TAT", wherein "TAT" represents the
amino acid sequence of a hypothetical TAT polypeptide of interest,
"Comparison Protein" represents the amino acid sequence of a
polypeptide against which the "TAT" polypeptide of interest is
being compared, and "X, "Y" and "Z" each represent different
hypothetical amino acid residues. Unless specifically stated
otherwise, all % amino acid sequence identity values used herein
are obtained as described in the immediately preceding paragraph
using the ALIGN-2 computer program.
[0528] "TAT variant polynucleotide" or "TAT variant nucleic acid
sequence" means a nucleic acid molecule which encodes a TAT
polypeptide, preferably an active TAT polypeptide, as defined
herein and which has at least about 80% nucleic acid sequence
identity with a nucleotide acid sequence encoding a full-length
native sequence TAT polypeptide sequence as disclosed herein, a
full-length native sequence TAT polypeptide sequence lacking the
signal peptide as disclosed herein, an extracellular domain of a
TAT polypeptide, with or without the signal peptide, as disclosed
herein or any other fragment of a full-length TAT polypeptide
sequence as disclosed herein (such as those encoded by a nucleic
acid that represents only a portion of the complete coding sequence
for a full-length TAT polypeptide). Ordinarily, a TAT variant
polynucleotide will have at least about 80% nucleic acid sequence
identity, alternatively at least about 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% nucleic acid sequence identity with a nucleic acid sequence
encoding a full-length native sequence TAT polypeptide sequence as
disclosed herein, a full-length native sequence TAT polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a TAT polypeptide, with or without the
signal sequence, as disclosed herein or any other fragment of a
full-length TAT polypeptide sequence as disclosed herein. Variants
do not encompass the native nucleotide sequence.
[0529] Ordinarily, TAT variant polynucleotides are at least about 5
nucleotides in length, alternatively at least about 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,
165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250,
260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,
390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,
520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,
650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,
780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900,
910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in
length, wherein in this context the term "about" means the
referenced nucleotide sequence length plus or minus 10% of that
referenced length.
[0530] "Percent (%) nucleic acid sequence identity" with respect to
TAT-encoding nucleic acid sequences identified herein is defined as
the percentage of nucleotides in a candidate sequence that are
identical with the nucleotides in the TAT nucleic acid sequence of
interest, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity.
Alignment for purposes of determining percent nucleic acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software.
[0531] For purposes herein, however, % nucleic acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2, wherein the complete source code for the
ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence
comparison computer program was authored by Genentech, Inc. and the
source code shown in Table 1 below has been filed with user
documentation in the U.S. Copyright Office, Washington D.C., 20559,
where it is registered under U.S. Copyright Registration No.
TXU510087. The ALIGN-2 program is publicly available through
Genentech, Inc., South San Francisco, Calif. or may be compiled
from the source code provided in Table 1 below. The ALIGN-2 program
should be compiled for use on a UNIX operating system, preferably
digital UNIX V4.0D. All sequence comparison parameters are set by
the ALIGN-2 program and do not vary.
[0532] In situations where ALIGN-2 is employed for nucleic acid
sequence comparisons, the % nucleic acid sequence identity of a
given nucleic acid sequence C to, with, or against a given nucleic
acid sequence D (which can alternatively be phrased as a given
nucleic acid sequence C that has or comprises a certain % nucleic
acid sequence identity to, with, or against a given nucleic acid
sequence D) is calculated as follows: 100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of C and D, and where Z is the total number of nucleotides in D. It
will be appreciated that where the length of nucleic acid sequence
C is not equal to the length of nucleic acid sequence D, the %
nucleic acid sequence identity of C to D will not equal the %
nucleic acid sequence identity of D to C. As examples of % nucleic
acid sequence identity calculations, Tables 4 and 5, demonstrate
how to calculate the % nucleic acid sequence identity of the
nucleic acid sequence designated "Comparison DNA" to the nucleic
acid sequence designated "TAT-DNA", wherein "TAT-DNA" represents a
hypothetical TAT-encoding nucleic acid sequence of interest,
"Comparison DNA" represents the nucleotide sequence of a nucleic
acid molecule against which the "TAT-DNA" nucleic acid molecule of
interest is being compared, and "N", "L" and "V" each represent
different hypothetical nucleotides. Unless specifically stated
otherwise, all % nucleic acid sequence identity values used herein
are obtained as described in the immediately preceding paragraph
using the ALIGN-2 computer program.
[0533] In other embodiments, TAT variant polynucleotides are
nucleic acid molecules that encode a TAT polypeptide and which are
capable of hybridizing, preferably under stringent hybridization
and wash conditions, to nucleotide sequences encoding a full-length
TAT polypeptide as disclosed herein. TAT variant polypeptides may
be those that are encoded by a TAT variant polynucleotide.
[0534] The term "full-length coding region" when used in reference
to a nucleic acid encoding a TAT polypeptide refers to the sequence
of nucleotides which encode the full-length TAT polypeptide of the
invention (which is often shown between start and stop codons,
inclusive thereof, in the accompanying figures). The term
"full-length coding region" when used in reference to an ATCC
deposited nucleic acid refers to the TAT polypeptide-encoding
portion of the cDNA that is inserted into the vector deposited with
the ATCC (which is often shown between start and stop codons,
inclusive thereof, in the accompanying figures).
[0535] "Isolated," when used to describe the various TAT
polypeptides disclosed herein, means polypeptide that has been
identified and separated and/or recovered from a component of its
natural environment. Contaminant components of its natural
environment are materials that would typically interfere with
diagnostic or therapeutic uses for the polypeptide, and may include
enzymes, hormones, and other proteinaceous or non-proteinaceous
solutes. In preferred embodiments, the polypeptide will be purified
(1) to a degree sufficient to obtain at least 15 residues of
N-terminal or internal amino acid sequence by use of a spinning cup
sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or
reducing conditions using Coomassie blue or, preferably, silver
stain. Isolated polypeptide includes polypeptide in situ within
recombinant cells, since at least one component of the TAT
polypeptide natural environment will not be present. Ordinarily,
however, isolated polypeptide will be prepared by at least one
purification step.
[0536] An "isolated" TAT polypeptide-encoding nucleic acid or other
polypeptide-encoding nucleic acid is a nucleic acid molecule that
is identified and separated from at least one contaminant nucleic
acid molecule with which it is ordinarily associated in the natural
source of the polypeptide-encoding nucleic acid. An isolated
polypeptide-encoding nucleic acid molecule is other than in the
form or setting in which it is found in nature. Isolated
polypeptide-encoding nucleic acid molecules therefore are
distinguished from the specific polypeptide-encoding nucleic acid
molecule as it exists in natural cells. However, an isolated
polypeptide-encoding nucleic acid molecule includes
polypeptide-encoding nucleic acid molecules contained in cells that
ordinarily express the polypeptide where, for example, the nucleic
acid molecule is in a chromosomal location different from that of
natural cells.
[0537] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0538] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0539] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured DNA to reanneal when complementary strands
are present in an environment below their melting temperature. The
higher the degree of desired homology between the probe and
hybridizable sequence, the higher the relative temperature which
can be used. As a result, it follows that higher relative
temperatures would tend to make the reaction conditions more
stringent, while lower temperatures less so. For additional details
and explanation of stringency of hybridization reactions, see
Ausubel et al., Current Protocols in Molecular Biology, Wiley
Interscience Publishers, (1995).
[0540] "Stringent conditions" or "high stringency conditions", as
defined herein, may be identified by those that: (1) employ low
ionic strength and high temperature for washing, for example 0.015
M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl
sulfate at 50.degree. C.; (2) employ during hybridization a
denaturing agent, such as formamide, for example, 50% (v/v)
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; or
(3) overnight hybridization in a solution that employs 50%
formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM
sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times.
Denhardt's solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1%
SDS, and 10% dextran sulfate at 42.degree. C., with a 10 minute
wash at 42.degree. C. in 0.2.times.SSC (sodium chloride/sodium
citrate) followed by a 10 minute high-stringency wash consisting of
0.1.times.SSC containing EDTA at 55.degree. C.
[0541] "Moderately stringent conditions" may be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and hybridization conditions (e.g.,
temperature, ionic strength and % SDS) less stringent that those
described above. An example of moderately stringent conditions is
overnight incubation at 37.degree. C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate
factors such as probe length and the like.
[0542] The term "epitope tagged" when used herein refers to a
chimeric polypeptide comprising a TAT polypeptide or anti-TAT
antibody fused to a "tag polypeptide". The tag polypeptide has
enough residues to provide an epitope against which an antibody can
be made, yet is short enough such that it does not interfere with
activity of the polypeptide to which it is fused. The tag
polypeptide preferably also is fairly unique so that the antibody
does not substantially cross-react with other epitopes. Suitable
tag polypeptides generally have at least six amino acid residues
and usually between about 8 and 50 amino acid residues (preferably,
between about 10 and amino acid residues).
[0543] "Active" or "activity" for the purposes herein refers to
form(s) of a TAT polypeptide which retain a biological and/or an
immunological activity of native or naturally-occurring TAT,
wherein "biological" activity refers to a biological function
(either inhibitory or stimulatory) caused by a native or
naturally-occurring TAT other than the ability to induce the
production of an antibody against an antigenic epitope possessed by
a native or naturally-occurring TAT and an "immunological" activity
refers to the ability to induce the production of an antibody
against an antigenic epitope possessed by a native or
naturally-occurring TAT.
[0544] The term "antagonist" is used in the broadest sense, and
includes any molecule that partially or fully blocks, inhibits, or
neutralizes a biological activity of a native TAT polypeptide
disclosed herein. In a similar manner, the term "agonist" is used
in the broadest sense and includes any molecule that mimics a
biological activity of a native TAT polypeptide disclosed herein.
Suitable agonist or antagonist molecules specifically include
agonist or antagonist antibodies or antibody fragments, fragments
or amino acid sequence variants of native TAT polypeptides,
peptides, antisense oligonucleotides, small organic molecules, etc.
Methods for identifying agonists or antagonists of a TAT
polypeptide may comprise contacting a TAT polypeptide with a
candidate agonist or antagonist molecule and measuring a detectable
change in one or more biological activities normally associated
with the TAT polypeptide.
[0545] "Treating" or "treatment" or "alleviation" refers to both
therapeutic treatment and prophylactic or preventative measures,
wherein the object is to prevent or slow down (lessen) the targeted
pathologic condition or disorder. Those in need of treatment
include those already with the disorder as well as those prone to
have the disorder or those in whom the disorder is to be prevented.
A subject or mammal is successfully "treated" for a TAT
polypeptide-expressing cancer if, after receiving a therapeutic
amount of an anti-TAT antibody, TAT binding oligopeptide or TAT
binding organic molecule according to the methods of the present
invention, the patient shows observable and/or measurable reduction
in or absence of one or more of the following: reduction in the
number of cancer cells or absence of the cancer cells; reduction in
the tumor size; inhibition (i.e., slow to some extent and
preferably stop) of cancer cell infiltration into peripheral organs
including the spread of cancer into soft tissue and bone;
inhibition (i.e., slow to some extent and preferably stop) of tumor
metastasis; inhibition, to some extent, of tumor growth; and/or
relief to some extent, one or more of the symptoms associated with
the specific cancer; reduced morbidity and mortality, and
improvement in quality of life issues. To the extent the anti-TAT
antibody or TAT binding oligopeptide may prevent growth and/or kill
existing cancer cells, it may be cytostatic and/or cytotoxic.
Reduction of these signs or symptoms may also be felt by the
patient.
[0546] The above parameters for assessing successful treatment and
improvement in the disease are readily measurable by routine
procedures familiar to a physician. For cancer therapy, efficacy
can be measured, for example, by assessing the time to disease
progression (TTP) and/or determining the response rate (RR).
Metastasis can be determined by staging tests and by bone scan and
tests for calcium level and other enzymes to determine spread to
the bone. CT scans can also be done to look for spread to the
pelvis and lymph nodes in the area. Chest X-rays and measurement of
liver enzyme levels by known methods are used to look for
metastasis to the lungs and liver, respectively. Other routine
methods for monitoring the disease include transrectal
ultrasonography (TRUS) and transrectal needle biopsy (TRNB).
[0547] For bladder cancer, which is a more localized cancer,
methods to determine progress of disease include urinary cytologic
evaluation by cystoscopy, monitoring for presence of blood in the
urine, visualization of the urothelial tract by sonography or an
intravenous pyelogram, computed tomography (CT) and magnetic
resonance imaging (MRI). The presence of distant metastases can be
assessed by CT of the abdomen, chest x-rays, or radionuclide
imaging of the skeleton.
[0548] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time. "Intermittent" administration is treatment that is
not consecutively done without interruption, but rather is cyclic
in nature.
[0549] "Mammal" for purposes of the treatment of, alleviating the
symptoms of or diagnosis of a cancer refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, cats,
cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the
mammal is human.
[0550] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0551] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers which are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.RTM., polyethylene glycol (PEG), and PLURONICS.RTM..
[0552] By "solid phase" or "solid support" is meant a non-aqueous
matrix to which an antibody, TAT binding oligopeptide or TAT
binding organic molecule of the present invention can adhere or
attach. Examples of solid phases encompassed herein include those
formed partially or entirely of glass (e.g., controlled pore
glass), polysaccharides (e.g., agarose), polyacrylamides,
polystyrene, polyvinyl alcohol and silicones. In certain
embodiments, depending on the context, the solid phase can comprise
the well of an assay plate; in others it is a purification column
(e.g., an affinity chromatography column). This term also includes
a discontinuous solid phase of discrete particles, such as those
described in U.S. Pat. No. 4,275,149.
[0553] A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant which is useful for
delivery of a drug (such as a TAT polypeptide, an antibody thereto
or a TAT binding oligopeptide) to a mammal. The components of the
liposome are commonly arranged in a bilayer formation, similar to
the lipid arrangement of biological membranes.
[0554] A "small" molecule or "small" organic molecule is defined
herein to have a molecular weight below about 500 Daltons.
[0555] An "effective amount" of a polypeptide, antibody, TAT
binding oligopeptide, TAT binding organic molecule or an agonist or
antagonist thereof as disclosed herein is an amount sufficient to
carry out a specifically stated purpose. An "effective amount" may
be determined empirically and in a routine manner, in relation to
the stated purpose.
[0556] The term "therapeutically effective amount" refers to an
amount of an antibody, polypeptide, TAT binding oligopeptide, TAT
binding organic molecule or other drug effective to "treat" a
disease or disorder in a subject or mammal. In the case of cancer,
the therapeutically effective amount of the drug may reduce the
number of cancer cells; reduce the tumor size; inhibit (i.e., slow
to some extent and preferably stop) cancer cell infiltration into
peripheral organs; inhibit (i.e., slow to some extent and
preferably stop) tumor metastasis; inhibit, to some extent, tumor
growth; and/or relieve to some extent one or more of the symptoms
associated with the cancer. See the definition herein of
"treating". To the extent the drug may prevent growth and/or kill
existing cancer cells, it may be cytostatic and/or cytotoxic.
[0557] A "growth inhibitory amount" of an anti-TAT antibody, TAT
polypeptide, TAT binding oligopeptide or TAT binding organic
molecule is an amount capable of inhibiting the growth of a cell,
especially tumor, e.g., cancer cell, either in vitro or in vivo. A
"growth inhibitory amount" of an anti-TAT antibody, TAT
polypeptide, TAT binding oligopeptide or TAT binding organic
molecule for purposes of inhibiting neoplastic cell growth may be
determined empirically and in a routine manner.
[0558] A "cytotoxic amount" of an anti-TAT antibody, TAT
polypeptide, TAT binding oligopeptide or TAT binding organic
molecule is an amount capable of causing the destruction of a cell,
especially tumor, e.g., cancer cell, either in vitro or in vivo. A
"cytotoxic amount" of an anti-TAT antibody, TAT polypeptide, TAT
binding oligopeptide or TAT binding organic molecule for purposes
of inhibiting neoplastic cell growth may be determined empirically
and in a routine manner.
[0559] The term "antibody" is used in the broadest sense and
specifically covers, for example, single anti-TAT monoclonal
antibodies (including agonist, antagonist, and neutralizing
antibodies), anti-TAT antibody compositions with polyepitopic
specificity, polyclonal antibodies, single chain anti-TAT
antibodies, and fragments of anti-TAT antibodies (see below) as
long as they exhibit the desired biological or immunological
activity. The term "immunoglobulin" (Ig) is used interchangeable
with antibody herein.
[0560] An "isolated antibody" is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0561] The basic 4-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains (an IgM antibody consists of 5 of the
basic heterotetramer unit along with an additional polypeptide
called J chain, and therefore contain 10 antigen binding sites,
while secreted IgA antibodies can polymerize to form polyvalent
assemblages comprising 2-5 of the basic 4-chain units along with J
chain). In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to a H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
domain (V.sub.H) followed by three constant domains (C.sub.H) for
each of the .alpha. and .gamma. chains and four C.sub.H domains for
.mu. and .epsilon. isotypes. Each L chain has at the N-terminus, a
variable domain (V.sub.L) followed by a constant domain (C.sub.L)
at its other end. The V.sub.L is aligned with the V.sub.H and the
C.sub.L is aligned with the first constant domain of the heavy
chain (C.sub.H1). Particular amino acid residues are believed to
form an interface between the light chain and heavy chain variable
domains. The pairing of a V.sub.H and V.sub.L together forms a
single antigen-binding site. For the structure and properties of
the different classes of antibodies, see, e.g., Basic and Clinical
Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and
Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn.,
1994, page 71 and Chapter 6.
[0562] The L chain from any vertebrate species can be assigned to
one of two clearly distinct types, called kappa and lambda, based
on the amino acid sequences of their constant domains. Depending on
the amino acid sequence of the constant domain of their heavy
chains (C.sub.H), immunoglobulins can be assigned to different
classes or isotypes. There are five classes of immunoglobulins:
IgA, IgD, IgE, IgG, and IgM, having heavy chains designated
.alpha., .delta., .epsilon., .gamma., and .mu., respectively. The
.gamma. and .alpha. classes are further divided into subclasses on
the basis of relatively minor differences in C.sub.H sequence and
function, e.g., humans express the following subclasses: IgG1,
IgG2, IgG3, IgG4, IgA1, and IgA2.
[0563] The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among
antibodies. The V domain mediates antigen binding and define
specificity of a particular antibody for its particular antigen.
However, the variability is not evenly distributed across the
10-amino acid span of the variable domains. Instead, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-12 amino
acids long. The variable domains of native heavy and light chains
each comprise four FRs, largely adopting a .beta.-sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The hypervariable regions in each chain are
held together in close proximity by the FRs and, With the
hypervariable regions from the other chain, contribute to the
formation of the antigen-binding site of antibodies (see Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). The constant domains are not involved directly in binding
an antibody to an antigen, but exhibit various effector functions,
such as participation of the antibody in antibody dependent
cellular cytotoxicity (ADCC).
[0564] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g. around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)
in the V.sub.L, and around about 1-35 (H1), 50-65 (H2) and 95-102
(H3) in the V.sub.H; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the V.sub.L, and 26-32 (H1), 53-55 (H2) and
96-101 (H3) in the V.sub.H; Chothia and Lesk J. Mol. Biol.
196:901-917 (1987)).
[0565] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations which include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they may be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies useful in the present invention may be
prepared by the hybridoma methodology first described by Kohler et
al., Nature, 256:495 (1975), or may be made using recombinant DNA
methods in bacterial, eukaryotic animal or plant cells (see, e.g.,
U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 352:624-628 (1991) and Marks
et al., J. Mol. Biol., 222:581-597 (1991), for example.
[0566] The monoclonal antibodies herein include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.
Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of
interest herein include "primatized" antibodies comprising variable
domain antigen-binding sequences derived from a non-human primate
(e.g. Old World Monkey, Ape etc), and human constant region
sequences.
[0567] An "intact" antibody is one which comprises an
antigen-binding site as well as a C.sub.L and at least heavy chain
constant domains, C.sub.H1, C.sub.H2 and C.sub.H3. The constant
domains may be native sequence constant domains (e.g. human native
sequence constant domains) or amino acid sequence variant thereof.
Preferably, the intact antibody has one or more effector
functions.
[0568] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies (see
U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng.
8(10): 1057-1062 [1995]); single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments.
[0569] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, and a residual
"Fc" fragment, a designation reflecting the ability to crystallize
readily. The Fab fragment consists of an entire L chain along with
the variable region domain of the H chain (V.sub.H), and the first
constant domain of one heavy chain (C.sub.H1). Each Fab fragment is
monovalent with respect to antigen binding, i.e., it has a single
antigen-binding site. Pepsin treatment of an antibody yields a
single large F(ab').sub.2 fragment which roughly corresponds to two
disulfide linked Fab fragments having divalent antigen-binding
activity and is still capable of cross-linking antigen. Fab'
fragments differ from Fab fragments by having additional few
residues at the carboxy terminus of the C.sub.H1 domain including
one or more cysteines from the antibody hinge region. Fab'-SH is
the designation herein for Fab' in which the cysteine residue(s) of
the constant domains bear a free thiol group. F(ab').sub.2 antibody
fragments originally were produced as pairs of Fab' fragments which
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0570] The Fc fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region, which
region is also the part recognized by Fc receptors (FcR) found on
certain types of cells.
[0571] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This fragment
consists of a dimer of one heavy- and one light-chain variable
region domain in tight, non-covalent association. From the folding
of these two domains emanate six hypervariable loops (3 loops each
from the H and L chain) that contribute the amino acid residues for
antigen binding and confer antigen binding specificity to the
antibody. However, even a single variable domain (or half of an Fv
comprising only three CDRs specific for an antigen) has the ability
to recognize and bind antigen, although at a lower affinity than
the entire binding site.
[0572] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody fragments that comprise the V.sub.H and V.sub.L antibody
domains connected into a single polypeptide chain. Preferably, the
sFv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994); Borrebaeck 1995, infra.
[0573] The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments (see preceding paragraph)
with short linkers (about 5-10 residues) between the V.sub.H and
V.sub.L domains such that inter-chain but not intra-chain pairing
of the V domains is achieved, resulting in a bivalent fragment,
i.e., fragment having two antigen-binding sites. Bispecific
diabodies are heterodimers of two "crossover" sFv fragments in
which the V.sub.H and V.sub.L domains of the two antibodies are
present on different polypeptide chains. Diabodies are described
more fully in, for example, EP 404,097; WO 93/11161; and Hollinger
et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0574] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from the
non-human antibody. For the most part, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from
a hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit or non-human primate having the desired
antibody specificity, affinity, and capability. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525(1986); Riechmann et al., Nature 332:323-329 (1988); and
Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0575] A "species-dependent antibody," e.g., a mammalian anti-human
IgE antibody, is an antibody which has a stronger binding affinity
for an antigen from a first mammalian species than it has for a
homologue of that antigen from a second mammalian species.
Normally, the species-dependent antibody "bind specifically" to a
human antigen (i.e., has a binding affinity (Kd) value of no more
than about 1.times.10.sup.-7 M, preferably no more than about
1.times.10.sup.-8 and most preferably no more than about
1.times.10.sup.-9 M) but has a binding affinity for a homologue of
the antigen from a second non-human mammalian species which is at
least about 50 fold, or at least about 500 fold, or at least about
1000 fold, weaker than its binding affinity for the human antigen.
The species-dependent antibody can be of any of the various types
of antibodies as defined above, but preferably is a humanized or
human antibody.
[0576] A "TAT binding oligopeptide" is an oligopeptide that binds,
preferably specifically, to a TAT polypeptide as described herein.
TAT binding oligopeptides may be chemically synthesized using known
oligopeptide synthesis methodology or may be prepared and purified
using recombinant technology. TAT binding oligopeptides are usually
at least about 5 amino acids in length, alternatively at least
about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in
length or more, wherein such oligopeptides that are capable of
binding, preferably specifically, to a TAT polypeptide as described
herein. TAT binding oligopeptides may be identified without undue
experimentation using well known techniques. In this regard, it is
noted that techniques for screening oligopeptide libraries for
oligopeptides that are capable of specifically binding to a
polypeptide target are well known in the art (see, e.g., U.S. Pat.
Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409,
5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506
and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A.,
81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A.,
82:178-182 (1985); Geysen et al., in Synthetic Peptides as
Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth.,
102:259-274(1987); Schoofs et al., J. Immunol., 140:611-616(1988),
Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378;
Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et
al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol.
Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci.
USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol.,
2:668).
[0577] A "TAT binding organic molecule" is an organic molecule
other than an oligopeptide or antibody as defined herein that
binds, preferably specifically, to a TAT polypeptide as described
herein. TAT binding organic molecules may be identified and
chemically synthesized using known methodology (see, e.g., PCT
Publication Nos. WO00/00823 and WO0/39585). TAT binding organic
molecules are usually less than about 2000 daltons in size,
alternatively less than about 1500, 750, 500, 250 or 200 daltons in
size, wherein such organic molecules that are capable of binding,
preferably specifically, to a TAT polypeptide as described herein
may be identified without undue experimentation using well known
techniques. In this regard, it is noted that techniques for
screening organic molecule libraries for molecules that are capable
of binding to a polypeptide target are well known in the art (see,
e.g., PCT Publication Nos. WO00/00823 and WO0/39585).
[0578] An antibody, oligopeptide or other organic molecule "which
binds" an antigen of interest, e.g. a tumor-associated polypeptide
antigen target, is one that binds the antigen with sufficient
affinity such that the antibody, oligopeptide or other organic
molecule is useful as a diagnostic and/or therapeutic agent in
targeting a cell or tissue expressing the antigen, and does not
significantly cross-react with other proteins. In such embodiments,
the extent of binding of the antibody, oligopeptide or other
organic molecule to a "non-target" protein will be less than about
10% of the binding of the antibody, oligopeptide or other organic
molecule to its particular target protein as determined by
fluorescence activated cell sorting (FACS) analysis or
radioimmunoprecipitation (RIA). With regard to the binding of an
antibody, oligopeptide or other organic molecule to a target
molecule, the term "specific binding" or "specifically binds to" or
is "specific for" a particular polypeptide or an epitope on a
particular polypeptide target means binding that is measurably
different from a non-specific interaction. Specific binding can be
measured, for example, by determining binding of a molecule
compared to binding of a control molecule, which generally is a
molecule of similar structure that does not have binding activity.
For example, specific binding can be determined by competition with
a control molecule that is similar to the target, for example, an
excess of non-labeled target. In this case, specific binding is
indicated if the binding of the labeled target to a probe is
competitively inhibited by excess unlabeled target. The term
"specific binding" or "specifically binds to" or is "specific for"
a particular polypeptide or an epitope on a particular polypeptide
target as used herein can be exhibited, for example, by a molecule
having a Kd for the target of at least about 10.sup.-4 M,
alternatively at least about 10.sup.-5 M, alternatively at least
about 10.sup.-6 M, alternatively at least about 10.sup.-7 M,
alternatively at least about 10.sup.-8 M, alternatively at least
about 10.sup.-9 M, alternatively at least about 10.sup.-10 M,
alternatively at least about 10.sup.-11 M, alternatively at least
about 10.sup.-12 M, or greater. In one embodiment, the term
"specific binding" refers to binding where a molecule binds to a
particular polypeptide or epitope on a particular polypeptide
without substantially binding to any other polypeptide or
polypeptide epitope.
[0579] An antibody, oligopeptide or other organic molecule that
"inhibits the growth of tumor cells expressing a TAT polypeptide"
or a "growth inhibitory" antibody, oligopeptide or other organic
molecule is one which results in measurable growth inhibition of
cancer cells expressing or overexpressing the appropriate TAT
polypeptide. The TAT polypeptide may be a transmembrane polypeptide
expressed on the surface of a cancer cell or may be a polypeptide
that is produced and secreted by a cancer cell. Preferred growth
inhibitory anti-TAT antibodies, oligopeptides or organic molecules
inhibit growth of TAT-expressing tumor cells by greater than 20%,
preferably from about 20% to about 50%, and even more preferably,
by greater than 50% (e.g., from about 50% to about 100%) as
compared to the appropriate control, the control typically being
tumor cells not treated with the antibody, oligopeptide or other
organic molecule being tested. In one embodiment, growth inhibition
can be measured at an antibody concentration of about 0.1 to 30
.mu.g/ml or about 0.5 nM to 200 nM in cell culture, where the
growth inhibition is determined 1-10 days after exposure of the
tumor cells to the antibody. Growth inhibition of tumor cells in
vivo can be determined in various ways such as is described in the
Experimental Examples section below. The antibody is growth
inhibitory in vivo if administration of the anti-TAT antibody at
about 1 .mu.g/kg to about 100 mg/kg body weight results in
reduction in tumor size or tumor cell proliferation within about 5
days to 3 months from the first administration of the antibody,
preferably within about 5 to 30 days.
[0580] An antibody, oligopeptide or other organic molecule which
"induces apoptosis" is one which induces programmed cell death as
determined by binding of annexin V, fragmentation of DNA, cell
shrinkage, dilation of endoplasmic reticulum, cell fragmentation,
and/or formation of membrane vesicles (called apoptotic bodies).
The cell is usually one which overexpresses a TAT polypeptide.
Preferably the cell is a tumor cell, e.g., a prostate, breast,
ovarian, stomach, endometrial, lung, kidney, colon, bladder cell.
Various methods are available for evaluating the cellular events
associated with apoptosis. For example, phosphatidyl serine (PS)
translocation can be measured by annexin binding; DNA fragmentation
can be evaluated through DNA laddering; and nuclear/chromatin
condensation along with DNA fragmentation can be evaluated by any
increase in hypodiploid cells. Preferably, the antibody,
oligopeptide or other organic molecule which induces apoptosis is
one which results in about 2 to 50 fold, preferably about 5 to 50
fold, and most preferably about 10 to 50 fold, induction of annexin
binding relative to untreated cell in an annexin binding assay.
[0581] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: C1q binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g., B cell receptor); and B cell activation.
[0582] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g., Natural
Killer (NIK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are absolutely
required for such killing. The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay, such as
that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be
performed. Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al. (USA) 95:652-656 (1998).
[0583] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native
sequence human FcR. Moreover, a preferred FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron,
Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in
Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer of maternal IgGs to the fetus (Guyer
et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.
24:249 (1994)).
[0584] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The effector cells may be isolated from a
native source, e.g., from blood.
[0585] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[0586] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include, but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include squamous cell cancer (e.g., epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung and squamous carcinoma
of the lung, cancer of the peritoneum, hepatocellular cancer,
gastric or stomach cancer including gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, cancer of the urinary tract,
hepatoma, breast cancer, colon cancer, rectal cancer, colorectal
cancer, endometrial or uterine carcinoma, salivary gland carcinoma,
kidney or renal cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,
melanoma, multiple myeloma and B-cell lymphoma, brain, as well as
head and neck cancer, and associated metastases.
[0587] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer.
[0588] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues.
[0589] An antibody, oligopeptide or other organic molecule which
"induces cell death" is one which causes a viable cell to become
nonviable. The cell is one which expresses a TAT polypeptide,
preferably a cell that overexpresses a TAT polypeptide as compared
to a normal cell of the same tissue type. The TAT polypeptide may
be a transmembrane polypeptide expressed on the surface of a cancer
cell or may be a polypeptide that is produced and secreted by a
cancer cell. Preferably, the cell is a cancer cell, e.g., a breast,
ovarian, stomach, endometrial, salivary gland, lung, kidney, colon,
thyroid, pancreatic or bladder cell. Cell death in vitro may be
determined in the absence of complement and immune effector cells
to distinguish cell death induced by antibody-dependent
cell-mediated cytotoxicity (ADCC) or complement dependent
cytotoxicity (CDC). Thus, the assay for cell death may be performed
using heat inactivated serum (i.e., in the absence of complement)
and in the absence of immune effector cells. To determine whether
the antibody, oligopeptide or other organic molecule is able to
induce cell death, loss of membrane integrity as evaluated by
uptake of propidium iodide (PI), trypan blue (see Moore et al.
Cytotechnology 17:1-11 (1995)) or 7AAD can be assessed relative to
untreated cells. Preferred cell death-inducing antibodies,
oligopeptides or other organic molecules are those which induce PI
uptake in the PI uptake assay in BT474 cells.
[0590] A "TAT-expressing cell" is a cell which expresses an
endogenous or transfected TAT polypeptide either on the cell
surface or in a secreted form. A "TAT-expressing cancer" is a
cancer comprising cells that have a TAT polypeptide present on the
cell surface or that produce and secrete a TAT polypeptide. A
"TAT-expressing cancer" optionally produces sufficient levels of
TAT polypeptide on the surface of cells thereof, such that an
anti-TAT antibody, oligopeptide or other organic molecule can bind
thereto and have a therapeutic effect with respect to the cancer.
In another embodiment, a "TAT-expressing cancer" optionally
produces and secretes sufficient levels of TAT polypeptide, such
that an anti-TAT antibody, oligopeptide or other organic molecule
antagonist can bind thereto and have a therapeutic effect with
respect to the cancer. With regard to the latter, the antagonist
may be an antisense oligonucleotide which reduces, inhibits or
prevents production and secretion of the secreted TAT polypeptide
by tumor cells. A cancer which "overexpresses" a TAT polypeptide is
one which has significantly higher levels of TAT polypeptide at the
cell surface thereof, or produces and secretes, compared to a
noncancerous cell of the same tissue type. Such overexpression may
be caused by gene amplification or by increased transcription or
translation. TAT polypeptide overexpression may be determined in a
diagnostic or prognostic assay by evaluating increased levels of
the TAT protein present on the surface of a cell, or secreted by
the cell (e.g., via an immunohistochemistry assay using anti-TAT
antibodies prepared against an isolated TAT polypeptide which may
be prepared using recombinant DNA technology from an isolated
nucleic acid encoding the TAT polypeptide; FACS analysis, etc.).
Alternatively, or additionally, one may measure levels of TAT
polypeptide-encoding nucleic acid or mRNA in the cell, e.g., via
fluorescent in situ hybridization using a nucleic acid based probe
corresponding to a TAT-encoding nucleic acid or the complement
thereof; (FISH; see WO98/45479 published October, 1998), Southern
blotting, Northern blotting, or polymerase chain reaction (PCR)
techniques, such as real time quantitative PCR (RT-PCR). One may
also study TAT polypeptide overexpression by measuring shed antigen
in a biological fluid such as serum, e.g, using antibody-based
assays (see also, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12,
1990; WO91/05264 published Apr. 18, 1991; U.S. Pat. No. 5,401,638
issued Mar. 28, 1995; and Sias et al., J. Immunol. Methods
132:73-80 (1990)). Aside from the above assays, various in vivo
assays are available to the skilled practitioner. For example, one
may expose cells within the body of the patient to an antibody
which is optionally labeled with a detectable label, e.g., a
radioactive isotope, and binding of the antibody to cells in the
patient can be evaluated, e.g., by external scanning for
radioactivity or by analyzing a biopsy taken from a patient
previously exposed to the antibody.
[0591] As used herein, the term "immunoadhesin" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein (an "adhesin") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with the desired
binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is "heterologous"), and an
immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a contiguous amino acid
sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the
immunoadhesin may be obtained from any immunoglobulin, such as
IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and
IgA-2), IgE, IgD or IgM.
[0592] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the antibody, oligopeptide or other organic molecule so as to
generate a "labeled" antibody, oligopeptide or other organic
molecule. The label may be detectable by itself (e.g. radioisotope
labels or fluorescent labels) or, in the case of an enzymatic
label, may catalyze chemical alteration of a substrate compound or
composition which is detectable.
[0593] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents e.g.
methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, and the various antitumor or anticancer
agents disclosed below. Other cytotoxic agents are described below.
A tumoricidal agent causes destruction of tumor cells.
[0594] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
a TAT-expressing cancer cell, either in vitro or in vivo. Thus, the
growth inhibitory agent may be one which significantly reduces the
percentage of TAT-expressing cells in S phase. Examples of growth
inhibitory agents include agents that block cell cycle progression
(at a place other than S phase), such as agents that induce G1
arrest and M-phase arrest. Classical M-phase blockers include the
vincas (vincristine and vinblastine), taxanes, and topoisomerase II
inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C. Further information can be
found in The Molecular Basis of Cancer, Mendelsohn and Israel,
eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and
antineoplastic drugs" by Murakami et al. (W B Saunders:
Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and
docetaxel) are anticancer drugs both derived from the yew tree.
Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer), derived from the
European yew, is a semisynthetic analogue of paclitaxel
(TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and docetaxel
promote the assembly of microtubules from tubulin dimers and
stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0595] "Doxorubicin" is an anthracycline antibiotic. The full
chemical name of doxorubicin is
(8S-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexapyranosyl)oxy]-7,-
8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-napht-
hacenedione.
[0596] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-.alpha. and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-.beta.; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growth
factor-I and -II; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon -.alpha., -.beta., and -.gamma.;
colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such
as TNF-.alpha. or TNF-.beta.; and other polypeptide factors
including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0597] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products. TABLE-US-00001
TABLE 2 TAT XXXXXXXXXXXXXXX (Length = 15 amino acids) Comparison
XXXXXYYYYYYY (Length = 12 amino Protein acids) % amino acid
sequence identity = (the number of identically matching amino acid
residues between the two polypeptide sequences as determined by
ALIGN-2) divided by (the total number of amino acid residues of the
TAT poly- peptide) = 5 divided by 15 = 33.3%
[0598] TABLE-US-00002 TABLE 3 TAT XXXXXXXXXX (Length = 10 amino
acids) Comparison XXXXXYYYYYYZZYZ (Length = 15 amino Protein acids)
% amino acid sequence identity = (the number of identically
matching amino acid residues between the two polypeptide sequences
as determined by ALIGN-2) divided by (the total number of amino
acid residues of the TAT polypep- tide) = 5 divided by 10 = 50%
[0599] TABLE-US-00003 TABLE 4 TAT-DNA NNNNNNNNNNNNNN (Length = 14
nu- cleotides) Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nu-
cleotides) % nucleic acid sequence identity = (the number of
identically matching nucleotides between the two nucleic acid
sequences as deter- mined by ALIGN-2) divided by (the total number
of nucleotides of the TAT-DNA nucleic acid sequence) = 6 divided by
14 = 42.9%
[0600] TABLE-US-00004 TABLE 5 TAT-DNA NNNNNNNNNNNN (Length = 12
nucle- otides) Comparison DNA NNNNLLLVV (Length = 9 nucle- otides)
% nucleic acid sequence identity = (the number of identically
matching nucleotides between the two nucleic acid sequences as
deter- mined by ALIGN-2) divided by (the total number of
nucleotides of the TAT-DNA nucleic acid sequence) = 4 divided by 12
= 33.3%
II. Compositions and Methods of the Invention
[0601] A. Anti-TAT Antibodies
[0602] In one embodiment, the present invention provides anti-TAT
antibodies which may find use herein as therapeutic and/or
diagnostic agents. Exemplary antibodies include polyclonal,
monoclonal, humanized, bispecific, and heteroconjugate
antibodies.
1. Polyclonal Antibodies
[0603] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen (especially when synthetic peptides are used)
to a protein that is immunogenic in the species to be immunized.
For example, the antigen can be conjugated to keyhole limpet
hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean
trypsin inhibitor, using a bifunctional or derivatizing agent,
e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through
cysteine residues), N-hydroxysuccinimide (through lysine residues),
glutaraldehyde, succinic anhydride, SOCl.sub.2, or
R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are different alkyl
groups.
[0604] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later, the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later, the animals
are bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Conjugates also can be made in
recombinant cell culture as protein fusions. Also, aggregating
agents such as alum are suitably used to enhance the immune
response.
[0605] 2. Monoclonal Antibodies
[0606] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler et al., Nature, 256:495 (1975), or may be
made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
[0607] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as described above to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
After immunization, lymphocytes are isolated and then fused with a
myeloma cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal
Antibodies: Principles and Practice, pp. 59-103 (Academic Press,
1986)).
[0608] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium which medium preferably contains one or
more substances that inhibit the growth or survival of the unfused,
parental myeloma cells (also referred to as fusion partner). For
example, if the parental myeloma cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the selective
culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium), which
substances prevent the growth of HGPRT-deficient cells.
[0609] Preferred fusion partner myeloma cells are those that fuse
efficiently, support stable high-level production of antibody by
the selected antibody-producing cells, and are sensitive to a
selective medium that selects against the unfused parental cells.
Preferred myeloma cell lines are murine myeloma lines, such as
those derived from MOPC-21 and MPC-11 mouse tumors available from
the Salk Institute Cell Distribution Center, San Diego, Calif. USA,
and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the
American Type Culture Collection, Manassas, Va., USA. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, pp. 51-63 (Marcel Dekker,
Inc., New York, 1987)).
[0610] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunosorbent assay
(ELISA).
[0611] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis described in
Munson et al., Anal. Biochem., 107:220 (1980).
[0612] Once hybridoma cells that produce antibodies of the desired
specificity, affinity, and/or activity are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media forthis purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal e.g, by i.p. injection of the cells
into mice.
[0613] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional antibody purification procedures such as, for
example, affinity chromatography (e.g., using protein A or protein
G-Sepharose) or ion-exchange chromatography, hydroxylapatite
chromatography, gel electrophoresis, dialysis, etc.
[0614] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce antibody protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells. Review
articles on recombinant expression in bacteria of DNA encoding the
antibody include Skerra et al., Curr. Opinion in Immunol.,
5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188
(1992).
[0615] In a further embodiment, monoclonal antibodies or antibody
fragments can be isolated from antibody phage libraries generated
using the techniques described in McCafferty et al., Nature,
348:552-554(1990). Clackson et al., Nature, 352:624-628(1991) and
Marks et al., J. Mol. Biol., 222:581-597(1991) describe the
isolation of murine and human antibodies, respectively, using phage
libraries. Subsequent publications describe the production of high
affinity (nM range) human antibodies by chain shuffling (Marks et
al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0616] The DNA that encodes the antibody may be modified to produce
chimeric or fusion antibody polypeptides, for example, by
substituting human heavy chain and light chain constant domain
(C.sub.H and C.sub.L) sequences for the homologous murine sequences
(U.S. Pat. No. 4,816,567; and Morrison, et al., Proc. Natl. Acad.
Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding
sequence with all or part of the coding sequence for a
non-immunoglobulin polypeptide (heterologous polypeptide). The
non-immunoglobulin polypeptide sequences can substitute for the
constant domains of an antibody, or they are substituted for the
variable domains of one antigen-combining site of an antibody to
create a chimeric bivalent antibody comprising one
antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0617] 3. Human and Humanized Antibodies
[0618] The anti-TAT antibodies of the invention may further
comprise humanized antibodies or human antibodies. Humanized forms
of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived
from non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In some instances, Fv framework
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Humanized antibodies may also comprise residues
which are found neither in the recipient antibody nor in the
imported CDR or framework sequences. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)].
[0619] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers [Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0620] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity and HAMA response (human anti-mouse antibody)
when the antibody is intended for human therapeutic use. According
to the so-called "best-fit" method, the sequence of the variable
domain of a rodent antibody is screened against the entire library
of known human variable domain sequences. The human V domain
sequence which is closest to that of the rodent is identified and
the human framework region (FR) within it accepted for the
humanized antibody (Sims et al., J. Immunol. 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chains. The same framework may be used for several different
humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA,
89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).
[0621] It is further important that antibodies be humanized with
retention of high binding affinity for the antigen and other
favorable biological properties. To achieve this goal, according to
a preferred method, humanized antibodies are prepared by a process
of analysis of the parental sequences and various conceptual
humanized products using three-dimensional models of the parental
and humanized sequences. Three-dimensional immunoglobulin models
are commonly available and are familiar to those skilled in the
art. Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0622] Various forms of a humanized anti-TAT antibody are
contemplated. For example, the humanized antibody may be an
antibody fragment, such as a Fab, which is optionally conjugated
with one or more cytotoxic agent(s) in order to generate an
immunoconjugate. Alternatively, the humanized antibody may be an
intact antibody, such as an intact IgG1 antibody.
[0623] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array into such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258(1993); Bruggemann et al., Year in Immuno.
7:33(1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of
GenPharm); U.S. Pat. No. 5,545,807; and WO 97/17852.
[0624] Alternatively, phage display technology (McCafferty et al.,
Nature 348:552-553 [1990]) can be used to produce human antibodies
and antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B-cell. Phage display can be performed in a variety of formats,
reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J.,
Current Opinion in Structural Biology 3:564-571 (1993). Several
sources of V-gene segments can be used for phage display. Clackson
et al., Nature, 352:624-628(1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated essentially following the techniques described by
Marks et al., J. Mol. Biol. 222:581-597(1991), or Griffith et al.,
EMBO J. 12:725-734(1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905.
[0625] As discussed above, human antibodies may also be generated
by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and
5,229,275).
[0626] 4. Antibody Fragments
[0627] In certain circumstances there are advantages of using
antibody fragments, rather than whole antibodies. The smaller size
of the fragments allows for rapid clearance, and may lead to
improved access to solid tumors.
[0628] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments. Antibody fragments can be isolated from
the antibody phage libraries discussed above. Alternatively,
Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Fab and F(ab').sub.2 fragment with increased in
vivo half-life comprising a salvage receptor binding epitope
residues are described in U.S. Pat. No. 5,869,046. Other techniques
for the production of antibody fragments will be apparent to the
skilled practitioner. In other embodiments, the antibody of choice
is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat.
No. 5,571,894; and U.S. Pat. No. 5,587,458. Fv and sFv are the only
species with intact combining sites that are devoid of constant
regions; thus, they are suitable for reduced nonspecific binding
during in vivo use. sFv fusion proteins may be constructed to yield
fusion of an effector protein at either the amino or the carboxy
terminus of an sFv. See Antibody Engineering, ed. Borrebaeck,
supra. The antibody fragment may also be a "linear antibody", e.g.,
as described in U.S. Pat. No. 5,641,870 for example. Such linear
antibody fragments may be monospecific or bispecific.
[0629] 5. Bispecific Antibodies
[0630] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of a TAT
protein as described herein. Other such antibodies may combine a
TAT binding site with a binding site for another protein.
Alternatively, an anti-TAT arm may be combined with an arm which
binds to a triggering molecule on a leukocyte such as a T-cell
receptor molecule (e.g. CD3), or Fc receptors for IgG (Fc.gamma.R),
such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII
(CD16), so as to focus and localize cellular defense mechanisms to
the TAT-expressing cell. Bispecific antibodies may also be used to
localize cytotoxic agents to cells which express TAT. These
antibodies possess a TAT-binding arm and an arm which binds the
cytotoxic agent (e.g., saporin, anti-interferon-.alpha., vinca
alkaloid, ricin A chain, methotrexate or radioactive isotope
hapten). Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g., F(ab').sub.2 bispecific
antibodies).
[0631] WO 96/16673 describes a bispecific
anti-ErbB2/anti-Fc.gamma.RIII antibody and U.S. Pat. No. 5,837,234
discloses a bispecific anti-ErbB2/anti-Fc.gamma.RI antibody. A
bispecific anti-ErbB2/Fc.alpha. antibody is shown in WO98/02463.
U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3
antibody.
[0632] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the co-expression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et at., Nature 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J. 10:3655-3659
(1991).
[0633] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences.
Preferably, the fusion is with an Ig heavy chain constant domain,
comprising at least part of the hinge, C.sub.H2, and C.sub.H3
regions. It is preferred to have the first heavy-chain constant
region (C.sub.H1) containing the site necessary for light chain
bonding, present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host cell. This
provides for greater flexibility in adjusting the mutual
proportions of the three polypeptide fragments in embodiments when
unequal ratios of the three polypeptide chains used in the
construction provide the optimum yield of the desired bispecific
antibody. It is, however, possible to insert the coding sequences
for two or all three polypeptide chains into a single expression
vector when the expression of at least two polypeptide chains in
equal ratios results in high yields or when the ratios have no
significant affect on the yield of the desired chain
combination.
[0634] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology 121:210 (1986).
[0635] According to another approach described in U.S. Pat. No.
5,731,168, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers which are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the C.sub.H3 domain. In this method,
one or more small amino acid side chains from the interface of the
first antibody molecule are replaced with larger side chains (e.g.,
tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to the large side chain(s) are created on the
interface of the second antibody molecule by replacing large amino
acid side chains with smaller ones (e.g., alanine or threonine).
This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as
homodimers.
[0636] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0637] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science 229:81 (1985) describe a procedure
wherein intact antibodies are proteolytically cleaved to generate
F(ab').sub.2 fragments. These fragments are reduced in the presence
of the dithiol complexing agent, sodium arsenite, to stabilize
vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0638] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
ErbB2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets. Various techniques for making and isolating
bispecific antibody fragments directly from recombinant cell
culture have also been described. For example, bispecific
antibodies have been produced using leucine zippers. Kostelny et
al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper
peptides from the Fos and Jun proteins were linked to the Fab'
portions of two different antibodies by gene fusion. The antibody
homodimers were reduced at the hinge region to form monomers and
then re-oxidized to form the antibody heterodimers. This method can
also be utilized for the production of antibody homodimers. The
"diabody" technology described by Hollinger et al., Proc. Natl.
Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative
mechanism for making bispecific antibody fragments. The fragments
comprise a V.sub.H connected to a V.sub.L by a linker which is too
short to allow pairing between the two domains on the same chain.
Accordingly, the V.sub.H and V.sub.L domains of one fragment are
forced to pair with the complementary V.sub.L and V.sub.H domains
of another fragment, thereby forming two antigen-binding sites.
Another strategy for making bispecific antibody fragments by the
use of single-chain Fv (sFv) dimers has also been reported. See
Gruber et al., J. Immunol., 152:5368 (1994).
[0639] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0640] 6. Heteroconjugate Antibodies
[0641] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells [U.S.
Pat. No. 4,676,980], and for treatment of HIV infection [WO
91/00360; WO 92/200373; EP 03089]. It is contemplated that the
antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0642] 7. Multivalent Antibodies
[0643] A multivalent antibody may be internalized (and/or
catabolized) faster than a bivalent antibody by a cell expressing
an antigen to which the antibodies bind. The antibodies of the
present invention can be multivalent antibodies (which are other
than of the IgM class) with three or more antigen binding sites
(e.g. tetravalent antibodies), which can be readily produced by
recombinant expression of nucleic acid encoding the polypeptide
chains of the antibody. The multivalent antibody can comprise a
dimerization domain and three or more antigen binding sites. The
preferred dimerization domain comprises (or consists of) an Fc
region or a hinge region. In this scenario, the antibody will
comprise an Fc region and three or more antigen binding sites
amino-terminal to the Fc region. The preferred multivalent antibody
herein comprises (or consists of) three to about eight, but
preferably four, antigen binding sites. The multivalent antibody
comprises at least one polypeptide chain (and preferably two
polypeptide chains), wherein the polypeptide chain(s) comprise two
or more variable domains. For instance, the polypeptide chain(s)
may comprise VD1-(X1).sub.n-VD2-(X2).sub.n-Fc, wherein VD1 is a
first variable domain, VD2 is a second variable domain, Fc is one
polypeptide chain of an Fc region, X1 and X2 represent an amino
acid or polypeptide, and n is 0 or 1. For instance, the polypeptide
chain(s) may comprise: VH--CH1-flexible linker-VH--CH1-Fc region
chain; or VH--CH1-VH--CH1-Fc region chain. The multivalent antibody
herein preferably further comprises at least two (and preferably
four) light chain variable domain polypeptides. The multivalent
antibody herein may, for instance, comprise from about two to about
eight light chain variable domain polypeptides. The light chain
variable domain polypeptides contemplated here comprise a light
chain variable domain and, optionally, further comprise a CL
domain.
[0644] 8. Effector Function Engineering
[0645] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g., so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.
J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.,
Cancer Research 53:2560-2565(1993). Alternatively, an antibody can
be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design 3:219-230 (1989). To increase the
serum half life of the antibody, one may incorporate a salvage
receptor binding epitope into the antibody (especially an antibody
fragment) as described in U.S. Pat. No. 5,739,277, for example. As
used herein, the term "salvage receptor binding epitope" refers to
an epitope of the Fc region of an IgG molecule (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, or IgG.sub.4) that is responsible for
increasing the in vivo serum half-life of the IgG molecule.
[0646] 9. Immunoconjugates
[0647] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g.,
an enzymatically active toxin of bacterial, fungal, plant, or
animal origin, or fragments thereof), or a radioactive isotope
(i.e., a radio conjugate).
[0648] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re. Conjugates of the antibody and cytotoxic
agent are made using a variety of bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate
(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCL), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutareldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al.,
Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0649] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, a trichothene, and
CC1065, and the derivatives of these toxins that have toxin
activity, are also contemplated herein.
Maytansine and maytansinoids
[0650] In one preferred embodiment, an anti-TAT antibody (full
length or fragments) of the invention is conjugated to one or more
maytansinoid molecules.
[0651] Maytansinoids are mitotic inhibitors which act by inhibiting
tubulin polymerization. Maytansine was first isolated from the east
African shrub Maytenus serrata (U.S. Pat. No. 3,896,111).
Subsequently, it was discovered that certain microbes also produce
maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S.
Pat. No. 4,151,042). Synthetic maytansinol and derivatives and
analogues thereof are disclosed, for example, in U.S. Pat. Nos.
4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757;
4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929;
4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219;
4,450,254; 4,362,663; and 4,371,533, the disclosures of which are
hereby expressly incorporated by reference.
Maytansinoid-Antibody Conjugates
[0652] In an attempt to improve their therapeutic index, maytansine
and maytansinoids have been conjugated to antibodies specifically
binding to tumor cell antigens. Immunoconjugates containing
maytansinoids and their therapeutic use are disclosed, for example,
in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0425
235 B1, the disclosures of which are hereby expressly incorporated
by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described immunoconjugates comprising a maytansinoid
designated DM1 linked to the monoclonal antibody C242 directed
against human colorectal cancer. The conjugate was found to be
highly cytotoxic towards cultured colon cancer cells, and showed
antitumor activity in an in vivo tumor growth assay. Chari et al.,
Cancer Research 52:127-131 (1992) describe immunoconjugates in
which a maytansinoid was conjugated via a disulfide linker to the
murine antibody A7 binding to an antigen on human colon cancer cell
lines, or to another murine monoclonal antibody TA.1 that binds the
HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoid
conjugate was tested in vitro on the human breast cancer cell line
SK-BR-3, which expresses 3.times.10.sup.5 HER-2 surface antigens
per cell. The drug conjugate achieved a degree of cytotoxicity
similar to the free maytansonid drug, which could be increased by
increasing the number of maytansinoid molecules per antibody
molecule. The A7-maytansinoid conjugate showed low systemic
cytotoxicity in mice.
Anti-Tat Polypeptide Antibody-Maytansinoid Conjugates
(Immunoconjugates)
[0653] Anti-TAT antibody-maytansinoid conjugates are prepared by
chemically linking an anti-TAT antibody to a maytansinoid molecule
without significantly diminishing the biological activity of either
the antibody or the maytansinoid molecule. An average of 3-4
maytansinoid molecules conjugated per antibody molecule has shown
efficacy in enhancing cytotoxicity of target cells without
negatively affecting the function or solubility of the antibody,
although even one molecule of toxin/antibody would be expected to
enhance cytotoxicity over the use of naked antibody. Maytansinoids
are well known in the art and can be synthesized by known
techniques or isolated from natural sources. Suitable maytansinoids
are disclosed, for example, in U.S. Pat. No. 5,208,020 and in the
other patents and nonpatent publications referred to hereinabove.
Preferred maytansinoids are maytansinol and maytansinol analogues
modified in the aromatic ring or at other positions of the
maytansinol molecule, such as various maytansinol esters.
[0654] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and
Chari et al., Cancer Research 52:127-131 (1992). The linking groups
include disufide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups, or esterase labile
groups, as disclosed in the above-identified patents, disulfide and
thioether groups being preferred.
[0655] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 [1978]) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage.
[0656] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hyrdoxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. In a
preferred embodiment, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
Calicheamicin
[0657] Another immunoconjugate of interest comprises an anti-TAT
antibody conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,
5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma..sub.1.sup.I, .alpha..sub.2.sup.I,
.alpha..sub.3.sup.I, N-acetyl-.gamma..sub.1.sup.I, PSAG and
.differential..sup.I.sub.1, (Hinman et al., Cancer Research
53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928
(1998) and the aforementioned U.S. patents to American Cyanamid).
Another anti-tumor drug that the antibody can be conjugated is QFA
which is an antifolate. Both calicheamicin and QFA have
intracellular sites of action and do not readily cross the plasma
membrane. Therefore, cellular uptake of these agents through
antibody mediated internalization greatly enhances their cytotoxic
effects.
Other Cytotoxic Agents
[0658] Other antitumor agents that can be conjugated to the
anti-TAT antibodies of the invention include BCNU, streptozoicin,
vincristine and 5-fluorouracil, the family of agents known
collectively LL-E33288 complex described in U.S. Pat. Nos.
5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296).
[0659] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0660] The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0661] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
anti-TAT antibodies. Examples include At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu.
When the conjugate is used for diagnosis, it may comprise a
radioactive atom for scintigraphic studies, for example tc.sup.99m
or I.sup.123, or a spin label for nuclear magnetic resonance (NMR)
imaging (also known as magnetic resonance imaging, mri), such as
iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,
nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[0662] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
tc.sup.99m or I.sup.123, .Re.sup.186, Re.sup.188 and In.sup.111 can
be attached via a cysteine residue in the peptide. Yttrium-90 can
be attached via a lysine residue. The IODOGEN method (Fraker et al
(1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to
incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes other
methods in detail.
[0663] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098(1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat.
No. 5,208,020) may be used.
[0664] Alternatively, a fusion protein comprising the anti-TAT
antibody and cytotoxic agent may be made, e.g., by recombinant
techniques or peptide synthesis. The length of DNA may comprise
respective regions encoding the two portions of the conjugate
either adjacent one another or separated by a region encoding a
linker peptide which does not destroy the desired properties of the
conjugate.
[0665] In yet another embodiment, the antibody may be conjugated to
a "receptor" (such streptavidin) for utilization in tumor
pre-targeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) which is conjugated to
a cytotoxic agent (e.g., a radionucleotide).
[0666] 10. Immunoliposomes
[0667] The anti-TAT antibodies disclosed herein may also be
formulated as immunoliposomes. A "liposome" is a small vesicle
composed of various types of lipids, phospholipids and/or
surfactant which is useful for delivery of a drug to a mammal. The
components of the liposome are commonly arranged in a bilayer
formation, similar to the lipid arrangement of biological
membranes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and
4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
[0668] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al., J. National Cancer Inst.
81(19):1484 (1989).
[0669] B. TAT Binding Oligopeptides
[0670] TAT binding oligopeptides of the present invention are
oligopeptides that bind, preferably specifically, to a TAT
polypeptide as described herein. TAT binding oligopeptides may be
chemically synthesized using known oligopeptide synthesis
methodology or may be prepared and purified using recombinant
technology. TAT binding oligopeptides are usually at least about 5
amino acids in length, alternatively at least about 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, or 100 aminoacids in length or more, wherein such
oligopeptides that are capable of binding, preferably specifically,
to a TAT polypeptide as described herein. TAT binding oligopeptides
may be identified without undue experimentation using well known
techniques. In this regard, it is noted that techniques for
screening oligopeptide libraries for oligopeptides that are capable
of specifically binding to a polypeptide target are well known in
the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871,
4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT
Publication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc.
Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc.
Natl. Acad. Sci. U.S.A., 82:178-182(1985); Geysen et al., in
Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J.
Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol.,
140:611-616(1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad.
Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry,
30:10832; Clackson, T. et al. (1991) Nature, 352:624; Marks, J. D.
et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991)
Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991)
Current Opin. Biotechnol., 2:668).
[0671] In this regard, bacteriophage (phage) display is one well
known technique which allows one to screen large oligopeptide
libraries to identify member(s) of those libraries which are
capable of specifically binding to a polypeptide target. Phage
display is a technique by which variant polypeptides are displayed
as fusion proteins to the coat protein on the surface of
bacteriophage particles (Scott, J. K. and Smith, G. P. (1990)
Science 249: 386). The utility of phage display lies in the fact
that large libraries of selectively randomized protein variants (or
randomly cloned cDNAs) can be rapidly and efficiently sorted for
those sequences that bind to a target molecule with high affinity.
Display of peptide (Cwirla, S. E. et al. (1990) Proc. Natl. Acad.
Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991)
Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352:
624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A.
S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on
phage have been used for screening millions of polypeptides or
oligopeptides for ones with specific binding properties (Smith, G.
P. (1991) Current Opin. Biotechnol., 2:668). Sorting phage
libraries of random mutants requires a strategy for constructing
and propagating a large number of variants, a procedure for
affinity purification using the target receptor, and a means of
evaluating the results of binding enrichments. U.S. Pat. Nos.
5,223,409, 5,403,484, 5,571,689, and 5,663,143.
[0672] Although most phage display methods have used filamentous
phage, lambdoid phage display systems (WO 95/34683; U.S. Pat. No.
5,627,024), T4 phage display systems (Ren, Z-J. et al. (1998) Gene
215:439; Zhu, Z. (1997) CAN 33:534; Jiang, J. et al. (1997) can
128:44380; Ren, Z-J. et al. (1997) CAN 127:215644; Ren, Z-J. (1996)
Protein Sci. 5:1833; Efimov, V. P. et al. (1995) Virus Genes
10:173) and T7 phage display systems (Smith, G. P. and Scott, J. K.
(1993) Methods in Enzymology, 217, 228-257; U.S. Pat. No.
5,766,905) are also known.
[0673] Many other improvements and variations of the basic phage
display concept have now been developed.
[0674] These improvements enhance the ability of display systems to
screen peptide libraries for binding to selected target molecules
and to display functional proteins with the potential of screening
these proteins for desired properties. Combinatorial reaction
devices for phage display reactions have been developed (WO
98/14277) and phage display libraries have been used to analyze and
control bimolecular interactions (WO 98/20169; WO 98/20159) and
properties of constrained helical peptides (WO 98/20036). WO
97/35196 describes a method of isolating an affinity ligand in
which a phage display library is contacted with one solution in
which the ligand will bind to a target molecule and a second
solution in which the affinity ligand will not bind to the target
molecule, to selectively isolate binding ligands. WO 97/46251
describes a method of biopanning a random phage display library
with an affinity purified antibody and then isolating binding
phage, followed by a micropanning process using microplate wells to
isolate high affinity binding phage. The use of Staphlylococcus
aureus protein A as an affinity tag has also been reported (L1 et
al. (1998) Mol Biotech., 9:187). WO 97/47314 describes the use of
substrate subtraction libraries to distinguish enzyme specificities
using a combinatorial library which may be a phage display library.
A method for selecting enzymes suitable for use in detergents using
phage display is described in WO 97/09446. Additional methods of
selecting specific binding proteins are described in U.S. Pat. Nos.
5,498,538, 5,432,018, and WO 98/15833.
[0675] Methods of generating peptide libraries and screening these
libraries are also disclosed in U.S. Pat. Nos. 5,723,286,
5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018,
5,698,426, 5,763,192, and 5,723,323.
[0676] C. TAT Binding Organic Molecules
[0677] TAT binding organic molecules are organic molecules other
than oligopeptides or antibodies as defined herein that bind,
preferably specifically, to a TAT polypeptide as described herein.
TAT binding organic molecules may be identified and chemically
synthesized using known methodology (see, e.g., PCT Publication
Nos. WO00/00823 and WO00/39585). TAT binding organic molecules are
usually less than about 2000 daltons in size, alternatively less
than about 1500, 750, 500, 250 or 200 daltons in size, wherein such
organic molecules that are capable of binding, preferably
specifically, to a TAT polypeptide as described herein may be
identified without undue experimentation using well known
techniques. In this regard, it is noted that techniques for
screening organic molecule libraries for molecules that are capable
of binding to a polypeptide target are well known in the art (see,
e.g., PCT Publication Nos. WO00/00823 and WO00/39585). TAT binding
organic molecules may be, for example, aldehydes, ketones, oximes,
hydrazones, semicarbazones, carbazides, primary amines, secondary
amines, tertiary amines, N-substituted hydrazines, hydrazides,
alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids,
esters, amides, ureas, carbamates, carbonates, ketals, thioketals,
acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides,
alkyl sulfonates, aromatic compounds, heterocyclic compounds,
anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines,
oxazolines, thiazolidines, thiazolines, enamines, sulfonamides,
epoxides, aziridines, isocyanates, sulfonyl chlorides, diazo
compounds, acid chlorides, or the like.
[0678] D. Screening for Anti-TAT Antibodies, TAT Binding
Oligopeptides and TAT Binding Organic Molecules With the Desired
Properties
[0679] Techniques for generating antibodies, oligopeptides and
organic molecules that bind to TAT polypeptides have been described
above. One may further select antibodies, oligopeptides or other
organic molecules with certain biological characteristics, as
desired.
[0680] The growth inhibitory effects of an anti-TAT antibody,
oligopeptide or other organic molecule of the invention may be
assessed by methods known in the art, e.g., using cells which
express a TAT polypeptide either endogenously or following
transfection with the TAT gene. For example, appropriate tumor cell
lines and TAT-transfected cells may treated with an anti-TAT
monoclonal antibody, oligopeptide or other organic molecule of the
invention at various concentrations for a few days (e.g., 2-7) days
and stained with crystal violet or MTT or analyzed by some other
colorimetric assay. Another method of measuring proliferation would
be by comparing .sup.3H-thymidine uptake by the cells treated in
the presence or absence an anti-TAT antibody, TAT binding
oligopeptide or TAT binding organic molecule of the invention.
After treatment, the cells are harvested and the amount of
radioactivity incorporated into the DNA quantitated in a
scintillation counter. Appropriate positive controls include
treatment of a selected cell line with a growth inhibitory antibody
known to inhibit growth of that cell line. Growth inhibition of
tumor cells in vivo can be determined in various ways known in the
art. Preferably, the tumor cell is one that overexpresses a TAT
polypeptide. Preferably, the anti-TAT antibody, TAT binding
oligopeptide or TAT binding organic molecule will inhibit cell
proliferation of a TAT-expressing tumor cell in vitro or in vivo by
about 25-100% compared to the untreated tumor cell, more
preferably, by about 30-100%, and even more preferably by about
50-100% or 70-100%, in one embodiment, at an antibody concentration
of about 0.5 to 30 .mu.g/ml. Growth inhibition can be measured at
an antibody concentration of about 0.5 to 30 .mu.g/ml or about 0.5
nM to 200 nM in cell culture, where the growth inhibition is
determined 1-10 days after exposure of the tumor cells to the
antibody. The antibody is growth inhibitory in vivo if
administration of the anti-TAT antibody at about 1 .mu.g/kg to
about 100 mg/kg body weight results in reduction in tumor size or
reduction of tumor cell proliferation within about 5 days to 3
months from the first administration of the antibody, preferably
within about 5 to 30 days.
[0681] To select for an anti-TAT antibody, TAT binding oligopeptide
or TAT binding organic molecule which induces cell death, loss of
membrane integrity as indicated by, e.g., propidium iodide (PI),
trypan blue or 7AAD uptake may be assessed relative to control. A
PI uptake assay can be performed in the absence of complement and
immune effector cells. TAT polypeptide-expressing tumor cells are
incubated with medium alone or medium containing the appropriate
anti-TAT antibody (e.g, at about 10 .mu.g/ml), TAT binding
oligopeptide or TAT binding organic molecule. The cells are
incubated for a 3 day time period. Following each treatment, cells
are washed and aliquoted into 35 mm strainer-capped 12.times.75
tubes (1 ml per tube, 3 tubes per treatment group) for removal of
cell clumps. Tubes then receive PI (10 .mu.g/ml). Samples may be
analyzed using a FACSCAN.RTM. flow cytometer and FACSCONVERT.RTM.
CellQuest software (Becton Dickinson). Those anti-TAT antibodies,
TAT binding oligopeptides or TAT binding organic molecules that
induce statistically significant levels of cell death as determined
by PI uptake may be selected as cell death-inducing anti-TAT
antibodies, TAT binding oligopeptides or TAT binding organic
molecules.
[0682] To screen for antibodies, oligopeptides or other organic
molecules which bind to an epitope on a TAT polypeptide bound by an
antibody of interest, a routine cross-blocking assay such as that
described in Antibodies, A Laboratory Manual, Cold Spring Harbor
Laboratory, Ed Harlow and David Lane (1988), can be performed. This
assay can be used to determine if a test antibody, oligopeptide or
other organic molecule binds the same site or epitope as a known
anti-TAT antibody. Alternatively, or additionally, epitope mapping
can be performed by methods known in the art. For example, the
antibody sequence can be mutagenized such as by alanine scanning,
to identify contact residues. The mutant antibody is initailly
tested for binding with polyclonal antibody to ensure proper
folding. In a different method, peptides corresponding to different
regions of a TAT polypeptide can be used in competition assays with
the test antibodies or with a test antibody and an antibody with a
characterized or known epitope.
[0683] E. Antibody Dependent Enzyme Mediated Prodrug Therapy
(ADEPT)
[0684] The antibodies of the present invention may also be used in
ADEPT by conjugating the antibody to a prodrug-activating enzyme
which converts a prodrug (e.g., a peptidyl chemotherapeutic agent,
see WO81/01145) to an active anti-cancer drug. See, for example, WO
88/07378 and U.S. Pat. No. 4,975,278.
[0685] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such a way so
as to covert it into its more active, cytotoxic form.
[0686] Enzymes that are useful in the method of this invention
include, but are not limited to, alkaline phosphatase useful for
converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful for converting non-toxic
5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;
proteases, such as serratia protease, thermolysin, subtilisin,
carboxypeptidases and cathepsins (such as cathepsins B and L), that
are useful for converting peptide-containing prodrugs into free
drugs; D-alanylcarboxypeptidases, useful for converting prodrugs
that contain D-amino acid substituents; carbohydrate-cleaving
enzymes such as .beta.-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; .beta.-lactamase
useful for converting drugs derivatized with .beta.-lactams into
free drugs; and penicillin amidases, such as penicillin V amidase
or penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes", can be used
to convert the prodrugs of the invention into free active drugs
(see, e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzyme
conjugates can be prepared as described herein for delivery of the
abzyme to a tumor cell population.
[0687] The enzymes of this invention can be covalently bound to the
anti-TAT antibodies by techniques well known in the art such as the
use of the heterobifunctional crosslinking reagents discussed
above. Alternatively, fusion proteins comprising at least the
antigen binding region of an antibody of the invention linked to at
least a functionally active portion of an enzyme of the invention
can be constructed using recombinant DNA techniques well known in
the art (see, e.g., Neuberger et al., Nature 312:604-608
(1984).
[0688] F. Full-Length TAT Polypeptides
[0689] The present invention also provides newly identified and
isolated nucleotide sequences encoding polypeptides referred to in
the present application as TAT polypeptides. In particular, cDNAs
(partial and full-length) encoding various TAT polypeptides have
been identified and isolated, as disclosed in further detail in the
Examples below.
[0690] As disclosed in the Examples below, various cDNA clones have
been deposited with the ATCC. The actual nucleotide sequences of
those clones can readily be determined by the skilled artisan by
sequencing of the deposited clone using routine methods in the art.
The predicted amino acid sequence can be determined from the
nucleotide sequence using routine skill. For the TAT polypeptides
and encoding nucleic acids described herein, in some cases,
Applicants have identified what is believed to be the reading frame
best identifiable with the sequence information available at the
time.
[0691] G. Anti-TAT Antibody and TAT Polypeptide Variants
[0692] In addition to the anti-TAT antibodies and full-length
native sequence TAT polypeptides described herein, it is
contemplated that anti-TAT antibody and TAT polypeptide variants
can be prepared. Anti-TAT antibody and TAT polypeptide variants can
be prepared by introducing appropriate nucleotide changes into the
encoding DNA, and/or by synthesis of the desired antibody or
polypeptide. Those skilled in the art will appreciate that amino
acid changes may alter post-translational processes of the anti-TAT
antibody or TAT polypeptide, such as changing the number or
position of glycosylation sites or altering the membrane anchoring
characteristics.
[0693] Variations in the anti-TAT antibodies and TAT polypeptides
described herein, can be made, for example, using any of the
techniques and guidelines for conservative and non-conservative
mutations set forth, for instance, in U.S. Pat. No. 5,364,934.
Variations may be a substitution, deletion or insertion of one or
more codons encoding the antibody or polypeptide that results in a
change in the amino acid sequence as compared with the native
sequence antibody or polypeptide. Optionally the variation is by
substitution of at least one amino acid with any other amino acid
in one or more of the domains of the anti-TAT antibody or TAT
polypeptide. Guidance in determining which amino acid residue may
be inserted, substituted or deleted without adversely affecting the
desired activity may be found by comparing the sequence of the
anti-TAT antibody or TAT polypeptide with that of homologous known
protein molecules and minimizing the number of amino acid sequence
changes made in regions of high homology. Amino acid substitutions
can be the result of replacing one amino acid with another amino
acid having similar structural and/or chemical properties, such as
the replacement of a leucine with a serine, i.e., conservative
amino acid replacements. Insertions or deletions may optionally be
in the range of about 1 to 5 amino acids. The variation allowed may
be determined by systematically making insertions, deletions or
substitutions of amino acids in the sequence and testing the
resulting variants for activity exhibited by the full-length or
mature native sequence.
[0694] Anti-TAT antibody and TAT polypeptide fragments are provided
herein. Such fragments may be truncated at the N-terminus or
C-terminus, or may lack internal residues, for example, when
compared with a full length native antibody or protein. Certain
fragments lack amino acid residues that are not essential for a
desired biological activity of the anti-TAT antibody or TAT
polypeptide.
[0695] Anti-TAT antibody and TAT polypeptide fragments may be
prepared by any of a number of conventional techniques. Desired
peptide fragments may be chemically synthesized. An alternative
approach involves generating antibody or polypeptide fragments by
enzymatic digestion, e.g., by treating the protein with an enzyme
known to cleave proteins at sites defined by particular amino acid
residues, or by digesting the DNA with suitable restriction enzymes
and isolating the desired fragment. Yet another suitable technique
involves isolating and amplifying a DNA fragment encoding a desired
antibody or polypeptide fragment, by polymerase chain reaction
(PCR). Oligonucleotides that define the desired termini of the DNA
fragment are employed at the 5' and 3' primers in the PCR.
Preferably, anti-TAT antibody and TAT polypeptide fragments share
at least one biological and/or immunological activity with the
native anti-TAT antibody or TAT polypeptide disclosed herein.
[0696] In particular embodiments, conservative substitutions of
interest are shown in Table 6 under the heading of preferred
substitutions. If such substitutions result in a change in
biological activity, then more substantial changes, denominated
exemplary substitutions in Table 6, or as further described below
in reference to amino acid classes, are introduced and the products
screened. TABLE-US-00005 TABLE 6 Original Exemplary Preferred
Residue Substitutions Substitutions Ala (A) val; leu; ile val Arg
(R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu
glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro;
ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala;
leu phe; norleucine Leu (L) norleucine; ile; val; ile met; ala; phe
Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu;
val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser
ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V)
ile; leu; met; phe; leu ala; norleucine
[0697] Substantial modifications in function or immunological
identity of the anti-TAT antibody or TAT polypeptide are
accomplished by selecting substitutions that differ significantly
in their effect on maintaining (a) the structure of the polypeptide
backbone in the area of the substitution, for example, as a sheet
or helical conformation, (b) the charge or hydrophobicity of the
molecule at the target site, or (c) the bulk of the side chain.
Naturally occurring residues are divided into groups based on
common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
[0698] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, more preferably, into the remaining (non-conserved)
sites.
[0699] The variations can be made using methods known in the art
such as oligonucleotide-mediated (site-directed) mutagenesis,
alanine scanning, and PCR mutagenesis. Site-directed mutagenesis
[Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al.,
Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et
al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells
et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or
other known techniques can be performed on the cloned DNA to
produce the anti-TAT antibody or TAT polypeptide variant DNA.
[0700] Scanning amino acid analysis can also be employed to
identify one or more amino acids along a contiguous sequence. Among
the preferred scanning amino acids are relatively small, neutral
amino acids. Such amino acids include alanine, glycine, serine, and
cysteine. Alanine is typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the
beta-carbon and is less likely to alter the main-chain conformation
of the variant [Cunningham and Wells, Science, 244:1081-1085
(1989)]. Alanine is also typically preferred because it is the most
common amino acid. Further, it is frequently found in both buried
and exposed positions [Creighton, The Proteins, (W.H. Freeman &
Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine
substitution does not yield adequate amounts of variant, an
isoteric amino acid can be used.
[0701] Any cysteine residue not involved in maintaining the proper
conformation of the anti-TAT antibody or TAT polypeptide also may
be substituted, generally with serine, to improve the oxidative
stability of the molecule and prevent aberrant crosslinking.
Conversely, cysteine bond(s) may be added to the anti-TAT antibody
or TAT polypeptide to improve its stability (particularly where the
antibody is an antibody fragment such as an Fv fragment).
[0702] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody (e.g., a humanized or human antibody). Generally,
the resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g., 6-7
sites) are mutated to generate all possible amino substitutions at
each site. The antibody variants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g., binding affinity) as herein disclosed. In order to
identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identify
hypervariable region residues contributing significantly to antigen
binding. Alternatively, or additionally, it may be beneficial to
analyze a crystal structure of the antigen-antibody complex to
identify contact points between the antibody and human TAT
polypeptide. Such contact residues and neighboring residues are
candidates for substitution according to the techniques elaborated
herein. Once such variants are generated, the panel of variants is
subjected to screening as described herein and antibodies with
superior properties in one or more relevant assays may be selected
for further development.
[0703] Nucleic acid molecules encoding amino acid sequence variants
of the anti-TAT antibody are prepared by a variety of methods known
in the art. These methods include, but are not limited to,
isolation from a natural source (in the case of naturally occurring
amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of the anti-TAT antibody.
[0704] H. Modifications of Anti-TAT Antibodies and TAT
Polypeptides
[0705] Covalent modifications of anti-TAT antibodies and TAT
polypeptides are included within the scope of this invention. One
type of covalent modification includes reacting targeted amino acid
residues of an anti-TAT antibody or TAT polypeptide with an organic
derivatizing agent that is capable of reacting with selected side
chains or the N- or C-terminal residues of the anti-TAT antibody or
TAT polypeptide. Derivatization with bifunctional agents is useful,
for instance, for crosslinking anti-TAT antibody or TAT polypeptide
to a water-insoluble support matrix or surface for use in the
method for purifying anti-TAT antibodies, and vice-versa. Commonly
used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0706] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .alpha.-amino groups of lysine, arginine, and
histidine side chains [T. E. Creighton, Proteins: Structure and
Molecular Properties, W.H. Freeman & Co., San Francisco, pp.
79-86 (1983)], acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
[0707] Another type of covalent modification of the anti-TAT
antibody or TAT polypeptide included within the scope of this
invention comprises altering the native glycosylation pattern of
the antibody or polypeptide. "Altering the native glycosylation
pattern" is intended for purposes herein to mean deleting one or
more carbohydrate moieties found in native sequence anti-TAT
antibody or TAT polypeptide (either by removing the underlying
glycosylation site or by deleting the glycosylation by chemical
and/or enzymatic means), and/or adding one or more glycosylation
sites that are not present in the native sequence anti-TAT antibody
or TAT polypeptide. In addition, the phrase includes qualitative
changes in the glycosylation of the native proteins, involving a
change in the nature and proportions of the various carbohydrate
moieties present.
[0708] Glycosylation of antibodies and other polypeptides is
typically either N-linked or O-linked. N-linked refers to the
attachment of the carbohydrate moiety to the side chain of an
asparagine residue. The tripeptide sequences asparagine-X-serine
and asparagine-X-threonine, where X is any amino acid except
proline, are the recognition sequences for enzymatic attachment of
the carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars N-aceylgalactosamine,
galactose, or xylose to a hydroxyamino acid, most commonly serine
or threonine, although 5-hydroxyproline or 5-hydroxylysine may also
be used.
[0709] Addition of glycosylation sites to the anti-TAT antibody or
TAT polypeptide is conveniently accomplished by altering the amino
acid sequence such that it contains one or more of the
above-described tripeptide sequences (for N-linked glycosylation
sites). The alteration may also be made by the addition of, or
substitution by, one or more serine or threonine residues to the
sequence of the original anti-TAT antibody or TAT polypeptide (for
O-linked glycosylation sites). The anti-TAT antibody or TAT
polypeptide amino acid sequence may optionally be altered through
changes at the DNA level, particularly by mutating the DNA encoding
the anti-TAT antibody or TAT polypeptide at preselected bases such
that codons are generated that will translate into the desired
amino acids.
[0710] Another means of increasing the number of carbohydrate
moieties on the anti-TAT antibody or TAT polypeptide is by chemical
or enzymatic coupling of glycosides to the polypeptide. Such
methods are described in the art, e.g., in WO 87/05330 published 11
Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp.
259-306 (1981).
[0711] Removal of carbohydrate moieties present on the anti-TAT
antibody or TAT polypeptide may be accomplished chemically or
enzymatically or by mutational substitution of codons encoding for
amino acid residues that serve as targets for glycosylation.
Chemical deglycosylation techniques are known in the art and
described, for instance, by Hakimuddin, et al., Arch. Biochem.
Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131
(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides
can be achieved by the use of a variety of endo- and
exo-glycosidases as described by Thotakura et al., Meth. Enzymol.,
138:350 (1987).
[0712] Another type of covalent modification of anti-TAT antibody
or TAT polypeptide comprises linking the antibody or polypeptide to
one of a variety of nonproteinaceous polymers, e.g., polyethylene
glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the
manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337. The antibody or polypeptide also
may be entrapped in microcapsules prepared, for example, by
coacervation techniques or by interfacial polymerization (for
example, hydroxymethylcellulose or gelatin-microcapsules and
poly-(methylmethacylate) microcapsules, respectively), in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules), or
in macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).
[0713] The anti-TAT antibody or TAT polypeptide of the present
invention may also be modified in a way to form chimeric molecules
comprising an anti-TAT antibody or TAT polypeptide fused to
another, heterologous polypeptide or amino acid sequence.
[0714] In one embodiment, such a chimeric molecule comprises a
fusion of the anti-TAT antibody or TAT polypeptide with a tag
polypeptide which provides an epitope to which an anti-tag antibody
can selectively bind. The epitope tag is generally placed at the
amino- or carboxyl-terminus of the anti-TAT antibody or TAT
polypeptide. The presence of such epitope-tagged forms of the
anti-TAT antibody or TAT polypeptide can be detected using an
antibody against the tag polypeptide. Also, provision of the
epitope tag enables the anti-TAT antibody or TAT polypeptide to be
readily purified by affinity purification using an anti-tag
antibody or another type of affinity matrix that binds to the
epitope tag. Various tag polypeptides and their respective
antibodies are well known in the art. Examples include
poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly)
tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et
al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the
8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al.,
Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes
Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et
al., Protein Engineering, 3(6):547-553 (1990)]. Other tag
polypeptides include the Flag-peptide [Hopp et al., BioTechnology,
6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al.,
Science, 255:192-194 (1992)]; an .alpha.-tubulin epitope peptide
[Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the
T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl.
Acad. Sci. USA, 87:6393-6397 (1990)].
[0715] In an alternative embodiment, the chimeric molecule may
comprise a fusion of the anti-TAT antibody or TAT polypeptide with
an immunoglobulin or a particular region of an immunoglobulin. For
a bivalent form of the chimeric molecule (also referred to as an
"immunoadhesin"), such a fusion could be to the Fc region of an IgG
molecule. The Ig fusions preferably include the substitution of a
soluble (transmembrane domain deleted or inactivated) form of an
anti-TAT antibody or TAT polypeptide in place of at least one
variable region within an Ig molecule. In a particularly preferred
embodiment, the immunoglobulin fusion includes the hinge, CH.sub.2
and CH.sub.3, or the hinge, CH.sub.1, CH.sub.2 and CH.sub.3 regions
of an IgG1 molecule. For the production of immunoglobulin fusions
see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0716] I. Preparation of Anti-TAT Antibodies and TAT
Polypeptides
[0717] The description below relates primarily to production of
anti-TAT antibodies and TAT polypeptides by culturing cells
transformed or transfected with a vector containing anti-TAT
antibody- and TAT polypeptide-encoding nucleic acid. It is, of
course, contemplated that alternative methods, which are well known
in the art, may be employed to prepare anti-TAT antibodies and TAT
polypeptides. For instance, the appropriate amino acid sequence, or
portions thereof, may be produced by direct peptide synthesis using
solid-phase techniques [see, e.g., Stewart et al., Solid-Phase
Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969);
Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro
protein synthesis may be performed using manual techniques or by
automation. Automated synthesis may be accomplished, for instance,
using an Applied Biosystems Peptide Synthesizer (Foster City,
Calif.) using manufacturer's instructions. Various portions of the
anti-TAT antibody or TAT polypeptide may be chemically synthesized
separately and combined using chemical or enzymatic methods to
produce the desired anti-TAT antibody or TAT polypeptide.
[0718] 1. Isolation of DNA Encoding Anti-TAT Antibody or TAT
Polypeptide
[0719] DNA encoding anti-TAT antibody or TAT polypeptide may be
obtained from a cDNA library prepared from tissue believed to
possess the anti-TAT antibody or TAT polypeptide mRNA and to
express it at a detectable level. Accordingly, human anti-TAT
antibody or TAT polypeptide DNA can be conveniently obtained from a
cDNA library prepared from human tissue. The anti-TAT antibody- or
TAT polypeptide-encoding gene may also be obtained from a genomic
library or by known synthetic procedures (e.g., automated nucleic
acid synthesis).
[0720] Libraries can be screened with probes (such as
oligonucleotides of at least about 20-80 bases) designed to
identify the gene of interest or the protein encoded by it.
Screening the cDNA or genomic library with the selected probe may
be conducted using standard procedures, such as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual (New York:
Cold Spring Harbor Laboratory Press, 1989). An alternative means to
isolate the gene encoding anti-TAT antibody or TAT polypeptide is
to use PCR methodology [Sambrook et al., supra; Dieffenbach et al.,
PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory
Press, 1995)].
[0721] Techniques for screening a cDNA library are well known in
the art. The oligonucleotide sequences selected as probes should be
of sufficient length and sufficiently unambiguous that false
positives are minimized. The oligonucleotide is preferably labeled
such that it can be detected upon hybridization to DNA in the
library being screened. Methods of labeling are well known in the
art, and include the use of radiolabels like .sup.32P-labeled ATP,
biotinylation or enzyme labeling. Hybridization conditions,
including moderate stringency and high stringency, are provided in
Sambrook et al., supra.
[0722] Sequences identified in such library screening methods can
be compared and aligned to other known sequences deposited and
available in public databases such as GenBank or other private
sequence databases. Sequence identity (at either the amino acid or
nucleotide level) within defined regions of the molecule or across
the full-length sequence can be determined using methods known in
the art and as described herein.
[0723] Nucleic acid having protein coding sequence may be obtained
by screening selected cDNA or genomic libraries using the deduced
amino acid sequence disclosed herein for the first time, and, if
necessary, using conventional primer extension procedures as
described in Sambrook et al., supra, to detect precursors and
processing intermediates of mRNA that may not have been
reverse-transcribed into cDNA.
[0724] 2. Selection and Transformation of Host Cells
[0725] Host cells are transfected or transformed with expression or
cloning vectors described herein for anti-TAT antibody or TAT
polypeptide production and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences. The culture conditions, such as media, temperature, pH
and the like, can be selected by the skilled artisan without undue
experimentation. In general, principles, protocols, and practical
techniques for maximizing the productivity of cell cultures can be
found in Mammalian Cell Biotechnology: a Practical Approach, M.
Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.
[0726] Methods of eukaryotic cell transfection and prokaryotic cell
transformation are known to the ordinarily skilled artisan, for
example, CaCl.sub.2, CaPO.sub.4, liposome-mediated and
electroporation. Depending on the host cell used, transformation is
performed using standard techniques appropriate to such cells. The
calcium treatment employing calcium chloride, as described in
Sambrook et al., supra, or electroporation is generally used for
prokaryotes. Infection with Agrobacterium tumefaciens is used for
transformation of certain plant cells, as described by Shaw et al.,
Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For
mammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology, 52:456-457
(1978) can be employed. General aspects of mammalian cell host
system transfections have been described in U.S. Pat. No.
4,399,216. Transformations into yeast are typically carried out
according to the method of Van Solingen et al., J Bact., 130:946
(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829
(1979). However, other methods for introducing DNA into cells, such
as by nuclear microinjection, electroporation, bacterial protoplast
fusion with intact cells, or polycations, e.g., polybrene,
polyornithine, may also be used. For various techniques for
transforming mammalian cells, see Keown et al., Methods in
Enzymology, 185:527-537 (1990) and Mansour et al., Nature,
336:348-352 (1988).
[0727] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as E. coli. Various E. coli
strains are publicly available, such as E. coli K12 strain MM294
(ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110
(ATCC 27,325) and K5772 (ATCC 53,635). Other suitable prokaryotic
host cells include Enterobacteriaceae such as Escherichia, e.g., E.
coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. These examples are illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host
because it is a common host strain for recombinant DNA product
fermentations. Preferably, the host cell secretes minimal amounts
of proteolytic enzymes. For example, strain W3110 may be modified
to effect a genetic mutation in the genes encoding proteins
endogenous to the host, with examples of such hosts including E.
coli W3110 strain 1A2, which has the complete genotype tonA; E.
coli W3110 strain 9E4, which has the complete genotype tonA ptr3;
E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete
genotype tonA ptr3phoA E15 (argF-lac) 169 degP ompT kan.sup.r; E.
coli W3110 strain 37D6, which has the complete genotype tonA ptr3
phoA E15 (argF-lac) 169 degP ompT rbs7 ilvG kan.sup.r; E. coli
W3110 strain 40B4, which is strain 37D6 with a non-kanamycin
resistant degP deletion mutation; and an E. coli strain having
mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783
issued 7 Aug. 1990. Alternatively, in vitro methods of cloning,
e.g., PCR or other nucleic acid polymerase reactions, are
suitable.
[0728] Full length antibody, antibody fragments, and antibody
fusion proteins can be produced in bacteria, in particular when
glycosylation and Fc effector function are not needed, such as when
the therapeutic antibody is conjugated to a cytotoxic agent (e.g.,
a toxin) and the immunoconjugate by itself shows effectiveness in
tumor cell destruction. Full length antibodies have greater half
life in circulation. Production in E. coli is faster and more cost
efficient. For expression of antibody fragments and polypeptides in
bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S.
Pat. No. 5,789,199 (Joly et al.), and U.S. Pat. No. 5,840,523
(Simmons et al.) which describes translation initiation regio (TIR)
and signal sequences for optimizing expression and secretion, these
patents incorporated herein by reference. After expression, the
antibody is isolated from the E. coli cell paste in a soluble
fraction and can be purified through, e.g., a protein A or G column
depending on the isotype. Final purification can be carried out
similar to the process for purifying antibody expressed e.g, in CHO
cells.
[0729] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for anti-TAT antibody- or TAT polypeptide-encoding vectors.
Saccharomyces cerevisiae is a commonly used lower eukaryotic host
microorganism. Others include Schizosaccharomyces pombe (Beach and
Nurse, Nature, 290: 140 [1981]; EP 139,383 published 2 May 1985);
Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,
Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis
(MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol.,
154(2):737-742 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus
(ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC
56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,
Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus;
yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et
al., J. Basic Microbiol., 28:265-278 [1988]); Candida, Trichoderma
reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl.
Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as
Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990);
and filamentous fungi such as, e.g., Neurospora, Penicillium,
Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus
hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res.
Commun., 112:284-289 [1983]; Tilburn et al., Gene, 26:205-221
[1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474
[1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]).
Methylotropic yeasts are suitable herein and include, but are not
limited to, yeast capable of growth on methanol selected from the
genera consisting of Hansenula, Candida, Kloeckera, Pichia,
Saccharomyces, Torulopsis, and Rhodotorula. A list of specific
species that are exemplary of this class of yeasts may be found in
C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).
[0730] Suitable host cells for the expression of glycosylated
anti-TAT antibody or TAT polypeptide are derived from multicellular
organisms. Examples of invertebrate cells include insect cells such
as Drosophila S2 and Spodoptera Sf9, as well as plant cells, such
as cell cultures of cotton, corn, potato, soybean, petunia, tomato,
and tobacco. Numerous baculoviral strains and variants and
corresponding permissive insect host cells from hosts such as
Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito),
Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly),
and Bombyx mori have been identified. A variety of viral strains
for transfection are publicly available, e.g., the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses may be used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells.
[0731] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7,
ATCCCRL 1651); human embryonic kidney line (293 or 293 cells
subcloned for growth in suspension culture, Graham et al., J. Gen
Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL
70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);
human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney
cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC
CRL 1442); human lung cells (Wi 38, ATCC CCL 75); human liver cells
(Hep G2, BB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);
TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982));
MRC5 cells; FS4 cells; and ahumanhepatoma line (Hep G2).
[0732] Host cells are transformed with the above-described
expression or cloning vectors for anti-TAT antibody or TAT
polypeptide production and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
[0733] 3. Selection and Use of a Replicable Vector
[0734] The nucleic acid (e.g., cDNA or genomic DNA) encoding
anti-TAT antibody or TAT polypeptide may be inserted into a
replicable vector for cloning (amplification of the DNA) or for
expression. Various vectors are publicly available. The vector may,
for example, be in the form of a plasmid, cosmid, viral particle,
or phage. The appropriate nucleic acid sequence may be inserted
into the vector by a variety of procedures. In general, DNA is
inserted into an appropriate restriction endonuclease site(s) using
techniques known in the art. Vector components generally include,
but are not limited to, one or more of a signal sequence, an origin
of replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence. Construction of
suitable vectors containing one or more of these components employs
standard ligation techniques which are known to the skilled
artisan.
[0735] The TAT may be produced recombinantly not only directly, but
also as a fusion polypeptide with a heterologous polypeptide, which
may be a signal sequence or other polypeptide having a specific
cleavage site at the N-terminus of the mature protein or
polypeptide. In general, the signal sequence may be a component of
the vector, or it may be a part of the anti-TAT antibody- or TAT
polypeptide-encoding DNA that is inserted into the vector. The
signal sequence may be a prokaryotic signal sequence selected, for
example, from the group of the alkaline phosphatase, penicillinase,
lpp, or heat-stable enterotoxin II leaders. For yeast secretion the
signal sequence may be, e.g., the yeast invertase leader, alpha
factor leader (including Saccharomyces and Kluyveromyces
.alpha.-factor leaders, the latter described in U.S. Pat. No.
5,010,182), or acid phosphatase leader, the C. albicans
glucoamylase leader (EP 362,179 published 4 Apr. 1990), or the
signal described in WO 90/13646 published 15 Nov. 1990. In
mammalian cell expression, mammalian signal sequences may be used
to direct secretion of the protein, such as signal sequences from
secreted polypeptides of the same or related species, as well as
viral secretory leaders.
[0736] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria, the
2.mu. plasmid origin is suitable for yeast, and various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells.
[0737] Expression and cloning vectors will typically contain a
selection gene, also termed a selectable marker.
[0738] Typical selection genes encode proteins that (a) confer
resistance to antibiotics or other toxins, e.g., ampicillin,
neomycin, methotrexate, or tetracycline, (b) complement auxotrophic
deficiencies, or (c) supply critical nutrients not available from
complex media, e.g., the gene encoding D-alanine racemase for
Bacilli.
[0739] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the anti-TAT antibody- or TAT polypeptide-encoding
nucleic acid, such as DHFR or thymidine kinase. An appropriate host
cell when wild-type DHFR is employed is the CHO cell line deficient
in DHFR activity, prepared and propagated as described by Urlaub et
al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable
selection gene for use in yeast is the trp1 gene present in the
yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979);
Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:
157 (1980)]. The trp1 gene provides a selection marker for a mutant
strain of yeast lacking the ability to grow in tryptophan, for
example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12
(1977)].
[0740] Expression and cloning vectors usually contain a promoter
operably linked to the anti-TAT antibody- or TAT
polypeptide-encoding nucleic acid sequence to direct mRNA
synthesis. Promoters recognized by a variety of potential host
cells are well known. Promoters suitable for use with prokaryotic
hosts include the .beta.-lactamase and lactose promoter systems
[Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature,
281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter
system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and
hybrid promoters such as the tac promoter [deBoer et al., Proc.
Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in
bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA encoding anti-TAT antibody or
TAT polypeptide.
[0741] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman
et al., J. Biol. Chem., 255:2073 (1980)] or otherglycolytic enzymes
[Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland,
Biochemistry, 17:4900 (1978)], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0742] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657.
[0743] Anti-TAT antibody or TAT polypeptide transcription from
vectors in mammalian host cells is controlled, for example, by
promoters obtained from the genomes of viruses such as polyoma
virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), from heterologous mammalian promoters,
e.g., the actin promoter or an immunoglobulin promoter, and from
heat-shock promoters, provided such promoters are compatible with
the host cell systems.
[0744] Transcription of a DNA encoding the anti-TAT antibody or TAT
polypeptide by higher eukaryotes may be 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. Many enhancer sequences are
now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. The enhancer may be spliced into the vector at a
position 5' or 3' to the anti-TAT antibody or TAT polypeptide
coding sequence, but is preferably located at a site 5' from the
promoter.
[0745] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding anti-TAT
antibody or TAT polypeptide.
[0746] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of anti-TAT antibody or TAT polypeptide
in recombinant vertebrate cell culture are described in Gething et
al., Nature, 293:620-625 (1981); Mantei et al., Nature,
281:40-46(1979); EP 117,060; and EP 117,058.
[0747] 4. Culturing the Host Cells
[0748] The host cells used to produce the anti-TAT antibody or TAT
polypeptide of this invention may be cultured in a variety of
media. Commercially available media such as Ham's F10 (Sigma),
Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and
Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for
culturing the host cells. In addition, any of the media described
in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.
Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;
4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or
U.S. Pat. Re. No. 30,985 may be used as culture media for the host
cells. Any of these media may be supplemented as necessary with
hormones and/or other growth factors (such as insulin, transferrin,
or epidermal growth factor), salts (such as sodium chloride,
calcium, magnesium, and phosphate), buffers (such as HEPES),
nucleotides (such as adenosine and thymidine), antibiotics (such as
GENTAMYCIN.TM. drug), trace elements (defined as inorganic
compounds usually present at final concentrations in the micromolar
range), and glucose or an equivalent energy source. Any other
necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. 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 the ordinarily skilled artisan.
[0749] 5. Detecting Gene Amplification/Expression
[0750] Gene amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
Northern blotting to quantitate the transcription of mRNA [Thomas,
Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Alternatively,
antibodies may be employed that can recognize specific duplexes,
including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and
the assay may be carried out where the duplex is bound to a
surface, so that upon the formation of duplex on the surface, the
presence of antibody bound to the duplex can be detected.
[0751] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene product. Antibodies
useful for immunohistochemical staining and/or assay of sample
fluids may be either monoclonal or polyclonal, and may be prepared
in any mammal. Conveniently, the antibodies may be prepared against
a native sequence TAT polypeptide or against a synthetic peptide
based on the DNA sequences provided herein or against exogenous
sequence fused to TAT DNA and encoding a specific antibody
epitope.
[0752] 6. Purification of Anti-TAT Antibody and TAT Polypeptide
[0753] Forms of anti-TAT antibody and TAT polypeptide may be
recovered from culture medium or from host cell lysates. If
membrane-bound, it can be released from the membrane using a
suitable detergent solution (e.g. Triton-X 100) or by enzymatic
cleavage. Cells employed in expression of anti-TAT antibody and TAT
polypeptide can be disrupted by various physical or chemical means,
such as freeze-thaw cycling, sonication, mechanical disruption, or
cell lysing agents.
[0754] It may be desired to purify anti-TAT antibody and TAT
polypeptide from recombinant cell proteins or polypeptides. The
following procedures are exemplary of suitable purification
procedures: by fractionation on an ion-exchange column; ethanol
precipitation; reverse phase HPLC; chromatography on silica or on a
cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;
ammonium sulfate precipitation; gel filtration using, for example,
Sephadex G-75; protein A Sepharose columns to remove contaminants
such as IgG; and metal chelating columns to bind epitope-tagged
forms of the anti-TAT antibody and TAT polypeptide. Various methods
of protein purification may be employed and such methods are known
in the art and described for example in Deutscher, Methods in
Enzymology, 182 (1990); Scopes, Protein Purification: Principles
and Practice, Springer-Verlag, New York (1982). The purification
step(s) selected will depend, for example, on the nature of the
production process used and the particular anti-TAT antibody or TAT
polypeptide produced.
[0755] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, are removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology
10:163-167 (1992) describe a procedure for isolating antibodies
which are secreted to the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can be removed by centrifugation. Where the antibody is
secreted into the medium, supernatants from such expression systems
are generally first concentrated using a commercially available
protein concentration filter, for example, an Amicon or Millipore
Pellicon ultrafiltration unit. A protease inhibitor such as PMSF
may be included in any of the foregoing steps to inhibit
proteolysis and antibiotics may be included to prevent the growth
of adventitious contaminants.
[0756] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2 or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a C.sub.H3 domain, the Bakerbond
ABX.TM. resin (J. T. Baker, Phillipsburg, N.J.) is useful for
purification. Other techniques for protein purification such as
fractionation on an ion-exchange column, ethanol precipitation,
Reverse Phase HPLC, chromatography on silica, chromatography on
heparin SEPHAROSE.TM. chromatography on an anion or cation exchange
resin (such as a polyaspartic acid column), chromatofocusing,
SDS-PAGE, and ammonium sulfate precipitation are also available
depending on the antibody to be recovered.
[0757] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25M salt).
[0758] J. Pharmaceutical Formulations
[0759] Therapeutic formulations of the anti-TAT antibodies, TAT
binding oligopeptides, TAT binding organic molecules and/or TAT
polypeptides used in accordance with the present invention are
prepared for storage by mixing the antibody, polypeptide,
oligopeptide or organic molecule having the desired degree of
purity with optional pharmaceutically acceptable carriers,
excipients or stabilizers (Remington's Pharmaceutical Sciences 16th
edition, Osol, A. Ed. (1980)), in the form of lyophilized
formulations or aqueous solutions. Acceptable carriers, excipients,
or stabilizers are nontoxic to recipients at the dosages and
concentrations employed, and include buffers such as acetate, Tris,
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
tonicifiers such as trehalose and sodium chloride; sugars such as
sucrose, mannitol, trehalose or sorbitol; surfactant such as
polysorbate; salt-forming counter-ions such as sodium; metal
complexes (e.g., Zn-protein complexes); and/or non-ionic
surfactants such as TWEEN.RTM., PLURONICS.RTM. or polyethylene
glycol (PEG). The antibody preferably comprises the antibody at a
concentration of between 5-200 mg/ml, preferably between 10-100
mg/ml.
[0760] The formulations herein may also contain more than one
active compound as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. For example, in addition to an
anti-TAT antibody, TAT binding oligopeptide, or TAT binding organic
molecule, it may be desirable to include in the one formulation, an
additional antibody, e.g., a second anti-TAT antibody which binds a
different epitope on the TAT polypeptide, or an antibody to some
other target such as a growth factor that affects the growth of the
particular cancer. Alternatively, or additionally, the composition
may further comprise a chemotherapeutic agent, cytotoxic agent,
cytokine, growth inhibitory agent, anti-hormonal agent, and/or
cardioprotectant. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0761] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences,
16th edition, Osol, A. Ed. (1980).
[0762] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi-permeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.RTM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0763] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0764] K. Diagnosis and Treatment with Anti-TAT Antibodies, TAT
Binding Oligopeptides and TAT Binding Organic Molecules
[0765] To determine TAT expression in the cancer, various
diagnostic assays are available. In one embodiment, TAT polypeptide
overexpression may be analyzed by immunohistochemistry (IHC).
Parrafin embedded tissue sections from a tumor biopsy may be
subjected to the IHC assay and accorded a TAT protein staining
intensity criteria as follows:
[0766] Score 0--no staining is observed or membrane staining is
observed in less than 10% of tumor cells.
[0767] Score 1+--a faint/barely perceptible membrane staining is
detected in more than 10% of the tumor cells. The cells are only
stained in part of their membrane.
[0768] Score 2+--a weak to moderate complete membrane staining is
observed in more than 10% of the tumor cells.
[0769] Score 3+--a moderate to strong complete membrane staining is
observed in more than 10% of the tumor cells.
[0770] Those tumors with 0 or 1+ scores for TAT polypeptide
expression may be characterized as not overexpressing TAT, whereas
those tumors with 2+ or 3+ scores may be characterized as
overexpressing TAT.
[0771] Alternatively, or additionally, FISH assays such as the
INFORM.RTM. (sold by Ventana, Ariz.) or PATHVISION.RTM. (Vysis,
Ill.) may be carried out on formalin-fixed, paraffin-embedded tumor
tissue to determine the extent (if any) of TAT overexpression in
the tumor.
[0772] TAT overexpression or amplification may be evaluated using
an in vivo diagnostic assay, e.g., by administering a molecule
(such as an antibody, oligopeptide or organic molecule) which binds
the molecule to be detected and is tagged with a detectable label
(e.g., a radioactive isotope or a fluorescent label) and externally
scanning the patient for localization of the label.
[0773] As described above, the anti-TAT antibodies, oligopeptides
and organic molecules of the invention have various non-therapeutic
applications. The anti-TAT antibodies, oligopeptides and organic
molecules of the present invention can be useful for diagnosis and
staging of TAT polypeptide-expressing cancers (e.g., in
radioimaging). The antibodies, oligopeptides and organic molecules
are also useful for purification or immunoprecipitation of TAT
polypeptide from cells, for detection and quantitation of TAT
polypeptide in vitro, e.g., in an ELISA or a Western blot, to kill
and eliminate TAT-expressing cells from a population of mixed cells
as a step in the purification of other cells.
[0774] Currently, depending on the stage of the cancer, cancer
treatment involves one or a combination of the following therapies:
surgery to remove the cancerous tissue, radiation therapy, and
chemotherapy. Anti-TAT antibody, oligopeptide or organic molecule
therapy may be especially desirable in elderly patients who do not
tolerate the toxicity and side effects of chemotherapy well and in
metastatic disease where radiation therapy has limited usefulness.
The tumor targeting anti-TAT antibodies, oligopeptides and organic
molecules of the invention are useful to alleviate TAT-expressing
cancers upon initial diagnosis of the disease or during relapse.
For therapeutic applications, the anti-TAT antibody, oligopeptide
or organic molecule can be used alone, or in combination therapy
with, e.g., hormones, antiangiogens, or radiolabelled compounds, or
with surgery, cryotherapy, and/or radiotherapy. Anti-TAT antibody,
oligopeptide or organic molecule treatment can be administered in
conjunction with other forms of conventional therapy, either
consecutively with, pre- or post-conventional therapy.
Chemotherapeutic drugs such as TAXOTERE.RTM. (docetaxel),
TAXOL.RTM. (palictaxel), estramustine and mitoxantrone are used in
treating cancer, in particular, in good risk patients. In the
present method of the invention for treating or alleviating cancer,
the cancer patient can be administered anti-TAT antibody,
oligopeptide or organic molecule in conjuction with treatment with
the one or more of the preceding chemotherapeutic agents. In
particular, combination therapy with palictaxel and modified
derivatives (see, e.g., EP0600517) is contemplated. The anti-TAT
antibody, oligopeptide or organic molecule will be administered
with a therapeutically effective dose of the chemotherapeutic
agent. In another embodiment, the anti-TAT antibody, oligopeptide
or organic molecule is administered in conjunction with
chemotherapy to enhance the activity and efficacy of the
chemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk
Reference (PDR) discloses dosages of these agents that have been
used in treatment of various cancers. The dosing regimen and
dosages of these aforementioned chemotherapeutic drugs that are
therapeutically effective will depend on the particular cancer
being treated, the extent of the disease and other factors familiar
to the physician of skill in the art and can be determined by the
physician.
[0775] In one particular embodiment, a conjugate comprising an
anti-TAT antibody, oligopeptide or organic molecule conjugated with
a cytotoxic agent is administered to the patient. Preferably, the
immunoconjugate bound to the TAT protein is internalized by the
cell, resulting in increased therapeutic efficacy of the
immunoconjugate in killing the cancer cell to which it binds. In a
preferred embodiment, the cytotoxic agent targets or interferes
with the nucleic acid in the cancer cell. Examples of such
cytotoxic agents are described above and include maytansinoids,
calicheamicins, ribonucleases and DNA endonucleases.
[0776] The anti-TAT antibodies, oligopeptides, organic molecules or
toxin conjugates thereof are administered to a human patient, in
accord with known methods, such as intravenous administration,
e.g., as a bolus or by continuous infusion over a period of time,
by intramuscular, intraperitoneal, intracerobrospinal,
subcutaneous, intra-articular, intrasynovial, intrathecal, oral,
topical, or inhalation routes. Intravenous or subcutaneous
administration of the antibody, oligopeptide or organic molecule is
preferred.
[0777] Other therapeutic regimens may be combined with the
administration of the anti-TAT antibody, oligopeptide or organic
molecule. The combined administration includes co-administration,
using separate formulations or a single pharmaceutical formulation,
and consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities. Preferably such
combined therapy results in a synergistic therapeutic effect.
[0778] It may also be desirable to combine administration of the
anti-TAT antibody or antibodies, oligopeptides or organic
molecules, with administration of an antibody directed against
another tumor antigen associated with the particular cancer.
[0779] In another embodiment, the therapeutic treatment methods of
the present invention involves the combined administration of an
anti-TAT antibody (or antibodies), oligopeptides or organic
molecules and one or more chemotherapeutic agents or growth
inhibitory agents, including co-administration of cocktails of
different chemotherapeutic agents. Chemotherapeutic agents include
estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil,
melphalan, cyclophosphamide, hydroxyurea and hydroxyureataxanes
(such as paclitaxel and doxetaxel) and/or anthracycline
antibiotics. Preparation and dosing schedules for such
chemotherapeutic agents may be used according to manufacturers'
instructions or as determined empirically by the skilled
practitioner. Preparation and dosing schedules for such
chemotherapy are also described in Chemotherapy Service Ed., M. C.
Perry, Williams & Wilkins, Baltimore, Md. (1992).
[0780] The antibody, oligopeptide or organic molecule may be
combined with an anti-hormonal compound; e.g., an anti-estrogen
compound such as tamoxifen; an anti-progesterone such as
onapristone (see, EP 616 812); or an anti-androgen such as
flutamide, in dosages known for such molecules. Where the cancer to
be treated is androgen independent cancer, the patient may
previously have been subjected to anti-androgen therapy and, after
the cancer becomes androgen independent, the anti-TAT antibody,
oligopeptide or organic molecule (and optionally other agents as
described herein) may be administered to the patient.
[0781] Sometimes, it may be beneficial to also co-administer a
cardioprotectant (to prevent or reduce myocardial dysfunction
associated with the therapy) or one or more cytokines to the
patient. In addition to the above therapeutic regimes, the patient
may be subjected to surgical removal of cancer cells and/or
radiation therapy, before, simultaneously with, or post antibody,
oligopeptide or organic molecule therapy. Suitable dosages for any
of the above co-administered agents are those presently used and
may be lowered due to the combined action (synergy) of the agent
and anti-TAT antibody, oligopeptide or organic molecule.
[0782] For the prevention or treatment of disease, the dosage and
mode of administration will be chosen by the physician according to
known criteria. The appropriate dosage of antibody, oligopeptide or
organic molecule will depend on the type of disease to be treated,
as defined above, the severity and course of the disease, whether
the antibody, oligopeptide or organic molecule is administered for
preventive or therapeutic purposes, previous therapy, the patient's
clinical history and response to the antibody, oligopeptide or
organic molecule, and the discretion of the attending physician.
The antibody, oligopeptide or organic molecule is suitably
administered to the patient at one time or over a series of
treatments. Preferably, the antibody, oligopeptide or organic
molecule is administered by intravenous infusion or by subcutaneous
injections. Depending on the type and severity of the disease,
about 1 .mu.g/kg to about 50 mg/kg body weight (e.g., about 0.1-15
mg/kg/dose) of antibody can be an initial candidate dosage for
administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. A dosing
regimen can comprise administering an initial loading dose of about
4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of
the anti-TAT antibody. However, other dosage regimens may be
useful. A typical daily dosage might range from about 1 .mu.g/kg to
100 mg/kg or more, depending on the factors mentioned above. For
repeated administrations over several days or longer, depending on
the condition, the treatment is sustained until a desired
suppression of disease symptoms occurs. The progress of this
therapy can be readily monitored by conventional methods and assays
and based on criteria known to the physician or other persons of
skill in the art.
[0783] Aside from administration of the antibody protein to the
patient, the present application contemplates administration of the
antibody by gene therapy. Such administration of nucleic acid
encoding the antibody is encompassed by the expression
"administering a therapeutically effective amount of an antibody".
See, for example, WO96/07321 published Mar. 14, 1996 concerning the
use of gene therapy to generate intracellular antibodies.
[0784] There are two major approaches to getting the nucleic acid
(optionally contained in a vector) into the patient's cells; in
vivo and ex vivo. For in vivo delivery the nucleic acid is injected
directly into the patient, usually at the site where the antibody
is required. For ex vivo treatment, the patient's cells are
removed, the nucleic acid is introduced into these isolated cells
and the modified cells are administered to the patient either
directly or, for example, encapsulated within porous membranes
which are implanted into the patient (see, e.g., U.S. Pat. Nos.
4,892,538 and 5,283,187). There are a variety of techniques
available for introducing nucleic acids into viable cells. The
techniques vary depending upon whether the nucleic acid is
transferred into cultured cells in vitro, or in vivo in the cells
of the intended host. Techniques suitable for the transfer of
nucleic acid into mammalian cells in vitro include the use of
liposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, the calcium phosphate precipitation method, etc. A
commonly used vector for ex vivo delivery of the gene is a
retroviral vector.
[0785] The currently preferred in vivo nucleic acid transfer
techniques include transfection with viral vectors (such as
adenovirus, Herpes simplex I virus, or adeno-associated virus) and
lipid-based systems (useful lipids for lipid-mediated transfer of
the gene are DOTMA, DOPE and DC-Chol, for example). For review of
the currently known gene marking and gene therapy protocols see
Anderson et al., Science 256:808-813 (1992). See also WO 93/25673
and the references cited therein.
[0786] The anti-TAT antibodies of the invention can be in the
different forms encompassed by the definition of "antibody" herein.
Thus, the antibodies include full length or intact antibody,
antibody fragments, native sequence antibody or amino acid
variants, humanized, chimeric or fusion antibodies,
immunoconjugates, and functional fragments thereof. In fusion
antibodies an antibody sequence is fused to a heterologous
polypeptide sequence. The antibodies can be modified in the Fc
region to provide desired effector functions. As discussed in more
detail in the sections herein, with the appropriate Fc regions, the
naked antibody bound on the cell surface can induce cytotoxicity,
e.g., via antibody-dependent cellular cytotoxicity (ADCC) or by
recruiting complement in complement dependent cytotoxicity, or some
other mechanism. Alternatively, where it is desirable to eliminate
or reduce effector function, so as to minimize side effects or
therapeutic complications, certain other Fc regions may be
used.
[0787] In one embodiment, the antibody competes for binding or bind
substantially to, the same epitope as the antibodies of the
invention. Antibodies having the biological characteristics of the
present anti-TAT antibodies of the invention are also contemplated,
specifically including the in vivo tumor targeting and any cell
proliferation inhibition or cytotoxic characteristics.
[0788] Methods of producing the above antibodies are described in
detail herein.
[0789] The present anti-TAT antibodies, oligopeptides and organic
molecules are useful for treating a TAT-expressing cancer or
alleviating one or more symptoms of the cancer in a mammal. Such a
cancer includes prostate cancer, cancer of the urinary tract, lung
cancer, breast cancer, colon cancer and ovarian cancer, more
specifically, prostate adenocarcinoma, renal cell carcinomas,
colorectal adenocarcinomas, lung adenocarcinomas, lung squamous
cell carcinomas, and pleural mesothelioma. The cancers encompass
metastatic cancers of any of the preceding. The antibody,
oligopeptide or organic molecule is able to bind to at least a
portion of the cancer cells that express TAT polypeptide in the
mammal. In a preferred embodiment, the antibody, oligopeptide or
organic molecule is effective to destroy or kill TAT-expressing
tumor cells or inhibit the growth of such tumor cells, in vitro or
in vivo, upon binding to TAT polypeptide on the cell. Such an
antibody includes a naked anti-TAT antibody (not conjugated to any
agent). Naked antibodies that have cytotoxic or cell growth
inhibition properties can be further harnessed with a cytotoxic
agent to render them even more potent in tumor cell destruction.
Cytotoxic properties can be conferred to an anti-TAT antibody by,
e.g., conjugating the antibody with a cytotoxic agent, to form an
immunoconjugate as described herein. The cytotoxic agent or a
growth inhibitory agent is preferably a small molecule. Toxins such
as calicheamicin or a maytansinoid and analogs or derivatives
thereof, are preferable.
[0790] The invention provides a composition comprising an anti-TAT
antibody, oligopeptide or organic molecule of the invention, and a
carrier. For the purposes of treating cancer, compositions can be
administered to the patient in need of such treatment, wherein the
composition can comprise one or more anti-TAT antibodies present as
an immunoconjugate or as the naked antibody. In a further
embodiment, the compositions can comprise these antibodies,
oligopeptides or organic molecules in combination with other
therapeutic agents such as cytotoxic or growth inhibitory agents,
including chemotherapeutic agents. The invention also provides
formulations comprising an anti-TAT antibody, oligopeptide or
organic molecule of the invention, and a carrier. In one
embodiment, the formulation is a therapeutic formulation comprising
a pharmaceutically acceptable carrier.
[0791] Another aspect of the invention is isolated nucleic acids
encoding the anti-TAT antibodies. Nucleic acids encoding both the H
and L chains and especially the hypervariable region residues,
chains which encode the native sequence antibody as well as
variants, modifications and humanized versions of the antibody, are
encompassed.
[0792] The invention also provides methods useful for treating a
TAT polypeptide-expressing cancer or alleviating one or more
symptoms of the cancer in a mammal, comprising administering a
therapeutically effective amount of an anti-TAT antibody,
oligopeptide or organic molecule to the mammal. The antibody,
oligopeptide or organic molecule therapeutic compositions can be
administered short term (acute) or chronic, or intermittent as
directed by physician. Also provided are methods of inhibiting the
growth of, and killing a TAT polypeptide-expressing cell.
[0793] The invention also provides kits and articles of manufacture
comprising at least one anti-TAT antibody, oligopeptide or organic
molecule. Kits containing anti-TAT antibodies, oligopeptides or
organic molecules find use, e.g., for TAT cell killing assays, for
purification or immunoprecipitation of TAT polypeptide from cells.
For example, for isolation and purification of TAT, the kit can
contain an anti-TAT antibody, oligopeptide or organic molecule
coupled to beads (e.g., sepharose beads). Kits can be provided
which contain the antibodies, oligopeptides or organic molecules
for detection and quantitation of TAT in vitro, e.g., in an ELISA
or a Western blot. Such antibody, oligopeptide or organic molecule
useful for detection may be provided with a label such as a
fluorescent or radiolabel.
[0794] L. Articles of Manufacture and Kits
[0795] Another embodiment of the invention is an article of
manufacture containing materials useful for the treatment of
anti-TAT expressing cancer. The article of manufacture comprises a
container and a label or package insert on or associated with the
container. Suitable containers include, for example, bottles,
vials, syringes, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is effective for treating the cancer condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an anti-TAT antibody, oligopeptide or
organic molecule of the invention. The label or package insert
indicates that the composition is used for treating cancer. The
label or package insert will further comprise instructions for
administering the antibody, oligopeptide or organic molecule
composition to the cancer patient. Additionally, the article of
manufacture may further comprise a second container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0796] Kits are also provided that are useful for various purposes,
e.g., for TAT-expressing cell killing assays, for purification or
immunoprecipitation of TAT polypeptide from cells. For isolation
and purification of TAT polypeptide, the kit can contain an
anti-TAT antibody, oligopeptide or organic molecule coupled to
beads (e.g., sepharose beads). Kits can be provided which contain
the antibodies, oligopeptides or organic molecules for detection
and quantitation of TAT polypeptide in vitro, e.g., in an ELISA or
a Western blot. As with the article of manufacture, the kit
comprises a container and a label or package insert on or
associated with the container. The container holds a composition
comprising at least one anti-TAT antibody, oligopeptide or organic
molecule of the invention. Additional containers may be included
that contain, e.g., diluents and buffers, control antibodies. The
label or package insert may provide a description of the
composition as well as instructions for the intended in vitro or
diagnostic use.
[0797] M. Uses for TAT Polypeptides and TAT-Polypeptide Encoding
Nucleic Acids
[0798] Nucleotide sequences (or their complement) encoding TAT
polypeptides have various applications in the art of molecular
biology, including uses as hybridization probes, in chromosome and
gene mapping and in the generation of anti-sense RNA and DNA
probes. TAT-encoding nucleic acid will also be useful for the
preparation of TAT polypeptides by the recombinant techniques
described herein, wherein those TAT polypeptides may find use, for
example, in the preparation of anti-TAT antibodies as described
herein.
[0799] The full-length native sequence TAT gene, or portions
thereof, may be used as hybridization probes for a cDNA library to
isolate the full-length TAT cDNA or to isolate still other cDNAs
(for instance, those encoding naturally-occurring variants of TAT
or TAT from other species) which have a desired sequence identity
to the native TAT sequence disclosed herein. Optionally, the length
of the probes will be about 20 to about 50 bases. The hybridization
probes may be derived from at least partially novel regions of the
full length native nucleotide sequence wherein those regions may be
determined without undue experimentation or from genomic sequences
including promoters, enhancer elements and introns of native
sequence TAT. By way of example, a screening method will comprise
isolating the coding region of the TAT gene using the known DNA
sequence to synthesize a selected probe of about 40 bases.
Hybridization probes may be labeled by a variety of labels,
including radionucleotides such as .sup.32P or .sup.35S, or
enzymatic labels such as alkaline phosphatase coupled to the probe
via avidin/biotin coupling systems. Labeled probes having a
sequence complementary to that of the TAT gene of the present
invention can be used to screen libraries of human cDNA, genomic
DNA or mRNA to determine which members of such libraries the probe
hybridizes to. Hybridization techniques are described in further
detail in the Examples below. Any EST sequences disclosed in the
present application may similarly be employed as probes, using the
methods disclosed herein.
[0800] Other useful fragments of the TAT-encoding nucleic acids
include antisense or sense oligonucleotides comprising a
singe-stranded nucleic acid sequence (either RNA or DNA) capable of
binding to target TAT mRNA (sense) or TAT DNA (antisense)
sequences. Antisense or sense oligonucleotides, according to the
present invention, comprise a fragment of the coding region of TAT
DNA. Such a fragment generally comprises at least about 14
nucleotides, preferably from about 14 to 30 nucleotides. The
ability to derive an antisense or a sense oligonucleotide, based
upon a cDNA sequence encoding a given protein is described in, for
example, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der
Krol et al. (Biotechniques 6:958, 1988).
[0801] Binding of antisense or sense oligonucleotides to target
nucleic acid sequences results in the formation of duplexes that
block transcription or translation of the target sequence by one of
several means, including enhanced degradation of the duplexes,
premature termination of transcription or translation, or by other
means. Such methods are encompassed by the present invention. The
antisense oligonucleotides thus may be used to block expression of
TAT proteins, wherein those TAT proteins may play a role in the
induction of cancer in mammals. Antisense or sense oligonucleotides
further comprise oligonucleotides having modified
sugar-phosphodiester backbones (or other sugar linkages, such as
those described in WO 91/06629) and wherein such sugar linkages are
resistant to endogenous nucleases. Such oligonucleotides with
resistant sugar linkages are stable in vivo (i.e., capable of
resisting enzymatic degradation) but retain sequence specificity to
be able to bind to target nucleotide sequences.
[0802] Preferred intragenic sites for antisense binding include the
region incorporating the translation initiation/start codon
(5'-AUG/5'-ATG) or termination/stop codon (5'-UAA, 5'-UAG and
5-UGA/5'-TAA, 5'-TAG and 5'-TGA) of the open reading frame (ORF) of
the gene. These regions refer to a portion of the mRNA or gene that
encompasses from about 25 to about 50 contiguous nucleotides in
either direction (i.e., 5' or 3') from a translation initiation or
termination codon. Other preferred regions for antisense binding
include: introns; exons; intron-exon junctions; the open reading
frame (ORF) or "coding region," which is the region between the
translation initiation codon and the translation termination codon;
the 5' cap of an mRNA which comprises an N7-methylated guanosine
residue joined to the 5'-most residue of the mRNA via a 5'-5'
triphosphate linkage and includes 5' cap structure itself as well
as the first 50 nucleotides adjacent to the cap; the 5'
untranslated region (5UTR), the portion of an mRNA in the 5'
direction from the translation initiation codon, and thus including
nucleotides between the 5' cap site and the translation initiation
codon of an mRNA or corresponding nucleotides on the gene; and the
3' untranslated region (3'UTR), the portion of an mRNA in the 3'
direction from the translation termination codon, and thus
including nucleotides between the translation termination codon and
3' end of an mRNA or corresponding nucleotides on the gene.
[0803] Specific examples of preferred antisense compounds useful
for inhibiting expression of TAT proteins include oligonucleotides
containing modified backbones or non-natural internucleoside
linkages. Oligonucleotides having modified backbones include those
that retain a phosphorus atom in the backbone and those that do not
have a phosphorus atom in the backbone. For the purposes of this
specification, and as sometimes referenced in the art, modified
oligonucleotides that do not have a phosphorus atom in their
internucleoside backbone can also be considered to be
oligonucleosides. Preferred modified oligonucleotide backbones
include, for example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates, 5'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and amino alkylphosphoramidates, thionophosphoramidates, thiono
alkylphosphonates, thionoalkylphosphotriesters, selenophosphates
and borano-phosphates having normal 3'-5' linkages, 2'-5' linked
analogs of these, and those having inverted polarity wherein one or
more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2'
linkage. Preferred oligonucleotides having inverted polarity
comprise a single 3' to 3' linkage at the 3'-most internucleotide
linkage i.e. a single inverted nucleoside residue which may be
abasic (the nucleobase is missing or has a hydroxyl group in place
thereof). Various salts, mixed salts and free acid forms are also
included. Representative United States patents that teach the
preparation of phosphorus-containing linkages include, but are not
limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;
5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899;
5,721,218; 5,672,697 and 5,625,050, each of which is herein
incorporated by reference.
[0804] Preferred modified oligonucleotide backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; riboacetyl backbones; alkene containing backbones;
sulfamate backbones; methyleneimino and methylenehydrazino
backbones; sulfonate and sulfonamide backbones; amide backbones;
and others having mixed N, O, S and CH.sub.2 component parts.
Representative United States patents that teach the preparation of
such oligonucleosides include, but are not limited to,. U.S. Pat.
Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;
5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;
5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;
5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;
5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and
5,677,439, each of which is herein incorporated by reference.
[0805] In other preferred antisense oligonucleotides, both the
sugar and the internucleoside linkage, i.e., the backbone, of the
nucleotide units are replaced with novel groups. The base units are
maintained for hybridization with an appropriate nucleic acid
target compound. One such oligomeric compound, an oligonucleotide
mimetic that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar-backbone of an oligonucleotide is replaced
with an amide containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are
bound directly or indirectly to aza nitrogen atoms of the amide
portion of the backbone. Representative United States patents that
teach the preparation of PNA compounds include, but are not limited
to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of
which is herein incorporated by reference. Further teaching of PNA
compounds can be found in Nielsen et al., Science, 1991, 254,
1497-1500.
[0806] Preferred antisense oligonucleotides incorporate
phosphorothioate backbones and/or heteroatom backbones, and in
particular --CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--]
described in the above referenced U.S. Pat. No. 5,489,677, and the
amide backbones of the above referenced U.S. Pat. No. 5,602,240.
Also preferred are antisense oligonucleotides having morpholino
backbone structures of the above-referenced U.S. Pat. No.
5,034,506.
[0807] Modified oligonucleotides may also contain one or more
substituted sugar moieties. Preferred oligonucleotides comprise one
of the following at the 2' position: OH; F; O-alkyl, S-alkyl, or
N-alkyl; O-alkenyl, S-alkeynyl, or N-alkenyl; O-alkynyl, S-alkynyl
or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and
alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10
alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly
preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3,
O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10. Other preferred antisense oligonucleotides
comprise one of the following at the 2' position: C.sub.1 to
C.sub.10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl,
alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl,
Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2 CH.sub.13,
ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl,
heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted
silyl, an RNA cleaving group, a reporter group, an intercalator, a
group for improving the pharmacokinetic properties of an
oligonucleotide, or a group for improving the pharmacodynamic
properties of an oligonucleotide, and other substituents having
similar properties. A preferred modification includes
2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).
[0808] A further preferred modification includes Locked Nucleic
Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or
4' carbon atom of the sugar ring thereby forming a bicyclic sugar
moiety. The linkage is preferably a methelyne (--CH.sub.2--).sub.n
group bridging the 2' oxygen atom and the 4' carbon atom wherein n
is 1 or 2. LNAs and preparation thereof are described in WO
98/39352 and WO 99/14226.
[0809] Other preferred modifications include
2'-methoxy(2'-O--CH.sub.3),
2'-aminopropoxy(2'-OCH.sub.2CH.sub.2CH.sub.2 NH.sub.2), 2'-allyl
(2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O-CH.sub.2--CH.dbd.CH.sub.2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. A preferred 2'-arabino modification is 2'-F. Similar
modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Oligonucleotides may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative United States patents that
teach the preparation of such modified sugar structures include,
but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;
5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;
5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747;
and 5,700,920, each of which is herein incorporated by reference in
its entirety.
[0810] Oligonucleotides may also include nucleobase (often referred
to in the art simply as "base") modifications or substitutions. As
used herein, "unmodified" or "natural" nucleobases include the
purine bases adenine (A) and guanine (G), and the pyrimidine bases
thymine (T), cytosine (C) and uracil (U). Modified nucleobases
include other synthetic and natural nucleobases such as
5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 2-thiouracil, 2-thiothymine and
2-thiocytosine, 5-halouracil and cytosine, 5-propynyl
(--C.dbd.C--CH.sub.3 or --CH.sub.2--C.dbd.CH) uracil and cytosine
and other alkynyl derivatives of pyrimidine bases, 6-azo uracil,
cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,
8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other
8-substituted adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,
8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine
and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases
include tricyclic pyrimidines such as phenoxazine
cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
phenothiazine cytidine
(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a
substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
Modified nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, and those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613. Certain of
these nucleobases are particularly useful for increasing the
binding affinity of the oligomeric compounds of the invention.
These include 5-substituted pyrimidines, 6-azapyrimidines and N-2,
N-6 and O-6 substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2.degree. C. (Sanghvi et al, Antisense Research
and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are
preferred base substitutions, even more particularly when combined
with 2'-O-methoxyethyl sugar modifications. Representative United
States patents that teach the preparation of modified nucleobases
include, but are not limited to: U.S. Pat. No. 3,687,808, as well
as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273;
5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177;
5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617;
5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,681,941 and
5,750,692, each of which is herein incorporated by reference.
[0811] Another modification of antisense oligonucleotides
chemically linking to the oligonucleotide one or more moieties or
conjugates which enhance the activity, cellular distribution or
cellular uptake of the oligonucleotide. The compounds of the
invention can include conjugate groups covalently bound to
functional groups such as primary or secondary hydroxyl groups.
Conjugate groups of the invention include intercalators, reporter
molecules, polyamines, polyamides, polyethylene glycols,
polyethers, groups that enhance the pharmacodynamic properties of
oligomers, and groups that enhance the pharmacokinetic properties
of oligomers. Typical conjugates groups include cholesterols,
lipids, cation lipids, phospholipids, cationic phospholipids,
biotin, phenazine, folate, phenanthridine, anthraquinone, acridine,
fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance
the pharmacodynamic properties, in the context of this invention,
include groups that improve oligomer uptake, enhance oligomer
resistance to degradation, and/or strengthen sequence-specific
hybridization with RNA. Groups that enhance the pharmacokinetic
properties, in the context of this invention, include groups that
improve oligomer uptake, distribution, metabolism or excretion.
Conjugate moieties include but are not limited to lipid moieties
such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad.
Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al.,
Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,
hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3,
2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,
1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or
undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10,
1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;
Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of
the invention may also be conjugated to active drug substances, for
example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen,
fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,
dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,
folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,
indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
Oligonucleotide-drug conjugates and their preparation are described
in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15,
1999) and U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241,5,391,723;
5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;
5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;
5,599,923; 5,599,928 and 5,688,941, each of which is herein
incorporated by reference.
[0812] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an oligonucleotide.
The present invention also includes antisense compounds which are
chimeric compounds. "Chimeric" antisense compounds or "chimeras,"
in the context of this invention, are antisense compounds,
particularly oligonucleotides, which contain two or more chemically
distinct regions, each made up of at least one monomer unit, i.e.,
a nucleotide in the case of an oligonucleotide compound. These
oligonucleotides typically contain at least one region wherein the
oligonucleotide is modified so as to confer upon the
oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, and/or increased binding affinity for
the target nucleic acid. An additional region of the
oligonucleotide may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is
a cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. Activation of RNase H, therefore, results in cleavage of
the RNA target, thereby greatly enhancing the efficiency of
oligonucleotide inhibition of gene expression. Consequently,
comparable results can often be obtained with shorter
oligonucleotides when chimeric oligonucleotides are used, compared
to phosphorothioate deoxyoligonucleotides hybridizing to the same
target region. Chimeric antisense compounds of the invention may be
formed as composite structures of two or more oligonucleotides,
modified oligonucleotides, oligonucleosides and/or oligonucleotide
mimetics as described above. Preferred chimeric antisense
oligonucleotides incorporate at least one 2' modified sugar
(preferably 2'-O--(CH.sub.2).sub.2--O--CH.sub.3) at the 3' terminal
to confer nuclease resistance and a region with at least 4
contiguous 2'-H sugars to confer RNase H activity. Such compounds
have also been referred to in the art as hybrids or gapmers.
Preferred gapmers have a region of 2' modified sugars (preferably
2'-O--(CH.sub.2).sub.2--O--CH.sub.3) at the 3'-terminal and at the
5' terminal separated by at least one region having at least 4
contiguous 2'-H sugars and preferably incorporate phosphorothioate
backbone linkages. Representative United States patents that teach
the preparation of such hybrid structures include, but are not
limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007;
5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
5,652,355; 5,652,356; and 5,700,922, each of which is herein
incorporated by reference in its entirety.
[0813] The antisense compounds used in accordance with this
invention may be conveniently and routinely made through the
well-known technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed. It is well known to use similar techniques to prepare
oligonucleotides such as the phosphorothioates and alkylated
derivatives. The compounds of the invention may also be admixed,
encapsulated, conjugated or otherwise associated with other
molecules, molecule structures or mixtures of compounds, as for
example, liposomes, receptor targeted molecules, oral, rectal,
topical or other formulations, for assisting in uptake,
distribution and/or absorption. Representative United States
patents that teach the preparation of such uptake, distribution
and/or absorption assisting formulations include, but are not
limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;
5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;
4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;
5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;
5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;
5,580,575; and 5,595,756, each of which is herein incorporated by
reference.
[0814] Other examples of sense or antisense oligonucleotides
include those oligonucleotides which are covalently linked to
organic moieties, such as those described in WO 90/10048, and other
moieties that increases affinity of the oligonucleotide for a
target nucleic acid sequence, such as poly-(L-lysine). Further
still, intercalating agents, such as ellipticine, and alkylating
agents or metal complexes may be attached to sense or antisense
oligonucleotides to modify binding specificities of the antisense
or sense oligonucleotide for the target nucleotide sequence.
[0815] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, CaPO.sub.4-mediated DNA
transfection, electroporation, or by using gene transfer vectors
such as Epstein-Barr virus. In a preferred procedure, an antisense
or sense oligonucleotide is inserted into a suitable retroviral
vector. A cell containing the target nucleic acid sequence is
contacted with the recombinant retroviral vector, either in vivo or
ex vivo. Suitable retroviral vectors include, but are not limited
to, those derived from the murine retrovirus M-MuLV, N2 (a
retrovirus derived from M-MuLV), or the double copy vectors
designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
[0816] Sense or antisense oligonucleotides also may be introduced
into a cell containing the target nucleotide sequence by formation
of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors.
Preferably, conjugation of the ligand binding molecule does not
substantially interfere with the ability of the ligand binding
molecule to bind to its corresponding molecule or receptor, or
block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell.
[0817] Alternatively, a sense or an antisense oligonucleotide may
be introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the
cell by an endogenous lipase.
[0818] Antisense or sense RNA or DNA molecules are generally at
least about 5 nucleotides in length, alternatively at least about
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,
155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230,
240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,
370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490,
500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620,
630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750,
760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880,
890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000
nucleotides in length, wherein in this context the term "about"
means the referenced nucleotide sequence length plus or minus 10%
of that referenced length.
[0819] The probes may also be employed in PCR techniques to
generate a pool of sequences for identification of closely related
TAT coding sequences.
[0820] Nucleotide sequences encoding a TAT can also be used to
construct hybridization probes for mapping the gene which encodes
that TAT and for the genetic analysis of individuals with genetic
disorders. The nucleotide sequences provided herein may be mapped
to a chromosome and specific regions of a chromosome using known
techniques, such as in situ hybridization, linkage analysis against
known chromosomal markers, and hybridization screening with
libraries.
[0821] When the coding sequences for TAT encode a protein which
binds to another protein (example, where the TAT is a receptor),
the TAT can be used in assays to identify the other proteins or
molecules involved in the binding interaction. By such methods,
inhibitors of the receptor/ligand binding interaction can be
identified. Proteins involved in such binding interactions can also
be used to screen for peptide or small molecule inhibitors or
agonists of the binding interaction. Also, the receptor TAT can be
used to isolate correlative ligand(s). Screening assays can be
designed to find lead compounds that mimic the biological activity
of a native TAT or a receptor for TAT. Such screening assays will
include assays amenable to high-throughput screening of chemical
libraries, making them particularly suitable for identifying small
molecule drug candidates. Small molecules contemplated include
synthetic organic or inorganic compounds. The assays can be
performed in a variety of formats, including protein-protein
binding assays, biochemical screening assays, immunoassays and cell
based assays, which are well characterized in the art.
[0822] Nucleic acids which encode TAT or its modified forms can
also be used to generate either transgenic animals or "knock out"
animals which, in turn, are useful in the development and screening
of therapeutically useful reagents. A transgenic animal (e.g., a
mouse or rat) is an animal having cells that contain a transgene,
which transgene was introduced into the animal or an ancestor of
the animal at a prenatal, e.g., an embryonic stage. A transgene is
a DNA which is integrated into the genome of a cell from which a
transgenic animal develops. In one embodiment, cDNA encoding TAT
can be used to clone genomic DNA encoding TAT in accordance with
established techniques and the genomic sequences used to generate
transgenic animals that contain cells which express DNA encoding
TAT. Methods for generating transgenic animals, particularly
animals such as mice or rats, have become conventional in the art
and are described, for example, in U.S. Pat. Nos. 4,736,866 and
4,870,009. Typically, particular cells would be targeted for TAT
transgene incorporation with tissue-specific enhancers. Transgenic
animals that include a copy of a transgene encoding TAT introduced
into the germ line of the animal at an embryonic stage can be used
to examine the effect of increased expression of DNA encoding TAT.
Such animals can be used as tester animals for reagents thought to
confer protection from, for example, pathological conditions
associated with its overexpression. In accordance with this facet
of the invention, an animal is treated with the reagent and a
reduced incidence of the pathological condition, compared to
untreated animals bearing the transgene, would indicate a potential
therapeutic intervention for the pathological condition.
[0823] Alternatively, non-human homologues of TAT can be used to
construct a TAT "knock out" animal which has a defective or altered
gene encoding TAT as a result of homologous recombination between
the endogenous gene encoding TAT and altered genomic DNA encoding
TAT introduced into an embryonic stem cell of the animal. For
example, cDNA encoding TAT can be used to clone genomic DNA
encoding TAT in accordance with established techniques. A portion
of the genomic DNA encoding TAT can be deleted or replaced with
another gene, such as a gene encoding a selectable marker which can
be used to monitor integration. Typically, several kilobases of
unaltered flanking DNA (both at the 5' and 3' ends) are included in
the vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for
a description of homologous recombination vectors]. The vector is
introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced DNA has
homologously recombined with the endogenous DNA are selected [see
e.g., L1 et al., Cell, 69:915 (1992)]. The selected cells are then
injected into a blastocyst of an animal (e.g., a mouse or rat) to
form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas
and Embryonic Stem Cells. A Practical Approach, E. J. Robertson,
ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then
be implanted into a suitable pseudopregnant female foster animal
and the embryo brought to term to create a "knock out" animal.
Progeny harboring the homologously recombined DNA in their germ
cells can be identified by standard techniques and used to breed
animals in which all cells of the animal contain the homologously
recombined DNA. Knockout animals can be characterized for instance,
for their ability to defend against certain pathological conditions
and for their development of pathological conditions due to absence
of the TAT polypeptide.
[0824] Nucleic acid encoding the TAT polypeptides may also be used
in gene therapy. In gene therapy applications, genes are introduced
into cells in order to achieve in vivo synthesis of a
therapeutically effective genetic product, for example for
replacement of a defective gene. "Gene therapy" includes both
conventional gene therapy where a lasting effect is achieved by a
single treatment, and the administration of gene therapeutic
agents, which involves the one time or repeated administration of a
therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can
be used as therapeutic agents for blocking the expression of
certain genes in vivo. It has already been shown that short
antisense oligonucleotides can be imported into cells where they
act as inhibitors, despite their low intracellular concentrations
caused by their restricted uptake by the cell membrane. (Zamecnik
et al., Proc. Natl. Acad. Sci. USA 83:4143-4146 [1986]). The
oligonucleotides can be modified to enhance their uptake, e.g. by
substituting their negatively charged phosphodiester groups by
uncharged groups.
[0825] There are a variety of techniques available for introducing
nucleic acids into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene
transfer techniques include transfection with viral (typically
retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et al., Trends in Biotechnology 11, 205-210
[1993]). In some situations it is desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an
antibody specific for a cell surface membrane protein or the target
cell, a ligand for a receptor on the target cell, etc. Where
liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular
half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432
(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414
(1990). For review of gene marking and gene therapy protocols see
Anderson et al., Science 256, 808-813 (1992).
[0826] The nucleic acid molecules encoding the TAT polypeptides or
fragments thereof described herein are useful for chromosome
identification. In this regard, there exists an ongoing need to
identify new chromosome markers, since relatively few chromosome
marking reagents, based upon actual sequence data are presently
available. Each TAT nucleic acid molecule of the present invention
can be used as a chromosome marker.
[0827] The TAT polypeptides and nucleic acid molecules of the
present invention may also be used diagnostically for tissue
typing, wherein the TAT polypeptides of the present invention may
be differentially expressed in one tissue as compared to another,
preferably in a diseased tissue as compared to a normal tissue of
the same tissue type. TAT nucleic acid molecules will find use for
generating probes for PCR, Northern analysis, Southern analysis and
Western analysis.
[0828] This invention encompasses methods of screening compounds to
identify those that mimic the TAT polypeptide (agonists) or prevent
the effect of the TAT polypeptide (antagonists). Screening assays
for antagonist drug candidates are designed to identify compounds
that bind or complex with the TAT polypeptides encoded by the genes
identified herein, or otherwise interfere with the interaction of
the encoded polypeptides with other cellular proteins, including
e.g., inhibiting the expression of TAT polypeptide from cells. Such
screening assays will include assays amenable to high-throughput
screening of chemical libraries, making them particularly suitable
for identifying small molecule drug candidates.
[0829] The assays can be performed in a variety of formats,
including protein-protein binding assays, biochemical screening
assays, immunoassays, and cell-based assays, which are well
characterized in the art.
[0830] All assays for antagonists are common in that they call for
contacting the drug candidate with a TAT polypeptide encoded by a
nucleic acid identified herein under conditions and for a time
sufficient to allow these two components to interact.
[0831] In binding assays, the interaction is binding and the
complex formed can be isolated or detected in the reaction mixture.
In a particular embodiment, the TAT polypeptide encoded by the gene
identified herein or the drug candidate is immobilized on a solid
phase, e.g., on a microtiter plate, by covalent or non-covalent
attachments. Non-covalent attachment generally is accomplished by
coating the solid surface with a solution of the TAT polypeptide
and drying. Alternatively, an immobilized antibody, e.g., a
monoclonal antibody, specific for the TAT polypeptide to be
immobilized can be used to anchor it to a solid surface. The assay
is performed by adding the non-immobilized component, which may be
labeled by a detectable label, to the immobilized component, e.g.,
the coated surface containing the anchored component. When the
reaction is complete, the non-reacted components are removed, e.g.,
by washing, and complexes anchored on the solid surface are
detected. When the originally non-immobilized component carries a
detectable label, the detection of label immobilized on the surface
indicates that complexing occurred. Where the originally
non-immobilized component does not carry a label, complexing can be
detected, for example, by using a labeled antibody specifically
binding the immobilized complex.
[0832] If the candidate compound interacts with but does not bind
to a particular TAT polypeptide encoded by a gene identified
herein, its interaction with that polypeptide can be assayed by
methods well known for detecting protein-protein interactions. Such
assays include traditional approaches, such as, e.g.,
cross-linking, co-immunoprecipitation, and co-purification through
gradients or chromatographic columns. In addition, protein-protein
interactions can be monitored by using a yeast-based genetic system
described by Fields and co-workers (Fields and Song, Nature
(London), 340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci.
USA, 88:9578-9582 (1991)) as disclosed by Chevray and Nathans,
Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Many
transcriptional activators, such as yeast GAL4, consist of two
physically discrete modular domains, one acting as the DNA-binding
domain, the other one functioning as the transcription-activation
domain. The yeast expression system described in the foregoing
publications (generally referred to as the "two-hybrid system")
takes advantage of this property, and employs two hybrid proteins,
one in which the target protein is fused to the DNA-binding domain
of GAL4, and another, in which candidate activating proteins are
fused to the activation domain. The expression of a GAL1-lacZ
reporter gene under control of a GAL4-activated promoter depends on
reconstitution of GAL4 activity via protein-protein interaction.
Colonies containing interacting polypeptides are detected with a
chromogenic substrate for .beta.-galactosidase. A complete kit
(MATCHMAKER.TM.) for identifying protein-protein interactions
between two specific proteins using the two-hybrid technique is
commercially available from Clontech. This system can also be
extended to map protein domains involved in specific protein
interactions as well as to pinpoint amino acid residues that are
crucial for these interactions.
[0833] Compounds that interfere with the interaction of a gene
encoding a TAT polypeptide identified herein and other intra- or
extracellular components can be tested as follows: usually a
reaction mixture is prepared containing the product of the gene and
the intra- or extracellular component under conditions and for a
time allowing for the interaction and binding of the two products.
To test the ability of a candidate compound to inhibit binding, the
reaction is run in the absence and in the presence of the test
compound. In addition, a placebo may be added to a third reaction
mixture, to serve as positive control. The binding (complex
formation) between the test compound and the intra- or
extracellular component present in the mixture is monitored as
described hereinabove. The formation of a complex in the control
reaction(s) but not in the reaction mixture containing the test
compound indicates that the test compound interferes with the
interaction of the test compound and its reaction partner.
[0834] To assay for antagonists, the TAT polypeptide may be added
to a cell along with the compound to be screened for a particular
activity and the ability of the compound to inhibit the activity of
interest in the presence of the TAT polypeptide indicates that the
compound is an antagonist to the TAT polypeptide. Alternatively,
antagonists may be detected by combining the TAT polypeptide and a
potential antagonist with membrane-bound TAT polypeptide receptors
or recombinant receptors under appropriate conditions for a
competitive inhibition assay. The TAT polypeptide can be labeled,
such as by radioactivity, such that the number of TAT polypeptide
molecules bound to the receptor can be used to determine the
effectiveness of the potential antagonist. The gene encoding the
receptor can be identified by numerous methods known to those of
skill in the art, for example, ligand panning and FACS sorting.
Coligan et al., Current Protocols in Immun., 1(2): Chapter 5
(1991). Preferably, expression cloning is employed wherein
polyadenylated RNA is prepared from a cell responsive to the TAT
polypeptide and a cDNA library created from this RNA is divided
into pools and used to transfect COS cells or other cells that are
not responsive to the TAT polypeptide. Transfected cells that are
grown on glass slides are exposed to labeled TAT polypeptide. The
TAT polypeptide can be labeled by a variety of means including
iodination or inclusion of a recognition site for a site-specific
protein kinase. Following fixation and incubation, the slides are
subjected to autoradiographic analysis. Positive pools are
identified and sub-pools are prepared and re-transfected using an
interactive sub-pooling and re-screening process, eventually
yielding a single clone that encodes the putative receptor.
[0835] As an alternative approach for receptor identification,
labeled TAT polypeptide can be photoaffinity-linked with cell
membrane or extract preparations that express the receptor
molecule. Cross-linked material is resolved by PAGE and exposed to
X-ray film. The labeled complex containing the receptor can be
excised, resolved into peptide fragments, and subjected to protein
micro-sequencing. The amino acid sequence obtained from
micro-sequencing would be used to design a set of degenerate
oligonucleotide probes to screen a cDNA library to identify the
gene encoding the putative receptor.
[0836] In another assay for antagonists, mammalian cells or a
membrane preparation expressing the receptor would be incubated
with labeled TAT polypeptide in the presence of the candidate
compound. The ability of the compound to enhance or block this
interaction could then be measured.
[0837] More specific examples of potential antagonists include an
oligonucleotide that binds to the fusions of immunoglobulin with
TAT polypeptide, and, in particular, antibodies including, without
limitation, poly- and monoclonal antibodies and antibody fragments,
single-chain antibodies, anti-idiotypic antibodies, and chimeric or
humanized versions of such antibodies or fragments, as well as
human antibodies and antibody fragments. Alternatively, a potential
antagonist may be a closely related protein, for example, a mutated
form of the TAT polypeptide that recognizes the receptor but
imparts no effect, thereby competitively inhibiting the action of
the TAT polypeptide.
[0838] Another potential TAT polypeptide antagonist is an antisense
RNA or DNA construct prepared using antisense technology, where,
e.g., an antisense RNA or DNA molecule acts to block directly the
translation of mRNA by hybridizing to targeted mRNA and preventing
protein translation. 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 the mature TAT polypeptides
herein, 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); Deivan et
al., Science, 251:1360 (1991)), thereby preventing transcription
and the production of the TAT polypeptide. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into the TAT polypeptide
(antisense-Okano, 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 TAT polypeptide.
When antisense DNA is used, oligodeoxyribonucleotides derived from
the translation-initiation site, e.g., between about -10 and +10
positions of the target gene nucleotide sequence, are
preferred.
[0839] Potential antagonists include small molecules that bind to
the active site, the receptor binding site, or growth factor or
other relevant binding site of the TAT polypeptide, thereby
blocking the normal biological activity of the TAT polypeptide.
Examples of small molecules include, but are not limited to, small
peptides or peptide-like molecules, preferably soluble peptides,
and synthetic non-peptidyl organic or inorganic compounds.
[0840] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. Ribozymes act by sequence-specific
hybridization to the complementary target RNA, followed by
endonucleolytic cleavage. Specific ribozyme cleavage sites within a
potential RNA target can be identified by known techniques. For
further details see, e.g., Rossi, Current Biology, 4:469-471
(1994), and PCT publication No. WO 97/33551 (published Sep. 18,
1997).
[0841] Nucleic acid molecules in triple-helix formation used to
inhibit transcription should be single-stranded and composed of
deoxynucleotides. The base composition of these oligonucleotides is
designed such that it promotes triple-helix formation via Hoogsteen
base-pairing rules, which generally require sizeable stretches of
purines or pyrimidines on one strand of a duplex. For further
details see, e.g., PCT publication No. WO 97/33551, supra.
[0842] These small molecules can be identified by any one or more
of the screening assays discussed hereinabove and/or by any other
screening techniques well known for those skilled in the art.
[0843] Isolated TAT polypeptide-encoding nucleic acid can be used
herein for recombinantly producing TAT polypeptide using techniques
well known in the art and as described herein. In turn, the
produced TAT polypeptides can be employed for generating anti-TAT
antibodies using techniques well known in the art and as described
herein.
[0844] Antibodies specifically binding a TAT polypeptide identified
herein, as well as other molecules identified by the screening
assays disclosed hereinbefore, can be administered for the
treatment of various disorders, including cancer, in the form of
pharmaceutical compositions.
[0845] If the TAT polypeptide is intracellular and whole antibodies
are used as inhibitors, internalizing antibodies are preferred.
However, lipofections or liposomes can also be used to deliver the
antibody, or an antibody fragment, into cells. Where antibody
fragments are used, the smallest inhibitory fragment that
specifically binds to the binding domain of the target protein is
preferred. For example, based upon the variable-region sequences of
an antibody, peptide molecules can be designed that retain the
ability to bind the target protein sequence. Such peptides can be
synthesized chemically and/or produced by recombinant DNA
technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA,
90: 7889-7893 (1993).
[0846] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Alternatively, or in addition, the
composition may comprise an agent that enhances its function, such
as, for example, a cytotoxic agent, cytokine, chemotherapeutic
agent, or growth-inhibitory agent. Such molecules are suitably
present in combination in amounts that are effective for the
purpose intended.
[0847] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0848] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0849] Commercially available reagents referred to in the examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va.
Example 1
Tissue Expression Profiling Using GeneExpress.RTM.
[0850] A proprietary database containing gene expression
information (GeneExpress.RTM., Gene Logic Inc., Gaithersburg, Md.)
was analyzed in an attempt to identify polypeptides (and their
encoding nucleic acids) whose expression is significantly
upregulated in a particular tumor tissue(s) of interest as compared
to other tumor(s) and/or normal tissues. Specifically, analysis of
the GeneExpress.RTM. database was conducted using either software
available through Gene Logic Inc., Gaithersburg, Md., for use with
the GeneExpress.RTM. database or with proprietary software written
and developed at Genentech, Inc. for use with the GeneExpress.RTM.
database. The rating of positive hits in the analysis is based upon
several criteria including, for example, tissue specificity, tumor
specificity and expression level in normal essential and/or normal
proliferating tissues. The following is a list of molecules whose
tissue expression profile as determined from an analysis of the
GeneExpress.RTM. database evidences high tissue expression and
significant upregulation of expression in a specific tumor or
tumors as compared to other tumor(s) and/or normal tissues and
optionally relatively low expression in normal essential and/or
normal proliferating tissues. As such, the molecules listed below
are excellent polypeptide targets for the diagnosis and therapy of
cancer in mammals. TABLE-US-00006 upregulation of Molecule
expression in: as compared to: DNA96792 colon tumor normal colon
tissue (TAT239) DNA96792 rectum tumor normal rectum tissue (TAT239)
DNA96792 pancreas tumor normal pancreas tissue (TAT239) DNA96792
lung tumor normal lung tissue (TAT239) DNA96792 stomach tumor
normal stomach tissue (TAT239) DNA96792 esophagus tumor normal
esophagus (TAT239) tissue DNA96792 breast tumor normal breast
tissue (TAT239) DNA96792 uterus tumor normal uterus tissue (TAT239)
DNA225793 ovarian tumor normal ovarian tissue (TAT223) DNA225793
kidney tumor normal kidney tissue (TAT223) DNA227611 prostate tumor
normal prostate tissue (TAT175) DNA227611 colon tumor normal colon
tissue (TAT175) DNA227611 breast tumor normal breast tissue
(TAT175) DNA261021 breast tumor normal breast tissue (TAT208)
DNA260655 lung tumor normal lung tissue (TAT209) DNA260655 colon
tumor normal colon tissue (TAT209) DNA260655 breast tumor normal
breast tissue (TAT209) DNA260655 liver tumor normal liver tissue
(TAT209) DNA260655 ovarian tumor normal ovarian tissue (TAT209)
DNA260655 skin tumor normal skin tissue (TAT209) DNA260655 spleen
tumor normal spleen tissue (TAT209) DNA260655 myeloid tumor normal
myeloid tissue (TAT209) DNA260655 muscle tumor normal muscle tissue
(TAT209) DNA260655 bone tumor normal bone tissue (TAT209) DNA261001
bone tumor normal bone tissue (TAT181) DNA261001 lung tumor normal
lung tissue (TAT181) DNA266928 bone tumor normal bone tissue
(TAT182) DNA266928 lung tumor normal lung tissue (TAT182) DNA268035
breast tumor normal breast tissue (TAT222) DNA268035 colon tumor
normal colon tissue (TAT222) DNA268035 ovarian tumor normal ovarian
tissue (TAT222) DNA268035 uterine tumor normal uterine tissue
(TAT222) DNA77509 colon tumor normal colon tissue (TAT177) DNA87993
breast tumor normal breast tissue (TAT235) DNA87993 pancreatic
tumor normal pancreatic (TAT235) tissue DNA87993 lung tumor normal
lung tissue (TAT235) DNA87993 colon tumor normal colon tissue
(TAT235) DNA87993 rectum tumor normal rectum tissue (TAT235)
DNA87993 gallbladder tumor normal gallbladder (TAT235) tissue
DNA92980 bone tumor normal bone tissue (TAT234) DNA92980 breast
tumor normal breast tissue (TAT234) DNA92980 cervical tumor normal
cervical tissue (TAT234) DNA92980 colon tumor normal colon tissue
(TAT234) DNA92980 rectum tumor normal rectum tissue (TAT234)
DNA92980 endometrial tumor normal endometrial (TAT234) tissue
DNA92980 liver tumor normal liver tissue (TAT234) DNA92980 lung
tumor normal lung tissue (TAT234) DNA92980 ovarian tumor normal
ovarian tissue (TAT234) DNA92980 pancreatic tumor normal pancreatic
(TAT234) tissue DNA92980 skin tumor normal skin tissue (TAT234)
DNA92980 soft tissue tumor normal soft tissue (TAT234) DNA92980
stomach tumor normal stomach tissue (TAT234) DNA92980 bladder tumor
normal bladder tissue (TAT234) DNA92980 thyroid tumor normal
thyroid tissue (TAT234) DNA105792 bone tumor normal bone tissue
(TAT233) DNA105792 breast tumor normal breast tissue (TAT233)
DNA105792 endometrial tumor normal endometrial (TAT233) tissue
DNA105792 esophagus tumor normal esophagus (TAT233) tissue
DNA105792 kidney tumor normal kidney tissue (TAT233) DNA105792 lung
tumor normal lung tissue (TAT233) DNA105792 ovarian tumor normal
ovarian tissue (TAT233) DNA105792 pancreatic tumor normal
pancreatic (TAT233) tissue DNA105792 prostate tumor normal prostate
tissue (TAT233) DNA105792 soft tissue tumor normal soft tissue
(TAT233) DNA105792 stomach tumor normal stomach tissue (TAT233)
DNA105792 thyroid tumor normal thyroid tissue (TAT233) DNA105792
bladder tumor normal bladder tissue (TAT233) DNA105792 brain tumor
normal brain tissue (TAT233) DNA105792 Wilm's tumor normal
associated (TAT233) tissue DNA119474 uterine tumor normal uterine
tissue (TAT228) DNA119474 ovarian tumor normal ovarian tissue
(TAT228) DNA280351 squamous cell lung normal squamous cell (TAT248)
tumor lung tissue DNA280351 colon tumor normal colon tissue
(TAT248) DNA150648 liver tumor normal liver tissue (TAT232)
DNA150648 breast tumor normal breast tissue (TAT232) DNA150648
brain tumor normal brain tissue
(TAT232) DNA150648 lung tumor normal lung tissue (TAT232) DNA150648
colon tumor normal colon tissue (TAT232) DNA150648 rectum tumor
normal rectum tissue (TAT232) DNA150648 kidney tumor normal kidney
tissue (TAT232) DNA150648 bladder tumor normal bladder tissue
(TAT232) DNA179651 breast tumor normal breast tissue (TAT224)
DNA179651 cervical tumor normal cervical tissue (TAT224) DNA179651
colon tumor normal colon tissue (TAT224) DNA179651 rectum tumor
normal rectum tissue (TAT224) DNA179651 uterine tumor normal
uterine tissue (TAT224) DNA179651 lung tumor normal lung tissue
(TAT224) DNA179651 ovarian tumor normal ovarian tissue (TAT224)
DNA207698 breast tumor normal breast tissue (TAT237) DNA207698
colon tumor normal colon tissue (TAT237) DNA207698 ovarian tumor
normal ovarian tissue (TAT237) DNA207698 pancreatic tumor normal
pancreatic (TAT237) tissue DNA207698 stomach tumor normal stomach
tissue (TAT237) DNA225886 breast tumor normal breast tissue
(TAT236) DNA225886 colon tumor normal colon tissue (TAT236)
DNA225886 rectum tumor normal rectum tissue (TAT236) DNA225886
endometrial tumor normal endometrial (TAT236) tissue DNA225886 lung
tumor normal lung tissue (TAT236) DNA225886 ovarian tumor normal
ovarian tissue (TAT236) DNA225886 pancreas tumor normal pancreas
tissue (TAT236) DNA225886 prostate tumor normal prostate tissue
(TAT236) DNA225886 bladder tumor normal bladder tissue (TAT236)
DNA226717 glioma normal glial tissue (TAT185) DNA226717 brain tumor
normal brain tissue (TAT185) DNA227162 breast tumor normal breast
tissue (TAT225) DNA227162 endometrial tumor normal endometrial
(TAT225) tissue DNA227162 lung tumor normal lung tissue (TAT225)
DNA227162 ovarian tumor normal ovarian tissue (TAT225) DNA277804
breast tumor normal breast tissue (TAT247) DNA277804 endometrial
tumor normal endometrial (TAT247) tissue DNA277804 lung tumor
normal lung tissue (TAT247) DNA277804 ovarian tumor normal ovarian
tissue (TAT247) DNA233034 glioma normal glial tissue (TAT174)
DNA233034 brain tumor normal brain tissue (TAT174) DNA266920 glioma
normal glial tissue (TAT214) DNA266920 brain tumor normal brain
tissue (TAT214) DNA266921 glioma normal glial tissue (TAT220)
DNA266921 brain tumor normal brain tissue (TAT220) DNA266922 glioma
normal glial tissue (TAT221) DNA266922 brain tumor normal brain
tissue (TAT221) DNA234441 colon tumor normal colon tissue (TAT201)
DNA234441 rectum tumor normal rectum tissue (TAT201) DNA234834
breast tumor normal breast tissue (TAT179) DNA234834 colon tumor
normal colon tissue (TAT179) DNA234834 rectum tumor normal rectum
tissue (TAT179) DNA234834 prostate tumor normal prostate tissue
(TAT179) DNA234834 pancreatic tumor normal pancreatic (TAT179)
tissue DNA234834 endometrial tumor normal endometrial (TAT179)
tissue DNA234834 lung tumor normal lung tissue (TAT179) DNA234834
ovarian tumor normal ovarian tissue (TAT179) DNA247587 breast tumor
normal breast tissue (TAT216) DNA247587 lung tumor normal lung
tissue (TAT216) DNA247587 ovarian tumor normal ovarian tissue
(TAT216) DNA247587 pancreatic tumor normal pancreatic (TAT216)
tissue DNA247587 stomach tumor normal stomach tissue (TAT216)
DNA247587 urinary tumor normal urinary tissue (TAT216) DNA255987
breast tumor normal breast tissue (TAT218) DNA56041 lymphoid tumor
normal lymphoid tissue (TAT206) DNA257845 lymphoid tumor normal
lymphoid tissue (TAT374) DNA247476 bone tumor normal bone tissue
(TAT180) DNA247476 breast tumor normal breast tissue (TAT180)
DNA247476 colon tumor normal colon tissue (TAT180) DNA247476 rectum
tumor normal rectum tissue (TAT180) DNA247476 kidney tumor normal
kidney tissue (TAT180) DNA247476 lung tumor normal lung tissue
(TAT180) DNA247476 pancreatic tumor normal pancreatic (TAT180)
tissue DNA247476 prostate tumor normal prostate tissue (TAT180)
DNA247476 skin tumor normal skin tissue (TAT180) DNA247476 soft
tissue tumor normal soft tissue (TAT180) DNA247476 stomach tumor
normal stomach tissue (TAT180) DNA260990 bone tumor normal bone
tissue (TAT375) DNA260990 breast tumor normal breast tissue
(TAT375) DNA260990 colon tumor normal colon tissue (TAT375)
DNA260990 rectum tumor normal rectum tissue (TAT375) DNA260990
kidney tumor normal kidney tissue (TAT375) DNA260990 lung tumor
normal lung tissue (TAT375) DNA260990 pancreatic tumor normal
pancreatic (TAT375) tissue DNA260990 prostate tumor normal prostate
tissue (TAT375) DNA260990 skin tumor normal skin tissue
(TAT375)
DNA260990 soft tissue tumor normal soft tissue (TAT375) DNA260990
stomach tumor normal stomach tissue (TAT375) DNA261013 breast tumor
normal breast tissue (TAT176) DNA261013 colon tumor normal colon
tissue (TAT176) DNA261013 rectum tumor normal rectum tissue
(TAT176) DNA261013 lung tumor normal lung tissue (TAT176) DNA261013
ovarian tumor normal ovarian tissue (TAT176) DNA261013 stomach
tumor normal stomach tissue (TAT176) DNA262144 breast tumor normal
breast tissue (TAT184) DNA262144 colon tumor normal colon tissue
(TAT184) DNA262144 rectum tumor normal rectum tissue (TAT184)
DNA262144 endometrial tumor normal endometrial (TAT184) tissue
DNA262144 kidney tumor normal kidney tissue (TAT184) DNA262144 lung
tumor normal lung tissue (TAT184) DNA262144 ovarian tumor normal
ovarian tissue (TAT184) DNA267342 stroma associated normal
associated (TAT213)) with the following tissues, respectively
tumors: bone, breast, colon, rectum, lung, ovarian, pancreas, soft
tissue, bladder DNA267626 breast tumor normal breast tissue
(TAT217) DNA267626 colon tumor normal colon tissue (TAT217)
DNA267626 rectum tumor normal rectum tissue (TAT217) DNA267626
endometrial tumor normal endometrial (TAT217) tissue DNA267626 lung
tumor normal lung tissue (TAT217) DNA267626 pancreatic tumor normal
pancreatic (TAT217) tissue DNA268334 kidney tumor normal kidney
tissue (TAT202) DNA269238 kidney tumor normal kidney tissue
(TAT215) DNA272578 liver tumor normal liver tissue (TAT238)
DNA272578 lung tumor normal lung tissue (TAT238) DNA272578 ovarian
tumor normal ovarian tissue (TAT238) DNA304853 breast tumor normal
breast tissue (TAT376) DNA304853 colon tumor normal colon tissue
(TAT376) DNA304853 rectum tumor normal rectum tissue (TAT376)
DNA304853 prostate tumor normal prostate tissue (TAT376) DNA304853
pancreatic tumor normal pancreatic (TAT376) tissue DNA304853
endometrial tumor normal endometrial (TAT376) tissue DNA304853 lung
tumor normal lung tissue (TAT376) DNA304853 ovarian tumor normal
ovarian tissue (TAT376) DNA304854 breast tumor normal breast tissue
(TAT377) DNA304854 colon tumor normal colon tissue (TAT377)
DNA304854 rectum tumor normal rectum tissue (TAT377) DNA304854
prostate tumor normal prostate tissue (TAT377) DNA304854 pancreatic
tumor normal pancreatic (TAT377) tissue DNA304854 endometrial tumor
normal endometrial (TAT377) tissue DNA304854 lung tumor normal lung
tissue (TAT377) DNA304854 ovarian tumor normal ovarian tissue
(TAT377) DNA304855 breast tumor normal breast tissue (TAT378)
DNA304855 colon tumor normal colon tissue (TAT378) DNA304855 rectum
tumor normal rectum tissue (TAT378) DNA304855 prostate tumor normal
prostate tissue (TAT378) DNA304855 pancreatic tumor normal
pancreatic (TAT378) tissue DNA304855 endometrial tumor normal
endometrial (TAT378) tissue DNA304855 lung tumor normal lung tissue
(TAT378) DNA304855 ovarian tumor normal ovarian tissue (TAT378)
DNA287971 bone tumor normal bone tissue (TAT379) DNA287971 breast
tumor normal breast tissue (TAT379) DNA287971 colon tumor normal
colon tissue (TAT379) DNA287971 rectum tumor normal rectum tissue
(TAT379) DNA287971 kidney tumor normal kidney tissue (TAT379)
DNA287971 lung tumor normal lung tissue (TAT379) DNA287971
pancreatic tumor normal pancreatic (TAT379) tissue DNA287971
prostate tumor normal prostate tissue (TAT379) DNA287971 skin tumor
normal skin tissue (TA1379) DNA287971 soft tissue tumor normal soft
tissue (TAT379) DNA287971 stomach tumor normal stomach tissue
(TAT379)
Example 2
Microarray Analysis to Detect Upregulation of TAT Polypeptides in
Cancerous Tumors
[0851] Nucleic acid microarrays, often containing thousands of gene
sequences, are useful for identifying differentially expressed
genes in diseased tissues as compared to their normal counterparts.
Using nucleic acid microarrays, test and control mRNA samples from
test and control tissue samples are reverse transcribed and labeled
to generate cDNA probes. The cDNA probes are then hybridized to an
array of nucleic acids immobilized on a solid support. The array is
configured such that the sequence and position of each member of
the array is known. For example, a selection of genes known to be
expressed in certain disease states may be arrayed on a solid
support. Hybridization of a labeled probe with a particular array
member indicates that the sample from which the probe was derived
expresses that gene. If the hybridization signal of a probe from a
test (disease tissue) sample is greater than hybridization signal
of a probe from a control (normal tissue) sample, the gene or genes
overexpressed in the disease tissue are identified. The implication
of this result is that an overexpressed protein in a diseased
tissue is useful not only as a diagnostic marker for the presence
of the disease condition, but also as a therapeutic target for
treatment of the disease condition.
[0852] The methodology of hybridization of nucleic acids and
microarray technology is well known in the art. In one example, the
specific preparation of nucleic acids for hybridization and probes,
slides, and hybridization conditions are all detailed in PCT Patent
Application Serial No. PCT/US01/10482, filed on Mar. 30, 2001 and
which is herein incorporated by reference.
[0853] In the present example, cancerous tumors derived from
various human tissues were studied for upregulated gene expression
relative to cancerous tumors from different tissue types and/or
non-cancerous human tissues in an attempt to identify those
polypeptides which are overexpressed in a particular cancerous
tumor(s). In certain experiments, cancerous human tumor tissue and
non-cancerous human tumor tissue of the same tissue type (often
from the same patient) were obtained and analyzed for TAT
polypeptide expression. Additionally, cancerous human tumor tissue
from any of a variety of different human tumors was obtained and
compared to a "universal" epithelial control sample which was
prepared by pooling non-cancerous human tissues of epithelial
origin, including liver, kidney, and lung. mRNA isolated from the
pooled tissues represents a mixture of expressed gene products from
these different tissues. Microarray hybridization experiments using
the pooled control samples generated a linear plot in a 2-color
analysis. The slope of the line generated in a 2-color analysis was
then used to normalize the ratios of (test:control detection)
within each experiment. The normalized ratios from various
experiments were then compared and used to identify clustering of
gene expression. Thus, the pooled "universal control" sample not
only allowed effective relative gene expression determinations in a
simple 2-sample comparison, it also allowed multi-sample
comparisons across several experiments.
[0854] In the present experiments, nucleic acid probes derived from
the herein described TAT polypeptide-encoding nucleic acid
sequences were used in the creation of the microarray and RNA from
various tumor tissues were used for the hybridization thereto.
Below is shown the results of these experiments, demonstrating that
various TAT polypeptides of the present invention are significantly
overexpressed in various human tumor tissues as compared to their
normal counterpart tissue(s). Moreover, all of the molecules shown
below are significantly overexpressed in their specific tumor
tissue(s) as compared to in the "universal" epithelial control. As
described above, these data demonstrate that the TAT polypeptides
of the present invention are useful not only as diagnostic markers
for the presence of one or more cancerous tumors, but also serve as
therapeutic targets for the treatment of those tumors.
TABLE-US-00007 upregulation of Molecule expression in: as compared
to: DNA172500 renal cell normal kidney (renal (TAT219) carcinoma
cell) tissue
Example 3
Quantitative Analysis of TAT mRNA Expression
[0855] In this assay, a 5' nuclease assay (for example,
TaqMan.RTM.) and real-time quantitative PCR (for example, ABI Prizm
7700 Sequence Detection System.RTM. (Perkin Elmer, Applied
Biosystems Division, Foster City, Calif.)), were used to find genes
that are significantly overexpressed in a cancerous tumor or tumors
as compared to other cancerous tumors or normal non-cancerous
tissue. The 5' nuclease assay reaction is a fluorescent PCR-based
technique which makes use of the 5' exonuclease activity of Taq DNA
polymerase enzyme to monitor gene expression in real time. Two
oligonucleotide primers (whose sequences are based upon the gene or
EST sequence of interest) are used to generate an amplicon typical
of a PCR reaction. A third oligonucleotide, or probe, is designed
to detect nucleotide sequence located between the two PCR primers.
The probe is non-extendible by Taq DNA polymerase enzyme, and is
labeled with a reporter fluorescent dye and a quencher fluorescent
dye. Any laser-induced emission from the reporter dye is quenched
by the quenching dye when the two dyes are located close together
as they are on the probe. During the PCR amplification reaction,
the Taq DNA polymerase enzyme cleaves the probe in a
template-dependent manner. The resultant probe fragments
disassociate in solution, and signal from the released reporter dye
is free from the quenching effect of the second fluorophore. One
molecule of reporter dye is liberated for each new molecule
synthesized, and detection of the unquenched reporter dye provides
the basis for quantitative interpretation of the data.
[0856] The 5' nuclease procedure is run on a real-time quantitative
PCR device such as the ABI Prism 7700.TM. Sequence Detection. The
system consists of a thermocycler, laser, charge-coupled device
(CCD) camera and computer. The system amplifies samples in a
96-well format on a thermocycler. During amplification,
laser-induced fluorescent signal is collected in real-time through
fiber optics cables for all 96 wells, and detected at the CCD. The
system includes software for running the instrument and for
analyzing the data.
[0857] The starting material for the screen was mRNA isolated from
a variety of different cancerous tissues. The mRNA is quantitated
precisely, e.g., fluorometrically. As a negative control, RNA was
isolated from various normal tissues of the same tissue type as the
cancerous tissues being tested.
[0858] 5' nuclease assay data are initially expressed as Ct, or the
threshold cycle. This is defined as the cycle at which the reporter
signal accumulates above the background level of fluorescence. The
ACt values are used as quantitative measurement of the relative
number of starting copies of a particular target sequence in a
nucleic acid sample when comparing cancer mRNA results to normal
human mRNA results. As one Ct unit corresponds to 1 PCR cycle or
approximately a 2-fold relative increase relative to normal, two
units corresponds to a 4-fold relative increase, 3 units
corresponds to an 8-fold relative increase and so on, one can
quantitatively measure the relative fold increase in mRNA
expression between two or more different tissues. Using this
technique, the molecules listed below have been identified as being
significantly overexpressed in a particular tumor(s) as compared to
their normal non-cancerous counterpart tissue(s) (from both the
same and different tissue donors) and thus, represent excellent
polypeptide targets for the diagnosis and therapy of cancer in
mammals. TABLE-US-00008 upregulation of Molecule expression in: as
compared to: DNA261021 lung tumor normal lung tissue (TAT208)
DNA77509 colon tumor normal colon tissue (TAT177) DNA119474 ovarian
tumor normal ovarian tissue (TAT226) DNA179651 ovarian tumor normal
ovarian tissue (TAT224) DNA226717 glioma normal glial/brain
(TAT185) tissue DNA227162 ovarian tumor normal ovarian tissue
(TAT225) DNA277804 ovarian tumor normal ovarian tissue (TAT247)
DNA233034 glioma normal glial/brain (TAT174) tissue DNA266920
glioma normal glial/brain (TAT214) tissue DNA266921 glioma normal
glial/brain (TAT220) tissue DNA266922 glioma normal glial/brain
(TAT221) tissue DNA234441 colon tumor normal colon tissue (TAT201)
DNA234834 colon tumor normal colon tissue (TAT179) DNA247587
squamous cell lung normal squamous cell (TAT216) tumor lung tissue
DNA255987 breast tumor normal breast tissue (TAT218) DNA247476
colon tumor normal colon tissue (TAT180) DNA260990 colon tumor
normal colon tissue (TAT375) DNA261013 breast tumor normal breast
tissue (TAT176) DNA262144 kidney tumor normal kidney tissue
(TAT184) DNA267342 breast tumor normal breast tissue (TAT213)
DNA267626 breast tumor normal breast tissue (TAT217) DNA268334
kidney tumor normal kidney tissue (TAT202) DNA269238 kidney tumor
normal kidney tissue (TAT215) DNA87993 lung tumor normal lung
tissue (TAT235) DNA92980 ovarian tumor normal ovarian tissue
(TAT234) DNA105792 lung tumor normal lung tissue (TAT233) DNA207698
colon tumor normal colon tissue (TAT237) DNA225886 colon tumor
normal colon tissue (TAT236) DNA272578 ovarian tumor normal ovarian
tissue (TAT238) DNA304853 colon tumor normal colon tissue (TAT376)
DNA304854 colon tumor normal colon tissue (TAT377) DNA304855 colon
tumor normal colon tissue (TAT378) DNA287971 colon tumor normal
colon tissue (TAT379)
Example 4
In Situ Hybridization
[0859] In situ hybridization is a powerful and versatile technique
for the detection and localization of nucleic acid sequences within
cell or tissue preparations. It may be useful, for example, to
identify sites of gene expression, analyze the tissue distribution
of transcription, identify and localize viral infection, follow
changes in specific mRNA synthesis and aid in chromosome
mapping.
[0860] In situ hybridization was performed following an optimized
version of the protocol by Lu and Gillett, Cell Vision 1: 169-176
(1994), using PCR-generated .sup.33P-labeled riboprobes. Briefly,
formalin-fixed, paraffin-embedded human tissues were sectioned,
deparaffinized, deproteinated in proteinase K (20 g/ml) for 15
minutes at 37.degree. C., and further processed for in situ
hybridization as described by Lu and Gillett, supra. A [.sup.33-P]
UTP-labeled antisense riboprobe was generated from a PCR product
and hybridized at 55.degree. C. overnight. The slides were dipped
in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.
.sup.33P-Riboprobe Synthesis
[0861] 6.0 .mu.l (125 mCi) of .sup.33P-UTP (Amersham BF 1002,
SA<2000 Ci/mmol) were speed vac dried. To each tube containing
dried .sup.33P-UTP, the following ingredients were added:
[0862] 2.0 .mu.l 5.times. transcription buffer
[0863] 1.0 .mu.l DTT (100 mM)
[0864] 2.0 .mu.l NTP mix (2.5 mM: 10.mu.; each of 10 mM GTP, CTP
& ATP+10 .mu.l H.sub.2O)
[0865] 1.0 .mu.l UTP (50 .mu.M)
[0866] 1.0 .mu.l Rnasin
[0867] 1.0 .mu.l DNA template (1 .mu.g)
[0868] 1.0 .mu.l H.sub.2O
[0869] 1.0 .mu.l RNA polymerase (for PCR products T3=AS, T7=S,
usually)
[0870] The tubes were incubated at 37.degree. C. for one hour. 1.0
.mu.l RQ1 DNase were added, followed by incubation at 37.degree. C.
for 15 minutes. 90 .mu.l TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0)
were added, and the mixture was pipetted onto DE81 paper. The
remaining solution was loaded in a Microcon-50 ultrafiltration
unit, and spun using program 10 (6 minutes). The filtration unit
was inverted over a second tube and spun using program 2 (3
minutes). After the final recovery spin, 100 .mu.l TE were added. 1
.mu.l of the final product was pipetted on DE81 paper and counted
in 6 ml of Biofluor II.
[0871] The probe was run on a TBE/urea gel. 1-3 .mu.l of the probe
or 5 .mu.l of RNA Mrk III were added to 3 .mu.l of loading buffer.
After heating on a 95.degree. C. heat block for three minutes, the
probe was immediately placed on ice. The wells of gel were flushed,
the sample loaded, and run at 180-250 volts for 45 minutes. The gel
was wrapped in saran wrap and exposed to XAR film with an
intensifying screen in -70.degree. C. freezer one hour to
overnight.
.sup.33P-Hybridization
[0872] A. Pretreatment of Frozen Sections
[0873] The slides were removed from the freezer, placed on
aluminium trays and thawed at room temperature for 5 minutes. The
trays were placed in 55.degree. C. incubator for five minutes to
reduce condensation. The slides were fixed for 10 minutes in 4%
paraformaldehyde on ice in the fume hood, and washed in
0.5.times.SSC for 5 minutes, at room temperature (25 ml
20.times.SSC+975 ml SQ H.sub.2O). After deproteination in 0.5
.mu.g/ml proteinase K for 10 minutes at 37.degree. C. (12.5 .mu.l
of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), the
sections were washed in 0.5.times.SSC for 10 minutes at room
temperature. The sections were dehydrated in 70%, 95%, 100%
ethanol, 2 minutes each.
[0874] B. Pretreatment of Paraffin-Embedded Sections
[0875] The slides were deparaffinized, placed in SQ H.sub.2O, and
rinsed twice in 2.times.SSC at room temperature, for 5 minutes each
time. The sections were deproteinated in 20 .mu.g/ml proteinase K
(500 .mu.l of 10 mg/ml in 250 ml RNase-free RNase buffer;
37.degree. C., 15 minutes)-human embryo, or 8.times. proteinase K
(100 .mu.l in 250 ml Rnase buffer, 37.degree. C., 30
minutes)--formalin tissues. Subsequent rinsing in 0.5.times.SSC and
dehydration were performed as described above.
[0876] C. Prehybridization
[0877] The slides were laid out in a plastic box lined with Box
buffer (4.times.SSC, 50% formamide)-saturated filter paper.
[0878] D. Hybridization
[0879] 1.0.times.10.sup.6 cpm probe and 1.0 .mu.l tRNA (50 mg/ml
stock) per slide were heated at 95.degree. C. for 3 minutes. The
slides were cooled on ice, and 48 .mu.l hybridization buffer were
added per slide. After voitexing, 50 .mu.l .sup.33P mix were added
to 50 .mu.l prehybridization on slide. The slides were incubated
overnight at 55.degree. C.
[0880] E. Washes
[0881] Washing was done 2.times.10 minutes with 2.times.SSC, EDTA
at room temperature (400 ml 20.times.SSC+16 ml 0.25M EDTA,
V.sub.f=4 L), followed by RNaseA treatment at 37.degree. C. for 30
minutes (500 .mu.l of 10 mg/ml in 250 ml Rnase buffer=20 .mu.g/ml),
The slides were washed 2.times.10 minutes with 2.times.SSC, EDTA at
room temperature. The stringency wash conditions were as follows: 2
hours at 55.degree. C., 0.1.times.SSC, EDTA (20 ml 20.times.SSC+16
ml EDTA, V.sub.f=4 L).
[0882] F. Oligonucleotides
[0883] In situ analysis was performed on a variety of DNA sequences
disclosed herein. The oligonucleotides employed for these analyses
were obtained so as to be complementary to the nucleic acids (or
the complements thereof) as shown in the accompanying figures.
[0884] G. Results
[0885] In situ analysis was performed on a variety of DNA sequences
disclosed herein. The results from these analyses are as
follows.
(1) DNA119474 (TAT226)
[0886] Positive expression is observed in 2 of 3 non-small cell
lung carcinomsa, 2 of 3 pancreatic adenocarcinomas, 1 of 2
hepatocellular carcinomas and 2 of 3 endometrial adenocarcinomas.
In a separate analysis, 10 of 16 ovarian adenocarcinomas are
positive and 3 of 9 endometrial adenocarcinomas are positive. All
normal tissues examined are negative for expression.
(2) DNA179651 (TAT224)
[0887] In one analysis, expression is seen in 5 of 7 uterine
adenocarcinomas and in 7 of 16 ovarian adenocarcinomas. Two cases
of dysgerminoma are positive as is one case of a Brenner's
tumor.
[0888] In another analysis, 33 of 68 ovarian adenocarcinomas
(serous, mucinous, endometrioid, clear cell) are positive for
expression. Moderate to strong expression is seen in normal
endometrium (no other normal tissues) and normal ovarian stroma is
negative.
[0889] In yet another analysis, positive:expression is seen in 3/3
endometrial, 2/2 colorectal, 1/3 transitional cell, 3/3 lung and
1/2 ovarian cancers.
(3) DNA227162 (TAT225)
[0890] Expression is seen in the following tumors: 1 of 3 lung
cancers, 1 of 2 colon cancers, 1 of 1 pancreatic cancer, 2 of 3
transitional cell carcinomas, 3 of 3 endometrial carcinomas, 2 of 2
ovarian carcinomas and 2 of 3 malignant melanomas.
[0891] In a separate analysis, positive expression is seen in 6 of
9 uterine adenocarcinomas and 6 of 14 ovarian tumors.
[0892] With regard to expression in normal tissues, weak expression
is seen in one core of urothelium (superficial cell layer positive)
and one core of gall bladder mucosa. All other normal tissues are
negative for expression.
(4) DNA277804 (TAT247)
[0893] Expression is seen in the following tumors: 1 of 3 lung
cancers, 1 of 2 colon cancers, 1 of 1 pancreatic cancer, 2 of 3
transitional cell carcinomas, 3 of 3 endometrial carcinomas, 2 of 2
ovarian carcinomas and 2 of 3 malignant melanomas.
[0894] In a separate analysis, positive expression is seen in 6 of
9 uterine adenocarcinomas and 6 of 14 ovarian tumors.
[0895] With regard to expression in normal tissues, weak expression
is seen in one core of urothelium (superficial cell layer positive)
and one core of gall bladder mucosa. All other normal tissues are
negative for expression.
(5) DNA234441 (TAT201)
[0896] Weak (and inconsistent) expression is seen in normal kidney,
normal colon mucosa and normal gallbladder. Weak to moderate,
though somewhat inconsistent expression is seen in normal
gastrointestinal mucosa (esophagus, stomach, small intestine,
colon, anus). Significant expression in tumors is seen as follows:
11 of 12 colorectal adenocarcinomas, 4 of 4 gastric
adenocarcinomas, 6 of 8 metastatic adenocarcinomas, 4 of 4
esophageal cancers and 1 of 2 pancreatic adenocarcinomas.
(6) DNA234834 (TAT179)
[0897] With regard to normal tissues, it appears that there is a
weak signal in colon mucosa and breast epithelium. With regard to
tumor tissues, expression is seen in 1 of 2 non-small cell lung
carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2
hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2
ovarian carcinomas and 2 of 3 malignant melanomas.
[0898] In a separate analysis, 12 of 16 colorectal carcinomas are
positive for expression; 2 of 8 gastric adenocarcinoma are positive
for expression, 2 of 4 esophageal carcinomas are positive for
expression; 7 of 10 metastatic adenocarcinoma are positive for
expression and 1 of 2 cholangiocarcinomas are positive for
expression. Expression level is tumor tissues is consistently
higher than in normal tissues.
(7) DNA247587 (TAT216)
[0899] Expression is seen in 13 of 16 non-small cell lung
carcinomas. Expression is also seen in benign bronchial mucosa and
occasional activated pneumocytes. Moreover, 65 of 89 cases of
invasive breast cancer are positive for expression. Strong
expression is seen in normal skin and normal urothelium. Moderate
expression is seen in normal mammary epithelium and trophoblasts of
the placenta, weak expression in normal prostate and normal gall
bladder epithelium and distal renal tubules.
(8) DNA56041 (TAT206)
[0900] In non-malignant lymphoid tissue expression is seen in
occasional larger lymphoid cells within germinal centers and in
interfollicular regions. Positive cells account for less than 5% of
all lymphoid cells. In section of spleen scattered positive cells
are seen within the periarteriolar lymphoid sheath and in the
marginal zone.
[0901] In four cases of Hodgkin's disease Reed-Sternberg cells are
negative, positive signal is observed in scattered lymphocytes.
Three of four cases of follicular lymphoma are positive (weak to
moderate), four of six cases of diffuse large cell lymphoma are
positive (weak to moderate). Two cases of small lymphocytic
lymphoma show a weak signal in variable proportion of cells.
(9) DNA257845 (TAT374)
[0902] In non-malignant lymphoid tissue expression is seen in
occasional larger lymphoid cells within germinal centers and in
interfollicular regions. Positive cells account for less than 5% of
all lymphoid cells. In section of spleen scattered positive cells
are seen within the periarteriolar lymphoid sheath and in the
marginal zone.
[0903] In four cases of Hodgkin's disease Reed-Sternberg cells are
negative, positive signal is observed in scattered lymphocytes.
Three of four cases of follicular lymphoma are positive (weak to
moderate), four of six cases of diffuse large cell lymphoma are
positive (weak to moderate). Two cases of small lymphocytic
lymphoma show a weak signal in variable proportion of cells.
(10) DNA247476 (TAT180)
[0904] With regard to normal tissues, strong expression is seen in
prostatic epithelium and in a section of peripheral nerve. Moderate
expression is seen in renal glomeruli. Weak expression is seen in
bile duct epithelium and mammary epithelium. Two sections of
stomach show weak expression in a subset of gastric glands.
Sections of colon and small intestine show a signal in lamina
propria and/or submucosa, most likely in small autonomic nerve
fibers. Another independent ISH study fails to show expression in
peripheral nerves of prostatectomy sections, despite adequate
signal in prostatic epithelium.
[0905] In a separate analysis, 42 of 77 breast tumors are positive
(55%) for expression.
[0906] In yet another analysis, 8 of 11 breast cancers are positive
for expression.
[0907] In yet another analysis, expression is seen in 1/2 non-small
cell lung carcinomas, 1/3 colorectal adenocarcinomas, 2/3
pancreatic adenocarcinomas, 1/1 prostate cancers, 1/3 transitional
cell carcinomas, 3/3 renal cell carcinomas, 3/3 endometrial
adenocarcinomas, 1/2 ovarian adenocarcinomas and 1/3 malignant
melanomas.
[0908] In yet another analysis, expression is seen in 42 of 45
(93%) prostate cancers.
[0909] In yet another analysis, expression is seen in all of 23
primary and in 12 of 15 (80%) metastatic prostate cancers
analyzed.
[0910] In yet another analysis, expression is observed in the
following carcinomas as follows: pancreatic adenocarcinoma--b 2 of
2 cases are positive; colorectal adenocarcinoma--12 of 14 cases are
positive; gastric adenocarcinoma--6 of 8 cases are positive;
esophageal carcinoma--2 of 3 cases are positive;
cholangiocarcinoma--1 of 1 case is positive; metastatic
adenocarcinoma (ovary, liver, lymph node, diaphragm)--8 of 12 cases
are positive.
(11) DNA260990 (TAT375)
[0911] With regard to normal tissues, strong expression is seen in
prostatic epithelium and in a section of peripheral nerve. Moderate
expression is seen in renal glomeruli. Weak expression is seen in
bile duct epithelium and mammary epithelium. Two sections of
stomach show weak expression in a subset of gastric glands.
Sections of colon and small intestine show a signal in lamina
propria and/or submucosa, most likely in small autonomic nerve
fibers. Another independent ISH study fails to show expression in
peripheral nerves of prostatectomy sections, despite adequate
signal in prostatic epithelium.
[0912] In a separate analysis, 42 of 77 breast tumors are positive
(55%) for expression.
[0913] In yet another analysis, 8 of 11 breast cancers are positive
for expression.
[0914] In yet another analysis, expression is seen in 1/2 non-small
cell lung carcinomas, 1/3 colorectal adenocarcinomas, 2/3
pancreatic adenocarcinomas, 1/1 prostate cancers, 1/3 transitional
cell carcinomas, 3/3 renal cell carcinomas, 3/3 endometrial
adenocarcinomas, 1/2 ovarian adenocarcinomas and 1/3 malignant
melanomas.
[0915] In yet another analysis, expression is seen in 42 of 45
(93%) prostate cancers.
[0916] In yet another analysis, expression is seen in all of 23
primary and in 12 of 15 (80%) metastatic prostate cancers
analyzed.
[0917] In yet another analysis, expression is observed in the
following carcinomas as follows: pancreatic adenocarcinoma--2 of 2
cases are positive; colorectal adenocarcinoma--12 of 14 cases are
positive; gastric adenocarcinoma--6 of 8 cases are positive;
esophageal carcinoma--2 of 3 cases are positive;
cholangiocarcinoma--1 of 1 case is positive; metastatic
adenocarcinoma (ovary, liver, lymph node, diaphragm)--8 of 12 cases
are positive.
(12) DNA261013 (TAT176)
[0918] With regard to normal tissues, prostate epithelium shows a
weak positive signal. Also, one core of colonic mucosa shows a weak
signal in mucosal epithelium. Two cores of a testicular neoplasm
are positive.
[0919] In another analysis, 87 cases of infiltrating ductal breast
cancer are available for review. 40 cases are positive for
expression. Additionally, all tested cell lines (A549, SK-MES,
SKBR3, MDA231, MDA453, MDA175, MCF7) are positive for
expression.
[0920] In another analysis, there is no consistent expression in
benign colon, small intestinal, liver, pancreatic, gastric or
esophageal tissue. In malignant tumors expression is observed as
follows: colorectal adenocarcinoma: 10 of 14 cases are positive,
gastric adenocarcinoma: 4 of 8 cases are positive, esophageal
carcinoma: 3 of 4 cases are positive and metastatic adenocarcinoma:
8 of 11 cases are positive.
(13) DNA262144 (TAT184)
[0921] Two of 4 cases of non-small cell lung carcinoma are positive
for expression while no signal is observed in non-neoplastic lung.
In a separate analysis, three cases of non-small cell lung
carcinoma are positive
(14) DNA267342 (TAT213)
[0922] Expression is not observed in any of the normal adult
tissues tested. Seventy four cases of breast cancer are available
for review and 30 cases give a positive signal Expression localizes
to tumor-associated stroma.
[0923] In a separate analysis, expression is seen in a minority of
sarcomas; moderate and occasionally strong expression is seen in a
case of a synovial sarcoma, angiosarcoma, fibrosarcoma, gliosarcoma
and malignant fibrohistiocytoma. In most cases expression appears
to localize to the malignant cell population.
(15) DNA267626 (TAT217)
[0924] Expression is seen in 6 of 9 invasive breast cancers.
Expression is in most cases of moderate intensity, expression is
also seen in benign mammary epithelium and fibroadenoma. The large
sections included in this study show expression in 1 of 1
endometrial adenocarcinomas, in 2 of 3 invasive ductal breast
cancers, in benign renal tubules, in normal breast epithelium and
in epidermis. Sections of lung, brain, myometrium and eye are
negative.
(16) DNA268334 (TAT202)
[0925] No expression is seen in any of the adult, normal tissues
tested while expression is observed in 3 of 3 renal cell
carcinomas.
(17) DNA269238 (TAT215)
[0926] Tumor-associated vasculature was strongly positive in all
renal cell carcinomas tested (n=6), in all hepatocellular
carcinomas tested (n=3), in all gastric adenocarcinomas tested
(n=5), in all endometrial adenocarcinomas tested (n=3), in all
malignant melanomas tested (n=3), in all malignant lymphomas tested
(n=3), in all pancreatic adenocarcinomas tested (n=1), in all
esophageal carcinomas tested (n=4), in all cholangiocarcinomas
tested (n=2), in 93% of all non-small cell lung cancers tested
(n=15), in 86% of all invasive ductal breast cancers tested (n=88),
in 83% of all colorectal adenocarcinomas tested (n=12), in 67% of
all metastatic adenocarcinomas tested (n=6), in 75% of all
transitional cell carcinomas tested (n=4). While TAT215 expression
is also observed in endothelial components of various normal
non-cancerous tissues, the expression level is significantly lower
in these non-cancerous tissues as compared to their cancerous
counterparts and the expression pattern in the tumor tissues was
distinct from that in the normal tissues, thereby providing a means
for both therapy and diagnosis of the cancerous condition.
(18) DNA304853 (TAT376)
[0927] With regard to normal tissues, it appears that there is a
weak signal in colon mucosa and breast epithelium. With regard to
tumor tissues, expression is seen in 1 of 2 non-small cell lung
carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2
hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2
ovarian carcinomas and 2 of 3 malignant melanomas.
[0928] In a separate analysis, 12 of 16 colorectal carcinomas are
positive for expression; 2 of 8 gastric adenocarcinoma are positive
for expression, 2 of 4 esophageal carcinomas are positive for
expression; 7 of 10 metastatic adenocarcinoma are positive for
expression and 1 of 2 cholangiocarcinomas are positive for
expression. Expression level is tumor tissues is consistently
higher than in normal tissues.
(19) DNA304854 (TAT377)
[0929] With regard to normal tissues, it appears that there is a
weak signal in colon mucosa and breast epithelium. With regard to
tumor tissues, expression is seen in 1 of 2 non-small cell lung
carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2
hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2
ovarian carcinomas and 2 of 3 malignant melanomas.
[0930] In a separate analysis, 12 of 16 colorectal carcinomas are
positive for expression; 2 of 8 gastric adenocarcinoma are positive
for expression, 2 of 4 esophageal carcinomas are positive for
expression; 7 of 10 metastatic adenocarcinoma are positive for
expression and 1 of 2 cholangiocarcinomas are positive for
expression. Expression level is tumor tissues is consistently
higher than in normal tissues.
(20) DNA304855 (TAT378)
[0931] With regard to normal tissues, it appears that there is a
weak signal in colon mucosa and breast epithelium. With regard to
tumor tissues, expression is seen in 1 of 2 non-small cell lung
carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2
hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2
ovarian carcinomas and 2 of 3 malignant melanomas.
[0932] In a separate analysis, 12 of 16 colorectal carcinomas are
positive for expression; 2 of 8 gastric adenocarcinoma are positive
for expression, 2 of 4 esophageal carcinomas are positive for
expression; 7 of 10 metastatic adenocarcinoma are positive for
expression and 1 of 2 cholangiocarcinomas are positive for
expression. Expression level is tumor tissues is consistently
higher than in normal tissues.
(21) DNA287971 (TAT379)
[0933] With regard to normal tissues, strong expression is seen in
prostatic epithelium and in a section of peripheral nerve. Moderate
expression is seen in renal glomeruli. Weak expression is seen in
bile duct epithelium and mammary epithelium. Two sections of
stomach show weak expression in a subset of gastric glands.
Sections of colon and small intestine show a signal in lamina
propria and/or submucosa, most likely in small autonomic nerve
fibers. Another independent ISH study fails to show expression in
peripheral nerves of prostatectomy sections, despite adequate
signal in prostatic epithelium.
[0934] In a separate analysis, 42 of 77 breast tumors are positive
(55%) for expression.
[0935] In yet another analysis, 8 of 11 breast cancers are positive
for expression.
[0936] In yet another analysis, expression is seen in 1/2 non-small
cell lung carcinomas, 1/3 colorectal adenocarcinomas, 2/3
pancreatic adenocarcinomas, 1/1 prostate cancers, 1/3 transitional
cell carcinomas, 3/3 renal cell carcinomas, 3/3 endometrial
adenocarcinomas, 1/2 ovarian adenocarcinomas and 1/3 malignant
melanomas.
[0937] In yet another analysis, expression is seen in 42 of 45
(93%) prostate cancers.
[0938] In yet another analysis, expression is seen in all of 23
primary and in 12 of 15 (80%) metastatic prostate cancers
analyzed.
[0939] In yet another analysis, expression is observed in the
following carcinomas as follows: pancreatic adenocarcinoma--2 of 2
cases are positive; colorectal adenocarcinoma--12 of 14 cases are
positive; gastric adenocarcinoma--6 of 8 cases are positive;
esophageal carcinoma--2 of 3 cases are positive;
cholangiocarcinoma--1 of 1 case is positive; metastatic
adenocarcinoma (ovary, liver, lymph node, diaphragm)--8 of 12 cases
are positive.
Example 5
Verification and Analysis of Differential TAT Polypeptide
Expression by GEPIS
[0940] TAT polypeptides which may have been identified as a tumor
antigen as described in one or more of the above Examples were
analyzed and verified as follows. An expressed sequence tag (EST)
DNA database (LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto,
Calif.) was searched and interesting EST sequences were identified
by GEPIS. Gene expression profiling in silico (GEPIS) is a
bioinformatics tool developed at Genentech, Inc. that characterizes
genes of interest for new cancer therapeutic targets. GEPIS takes
advantage of large amounts of EST sequence and library information
to determine gene expression profiles. GEPIS is capable of
determining the expression profile of a gene based upon its
proportional correlation with the number of its occurrences in EST
databases, and it works by integrating the LIFESEQ.RTM. EST
relational database and Genentech proprietary information in a
stringent and statistically meaningful way. In this example, GEPIS
is used to identify and cross-validate novel tumor antigens,
although GEPIS can be configured to perform either very specific
analyses or broad screening tasks. For the initial screen, GEPIS is
used to identify EST sequences from the LIFESEQ.RTM. database that
correlate to expression in a particular tissue or tissues of
interest (often a tumor tissue of interest). The EST sequences
identified in this initial screen (or consensus sequences obtained
from aligning multiple related and overlapping EST sequences
obtained from the initial screen) were then subjected to a screen
intended to identify the presence of at least one transmembrane
domain in the encoded protein. Finally, GEPIS was employed to
generate a complete tissue expression profile for the various
sequences of interest. Using this type of screening bioinformatics,
various TAT polypeptides (and their encoding nucleic acid
molecules) were identified as being significantly overexpressed in
a particular type of cancer or certain cancers as compared to other
cancers and/or normal non-cancerous tissues. The rating of GEPIS
hits is based upon several criteria including, for example, tissue
specificity, tumor specificity and expression level in normal
essential and/or normal proliferating tissues. The following is a
list of molecules whose tissue expression profile as determined by
GEPIS evidences high tissue expression and significant upregulation
of expression in a specific tumor or tumors as compared to other
tumor(s) and/or normal tissues and optionally relatively low
expression in normal essential and/or normal proliferating tissues.
As such, the molecules listed below are excellent polypeptide
targets for the diagnosis and therapy of cancer in mammals.
TABLE-US-00009 upregulation of Molecule expression in: as compared
to: DNA67962 colon tumor normal colon tissue (TAT207) DNA67962
uterus tumor normal uterus tissue (TAT207) DNA67962 lung tumor
normal lung tissue (TAT207) DNA67962 prostate tumor normal prostate
tissue (TAT207) DNA67962 breast tumor normal breast tissue (TAT207)
DNA96792 colon tumor normal colon tissue (TAT239) DNA96792 rectum
tumor normal rectum tissue (TAT239) DNA96792 pancreas tumor normal
pancreas tissue (TAT239) DNA96792 lung tumor normal lung tissue
(TAT239) DNA96792 stomach tumor normal stomach tissue (TAT239)
DNA96792 esophagus tumor normal esophagus (TAT239) tissue DNA96792
breast tumor normal breast tissue (TAT239) DNA96792 uterus tumor
normal uterus tissue (TAT239) DNA96964 breast tumor normal breast
tissue (TAT193) DNA96964 brain tumor normal brain tissue (TAT193)
DNA142915 breast tumor normal breast tissue (TAT199) DNA142915
ovary tumor normal ovary tissue (TAT199) DNA142915 brain tumor
normal brain tissue (TAT199) DNA208551 prostate tumor normal
prostate tissue (TAT178) DNA208551 colon tumor normal colon tissue
(TAT178) DNA210159 prostate tumor normal prostate tissue (TAT198)
DNA210159 uterus tumor normal uterus tissue (TAT198) DNA210159
breast tumor normal breast tissue (TAT198) DNA210159 ovarian tumor
normal ovarian tissue (TAT198) DNA225706 adrenal tumor normal
adrenal tissue (TAT194) DNA225706 prostate tumor normal prostate
tissue (TAT194) DNA225706 breast tumor normal breast tissue
(TAT194) DNA225706 connective tissue normal connective (TAT194)
tumor tissue DNA225793 ovarian tumor normal ovarian tissue (TAT223)
DNA225793 fallopian tube normal fallopian tube (TAT223) tumor
tissue DNA225793 kidney tumor normal kidney tissue (TAT223)
DNA225796 breast tumor normal breast tissue (TAT196) DNA225943
liver tumor normal liver tissue (TAT195) DNA225943 lung tumor
normal lung tissue (TAT195) DNA225943 breast tumor normal breast
tissue (TAT195) DNA226283 uterine tumor normal uterine tissue
(TAT203) DNA226283 breast tumor normal breast tissue (TAT203)
DNA226283 squamous cell lung normal squamous cell (TAT203) tumor
lung tissue DNA226283 colon tumor normal colon tissue (TAT203)
DNA226283 ovarian tumor normal ovarian tissue (TAT203) DNA226589
brain tumor normal brain tissue (TAT200) DNA226589 colon tumor
normal colon tissue (TA1200) DNA226589 breast tumor normal breast
tissue (TAT200) DNA226589 prostate tumor normal prostate tissue
(TAT200) DNA226622 squamous cell lung normal squamous cell (TAT205)
tumor lung tissue DNA226622 kidney tumor normal kidney tissue
(TAT205) DNA226622 uterine tumor normal uterine tissue (TAT205)
DNA226622 breast tumor normal breast tissue (TAT205) DNA226622
colon tumor normal colon tissue (TAT205) DNA227545 breast tumor
normal breast tissue (TAT197) DNA227611 prostate tumor normal
prostate tissue (TAT175) DNA227611 colon tumor normal colon tissue
(TAT175) DNA227611 breast tumor normal breast tissue (TAT175)
DNA227611 uterine tumor normal uterine tissue (TAT175) DNA261021
prostate tumor normal prostate tissue (TAT208) DNA261021 colon
tumor normal colon tissue (TAT208) DNA261021 breast tumor normal
breast tissue (TAT208) DNA261021 uterine tumor normal uterine
tissue (TAT208) DNA260655 lung tumor normal lung tissue (TAT209)
DNA260655 colon tumor normal colon tissue (TAT209) DNA260655 breast
tumor normal breast tissue (TAT209) DNA260655 liver tumor normal
liver tissue (TAT209) DNA260655 ovarian tumor normal ovarian tissue
(TAT209) DNA260655 skin tumor normal skin tissue (TAT209) DNA260655
spleen tumor normal spleen tissue (TAT209) DNA260655 myeloid tumor
normal myeloid tissue (TAT209) DNA260655 muscle tumor normal muscle
tissue (TAT209) DNA260655 bone tumor normal bone tissue (TAT209)
DNA260945 brain tumor normal brain tissue (TAT192)
DNA260945 breast tumor normal breast tissue (TAT192) DNA260945
colon tumor normal colon tissue (TAT192) DNA260945 ovarian tumor
normal ovarian tissue (TAT192) DNA260945 pancreatic tumor normal
pancreatic (TAT192) tissue DNA261001 bone tumor normal bone tissue
(TAT181) DNA261001 lung tumor normal lung tissue (TAT181) DNA266928
bone tumor normal bone tissue (TAT182) DNA266928 lung tumor normal
lung tissue (TAT182) DNA268035 ovarian tumor normal ovarian tissue
(TAT222) DNA277797 breast tumor normal breast tissue (TAT212)
DNA277797 pancreatic tumor normal pancreatic (TAT212) tissue
DNA77509 colon tumor normal colon tissue (TAT177) DNA77509 testis
tumor normal testis tissue (TAT177) DNA87993 breast tumor normal
breast tissue (TAT235) DNA87993 prostate tumor normal prostate
tissue (TAT235) DNA87993 colon tumor normal colon tissue (TAT235)
DNA87993 ovarian tumor normal ovarian tissue (TAT235) DNA92980 bone
tumor normal bone tissue (TAT234) DNA92980 breast tumor normal
breast tissue (TAT234) DNA92980 cervical tumor normal cervical
tissue (TAT234) DNA92980 colon tumor normal colon tissue (TAT234)
DNA92980 rectum tumor normal rectum tissue (TAT234) DNA92980
endometrial tumor normal endometrial (TAT234) tissue DNA92980 liver
tumor normal liver tissue (TAT234) DNA92980 lung tumor normal lung
tissue (TA1234) DNA92980 ovarian tumor normal ovarian tissue
(TAT234) DNA92980 pancreatic tumor normal pancreatic (TAT234)
tissue DNA92980 skin tumor normal skin tissue (TAT234) DNA92980
soft tissue tumor normal soft tissue (TAT234) DNA92980 stomach
tumor normal stomach tissue (TAT234) DNA92980 bladder tumor normal
bladder tissue (TAT234) DNA92980 thyroid tumor normal thyroid
tissue (TAT234) DNA92980 esophagus tumor normal esophagus (TAT234)
tissue DNA92980 testis tumor normal testis tissue (TAT234)
DNA105792 adrenal tumor normal adrenal tissue (TAT233) DNA105792
breast tumor normal breast tissue (TAT233) DNA105792 endometrial
tumor normal endometrial (TAT233) tissue DNA105792 esophagus tumor
normal esophagus (TAT233) tissue DNA105792 kidney tumor normal
kidney tissue (TAT233) DNA105792 lung tumor normal lung tissue
(TAT233) DNA105792 ovarian tumor normal ovarian tissue (TAT233)
DNA105792 pancreatic tumor normal pancreatic (TAT233) tissue
DNA105792 prostate tumor normal prostate tissue (TAT233) DNA105792
soft tissue tumor normal soft tissue (TAT233) DNA105792 myeloid
tumor normal myeloid tissue (TAT233) DNA105792 thyroid tumor normal
thyroid tissue (TAT233) DNA105792 bladder tumor normal bladder
tissue (TAT233) DNA105792 brain tumor normal brain tissue (TAT233)
DNA105792 testis tumor normal testis tissue (TAT233) DNA119474
kidney tumor normal kidney tissue (TAT226) DNA119474 adrenal tumor
normal adrenal tissue (TAT226) DNA119474 uterine tumor normal
uterine tissue (TAT226) DNA119474 ovarian tumor normal ovarian
tissue (TAT226) DNA150491 squamous cell lung normal squamous cell
(TAT204) tumor lung tissue DNA150491 colon tumor normal colon
tissue (TAT204) DNA280351 squamous cell lung normal squamous cell
(TAT248) tumor lung tissue DNA280351 colon tumor normal colon
tissue (TAT248) DNA150648 liver tumor normal liver tissue (TAT232)
DNA150648 breast tumor normal breast tissue (TAT232) DNA150648
brain tumor normal brain tissue (TAT232) DNA150648 lung tumor
normal lung tissue (TAT232) DNA150648 colon tumor normal colon
tissue (TAT232) DNA150648 rectum tumor normal rectum tissue
(TAT232) DNA150648 kidney tumor normal kidney tissue (TAT232)
DNA150648 bladder tumor normal bladder tissue (TAT232) DNA179651
colon tumor normal colon tissue (TAT224) DNA179651 uterine tumor
normal uterine tissue (TAT224) DNA179651 lung tumor normal lung
tissue (TAT224) DNA179651 kidney tumor normal kidney tissue
(TAT224) DNA225886 breast tumor normal breast tissue (TAT236)
DNA225886 colon tumor normal colon tissue (TAT236) DNA225886 rectum
tumor normal rectum tissue (TAT236) DNA225886 ovarian tumor normal
ovarian tissue (TAT236) DNA225886 pancreas tumor normal pancreas
tissue (TAT236) DNA225886 prostate tumor normal prostate tissue
(TAT236) DNA225886 bladder tumor normal bladder tissue (TAT236)
DNA225886 testis tumor normal testis tissue (TAT236) DNA226717
glioma normal glial tissue (TAT185) DNA226717 brain tumor normal
brain tissue (TAT185) DNA227162 myeloid tumor normal myeloid tissue
(TAT225) DNA227162 uterine tumor normal uterine tissue (TAT225)
DNA227162 prostate tumor normal prostate tissue (TAT225) DNA277804
myeloid tumor normal myeloid tissue (TAT247) DNA277804 uterine
tumor normal uterine tissue
(TAT247) DNA277804 prostate tumor normal prostate tissue (TAT247)
DNA233034 glioma normal glial tissue (TAT174) DNA233034 brain tumor
normal brain tissue (TAT174) DNA233034 kidney tumor normal kidney
tissue (TAT174) DNA233034 adrenal tumor normal adrenal tissue
(TAT174) DNA266920 glioma normal glial tissue (TAT214) DNA266920
brain tumor normal brain tissue (TAT214) DNA266920 kidney tumor
normal kidney tissue (TAT214) DNA266920 adrenal tumor normal
adrenal tissue (TAT214) DNA266921 glioma normal glial tissue
(TAT220) DNA266921 brain tumor normal brain tissue (TAT220)
DNA266921 kidney tumor normal kidney tissue (TAT220) DNA266921
adrenal tumor normal adrenal tissue (TAT220) DNA266922 glioma
normal glial tissue (TAT221) DNA266922 brain tumor normal brain
tissue (TAT221) DNA266922 kidney tumor normal kidney tissue
(TAT221) DNA266922 adrenal tumor normal adrenal tissue (TAT221)
DNA234834 colon tumor normal colon tissue (TAT179) DNA234834
uterine tumor normal uterine tissue (TAT179) DNA234834 breast tumor
normal breast tissue (TAT179) DNA234834 prostate tumor normal
prostate tissue (TAT179) DNA247587 breast tumor normal breast
tissue (TAT216) DNA247587 prostate tumor normal prostate tissue
(TAT216) DNA247587 bladder tumor normal bladder tissue (TAT216)
DNA247587 lymphoid tumor normal lymphoid tissue (TAT216) DNA255987
brain tumor normal brain tissue (TAT218) DNA255987 breast tumor
normal breast tissue (TAT218) DNA247476 prostate tumor normal
prostate tissue (TAT180) DNA247476 pancreas tumor normal pancreas
tissue (TAT180) DNA247476 brain tumor normal brain tissue (TAT180)
DNA247476 stomach tumor normal stomach tissue (TAT180) DNA247476
bladder tumor normal bladder tissue (TAT180) DNA247476 soft tissue
tumor normal soft tissue (TAT180) DNA247476 skin tumor normal skin
tissue (TAT180) DNA247476 kidney tumor normal kidney tissue
(TAT180) DNA260990 prostate tumor normal prostate tissue (TAT375)
DNA260990 pancreas tumor normal pancreas tissue (TAT375) DNA260990
brain tumor normal brain tissue (TAT375) DNA260990 stomach tumor
normal stomach tissue (TAT375) DNA260990 bladder tumor normal
bladder tissue (TAT375) DNA260990 soft tissue tumor normal soft
tissue (TAT375) DNA260990 skin tumor normal skin tissue (TAT375)
DNA260990 kidney tumor normal kidney tissue (TAT375) DNA261013
prostate tumor normal prostate tissue (TAT176) DNA261013 colon
tumor normal colon tissue (TAT176) DNA261013 small intestine normal
small intestine (TAT176) tumor tissue DNA261013 pancreatic tumor
normal pancreatic (TAT176) tissue DNA261013 uterine tumor normal
uterine tissue (TAT176) DNA261013 ovarian tumor normal ovarian
tissue (TAT176) DNA261013 bladder tumor normal bladder tissue
(TAT176) DNA261013 stomach tumor normal stomach tissue (TAT176)
DNA267342 breast tumor normal breast tissue (TAT213) DNA267342
uterine tumor normal uterine tissue (TAT213) DNA267342 colon tumor
normal colon tissue (TAT213) DNA267342 kidney tumor normal kidney
tissue (TAT213) DNA267342 bladder tumor normal bladder tissue
(TAT213) DNA267342 bone tumor normal bone tissue (TAT213) DNA267342
ovarian tumor normal ovarian tissue (TAT213) DNA267342 pancreatic
tumor normal pancreatic (TAT213) tissue DNA267626 breast tumor
normal breast tissue (TAT217) DNA267626 colon tumor normal colon
tissue (TAT217) DNA267626 pancreatic tumor normal pancreatic
(TAT217) tissue DNA267626 ovarian tumor normal ovarian tissue
(TAT217) DNA268334 kidney tumor normal kidney tissue (TAT202)
DNA269238 colon tumor normal colon tissue (TAT215) DNA269238 kidney
tumor normal kidney tissue (TAT215) DNA269238 adrenal tumor normal
adrenal tissue (TAT215) DNA269238 bladder tumor normal bladder
tissue (TAT215) DNA272578 adrenal tumor normal adrenal tissue
(TAT238) DNA272578 lung tumor normal lung tissue (TAT238) DNA272578
ovarian tumor normal ovarian tissue (TAT238) DNA272578 uterine
tumor normal uterine tissue (TAT238) DNA304853 colon tumor normal
colon tissue (TAT376) DNA304853 uterine tumor normal uterine tissue
(TAT376) DNA304853 breast tumor normal breast tissue (TAT376)
DNA304853 prostate tumor normal prostate tissue (TAT376) DNA304854
colon tumor normal colon tissue (TAT377) DNA304854 uterine tumor
normal uterine tissue (TAT377) DNA304854 breast tumor normal breast
tissue (TAT377) DNA304854 prostate tumor normal prostate tissue
(TAT377) DNA304855 colon tumor normal colon tissue (TAT378)
DNA304855 uterine tumor normal uterine tissue (TAT378) DNA304855
breast tumor normal breast tissue (TAT378)
DNA304855 prostate tumor normal prostate tissue (TAT378) DNA287971
prostate tumor normal prostate tissue (TAT379) DNA287971 pancreas
tumor normal pancreas tissue (TAT379) DNA287971 brain tumor normal
brain tissue (TAT379) DNA287971 stomach tumor normal stomach tissue
(TAT379) DNA287971 bladder tumor normal bladder tissue (TAT379)
DNA287971 soft tissue tumor normal soft tissue (TAT379) DNA287971
skin tumor normal skin tissue (TAT379) DNA287971 kidney tumor
normal kidney tissue (TAT379)
Example 6
Use of TAT as a Hybridization Probe
[0941] The following method describes use of a nucleotide sequence
encoding TAT as a hybridization probe for, i.e., diagnosis of the
presence of a tumor in a mammal.
[0942] DNA comprising the coding sequence of full-length or mature
TAT as disclosed herein can also be employed as a probe to screen
for homologous DNAs (such as those encoding naturally-occurring
variants of TAT) in human tissue cDNA libraries or human tissue
genomic libraries.
[0943] Hybridization and washing of filters containing either
library DNAs is performed under the following high stringency
conditions. Hybridization of radiolabeled TAT-derived probe to the
filters is performed in a solution of 50% formamide, 5.times.SSC,
0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH
6.8, 2.times. Denhardt's solution, and 10% dextran sulfate at
42.degree. C. for 20 hours. Washing of the filters is performed in
an aqueous solution of 0.1.times.SSC and 0.1% SDS at 42.degree.
C.
[0944] DNAs having a desired sequence identity with the DNA
encoding full-length native sequence TAT can then be identified
using standard techniques known in the art.
Example 7
Expression of TAT in E. coli
[0945] This example illustrates preparation of an unglycosylated
form of TAT by recombinant expression in E. coli.
[0946] The DNA sequence encoding TAT is initially amplified using
selected PCR primers. The primers should contain restriction enzyme
sites which correspond to the restriction enzyme sites on the
selected expression vector. A variety of expression vectors may be
employed. An example of a suitable vector is pBR322 (derived from
E. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains
genes for ampicillin and tetracycline resistance. The vector is
digested with restriction enzyme and dephosphorylated. The PCR
amplified sequences are then ligated into the vector. The vector
will preferably include sequences which encode for an antibiotic
resistance gene, a trp promoter, a polyhis leader (including the
first six STII codons, polyhis sequence, and enterokinase cleavage
site), the TAT coding region, lambda transcriptional terminator,
and an argU gene.
[0947] The ligation mixture is then used to transform a selected E.
coli strain using the methods described in Sambrook et al., supra.
Transformants are identified by their ability to grow on LB plates
and antibiotic resistant colonies are then selected. Plasmid DNA
can be isolated and confirmed by restriction analysis and DNA
sequencing.
[0948] Selected clones can be grown overnight in liquid culture
medium such as LB broth supplemented with antibiotics. The
overnight culture may subsequently be used to inoculate a larger
scale culture. The cells are then grown to a desired optical
density, during which the expression promoter is turned on.
[0949] After culturing the cells for several more hours, the cells
can be harvested by centrifugation. The cell pellet obtained by the
centrifugation can be solubilized using various agents known in the
art, and the solubilized TAT protein can then be purified using a
metal chelating column under conditions that allow tight binding of
the protein.
[0950] TAT may be expressed in E. coli in a poly-His tagged form,
using the following procedure. The DNA encoding TAT is initially
amplified using selected PCR primers. The primers will contain
restriction enzyme sites which correspond to the restriction enzyme
sites on the selected expression vector, and other useful sequences
providing for efficient and reliable translation initiation, rapid
purification on a metal chelation column, and proteolytic removal
with enterokinase. The PCR-amplified, poly-His tagged sequences are
then ligated into an expression vector, which is used to transform
an E. coli host based on strain 52 (W3110 fuhA(tonA) Ion galE
rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LB
containing 50 mg/ml carbenicillin at 30.degree. C. with shaking
until an O.D.600 of 3-5 is reached. Cultures are then diluted
50-100 fold into CRAP media (prepared by mixing 3.57 g
(NH.sub.4).sub.2SO.sub.4, 0.71 g sodium citrate.2H.sub.2O, 1.07 g
KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500
mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7
mM MgSO.sub.4) and grown for approximately 20-30 hours at
30.degree. C. with shaking. Samples are removed to verify
expression by SDS-PAGE analysis, and the bulk culture is
centrifuged to pellet the cells. Cell pellets are frozen until
purification and refolding.
[0951] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets)
is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH
8 buffer. Solid sodium sulfite and sodium tetrathionate is added to
make final concentrations of 0.1 M and 0.02 M, respectively, and
the solution is stirred overnight at 4.degree. C. This step results
in a denatured protein with all cysteine residues blocked by
sulfitolization. The solution is centrifuged at 40,000 rpm in a
Beckman Ultracentifuge for 30 min. The supernatant is diluted with
3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM
Tris, pH 7.4) and filtered through 0.22 micron filters to clarify.
The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal
chelate column equilibrated in the metal chelate column buffer. The
column is washed with additional buffer containing 50 mM imidazole
(Calbiochem, Utrol grade), pH 7.4. The protein is eluted with
buffer containing 250 mM imidazole. Fractions containing the
desired protein are pooled and stored at 4.degree. C. Protein
concentration is estimated by its absorbance at 280 nm using the
calculated extinction coefficient based on its amino acid
sequence.
[0952] The proteins are refolded by diluting the sample slowly into
freshly prepared refolding buffer consisting of: 20 mM Tris, pH
8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM
EDTA. Refolding volumes are chosen so that the final protein
concentration is between 50 to 100 micrograms/ml. The refolding
solution is stirred gently at 4.degree. C. for 12-36 hours. The
refolding reaction is quenched by the addition of TFA to a final
concentration of 0.4% (pH of approximately 3). Before further
purification of the protein, the solution is filtered through a
0.22 micron filter and acetonitrile is added to 2-10% final
concentration. The refolded protein is chromatographed on a Poros
R1/H reversed phase column using a mobile buffer of 0.1% TFA with
elution with a gradient of acetonitrile from 10 to 80%. Aliquots of
fractions with A280 absorbance are analyzed on SDS polyacrylamide
gels and fractions containing homogeneous refolded protein are
pooled. Generally, the properly refolded species of most proteins
are eluted at the lowest concentrations of acetonitrile since those
species are the most compact with their hydrophobic interiors
shielded from interaction with the reversed phase resin. Aggregated
species are usually eluted at higher acetonitrile concentrations.
In addition to resolving misfolded forms of proteins from the
desired form, the reversed phase step also removes endotoxin from
the samples.
[0953] Fractions containing the desired folded TAT polypeptide are
pooled and the acetonitrile removed using a gentle stream of
nitrogen directed at the solution. Proteins are formulated into 20
mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by
dialysis or by gel filtration using G25 Superfine (Pharmacia)
resins equilibrated in the formulation buffer and sterile
filtered.
[0954] Certain of the TAT polypeptides disclosed herein have been
successfully expressed and purified using this technique(s).
Example 8
Expression of TAT in Mammalian Cells
[0955] This example illustrates preparation of a potentially
glycosylated form of TA T by recombinant expression in mammalian
cells.
[0956] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
is employed as the expression vector. Optionally, the TAT DNA is
ligated into pRK5 with selected restriction enzymes to allow
insertion of the TAT DNA using ligation methods such as described
in Sambrook et al., supra. The resulting vector is called
pRK5-TAT.
[0957] In one embodiment, the selected host cells may be 293 cells.
Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue
culture plates in medium such as DMEM supplemented with fetal calf
serum and optionally, nutrient components and/or antibiotics. About
10 .mu.g pRK5-TAT DNA is mixed with about 1 .mu.g DNA encoding the
VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved
in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl.sub.2. To
this mixture is added, dropwise, 500 .mu.l of 50 mM HEPES (pH
7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and a precipitate is allowed
to form for 10 minutes at 25.degree. C. The precipitate is
suspended and added to the 293 cells and allowed to settle for
about four hours at 37.degree. C. The culture medium is aspirated
off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The
293 cells are then washed with serum free medium, fresh medium is
added and the cells are incubated for about 5 days.
[0958] Approximately 24 hours after the transfections, the culture
medium is removed and replaced with culture medium (alone) or
culture medium containing 200 .mu.Ci/ml .sup.35S-cysteine and 200
.mu.Ci/ml .sup.35S-methionine. After a 12 hour incubation, the
conditioned medium is collected, concentrated on a spin filter, and
loaded onto a 15% SDS gel. The processed gel may be dried and
exposed to film for a selected period of time to reveal the
presence of TAT polypeptide. The cultures containing transfected
cells may undergo further incubation (in serum free medium) and the
medium is tested in selected bioassays.
[0959] In an alternative technique, TAT may be introduced into 293
cells transiently using the dextran sulfate method described by
Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293
cells are grown to maximal density in a spinner flask and 700 .mu.g
pRK5-TAT DNA is added. The cells are first concentrated from the
spinner flask by centrifugation and washed with PBS. The
DNA-dextran precipitate is incubated on the cell pellet for four
hours. The cells are treated with 20% glycerol for 90 seconds,
washed with tissue culture medium, and re-introduced into the
spinner flask containing tissue culture medium, 5 .mu.g/ml bovine
insulin and 0.1 .mu.g/ml bovine transferrin. After about four days,
the conditioned media is centrifuged and filtered to remove cells
and debris. The sample containing expressed TAT can then be
concentrated and purified by any selected method, such as dialysis
and/or column chromatography.
[0960] In another embodiment, TAT can be expressed in CHO cells.
The pRK5-TAT can be transfected into CHO cells using known reagents
such as CaPO.sub.4 or DEAE-dextran. As described above, the cell
cultures can be incubated, and the medium replaced with culture
medium (alone) or medium containing a radiolabel such as
.sup.35S-methionine. After determining the presence of TAT
polypeptide, the culture medium may be replaced with serum free
medium. Preferably, the cultures are incubated for about 6 days,
and then the conditioned medium is harvested. The medium containing
the expressed TAT can then be concentrated and purified by any
selected method.
[0961] Epitope-tagged TAT may also be expressed in host CHO cells.
The TAT may be subcloned out of the pRK5 vector. The subclone
insert can undergo PCR to fuse in frame with a selected epitope tag
such as a poly-his tag into a Baculovirus expression vector. The
poly-his tagged TAT insert can then be subcloned into a SV40 driven
vector containing a selection marker such as DHFR for selection of
stable clones. Finally, the CHO cells can be transfected (as
described above) with the SV40 driven vector. Labeling may be
performed, as described above, to verify expression. The culture
medium containing the expressed poly-His tagged TAT can then be
concentrated and purified by any selected method, such as by
Ni.sup.2+-chelate affinity chromatography.
[0962] TAT may also be expressed in CHO and/or COS cells by a
transient expression procedure or in CHO cells by another stable
expression procedure.
[0963] Stable expression in CHO cells is performed using the
following procedure. The proteins are expressed as an IgG construct
(immunoadhesin), in which the coding sequences for the soluble
forms (e.g. extracellular domains) of the respective proteins are
fused to an IgG1 constant region sequence containing the hinge,
C.sub.H2 and C.sub.H2 domains and/or is a poly-His tagged form.
[0964] Following PCR amplification, the respective DNAs are
subcloned in a CHO expression vector using standard techniques as
described in Ausubel et al., Current Protocols of Molecular
Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression
vectors are constructed to have compatible restriction sites 5' and
3' of the DNA of interest to allow the convenient shuttling of
cDNA's. The vector used expression in CHO cells is as described in
Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the
SV40 early promoter/enhancer to drive expression of the cDNA of
interest and dihydrofolate reductase (DHFR). DHFR expression
permits selection for stable maintenance of the plasmid following
transfection.
[0965] Twelve micrograms of the desired plasmid DNA is introduced
into approximately 10 million CHO cells using commercially
available transfection reagents Superfect.RTM. (Quiagen),
Dosper.RTM. or Fugene.RTM. (Boehringer Mannheim). The cells are
grown as described in Lucas et al., supra. Approximately
3.times.10.sup.7 cells are frozen in an ampule for further growth
and production as described below.
[0966] The ampules containing the plasmid DNA are thawed by
placement into water bath and mixed by vortexing. The contents are
pipetted into a centrifuge tube containing 10 mLs of media and
centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated
and the cells are resuspended in 10 mL of selective media (0.2
.mu.m filtered PS20 with 5% 0.2 .mu.m diafiltered fetal bovine
serum). The cells are then aliquoted into a 100 mL spinner
containing 90 mL of selective media. After 1-2 days, the cells are
transferred into a 250 mL spinner filled with 150 mL selective
growth medium and incubated at 37.degree. C. After another 2-3
days, 250 mL, 500 mL and 2000 mL spinners are seeded with
3.times.10.sup.5 cells/mL. The cell media is exchanged with fresh
media by centrifugation and resuspension in production medium.
Although any suitable CHO media may be employed, a production
medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992
may actually be used. A 3 L production spinner is seeded at
1.2.times.10.sup.6 cells/mL. On day 0, the cell number pH ie
determined. On day 1, the spinner is sampled and sparging with
filtered air is commenced. On day 2, the spinner is sampled, the
temperature shifted to 33.degree. C., and 30 mL of 500 g/L glucose
and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane
emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout
the production, the pH is adjusted as necessary to keep it at
around 7.2. After 10 days, or until the viability dropped below
70%, the cell culture is harvested by centrifugation and filtering
through a 0.22 .mu.m filter. The filtrate was either stored at
4.degree. C. or immediately loaded onto columns for
purification.
[0967] For the poly-His tagged constructs, the proteins are
purified using a Ni-NTA column (Qiagen). Before purification,
imidazole is added to the conditioned media to a concentration of 5
mM. The conditioned media is pumped onto a 6 ml Ni-NTA column
equilibrated in 20 nM Hepes, pH 7.4, buffer containing 0.3 M NaCl
and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4.degree. C.
After loading, the column is washed with additional equilibration
buffer and the protein eluted with equilibration buffer containing
0.25 M imidazole. The highly purified protein is subsequently
desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl
and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia)
column and stored at -80.degree. C.
[0968] Immunoadhesin (Fc-containing) constructs are purified from
the conditioned media as follows. The conditioned medium is pumped
onto a 5 ml Protein A column (Pharmacia) which had been
equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading,
the column is washed extensively with equilibration buffer before
elution with 100 mM citric acid, pH 3.5. The eluted protein is
immediately neutralized by collecting 1 ml fractions into tubes
containing 275 .mu.L of 1 M Tris buffer, pH 9. The highly purified
protein is subsequently desalted into storage buffer as described
above for the poly-His tagged proteins. The homogeneity is assessed
by SDS polyacrylamide gels and by N-terminal amino acid sequencing
by Edman degradation.
[0969] Certain of the TAT polypeptides disclosed herein have been
successfully expressed and purified using this technique(s).
Example 9
Expression of TAT in Yeast
[0970] The following method describes recombinant expression of TAT
in yeast.
[0971] First, yeast expression vectors are constructed for
intracellular production or secretion of TAT from the ADH2/GAPDH
promoter. DNA encoding TAT and the promoter is inserted into
suitable restriction enzyme sites in the selected plasmid to direct
intracellular expression of TAT. For secretion, DNA encoding TAT
can be cloned into the selected plasmid, together with DNA encoding
the ADH2/GAPDH promoter, a native TAT signal peptide or other
mammalian signal peptide, or, for example, a yeast alpha-factor or
invertase secretory signal/leader sequence, and linker sequences
(if needed) for expression of TAT.
[0972] Yeast cells, such as yeast strain AB110, can then be
transformed with the expression plasmids described above and
cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by precipitation with 10%
trichloroacetic acid and separation by SDS-PAGE, followed by
staining of the gels with Coomassie Blue stain.
[0973] Recombinant TAT can subsequently be isolated and purified by
removing the yeast cells from the fermentation medium by
centrifugation and then concentrating the medium using selected
cartridge filters. The concentrate containing TAT may further be
purified using selected column chromatography resins.
[0974] Certain of the TAT polypeptides disclosed herein have been
successfully expressed and purified using this technique(s).
Example 10
Expression of TAT in Baculovirus-Infected Insect Cells
[0975] The following method describes recombinant expression of TAT
in Baculovirus-infected insect cells.
[0976] The sequence coding for TAT is fused upstream of an epitope
tag contained within a baculovirus expression vector. Such epitope
tags include poly-his tags and immunoglobulin tags (like Fc regions
of IgG). A variety of plasmids may be employed, including plasmids
derived from commercially available plasmids such as pVL1393
(Novagen). Briefly, the sequence encoding TAT or the desired
portion of the coding sequence of TAT such as the sequence encoding
an extracellular domain of a transmembrane protein or the sequence
encoding the mature protein if the protein is extra cellular is
amplified by PCR with primers complementary to the 5' and 3'
regions. The 5' primer may incorporate flanking (selected)
restriction enzyme sites. The product is then digested with those
selected restriction enzymes and subcloned into the expression
vector.
[0977] Recombinant baculovirus is generated by co-transfecting the
above plasmid and BaculoGold.TM. virus DNA (Pharmingen) into
Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially available from GIBCO-BRL). After 4-5 days
of incubation at 28.degree. C., the released viruses are harvested
and used for further amplifications. Viral infection and protein
expression are performed as described by O'Reilley et al.,
Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford
University Press (1994).
[0978] Expressed poly-his tagged TAT can then be purified, for
example, by Ni.sup.2+-chelate affinity chromatography as follows.
Extracts are prepared from recombinant virus-infected Sf9 cells as
described by Rupert et al., Nature, 362:175-179(1993). Briefly, Sf9
cells are washed, resuspended in sonicationbuffer (25 mL Hepes, pH
7.9; 12.5 mM MgCl.sub.2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4
M KCl), and sonicated twice for 20 seconds on ice. The sonicates
are cleared by centrifugation, and the supernatant is diluted
50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%
glycerol, pH 7.8) and filtered through a 0.45 .mu.m filter. A
Ni.sup.2+-NTA agarose column (commercially available from Qiagen)
is prepared with a bed volume of 5 mL, washed with 25 mL of water
and equilibrated with 25 mL of loading buffer. The filtered cell
extract is loaded onto the column at 0.5 mL per minute. The column
is washed to baseline A.sub.280 with loading buffer, at which point
fraction collection is started. Next, the column is washed with a
secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol,
pH 6.0), which elutes nonspecifically bound protein. After reaching
A.sub.280 baseline again, the column is developed with a 0 to 500
mM Imidazole gradient in the secondary wash buffer. One mL
fractions are collected and analyzed by SDS-PAGE and silver
staining or Western blot with Ni.sup.2+-NTA-conjugated to alkaline
phosphatase (Qiagen). Fractions containing the eluted
His.sub.10-tagged TAT are pooled and dialyzed against loading
buffer.
[0979] Alternatively, purification of the IgG tagged (or Fc tagged)
TAT can be performed using known chromatography techniques,
including for instance, Protein A or protein G column
chromatography.
[0980] Certain of the TAT polypeptides disclosed herein have been
successfully expressed and purified using this technique(s).
Example 11
Preparation of Antibodies that Bind TAT
[0981] This example illustrates preparation of monoclonal
antibodies which can specifically bind TAT.
[0982] Techniques for producing the monoclonal antibodies are known
in the art and are described, for instance, in Goding, supra.
Immunogens that may be employed include purified TAT, fusion
proteins containing TAT, and cells expressing recombinant TAT on
the cell surface. Selection of the immunogen can be made by the
skilled artisan without undue experimentation.
[0983] Mice, such as Balb/c, are immunized with the TAT immunogen
emulsified in complete Freund's adjuvant and injected
subcutaneously or intraperitoneally in an amount from 1-100
micrograms. Alternatively, the immunogen is emulsified in MPL-TDM
adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and
injected into the animal's hind foot pads. The immunized mice are
then boosted 10 to 12 days later with additional immunogen
emulsified in the selected adjuvant. Thereafter, for several weeks,
the mice may also be boosted with additional immunization
injections. Serum samples may be periodically obtained from the
mice by retro-orbital bleeding for testing in ELISA assays to
detect anti-TAT antibodies.
[0984] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a final
intravenous injection of TAT. Three to four days later, the mice
are sacrificed and the spleen cells are harvested. The spleen cells
are then fused (using 35% polyethylene glycol) to a selected murine
myeloma cell line such as P3X63AgU.1, available from ATCC, No. CRL
1597. The fusions generate hybridoma cells which can then be plated
in 96 well tissue culture plates containing HAT (hypoxanthine,
aminopterin, and thymidine) medium to inhibit proliferation of
non-fused cells, myeloma hybrids, and spleen cell hybrids.
[0985] The hybridoma cells will be screened in an ELISA for
reactivity against TAT. Determination of "positive" hybridoma cells
secreting the desired monoclonal antibodies against TAT is within
the skill in the art.
[0986] The positive hybridoma cells can be injected
intraperitoneally into syngeneic Balb/c mice to produce ascites
containing the anti-TAT monoclonal antibodies. Alternatively, the
hybridoma cells can be grown in tissue culture flasks or roller
bottles. Purification of the monoclonal antibodies produced in the
ascites can be accomplished using ammonium sulfate precipitation,
followed by gel exclusion chromatography. Alternatively, affinity
chromatography based upon binding of antibody to protein A or
protein G can be employed.
Example 12
Purification of TAT Polypeptides Using Specific Antibodies
[0987] Native or recombinant TAT polypeptides may be purified by a
variety of standard techniques in the art of protein purification.
For example, pro-TAT polypeptide, mature TAT polypeptide, or
pre-TAT polypeptide is purified by immunoaffinity chromatography
using antibodies specific for the TAT polypeptide of interest. In
general, an immunoaffinity column is constructed by covalently
coupling the anti-TAT polypeptide antibody to an activated
chromatographic resin.
[0988] Polyclonal immunoglobulins are prepared from immune sera
either by precipitation with ammonium sulfate or by purification on
immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway,
N.J.). Likewise, monoclonal antibodies are prepared from mouse
ascites fluid by ammonium sulfate precipitation or chromatography
on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a chromatographic resin such as
CnBr-activated SEPHAROSE.TM. (Pharmacia LKB Biotechnology). The
antibody is coupled to the resin, the resin is blocked, and the
derivative resin is washed according to the manufacturer's
instructions.
[0989] Such an immunoaffinity column is utilized in the
purification of TAT polypeptide by preparing a fraction from cells
containing TAT polypeptide in a soluble form. This preparation is
derived by solubilization of the whole cell or of a subcellular
fraction obtained via differential centrifugation by the addition
of detergent or by other methods well known in the art.
Alternatively, soluble TAT polypeptide containing a signal sequence
may be secreted in useful quantity into the medium in which the
cells are grown.
[0990] A soluble TAT polypeptide-containing preparation is passed
over the immunoaffinity column, and the column is washed under
conditions that allow the preferential absorbance of TAT
polypeptide (e.g., high ionic strength buffers in the presence of
detergent). Then, the column is eluted under conditions that
disrupt antibody/TAT polypeptide binding (e.g., a low pH buffer
such as approximately pH 2-3, or a high concentration of a
chaotrope such as urea or thiocyanate ion), and TAT polypeptide is
collected.
Example 13
In Vitro Tumor Cell Killing Assay
[0991] Mammalian cells expressing the TAT polypeptide of interest
may be obtained using standard expression vector and cloning
techniques. Alternatively, many tumor cell lines expressing TAT
polypeptides of interest are publicly available, for example,
through the ATCC and can be routinely identified using standard
ELISA or FACS analysis. Anti-TAT polypeptide monoclonal antibodies
(and toxin conjugated derivatives thereof) may then be employed in
assays to determine the ability of the antibody to kill TAT
polypeptide expressing cells in vitro.
[0992] For example, cells expressing the TAT polypeptide of
interest are obtained as described above and plated into 96 well
dishes. In one analysis, the antibody/toxin conjugate (or naked
antibody) is included throughout the cell incubation for a period
of 4 days. In a second independent analysis, the cells are
incubated for 1 hour with the antibody/toxin conjugate (or naked
antibody) and then washed and incubated in the absence of
antibody/toxin conjugate for a period of 4 days. Cell viability is
then measured using the CellTiter-Glo Luminescent Cell Viability
Assay from Promega (Cat# G7571). Untreated cells serve as a
negative control.
Example 14
In Vivo Tumor Cell Killing Assay
[0993] To test the efficacy of conjugated or unconjugated anti-TAT
polypeptide monoclonal antibodies, anti-TAT antibody is injected
intraperitoneally into nude mice 24 hours prior to receiving tumor
promoting cells subcutaneously in the flank. Antibody injections
continue twice per week for the remainder of the study. Tumor
volume is then measured twice per week.
[0994] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0995] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the construct deposited, since the deposited embodiment is intended
as a single illustration of certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Sequence CWU 1
1
120 1 3781 DNA Homo Sapien 1 ctccgggtcc ccaggggctg cgccgggccg
gcctggcaag ggggacgagt 50 cagtggacac tccaggaaga gcggccccgc
ggggggcgat gaccgtgcgc 100 tgaccctgac tcactccagg tccggaggcg
ggggcccccg gggcgactcg 150 ggggcggacc gcggggcgga gctgccgccc
gtgagtccgg ccgagccacc 200 tgagcccgag ccgcgggaca ccgtcgctcc
tgctctccga atgctgcgca 250 ccgcgatggg cctgaggagc tggctcgccg
ccccatgggg cgcgctgccg 300 cctcggccac cgctgctgct gctcctgctg
ctgctgctcc tgctgcagcc 350 gccgcctccg acctgggcgc tcagcccccg
gatcagcctg cctctgggct 400 ctgaagagcg gccattcctc agattcgaag
ctgaacacat ctccaactac 450 acagcccttc tgctgagcag ggatggcagg
accctgtacg tgggtgctcg 500 agaggccctc tttgcactca gtagcaacct
cagcttcctg ccaggcgggg 550 agtaccagga gctgctttgg ggtgcagacg
cagagaagaa acagcagtgc 600 agcttcaagg gcaaggaccc acagcgcgac
tgtcaaaact acatcaagat 650 cctcctgccg ctcagcggca gtcacctgtt
cacctgtggc acagcagcct 700 tcagccccat gtgtacctac atcaacatgg
agaacttcac cctggcaagg 750 gacgagaagg ggaatgtcct cctggaagat
ggcaagggcc gttgtccctt 800 cgacccgaat ttcaagtcca ctgccctggt
ggttgatggc gagctctaca 850 ctggaacagt cagcagcttc caagggaatg
acccggccat ctcgcggagc 900 caaagccttc gccccaccaa gaccgagagc
tccctcaact ggctgcaaga 950 cccagctttt gtggcctcag cctacattcc
tgagagcctg ggcagcttgc 1000 aaggcgatga tgacaagatc tactttttct
tcagcgagac tggccaggaa 1050 tttgagttct ttgagaacac cattgtgtcc
cgcattgccc gcatctgcaa 1100 gggcgatgag ggtggagagc gggtgctaca
gcagcgctgg acctccttcc 1150 tcaaggccca gctgctgtgc tcacggcccg
acgatggctt ccccttcaac 1200 gtgctgcagg atgtcttcac gctgagcccc
agcccccagg actggcgtga 1250 cacccttttc tatggggtct tcacttccca
gtggcacagg ggaactacag 1300 aaggctctgc cgtctgtgtc ttcacaatga
aggatgtgca gagagtcttc 1350 agcggcctct acaaggaggt gaaccgtgag
acacagcagt ggtacaccgt 1400 gacccacccg gtgcccacac cccggcctgg
agcgtgcatc accaacagtg 1450 cccgggaaag gaagatcaac tcatccctgc
agctcccaga ccgcgtgctg 1500 aacttcctca aggaccactt cctgatggac
gggcaggtcc gaagccgcat 1550 gctgctgctg cagccccagg ctcgctacca
gcgcgtggct gtacaccgcg 1600 tccctggcct gcaccacacc tacgatgtcc
tcttcctggg cactggtgac 1650 ggccggctcc acaaggcagt gagcgtgggc
ccccgggtgc acatcattga 1700 ggagctgcag atcttctcat cgggacagcc
cgtgcagaat ctgctcctgg 1750 acacccacag ggggctgctg tatgcggcct
cacactcggg cgtagtccag 1800 gtgcccatgg ccaactgcag cctgtaccgg
agctgtgggg actgcctcct 1850 cgcccgggac ccctactgtg cttggagcgg
ctccagctgc aagcacgtca 1900 gcctctacca gcctcagctg gccaccaggc
cgtggatcca ggacatcgag 1950 ggagccagcg ccaaggacct ttgcagcgcg
tcttcggttg tgtccccgtc 2000 ttttgtacca acaggggaga agccatgtga
gcaagtccag ttccagccca 2050 acacagtgaa cactttggcc tgcccgctcc
tctccaacct ggcgacccga 2100 ctctggctac gcaacggggc ccccgtcaat
gcctcggcct cctgccacgt 2150 gctacccact ggggacctgc tgctggtggg
cacccaacag ctgggggagt 2200 tccagtgctg gtcactagag gagggcttcc
agcagctggt agccagctac 2250 tgcccagagg tggtggagga cggggtggca
gaccaaacag atgagggtgg 2300 cagtgtaccc gtcattatca gcacatcgcg
tgtgagtgca ccagctggtg 2350 gcaaggccag ctggggtgca gacaggtcct
actggaagga gttcctggtg 2400 atgtgcacgc tctttgtgct ggccgtgctg
ctcccagttt tattcttgct 2450 ctaccggcac cggaacagca tgaaagtctt
cctgaagcag ggggaatgtg 2500 ccagcgtgca ccccaagacc tgccctgtgg
tgctgccccc tgagacccgc 2550 ccactcaacg gcctagggcc ccctagcacc
ccgctcgatc accgagggta 2600 ccagtccctg tcagacagcc ccccgggggc
ccgagtcttc actgagtcag 2650 agaagaggcc actcagcatc caagacagct
tcgtggaggt atccccagtg 2700 tgcccccggc cccgggtccg ccttggctcg
gagatccgtg actctgtggt 2750 gtgagagctg acttccagag gacgctgccc
tggcttcagg ggctgtgaat 2800 gctcggagag ggtcaactgg acctcccctc
cgctctgctc ttcgtggaac 2850 acgaccgtgg tgcccggccc ttgggagcct
tggagccagc tggcctgctg 2900 ctctccagtc aagtagcgaa gctcctacca
cccagacacc caaacagccg 2950 tggccccaga ggtcctggcc aaatatgggg
gcctgcctag gttggtggaa 3000 cagtgctcct tatgtaaact gagccctttg
tttaaaaaac aattccaaat 3050 gtgaaactag aatgagaggg aagagatagc
atggcatgca gcacacacgg 3100 ctgctccagt tcatggcctc ccaggggtgc
tggggatgca tccaaagtgg 3150 ttgtctgaga cagagttgga aaccctcacc
aactggcctc ttcaccttcc 3200 acattatccc gctgccaccg gctgccctgt
ctcactgcag attcaggacc 3250 agcttgggct gcgtgcgttc tgccttgcca
gtcagccgag gatgtagttg 3300 ttgctgccgt cgtcccacca cctcagggac
cagagggcta ggttggcact 3350 gcggccctca ccaggtcctg ggctcggacc
caactcctgg acctttccag 3400 cctgtatcag gctgtggcca cacgagagga
cagcgcgagc tcaggagaga 3450 tttcgtgaca atgtacgcct ttccctcaga
attcagggaa gagactgtcg 3500 cctgccttcc tccgttgttg cgtgagaacc
cgtgtgcccc ttcccaccat 3550 atccaccctc gctccatctt tgaactcaaa
cacgaggaac taactgcacc 3600 ctggtcctct ccccagtccc cagttcaccc
tccatccctc accttcctcc 3650 actctaaggg atatcaacac tgcccagcac
aggggccctg aatttatgtg 3700 gtttttatac attttttaat aagatgcact
ttatgtcatt ttttaataaa 3750 gtctgaagaa ttactgttta aaaaaaaaaa a 3781
2 2010 DNA Homo Sapien 2 ggaaaggctg agtctccagc tcaaggtcaa
aacgtccaag gccgaaagcc 50 ctccagtttc ccctggacgc cttgctcctg
cttctgctac gaccttctgg 100 ggaaaacgaa tttctcattt tcttcttaaa
ttgccatttt cgctttagga 150 gatgaatgtt ttcctttggc tgttttggca
atgactctga attaaagcga 200 tgctaacgcc tcttttcccc ctaattgtta
aaagctatgg actgcaggaa 250 gatggcccgc ttctcttaca gtgtgatttg
gatcatggcc atttctaaag 300 tctttgaact gggattagtt gccgggctgg
gccatcagga atttgctcgt 350 ccatctcggg gatacctggc cttcagagat
gacagcattt ggccccagga 400 ggagcctgca attcggcctc ggtcttccca
gcgtgtgccg cccatgggga 450 tacagcacag taaggagcta aacagaacct
gctgcctgaa tgggggaacc 500 tgcatgctgg ggtccttttg tgcctgccct
ccctccttct acggacggaa 550 ctgtgagcac gatgtgcgca aagagaactg
tgggtctgtg ccccatgaca 600 cctggctgcc caagaagtgt tccctgtgta
aatgctggca cggtcagctc 650 cgctgctttc ctcaggcatt tctacccggc
tgtgatggcc ttgtgatgga 700 tgagcacctc gtggcttcca ggactccaga
actaccaccg tctgcacgta 750 ctaccacttt tatgctagtt ggcatctgcc
tttctataca aagctactat 800 taatcgacat tgacctattt ccagaaatac
aattttagat atcatgcaaa 850 tttcatgacc agtaaaggct gctgctacaa
tgtcctaact gaaagatgat 900 catttgtagt tgccttaaaa taatgaatac
atttccaaaa tggtctctaa 950 catttcctta cagaactact tcttacttct
ttgccctgcc ctctcccaaa 1000 aaactacttc ttttttcaaa agaaagtcag
ccatatctcc attgtgccta 1050 agtccagtgt ttcttttttt tttttttttg
agacggagtc tcactctgtc 1100 acccaggctg gactgcaatg acgcgatctt
ggttcactgc aacctccgca 1150 tccggggttc aagccattct cctgcctcag
cctcccaagt aactgggatt 1200 acaggcatgt gtcaccatgc ccagctaatt
tttttgtatt tttagtagag 1250 atgggggttt caccatattg gccagtctgg
tctcgaactc ctgaccttgt 1300 gatccactcg cctcagcctc tcgaagtgct
gagattacac acgtgagcaa 1350 ctgtgcaagg cctggtgttt cttgatacat
gtaattctac caaggtcttc 1400 ttaatatgtt cttttaaatg attgaattat
atgttcagat tattggagac 1450 taattctaat gtggacctta gaatacagtt
ttgagtagag ttgatcaaaa 1500 tcaattaaaa tagtctcttt aaaaggaaag
aaaacatctt taaggggagg 1550 aaccagagtg ctgaaggaat ggaagtccat
ctgcgtgtgt gcagggagac 1600 tgggtaggaa agaggaagca aatagaagag
agaggttgaa aaacaaaatg 1650 ggttacttga ttggtgatta ggtggtggta
gagaagcaag taaaaaggct 1700 aaatggaagg gcaagtttcc atcatctata
gaaagctata taagacaaga 1750 actccccttt ttttcccaaa ggcattataa
aaagaatgaa gcctccttag 1800 aaaaaaaatt atacctcaat gtccccaaca
agattgctta ataaattgtg 1850 tttcctccaa gctattcaat tcttttaact
gttgtagaag acaaaatgtt 1900 cacaatatat ttagttgtaa accaagtgat
caaactacat attgtaaagc 1950 ccatttttaa aatacattgt atatatgtgt
atgcacagta aaaatggaaa 2000 ctatattgaa 2010 3 549 DNA Homo Sapien 3
gccaggaggg agagccttcc ccaagcaaac aatccagagc agctgtgcaa 50
acaacggtgc ataaatgagg cctcctggac catgaagcga gtcctgagct 100
gcgtcccgga gcccacggtg gtcatggctg ccagagcgct ctgcatgctg 150
gggctggtcc tggccttgct gtcctccagc tctgctgagg agtacgtggg 200
cctgtctgca aaccagtgtg ccgtgccagc caaggacagg gtggactgcg 250
gctaccccca tgtcaccccc aaggagtgca acaaccgggg ctgctgcttt 300
gactccagga tccctggagt gccttggtgt ttcaagcccc tgcaggaagc 350
agaatgcacc ttctgaggca cctccagctg cccccggccg ggggatgcga 400
ggctcggagc acccttgccc ggctgtgatt gctgccaggc actgttcatc 450
tcagcttttc tgtccctttg ctcccggcaa gcgcttctgc tgaaagttca 500
tatctggagc ctgatgtctt aacgaataaa ggtcccatgc tccacccga 549 4 1424
DNA Homo Sapien 4 gaccagactc gtctcaggcc agttgcagcc ttctcagcca
aacgccgacc 50 aaggaaaact cactaccatg agaattgcag tgatttgctt
ttgcctccta 100 ggcatcacct gtgccatacc agttaaacag gctgattctg
gaagttctga 150 ggaaaagcag ctttacaaca aatacccaga tgctgtggcc
acatggctaa 200 accctgaccc atctcagaag cagaatctcc tagccccaca
gaatgctgtg 250 tcctctgaag aaaccaatga ctttaaacaa gagacccttc
caagtaagtc 300 caacgaaagc catgaccaca tggatgatat ggatgatgaa
gatgatgatg 350 accatgtgga cagccaggac tccattgact cgaacgactc
tgatgatgta 400 gatgacactg atgattctca ccagtctgat gagtctcacc
attctgatga 450 atctgatgaa ctggtcactg attttcccac ggacctgcca
gcaaccgaag 500 ttttcactcc agttgtcccc acagtagaca catatgatgg
ccgaggtgat 550 agtgtggttt atggactgag gtcaaaatct aagaagtttc
gcagacctga 600 catccagtac cctgatgcta cagacgagga catcacctca
cacatggaaa 650 gcgaggagtt gaatggtgca tacaaggcca tccccgttgc
ccaggacctg 700 aacgcgcctt ctgattggga cagccgtggg aaggacagtt
atgaaacgag 750 tcagctggat gaccagagtg ctgaaaccca cagccacaag
cagtccagat 800 tatataagcg gaaagccaat gatgagagca atgagcattc
cgatgtgatt 850 gatagtcagg aactttccaa agtcagccgt gaattccaca
gccatgaatt 900 tcacagccat gaagatatgc tggttgtaga ccccaaaagt
aaggaagaag 950 ataaacacct gaaatttcgt atttctcatg aattagatag
tgcatcttct 1000 gaggtcaatt aaaaggagaa aaaatacaat ttctcacttt
gcatttagtc 1050 aaaagaaaaa atgctttata gcaaaatgaa agagaacatg
aaatgcttct 1100 ttctcagttt attggttgaa tgtgtatcta tttgagtctg
gaaataacta 1150 atgtgtttga taattagttt agtttgtggc ttcatggaaa
ctccctgtaa 1200 actaaaagct tcagggttat gtctatgttc attctataga
agaaatgcaa 1250 actatcactg tattttaata tttgttattc tctcatgaat
agaaatttat 1300 gtagaagcaa acaaaatact tttacccact taaaaagaga
atataacatt 1350 ttatgtcact ataatctttt gttttttaag ttagtgtata
ttttgttgtg 1400 attatctttt tgtggtgtga ataa 1424 5 1166 DNA Homo
Sapien unsure 721-761 unknown base 5 cggacgcgtg ggcggaggga
agaggaccgc aaaccaaccc aggacccgct 50 cagttccacg cgcggcagcc
ctccgtgcgc gcaggctcgg tatgagccgc 100 acagcctaca cggtgggagc
cctgcttctc ctcttgggga ccctgctgcc 150 ggctgctgaa gggaaaaaga
aagggtccca aggtgccatc cccccgccag 200 acaaggccca gcacaatgac
tcagagcaga ctcagtcgcc ccagcagcct 250 ggctccagga accgggggcg
gggccaaggg cggggcactg ccatgcccgg 300 ggaggaggtg ctggagtcca
gccaagaggc cctgcatgtg acggagcgca 350 aatacctgaa gcgagactgg
tgcaaaaccc agccgcttaa gcagaccatc 400 cacgaggaag gctgcaacag
tcgcaccatc atcaaccgct tctgttacgg 450 ccagtgcaac tctttctaca
tccccaggca catccggaag gaggaaggtt 500 cctttcagtc ctgctccttc
tgcaagccca agaaattcac taccatgatg 550 gtcacactca actgccctga
actacagcca cctaccaaga agaagagagt 600 cacacgtgtg aagcagtgtc
gttgcatatc catcgatttg gattaagcca 650 aatccaggtg cacccagcat
gtcctaggaa tgcagcccca ggaagtccca 700 gacctaaaac aaccagattc
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 750 nnnnnnnnnn nagacttacg
atgcatgtat acaaacgaat agcagataat 800 gatgactagt tcacacataa
agtcctttta aggagaaaat ctaaaatgaa 850 aagtggataa acagaacatt
tataagtgat cagttaatgc ctaagagtga 900 aagtagttct attgacattc
ctcaagatat ttaatatcaa ctgcattatg 950 tattatgtct gcttaaatca
tttaaaaacg gcaaagaatt atatagacta 1000 tgaggtacct tgctgtgtag
gaggatgaaa ggggagttga tagtctcata 1050 aaactaattt ggcttcaagt
ttcatgaatc tgtaactaga atttaatttt 1100 caccccaata atgttctata
tagcctttgc taaagagcaa ctaataaatt 1150 aaacctattc tttcaa 1166 6 2279
DNA Homo Sapien 6 cggacctgaa cccctaaaag cggaaccgcc tcccgccctc
gccatcgcgg 50 agctgagtcg ccggcggcgg tggctgctgc cagacccgga
gtttcctctt 100 tcactggatg gagctgaact ttgggcggcc agagcagcac
agctgtccgg 150 ggatcgctgc atgctgagct ccctcggcaa gacccagcgg
cggctcggga 200 tttttttggg ggggcgggga ccagccccgc gccggcacca
tgttcctggc 250 gaccctgtac ttcgcgctgc cgctcttgga cttgctcctg
tcggccgaag 300 tgagcggcgg agaccgcctg gattgcgtga aagccagtga
tcagtgcctg 350 aaggagcaga gctgcagcac caagtaccgc acgctaaggc
agtgcgtggc 400 gggcaaggag accaacttca gcctggcatc cggcctggag
gccaaggatg 450 agtgccgcag cgccatggag gccctgaagc agaagtcgct
ctacaactgc 500 cgctgcaagc ggggtatgaa gaaggagaag aactgcctgc
gcatttactg 550 gagcatgtac cagagcctgc agggaaatga tctgctggag
gattccccat 600 atgaaccagt taacagcaga ttgtcagata tattccgggt
ggtcccattc 650 atatcagtgg agcacattcc caaagggaac aactgcctgg
atgcagcgaa 700 ggcctgcaac ctcgacgaca tttgcaagaa gtacaggtcg
gcgtacatca 750 ccccgtgcac caccagcgtg tccaatgatg tctgcaaccg
ccgcaagtgc 800 cacaaggccc tccggcagtt ctttgacaag gtcccggcca
agcacagcta 850 cggaatgctc ttctgctcct gccgggacat cgcctgcaca
gagcggaggc 900 gacagaccat cgtgcctgtg tgctcctatg aagagaggga
gaagcccaac 950 tgtttgaatt tgcaggactc ctgcaagacg aattacatct
gcagatctcg 1000 ccttgcggat ttttttacca actgccagcc agagtcaagg
tctgtcagca 1050 gctgtctaaa ggaaaactac gctgactgcc tcctcgccta
ctcggggctt 1100 attggcacag tcatgacccc caactacata gactccagta
gcctcagtgt 1150 ggccccatgg tgtgactgca gcaacagtgg gaacgaccta
gaagagtgct 1200 tgaaattttt gaatttcttc aaggacaata catgtcttaa
aaatgcaatt 1250 caagcctttg gcaatggctc cgatgtgacc gtgtggcagc
cagccttccc 1300 agtacagacc accactgcca ctaccaccac tgccctccgg
gttaagaaca 1350 agcccctggg gccagcaggg tctgagaatg aaattcccac
tcatgttttg 1400 ccaccgtgtg caaatttaca ggcacagaag ctgaaatcca
atgtgtcggg 1450 caatacacac ctctgtattt ccaatggtaa ttatgaaaaa
gaaggtctcg 1500 gtgcttccag ccacataacc acaaaatcaa tggctgctcc
tccaagctgt 1550 ggtctgagcc cactgctggt cctggtggta accgctctgt
ccaccctatt 1600 atctttaaca gaaacatcat agctgcatta aaaaaataca
atatggacat 1650 gtaaaaagac aaaaaccaag ttatctgttt cctgttctct
tgtatagctg 1700 aaattccagt ttaggagctc agttgagaaa cagttccatt
caactggaac 1750 attttttttt tttcctttta agaaagcttc ttgtgatcct
tcggggcttc 1800 tgtgaaaaac ctgatgcagt gctccatcca aactcagaag
gctttgggat 1850 atgctgtatt ttaaagggac agtttgtaac ttgggctgta
aagcaaactg 1900 gggctgtgtt ttcgatgatg atgatgatca tgatgatgat
catcatgatc 1950 atgatgatga tcatcatgat catgatgatg attttaacag
ttttacttct 2000 ggcctttcct agctagagaa ggagttaata tttctaaggt
aactcccata 2050 tctcctttaa tgacattgat ttctaatgat ataaatttca
gcctacattg 2100 atgccaagct tttttgccac aaagaagatt cttaccaaga
gtgggctttg 2150 tggaaacagc tggtactgat gttcaccttt atatatgtac
tagcattttc 2200 cacgctgatg tttatgtact gtaaacagtt ctgcactctt
gtacaaaaga 2250 aaaaacacct gtcacatcca aatataaaa 2279 7 562 DNA Homo
Sapien 7 atgcagcacc gaggcttcct cctcctcacc ctcctcgccc tgctggcgct 50
cacctccgcg gtcgccaaaa agaaagataa ggtgaagaag ggcggcccgg 100
ggagcgagtg cgctgagtgg gcctgggggc cctgcacccc cagcagcaag 150
gattgcggcg tgggtttccg cgagggcacc tgcggggccc agacccagcg 200
catccggtgc agggtgccct gcaactggaa gaaggagttt ggagccgact 250
gcaagtacaa gtttgagaac tggggtgcgt gtgatggggg cacaggcacc 300
aaagtccgcc aaggcaccct gaagaaggcg cgctacaatg ctcagtgcca 350
ggagaccatc cgcgtcacca agccctgcac ccccaagacc aaagcaaagg 400
ccaaagccaa gaaagggaag ggaaaggact agacgccaag cctggatgcc 450
aaggagcccc tggtgtcaca tggggcctgg cccacgccct ccctctccca 500
ggcccgagat gtgacccacc agtgccttct gtctgctcgt tagctttaat 550
caatcatgcc cc 562 8 1524 DNA Homo Sapien 8 gcggcagcag cgcgggcccc
agcagcctcg gcagccacag ccgctgcagc 50 cggggcagcc tccgctgctg
tcgcctcctc tgatgcgctt gccctctccc 100 ggccccggga ctccgggaga
atgtgggtcc taggcatcgc ggcaactttt 150 tgcggattgt tcttgcttcc
aggctttgcg ctgcaaatcc agtgctacca 200 gtgtgaagaa ttccagctga
acaacgactg ctcctccccc gagttcattg 250 tgaattgcac ggtgaacgtt
caagacatgt gtcagaaaga agtgatggag 300 caaagtgccg ggatcatgta
ccgcaagtcc tgtgcatcat cagcggcctg 350 tctcatcgcc tctgccgggt
accagtcctt ctgctcccca gggaaactga 400 actcagtttg catcagctgc
tgcaacaccc ctctttgtaa
cgggccaagg 450 cccaagaaaa ggggaagttc tgcctcggcc ctcaggccag
ggctccgcac 500 caccatcctg ttcctcaaat tagccctctt ctcggcacac
tgctgaagct 550 gaaggagatg ccaccccctc ctgcattgtt cttccagccc
tcgcccccaa 600 ccccccacct ccctgagtga gtttcttctg ggtgtccttt
tattctgggt 650 agggagcggg agtccgtgtt ctcttttgtt cctgtgcaaa
taatgaaaga 700 gctcggtaaa gcattctgaa taaattcagc ctgactgaat
tttcagtatg 750 tacttgaagg aaggaggtgg agtgaaagtt cacccccatg
tctgtgtaac 800 cggagtcaag gccaggctgg cagagtcagt ccttagaagt
cactgaggtg 850 ggcatctgcc ttttgtaaag cctccagtgt ccattccatc
cctgatgggg 900 gcatagtttg agactgcaga gtgagagtga cgttttctta
gggctggagg 950 gccagttccc actcaaggct ccctcgcttg acattcaaac
ttcatgctcc 1000 tgaaaaccat tctctgcagc agaattggct ggtttcgcgc
ctgagttggg 1050 ctctagtgac tcgagactca atgactggga cttagactgg
ggctcggcct 1100 cgctctgaaa agtgcttaag aaaatcttct cagttctcct
tgcagaggac 1150 tggcgccggg acgcgaagag caacgggcgc tgcacaaagc
gggcgctgtc 1200 ggtggtggag tgcgcatgta cgcgcaggcg cttctcgtgg
ttggcgtgct 1250 gcagcgacag gcggcagcac agcacctgca cgaacacccg
ccgaaactgc 1300 tgcgaggaca ccgtgtacag gagcgggttg atgaccgagc
tgaggtagaa 1350 aaacgtctcc gagaagggga ggaggatcat gtacgcccgg
aagtaggacc 1400 tcgtccagtc gtgcttgggt ttggccgcag ccatgatcct
ccgaatctgg 1450 ttgggcatcc agcatacggc caatgtcaca acaatcagcc
ctgggcagac 1500 acgagcagga gggagagaca gaga 1524 9 1253 DNA Homo
Sapien 9 caccctccgt ggcaaggcga ggccccgggg gcgggccggg gtcaccacgc 50
ctgccccagg gaaccgcaca gacggtactc acccttcttg cgatgatgtg 100
agatgataaa atgcctacat gatgagatga agtgagatga aaaacatagg 150
ccttgtgatg gaatgggaaa ttccagagat aatttgcacg tgcgctaagc 200
tgcggctacc cccgcaagca accttccaag tccttcgtgg caatggtgct 250
tccgtgggga ccgtgctcat gttccgctgc ccctccaacc accagatggt 300
ggggtctggg ctcctcacct gcacctggaa ggggagcatc gctgagtggt 350
cttcagggtc cccagtgtgc aaactggtgc caccacacga gacctttggc 400
ttcaaggtgg ccgtgatcgc ctccattgtg agctgtgcca tcatcctgct 450
catgtccatg gccttcctca cctgctgcct cctcaagtgc gtgaagaaga 500
gcaagcggcg gcgctccaac aggtcagccc agctgtggtc ccagctgaaa 550
gatgaggact tggagacggt gcaggccgca taccttggcc tcaagcactt 600
caacaaaccc gtgagcgggc ccagccaggc gcacgacaac cacagcttca 650
ccacagacca tggtgagagc accagcaagc tggccagtgt gacccgcagc 700
gtggacaagg accctgggat ccccagagct ctaagcctca gtggctcctc 750
cagctcaccc caagcccagg tgatggtgca catggcaaac cccagacagc 800
ccctgcctgc ctctgggctg gccacaggaa tgccacaaca gcccgcagca 850
tatgccctag ggtgaccacg cagtgaggct ggtgcccatg ctccacactg 900
ggaggccagg ctgaccccac cagccagtca gctacaactc cacatcaact 950
ccacatgcgc ccagctcgag actgatgagt ggaatcagct tccaggtgta 1000
gggacccctt gaggggccga gctgacatcc aaggctgagg accccagtgg 1050
ggagtgttct gttccggcat atcctggccg taacgatttt tatagttatg 1100
gactacttga aaccactact gagggtaatt tactagctgt ggcctcccac 1150
taactagcat tcctttaaag agactgggaa atgttttaag caaatctagt 1200
tttgtataat aaaataagaa aatagcaata aacttctttt cagcaactac 1250 aaa
1253 10 5542 DNA Homo Sapien 10 ctgactgcac tggtgatggt ccctggcaat
ccaacctggc accatcgcag 50 ttggagtact atgcatcttc accagatgaa
aaggctctag tagaagctgc 100 tgcaaggatt ggtattgtgt ttattggcaa
ttctgaagaa actatggagg 150 ttaaaactct tggaaaactg gaacggtaca
aactgcttca tattctggaa 200 tttgattcag atcgtaggag aatgagtgta
attgttcagg caccttcagg 250 tgagaagtta ttatttgcta aaggagctga
gtcatcaatt ctccctaaat 300 gtataggtgg agaaatagaa aaaaccagaa
ttcatgtaga tgaatttgct 350 ttgaaagggc taagaactct gtgtatagca
tatagaaaat ttacatcaaa 400 agagtatgag gaaatagata aacgcatatt
tgaagccagg actgccttgc 450 agcagcggga agagaaattg gcagctgttt
tccagttcat agagaaagac 500 ctgatattac ttggagccac agcagtagaa
gacagactac aagataaagt 550 tcgagaaact attgaagcat tgagaatggc
tggtatcaaa gtatgggtac 600 ttactgggga taaacatgaa acagctgtta
gtgtgagttt atcatgtggc 650 cattttcata gaaccatgaa catccttgaa
cttataaacc agaaatcaga 700 cagcgagtgt gctgaacaat tgaggcagct
tgccagaaga attacagagg 750 atcatgtgat tcagcatggg ctggtagtgg
atgggaccag cctatctctt 800 gcactcaggg agcatgaaaa actatttatg
gaagtttgca gaaattgttc 850 agctgtatta tgctgtcgta tggctccact
gcagaaagca aaagtaataa 900 gactaataaa aatatcacct gagaaaccta
taacattggc tgttggtgat 950 ggtgctaatg acgtaagcat gatacaagaa
gcccatgttg gcataggaat 1000 catgggtaaa gaaggaagac aggctgcaag
aaacagtgac tatgcaatag 1050 ccagatttaa gttcctctcc aaattgcttt
ttgttcatgg tcatttttat 1100 tatattagaa tagctaccct tgtacagtat
tttttttata agaatgtgtg 1150 ctttatcaca ccccagtttt tatatcagtt
ctactgtttg ttttctcagc 1200 aaacattgta tgacagcgtg tacctgactt
tatacaatat ttgttttact 1250 tccctaccta ttctgatata tagtcttttg
gaacagcatg tagaccctca 1300 tgtgttacaa aataagccca ccctttatcg
agacattagt aaaaaccgcc 1350 tcttaagtat taaaacattt ctttattgga
ccatcctggg cttcagtcat 1400 gcctttattt tcttttttgg atcctattta
ctaataggga aagatacatc 1450 tctgcttgga aatggccaga tgtttggaaa
ctggacattt ggcactttgg 1500 tcttcacagt catggttatt acagtcacag
taaagatggc tctggaaact 1550 catttttgga cttggatcaa ccatctcgtt
acctggggat ctattatatt 1600 ttattttgta ttttccttgt tttatggagg
gattctctgg ccatttttgg 1650 gctcccagaa tatgtatttt gtgtttattc
agctcctgtc aagtggttct 1700 gcttggtttg ccataatcct catggttgtt
acatgtctat ttcttgatat 1750 cataaagaag gtctttgacc gacacctcca
ccctacaagt actgaaaagg 1800 cacagcttac tgaaacaaat gcaggtatca
agtgcttgga ctccatgtgc 1850 tgtttcccgg aaggagaagc agcgtgtgca
tctgttggaa gaatgctgga 1900 acgagttata ggaagatgta gtccaaccca
catcagcaga tcatggagtg 1950 catcggatcc tttctatacc aacgacagga
gcatcttgac tctctccaca 2000 atggactcat ctacttgtta aaggggcagt
agtactttgt gggagccagt 2050 tcacctcctt tcctaaaatt cagtgtgatc
accctgttaa tggccacact 2100 agctctgaaa ttaatttcca aaatctttgt
agtagttcat acccactcag 2150 agttataatg gcaaacaaac agaaagcatt
agtacaagcc cctcccaaca 2200 cccttaattt gaatctgaac atgttaaaat
ttgagaataa agagacattt 2250 ttcatctctt tgtctggttt gtcccttgtg
cttatgggac tcctaatggc 2300 atttcagtct gttgctgagg ccattatatt
ttaatataaa tgtagaaaaa 2350 agagagaaat cttagtaaag agtatttttt
agtattagct tgattattga 2400 ctcttctatt taaatctgct tctgtaaatt
atgctgaaag tttgccttga 2450 gaactctatt tttttattag agttatattt
aaagcttttc atgggaaaag 2500 ttaatgtgaa tactgaggaa ttttggtccc
tcagtgacct gtgttgttaa 2550 ttcattaatg cattctgagt tcacagagca
aattaggaga atcatttcca 2600 accattattt actgcagtat ggggagtaaa
tttataccaa ttcctctaac 2650 tgtactgtaa cacagcctgt aaagttagcc
atataaatgc aagggtatat 2700 catatataca aatcaggaat caggtccgtt
caccgaactt caaattgatg 2750 tttactaata tttttgtgac agagtataaa
gaccctatag tgggtaaatt 2800 agatactatt agcatattat taatttaatg
tctttatcat tggatctttt 2850 gcatgcttta atctggttaa catatttaaa
tttgcttttt ttctctttac 2900 ctgaaggctc tgtgtatagt atttcatgac
atcgttgtac agtttaacta 2950 tcaataaaaa gtttggacag tatttaaata
ttgcaaatat gtttaattat 3000 acaaatcaga atagtatggg taattaaatg
aatacaaaaa gaagagcctc 3050 tttctgcagc cgacttagac atgctcttcc
ctttctataa gctagatttt 3100 agaataaagg gtttcagtta ataatcttat
tttcaggtta tgtcatctaa 3150 cttatagcaa actaccacaa tacagtgagt
tctgccagtg tcccagtaca 3200 aggcatattt caggtgtggc tgtggaatgt
aaaaatgctc aacttgtatc 3250 aggtaatgtt agcaataaat taaatgctaa
gaatgattaa tcgggtacat 3300 gttactgtaa ttaactcatt gcacttcaaa
acctaacttc catcctgaat 3350 ttatcaagta gttcagtatt gtcatttgtt
tttgttttat tgaaaagtaa 3400 tgttgtctta agatttagaa gtgattatta
gcttgagaac tattacccag 3450 ctctaagcaa ataatgattg tatacatatt
aagataatgg ttaaatgcgg 3500 ttttaccaag ttttcccttg aaaatgtaat
tcctttatgg agatttattg 3550 tgcagcccta agcttccttc ccatttcatg
aatataaggc ttctagaatt 3600 ggactggcag gggaaagaat ggtagagaca
gaaattaaga ctttatcctt 3650 gtttgcttgt aaactattat tttcttgcta
atgtaacatt tgtctgttcc 3700 agtgatgtaa ggatattaag ttattaagct
aaatattaat tttcaaaaat 3750 agtccttctt taacttagat atttcatagc
tggatttagg aagatctgtt 3800 attctggaag tactaaaaag aataatacaa
cgtacaatgt ctgcattcac 3850 taattcatgt tccagaagag gaaataatga
agatatactc agtagagtac 3900 taggtgggag gatatggaaa tttgctcata
aaatctctta taaaacgtgc 3950 atataacaaa atgacaccca gtaggcctgc
attacattta catgaccgtg 4000 tttatttgcc atcaaataaa ctgagtactg
acaccagaca aagactccaa 4050 agtcataaaa tagcctatga ccaactgcag
caagacagga ggtcagctcg 4100 cctataatgg tgcttaaagt gtgattgatg
taattttctg tactcaccat 4150 ttgaagttag ttaaggagaa ctttattttt
ttaaaaaaag taaatggcaa 4200 ccactagtgt gctcatcctg aactgttact
ccaaatccac tccgttttta 4250 aagcaaaatt atcttgtgat tttaagaaaa
gagttttcta tttatttaag 4300 aaagtaacaa tgcagtctgc aagctttcag
tagttttcta gtgctatatt 4350 catcctgtaa aactcttact acgtaaccag
taatcacaag gaaagtgtcc 4400 cctttgcata tttctttaaa attctttctt
tggaaagtat gatgttgata 4450 attaacttac ccttatctgc caaaaccaga
gcaaaatgct aaatacgtta 4500 ttgctaatca gtggtctcaa atcgatttgc
ctccctttgc ctcgtctgag 4550 ggctgtaagc ctgaagatag tggcaagcac
caagtcagtt tccaaaattg 4600 cccctcagct gctttaagtg actcagcacc
ctgcctcagc ttcagcaggc 4650 gtaggctcac cctgggcgga gcaaagtatg
ggccagggag aactacagct 4700 acgaagacct gctgtcgagt tgagaaaagg
ggagaattta tggtctgaat 4750 tttctaactg tcctctttct tgggtctaaa
gctcataata cacaaaggct 4800 tccagacctg agccacaccc aggccctatc
ctgaacagga gactaaacag 4850 aggcaaatca accctaggaa atacttgcat
tctgccctac ggttagtacc 4900 aggactgagg tcatttctac tggaaaagat
tgtgagattg aacttatctg 4950 atcgcttgag actcctaata ggcaggagtc
aaggccacta gaaaattgac 5000 agttaagagc caaaagtttt taaaatatgc
tactctgaaa aatctcgtga 5050 aggctgtagg aaaagggaga atcttccatg
ttggtgtttt tcctgtaaag 5100 atcagtttgg ggtatgatat aagcaggtat
taataaaaat aacacaccaa 5150 agagttacgt aaaacatgtt ttattaattt
tggtccccac gtacagacat 5200 tttatttcta ttttgaaatg agttatctat
tttcataaaa gtaaaacact 5250 attaaagtgc tgttttatgt gaaataactt
gaatgttgtt cctataaaaa 5300 atagatcata actcatgata tgtttgtaat
catggtaatt tagattttta 5350 tgaggaatga gtatctggaa atattgtagc
aatacttggt ttaaaatttt 5400 ggacctgaga cactgtggct gtctaatgta
atcctttaaa aattctctgc 5450 attgtcagta aatgtagtat attattgtac
agctactcat aattttttaa 5500 agtttatgaa gttatattta tcaaataaaa
actttcctat at 5542 11 6155 DNA Homo Sapien 11 atgtgggaag aagaagacat
tgctattctg ttcaataaag aaccaggaaa 50 aacagagaat attgaaaata
atctaagttc caaccataga agaagctgca 100 gaagaagtga agaaagtgat
gatgatttgg attttgatat tggtttagaa 150 aacacaggag gagaccctca
aattctgaga tttatttcag acttccttgc 200 ttttttggtt ctctacaatt
tcatcattcc aatttcatta tatgtgacag 250 tcgaaatgca gaaatttctt
ggatcatttt ttattggctg ggatcttgat 300 ctgtatcatg aagaatcaga
tcagaaagct caagtcaata cttccgatct 350 gaatgaagag cttggacagg
tagagtacgt gtttacagat aaaactggta 400 cactgacaga aaatgagatg
cagtttcggg aatgttcaat taatggcatg 450 aaataccaag aaattaatgg
tagacttgta cccgaaggac caacaccaga 500 ctcttcagaa ggaaacttat
cttatcttag tagtttatcc catcttaaca 550 acttatccca tcttacaacc
agttcctctt tcagaaccag tcctgaaaat 600 gaaactgaac taattaaaga
acatgatctc ttctttaaag cagtcagtct 650 ctgtcacact gtacagatta
gcaatgttca aactgactgc actggtgatg 700 gtccctggca atccaacctg
gcaccatcgc agttggagta ctatgcatct 750 tcaccagatg aaaaggctct
agtagaagct gctgcaaggt acaaactgct 800 tcatattctg gaatttgatt
cagatcgtag gagaatgagt gtaattgttc 850 aggcaccttc aggtgagaag
ttattatttg ctaaaggagc tgagtcatca 900 attctcccta aatgtatagg
tggagaaata gaaaaaacca gaattcatgt 950 agatgaattt gctttgaaag
ggctaagaac tctgtgtata gcatatagaa 1000 aatttacatc aaaagagtat
gaggaaatag ataaacgcat atttgaagcc 1050 aggactgcct tgcagcagcg
ggaagagaaa ttggcagctg ttttccagtt 1100 catagagaaa gacctgatat
tacttggagc cacagcagta gaagacagac 1150 tacaagataa agttcgagaa
actattgaag cattgagaat ggctggtatc 1200 aaagtatggg tacttactgg
ggataaacat gaaacagctg ttagtgtgag 1250 tttatcatgt ggccattttc
atagaaccat gaacatcctt gaacttataa 1300 accagaaatc agacagcgag
tgtgctgaac aattgaggca gcttgccaga 1350 agaattacag aggatcatgt
gattcagcat gggctggtag tggatgggac 1400 cagcctatct cttgcactca
gggagcatga aaaactattt atggaagttt 1450 gcagaaattg ttcagctgta
ttatgctgtc gtatggctcc actgcagaaa 1500 gcaaaagtaa taagactaat
aaaaatatca cctgagaaac ctataacatt 1550 ggctgttggt gatggtgcta
atgacgtaag catgatacaa gaagcccatg 1600 ttggcatagg aatcatgggt
aaagaaggaa gacaggctgc aagaaacagt 1650 gactatgcaa tagccagatt
taagttcctc tccaaattgc tttttgttca 1700 tggtcatttt tattatatta
gaatagctac ccttgtacag tatttttttt 1750 ataagaatgt gtgctttatc
acaccccagt ttttatatca gttctactgt 1800 ttgttttctc agcaaacatt
gtatgacagc gtgtacctga ctttatacaa 1850 tatttgtttt acttccctac
ctattctgat atatagtctt ttggaacagc 1900 atgtagaccc tcatgtgtta
caaaataagc ccacccttta tcgagacatt 1950 agtaaaaacc gcctcttaag
tattaaaaca tttctttatt ggaccatcct 2000 gggcttcagt catgccttta
ttttcttttt tggatcctat ttactaatag 2050 ggaaagatac atctctgctt
ggaaatggcc agatgtttgg aaactggaca 2100 tttggcactt tggtcttcac
agtcatggtt attacagtca cagtaaagat 2150 ggctctggaa actcattttt
ggacttggat caaccatctc gttacctggg 2200 gatctattat attttatttt
gtattttcct tgttttatgg agggattctc 2250 tggccatttt tgggctccca
gaatatgtat tttgtgttta ttcagctcct 2300 gtcaagtggt tctgcttggt
ttgccataat cctcatggtt gttacatgtc 2350 tatttcttga tatcataaag
aaggtctttg accgacacct ccaccctaca 2400 agtactgaaa aggcacagct
tactgaaaca aatgcaggta tcaagtgctt 2450 ggactccatg tgctgtttcc
cggaaggaga agcagcgtgt gcatctgttg 2500 gaagaatgct ggaacgagtt
ataggaagat gtagtccaac ccacatcagc 2550 agatcatgga gtgcatcgga
tcctttctat accaacgaca ggagcatctt 2600 gactctctcc acaatggact
catctacttg ttaaaggggc agtagtactt 2650 tgtgggagcc agttcacctc
ctttcctaaa attcagtgtg atcaccctgt 2700 taatggccac actagctctg
aaattaattt ccaaaatctt tgtagtagtt 2750 catacccact cagagttata
atggcaaaca aacagaaagc attagtacaa 2800 gcccctccca acacccttaa
tttgaatctg aacatgttaa aatttgagaa 2850 taaagagaca tttttcatct
ctttgtctgg tttgtccctt gtgcttatgg 2900 gactcctaat ggcatttcag
tctgttgctg aggccattat attttaatat 2950 aaatgtagaa aaaagagaga
aatcttagta aagagtattt tttagtatta 3000 gcttgattat tgactcttct
atttaaatct gcttctgtaa attatgctga 3050 aagtttgcct tgagaactct
atttttttat tagagttata tttaaagctt 3100 ttcatgggaa aagttaatgt
gaatactgag gaattttggt ccctcagtga 3150 cctgtgttgt taattcatta
atgcattctg agttcacaga gcaaattagg 3200 agaatcattt ccaaccatta
tttactgcag tatggggagt aaatttatac 3250 caattcctct aactgtactg
taacacagcc tgtaaagtta gccatataaa 3300 tgcaagggta tatcatatat
acaaatcagg aatcaggtcc gttcaccgaa 3350 cttcaaattg atgtttacta
atatttttgt gacagagtat aaagacccta 3400 tagtgggtaa attagatact
attagcatat tattaattta atgtctttat 3450 cattggatct tttgcatgct
ttaatctggt taacatattt aaatttgctt 3500 tttttctctt tacctgaagg
ctctgtgtat agtatttcat gacatcgttg 3550 tacagtttaa ctatcaataa
aaagtttgga cagtatttaa atattgcaaa 3600 tatgtttaat tatacaaatc
agaatagtat gggtaattaa atgaatacaa 3650 aaagaagagc ctctttctgc
agccgactta gacatgctct tccctttcta 3700 taagctagat tttagaataa
agggtttcag ttaataatct tattttcagg 3750 ttatgtcatc taacttatag
caaactacca caatacagtg agttctgcca 3800 gtgtcccagt acaaggcata
tttcaggtgt ggctgtggaa tgtaaaaatg 3850 ctcaacttgt atcaggtaat
gttagcaata aattaaatgc taagaatgat 3900 taatcgggta catgttactg
taattaactc attgcacttc aaaacctaac 3950 ttccatcctg aatttatcaa
gtagttcagt attgtcattt gtttttgttt 4000 tattgaaaag taatgttgtc
ttaagattta gaagtgatta ttagcttgag 4050 aactattacc cagctctaag
caaataatga ttgtatacat attaagataa 4100 tggttaaatg cggttttacc
aagttttccc ttgaaaatgt aattccttta 4150 tggagattta ttgtgcagcc
ctaagcttcc ttcccatttc atgaatataa 4200 ggcttctaga attggactgg
caggggaaag aatggtagag acagaaatta 4250 agactttatc cttgtttgct
tgtaaactat tattttcttg ctaatgtaac 4300 atttgtctgt tccagtgatg
taaggatatt aagttattaa gctaaatatt 4350 aattttcaaa aatagtcctt
ctttaactta gatatttcat agctggattt 4400 aggaagatct gttattctgg
aagtactaaa aagaataata caacgtacaa 4450 tgtctgcatt cactaattca
tgttccagaa gaggaaataa tgaagatata 4500 ctcagtagag tactaggtgg
gaggatatgg
aaatttgctc ataaaatctc 4550 ttataaaacg tgcatataac aaaatgacac
ccagtaggcc tgcattacat 4600 ttacatgacc gtgtttattt gccatcaaat
aaactgagta ctgacaccag 4650 acaaagactc caaagtcata aaatagccta
tgaccaactg cagcaagaca 4700 ggaggtcagc tcgcctataa tggtgcttaa
agtgtgattg atgtaatttt 4750 ctgtactcac catttgaagt tagttaagga
gaactttatt tttttaaaaa 4800 aagtaaatgg caaccactag tgtgctcatc
ctgaactgtt actccaaatc 4850 cactccgttt ttaaagcaaa attatcttgt
gattttaaga aaagagtttt 4900 ctatttattt aagaaagtaa caatgcagtc
tgcaagcttt cagtagtttt 4950 ctagtgctat attcatcctg taaaactctt
actacgtaac cagtaatcac 5000 aaggaaagtg tcccctttgc atatttcttt
aaaattcttt ctttggaaag 5050 tatgatgttg ataattaact tacccttatc
tgccaaaacc agagcaaaat 5100 gctaaatacg ttattgctaa tcagtggtct
caaatcgatt tgcctccctt 5150 tgcctcgtct gagggctgta agcctgaaga
tagtggcaag caccaagtca 5200 gtttccaaaa ttgcccctca gctgctttaa
gtgactcagc accctgcctc 5250 agcttcagca ggcgtaggct caccctgggc
ggagcaaagt atgggccagg 5300 gagaactaca gctacgaaga cctgctgtcg
agttgagaaa aggggagaat 5350 ttatggtctg aattttctaa ctgtcctctt
tcttgggtct aaagctcata 5400 atacacaaag gcttccagac ctgagccaca
cccaggccct atcctgaaca 5450 ggagactaaa cagaggcaaa tcaaccctag
gaaatacttg cattctgccc 5500 tacggttagt accaggactg aggtcatttc
tactggaaaa gattgtgaga 5550 ttgaacttat ctgatcgctt gagactccta
ataggcagga gtcaaggcca 5600 ctagaaaatt gacagttaag agccaaaagt
ttttaaaata tgctactctg 5650 aaaaatctcg tgaaggctgt aggaaaaggg
agaatcttcc atgttggtgt 5700 ttttcctgta aagatcagtt tggggtatga
tataagcagg tattaataaa 5750 aataacacac caaagagtta cgtaaaacat
gttttattaa ttttggtccc 5800 cacgtacaga cattttattt ctattttgaa
atgagttatc tattttcata 5850 aaagtaaaac actattaaag tgctgtttta
tgtgaaataa cttgaatgtt 5900 gttcctataa aaaatagatc ataactcatg
atatgtttgt aatcatggta 5950 atttagattt ttatgaggaa tgagtatctg
gaaatattgt agcaatactt 6000 ggtttaaaat tttggacctg agacactgtg
gctgtctaat gtaatccttt 6050 aaaaattctc tgcattgtca gtaaatgtag
tatattattg tacagctact 6100 cataattttt taaagtttat gaagttatat
ttatcaaata aaaactttcc 6150 tatat 6155 12 1372 DNA Homo Sapien 12
gcacgagggc gcttttgtct ccggtgagtt ttgtggcggg aagcttctgc 50
gctggtgctt agtaaccgac tttcctccgg actcctgcac gacctgctcc 100
tacagccggc gatccactcc cggctgttcc cccggagggt ccagaggcct 150
ttcagaagga gaaggcagct ctgtttctct gcagaggagt agggtccttt 200
cagccatgaa gcatgtgttg aacctctacc tgttaggtgt ggtactgacc 250
ctactctcca tcttcgttag agtgatggag tccctagaag gcttactaga 300
gagcccatcg cctgggacct cctggaccac cagaagccaa ctagccaaca 350
cagagcccac caagggcctt ccagaccatc catccagaag catgtgataa 400
gacctccttc catactggcc atattttgga acactgacct agacatgtcc 450
agatgggagt cccattccta gcagacaagc tgagcaccgt tgtaaccaga 500
gaactattac taggccttga agaacctgtc taactggatg ctcattgcct 550
gggcaaggcc tgtttaggcc ggttgcggtg gctcatgcct gtaatcctag 600
cactttggga ggctgaggtg ggtggatcac ctgaggtcag gagttcgaga 650
ccagcctcgc caacatggcg aaaccccatc tctactaaaa atacaaaagt 700
tagctgggtg tggtggcaga ggcctgtaat cccagttcct tgggaggctg 750
aggcgggaga attgcttgaa cccggggacg gaggttgcag tgaaccgaga 800
tcgcactgct gtacccagcc tgggccacag tgcaagactc catctcaaaa 850
aaaaaaagaa aagaaaaagc ctgtttaatg cacaggtgtg agtggattgc 900
ttatggctat gagataggtt gatctcgccc ttaccccggg gtctggtgta 950
tgctgtgctt tcctcagcag tatggctctg acatctctta gatgtcccaa 1000
cttcagctgt tgggagatgg tgatattttc aaccctactt cctaaacatc 1050
tgtctggggt tcctttagtc ttgaatgtct tatgctcaat tatttggtgt 1100
tgagcctctc ttccacaaga gctcctccat gtttggatag cagttgaaga 1150
ggttgtgtgg gtgggctgtt gggagtgagg atggagtgtt cagtgcccat 1200
ttctcatttt acattttaaa gtcgttcctc caacatagtg tgtattggtc 1250
tgaagggggt ggtgggatgc caaagcctgc tcaagttatg gacattgtgg 1300
ccaccatgtg gcttaaatga ttttttctaa ctaataaagt ggaatatata 1350
tttcaaaaaa aaaaaaaaaa aa 1372 13 770 DNA Homo Sapien unsure 45,
611, 715 unknown base 13 atacgactca ctatagggcg aattgggtac
cgggcccccc ctcgngtcga 50 cggtatcgat aagcttgata tcgaattcgg
ccacactggc cggatcctct 100 agagatccct cgacctcgac ccacgcgtcc
gcccacgcgt ccgatgtgcc 150 tctgggcaaa gaagcagagc taacgaggaa
agggatttaa agagtttttc 200 ttgggtgttt gtcaaacttt tattccctgt
ctgtgtgcag aggggattca 250 acttcaattt ttctgcagtg gctctgagtc
cagcccctta cttaaagatc 300 tggaaagcat gaagactggg ctttttttcc
tatgtctctt gggaactgca 350 gctgcaatcc cgacaaatgc aagattatta
tctgatcatt ccaaaccaac 400 tgctgaaacg gtagcacccg acaacactgc
aatccccagt ttaagggctg 450 aagatgaaga aaatgaaaaa gaaacagcag
tatccacaga agacgattcc 500 caccataagg ctgaaaaatc atcagtacta
aagtcaaaag aggaaagcca 550 tgaacagtca gcagaacagg gcaagagttc
tagccaagag ctgggattga 600 aggatcaaga ngacagtgat ggtgacttaa
gtgtgaattt ggagtatgca 650 ccaactgaag gtacattgga cataaaagaa
gatatgagtg agcctcagga 700 gaaaaactct caganacact gattttttgg
ctcctggggt agttccttcc 750 agattctacc acagaagttt 770 14 1187 DNA
Homo Sapien 14 cgcgggccat ggctccctgg gcggaggccg agcactcggc
gctgaacccg 50 ctgcgcgcgg tgtggctcac gctgaccgcc gccttcctgc
tgaccctact 100 gctgcagctc ctgccgcccg gcctgctccc gggctgcgcg
atcttccagg 150 acctgatccg ctatgggaaa accaagtgtg gggagccgtc
gcgccccgcc 200 gcctgccgag cctttgatgt ccccaagaga tatttttccc
acttttatat 250 catctcagtg ctgtggaatg gcttcctgct ttggtgcctt
actcaatctc 300 tgttcctggg agcacctttt ccaagctggc ttcatggttt
gctcagaatt 350 ctcggggcgg cacagttcca gggaggggag ctggcactgt
ctgcattctt 400 agtgctagta tttctgtggc tgcacagctt acgaagactc
ttcgagtgcc 450 tctacgtcag tgtcttctcc aatgtcatga ttcacgtcgt
gcagtactgt 500 tttggacttg tctattatgt ccttgttggc ctaactgtgc
tgagccaagt 550 gccaatggat ggcaggaatg cctacataac agggaaaaat
ctattgatgc 600 aagcacggtg gttccatatt cttgggatga tgatgttcat
ctggtcatct 650 gcccatcagt ataagtgcca tgttattctc ggcaatctca
ggaaaaataa 700 agcaggagtg gtcattcact gtaaccacag gatcccattt
ggagactggt 750 ttgaatatgt ttcttcccct aactacttag cagagctgat
gatctacgtt 800 tccatggccg tcacctttgg gttccacaac ttaacttggt
ggctagtggt 850 gacaaatgtc ttctttaatc aggccctgtc tgcctttctc
agccaccaat 900 tctacaaaag caaatttgtc tcttacccga agcataggaa
agctttccta 950 ccatttttgt tttaagttaa cctcagtcat gaagaatgca
aaccaggtga 1000 tggtttcaat gcctaaggac agtgaagtct ggagcccaaa
gtacagtttc 1050 agcaaagctg tttgaaactc tccattccat ttctataccc
cacaagtttt 1100 cactgaatga gcatggcagt gccactcaat aaaatgaatc
tccaaagtat 1150 cttcaaagaa taaatactaa tggcaaaaaa aaaaaaa 1187 15
1840 DNA Homo Sapien 15 tccacacaca caaaaaacct gcgcgtgagg ggggaggaaa
agcagggcct 50 ttaaaaaggc aatcacaaca acttttgctg ccaggatgcc
cttgctttgg 100 ctgagaggat ttctgttggc aagttgctgg attatagtga
ggagttcccc 150 caccccagga tccgaggggc acagcgcggc ccccgactgt
ccgtcctgtg 200 cgctggccgc cctcccaaag gatgtaccca actctcagcc
agagatggtg 250 gaggccgtca agaagcacat tttaaacatg ctgcacttga
agaagagacc 300 cgatgtcacc cagccggtac ccaaggcggc gcttctgaac
gcgatcagaa 350 agcttcatgt gggcaaagtc ggggagaacg ggtatgtgga
gatagaggat 400 gacattggaa ggagggcaga aatgaatgaa cttatggagc
agacctcgga 450 gatcatcacg tttgccgagt caggaacagc caggaagacg
ctgcacttcg 500 agatttccaa ggaaggcagt gacctgtcag tggtggagcg
tgcagaagtc 550 tggctcttcc taaaagtccc caaggccaac aggaccagga
ccaaagtcac 600 catccgcctc ttccagcagc agaagcaccc gcagggcagc
ttggacacag 650 gggaagaggc cgaggaagtg ggcttaaagg gggagaggag
tgaactgttg 700 ctctctgaaa aagtagtaga cgctcggaag agcacctggc
atgtcttccc 750 tgtctccagc agcatccagc ggttgctgga ccagggcaag
agctccctgg 800 acgttcggat tgcctgtgag cagtgccagg agagtggcgc
cagcttggtt 850 ctcctgggca agaagaagaa gaaagaagag gagggggaag
ggaaaaagaa 900 gggcggaggt gaaggtgggg caggagcaga tgaggaaaag
gagcagtcgc 950 acagaccttt cctcatgctg caggcccggc agtctgaaga
ccaccctcat 1000 cgccggcgtc ggcggggctt ggagtgtgat ggcaaggtca
acatctgctg 1050 taagaaacag ttctttgtca gtttcaagga catcggctgg
aatgactgga 1100 tcattgctcc ctctggctat catgccaact actgcgaggg
tgagtgcccg 1150 agccatatag caggcacgtc cgggtcctca ctgtccttcc
actcaacagt 1200 catcaaccac taccgcatgc ggggccatag cccctttgcc
aacctcaaat 1250 cgtgctgtgt gcccaccaag ctgagaccca tgtccatgtt
gtactatgat 1300 gatggtcaaa acatcatcaa aaaggacatt cagaacatga
tcgtggagga 1350 gtgtgggtgc tcatagagtt gcccagccca gggggaaagg
gagcaagagt 1400 tgtccagaga agacagtggc aaaatgaaga aatttttaag
gtttctgagt 1450 taaccagaaa aatagaaatt aaaaacaaaa caaaacaaaa
aaaaaaacaa 1500 aaaaaaacaa aagtaaatta aaaacaaacc tgatgaaaca
gatgaaacag 1550 atgaaggaag atgtggaaat cttagcctgc cttagccagg
gctcagagat 1600 gaagcagtga agagacagat tgggagggaa agggagaatg
gtgtaccctt 1650 tatttcttct gaaatcacac tgatgacatc agttgtttaa
acggggtatt 1700 gtcctttccc cccttgaggt tcccttgtga gcttgaatca
accaatctga 1750 tctgcagtag tgtggactag aacaacccaa atagcatcta
gaaagccatg 1800 agtttgaaag ggcccatcac aggcactttc ctagcctaat 1840 16
1771 DNA Homo Sapien 16 gcggagaagc cgggagcgcg gggctcagtc ggggggcggc
ggcggcggcg 50 gctccgggga tggcggcggc tccgctgctg ctgctgctgc
tgctcgtgcc 100 cgtgccgctg ctgccgctgc tggcccaagg gcccggaggg
gcgctgggaa 150 accggcatgc ggtgtactgg aacagctcca accagcacct
gcggcgagag 200 ggctacaccg tgcaggtgaa cgtgaacgac tatctggata
tttactgccc 250 gcactacaac agctcggggg tgggccccgg ggcgggaccg
gggcccggag 300 gcggggcaga gcagtacgtg ctgtacatgg tgagccgcaa
cggctaccgc 350 acctgcaacg ccagccaggg cttcaagcgc tgggagtgca
accggccgca 400 cgccccgcac agccccatca agttctcgga gaagttccag
cgctacagcg 450 ccttctctct gggctacgag ttccacgccg gccacgagta
ctactacatc 500 tccacgccca ctcacaacct gcactggaag tgtctgagga
tgaaggtgtt 550 cgtctgctgc gcctccacat cgcactccgg ggagaagccg
gtccccactc 600 tcccccagtt caccatgggc cccaatgtga agatcaacgt
gctggaagac 650 tttgagggag agaaccctca ggtgcccaag cttgagaaga
gcatcagcgg 700 gaccagcccc aaacgggaac acctgcccct ggccgtgggc
atcgccttct 750 tcctcatgac gttcttggcc tcctagctct gccccctccc
ctgggggggg 800 agagatgggg cggggcttgg aaggagcagg gagcctttgg
cctctccaag 850 ggaagcctag tgggcctaga cccctcctcc catggctaga
agtggggcct 900 gcaccataca tctgtgtccg ccccctctac cccttccccc
cacgtagggc 950 actgtagtgg accaagcacg gggacagcca tgggtcccgg
gcggccttgt 1000 ggctctggta atgtttggta ccaaacttgg gggccaaaaa
gggcagtgct 1050 caggactccc tggcccctgg tacctttccc tgactcctgg
tgccctctcc 1100 ctttgtcccc ccagagagac atatgccccc agagagagca
aatcgaagcg 1150 tgggaggcac ccccattgct ctcctccagg ggcagaacat
ggggagggga 1200 ctagatgggc aaggggcagc actgcctgct gcttccttcc
cctgtttaca 1250 gcaataaagc acgtcctcct cccccactcc cacttccagg
attgtggttt 1300 ggattgaaac caagtttaca agtagacacc cctggggggg
cgggcagtgg 1350 acaaggatgg caaggggtgg gcattggggt gccaggcagg
catgtacaga 1400 ctctatatct ctatatataa tgtacagaca gacagagtcc
cttccctctt 1450 taaccccctg acctttcttg acttcccctt cagcttcaga
ccccttcccc 1500 accaggctta ggccccccca caccttgggg ggacccccct
ggcccctctt 1550 ttgtcttctg tgaagacagg acctatgcaa cgcacagaca
cttttggaga 1600 ccgtaaaaca acagcgcccc ctcccttcca gccctgagcc
gggaaccatc 1650 tcccaggacc ttgccctgct caccctatgt ggtcccacct
atcctcctgg 1700 gcctttttca agtgctttgg ctgtgacttt catactctgc
tcttagtcta 1750 aaaaaaataa actggagata a 1771 17 4126 DNA Homo
sapien 17 cgctcgccat gggccactcc ccacctgtcc tgcctttgtg tgcctctgtg 50
tctttgctgg gtggcctgac ctttggttat gaactggcag tcatatcagg 100
tgccctgctg ccactgcagc ttgactttgg gctaagctgc ttggagcagg 150
agttcctggt gggcagcctg ctcctggggg ctctcctcgc ctccctggtt 200
ggtggcttcc tcattgactg ctatggcagg aagcaagcca tcctcgggag 250
caacttggtg ctgctggcag gcagcctgac cctgggcctg gctggttccc 300
tggcctggct ggtcctgggc cgcgctgtgg ttggcttcgc catttccctc 350
tcctccatgg cttgctgtat ctacgtgtca gagctggtgg ggccacggca 400
gcggggagtg ctggtgtccc tctatgaggc aggcatcacc gtgggcatcc 450
tgctctccta tgccctcaac tatgcactgg ctggtacccc ctggggatgg 500
aggcacatgt tcggctgggc cactgcacct gctgtcctgc aatccctcag 550
cctcctcttc ctccctgctg gtacagatga gactgcaaca cacaaggacc 600
tcatcccact ccagggaggt gaggccccca agctgggccc ggggaggcca 650
cggtactcct ttctggacct cttcagggca cgcgataaca tgcgaggccg 700
gaccacagtg ggcctggggc tggtgctctt ccagcaacta acagggcagc 750
ccaacgtgct gtgctatgcc tccaccatct tcagctccgt tggtttccat 800
gggggatcct cagccgtgct ggcctctgtg gggcttggcg cagtgaaggt 850
ggcagctacc ctgaccgcca tggggctggt ggaccgtgca ggccgcaggg 900
ctctgttgct agctggctgt gccctcatgg ccctgtccgt cagtggcata 950
ggcctcgtca gctttgccgt gcccatggac tcaggcccaa gctgtctggc 1000
tgtgcccaat gccaccgggc agacaggcct ccctggagac tctggcctgc 1050
tgcaggactc ctctctacct cccattccaa ggaccaatga ggaccaaagg 1100
gagccaatct tgtccactgc taagaaaacc aagccccatc ccagatctgg 1150
agacccctca gcccctcctc ggctggccct gagctctgcc ctccctgggc 1200
cccctctgcc cgctcggggg catgcactgc tgcgctggac cgcactgctg 1250
tgcctgatgg tctttgtcag tgccttctcc tttgggtttg ggccagtgac 1300
ctggcttgtc ctcagcgaga tctaccctgt ggagatacga ggaagagcct 1350
tcgccttctg caacagcttc aactgggcgg ccaacctctt catcagcctc 1400
tccttcctcg atctcattgg caccatcggc ttgtcctgga ccttcctgct 1450
ctacggactg accgctgtcc tcggcctggg cttcatctat ttatttgttc 1500
ctgaaacaaa aggccagtcg ttggcagaga tagaccagca gttccagaag 1550
agacggttca ccctgagctt tggccacagg cagaactcca ctggcatccc 1600
gtacagccgc atcgagatct ctgcggcctc ctgaggaatc cgtctgcctg 1650
gaaattctgg aactgtggct ttggcagacc atctccagca tcctgcttcc 1700
taggccccag agcacaagtt ccagctggtc ttttgggagt ggcccctgcc 1750
cccaaaggtg gtctgctttt gctggggtaa aaaggatgaa agtctgagaa 1800
tgcccaactc ttcattttga gtctcaggcc ctgaaggttc ctgaggatct 1850
agcttcatgc ctcagtttcc ccattgactt gcacatctct gcagtattta 1900
taagaagaat attctatgaa gtctttgttg caccatggac ttttctcaaa 1950
gaatctcaag ggtaccaatc ctggcaggaa gtctctcccg atatcacccc 2000
taaatccaaa tgaggatatc atcttttcta atctcttttt tcaactggct 2050
gggacatttt cggaaggggg aagtctcttt ttttactctt atcatttttt 2100
ttttttgagg tggagtctca ttctgttgcc caggctggcc tgatcttggc 2150
tcactgcaac ctccacctcc tgagttcaag cgattcttgt gcctcagcct 2200
cctaagcagc tgggactaca ggcgcatgca accataccca gctaatttat 2250
ttttagcaga gatggggttt cactgtgttg gccaggctgg tcgtgaactc 2300
ctgagctcaa gtgatccacc cacctcagcc tcccagagtg ctaggattac 2350
aggccttttg actcttttat ctgagtttta ttgacccctc taattctctt 2400
acccagaata tttatccttc accagcaact ctgactcttt gacgggaggc 2450
ctcagttcta gtccttggtc tgctggtgtc attgctgtag gaatgaccac 2500
gggcctcagt ttccccattt gtataatggg aagcctgtac caggtcattc 2550
ttaagatttc tcctgactcc agtgagctgg aattctaaat gctggtctag 2600
gagctgtctc caggatggtg caggatggct ttgcggaaag gagatgggtt 2650
tggaggccaa caaacctgct tgtcaatatt gcctttgcct cttggcagcc 2700
cttgaacttg agtaaataac aactccctga acctcagttt cctcatctgc 2750
agaatgggga taattatgtc ccaggggtat atttagaccc tgtttccttt 2800
caggagggtc cccagctggt ccagggcctg ggaaatttct acttatcctc 2850
attacccagg tccctccttt ggaccctgta aagggtcagg gtgaatcaga 2900
tgggggactg agcaagtagc tatgactgca gatcatgtaa ggaagggact 2950
gacaagaagc tcccagatgc tggggagaat gaagagctaa aatagatcct 3000
aggtgctgga tgctttgtca tccatgcgtg cacatatggg tgctggcaga 3050
gcccccaagg actctggcct ctcgagttct cctatcttct ccattctaga 3100
tgcttccctt gtatccagtg atgtgctgga gctggctttg ccaagcttgt 3150
gagagctggt tgctacattt tcaggatttt tacaagttgg taaacacagc 3200
cattataaaa aattaaatga tttaaattta taattaagta aattacatta 3250
aaacaaaaaa attatactca aaattcatta cttaatttta ctacctgtta 3300
ctattatctg tgcttttgag gctatttcta catagtaact cttatggaga 3350
cctaggggag acaccgcgca tctcttcctg attccccact caatgacatc 3400
atgttagtct ttggttgctt aactggctgt ggggagtgtt tttgtatcac 3450
aaagattaga gaggactaca catcagggct tgatttattg tttgttgatt 3500
ttctagactt cagaacatgc tggataaaat gtcagtaatg caaattaaac 3550
tttaaagtat gtcttgtttg tagccaatac atggtgtata gcaccaaaaa 3600
atggagggat tattcttcca gtagttgaac actgtcatcc gtttcagctg 3650
acagctgctc aaatcattta agaaggagtt ctgacattca
ttttcattgt 3700 tttacttttg tcttcctcac tagtgtaaac aaaaatttca
accagcattc 3750 atgccgaacc tatacccatt cttcagtgcc tagctgtaca
gttatcaggg 3800 atttttattt gtagtctaat tttgtcaaat catggccaaa
tcgcagtgat 3850 agttgacttt ggatacaagg tttggcaaaa aaaaaaatat
taacaaaata 3900 ttctgtaaga atcaattgtc tatatggaat ttaggataaa
gaatatttac 3950 aataaagaat atttacaata aagagtttat tattatttgt
aagttgtgtg 4000 caacaaacat accctttatc tctgtaaaat ttatacacac
aaaaattaac 4050 aaaagattct gtaagaatta attggctata tggaatttag
gatagaatat 4100 ttacaataaa gagtatttac aataaa 4126 18 5615 DNA Homo
Sapien unsure 429 unknown base 18 gcttcagtcc cgcgaccgaa gcagggcgcg
cagcagcgct gagtgccccg 50 gaacgtgcgt cgcgccccca gtgtccgtcg
cgtccgccgc gccccgggcg 100 gggatggggc ggccagactg agcgccgcac
ccgccatcca gacccgccgg 150 ccctagccgc agtccctcca gccgtggccc
cagcgcgcac gggcgatggc 200 gaaggcgacg tccggtgccg cggggctgcg
tctgctgttg ctgctgctgc 250 tgccgctgct aggcaaagtg gcattgggcc
tctacttctc gagggatgct 300 tactgggaga agctgtatgt ggaccaggcg
gccggcacgc ccttgctgta 350 cgtccatgcc ctgcgggacg cccctgagga
ggtgcccagc ttccgcctgg 400 gccagcatct ctacggcacg taccgcacnc
ggctgcatga gaacaactgg 450 atctgcatcc aggaggacac cggcctcctc
taccttaacc ggagcctgga 500 ccatagctcc tgggagaagc tcagtgtccg
caaccgcggc tttcccctgc 550 tcaccgtcta cctcaaggtc ttcctgtcac
ccacatccct tcgtgagggc 600 gagtgccagt ggccaggctg tgcccgcgta
tacttctcct tcttcaacac 650 ctcctttcca gcctgcagct ccctcaagcc
ccgggagctc tgcttcccag 700 agacaaggcc ctccttccgc attcgggaga
accgaccccc aggcaccttc 750 caccagttcc gcctgctgcc tgtgcagttc
ttgtgcccca acatcagcgt 800 ggcctacagg ctcctggagg gtgagggtct
gcccttccgc tgcgccccgg 850 acagcctgga ggtgagcacg cgctgggccc
tggaccgcga gcagcgggag 900 aagtacgagc tggtggccgt gtgcaccgtg
cacgccggcg cgcgcgagga 950 ggtggtgatg gtgcccttcc cggtgaccgt
gtacgacgag gacgactcgg 1000 cgcccacctt ccccgcgggc gtcgacaccg
ccagcgccgt ggtggagttc 1050 aagcggaagg aggacaccgt ggtggccacg
ctgcgtgtct tcgatgcaga 1100 cgtggtacct gcatcagggg agctggtgag
gcggtacaca agcacgctgc 1150 tccccgggga cacctgggcc cagcagacct
tccgggtgga acactggccc 1200 aacgagacct cggtccaggc caacggcagc
ttcgtgcggg cgaccgtaca 1250 tgactatagg ctggttctca accggaacct
ctccatctcg gagaaccgca 1300 ccatgcagct ggcggtgctg gtcaatgact
cagacttcca gggcccagga 1350 gcgggcgtcc tcttgctcca cttcaacgtg
tcggtgctgc cggtcagcct 1400 gcacctgccc agtacctact ccctctccgt
gagcaggagg gctcgccgat 1450 ttgcccagat cgggaaagtc tgtgtggaaa
actgccaggc gttcagtggc 1500 atcaacgtcc agtacaagct gcattcctct
ggtgccaact gcagcacgct 1550 aggggtggtc acctcagccg aggacacctc
ggggatcctg tttgtgaatg 1600 acaccaaggc cctgcggcgg cccaagtgtg
ccgaacttca ctacatggtg 1650 gtggccaccg accagcagac ctctaggcag
gcccaggccc agctgcttgt 1700 aacagtggag gggtcatatg tggccgagga
ggcgggctgc cccctgtcct 1750 gtgcagtcag caagagacgg ctggagtgtg
aggagtgtgg cggcctgggc 1800 tccccaacag gcaggtgtga gtggaggcaa
ggagatggca aagggatcac 1850 caggaacttc tccacctgct ctcccagcac
caagacctgc cccgacggcc 1900 actgcgatgt tgtggagacc caagacatca
acatttgccc tcaggactgc 1950 ctccggggca gcattgttgg gggacacgag
cctggggagc cccgggggat 2000 taaagctggc tatggcacct gcaactgctt
ccctgaggag gagaagtgct 2050 tctgcgagcc cgaagacatc caggatccac
tgtgcgacga gctgtgccgc 2100 acggtgatcg cagccgctgt cctcttctcc
ttcatcgtct cggtgctgct 2150 gtctgccttc tgcatccact gctaccacaa
gtttgcccac aagccaccca 2200 tctcctcagc tgagatgacc ttccggaggc
ccgcccaggc cttcccggtc 2250 agctactcct cttccggtgc ccgccggccc
tcgctggact ccatggagaa 2300 ccaggtctcc gtggatgcct tcaagatcct
ggaggatcca aagtgggaat 2350 tccctcggaa gaacttggtt cttggaaaaa
ctctaggaga aggcgaattt 2400 ggaaaagtgg tcaaggcaac ggccttccat
ctgaaaggca gagcagggta 2450 caccacggtg gccgtgaaga tgctgaaaga
gaacgcctcc ccgagtgagc 2500 ttcgagacct gctgtcagag ttcaacgtcc
tgaagcaggt caaccaccca 2550 catgtcatca aattgtatgg ggcctgcagc
caggatggcc cgctcctcct 2600 catcgtggag tacgccaaat acggctccct
gcggggcttc ctccgcgaga 2650 gccgcaaagt ggggcctggc tacctgggca
gtggaggcag ccgcaactcc 2700 agctccctgg accacccgga tgagcgggcc
ctcaccatgg gcgacctcat 2750 ctcatttgcc tggcagatct cacaggggat
gcagtatctg gccgagatga 2800 agctcgttca tcgggacttg gcagccagaa
acatcctggt agctgagggg 2850 cggaagatga agatttcgga tttcggcttg
tcccgagatg tttatgaaga 2900 ggattcctac gtgaagagga gccagggtcg
gattccagtt aaatggatgg 2950 caattgaatc cctttttgat catatctaca
ccacgcaaag tgatgtatgg 3000 tcttttggtg tcctgctgtg ggagatcgtg
accctagggg gaaaccccta 3050 tcctgggatt cctcctgagc ggctcttcaa
ccttctgaag accggccacc 3100 ggatggagag gccagacaac tgcagcgagg
agatgtaccg cctgatgctg 3150 caatgctgga agcaggagcc ggacaaaagg
ccggtgtttg cggacatcag 3200 caaagacctg gagaagatga tggttaagag
gagagactac ttggaccttg 3250 cggcgtccac tccatctgac tccctgattt
atgacgacgg cctctcagag 3300 gaggagacac cgctggtgga ctgtaataat
gcccccctcc ctcgagccct 3350 cccttccaca tggattgaaa acaaactcta
tggcatgtca gacccgaact 3400 ggcctggaga gagtcctgta ccactcacga
gagctgatgg cactaacact 3450 gggtttccaa gatatccaaa tgatagtgta
tatgctaact ggatgctttc 3500 accctcagcg gcaaaattaa tggacacgtt
tgatagttaa catttctttg 3550 tgaaaggtaa tggactcaca aggggaagaa
acatgctgag aatggaaagt 3600 ctaccggccc tttctttgtg aacgtcacat
tggccgagcc gtgttcagtt 3650 cccaggtggc agactcgttt ttggtagttt
gttttaactt ccaaggtggt 3700 tttacttctg atagccggtg attttccctc
ctagcagaca tgccacaccg 3750 ggtaagagct ctgagtctta gtggttaagc
attcctttct cttcagtgcc 3800 cagcagcacc cagtgttggt ctgtgtccat
cagtgaccac caacattctg 3850 tgttcacatg tgtgggtcca acacttacta
cctggtgtat gaaattggac 3900 ctgaactgtt ggatttttct agttgccgcc
aaacaaggca aaaaaattta 3950 aacatgaagc acacacacaa aaaaggcagt
aggaaaaatg ctggccctga 4000 tgacctgtcc ttattcagaa tgagagactg
cggggggggc ctgggggtag 4050 tgtcaatgcc cctccagggc tggaggggaa
gaggggcccc gaggatgggc 4100 ctgggctcag cattcgagat cttgagaatg
atttttttta aatcatgcaa 4150 cctttcctta ggaagacatt tggttttcat
catgattaag atgattccta 4200 gatttagcac aatggagaga ttccatgcca
tctttactat gtggatggtg 4250 gtatcaggga agagggctca caagacacat
ttgtcccccg ggcccaccac 4300 atcatcctca cgtgttcggt actgagcagc
cactacccct gatgagaaca 4350 gtatgaagaa agggggctgt tggagtccca
gaattgctga cagcagaggc 4400 tttgctgctg tgaatcccac ctgccaccag
cctgcagcac accccacagc 4450 caagtagagg cgaaacgagt ggctcatcct
acctgttagg agcaggtagg 4500 gcttgtactc actttaattt gaatcttatc
aacttactca taaagggaca 4550 ggctagctag ctgtgtcaga agtagcaatg
acaatgacca aggactgcta 4600 cacctctgat tacaattctg atgtgaaaaa
gatggtgttt ggctcttata 4650 gagcctgtgt gaaaggccca tggatcagct
cttcctgtgt ttgtaattta 4700 atgctgctac aagatgtttc tgtttcttag
attctgacca tgactcataa 4750 gcttcttgtc attcttcatt gcttgtttgt
ggtcacagat gcacaacact 4800 cctccagtct tgtgggggca gcttttggga
agtctcagca gctcttctgg 4850 ctgtgttgtc agcactgtaa cttcgcagaa
aagagtcgga ttaccaaaac 4900 actgcctgct cttcagactt aaagcactga
taggacttaa aatagtctca 4950 ttcaaatact gtattttata taggcatttc
acaaaaacag caaaattgtg 5000 gcattttgtg aggccaaggc ttggatgcgt
gtgtaataga gccttatggt 5050 gtgtgcgcac acacccagag gagagtttga
aaaatgctta ttggacacgt 5100 aacctggctc taatttgggc tgtttttcag
atacactgtg ataagttctt 5150 ttacaaatat ctatagacat ggtaaacttt
tggttttcag atatgcttaa 5200 tgatagtctt actaaatgca gaaataagaa
taaactttct caaattatta 5250 aaaatgccta cacagtaagt gtgaattgct
gcaacaggtt tgttctcagg 5300 agggtaagaa ctccaggtct aaacagctga
cccagtgatg gggaatttat 5350 ccttgaccaa tttatccttg accaataacc
taattgtcta ttcctgagtt 5400 ataaaggtcc ccatccttat tagctctact
ggaattttca tacacgtaaa 5450 tgcagaagtt actaagtatt aagtattact
gagtattaag tagtaatctg 5500 tcagttatta aaatttgtaa aatctattta
tgaaaggtca ttaaaccaga 5550 tcatgttcct ttttttgtaa tcaaggtgac
taagaaaatc agttgtgtaa 5600 ataaaatcat gtatc 5615 19 4315 DNA Homo
Sapien 19 tgagagccaa gcaaagaaca ttaaggaagg aaggaggaat gaggctggat 50
acggtgcagt gaaaaaggca cttccaagag tggggcactc actacgcaca 100
gactcgacgg tgccatcagc atgagaactt accgctactt cttgctgctc 150
ttttgggtgg gccagcccta cccaactctc tcaactccac tatcaaagag 200
gactagtggt ttcccagcaa agaaaagggc cctggagctc tctggaaaca 250
gcaaaaatga gctgaaccgt tcaaaaagga gctggatgtg gaatcagttc 300
tttctcctgg aggaatacac aggatccgat tatcagtatg tgggcaagtt 350
acattcagac caggatagag gagatggatc acttaaatat atcctttcag 400
gagatggagc aggagatctc ttcattatta atgaaaacac aggcgacata 450
caggccacca agaggctgga cagggaagaa aaacccgttt acatccttcg 500
agctcaagct ataaacagaa ggacagggag acccgtggag cccgagtctg 550
aattcatcat caagatccat gacatcaatg acaatgaacc aatattcacc 600
aaggaggttt acacagccac tgtccctgaa atgtctgatg tcggtacatt 650
tgttgtccaa gtcactgcga cggatgcaga tgatccaaca tatgggaaca 700
gtgctaaagt tgtctacagt attctacagg gacagcccta tttttcagtt 750
gaatcagaaa caggtattat caagacagct ttgctcaaca tggatcgaga 800
aaacagggag cagtaccaag tggtgattca agccaaggat atgggcggcc 850
agatgggagg attatctggg accaccaccg tgaacatcac actgactgat 900
gtcaacgaca accctccccg attcccccag agtacatacc agtttaaaac 950
tcctgaatct tctccaccgg ggacaccaat tggcagaatc aaagccagcg 1000
acgctgatgt gggagaaaat gctgaaattg agtacagcat cacagacggt 1050
gaggggctgg atatgtttga tgtcatcacc gaccaggaaa cccaggaagg 1100
gattataact gtcaaaaagc tcttggactt tgaaaagaag aaagtgtata 1150
cccttaaagt ggaagcctcc aatccttatg ttgagccacg atttctctac 1200
ttggggcctt tcaaagattc agccacggtt agaattgtgg tggaggatgt 1250
agatgagcca cctgtcttca gcaaactggc ctacatctta caaataagag 1300
aagatgctca gataaacacc acaataggct ccgtcacagc ccaagatcca 1350
gatgctgcca ggaatcctgt caagtactct gtagatcgac acacagatat 1400
ggacagaata ttcaacattg attctggaaa tggttcgatt tttacatcga 1450
aacttcttga ccgagaaaca ctgctatggc acaacattac agtgatagca 1500
acagagatca ataatccaaa gcaaagtagt cgagtacctc tatatattaa 1550
agttctagat gtcaatgaca acgccccaga atttgctgag ttctatgaaa 1600
cttttgtctg tgaaaaagca aaggcagatc agttgattca gaccctgcat 1650
gctgttgaca aggatgaccc ttatagtgga caccaatttt cgttttcctt 1700
ggcccctgaa gcagccagtg gctcaaactt taccattcaa gacaacaaag 1750
acaacacggc gggaatctta actcggaaaa atggctataa tagacacgag 1800
atgagcacct atctcttgcc tgtggtcatt tcagacaacg actacccagt 1850
tcaaagcagc actgggacag tgactgtccg ggtctgtgca tgtgaccacc 1900
acgggaacat gcaatcctgc catgcggagg cgctcatcca ccccacggga 1950
ctgagcacgg gggctctggt tgccatcctt ctgtgcatcg tgatcctact 2000
agtgacagtg gtgctgtttg cagctctgag gcggcagcga aaaaaagagc 2050
ctttgatcat ttccaaagag gacatcagag ataacattgt cagttacaac 2100
gacgaaggtg gtggagagga ggacacccag gcttttgata tcggcaccct 2150
gaggaatcct gaagccatag aggacaacaa attacgaagg gacattgtgc 2200
ccgaagccct tttcctaccc cgacggactc caacagctcg cgacaacacc 2250
gatgtcagag atttcattaa ccaaaggtta aaggaaaatg acacggaccc 2300
cactgccccg ccatacgact ccttggccac ttacgcctat gaaggcactg 2350
gctccgtggc ggattccctg agctcgctgg agtcagtgac cacggatgca 2400
gatcaagact atgattacct tagtgactgg ggacctcgat tcaaaaagct 2450
tgcagatatg tatggaggag tggacagtga caaagactcc taatctgttg 2500
cctttttcat tttccaatac gacactgaaa tatgtgaagt ggctatttct 2550
ttatatttat ccactactcc gtgaaggctt ctctgttcta cccgttccaa 2600
aagccaatgg ctgcagtccg tgtggatcca atgttagaga cttttttcta 2650
gtacactttt atgagcttcc aaggggcaaa tttttatttt ttagtgcatc 2700
cagttaacca agtcagccca acaggcaggt gccggagggg aggacaggga 2750
acagtatttc cacttgttct cagggcagcg tgcccgcttc cgctgtcctg 2800
gtgttttact acactccatg tcaggtcagc caactgccct aactgtacat 2850
ttcacaggct aatgggataa aggactgtgc tttaaagata aaaatatcat 2900
catagtaaaa gaaatgaggg catatcggct cacaaagaga taaactacat 2950
aggggtgttt atttgtgtca caaagaattt aaaataacac ttgcccatgc 3000
tatttgttct tcaagaactt tctctgccat caactactat tcaaaacctc 3050
aaatccaccc atatgttaaa attctcatta ctcttaagga atagaagcaa 3100
attaaacggt aacatccaaa agcaaccaca aacctagtac gacttcattc 3150
cttccactaa ctcatagttt gttatatcct agactagaca tgcgaaagtt 3200
tgcctttgta ccatataaag ggggagggaa atagctaata atgttaacca 3250
aggaaatata ttttaccata catttaaagt tttggccacc acatgtatca 3300
cgggtcactt gaaattcttt cagctatcag taggctaatg tcaaaattgt 3350
ttaaaaattc ttgaaagaat tttcctgaga caaattttaa cttcttgtct 3400
atagttgtca gtattattct actatactgt acatgaaagt agcagtgtga 3450
agtacaataa ttcatattct tcatatcctt cttacacgac taagttgaat 3500
tagtaaagtt agattaaata aaacttaaat ctcactctag gagttcagtg 3550
gagaggttag agccagccac acttgaacct aataccctgc ccttgacatc 3600
tggaaacctc tacatattta tataacgtga tacatttgga taaacaacat 3650
tgagattatg atgaaaacct acatattcca tgtttggaag acccttggaa 3700
gaggaaaatt ggattccctt aaacaaaagt gtttaagatt gtaattaaaa 3750
tgatagttga ttttcaaaag cattaatttt ttttcattgt ttttaacttt 3800
gctttcatga ccatcctgcc atccttgact ttgaactaat gataaagtaa 3850
tgatctcaaa ctatgacaga aaagtaatgt aaaatccatc caatctatta 3900
tttctctaat tatgcaatta gcctcatagt tattatccag aggacccaac 3950
tgaactgaac taatccttct ggcagattca aatcgtttat ttcacacgct 4000
gttctaatgg cacttatcat tagaatctta ccttgtgcag tcatcagaaa 4050
ttccagcgta ctataatgaa aacatccttg ttttgaaaac ctaaaagaca 4100
ggctctgtat atatatatac ttaagaatat gctgacttca cttattagtc 4150
ttagggattt attttcaatt aatattaatt ttctacaaat aattttagtg 4200
tcatttccat ttggggatat tgtcatatca gcacatattt tctgtttgga 4250
aacacactgt tgtttagtta agttttaaat aggtgtatta cccaagaagt 4300
aaagatggaa acgtt 4315 20 2521 DNA Homo Sapien 20 cggtggaggc
cacagacacc tcaaacctgg attccacaat tctacgttaa 50 gtgttggagt
ttttattact ctgctgtagg aaagcctttg ccaatgctta 100 caaggaactg
tttatccctg cttctctggg ttctgtttga tggaggtctc 150 ctaacaccac
tacaaccaca gccacagcag actttagcca cagagccaag 200 agaaaatgtt
atccatctgc caggacaacg gtcacatttc caacgtgtta 250 aacgtggctg
ggtatggaat caattttttg tgctggaaga atacgtgggc 300 tccgagcctc
agtatgtggg aaagctccat tccgacttag acaagggaga 350 gggcactgtg
aaatacaccc tctcaggaga tggcgctggc accgttttta 400 ccattgatga
aaccacaggg gacattcatg caataaggag cctagataga 450 gaagagaaac
ctttctacac tcttcgtgct caggctgtgg acatagaaac 500 cagaaagccc
ctggagcctg aatcagaatt catcatcaaa gtgcaggata 550 ttaatgataa
tgagccaaag tttttggatg gaccttatgt tgctactgtt 600 ccagaaatgt
ctcctgtggg tgcatatgta ctccaggtca aggccacaga 650 tgcagatgac
ccgacctatg gaaacagtgc cagagtcgtt tacagcattc 700 ttcagggaca
accttatttc tctattgatc ccaagacagg tgttattaga 750 acagctttgc
caaacatgga cagagaagtc aaagaacaat atcaagtact 800 catccaagcc
aaggatatgg gaggacagct tggaggatta gccggaacaa 850 caatagtcaa
catcactctc accgatgtca atgacaatcc acctcgattc 900 cccaaaagca
tcttccactt gaaagttcct gagtcttccc ctattggttc 950 agctattgga
agaataagag ctgtggatcc tgattttgga caaaatgcag 1000 aaattgaata
caatattgtt ccaggagatg ggggaaattt gtttgacatc 1050 gtcacagatg
aggatacaca agagggagtc atcaaattga aaaagccttt 1100 agattttgaa
acaaagaagg catacacttt caaagttgag gcttccaacc 1150 ttcaccttga
ccaccggttt cactcggcgg gccctttcaa agacacagct 1200 acggtgaaga
tcagcgtgct ggacgtagat gagccaccgg ttttcagcaa 1250 gccgctctac
accatggagg tttatgaaga cactccggta gggaccatca 1300 ttggcgctgt
cactgctcaa gacctggatg taggcagcgg tgctgttagg 1350 tacttcatag
attggaagag tgatggggac agctacttta caatagatgg 1400 aaatgaagga
accatcgcca ctaatgaatt actagacaga gaaagcactg 1450 cgcagtataa
tttctccata attgcgagta aagttagtaa ccctttattg 1500 accagcaaag
tcaatatact gattaatgtc ttagatgtaa atgaatttcc 1550 tccagaaata
tctgtgccat atgagacagc cgtgtgtgaa aatgccaagc 1600 caggacagat
aattcagata gtcagtgctg cagaccgaga tctttcacct 1650 gctgggcaac
aattctcctt tagattatca cctgaggctg ctatcaaacc 1700 aaattttaca
gttcgtgact tcagaaacaa cacagcgggg attgaaaccc 1750 gaagaaatgg
atacagccgc aggcagcaag agttgtattt cctccctgtt 1800 gtaatagaag
acagcagcta ccctgtccag agcagcacaa acacaatgac 1850 tattcgagtc
tgtagatgtg actctgatgg caccatcctg tcttgtaatg 1900 tggaagcaat
ttttctacct gtaggactta gcactggggc gttgattgca 1950 attctactat
gcattgttat actcttagcc atagttgtac tgtatgtagc 2000
actgcgaagg cagaagaaaa agcacaccct gatgacctct aaagaagaca 2050
tcagagacaa cgtcatccat tacgatgatg aaggaggtgg ggaggaagat 2100
acccaggctt tcgacatcgg ggctctgaga aacccaaaag tgattgagga 2150
gaacaaaatt cgcagggata taaaaccaga ctctctctgt ttacctcgtc 2200
agagaccacc catggaagat aacacagaca taagggattt cattcatcaa 2250
aggctacagg aaaatgatgt agatccaact gccccaccaa tcgattcact 2300
ggccacatat gcctacgaag ggagtgggtc cgtggcagag tccctcagct 2350
ctatagactc tctcaccaca gaagccgacc aggactatga ctatctgaca 2400
gactggggac cccgctttaa agtcttggca gacatgtttg gcgaagaaga 2450
gagttataac cctgataaag tcacttaagg gagtcgtgga ggctaaaata 2500
caaccgagag gggagatttt t 2521 21 736 DNA Homo Sapien 21 ggctctcacc
ctcctctcct gcagctccag ctctgtgctc tgcctctgag 50 gagaccatgg
cccggcctct gtgtaccctg ctactcctga tggctaccct 100 ggctggggct
ctggcctcga gctccaagga ggagaatagg ataatcccag 150 gtggcatcta
tgatgcagac ctcaatgatg agtgggtaca gcgtgccctt 200 cacttcgcca
tcagcgagta caacaaggcc accgaagatg agtactacag 250 acgcccgctg
caggtgctgc gagccaggga gcagaccttt gggggggtga 300 attacttctt
cgacgtagag gtgggccgca ccatatgtac caagtcccag 350 cccaacttgg
acacctgtgc cttccatgaa cagccagaac tgcagaagaa 400 acagttatgc
tctttcgaga tctacgaagt tccctgggag gacagaatgt 450 ccctggtgaa
ttccaggtgt caagaagcct aggggtctgt gccaggccag 500 tcacaccgac
caccacccac tcccaccccc tgtagtgctc ccacccctgg 550 actggtggcc
cccaccctgc gggaggcctc cccatgtgcc tgtgccaaga 600 gacagacaga
gaaggctgca ggagtccttt gttgctcagc agggcgctct 650 gccctccctc
cttccttctt gcttctaata gacctggtac atggtacaca 700 cacccccacc
tcctgcaatt aaacagtagc atcgcc 736 22 2025 DNA Homo Sapien 22
ggcagcggtg gcaggggctg caggagcaag tgaccaggag caggactggg 50
gacaggcctg atcgcccctg cacgaaccag acccttcgcc gccctcacga 100
tgactacctc tccgatcctg cagctgctgc tgcggctctc actgtgcggg 150
ctgctgctcc agagggcgga gacaggctct aaggggcaga cggcggggga 200
gctgtaccag cgctgggaac ggtaccgcag ggagtgccag gagaccttgg 250
cagccgcgga accgccttca ggcctcgcct gtaacgggtc cttcgatatg 300
tacgtctgct gggactatgc tgcacccaat gccactgccc gtgcgtcctg 350
cccctggtac ctgccctggc accaccatgt ggctgcaggt ttcgtcctcc 400
gccagtgtgg cagtgatggc caatggggac tttggagaga ccatacacaa 450
tgtgagaacc cagagaagaa tgaggccttt ctggaccaaa ggctcatctt 500
ggagcggttg caggtcatgt acactgtcgg ctactccctg tctctcgcca 550
cactgctgct agccctgctc atcttgagtt tgttcaggcg gctacattgc 600
actagaaact atatccacat caacctgttc acgtctttca tgctgcgagc 650
tgcggccatt ctcagccgag accgtctgct acctcgacct ggcccctacc 700
ttggggacca ggcccttgcg ctgtggaacc aggccctcgc tgcctgccgc 750
acggcccaga tcgtgaccca gtactgcgtg ggtgccaact acacgtggct 800
gctggtggag ggcgtctacc tgcacagtct cctggtgctc gtgggaggct 850
ccgaggaggg ccacttccgc tactacctgc tcctcggctg gggggccccc 900
gcgcttttcg tcattccctg ggtgatcgtc aggtacctgt acgagaacac 950
gcagtgctgg gagcgcaacg aagtcaaggc catttggtgg attatacgga 1000
cccccatcct catgaccatc ttgattaatt tcctcatttt tatccgcatt 1050
cttggcattc tcctgtccaa gctgaggaca cggcaaatgc gctgccggga 1100
ttaccggctg aggctggctc gctccacgct gacgctggtg cccctgctgg 1150
gtgtccacga ggtggtgttt gctcccgtga cagaggaaca ggcccggggc 1200
gccctgcgct tcgccaagct cggctttgag atcttcctca gctccttcca 1250
gggcttcctg gtcagcgtcc tctactgctt catcaacaag gaggtgcagt 1300
cggagatccg ccgtggctgg caccactgcc gcctgcgccg cagcctgggc 1350
gaggagcaac gccagctccc ggagcgcgcc ttccgggccc tgccctccgg 1400
ctccggcccg ggcgaggtcc ccaccagccg cggcttgtcc tcggggaccc 1450
tcccagggcc tgggaatgag gccagccggg agttggaaag ttactgctag 1500
ggggcgggat ccccgtgtct gttcagttag catggattta ttgagtgcca 1550
actgcgtgcc aggcccagta cggaggacgc tggggaaatg gtgaaggaaa 1600
cagaaaaaag gtccctgccc ttctggagat gacaactgag tggggaaaac 1650
agaccgtgaa cacaaaacat caagttccac acacgctatg gaatggttat 1700
gaagggaagc gagaaggggg cctagggtgg tctgggaggc gtctccaagg 1750
aggtgacact taagccatcc ccgaaagagg tgaaagagat cactttgggg 1800
agagctggag aacaggattc taggcggaag cgatagcata ggcaaaggcc 1850
cttgggcagg aaggcgctca gccttggctg gagtagaatt aagtcagagc 1900
caacaggttg gggagagaca gagaagtggg caggggcacc caagttggga 1950
tttcatttca ggtgcattgg agattcttag gagtgtctct tgggggtaat 2000
attttatttt ttaaaaaatg aggat 2025 23 3168 DNA Homo Sapien 23
gccagagcgt gagccgcgac ctccgcgcag gtggtcgcgc cggtctccgc 50
ggaaatgttg tccaaagttc ttccagtcct cctaggcatc ttattgatcc 100
tccagtcgag ggtcgaggga cctcagactg aatcaaagaa tgaagcctct 150
tcccgtgatg ttgtctatgg cccccagccc cagcctctgg aaaatcagct 200
cctctctgag gaaacaaagt caactgagac tgagactggg agcagagttg 250
gcaaactgcc agaagcctct cgcatcctga acactatcct gagtaattat 300
gaccacaaac tgcgccctgg cattggagag aagcccactg tggtcactgt 350
tgagatcgcc gtcaacagcc ttggtcctct ctctatccta gacatggaat 400
acaccattga catcatcttc tcccagacct ggtacgacga acgcctctgt 450
tacaacgaca cctttgagtc tcttgttctg aatggcaatg tggtgagcca 500
gctatggatc ccggacacct tttttaggaa ttctaagagg acccacgagc 550
atgagatcac catgcccaac cagatggtcc gcatctacaa ggatggcaag 600
gtgttgtaca caattaggat gaccattgat gccggatgct cactccacat 650
gctcagattt ccaatggatt ctcactcttg ccctctatct ttctctagct 700
tttcctatcc tgagaatgag atgatctaca agtgggaaaa tttcaagctt 750
gaaatcaatg agaagaactc ctggaagctc ttccagtttg attttacagg 800
agtgagcaac aaaactgaaa taatcacaac cccagttggt gacttcatgg 850
tcatgacgat tttcttcaat gtgagcaggc ggtttggcta tgttgccttt 900
caaaactatg tcccttcttc cgtgaccacg atgctctcct gggtttcctt 950
ttggatcaag acagagtctg ctccagcccg gacctctcta gggatcacct 1000
ctgttctgac catgaccacg ttgggcacct tttctcgtaa gaatttcccg 1050
cgtgtctcct atatcacagc cttggatttc tatatcgcca tctgcttcgt 1100
cttctgcttc tgcgctctgt tggagtttgc tgtgctcaac ttcctgatct 1150
acaaccagac aaaagcccat gcttctccta aactccgcca tcctcgtatc 1200
aatagccgtg cccatgcccg tacccgtgca cgttcccgag cctgtgcccg 1250
ccaacatcag gaagcttttg tgtgccagat tgtcaccact gagggaagtg 1300
atggagagga gcgcccgtct tgctcagccc agcagccccc tagcccaggt 1350
agccctgagg gtccccgcag cctctgctcc aagctggcct gctgtgagtg 1400
gtgcaagcgt tttaagaagt acttctgcat ggtccccgat tgtgagggca 1450
gtacctggca gcagggccgc ctctgcatcc atgtctaccg cctggataac 1500
tactcgagag ttgttttccc agtgactttc ttcttcttca atgtgctcta 1550
ctggcttgtt tgccttaact tgtaggtacc agctggtacc ctgtggggca 1600
acctctccag ttccccagga ggtccaagcc ccttgccaag ggagttgggg 1650
gaaagcagca gcagcagcag gagcgactag agtttttcct gccccattcc 1700
ccaaacagaa gcttgcagag ggtttgtctt tgctgcccct ctcccctacc 1750
tggcccattc actgagtctt ctcagcagac catttcaaat tattaataaa 1800
tgggccacct ccctcttctt caaggagcat ccgtgatgct cagtgttcaa 1850
aaccacagcc acttagtgat cagctcccta aaaccatgcc taagtacagg 1900
cggattagct atcttccaac aatgctgacc accagacaat tactgcattt 1950
ttccagaagc ccactattgc ctttgtagtg ctttcggccc agttctggcc 2000
tcagcctcaa agtgcaccga ctagttgctt gcctatacct ggcacctcat 2050
taagatgctg ggcagcagta taacaggagg aagagatccc tctcctttgg 2100
tcagattatt atgttctcag ttctctctcc ctgctacccc tttctctgca 2150
gatagataga cactggcatt atccctttag gaagaggggg gggcagcaag 2200
agagcctatt tgggacagca ttcctctctc tctgctgctg tgacatctcc 2250
ctctccttgc tggctccatc tttcgtctgc actaccaatt caatgccctt 2300
catccaatgg gtatctattt ttgtgtgtga ttatagtaac tactccctgc 2350
tttatatgcc accctcttcc ttctctttga cccctgtgac tctttctgta 2400
actttcccag tgacttcccc tagccctgac ccaggcacta ggccttggtg 2450
acttcctggg gccaagaaac taaggaaact cggctttgca acaggcatta 2500
ctcgccattg attggtgccc acccagggca cactgtcgga gttctatcac 2550
ttgcttgacc cctggaccca taaaccagtc cactgttata cccggggcac 2600
tctaaccatc acaatcaatc aatcaaattc ccttaaattt gtatggcact 2650
ggaactttgg caaagcactt ttgacaagtt gtgtctgatt ggagcttcat 2700
gatagccttg tgacatcttt agggcaggat tcttatcccc attttgcaga 2750
tgaaaaccct gagtcacaga tttctgtggg actgtggatc tcactggaag 2800
ctatccaaga gcccactgtc accttctaga ccacatgata gggctagaca 2850
gctcagttca ccatgattct cttctgtcac ctctgctggc acaccagtgg 2900
caaggcccag aatggcgacc tctctttagc tcaatttctg ggcctgaggt 2950
gctcagactg cccccaagat caaatctctc ctggctgtag taacccagtg 3000
gaatgaattt ggacatgccc caatgcttct atatgctaag tgaaatctgt 3050
gtctgtaatt tgttgggggg tggatagggt ggggtctcca tctacttttt 3100
gtcaccatca tctgaaatgg ggaaatatgt aaataaatat atcagcaaag 3150
caaaaagaaa aaaaaaaa 3168 24 2837 DNA Homo Sapien 24 tatcacagga
ttgtctactt cagcaatagc aactaatgga tttgtaagag 50 gaggaggagc
atattattta atatctagaa gtctagggcc agaatttggt 100 ggtgcaattg
gtctaatctt cgcctttgcc aacgctgttg cagttgctat 150 gtatgtggtt
ggatttgcag aaaccgtggt ggagttgctt aaggaacatt 200 ccatacttat
gatagatgaa atcaatgata tccgaattat tggagccatt 250 acagtcgtga
ttcttttagg tatctcagta gctggaatgg agtgggaagc 300 aaaagctcag
attgttcttt tggtgatcct acttcttgct attggtgatt 350 tcgtcatagg
aacatttatc ccactggaga gcaagaagcc aaaagggttt 400 tttggttata
aatctgaaat atttaatgag aactttgggc ccgattttcg 450 agaggaagag
actttctttt ctgtatttgc catctttttt cctgctgcaa 500 ctggtattct
ggctggagca aatatctcag gtgatcttgc agatcctcag 550 tcagccatac
ccaaaggaac actcctagcc attttaatta ctacattggt 600 ttacgtagga
attgcagtat ctgtaggttc ttgtgttgtt cgagatgcca 650 ctggaaacgt
taatgacact atcgtaacag agctaacaaa ctgtacttct 700 gcagcctgca
aattaaactt tgatttttca tcttgtgaaa gcagtccttg 750 ttcctatggc
ctaatgaaca acttccaggt aatgagtatg gtgtcaggat 800 ttacaccact
aatttctgca ggtatatttt cagccactct ttcttcagca 850 ttagcatccc
tagtgagtgc tcccaaaata tttcaggctc tatgtaagga 900 caacatctac
ccagctttcc agatgtttgc taaaggttat gggaaaaata 950 atgaacctct
tcgtggctac atcttaacat tcttaattgc acttggattc 1000 atcttaattg
ctgaactgaa tgttattgca ccaattatct caaacttctt 1050 ccttgcatca
tatgcattga tcaatttttc agtattccat gcatcacttg 1100 caaaatctcc
aggatggcgt cctgcattca aatactacaa catgtggata 1150 tcacttcttg
gagcaattct ttgttgcata gtaatgttcg tcattaactg 1200 gtgggctgca
ttgctaacat atgtgatagt ccttgggctg tatatttatg 1250 ttacctacaa
aaaaccagat gtgaattggg gatcctctac acaagccctg 1300 acttacctga
atgcactgca gcattcaatt cgtctttctg gagtggaaga 1350 ccacgtgaaa
aactttaggc cacagtgtct tgttatgaca ggtgctccaa 1400 actcacgtcc
agctttactt catcttgttc atgatttcac aaaaaatgtt 1450 ggtttgatga
tctgtggcca tgtacatatg ggtcctcgaa gacaagccat 1500 gaaagagatg
tccatcgatc aagccaaata tcagcgatgg cttattaaga 1550 acaaaatgaa
ggcattttat gctccagtac atgcagatga cttgagagaa 1600 ggtgcacagt
atttgatgca ggctgctggt cttggtcgta tgaagccaaa 1650 cacacttgtc
cttggattta agaaagattg gttgcaagca gatatgaggg 1700 atgtggatat
gtatataaac ttatttcatg atgcttttga catacaatat 1750 ggagtagtgg
ttattcgcct aaaagaaggt ctggatatat ctcatcttca 1800 aggacaagaa
gaattattgt catcacaaga gaaatctcct ggcaccaagg 1850 atgtggtagt
aagtgtggaa tatagtaaaa agtccgattt agatacttcc 1900 aaaccactca
gtgaaaaacc aattacacac aaagttgagg aagaggatgg 1950 caagactgca
actcaaccac tgttgaaaaa agaatccaaa ggccctattg 2000 tgcctttaaa
tgtagctgac caaaagcttc ttgaagctag tacacagttt 2050 cagaaaaaac
aaggaaagaa tactattgat gtctggtggc tttttgatga 2100 tggaggtttg
accttattga taccttacct tctgacgacc aagaaaaaat 2150 ggaaagactg
taagatcaga gtattcattg gtggaaagat aaacagaata 2200 gaccatgacc
ggagagcgat ggctactttg cttagcaagt tccggataga 2250 cttttctgat
atcatggttc taggagatat caataccaaa ccaaagaaag 2300 aaaatattat
agcttttgag gaaatcattg agccatacag acttcatgaa 2350 gatgataaag
agcaagatat tgcagataaa atgaaagaag atgaaccatg 2400 gcgaataaca
gataatgagc ttgaacttta taagaccaag acataccggc 2450 agatcaggtt
aaatgagtta ttaaaggaac attcaagcac agctaatatt 2500 attgtcatga
gtctcccagt tgcacgaaaa ggtgctgtgt ctagtgctct 2550 ctacatggca
tggttagaag ctctatctaa ggacctacca ccaatcctcc 2600 tagttcgtgg
gaatcatcag agtgtcctta ccttctattc ataaatgttc 2650 tatacagtgg
acagccctcc agaatggtac ttcagtgcct agtgtagtaa 2700 cctgaaatct
tcaatgacac attaacatca caatggcgaa tggtgacttt 2750 tctttcacga
tttcattaat ttgaaagcac acaggaaagc ttgctccatt 2800 gataacgtgt
atggagactt cggttttagt caattcc 2837 25 4709 DNA Homo Sapien 25
gagcttgtcc agacgaagcc tcgcagggat gggttggagc ctgggccgtg 50
cttcgctcag gcagcgtttg aggcagaccc agcagggtcc tcctggggcc 100
ttcctgcctt tgaactgcgg tggcgggcgg gcgcacggtc tcctgtacgc 150
cctagactag gggccgccat ctccatggcc acggccgtga gccggccctg 200
cgccggcagg tcgcgggaca tactgtggcg cgttttgggc tggaggatag 250
ttgcaagtat tgtttggtca gtgctatttc tacccatctg caccacagta 300
tttataattt tcagcaggat tgatttgttt catcctatac agtggctgtc 350
tgattctttc agtgacctgt atagttccta tgtaatcttt tacttcctgc 400
tgctgtcagt ggtaataata ataataagta ttttcaatgt ggagttctat 450
gcagttgtgc cttctattcc ttgctccaga ctagctctga tagggaagat 500
cattcatcct cagcaactca tgcactcatt tattcatgct gcaatgggaa 550
tggtgatggc ctggtgtgct gcagtgataa cccagggcca gtacagcttt 600
cttgtggttc cctgcactgg tactaacagc tttggtagcc ctgctgcgca 650
aacctgctta aatgaatatc atcttttttt cctactgact ggagcattta 700
tgggctatag ctatagcctc ctgtattttg ttaacaacat gaactatctt 750
ccatttccca tcatacagca atacaagttc ttgcgtttta ggagatctct 800
gctcttatta gttaaacaca gttgtgtgga atcactgttc ctggttagaa 850
atttctgcat tttatattat tttcttggct atattcccaa agcttggatt 900
agcactgcta tgaaccttca catagatgag caggttcata ggccacttga 950
cacagtgagt ggcctcttaa atctctcgtt actctaccat gtctggctgt 1000
gtggtgtctt tctcctgacg acttggtatg tctcatggat actcttcaaa 1050
atctatgcca cagaggctca tgtgtttcct gttcaaccac catttgcaga 1100
agggtcagat gagtgccttc caaaagtgtt aaatagcaat cctcccccca 1150
tcataaagta tttagccttg caggacctga tgttgctttc tcaatattct 1200
ccttcacgaa gacaagaagt tttcagcctc agccaaccag gtggacatcc 1250
ccacaattgg acagccattt caagggagtg tttgaatctt ttaaatggta 1300
tgactcagaa actgattctc tatcaagaag ctgctgctac gaatgggaga 1350
gtgtcttcat cttacccagt ggaacctaag aaattaaatt ctccagaaga 1400
aactgctttt cagacaccaa aatctagcca gatgcctcgg ccttcagtgc 1450
caccattagt taaaacatca ctgttttctt caaaattatc tacacctgat 1500
gttgtgagcc catttgggac cccatttggc tctagtgtaa tgaatcggat 1550
ggctggaatt tttgatgtaa acacctgcta tgggtcaccg caaagtcctc 1600
agctaataag aagggggcca agattgtgga catcagcttc tgatcagcaa 1650
atgactgaat tttctaatcc ttctccatct acctctatta gtgctgaggg 1700
taagacaatg agacaaccca gtgtgattta ttcatggatt cagaataaac 1750
gtgaacagat taagaatttc ttgtcaaaac gggtgctgat aatgtatttt 1800
ttcagtaagc acccagaggc ctccattcag gctgtttttt cagatgccca 1850
aatgcatatt tgggcattag aaggtctgtc gcacttagta gcagcatcat 1900
ttacagagga tagatttgga gttgtccaga cgacactacc agctatcctt 1950
aatactttgt tgacactgca agaggcagtc gacaagtact ttaagcttcc 2000
tcatgcttcc agtaaaccac cccggatttc aggaagcctt gtggacactt 2050
catataaaac attaagattt gcattcagag catcactgaa aactgccatc 2100
tatcgaataa ctactacatt tggtgaacat ctgaatgctg tgcaagcatc 2150
tgcagaacat cagaaaagac ttcaacagtt cttggagttc aaagaatagt 2200
taagtaatat aaactgtgtt cattacactg ctgatacaac tacagatggg 2250
acagtaaatg ttcagcattc ttggatcaga agaaaacgga ctaattagat 2300
gcttcctttg tcgtggtggt tgctttgaaa actatacttt aatgggagaa 2350
atcatggaaa gaaattctca acagaataac tgaaaactgc cttttctgta 2400
ccgattgctt tttgtgtgtg tggtataata aaatctttat tcaattttac 2450
agaagcattg atggcagtcg aaatgtctct agctcatata acttaatagt 2500
aataactaaa aaacttttag aatttacttt tgaaaggagg gaagccagtt 2550
ctgaaatgag tataggttga tttcatagtc ttcttaatta agagtttagc 2600
tctttgtaaa ctcaaaatac ataaactttt taagtgtagt ttcatttact 2650
gaaggataaa aatggtaaca gtgcagcaat attcacaaaa aatattgtct 2700
aacggacata ttttgttaat ctgttaggtt gggtttttgt ttccagggac 2750
aaattaaatt tgtatgatta cccaaaaaag ggtctcagtt tacagatgct 2800
aactctatat aaaggaatgt ggaaaaactc agttcttaag ttacaagatt 2850
aaaaattcac atttggtctt taagaaacaa ttgactgaca tctatgaatt 2900
tattttgtat catgctagta aacacgaagt attaatgtat gggtattttc 2950
ccagctagtt ttgctttctt tttctggagc aaaacattaa gtgattgcag 3000
agtttttcaa gcaagagaaa aaggtttgca aaaaaaccca ggaaatgttc 3050
ccttttttcc ccaccattca tcttcattag atcaaattct gtgaaacttg 3100
tctggtctct caaagggagc agcctctgta gtgttaaatg gctaattaaa 3150
ataggaagat ctttatagcc agaaacaact tagtcatcaa atagcaagtg 3200
aaaccaaaac gtcagaggga ttactgtact tggaagtatg ttgtgtgtcc 3250
caaatgtgaa cgaagtattg ttagaattta ttagatcagc ttctttggag 3300
atcaaagatt ggaaatccta gtcatagata ttcactggac tggctttgga 3350
ctgaaatgct cctttgtaat tcttttccta ttgtcttttc cttctagtgt 3400
cccaaaatat tttctttaaa gtcagcacag tactgtatat gaatctttaa 3450
tgtggtatca tatatgtcta cttttgtctg attcatcgat gtattatatc 3500
tttataattg aatattttag ctccgggtcc tgttgcccct tcaagcagta 3550
catgccaaat tataaatagg tgctactggc cttgagcata tcactgtggg 3600
acagttcccc aattgtcaag tgtttagata tgtagactat tgccatttgt 3650
ttttttgttt tggttttgct ttgtgtctga agctgaattg atttcttttt 3700
tttgaatgtg aaagttgaat ttcaaacgta gtcatttctt acagatggcc 3750
aagacagaaa attgtggcta ggttgactga gaactgttgt cttccatgta 3800
ttaacacaat taagcttttt atattccact ctctgtgctg accctggctg 3850
aggcattttg ggagacaagg actctgaatc ttctgcttcc attaaagaag 3900
aactgtgata ttcaacattg gatttctgag aataaagata ggatgattcc 3950
tttgaacttt gacttacttg tataaaatgt ccagctaggt taggtttttg 4000
ccatttccta tatactttgg gtaaagctac atttgatgag caatgtgaat 4050
gtttctgaga atgttcattc ctgttttctc ttaagagaat gtgctgtgta 4100
ctaaatacag gccacatagt gtctgcctgt tgaagatctg gaaactgcct 4150
ccccagatct gtattgtatt tggtaggtaa gggggtcagt ttctttttct 4200
cattgtgtgt tgataatcta cacaccatct gttggaacca gggtgttatt 4250
atggggaact cctcctgtgt actaggagga ggaccttagg gagaccaaga 4300
ggagagaagc atttcctttg atgaagtcac atcctgtcta tgagcccact 4350
aatgctgtaa cattggcctg aaagagagtg ttctttaaaa gcctttctcg 4400
gctgttagta taaaaacatg atggtatcag ctcttagcat gtttgcttga 4450
cccttatgga aggtataaat ccacagaact tccttcccag agaactggga 4500
aattgtccta gaaataaacc ttgtacagtt gagtggacat ggataagcaa 4550
caatttgtta ctttgcagga tttgttcctt ggtaattgtt tggtgtgtca 4600
tcctgtaaat attcatgata gtctgtttat atccttttgt atatcgttga 4650
tactggattg ggtagaaaaa taaattggca atttaaaaaa atggaacagt 4700
taattgaaa 4709 26 6310 DNA Homo Sapien 26 gatgggggcc ccgtttgtct
gggccttggg ccttttgatg ctgcagatgc 50 tgctctttgt ggctggggaa
cagggcacac aggatatcac cgatgccagc 100 gaaagggggc tccacatgca
gaagctgggg tctgggtcag tgcaggctgc 150 gctggcggag ctggtggccc
tgccctgtct ctttaccctg cagccacggc 200 caagcgcagc ccgagatgcc
cctcggataa agtggaccaa ggtgcggact 250 gcgtcgggcc agcgacagga
cttgcccatc ctggtggcca aggacaatgt 300 cgtgagggtg gccaaaagct
ggcagggacg agtgtcactg ccttcctacc 350 cccggcgccg agccaacgcc
acgctacttc tggggccact gagggccagt 400 gactctgggc tgtaccgctg
ccaggtggtg aggggcatcg aggatgagca 450 ggacctggtg cccttggagg
tgacaggtgt tgtgttccac taccgatcag 500 cccgggaccg ctatgcactg
accttcgctg aggcccagga ggcctgccgt 550 ctcagctcag ccatcattgc
agcccctcgg catctacagg ctgcctttga 600 ggatggcttt gacaactgtg
atgctggctg gctctctgac cgcactgttc 650 ggtatcctat cacccagtcc
cgtcctggtt gctatggcga ccgtagcagc 700 cttccagggg ttcggagcta
tgggaggcgc aacccacagg aactctacga 750 tgtgtattgc tttgcccggg
agctgggggg cgaggtcttc tacgtgggcc 800 cggcccgccg cctgacactg
gccggcgcgc gtgcacagtg ccgccgccag 850 ggtgccgcgc tggcctcggt
gggacagctg cacctggcct ggcatgaggg 900 cctggaccag tgcgacccgg
gctggctggc cgacggcagc gtgcgctacc 950 cgatccagac gccgcgccgg
cgctgcgggg gcccagcccc gggcgtgcgc 1000 accgtctacc gcttcgctaa
ccggaccggc ttcccctcac ccgccgagcg 1050 cttcgacgcc tactgcttcc
gagctcatca ccccacgtca caacatggag 1100 acctagagac cccatcctct
ggggatgagg gggagattct gtcagcagag 1150 gggcccccag ttagagaact
ggagcccacc ctggaggagg aagaggtggt 1200 cacccctgac ttccaggagc
ctctggtgtc cagtggggaa gaagaaaccc 1250 tgattttgga ggagaagcag
gagtctcaac agaccctcag ccctacccct 1300 ggggacccca tgctggcctc
atggcccact ggggaagtgt ggctaagcac 1350 ggtggccccc agccctagcg
acatgggggc aggcactgca gcaagttcac 1400 acacggaggt ggccccaact
gaccctatgc ctaggagaag ggggcgcttc 1450 aaagggttga atgggcgcta
cttccagcag caggaaccgg agccggggct 1500 gcaagggggg atggaggcca
gcgcccagcc ccccacctca gaggctgcag 1550 tgaaccaaat ggagcctccg
ttggccatgg cagtcacaga gatgttgggc 1600 agtggccaga gccggagccc
ctgggctgat ctgaccaatg aggtggatat 1650 gcctggagct ggttctgctg
gtggcaagag ctccccagag ccctggctgt 1700 ggccccctac catggtccca
cccagcatct caggccacag cagggcccct 1750 gtcctggagc tagagaaagc
cgagggcccc agtgccaggc cagccacccc 1800 agacctgttt tggtccccct
tggaggccac tgtctcagct cccagccctg 1850 ccccctggga ggcattccct
gtggccacct ccccagatct ccctatgatg 1900 gccatgctgc gtggtcccaa
agagtggatg ctaccacacc ccacccccat 1950 ctccaccgag gccaatagag
ttgaggcaca tggtgaggcc accgccacgg 2000 ctccaccctc ccctgctgca
gagaccaagg tgtattccct gcctctctct 2050 ttgaccccaa caggacaggg
tggagaggcc atgcccacaa cacctgagtc 2100 ccccagggca gacttcagag
aaactgggga gaccagccct gctcaggtca 2150 acaaagctga gcactccagc
tccagcccat ggccttctgt aaacaggaat 2200 gtggctgtag gttttgtccc
cactgagact gccactgagc caacgggcct 2250 caggggtatc ccggggtctg
agtctggggt cttcgacaca gcagaaagcc 2300 ccacttctgg cttgcaggcc
actgtagatg aggtgcagga cccctggccc 2350 tcagtgtaca gcaaagggct
ggatgcaagt tccccatctg cccccctggg 2400 gagccctgga gtcttcttgg
tacccaaagt caccccaaat ttggagcctt 2450 gggttgctac agatgaagga
cccactgtga atcccatgga ttccacagtc 2500 acgccggccc ccagtgatgc
tagtggaatt tgggaacctg gatcccaggt 2550 gtttgaagaa gccgaaagca
ccaccttgag ccctcaggtg gccctggata 2600 caagcattgt gacgcccctc
acgaccctgg agcaggggga caaggttgga 2650 gttccagcca tgtctacact
gggctcctca agctcccaac cccacccaga 2700 gccagaggat caggtggaga
cccagggaac atcaggagct tcagtgcctc 2750 cgcatcagag cagtccccta
gggaaaccgg ctgttcctcc tgggacaccg 2800 actgcagcca gtgtgggcga
gtctgcctca gtttcctcag gggagcctac 2850 ggtaccgtgg gacccctcca
gcaccctgct gcctgtcacc ctgggcatag 2900 aggacttcga actggaggtc
ctggcaggga gcccgggtgt agagagcttc 2950 tgggaggagg tggcaagtgg
agaggagcca gccctgccag ggacccctat 3000 gaatgcaggt gcggaggagg
tgcactcaga tccctgtgag aacaaccctt 3050 gtcttcatgg agggacatgt
aatgccaatg gcaccatgta tggctgtagc 3100 tgtgatcagg gcttcgccgg
ggagaactgt gagattgaca ttgatgactg 3150 cctctgcagc ccctgtgaga
atggaggcac ctgtattgat gaggtcaatg 3200 gctttgtctg cctttgcctc
cccagctatg ggggcagctt ttgtgagaaa 3250 gacaccgagg gctgtgaccg
cggctggcat aagttccagg gccactgtta 3300 ccgctatttt gcccaccgga
gggcatggga agatgccgag aaggactgcc 3350 gccgccgctc cggccacctg
accagcgtcc actcaccgga ggaacacagc 3400 ttcattaata gctttgggca
tgaaaacacg tggatcggcc tgaacgacag 3450 gatcgtggag agagatttcc
agtggacgga caacaccggg ctgcaatttg 3500 agaactggcg agagaaccag
ccggacaatt tcttcgcggg tggcgaggac 3550 tgtgtggtga tggtggcgca
tgaaagcggg cgctggaacg atgtcccctg 3600 caactacaac ctaccctatg
tctgcaagaa gggcacagtg ctctgtggtc 3650 cccctccggc agtggagaat
gcctcactca tcggtgcccg caaggccaag 3700 aacaatgtcc atgccactgt
aaggtaccag tgcaatgaag gatttgccca 3750 gcaccatgtg gtcaccattc
gatgccggag caatggcaag tgggacaggc 3800 cccaaattgt ctgcaccaaa
cccagacgtt cacatcggat gcggggacac 3850 caccaccacc accaacacca
ccaccagcat caccaccaca aatcccgcaa 3900 ggagcgcaga aaacacaaga
aacacccaac ggaggactgg gagaaggacg 3950 aagggaattt ttgctgaaga
accagaaaaa agaaagcaca acacctttcc 4000 catgcctcct ctggagcctt
cgcctgggga gacagaaccc agagagaaac 4050 aagagagtcc agaagtccct
gaaccccaaa ctgttctcgc aaaaaaaata 4100 ttcctttgaa caaaggtctt
cttttccttt ttttacatac acaagatctt 4150 cttggcaggt ggagccaggt
gtctgaaaag ttcattctcg tctggctgaa 4200 ctctgggagt gtgtcccagc
tgagggaagc acaagtagca aagctcattg 4250 gtctggtctc ttgtttgcca
ggctgattga agcaggcctt gatgagggtg 4300 catgagtgta tgtttgcatt
cacatgaagg aattgctttt cacaccagaa 4350 attcagactt agtcaatgtt
ggctgaattc ctaaatccag gaagaagcct 4400 ggacgtaggg tcattagctt
tgggaataga aggctacaca gaagcacact 4450 gtttttgaac ttgacaacag
ctctcccttt accctggact tcagcccaag 4500 ttccgtcttt ggtcttggtg
gataaacaca cagtgtggag atcccacgta 4550 ctgcatttta gggatgtttt
taggacaacc tccctccatg ccttcagagt 4600 taggagtgag aatgatcaaa
gcaatatgta ggtgatggag ggagagtgta 4650 ttgctaaccc ttccaggtct
agtccagcgc tgagatttgg tggttctgca 4700 tgtgtgatga atctctttca
cacaaataga cgagaggata tttagggcta 4750 gatgagccca gatttcttcc
ccctccatct ctcagggaga caaagaacct 4800 ccttcctgga ccaaggaggt
gctgccaagt tttctagccc agtgcacata 4850 cccagtcctt aagcagacat
tggtagtgcc cctgccctgg gtcccactcc 4900 tgccccaccc cacccttgtc
cctggccatt gcctggtggt ctagaaacac 4950 ttaaaacttg aagtagtgac
acctacctgc ggtcatattg tagagagatg 5000 ctcagtgtta aaactgaaac
acacaaacac acacacacac acatttttct 5050 cttgtagatt ttaatttttt
aagtgggaaa gaactcacct tgccttcctc 5100 ccccaaatgt gcaacctgta
aaaggtctct ccacaccagg ggccaggatc 5150 cagttccctc atctctggca
ggaaagatcc acagcttttc ctccatgtct 5200 gttactcact ttcagcagtc
cgggtaaaat ctgtggatca gggttaaaaa 5250 agcaccgtgg agaatggccc
tcttcaggaa agaaaaataa gcaaatgaat 5300 ggtccaccta ggggttcagt
aaagaaagaa atgtgttaac tgagcctgaa 5350 tcccttctgg gaagtaataa
tgaccattga caactaagaa gtagacacca 5400 tgctaaagac ttacatacaa
tctccttgaa tcttctcaat agcccattga 5450 cttagaaact gttactttcc
cattttacac acagtgaaac tgaggctcag 5500 atataaagga aaggtactgg
cttgaagtca caaccacgac aggagtaagg 5550 atttggaata aggatttggt
cctgttttct ggaccaaatc cttactctgg 5600 ctctgcttac actttctctc
catcaccaaa tccttactcc aaatccagaa 5650 gtcagagcca actcccatct
tggttctgac ccaaatcctg ctctggactc 5700 tggagaggag attgaaatat
aattgcaccc tcatacacat ttaggaaatg 5750 gttaagaagt gtaaactgaa
cccttatcct tgtcttcaat cttcctccct 5800 gtagacatct atcttattat
ggttattatt cagaaaaccc agggatacag 5850 gtttgtcttc ttactttgat
aactcttctt agtttaaaat aataataata 5900 acacatcttt ggtcatctat
gtcacacaaa aattttcctt tgtttgcggg 5950 gggctgggga tgcagtgttt
tttggggggt cttggtttat gctccctgcc 6000 cttgagcccc tcagccgttt
gccctgcccc cacctcggct ccatggtggg 6050 agggggctct ggtcttttct
aaagtgggcg gtttgtcttt tgatctttcc 6100 cttttggatg tgcgtgtgtg
tctgcgtgtg ccatgtgcgt ggcacgcata 6150 tgagtgtgtg tgcgtgtgaa
cggctttggg tcctgctggt tttgctgtga 6200 gctgcagtgt tctgtgggtc
tgtggtatct gacactgtgg acattaatgt 6250 acttcttgga cattttaata
aattttttaa cagttcaaaa aaaaaaaaaa 6300 aaaaaaaaaa 6310 27 4577 DNA
Homo Sapien 27 actagagatg gcgggcgggc tgctctgaag agacctcggc
ggcggcggag 50 gaggagagaa gcgcagcgcc gcgccgcgcc ggggcccatg
tggggaggag 100 tcggagtcgc tgttgccgcc gccgcctgta gctgctggac
ccgagtggga 150 gtgaggggga aacggcagga tgaagttcgc cgagcacctc
tccgcgcaca 200 tcactcccga gtggaggaag caatacatcc agtatgaggc
tttcaaggat 250 atgctgtatt cagctcagga ccaggcacct tctgtggaag
ttacagatga 300 ggacacagta aagaggtatt ttgccaagtt tgaagagaag
tttttccaaa 350 cctgtgaaaa agaacttgcc aaaatcaaca cattttattc
agagaagctc 400 gcagaggctc agcgcaggtt tgctacactt cagaatgagc
ttcagtcatc 450 actggatgca cagaaagaaa gcactggtgt tactacgctg
cgacaacgca 500 gaaagccagt cttccacttg tcccatgagg aacgtgtcca
acatagaaat 550 attaaagacc ttaaactggc cttcagtgag ttctacctca
gtctaatcct 600 gctgcagaac tatcagaatc tgaattttac agggtttcga
aaaatcctga 650 aaaagcatga caagatcctg gaaacatctc gtggagcaga
ttggcgagtg 700 gctcacgtag aggtggcccc attttataca tgcaagaaaa
tcaaccagct 750 tatctctgaa actgaggctg tagtgaccaa tgaacttgaa
gatggtgaca 800 gacaaaaggc tatgaagcgt ttacgtgtcc cccctttggg
agctgctcag 850 cctgcaccag catggactac ttttagagtt ggcctatttt
gtggaatatt 900 cattgtactg aatattaccc ttgtgcttgc cgctgtattt
aaacttgaaa 950 cagatagaag tatatggccc ttgataagaa tctatcgggg
tggctttctt 1000 ctgattgaat tcctttttct actgggcatc aacacgtatg
gttggagaca 1050 ggctggagta aaccatgtac tcatctttga acttaatccg
agaagcaatt 1100 tgtctcatca acatctcttt gagattgctg gattcctcgg
gatattgtgg 1150 tgcctgagcc ttctggcatg cttctttgct ccaattagtg
tcatccccac 1200 atatgtgtat ccacttgccc tttatggatt tatggttttc
ttccttatca 1250 accccaccaa aactttctac tataaatccc ggttttggct
gcttaaactg 1300 ctgtttcgag tatttacagc ccccttccat aaggtaggct
ttgctgattt 1350 ctggctggcg gatcagctga acagcctgtc agtgatactg
atggacctgg 1400 aatatatgat ctgcttctac agtttggagc tcaaatggga
tgaaagtaag 1450 ggcctgttgc caaataattc agaagaatca ggaatttgcc
acaaatatac 1500 atatggtgtg cgggccattg ttcagtgcat tcctgcttgg
cttcgcttca 1550 tccagtgcct gcgccgatat cgagacacaa aaagggcctt
tcctcattta 1600 gttaatgctg gcaagtactc cacaactttc ttcatggtgg
cgtttgcagc 1650 cctttacagc actcacaaag aacgaggtca ctcggacact
atggtgttct 1700 tttacctgtg gattgtcttt tatatcatca gttcctgcta
taccctcatc 1750 tgggatctca agatggactg gggtctcttc gataagaatg
ctggagagaa 1800 cactttcctc cgggaagaga ttgtataccc ccaaaaagcc
tactactact 1850 gtgccataat agaggatgtg attctgcgct ttgcttggac
tatccaaatc 1900 tcgattacct ctacaacttt gttgcctcat tctggggaca
tcattgctac 1950 tgtctttgcc ccacttgagg ttttccggcg atttgtgtgg
aacttcttcc 2000 gcctggagaa tgaacatctg aataactgtg gtgaattccg
tgctgtgcgg 2050 gacatctctg tggcccccct gaacgcagat gatcagactc
tcctagaaca 2100 gatgatggac caggatgatg gggtacgaaa ccgccagaag
aatcggtcat 2150 ggaagtacaa ccagagcata tccctgcgcc ggcctcgcct
cgcttctcaa 2200 tccaaggctc gtgacactaa ggtattgata gaagacacag
atgatgaagc 2250 taacacttga attttctgaa gtctagctta acatctttgg
ttttcctact 2300 ctacaatcct ttcctcgacc aacgcaacct ctagtacctt
tccagccgaa 2350 aacaggagaa aacacataac acattttccg agctcttccg
gatcggatcc 2400 tatggactcc aaacaagctc actgtgtttc ttttcttttc
ttctggttta 2450 attttaattt tctattttca aaacaagtat ttacttcatt
tgccaatcag 2500 aggatgtttt aagaaacaaa acatagtatc ttatggattg
tttacaatca 2550 caaggacata gatacctatc aggatgaaga acaggcattg
caaggaccct 2600 ctgatgggac ggtactgaga tatctcggct tccgctcagc
ccggttttga 2650 atggttgaaa ccggacattg gtttttaaat tttttgtcag
tttatgtgga 2700 gaattttttt ctttccttca tacccagcgc aaaggcactg
gccgcacttg 2750 caggaaaagt gcaacttaaa gcagtacctt cattcatgaa
gctacttttt 2800 aatttgatgt aacttttctt attttgggaa gggttgctgg
gtgggtggga 2850 aatatgatgt atttgttaca catagttttc tcattattta
tgaaacttaa 2900 ccatacagaa tgatataact cctgtgcaat gaaggtgata
acagtaaaag 2950 tgatataact cctgtgcaat gaaggtgata acagtaaaag
aaggcagggg 3000 aaacttacgt tggatgacat ttatgagggt cagtcccaca
tacctctttc 3050 aggagacaac ttgcaccagt ttgacctttt cttttctttg
tttttatttt 3100 aagccaaagt ttcattgcta acttcttaag ttgctgctgc
tttagagtcc 3150 tgagcatatc tctcataaca aggaatccca cacttcacac
caccggctga 3200 atttcatgga agaggttctg ataatttttt taacttttta
aggaacagat 3250 gtggaataca ctggcccata tttcaacctt aacagctgaa
gctatgcctt 3300 attatgcatc cacatgtatg gtccctgtag cgtgaccttt
actagctctg 3350 aatcagaaga cagagctatt tcagaggctc tgtgtgccct
cactagatag 3400 tttttcttct gggttcaacc actttagcca gaatttgatc
aaattaaaag 3450 tctgtcatgg ggaaactata tttttgagca catggaacaa
attatacttc 3500 ctcattcata ttatgttgat acaaaagacc ttggcagcca
tttctcccag 3550 cagttttaaa ggatgaacat tggatttcat gccatcccat
agaaaacctg 3600 ttttaaaatt ttagggatct ttacttggtc atacatgaaa
agtacactgc 3650 ttagaaatta tagactatta tgatctgtcc acagtgccca
ttgtcacttc 3700 tttgtctcat ttcttccctt tgttccttag tcatccaaat
aagcctgaaa 3750 accataagag atattacttt attgaatatg gttggcatta
aatttagcat 3800 ttcattatct aacaaaatta atataaattc caggacatgg
taaaatgtgt 3850 tttaataacc cccagaccca aatgaaaatt tcaaagtcaa
taccagcaga 3900 ttcatgaaag taaatttagt cctataattt tcagcttaat
tataaacaaa 3950 ggaacaaata agtggaaggg cagctattac cattcgctta
gtcaaaacat 4000 tcggttactg ccctttaata cactcctatc atcagcactt
ccaccatgta 4050 ttacaagtct tgacccatcc ctgtcgtaac tccagtaaaa
gttactgtta 4100 ctagaaaatt tttatcaatt aactgacaaa tagtttcttt
ttaaagtagt 4150 ttcttccatc tttattctga ctagcttcca aaatgtgttc
cctttttgaa 4200 tcgaggtttt tttgttttgt tttgttttct gaaaaaatca
tacaactttg 4250 tgcttctatt gcttttttgt gttttgttaa gcatgtccct
tggcccaaat 4300 ggaagaggaa atgtttaatt aatgcttttt agtttaaata
aattgaatca 4350 tttataataa tcagtgttaa caatttagtg acccttggta
ggttaaaggt 4400 tgcattattt atacttgaga tttttttccc ctaactattc
tgttttttgt 4450 actttaaaac
tatgggggaa atatcactgg tctgtcaaga aacagcagta 4500 attattactg
agttaaattg aaaagtccag tggaccaggc atttcttata 4550 taaataaaat
tggtggtact aatgtgt 4577 28 2203 DNA Homo Sapien 28 cccttgctgg
acccgagtgg gagtgagggg gaaacggcag gatgaagttc 50 gccgagcacc
tctccgcgca catcactccc gagtggagga agcaatacat 100 ccagtatgag
gctttcaagg atatgctgta ttcagctcag gaccaggcac 150 cttctgtgga
agttacagat gaggacacag taaagaggta ttttgccaag 200 tttgaagaga
agtttttcca aacctgtgaa aaagaacttg ccaaaatcaa 250 cacattttat
tcagagaagc tcgcagaggc tcagcgcagg tttgctacac 300 ttcagaatga
gcttcagtca tcactggatg cacagaaaga aagcactggt 350 gttactacgc
tgcgacaacg cagaaagcca gtcttccact tgtcccatga 400 ggaacgtgtc
caacatagaa atattaaaga ccttaaactg gccttcagtg 450 agttctacct
cagtctaatc ctgctgcaga actatcagaa tctgaatttt 500 acagggtttc
gaaaaatcct gaaaaagcat gacaagatcc tggaaacatc 550 tcgtggagca
gattggcgag tggctcacgt agaggtggcc ccattttata 600 catgcaagaa
aatcaaccag cttatctctg aaactgaggc tgtagtgacc 650 aatgaacttg
aagatggtga cagacaaaag gctatgaagc gtttacgtgt 700 cccccctttg
ggagctgctc agcctgcacc agcatggact acttttagag 750 ttggcctatt
ttgtggaata ttcattgtac tgaatattac ccttgtgctt 800 gccgctgtat
ttaaacttga aacagataga agtatatggc ccttgataag 850 aatctatcgg
ggtggctttc ttctgattga attccttttt ctactgggca 900 tcaacacgta
tggttggaga caggctggag taaaccatgt actcatcttt 950 gaacttaatc
cgagaagcaa tttgtctcat caacatctct ttgagattgc 1000 tggattcctc
gggatattgt ggtgcctgag ccttctggca tgcttctttg 1050 ctccaattag
tgtcatcccc acatatgtgt atccacttgc cctttatgga 1100 tttatggttt
tcttccttat caaccccacc aaaactttct actataaatc 1150 ccggttttgg
ctgcttaaac tgctgtttcg agtatttaca gcccccttcc 1200 ataaggtagg
ctttgctgat ttctggctgg cggatcagct gaacagcctg 1250 tcagtgatac
tgatggacct ggaatatatg atctgcttct acagtttgga 1300 gctcaaatgg
gatgaaagta agggcctgtt gccaaataat tcagaagaat 1350 caggaatttg
ccacaaatat acatatggtg tgcgggccat tgttcagtgc 1400 attcctgctt
ggcttcgctt catccagtgc ctgcgccgat atcgagacac 1450 aaaaagggcc
tttcctcatt tagttaatgc tggcaaatac tccacaactt 1500 tcttcatggt
gacgtttgca gccctttaca gcactcacaa agaacgaggt 1550 cactcggaca
ctatggtgtt cttttacctg tggattgtct tttatatcat 1600 cagttcctgc
tataccctca tctgggatct caagatggac tggggtctct 1650 tcgataagaa
tgctggagag aacactttcc tccgggaaga gattgtatac 1700 ccccaaaaag
cctactacta ctgtgccata atagaggatg tgattctgcg 1750 ctttgcttgg
actatccaaa tctcgattac ctctacaact ttgttgcctc 1800 attctgggga
catcattgct actgtctttg ccccacttga ggttttccgg 1850 cgatttgtgt
ggaacttctt ccgcctggag aatgaacatc tgaataactg 1900 tggtgaattc
cgtgctgtgc gggacatctc tgtggccccc ctgaacgcag 1950 atgatcagac
tctcctagaa cagatgatgg accaggatga tggggtacga 2000 aaccgccaga
agaatcggtc atggaagtac aaccagagca tatccctgcg 2050 ccggcctcgc
ctcgcttctc aatccaaggc tcgtgacact aaggtattga 2100 tagaagacac
agatgatgaa gctaacactt gaattttctg aagtctagct 2150 taacatcttt
ggttttccta ctctacaatc ctttcctcga ccaacgcaag 2200 ggc 2203 29 3162
DNA Homo Sapien 29 gcgccctagc cctctttcgg ggatactggc cgaccccctc
ttccttttcc 50 cctttagtga aggcctcccc cgtcgccgcg cggcttcccg
gagccgactg 100 cagactccct cagcccggtg ttccccgcgt ccggacgccg
aggtcgcggc 150 ttcgcagaaa ctcgggcccc tccatccgcc ctcagaaaag
ggagcgatgt 200 tgatctcagg aagcacaaag ggaccttcct agctctgact
gaaccacgga 250 gctcaccctg gacagtatca ctccgtggag gaagactgtg
agactgtggc 300 tggaagccag attgtagcca cacatccgcc cctgccctac
cccagagccc 350 tggagcagca actggctgca gatcacagac acagtgagga
tatgagtgta 400 ggggtgagca cctcagcccc tctttcccca acctcgggca
caagcgtggg 450 catgtctacc ttctccatca tggactatgt ggtgttcgtc
ctgctgctgg 500 ttctctctct tgccattggg ctctaccatg cttgtcgtgg
ctggggccgg 550 catactgttg gtgagctgct gatggcggac cgcaaaatgg
gctgccttcc 600 ggtggcactg tccctgctgg ccaccttcca gtcagccgtg
gccatcctgg 650 gtgtgccgtc agagatctac cgatttggga cccaatattg
gttcctgggc 700 tgctgctact ttctggggct gctgatacct gcacacatct
tcatccccgt 750 tttctaccgc ctgcatctca ccagtgccta tgagtacctg
gagcttcgat 800 tcaataaaac tgtgcgagtg tgtggaactg tgaccttcat
ctttcagatg 850 gtgatctaca tgggagttgt gctctatgct ccgtcattgg
ctctcaatgc 900 agtgactggc tttgatctgt ggctgtccgt gctggccctg
ggcattgtct 950 gtaccgtcta tacagctctg ggtgggctga aggccgtcat
ctggacagat 1000 gtgttccaga cactggtcat gttcctcggg cagctggcag
ttatcatcgt 1050 ggggtcagcc aaggtgggcg gcttggggcg tgtgtgggcc
gtggcttccc 1100 agcacggccg catctctggg tttgagctgg atccagaccc
ctttgtgcgg 1150 cacaccttct ggaccttggc cttcgggggt gtcttcatga
tgctctcctt 1200 atacggggtg aaccaggctc aggtgcagcg gtacctcagt
tcccgcacgg 1250 agaaggctgc tgtgctctcc tgttatgcag tgttcccctt
ccagcaggtg 1300 tccctctgcg tgggctgcct cattggcctg gtcatgttcg
cgtattacca 1350 ggagtatccc atgagcattc agcaggctca ggcagcccca
gaccagttcg 1400 tcctgtactt tgtgatggat ctcctgaagg gcctgccagg
cctgccaggg 1450 ctcttcattg cctgcctctt cagcggctct ctcagcacta
tatcctctgc 1500 ttttaattca ttggcaactg ttacgatgga agacctgatt
cgaccttggt 1550 tccctgagtt ctctgaagcc cgggccatca tgctttccag
aggccttgcc 1600 tttggctatg ggctgctttg tctaggaatg gcctatattt
cctcccagat 1650 gggacctgtg ctgcaggcag caatcagcat ctttggcatg
gttgggggac 1700 cgctgctggg actcttctgc cttggaatgt tctttccatg
tgctaaccct 1750 cctggtgctg ttgtgggcct gttggctggg ctcgtcatgg
ccttctggat 1800 tggcatcggg agcatcgtga ccagcatggg cttcagcatg
ccaccctctc 1850 cctctaatgg gtccagcttc tccctgccca ccaatctaac
cgttgccact 1900 gtgaccacac tgatgccctt gactaccttc tccaagccca
cagggctgca 1950 gcggttctat tccttgtctt acttatggta cagtgctcac
aactccacca 2000 cagtgattgt ggtgggcctg attgtcagtc tactcactgg
gagaatgcga 2050 ggccggtccc tgaaccctgc aaccatttac ccagtgttgc
caaagctcct 2100 gtccctcctt ccgttgtcct gtcagaagcg gctccactgc
aggagctacg 2150 gccaggacca cctcgacact ggcctgtttc ctgagaagcc
gaggaatggt 2200 gtgctggggg acagcagaga caaggaggcc atggccctgg
atggcacagc 2250 ctatcagggg agcagctcca cctgcatcct ccaggagacc
tccctgtgat 2300 gttgactcag gaccccgcct ctgtcctcac tgtgccaggc
catagccaga 2350 ggccaccctg tagtacaggg atgagtcttg gtgtgttctg
cagggacagg 2400 cctggatgat ctagctcata ccaaaggacc ttgttctgag
aggttcttgc 2450 ctgcaggaga agctgtcaca tctcaagcat gtgaggcacc
gtttttctcg 2500 tcgcttgcca atctgttttt taaaggatca ggctcgtagg
gagcaggatc 2550 atgccagaaa tagggatgga agtgcatcct ctgggaaaaa
gataatggct 2600 tctgattcaa catagccata gtcctttgaa gtaagtggct
agaaacagca 2650 ctctggttat aattgcccca gggcctgatt caggactgac
tctccaccat 2700 aaaactggaa gctgcttccc ctgtagtccc catttcagta
ccagttctgc 2750 cagccacagt gagcccctat tattactttc agattgtctg
tgacactcaa 2800 gcccctctca tttttatctg tctacctcca ttctgaagag
ggaggttttg 2850 gtgtccctgg tcctctggga atagaagatc catttgtctt
tgtgtagagc 2900 aagcacgttt tccacctcac tgtctccatc ctccacctct
gagatggaca 2950 cttaagagac ggggcaaatg tggatccaag aaaccagggc
catgaccagg 3000 tccactgtgg agcagccatc tatctacctg actcctgagc
caggctgccg 3050 tggtgtcatt tctgtcatcc gtgctctgtt tccttttgga
gtttcttctc 3100 cacattatct ttgttcctgg ggaataaaaa ctaccattgg
acctaaaaaa 3150 aaaaaaaaaa aa 3162 30 1432 DNA Homo Sapien 30
gcgggcgccc agtgcaccgg aggaggtgag cgccaggtcg ccttcgcggc 50
ccggggacac aggcagggac gcgggagctg atgcggctgg accggccggg 100
gaaacagtat tttctggaag ggggcccctc tgaagcggtc caggatcctg 150
cacatggcgc tgaccggggc ctcagacccc tctgcagagg cagaggccaa 200
cggggagaag ccctttctgc tgcgggcatt gcagatcgcg ctggtggtct 250
ccctctactg ggtcacctcc atctccatgg tgttccttaa taagtacctg 300
ctggacagcc cctccctgcg gctggacacc cccatcttcg tcaccttcta 350
ccagtgcctg gtgaccacgc tgctgtgcaa aggcctcagc gctctggccg 400
cctgctgccc tggtgccgtg gacttcccca gcttgcgcct ggacctcagg 450
gtggcccgca gcgtcctgcc cctgtcggtg gtcttcatcg gcatgatcac 500
cttcaataac ctctgcctca agtacgtcgg tgtggccttc tacaatgtgg 550
gccgctcact caccaccgtc ttcaacgtgc tgctctccta cctgctgctc 600
aagcagacca cctccttcta tgccctgctc acctgcggta tcatcatcgg 650
gggcttctgg cttggtgtgg accaggaggg ggcagaaggc accctgtcgt 700
ggctgggcac cgtcttcggc gtgctggcta gcctctgtgt ctcgctcaac 750
gccatctaca ccacgaaggt gctcccggcg gtggacggca gcatctggcg 800
cctgactttc tacaacaacg tcaacgcctg catcctcttc ctgcccctgc 850
tcctgctgct cggggagctt caggccctgc gtgaccttgc ccagctgggc 900
agtgcccact tctgggggat gatgacgctg ggcggcctgt ttggctttgc 950
catcggctac gtgacaggac tgcagatcaa gttcaccagt ccgctgaccc 1000
acaatgtgtc gggcacggcc aaggcctgtg cccagacagt gctggccgtg 1050
ctctactacg aggagaccaa gagcttcctc tggtggacga gcaacatgat 1100
ggtgctgggc ggctcctccg cctacacctg ggtcaggggc tgggagatga 1150
agaagactcc ggaggagccc agccccaaag acagcgagaa gagcgccatg 1200
ggggtgtgag caccacaggc accctggatg gcccggcccc ggggcccgta 1250
cacaggcggg gccagcacag tagtgaaggc ggtctcctgg accccagaag 1300
cgtgctgtgg tgtggactgg gtgctactta tagacccaat cagaatacgg 1350
tggttgagaa ggaaccagtg tttacaagta atatcagaaa gttgaaggaa 1400
ccagtgttta caagtaatac cagaaagttg cc 1432 31 1094 DNA Homo Sapien 31
gcccttatcc tgcacatggc gctgaccggg gcctcagacc cctctgcaga 50
ggcagaggcc aacggggaga agccctttct gctgcgggca ttgcagatcg 100
cgctggtggt ctccctctac tgggtcacct ccatctccat ggtgttcctt 150
aataagtacc tgctggacag cccctccctg cggctggaca cccccatctt 200
cgtcaccttc taccagtgcc tggtgaccac gctgctgtgc aaaggcctca 250
gcgctctggc cgcctgctgc cctggtgccg tggacttccc cagcttgcgc 300
ctggacctca gggtggcccg cagcgtcctg cccctgtcgg tggtcttcat 350
cggcatgatc accttcaata acctctgcct caagtacgtc ggtgtggcct 400
tctacaatgt gggccgctca ctcaccaccg tcttcaacgt gctgctctcc 450
tacctgctgc tcaagcagac cacctccttc tatgccctgc tcacctgcgg 500
tatcatcatc gggggcttct ggcttggtgt ggaccaggag ggggcagaag 550
gcaccctgtc gtggctgggc accgtcttcg gcgtgctggc tagcctctgt 600
gtctcgctca acgccatcta caccacgaag gtgctcccgg cggtggacgg 650
cagcatctgg cgcctgactt tctacaacaa cgtcaacgcc tgcatcctct 700
tcctgcccct gctcctgctg ctcggggagc ttcaggccct gcgtgacttt 750
gcccagctgg gcagtgccca cttctggggg atgatgacgc tgggcggcct 800
gtttggcttt gccatcggct acgtgacagg actgcagatc aagttcacca 850
gtccgctgac ccacaatgtg tcgggcacgg ccaaggcctg tgcccagaca 900
gtgctggccg tgctctacta cgaggagacc aagagcttcc tctggtggac 950
gagcaacatg atggtgctgg gcggctcctc cgcctacacc tgggtcaggg 1000
gctgggagat gaagaagact ccggaggagc ccagccccaa agacagcgag 1050
aagagcgcca tgggggtgtg agcaccacag gcaccctgaa gggc 1094 32 900 DNA
Homo Sapien 32 ccgagcgcgg ggcaccgggg gcctcctgta taggcgggca
ccatgggctc 50 ctgctccggc cgctgcgcgc tcgtcgtcct ctgcgctttt
cagctggtcg 100 ccgccctgga gaggcaggtg tttgacttcc tgggctacca
gtgggcgccc 150 atcctggcca actttgtcca catcatcatc gtcatcctgg
gactcttcgg 200 caccatccag taccggctgc gctacgtcat ggtgtacacg
ctgtgggcag 250 ccgtctgggt cacctggaac gtcttcatca tctgcttcta
cctggaagtc 300 ggtggcctct tacaggacag cgagctactg accttcagcc
tctcccggca 350 tcgctcctgg tggcgtgagc gctggccagg ctgtctgcat
gaggaggtgc 400 cagcagtggg cctcggggcc ccccatggcc aggccctggt
gtcaggtgct 450 ggctgtgccc tggagcccag ctatgtggag gccctacaca
gtggcctgca 500 gatcctgatc gcgcttctgg gctttgtctg tggctgccag
gtggtcagcg 550 tgtttacgga ggaagaggac agctttgatt tcattggtgg
atttgatcca 600 tttcctctct accatgtcaa tgaaaagcca tccagtctct
tgtccaagca 650 ggtgtacttg cctgcgtaag tgaggaaaca gctgatcctg
ctcctgtggc 700 ctccagcctc agcgaccgac cagtgacaat gacaggagct
cccaggcctt 750 gggacgcgcc cccacccagc accccccagg cggccggcag
cacctgccct 800 gggttctaag tactggacac cagccagggc ggcagggcag
tgccacggct 850 ggctgcagcg tcaagagagt ttgtaatttc ctttctctta
aaaaaaaaaa 900 33 666 DNA Homo Sapien 33 ctcctgtata ggcgggcacc
atgggctcct gctccggccg ctgcgcgctc 50 gtcgtcctct gcgcttttca
gctggtcgcc gccctggaga ggcaggtgtt 100 tgacttcctg ggctaccagt
gggcgcccat cctggccaac tttgtccaca 150 tcatcatcgt catcctggga
ctcttcggca ccatccagta ccggctgcgc 200 tatgtcatgg tgtacacgct
gtgggcagcc gtctgggtca cctggaacgt 250 cttcatcatc tgcttctacc
tggaagtcgg tggcctctta aaggacagcg 300 agctactgac cttcagcctc
tcccggcatc gctcctggtg gcgtgagcgc 350 tggccaggct gtctgcatga
ggaggtgcca gcagtgggcc tcggggcccc 400 ccatggccag gccctggtgt
caggtgctgg ctgtgccctg gagcccagct 450 atgtggaggc cctacacagt
tgcctgcaga tcctgatcgc gcttctgggc 500 tttgtctgtg gctgccaggt
ggtcagcgtg tttacggagg aagaggacag 550 ctttgatttc attggtggat
ttgatccatt tcctctctac catgtcaatg 600 aaaagccatc cagtctcttg
tccaagcagg tgtacttgcc tgcgtaagtg 650 aggaaacagc tgatcc 666 34 582
DNA Homo sapien 34 ctcctgtata ggcgggcacc atgggctcct gctccggccg
ctgcgcgctc 50 gtcgtcctct gcgcttttca gctggtcgcc gccctggaga
ggcaggtgtt 100 tgacttcctg ggctaccagt gggcgcccat cctggccaac
tttgtccaca 150 tcatcatcgt catcctggga ctcttcggca ccatccagta
ccggctgcgc 200 tatgtcatgg tgtacacgct gtgggcagcc gtctgggtca
cctggaacgt 250 cttcatcatc tgcttctacc tggaagtcgg tggcctctta
aaggacagcg 300 agctactgac cttcagcctc tcccggcatc gctcctggtg
gcgtgagcgc 350 tggccaggct gtctgcatga ggaggtgcca gcagtgggcc
tcggggcccc 400 ccatggccag gccctggtgt caggtgctgg ctgtgccctg
gagcccagct 450 atgtggaggc cctacacagt tgcctgcaga tcctgatcgc
gcttctgggc 500 tttgtctgtg gctgccaggt ggtcagcgtg tttacggagg
aagaggacag 550 ctgcctgcgt aagtgaggaa acagctgatc ca 582 35 582 DNA
Homo Sapien 35 ctcctgtata ggcgggcacc atgggctcct gctccggccg
ctgcgcgctc 50 gtcgtcctct gcgcttttca gctggtcgcc gccctggaga
ggcaggtgtt 100 tgacttcctg ggctaccagt gggcgcccat cctggccaac
tttgtccaca 150 tcatcatcgt catcctggga ctcttcggca ccatccagta
ccggctgcgc 200 tacgtcatgg tgtacacgct gtgggcagcc gtctgggtca
cctggaacgt 250 cttcatcatc tgcttctacc tggaagtcgg tggcctctta
caggacagcg 300 agctactgac cttcagcctc tcccggcatc gctcctggtg
gcgtgagcgc 350 tggccaggct gtctgcatga ggaggtgcca gcagtgggcc
tcggggcccc 400 ccatggccag gccctggtgt caggtgctgg ctgtgccctg
gagcccagct 450 atgtggaggc cctacacagt ggcctgcaga tcctgatcgc
gcttctgggc 500 tttgtctgtg gctgccaggt ggtcagcgtg tttacggagg
aagaggacag 550 ctgcctgcgt aagtgaggaa acagctgatc ca 582 36 1546 DNA
Homo sapien 36 gcatggaaag tctttatttg agccccttag ctgatgtgga
atcagaagag 50 caaaaaggtc atcttcagag tggcctgggc tgggtccttt
tctctccagg 100 atagaaaagt ggtggtcact ttatccctag tagacatgct
gctgggcttt 150 atcgccccag cattcccatc ccctccagag ccccttgtca
ctccagacca 200 gcgagtgtgg gcctttatct ggactctgct tcctccctgg
ggacaccagg 250 tcttggagca agagaacttg gcaggctctc cccatggcag
tcttattcct 300 cctcctgttc ctatgtggaa ctccccaggc tgcagacaac
atgcaggcca 350 tctatgtggc cttgggggag gcagtagagc tgccatgtcc
ctcaccacct 400 actctacatg gggacgaaca cctgtcatgg ttctgcagcc
ctgcagcagg 450 ctccttcacc accctggtag cccaagtcca agtgggcagg
ccagccccag 500 accctggaaa accaggaagg gaatccaggc tcagactgct
ggggaactat 550 tctttgtggt tggagggatc caaagaggaa gatgccgggc
ggtactggtg 600 cgctgtgcta ggtcagcacc acaactacca gaactggagg
gtgtacgacg 650 tcttggtgct caaaggatcc cagttatctg caagggctgc
agatggatcc 700 ccctgcaatg tcctcctgtg ctctgtggtc cccagcagac
gcatggactc 750 tgtgacctgg caggaaggga agggtcccgt gaggggccgt
gttcagtcct 800 tctggggcag tgaggctgcc ctgctcttgg tgtgtcctgg
ggaggggctt 850 tctgagccca ggagccgaag accaagaatc atccgctgcc
tcatgactca 900 caacaaaggg gtcagcttta gcctggcagc ctccatcgat
gcttctcctg 950 ccctctgtgc cccttccacg ggctgggaca tgccttggat
tctgatgctg 1000 ctgctcacaa tgggccaggg agttgtcatc ctggccctca
gcatcgtgct 1050 ctggaggcag agggtccgtg gggctccagg cagaggaaac
cgaatgcggt 1100 gctacaactg tggtggaagc cccagcagtt cttgcaaaga
ggccgtgacc 1150 acctgtggcg agggcagacc ccagccaggc ctggaacaga
tcaagctacc 1200 tggaaacccc ccagtgacct tgattcacca acatccagcc
tgcgtcgcag 1250 cccatcattg caatcaagtg gagacagagt cggtgggaga
cgtgacttat 1300 ccagcccaca gggactgcta cctgggagac ctgtgcaaca
gcgccgtggc 1350 aagccatgtg gcccctgcag gcattttggc tgcagcagct
accgccctga 1400 cctgtctctt gccaggactg tggagcggat agggggagta
ggagtagaga 1450 agggaacaag ggagcaaggg aacaagggac atctgaacat
ctaatgtgag 1500 aagagaaaca tccttctgtg agtcattaaa atctatgaac cactct
1546 37 4619 DNA Homo Sapien 37 ctttagagaa aggaagggcc aaaactacga
cttggctttc tgaaacggaa 50 gcataaatgt tcttttcctc catttgtctg
gatctgagaa cctgcatttg 100 gtattagcta gtggaagcag tatgtatggt
tgaagtgcat tgctgcagct 150 ggtagcatga gtggtggcca ccagctgcag
ctggctgccc tctggccctg 200 gctgctgatg gctaccctgc aggcaggctt
tggacgcaca ggactggtac 250 tggcagcagc ggtggagtct gaaagatcag
cagaacagaa agctgttatc 300 agagtgatcc ccttgaaaat ggaccccaca
ggaaaactga atctcacttt 350 ggaaggtgtg tttgctggtg ttgctgaaat
aactccagca gaaggaaaat 400 taatgcagtc ccacccactg tacctgtgca
atgccagtga tgacgacaat 450 ctggagcctg gattcatcag catcgtcaag
ctggagagtc ctcgacgggc 500 cccccgcccc tgcctgtcac tggctagcaa
ggctcggatg gcgggtgagc 550 gaggagccag tgctgtcctc tttgacatca
ctgaggatcg agctgctgct 600 gagcagctgc agcagccgct ggggctgacc
tggccagtgg tgttgatctg 650 gggtaatgac gctgagaagc tgatggagtt
tgtgtacaag aaccaaaagg 700 cccatgtgag gattgagctg aaggagcccc
cggcctggcc agattatgat 750 gtgtggatcc taatgacagt ggtgggcacc
atctttgtga tcatcctggc 800 ttcggtgctg cgcatccggt gccgcccccg
ccacagcagg ccggatccgc 850 ttcagcagag aacagcctgg gccatcagcc
agctggccac caggaggtac 900 caggccagct gcaggcaggc ccggggtgag
tggccagact cagggagcag 950 ctgcagctca gcccctgtgt gtgccatctg
tctggaggag ttctctgagg 1000 ggcaggagct acgggtcatt tcctgcctcc
atgagttcca tcgtaactgt 1050 gtggacccct ggttacatca gcatcggact
tgccccctct gcgtgttcaa 1100 catcacagag ggagattcat tttcccagtc
cctgggaccc tctcgatctt 1150 accaagaacc aggtcgaaga ctccacctca
ttcgccagca tcccggccat 1200 gcccactacc acctccctgc tgcctacctg
ttgggccctt cccggagtgc 1250 agtggctcgg cccccacgac ctggtccctt
cctgccatcc caggagccag 1300 gcatgggccc tcggcatcac cgcttcccca
gagctgcaca tccccgggct 1350 ccaggagagc agcagcgcct ggcaggagcc
cagcacccct atgcacaagg 1400 ctggggaatg agccacctcc aatccacctc
acagcaccct gctgcttgcc 1450 cagtgcccct acgccgggcc aggccccctg
acagcagtgg atctggagaa 1500 agctattgca cagaacgcag tgggtacctg
gcagatgggc cagccagtga 1550 ctccagctca gggccctgtc atggctcttc
cagtgactct gtggtcaact 1600 gcacggacat cagcctacag ggggtccatg
gcagcagttc tactttctgc 1650 agctccctaa gcagtgactt tgacccccta
gtgtactgca gccctaaagg 1700 ggatccccag cgagtggaca tgcagcctag
tgtgacctct cggcctcgtt 1750 ccttggactc ggtggtgccc acaggggaaa
cccaggtttc cagccatgtc 1800 cactaccacc gccaccggca ccaccactac
aaaaagcggt tccagtggca 1850 tggcaggaag cctggcccag aaaccggagt
cccccagtcc aggcctccta 1900 ttcctcggac acagccccag ccagagccac
cttctcctga tcagcaagtc 1950 accggatcca actcagcagc cccttcgggg
cggctctcta acccacagtg 2000 ccccagggcc ctccctgagc cagcccctgg
cccagttgac gcctccagca 2050 tctgccccag taccagcagt ctgttcaact
tgcaaaaatc cagcctctct 2100 gcccgacacc cacagaggaa aaggcggggg
ggtccctccg agcccacccc 2150 tggctctcgg ccccaggatg caactgtgca
cccagcttgc cagatttttc 2200 cccattacac ccccagtgtg gcatatcctt
ggtccccaga ggcacacccc 2250 ttgatctgtg gacctccagg cctggacaag
aggctgctac cagaaacccc 2300 aggcccctgt tactcaaatt cacagccagt
gtggttgtgc ctgactcctc 2350 gccagcccct ggaaccacat ccacctgggg
aggggccttc tgaatggagt 2400 tctgacaccg cagagggcag gccatgccct
tatccgcact gccaggtgct 2450 gtcggcccag cctggctcag aggaggaact
cgaggagctg tgtgaacagg 2500 ctgtgtgaga tgttcaggcc tagctccaac
caagagtgtg ctccagatgt 2550 gtttgggccc tacctggcac agagtcctgc
tcctgggaaa ggaaaggacc 2600 acagcaaaca ccattctttt tgccgtactt
cctagaagca ctggaagagg 2650 actggtgatg gtggagggtg agagggtgcc
gtttcctgct ccagctccag 2700 accttgtctg cagaaaacat ctgcagtgca
gcaaatccat gtccagccag 2750 gcaaccagct gctgcctgtg gcgtgtgtgg
gctggatccc ttgaaggctg 2800 agtttttgag ggcagaaagc tagctatggg
tagccaggtg ttacaaaggt 2850 gctgctcctt ctccaacccc tacttggttt
ccctcacccc aagcctcatg 2900 ttcataccag ccagtgggtt cagcagaacg
catgacacct tatcacctcc 2950 ctccttgggt gagctctgaa caccagcttt
ggcccctcca cagtaaggct 3000 gctacatcag gggcaaccct ggctctatca
ttttcctttt ttgccaaaag 3050 gaccagtagc ataggtgagc cctgagcact
aaaaggaggg gtccctgaag 3100 ctttcccact atagtgtgga gttctgtccc
tgaggtgggt acagcagcct 3150 tggttcctct gggggttgag aataagaata
gtggggaggg aaaaactcct 3200 ccttgaagat ttcctgtctc agagtcccag
agaggtagaa aggaggaatt 3250 tctgctggac ttcatctggg cagaggaagg
atggaatgaa ggtagaaaag 3300 gcagaattac agctgagcgg ggacaacaaa
gagttcttct ctgggaaaag 3350 ttttgtctta gagcaaggat ggaaaatggg
gacaacaaag gaaaagcaaa 3400 gtgtgaccct tgggtttgga cagcccagag
gcccagctcc ccagtataag 3450 ccatacaggc cagggaccca caggagagtg
gattagagca caagtctggc 3500 ctcactgagt ggacaagagc tgatgggcct
catcagggtg acattcaccc 3550 cagggcagcc tgaccactct tggcccctca
ggcattatcc catttggaat 3600 gtgaatgtgg tggcaaagtg ggcagaggac
cccacctggg aacctttttc 3650 cctcagttag tggggagact agcacctagg
tacccacatg ggtatttata 3700 tctgaaccag acagacgctt gaatcaggca
ctatgttaag aaatatattt 3750 atttgctaat atatttatcc acaaatgtgg
tctggtcttg tggttttgtt 3800 ctgtcgtgac tgtcactcag ggtaacaacg
tcatctcttt ctacatcaag 3850 agaagtaaat tatttatgtt atcagaggct
aggctccgat tcatgaaagg 3900 atagggtaga gtagagggct tggcaataag
aactggtttg taagccccta 3950 aaagtgtggc ttagtgagat cagggaagga
gaaagcatga ctggattctt 4000 actgtgcttc agtcattatt attatactgt
tcacttcaca cattatcata 4050 cttcagtgac tyagaccttg ggcaaatact
ctgtgcctcg ctttttcagt 4100 ccataaaatg ggcctactta atagttgttg
caggacttac atgagataat 4150 agagtgtaga aaatatgttc caaagtggaa
agttttattc agtgatagaa 4200 aacatccaaa cctgtcacag agcccatctg
aacacagcat gggaccgcca 4250 acaagaagaa agcccgcccg gaagcagctc
aatcaggagg ctgggctgga 4300 atgacagcgc agcggggcct gaaactattt
atatcccaaa gctcctctca 4350 gataaacaca aatgactgcg ttctgcctgc
actcgggcta ttgcgaggac 4400 agagagctgg tgctccattg gcgtgaagtc
tccagggcca gaaggggcct 4450 ttgtcgcttc ctcacaaggc acaagttccc
cttctgcttc cccgagaaag 4500 gtttggtagg ggtggtggtt tagtgcctat
agaacaaggc atttcgcttc 4550 ctagacggtg aaatgaaagg gaaaaaaagg
acacctaatc tcctacaaat 4600 ggtctttagt aaaggaacc 4619 38 3510 DNA
Homo Sapien 38 gcagctctgg gggagctcgg agctcccgat cacggcttct
tgggggtagc 50 tacggctggg tgtgtagaac ggggccgggg ctggggctgg
gtcccctagt 100 ggagacccaa gtgcgagagg caagaactct gcagcttcct
gccttctggg 150 tcagttcctt attcaagtct gcagccggct cccagggaga
tctcggtgga 200 acttcagaaa cgctgggcag tctgcctttc aaccatgccc
ctgtccctgg 250 gagccgagat gtgggggcct gaggcctggc tgctgctgct
gctactgctg 300 gcatcattta caggccggtg ccccgcgggt gagctggaga
cctcagacgt 350 ggtaactgtg gtgctgggcc aggacgcaaa actgccctgc
ttctaccgag 400 gggactccgg cgagcaagtg gggcaagtgg catgggctcg
ggtggacgcg 450 ggcgaaggcg cccaggaact agcgctactg cactccaaat
acgggcttca 500 tgtgagcccg gcttacgagg gccgcgtgga gcagccgccg
cccccacgca 550 accccctgga cggctcagtg ctcctgcgca acgcagtgca
ggcggatgag 600 ggcgagtacg agtgccgggt cagcaccttc cccgccggca
gcttccaggc 650 gcggctgcgg ctccgagtgc tggtgcctcc cctgccctca
ctgaatcctg 700 gtccagcact agaagagggc cagggcctga ccctggcagc
ctcctgcaca 750 gctgagggca gcccagcccc cagcgtgacc tgggacacgg
aggtcaaagg 800 cacaacgtcc agccgttcct tcaagcactc ccgctctgct
gccgtcacct 850 cagagttcca cttggtgcct agccgcagca tgaatgggca
gccactgact 900 tgtgtggtgt cccatcctgg cctgctccag gaccaaagga
tcacccacat 950 cctccacgtg tccttccttg ctgaggcctc tgtgaggggc
cttgaagacc 1000 aaaatctgtg gcacattggc agagaaggag ctatgctcaa
gtgcctgagt 1050 gaagggcagc cccctccctc atacaactgg acacggctgg
atgggcctct 1100 gcccagtggg gtacgagtgg atggggacac tttgggcttt
cccccactga 1150 ccactgagca cagcggcatc tacgtctgcc atgtcagcaa
tgagttctcc 1200 tcaagggatt ctcaggtcac tgtggatgtt cttgaccccc
aggaagactc 1250 tgggaagcag gtggacctag tgtcagcctc ggtggtggtg
gtgggtgtga 1300 tcgccgcact cttgttctgc cttctggtgg tggtggtggt
gctcatgtcc 1350 cgataccatc ggcgcaaggc ccagcagatg acccagaaat
atgaggagga 1400 gctgaccctg accagggaga actccatccg gaggctgcat
tcccatcaca 1450 cggaccccag gagccagccg gaggagagtg tagggctgag
agccgagggc 1500 caccctgata gtctcaagga caacagtagc tgctctgtga
tgagtgaaga 1550 gcccgagggc cgcagttact ccacgctgac cacggtgagg
gagatagaaa 1600 cacagactga actgctgtct ccaggctctg ggcgggccga
ggaggaggaa 1650 gatcaggatg aaggcatcaa acaggccatg aaccattttg
ttcaggagaa 1700 tgggacccta cgggccaagc ccacgggcaa tggcatctac
atcaatgggc 1750 ggggacacct ggtctgaccc aggcctgcct cccttcccta
ggcctggctc 1800 cttctgttga catgggagat tttagctcat cttgggggcc
tccttaaaca 1850 cccccatttc ttgcggaaga tgctccccat cccactgact
gcttgacctt 1900 tacctccaac ccttctgttc atcgggaggg ctccaccaat
tgagtctctc 1950 ccaccatgca tgcaggtcac tgtgtgtgtg catgtgtgcc
tgtgtgagtg 2000 ttgactgact gtgtgtgtgt ggaggggtga ctgtccgtgg
aggggtgact 2050 gtgtccgtgg tgtgtattat gctgtcatat cagagtcaag
tgaactgtgg 2100 tgtatgtgcc acgggatttg agtggttgcg tgggcaacac
tgtcagggtt 2150 tggcgtgtgt gtcatgtggc tgtgtgtgac ctctgcctga
aaaagcaggt 2200 attttctcag accccagagc agtattaatg atgcagaggt
tggaggagag 2250 aggtggagac tgtggctcag acccaggtgt gcgggcatag
ctggagctgg 2300 aatctgcctc cggtgtgagg gaacctgtct cctaccactt
cggagccatg 2350 ggggcaagtg tgaagcagcc agtccctggg tcagccagag
gcttgaactg 2400 ttacagaagc cctctgccct ctggtggcct ctgggcctgc
tgcatgtaca 2450 tattttctgt aaatatacat gcgccgggag cttcttgcag
gaatactgct 2500 ccgaatcact tttaattttt ttcttttttt tttcttgccc
tttccattag 2550 ttgtattttt tatttatttt tatttttatt tttttttaga
gatggagtct 2600 cactatgttg ctcaggctgg ccttgaactc ctgggctcaa
gcaatcctcc 2650 tgcctcagcc tccctagtag ctgggacttt aagtgtacac
cactgtgcct 2700 gctttgaatc ctttacgaag agaaaaaaaa aattaaagaa
agcctttaga 2750 tttatccaat gtttactact gggattgctt aaagtgaggc
ccctccaaca 2800 ccagggggtt aattcctgtg attgtgaaag gggctacttc
caaggcatct 2850 tcatgcaggc agccccttgg gagggcacct gagagctggt
agagtctgaa 2900 attagggatg tgagcctcgt ggttactgag taaggtaaaa
ttgcatccac 2950 cattgtttgt gataccttag ggaattgctt ggacctggtg
acaagggctc 3000 ctgttcaata gtggtgttgg ggagagagag agcagtgatt
atagaccgag 3050 agagtaggag ttgaggtgag gtgaaggagg tgctgggggt
gagaatgtcg 3100 cctttccccc tgggttttgg atcactaatt caaggctctt
ctggatgttt 3150 ctctgggttg gggctggagt tcaatgaggt ttatttttag
ctggcccacc 3200 cagatacact cagccagaat acctagattt agtacccaaa
ctcttcttag 3250 tctgaaatct gctggatttc tggcctaagg gagaggctcc
catccttcgt 3300 tccccagcca gcctaggact tcgaatgtgg agcctgaaga
tctaagatcc 3350 taacatgtac attttatgta aatatgtgca tatttgtaca
taaaatgata 3400 ttctgttttt aaataaacag acaaaacttg aaaaaaaaaa
aaaaaaaaaa 3450 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 3500 aaaaaaaaaa 3510 39 2211 DNA Homo sapien 39
ttgggggttt attctcttcc cttctaactt gacagggtct tgctctgtca 50
ttcaggcaag agtgcagtag tgtgatcact tcttactgcc gcctcaagct 100
tccagcctca actcaagcaa tcctcccacc tcagccaccc aagtggctgg 150
gactacagat taagaatgac ccaaaataaa ttaaagcttt gttccaaagc 200
caatgtgtat actgaagtgc ctgatggagg atggggctgg gcggtagctg 250
tttcattttt cttcgttgaa gtcttcacct acggcatcat caagacattt 300
ggtgtcttct ttaatgactt aatggacagt tttaatgaat ccaatagcag 350
gatctcatgg ataatctcaa tctgtgtgtt tgtcttaaca ttttcagctc 400
ccctcgccac agtcctgagc aatcgtttcg gacaccgtct ggtagtgatg 450
ttgggggggc tacttgtcag caccgggatg gtggccgcct ccttctcaca 500
agaggtttct catatgtacg tcgccatcgg catcatctct ggtctgggat 550
actgctttag ttttctccca actgtaacca tcctatcaca atattttggc 600
aaaagacgtt ccatagtcac tgcagttgct tccacaggag aatgtttcgc 650
tgtgtttgct ttcgcaccag caatcatggc tctgaaggag cgcattggct 700
ggagatacag cctcctcttc gtgggcctac tacagttaaa cattgtcatc 750
ttcggagcac tgctcagacc catcattatc agaggaccag cgtcaccgaa 800
aatagtcatc caggaaaatc ggaaagaagc gcagtatatg cttgaaaatg 850
agaaaacacg aacctcaata gactccattg actcaggagt agaactaact 900
acctcaccta aaaatgtgcc tactcacact aacctggaac tggagccgaa 950
ggccgacatg cagcaggtcc tggtgaagac cagccccagg ccaagcgaaa 1000
agaaagcccc gctattagac ttctccattt tgaaagagaa aagttttatt 1050
tgttatgcat tatttggtct ctttgcaaca ctgggattct ttgcaccttc 1100
cttgtacatc attcctctgg gcattagtct gggcattgac caggaccgcg 1150
ctgctttttt attatctacg atggccattg cagaagtttt cggaaggatc 1200
ggagctggtt ttgtcctcaa cagggagccc attcgtaaga tttacattga 1250
gctcatctgc gtcatcttat tgactgtgtc tctgtttgcc tttacttttg 1300
ctactgaatt ctggggtcta atgtcatgca gcatattttt tgggtttatg 1350
gttggaacaa taggaggact cacattccac tgcttgctga agatgatgtc 1400
gtgggcattg cagaagatgt cttctgcagc tggggtctac atcttcattc 1450
agagcatagc aggactggct ggaccgcccc ttgcaggttt gttggtggac 1500
caaagtaaga tctacagcag ggccttctac tcctgcgcag ctggcatggc 1550
cctggctgct gtgtgcctcg ccctggtgag accgtgtaag atgggactgt 1600
gccagcgtca tcactcaggt gaaacaaagg tagtgagcca tcgtgggaag 1650
actttacagg acatacctga agactttctg gaaatggatc ttgcaaaaaa 1700
tgagcacaga gttcacgtgc aaatggagcc ggtatgacac actttcttac 1750
aacaacagcc actgtgttgg ctggagaggg atggggtggg cccaacgggg 1800
acacaaggag gcagaggagc taacccctct actccacttt caaaactaca 1850
ttttaaaggg aatgtgtatg tgaagagcac taccaacatc gcttttgttt 1900
tgttttgttt tgttttaagc tttttttttt tgcttgtttt taaagccaaa 1950
acaaaaaaca accaagcact cttccatata taaatctggc tgtattcagt 2000
agcaatacaa gagatatgta gaaagactct ttggttcaca ttccgatatt 2050
aaaatagtga catgaactgg caaagtggtt ttaaaagctt tcacgtggga 2100
taaatgattt tctttttttc ttttctttct tcctatggtc ttgtctgaat 2150
aaactactct cctgaataaa acaacatcca acccaggtca ttgaaatgaa 2200
attggccagt c 2211 40 685 DNA Homo Sapien 40 gatgtgctcc ttggagctgg
tgtgcagtgt cctgactgta agatcaagtc 50 caaacctgtt ttggaattga
ggaaacttct cttttgatct cagcccttgg 100 tggtccaggt cttcatgctg
ctgtgggtga tattactggt cctggctcct 150 gtcagtggac agtttgcaag
gacacccagg cccattattt tcctccagcc 200 tccatggacc acagtcttcc
aaggagagag agtgaccctc acttgcaagg 250 gatttcgctt ctactcacca
cagaaaacaa aatggtacca tcggtacctt 300 gggaaagaaa tactaagaga
aaccccagac aatatccttg aggttcagga 350 atctggagag tacagatgcc
aggcccaggg ctcccctctc agtagccctg 400 tgcacttgga tttttcttca
gagatgggat ttcctcatgc tgcccaggct 450 aatgttgaac tcctgggctc
aagtgatctg ctcacctagg cctctcaaag 500 cgctgggatt acagcttcgc
tgatcctgca agctccactt tctgtgtttg 550 aaggagactc tgtggttctg
aggtgccggg caaaggcgga agtaacactg 600 aataatacta tttacaagaa
tgataatgtc ctggcattcc ttaataaaag 650 aactgacttc caaaaaaaaa
aaaaaaaaaa aaaaa 685 41 5392 DNA Homo Sapien 41 aattcactaa
tgcattctgc tctttttgag agcacagctt ctcagatgtg 50 ctccttggag
ctggtgtgca gtgtcctgac tgtaagatca agtccaaacc 100 tgttttggaa
ttgaggaaac ttctcttttg atctcagccc ttggtggtcc 150 aggtcttcat
gctgctgtgg gtgatattac tggtcctggc tcctgtcagt 200 ggacagtttg
caaggacacc caggcccatt attttcctcc agcctccatg 250 gaccacagtc
ttccaaggag agagagtgac cctcacttgc aagggatttc 300 gcttctactc
accacagaaa acaaaatggt accatcggta cctcgggaaa 350 gaaatactaa
gagaaacccc agacaatatc cttgaggttc aggaatctgg 400 agagtacaga
tgccaggccc agggctcccc tctcagtagc cctgtgcact 450 tggatttttc
ttcagcttcg ctgatcctgc aagctccact ttctgtgttt 500 gaaggagact
ctgtggttct gaggtgccgg gcaaaggcgg aagtaacact 550 gaataatact
atttacaaga atgataatgt cctggcattc cttaataaaa 600 gaactgactt
ccatattcct catgcatgtc tcaaggacaa tggtgcatat 650 cgctgtactg
gatataagga aagttgttgc cctgtttctt ccaatacagt 700 caaaatccaa
gtccaagagc catttacacg tccagtgctg agagccagct 750 ccttccagcc
catcagcggg aacccagtga ccctgacctg tgagacccag 800 ctctctctag
agaggtcaga tgtcccgctc cggttccgct tcttcagaga 850 tgaccagacc
ctgggattag gctggagtct ctccccgaat ttccagatta 900 ctgccatgtg
gagtaaagat tcagggttct actggtgtaa ggcagcaaca 950 atgcctcaca
gcgtcatatc tgacagcccg agatcctgga tacaggtgca 1000 gatccctgca
tctcatcctg tcctcactct cagccctgaa aaggctctga 1050 attttgaggg
aaccaaggtg acacttcact gtgaaaccca ggaagattct 1100 ctgcgcactt
tgtacaggtt ttatcatgag ggtgtccccc tgaggcacaa 1150 gtcagtccgc
tgtgaaaggg gagcatccat cagcttctca
ctgactacag 1200 agaattcagg gaactactac tgcacagctg acaatggcct
tggcgccaag 1250 cccagtaagg ctgtgagcct ctcagtcact gttcccgtgt
ctcatcctgt 1300 cctcaacctc agctctcctg aggacctgat ttttgaggga
gccaaggtga 1350 cacttcactg tgaagcccag agaggttcac tccccatcct
gtaccagttt 1400 catcatgagg atgctgccct ggagcgtagg tcggccaact
ctgcaggagg 1450 agtggccatc agcttctctc tgactgcaga gcattcaggg
aactactact 1500 gcacagctga caatggcttt ggcccccagc gcagtaaggc
ggtgagcctc 1550 tccatcactg tccctgtgtc tcatcctgtc ctcaccctca
gctctgctga 1600 ggccctgact tttgaaggag ccactgtgac acttcactgt
gaagtccaga 1650 gaggttcccc acaaatccta taccagtttt atcatgagga
catgcccctg 1700 tggagcagct caacaccctc tgtgggaaga gtgtccttca
gcttctctct 1750 gactgaagga cattcaggga attactactg cacagctgac
aatggctttg 1800 gtccccagcg cagtgaagtg gtgagccttt ttgtcactgt
tccagtgtct 1850 cgccccatcc tcaccctcag ggttcccagg gcccaggctg
tggtggggga 1900 cctgctggag cttcactgtg aggccccgag aggctctccc
ccaatcctgt 1950 actggtttta tcatgaggat gtcaccctgg ggagcagctc
agccccctct 2000 ggaggagaag cttctttcaa cctctctctg actgcagaac
attctggaaa 2050 ctactcatgt gaggccaaca atggcctagt ggcccagcac
agtgacacaa 2100 tatcactcag tgttatagtt ccagtatctc gtcccatcct
caccttcagg 2150 gctcccaggg cccaggctgt ggtgggggac ctgctggagc
ttcactgtga 2200 ggccctgaga ggctcctccc caatcctgta ctggttttat
catgaagatg 2250 tcaccctggg taagatctca gccccctctg gaggaggggc
ctccttcaac 2300 ctctctctga ctacagaaca ttctggaatc tactcctgtg
aggcagacaa 2350 tggtccggag gcccagcgca gtgagatggt gacactgaaa
gttgcagttc 2400 cggtgtctcg cccggtcctc accctcaggg ctcccgggac
ccatgctgcg 2450 gtgggggacc tgctggagct tcactgtgag gccctgagag
gctctcccct 2500 gatcctgtac cggttttttc atgaggatgt caccctagga
aataggtcgt 2550 ccccctctgg aggagcgtcc ttaaacctct ctctgactgc
agagcactct 2600 ggaaactact cctgtgaggc cgacaatggc ctcggggccc
agcgcagtga 2650 gacagtgaca ctttatatca cagggctgac cgcgaacaga
agtggccctt 2700 ttgccacagg agtcgccggg ggcctgctca gcatagcagg
ccttgctgcg 2750 ggggcactgc tgctctactg ctggctctcg agaaaagcag
ggagaaagcc 2800 tgcctctgac cccgccagga gccctccaga ctcggactcc
caagagccca 2850 cctatcacaa tgtaccagcc tgggaagagc tgcaaccagt
gtacactaat 2900 gcaaatccta gaggagaaaa tgtggtttac tcagaagtac
ggatcatcca 2950 agagaaaaag aaacatgcag tggcctctga ccccaggcat
ctcaggaaca 3000 agggttcccc tatcatctac tctgaagtta aggtggcgtc
aaccccggtt 3050 tccggatccc tgttcttggc ttcctcagct cctcacagat
gagtccacac 3100 gtctctccaa ctgctgtttc agcctctgca ccccaaagtt
ccccttgggg 3150 gagaagcagc attgaagtgg gaagatttag gctgccccag
accatatcta 3200 ctggcctttg tttcacatgt cctcattctc agtctgacca
gaatgcaggg 3250 ccctgctgga ctgtcacctg tttcccagtt aaagccctga
ctggcaggtt 3300 ttttaatcca gtggcaaggt gctcccactc cagggcccag
cacatctcct 3350 ggattcctta gtgggcttca gctgtgattg ctgttctgag
tactgctctc 3400 atcacacccc cacagagggg gtcttaccac acaaagggag
agtgggcctt 3450 caggagatgc cgggctggcc taacagctca ggtgctccta
aactccgaca 3500 cagagttcct gctttgggtg gatgcatttc tcaattgtca
tcagcctggt 3550 ggggctactg cagtgtgctg ccaaatggga cagcacacag
cctgtgcaca 3600 tgggacatgt gatgggtctc cccacggggg ctgcatttca
cactcctcca 3650 cctgtctcaa actctaaggt cggcacttga caccaaggta
acttctctcc 3700 tgctcatgtg tcagtgtcta cctgcccaag taagtggctt
tcatacacca 3750 agtcccaagt tcttcccatc ctaacagaag taacccagca
agtcaaggcc 3800 aggaggacca ggggtgcaga cagaacacat actggaacac
aggaggtgct 3850 caattactat ttgactgact gactgaatga atgaatgaat
gaggaagaaa 3900 actgtgggta atcaaactgg cataaaatcc agtgcactcc
ctaggaaatc 3950 cgggaggtat tctggcttcc ctaagaaaca acggaagaga
aggagcttgg 4000 atgaggaaac tgttcagcaa gaggaagggc ttctcacact
ttcatgtgct 4050 tgtggatcac ctgaggatcc tgtgaaaata cagatactga
ttcagtgggt 4100 ctgtgtagag cctgagactg ccattctaac atgttcccag
gggatgctga 4150 tgctgctggc cctgggactg cactgcatgc atgtgaagcc
ctataggtct 4200 cagcagaggc ccatggagag ggaatgtgtg gctctggctg
cccagggccc 4250 aactcggttc acacggatcg tgctgctccc tggccagcct
ttggccacag 4300 caccaccagc tgctgttgct gagagagctt cttctctgtg
acatgttggc 4350 tttcatcagc caccctggga agcggaaagt agctgccact
atctttgttt 4400 ccccacctca ggcctcacac tttcccatga aaagggtgaa
tgtatataac 4450 ctgagccctc tccattcaga gttgttctcc catctctgag
caatgggatg 4500 ttctgttccg cttttatgat atccatcaca tcttatcttg
atctttgctc 4550 ccagtggatt gtacagtgat gacttttaag ccccacggcc
ctgaaataaa 4600 atccttccaa gggcattgga agctctctcc acctgaacca
tggcttttca 4650 tgcttccaag tgtcagggcc ttgcccagat agacagggct
gactctgctg 4700 ccccaacctt tcaaggagga aaccagacac ctgagacagg
agcctgtatg 4750 cagcccagtg cagccttgca gaggacaagg ctggaggcat
ttgtcatcac 4800 tacagatatg caactaaaat agacgtggag caagagaaat
gcattcccac 4850 cgaggccgct tttttaggcc tagttgaaag tcaagaagga
cagcagcaag 4900 cataggctca ggattaaaga aaaaaatctg ctcacagttt
gttctggagg 4950 tcacatcacc aacaaagctc acgccctatg cagttctgag
aaggtggagg 5000 caccaggctc aaaagaggaa atttagaatt tctcattggg
agagtaaggt 5050 acccccatcc cagaatgata actgcacagt ggcagaacaa
actccaccct 5100 aatgtgggtg gaccccatcc agtctgttga aggcctgagt
gtaacaaaag 5150 ggcttattct tcctcaagta agggggaact cctgctttgg
gctgggacat 5200 aagtttttct gctttcagac gcaaactgaa aaatggctct
tcttgggtct 5250 tgagcttgct ggcatatgga ctgaaagaaa ctatgctatt
ggatctcctg 5300 gatctccagc ttgctgactg cagatcttga gatatgtcag
cctctacagt 5350 cacaagagct aattcattct aataaaccaa tctttctgta aa 5392
42 626 DNA Homo Sapien 42 ggacctggga aggagcatag gacagggcaa
ggcgggataa ggaggggcac 50 cacagccctt aaggcacgag ggaacctcac
tgcgcatgct cctttggtgc 100 ccacctcagt gcgcatgttc actgggcgtc
ttcccatcgg ccccttcgcc 150 agtgtgggga acgcggcgga gctgtgagcc
ggcgactcgg gtccctgagg 200 tctggattct ttctccgcta ctgagacacg
gcggacacac acaaacacag 250 aaccacacag ccagtcccag gagcccagta
atggagagcc ccaaaaagaa 300 gaaccagcag ctgaaagtcg ggatcctaca
cctgggcagc agacagaaga 350 agatcaggat acagctgaga tcccagtgcg
cgacatggaa ggtgatctgc 400 aagagctgca tcagtcaaac accggggata
aatctggatt tgggttccgg 450 cgtcaaggtg aagataatac ctaaagagga
acactgtaaa atgccagaag 500 caggtgaaga gcaaccacaa gtttaaatga
agacaagctg aaacaacgca 550 agctggtttt atattagata tttgacttaa
actatctcaa taaagttttg 600 cagctttcac caaaaaaaaa aaaaaa 626 43 1505
DNA Homo Sapien 43 agcggctggc gagccggcgc cggccgagct gcgggagccg
cggagagcac 50 cagctgtcgc cgcgggagct gctccggccg caccatgcgg
gagctggcca 100 ttgagatcgg ggtgcgagcc ctgctcttcg gagtcttcgt
ttttacagag 150 tttttggatc cgttccagag agtcatccag ccagaagaga
tctggctcta 200 taaaaatcct ttggtgcaat cagataacat acctacccgc
ctcatgtttg 250 caatttcttt cctcacaccc ctggctgtta tttgtgtggt
gaaaattatc 300 cggcgaacag acaagactga aattaaggaa gccttcttag
cggtgtcctt 350 ggctcttgct ttgaatggag tctgcacaaa cactattaaa
ttaatagtgg 400 gaagacctcg cgccgatttc ttttaccgct gctttccaga
tggagtgatg 450 aactcggaaa tgcattgcac aggtgacccc gatctggtgt
ccgagggccg 500 caaaagcttc cccagcatcc attcctcctt tgccttttcg
ggccttggct 550 tcacgacgtt ctacttggcg ggcaagctgc actgcttcac
cgagagtggg 600 cggggaaaga gctggcggct ctgtgctgcc atcctgccct
tgtactgcgc 650 catgatgatt gccctgtccc gcatgtgcga ctacaagcat
cactggcaag 700 attcctttgt gggtggagtc atcgcgctca tttttgcata
catttgctac 750 agacagcact atcctcctct gggccaacac agcttgccat
aaaccctacg 800 ttagtctgcg agtttgccat aaaccctacg ttagtctgcg
agtcccagcc 850 tcactgaaga aagaggagag gcccacagct gacagcgcac
ccagcttgcc 900 tctggagggg atcaccgaag gcccggtatg accagtgtcc
tgggaggatg 950 gacactaagc cctgggcaca tctgccaccc tgacatcata
acacaataga 1000 aatggttttc tgtagtgtat ttttcatcag ttgtttctca
aagtcatcgt 1050 acttctgctt ctgtttcact gatggtgttc ctgctacttt
aaatgtctac 1100 ttccaacatc cttgaatttg caagtgaagg acaacaatct
ctgagagacg 1150 tgtggaagag gctgcgaagg tggggtttgg ggagcttcgc
cgattcgtct 1200 atctgaaatg tttgctgtaa cagccacctt cctatgtttt
catggttagt 1250 aaacataata aaacctccca tcgggaaaaa atacaaaatt
cattgattta 1300 ggaatatata tataatattc acatgtgtaa ttccccccct
ccctttagtg 1350 agggtaattc aagatccttc tcaactgctt tgtgcgactt
agactttatg 1400 ttgcagcaga cttttttatt ttacttatag cgcggaatcc
gtgtttcctc 1450 agaatcaggg aatccgcccg aaaatctgtt acaaaggccg
ccaagtgaca 1500 taact 1505 44 1850 DNA Homo Sapien 44 tccttgggtt
cgggtgaaag cgcctggggg ttcgtggcca tgatccccga 50 gctgctggag
aactgaaggc ggacagtctc ctgcgaaacc aggcaatggc 100 ggagctggag
tttgttcaga tcatcatcat cgtggtggtg atgatggtga 150 tggtggtggt
gatcacgtgc ctgctgagcc actacaagct gtctgcacgg 200 tccttcatca
gccggcacag ccaggggcgg aggagagaag atgccctgtc 250 ctcagaagga
tgcctgtggc cctcggagag cacagtgtca ggcaacggaa 300 tcccagagcc
gcaggtctac gccccgcctc ggcccaccga ccgcctggcc 350 gtgccgccct
tcgcccagcg ggagcgcttc caccgcttcc agcccaccta 400 tccgtacctg
cagcacgaga tcgacctgcc acccaccatc tcgctgtcag 450 acggggagga
gcccccaccc taccagggcc cctgcaccct ccagcttcgg 500 gaccccgagc
agcagctgga actgaaccgg gagtcggtgc gcgcaccccc 550 aaacagaacc
atcttcgaca gtgacctgat ggatagtgcc aggctgggcg 600 gcccctgccc
ccccagcagt aactcgggca tcagcgccac gtgctacggc 650 agcggcgggc
gcatggaggg gccgccgccc acctacagcg aggtcatcgg 700 ccactacccg
gggtcctcct tccagcacca gcagagcagt gggccgccct 750 ccttgctgga
ggggacccgg ctccaccaca cacacatcgc gcccctagag 800 agcgcagcca
tctggagcaa agagaaggat aaacagaaag gacaccctct 850 ctagggtccc
caggggggcc gggctggggc tgcgtaggtg aaaaggcaga 900 acactccgcg
cttcttagaa gaggagtgag aggaaggcgg ggggcgcagc 950 aacgcatcgt
gtggccctcc cctcccacct ccctgtgtat aaatatttac 1000 atgtgatgtc
tggtctgaat gcacaagcta agagagcttg caaaaaaaaa 1050 aagaaaaaag
aaaaaaaaaa accacgtttc tttgttgagc tgtgtcttga 1100 aggcaaaaga
aaaaaaattt ctacagtagt ctttcttgtt tctagttgag 1150 ctgcgtgcgt
gaatgcttat tttcttttgt ttatgataat ttcacttaac 1200 tttaaagaca
tatttgcaca aaacctttgt ttaaagatct gcaatattat 1250 atatataaat
atatataaga taagagaaac tgtatgtgcg agggcaggag 1300 tatttttgta
ttagaagagg cctattaaaa aaaaaagttg ttttctgaac 1350 tagaagagga
aaaaaatggc aatttttgag tgccaagtca gaaagtgtgt 1400 attaccttgt
aaagaaaaaa attacaaagc aggggtttag agttatttat 1450 ataaatgttg
agattttgca ctatttttta atataaatat gtcagtgctt 1500 gcttgatgga
aacttctctt gtgtctgttg agactttaag ggagaaatgt 1550 cggaatttca
gagtcgcctg acggcagagg gtgagccccc gtggagtctg 1600 cagagaggcc
ttggccagga gcggcgggct ttcccgaggg gccactgtcc 1650 ctgcagagtg
gatgcttctg cctagtgaca ggttatcacc acgttatata 1700 ttccctaccg
aaggagacac cttttccccc ctgacccaga acagccttta 1750 aatcacaagc
aaaataggaa agttaaccac ggaggcaccg agttccaggt 1800 agtggttttg
cctttcccaa aaatgaaaat aaactgttac cgaaggaatt 1850 45 806 DNA Homo
Sapien 45 gcccttcgga cagtctcctg cgaaaccagg caatggcgga gctggagttt 50
gttcagatca tcatcatcgt ggtggtgatg atggtgatgg tggtggtgat 100
cacgtgcctg ctgagccact acaagctgtc tgcacggtcc ttcatcagcc 150
ggcacagcca ggggcggagg agagaagatg ccctgtcctc agaaggatgc 200
ctgtggccct cggagagcac agtgtcaggc aacggaatcc cagagccgca 250
ggtctacgcc ccgcctcggc ccaccgaccg cctggccgtg ccgcccttcg 300
cccagcggga gcgcttccac cgcttccagc ccacctatcc gtacctgcag 350
cacgagatcg acctgccgcc caccatctcg ctgtcagacg gggaggagcc 400
cccaccctac cagggcccct gcaccctcca gcttcgggac cccgagcagc 450
agctggaact gaaccgggag tcggtgcgcg cacccccaaa cagaaccatc 500
ttcgacagtg acctgatgga tagtgccagg ctgggcggcc cctgcccccc 550
cagcagtaac tcgggcatca gcgccacgtg ctacggcagc ggcgggcgca 600
tggaggggcc gccgcccacc tacagcgagg tcatcggcca ctacccgggg 650
tcctccttcc agcaccagca gagcagtggg ccgccctcct tgctggaggg 700
gacccggctc caccacacac acatcgcgcc cctagagagc gcagccatct 750
ggagcaaaga gaaggataaa cagaaaggac accctctcta gggtccccag 800 aagggc
806 46 1982 DNA Homo Sapien 46 ggcgagaggc gggctgaggc ggcccagcgg
cggcaggtga ggcggaacca 50 accctcctgg ccatgggagg ggccgtggtg
gacgagggcc ccacaggcgt 100 caaggcccct gacggcggct ggggctgggc
cgtgctcttc ggctgtttcg 150 tcatcactgg cttctcctac gccttcccca
aggccgtcag tgtcttcttc 200 aaggagctca tacaggagtt tgggatcggc
tacagcgaca cagcctggat 250 ctcctccatc ctgctggcca tgctctacgg
gacaggtccg ctctgcagtg 300 tgtgcgtgaa ccgctttggc tgccggcccg
tcatgcttgt ggggggtctc 350 tttgcgtcgc tgggcatggt ggctgcgtcc
ttttgccgga gcatcatcca 400 ggtctacctc accactgggg tcatcacggg
gttgggtttg gcactcaact 450 tccagccctc gctcatcatg ctgaaccgct
acttcagcaa gcggcgcccc 500 atggccaacg ggctggcggc agcaggtagc
cctgtcttcc tgtgtgccct 550 gagcccgctg gggcagctgc tgcaggaccg
ctacggctgg cggggcggct 600 tcctcatcct gggcggcctg ctgctcaact
gctgcgtgtg tgccgcactc 650 atgaggcccc tggtggtcac ggcccagccg
ggctcggggc cgccgcgacc 700 ctcccggcgc ctgctagacc tgagcgtctt
ccgggaccgc ggctttgtgc 750 tttacgccgt ggccgcctcg gtcatggtgc
tggggctctt cgtcccgccc 800 gtgttcgtgg tgagctacgc caaggacctg
ggcgtgcccg acaccaaggc 850 cgccttcctg ctcaccatcc tgggcttcat
tgacatcttc gcgcggccgg 900 ccgcgggctt cgtggcgggg cttgggaagg
tgcggcccta ctccgtctac 950 ctcttcagct tctccatgtt cttcaacggc
ctcgcggacc tggcgggctc 1000 tacggcgggc gactacggcg gcctcgtggt
cttctgcatc ttctttggca 1050 tctcctacgg catggtgggg gccctgcagt
tcgaggtgct catggccatc 1100 gtgggcaccc acaagttctc cagtgccatt
ggcctggtgc tgctgatgga 1150 ggcggtggcc gtgctcgtcg ggcccccttc
gggaggcaaa ctcctggatg 1200 cgacccacgt ctacatgtac gtgttcatcc
tggcgggggc cgaggtgctc 1250 acctcctccc tgattttgct gctgggcaac
ttcttctgca ttaggaagaa 1300 gcccaaagag ccacagcctg aggtggcggc
cgcggaggag gagaagctcc 1350 acaagcctcc tgcagactcg ggggtggact
tgcgggaggt ggagcatttc 1400 ctgaaggctg agcctgagaa aaacggggag
gtggttcaca ccccggaaac 1450 aagtgtctga gtggctgggc ggggccggca
ggcacaggga ggaggtacag 1500 aagccggcaa cgcttgctat ttattttaca
aactggactg gctcaggcag 1550 ggccacggct gggctccagc tgccggccca
gcggatcgtc gcccgatcag 1600 tgttttgagg gggaaggtgg cggggtggga
accgtgtcat tccagagtgg 1650 atctgcggtg aagccaagcc gcaaggttac
aaggcatcct caccaggggc 1700 cccgcctgct gctcccaggt ggcctgcggc
cactgctatg ctcaaggacc 1750 tggaaaccca tgcttcgaga caacgtgact
ttaatgggag ggtgggtggg 1800 ccgcagacag gctggcaggg caggtgctgc
gtggggccct ctccagcccg 1850 tcctaccctg ggctcacatg gggcctgtgc
ccacccctct tgagtgtctt 1900 ggggacagct ctttccaccc ctggaagatg
gaaataaacc tgcgtgtggg 1950 tggagtgttc tcgtgccgaa ttcaaaaagc tt 1982
47 2171 DNA Homo Sapien 47 cccacgcgtc cgcccacgcg tccgccgggt
cctgcgcgct ccggactgag 50 gtggcgtccc tgggccggac ggcggtgtcc
cggcgtggcg ggaagccggc 100 actggagcgg gagcgcactg ggcgcgggac
cgggaggcgc agggaccgga 150 cggctcccga gtcgcccacc tgacggtacc
gagagggcgg cgcccctccg 200 agcagagccg tcccggccac tcccctggga
tctgacttgg ctcttgcggt 250 cgcgggcacc gtgaagccct ggggtgtgcg
tggctcctcc tggtaggcgc 300 cctttcccgg cgtccggctt ggggtggtgg
tggcgttgac tccagccccg 350 cctctccctg gagaggaggg ctccactcgc
tccttcggcc tcctcccctg 400 gggccgcagc gactcgggcc ggcttcctgc
ttccctgcct gccggcggtc 450 ccgctggcta gaagaagtct tcacttccca
ggagagccaa agcgtgtctg 500 gccctaggtg ggaaaagaac tggctgtgac
ctttgccctg acctggaagg 550 gcccagcctt gggctgaatg gcagcaccca
cgcccgcccg tccggtgctg 600 acccacctgc tggtggctct cttcggcatg
ggctcctggg ctgcggtcaa 650 tgggatctgg gtggagctac ctgtggtggt
caaagagctt ccagagggtt 700 ggagcctccc ctcttacgtc tctgtgcttg
tggctctggg gaacctgggt 750 ctgctggtgg tgaccctctg gaggaggctg
gccccaggaa aggacgagca 800 ggtccccatc cgggtggtgc aggtgctggg
catggtgggc acagccctgc 850 tggcctctct gtggcaccat gtggccccag
tggcaggaca gttgcattct 900 gtggccttct tagcactggc ctttgtgctg
gcactggcat gctgtgcctc 950 gaatgtcact ttcctgccct tcttgagcca
cctgccacct cgcttcttac 1000 ggtcattctt cctgggtcaa ggcctgagtg
ccctgctgcc ctgcgtgctg 1050 gccctagtgc agggtgtggg ccgcctcgag
tgcccgccag cccccatcaa 1100 cggcacccct ggccccccgc tcgacttcct
tgagcgtttt cccgccagca 1150 ccttcttctg ggcactgact gcccttctgg
tcgcttcagc tgctgccttc 1200 cagggtcttc tgctgctgtt gccgccacca
ccatctgtac ccacagggga 1250 gttaggatca ggcctccagg tgggagcccc
aggagcagag gaagaggtgg 1300 aagagtcctc
accactgcaa gagccaccaa gccaggcagc aggcaccacc 1350 cctggtccag
accctaaggc ctatcagctt ctatcagccc gcagtgcctg 1400 cctgctgggc
ctgttggccg ccaccaacgc gctgaccaat ggcgtgctgc 1450 ctgccgtgca
gagcttttcc tgcttaccct acgggcgtct ggcctaccac 1500 ctggctgtgg
tgctgggcag tgctgccaat cccctggcct gcttcctggc 1550 catgggtgtg
ctgtgcaggt ccttggcagg gctgggcggc ctctctctgc 1600 tgggcgtgtt
ctgtgggggc tacctgatgg cgctggcagt cctgagcccc 1650 tgcccgcccc
tggtgggcac ctcggcgggg gtggtcctcg tggtgctgtc 1700 gtgggtgctg
tgtcttggcg tgttctccta cgtgaaggtg gcagccagct 1750 ccctgctgca
tggcgggggc cggccggcat tgctggcagc cggcgtggcc 1800 atccaggtgg
gctctctgct cggcgctgtt gctatgttcc ccccgaccag 1850 catctatcac
gtgttccaca gcagaaagga ctgtgcagac ccctgtgact 1900 cctgagcctg
ggcaggtggg gaccccgctc cccaacacct gtctttccct 1950 caatgctgcc
accatgcctg agtgcctgca gcccaggagg cccgcacacc 2000 ggtacactcg
tggacaccta cacactccat aggagatcct ggctttccag 2050 ggtgggcaag
ggcaaggagc aggcttggag ccagggacca gtgggggctg 2100 tagggtaagc
ccctgagcct gggacctaca tgtggtttgc gtaataaaac 2150 atttgtattt
aaaaaaaaaa a 2171 48 1617 DNA Homo Sapien 48 gccagcacag ctgccctctg
gaccctgcgg accccagccg agccccttcc 50 tgagttccac aggcgcagcc
cccgggcggt cgggcggagg ggtccccggg 100 gcggtgccag gcgcaatcct
ggagggcggc cgggaggagg aggtgcgcgc 150 ggccatgcac accgtggcta
cgtccggacc caacgcgtcc tggggggcac 200 cggccaacgc ctccggctgc
ccgggctgtg gcgccaacgc ctcggacggc 250 ccagtccctt cgccgcgggc
cgtggacgcc tggctcgtgc cgctcttctt 300 cgcggcgctg atgctgctgg
gcctggtggg gaactcgctg gtcatctacg 350 tcatctgccg ccacaagccg
atgcggaccg tgaccaactt ctacatcgcc 400 aacctggcgg ccacggacgt
gaccttcctc ctgtgctgcg tccccttcac 450 ggccctgctg tacccgctgc
ccggctgggt gctgggcgac ttcatgtgca 500 agttcgtcaa ctacatccag
caggtctcgg tgcaggccac gtgtgccact 550 ctgaccgcca tgagtgtgga
ccgctggtac gtgacggtgt tcccgttgcg 600 cgccctgcac cgccgcacgc
cccgcctggc gctggctgtc agcctcagca 650 tctgggtagg ctctgcggcg
gtgtctgcgc cggtgctcgc cctgcaccgc 700 ctgtcacccg ggccgcgcgc
ctactgcagt gaggccttcc ccagccgcgc 750 cctggagcgc gccttcgcac
tgtacaacct gctggcgctg tacctgctgc 800 cgctgctcgc cacctgcgcc
tgctatgcgg ccatgctgcg ccacctgggc 850 cgggtcgccg tgcgccccgc
gcccgccgat agcgccctgc aggggcaggt 900 gctggcagag cgcgcaggcg
ccgtgcgggc caaggtctcg cggctggtgg 950 cggccgtggt cctgctcttc
gccgcctgct ggggccccat ccagctgttc 1000 ctggtgctgc aggcgctggg
ccccgcgggc tcctggcacc cacgcagcta 1050 cgccgcctac gcgcttaaga
cctgggctca ctgcatgtcc tacagcaact 1100 ccgcgctgaa cccgctgctc
tacgccttcc tgggctcgca cttccgacag 1150 gccttccgcc gcgtctgccc
ctgcgcgccg cgccgccccc gccgcccccg 1200 ccggcccgga ccctcggacc
ccgcagcccc acacgcggag ctgcaccgcc 1250 tggggtccca cccggccccc
gccagggcgc agaagccagg gagcagtggg 1300 ctggccgcgc gcgggctgtg
cgtcctgggg gaggacaacg cccctctttg 1350 agcggacccg gtgggaatcc
gagcggctcc ctcgggagcg gggactgctg 1400 gaacagcggc tattcttctg
ttattagtat tttttttact gtccaagatc 1450 aactgtggaa atattttggt
ctcttgtgac gttcggtgca gtttcgttgt 1500 gaagtttgct attgatattg
aaattatgac ttctgtgttt cctgaaatta 1550 aacatgtgtc aacacaggac
tttttggatc attccagaaa gtgtcagacg 1600 tttaaaaaaa aaaaaaa 1617 49
3095 DNA Homo Sapien 49 ggcgcggggc gccatggcac accgagcggc tccgtcttct
gctcctcaga 50 gagcccggct ggcggcctgg gatgacaaga tgtctggact
gcaatcctgc 100 acagttttga gagggagatg acttgagtgg ttggctttta
tctccacaac 150 aatgtccatg aacaattcca aacagctagt gtctcctgca
gctgcgcttc 200 tttcaaacac aacctgccag acggaaaacc ggctttccgt
atttttttca 250 gtaatcttca tgacagtggg aatcttgtca aacagccttg
ccatcgccat 300 tctcatgaag gcatatcaga gatttagaca gaagtccaag
gcatcgtttc 350 tgcttttggc cagcggcctg gtaatcactg atttctttgg
ccatctcatc 400 aatggagcca tagcagtatt tgtatatgct tctgataaag
aatggatccg 450 ctttgaccaa tcaaatgtcc tttgcagtat ttttggtatc
tgcatggtgt 500 tttctggtct gtgcccactt cttctaggca gtgtgatggc
cattgagcgg 550 tgtattggag tcacaaaacc aatatttcat tctacgaaaa
ttacatccaa 600 acatgtgaaa atgatgttaa gtggtgtgtg cttgtttgct
gttttcatag 650 ctttgctgcc catccttgga catcgagact ataaaattca
ggcgtcgagg 700 acctggtgtt tctacaacac agaagacatc aaagactggg
aagatagatt 750 ttatcttcta cttttttctt ttctggggct cttagccctt
ggtgtttcat 800 tgttgtgcaa tgcaatcaca ggaattacac ttttaagagt
taaatttaaa 850 agtcagcagc acagacaagg cagatctcat catttggaaa
tggtaatcca 900 gctcctggcg ataatgtgtg tctcctgtat ttgttggagc
ccatttctgg 950 ttacaatggc caacattgga ataaatggaa atcattctct
ggaaacctgt 1000 gaaacaacac tttttgctct ccgaatggca acatggaatc
aaatcttaga 1050 tccttgggta tatattcttc tacgaaaggc tgtccttaag
aatctctata 1100 agcttgccag tcaatgctgt ggagtgcatg tcatcagctt
acatatttgg 1150 gagcttagtt ccattaaaaa ttccttaaag gttgctgcta
tttctgagtc 1200 accagttgca gagaaatcag caagcaccta gcttaatagg
acagtaaatc 1250 tgtgtggggc tagaacaaaa attaagacat gtttggcaat
atttcagtta 1300 gttaaatacc tgtagcctaa ctggaaaatt caggcttcat
catgtagttt 1350 gaagatacta ttgtcagatt caggttttga aatttgtcaa
ataaacagga 1400 taactgtaca ttttcaactt gtttttgcca atgggaggta
gacacaataa 1450 aataatgcca tgggagtcac actgaaagca attttgagct
tatctgtctt 1500 atttatgctt tgagtgaatc atctgttgag gtctaatgcc
tctacttggc 1550 ctatttgcca gagaacatct taatgcagcc tgcatagtga
aatggttatt 1600 ttgagatcac cgctctgtag ctaaccctta taaactaggc
tcagtaaaat 1650 aaagcactct tattttttga tctggcctat tttgcccctc
attgtgtagc 1700 ctcaattaac acatgcatgg tcatgacacc cagaattcat
gatggtttgt 1750 tataacaacc tctgcatatt ccaggtctgg cagacaggtt
gcctgaccct 1800 gcaatcctat ctagaatggg cccattcttg tcacatttga
caaataggac 1850 tgcctacatt tattattatg aaggtcgatt gttgttggaa
gtgttttttc 1900 atgtcataga ttagcaattt tcaaataatt attttttctc
tgaaaatttt 1950 gtgtgtgatt gcacaataaa taatttttag agaaacaaag
gctctttctc 2000 agcacattga tgggcaacta gaattacagc agtttcaaac
tctaccatgg 2050 ataatgcaaa caaaccgaag ctacatgcca atgataggtg
caaagaatat 2100 tggcaaaagg tgctttacct tgagccatta tttgtgtcag
agaacaaaag 2150 aaacagaatc aatatataaa ttcaaagact atctgcagct
agtgtgtttc 2200 ttctttacac acatatacac acagacatca gaaaattctg
ttgagagcag 2250 gttcattaaa tttgtaagat ggcatattct aaagcctgtg
ctaccagtac 2300 taagagggga agactggcaa tttgccaagc acttggggat
tattataaca 2350 attaactagg agatcaagag ataataatct ctccccaaat
tttccaataa 2400 taattgagac tttttctttg cttgtttgtg taattcaacc
aaaagaattt 2450 caatacccat tcaaattgtc ctaggtctat cagaaattag
ggaaggtagt 2500 cctgctttat aataggaaaa tgtatttctg tataagattt
ctttgctttc 2550 attaaaaatg ggattcattt aaaaattaat ctttccctgt
taggctgatt 2600 tcagattctc taggaaatct ggtgaagtaa ccagaagact
ttcagatggt 2650 ttatttgctt tcagcagaga atttatttca tacagttact
taagagtgtt 2700 gatgtcttgt gaacagagat ataaggaacc attctccatc
cttccttatc 2750 atgctgggta caatgcttct atgaatattt ccatgtattt
tgactgggga 2800 gaggcatgga gaagaaactc tcattcaggg gctccaggat
ccttctcctt 2850 gaggcttcta aataaatggc agaattcttg ctgtattgcc
atgatgtcac 2900 cctggccatg tgtactgact tgaggagatc ttgcaacatg
gccatgtgca 2950 aggctttaag gagtgagaga gatgtgtaca tatcttagga
gggttatcta 3000 tgttatctga gtatatgttt gggtaaccaa attggtctta
aaaatgatgt 3050 taacccaaga agtagacatc aaaaattaaa aaaaaaaaaa aaaaa
3095 50 6476 DNA Homo Sapien 50 atgtcacgca tgagccggca tccagacaag
gacctggccc agggtccctt 50 caacacctgc tgtggctgca ccttaatggc
tagtcctgct aatctccctc 100 cgaacactca agcagctgca gaaagggccc
tttcccagag caggtggaag 150 agggtgcaag tgcccgcccc ggcatccctg
tcccctttcc cactggccat 200 ggcttcagtt gccttctgga tcagcatcct
gattggctgc gaggaacaga 250 ctctctgcag aggctggcgt agcccagtcg
gggatggctg tgctcatgtg 300 cctccccagg agcgagcgac cgcagaggca
gaccctccag ggcggtgcag 350 cacctccacg gcgtcgtcta ccatctgtgg
cctgtggcat ttgtccccac 400 ggctgcagct cctcccacct ctgcattcca
ggcagggaga agagtcgggc 450 aaaactgaga aggtgcttct ctggggaaga
gagggcctcc atgtgtggaa 500 acccggagtc ctgcagcccg atgtccacgg
cacctccaac ctggggaact 550 gctccttcct gcacggcctg gttacggctc
cctcttgtcc acggcgggcg 600 ggcgccgagc tgctgaattc tttaggaagt
cagtttgcca ttagcctttt 650 tgaagttcag agtggaactg agcccagcat
tacaggtgtg gccacgtcag 700 ggcagtgcag ggctatgcca ctgaagcatt
atctcctttt gctggtgggc 750 tgccaagcct ggggtgcagg gttggcctac
catggctgcc ctagcgagtg 800 tacctgctcc agggcctccc aggtggagtg
caccggggca cgcattgtgg 850 cggtgcccac ccctctgccc tggaacgcca
tgagcctgca gatcctcaac 900 acgcacatca ctgaactcaa tgagtccccg
ttcctcaata tttcagccct 950 catcgccctg aggattgaga agaatgagct
gtcgcgcatc acgcctgggg 1000 ccttccgaaa cctgggctcg ctgcgctatc
tcagcctcgc caacaacaag 1050 ctgcaggttc tgcccatcgg cctcttccag
ggcctggaca gccttgagtc 1100 tctccttctg tccagtaacc agctgttgca
gatccagccg gcccacttct 1150 cccagtgcag caacctcaag gagctgcagt
tgcacggcaa ccacctggaa 1200 tacatccctg acggagcctt cgaccacctg
gtaggactca cgaagctcaa 1250 tctgggcaag aatagcctca cccacatctc
acccagggtc ttccagcacc 1300 tgggcaatct ccaggtcctc cggctgtatg
agaacaggct cacggatatc 1350 cccatgggca cttttgatgg gcttgttaac
ctgcaggaac tggctctaca 1400 gcagaaccag attggactgc tctcccctgg
tctcttccac aacaaccaca 1450 acctccagag actctacctg tccaacaacc
acatctccca gctgccaccc 1500 agcatcttca tgcagctgcc ccagctcaac
cgtcttactc tctttgggaa 1550 ttccctgaag gagctctctc tggggatctt
cgggcccatg cccaacctgc 1600 gggagctttg gctctatgac aaccacatct
cttctctacc cgacaatgtc 1650 ttcagcaacc tccgccagtt gcaggtcctg
attcttagcc gcaatcagat 1700 cagcttcatc tccccgggtg ccttcaacgg
gctaacggag cttcgggagc 1750 tgtccctcca caccaacgca ctgcaggacc
tggacgggaa tgtcttccgc 1800 atgttggcca acctgcagaa catctccctg
cagaacaatc gcctcagaca 1850 gctcccaggg aatatcttcg ccaacgtcaa
tggcctcatg gccatccagc 1900 tgcagaacaa ccagctggag aacttgcccc
tcggcatctt cgatcacctg 1950 gggaaactgt gtgagctgcg gctgtatgac
aatccctgga ggtgtgactc 2000 agacatcctt ccgctccgca actggctcct
gctcaaccag cctaggttag 2050 ggacggacac tgtacctgtg tgtttcagcc
cagccaatgt ccgaggccag 2100 tccctcatta tcatcaatgt caacgttgct
gttccaagcg tccatgtacc 2150 tgaggtgcct agttacccag aaacaccatg
gtacccagac acacccagtt 2200 accctgacac cacatccgtc tcttctacca
ctgagctaac cagccctgtg 2250 gaagactaca ctgatctgac taccattcag
gtcactgatg accgcagcgt 2300 ttggggcatg acccatgccc atagcgggct
ggccattgcc gccattgtaa 2350 ttggcattgt cgccctggcc tgctccctgg
ctgcctgcgt cggctgttgc 2400 tgctgcaaga agaggagcca agctgtcctg
atgcagatga aggcacccaa 2450 tgagtgttaa agaggcaggc tggagcaggg
ctggggaatg atgggactgg 2500 aggacctggg aatttcatct ttctgcctcc
acccctgggt ccatggagct 2550 ttcccgtgat tgctctttct ggccctagat
aaaggtgtgc ctacctcttc 2600 ctgacttgcc tgattctccc gtagagaagc
aggtcgtgcc ggaccttcct 2650 acaatcagga agatagatcc aactggccat
ggcaaaagcc ctggggattt 2700 ccgattcata cccctgggct tccttcgaga
gggctcttcc tccaaatcct 2750 ccccacctgt cctccaagaa cagccttccc
tgcgcccagg ccccctccgg 2800 gcctctgtag actcagttag tccacagcct
gctcacttcg tgggaatagt 2850 tctccgctga gatagcccct ctcgcctaag
tattatgtaa gttgatttcc 2900 cttcttttgt ttctcttgtt tgtgctatgg
cttgacccag catgtcccct 2950 caaatgaaag ttctcccctt gattttctgc
tcctgaaggc agggtgagtt 3000 ctctcctcaa agaagacttc aaaccattta
actggtttct taagagccgt 3050 caatcagcct ggttttgggg atgctatgaa
agagagaagg aaaatcatgc 3100 cgctcagttc ctggagacag aagagccgtc
atcagtgtct cacttgtgat 3150 ttttatctgg aaaaggaaga aacaccccag
cacagcaagc tcagcctttt 3200 agagaaggat atttccaaac tgcaaacttt
gctttgaaaa gtttagccct 3250 ttaaggaatg aaatcatgta gaattttgga
cttctaaaaa cattaaaatc 3300 agcttattaa tacgggatag agaaagaaat
ctggtgcctg ggggtccctg 3350 tgttcacccc tagagtttgt tttaaaattt
ttaattgaag catgtgaagt 3400 gtacctgcag aaaagtggga acatgatagt
gtatggcttg gtggattttc 3450 acaaactgaa catacctgtg taatcagcat
ctagacccag acccagagcg 3500 tcacaaatat cccccatcct gggcttttcc
cagaggagat gggggcttct 3550 gaagatggac ttacctggga cctgcccccc
atgagccagg acggtccccc 3600 cacagtcagc ctgtgcaaag gccccgtggc
caggggtgga ggagaatatg 3650 tgggtgtgga caggatggga gactgtggcc
tgaacaggag attttattat 3700 atctggagac cctgagagac cctgagacct
ggggcaccct ggctggccag 3750 gtcagaagca tcctgactgc agaggtccgt
gcagccacac cctcttccct 3800 gccagcaagc tgtctgcggc tcatcggagg
cccctccgcc tggagccttc 3850 tatggacgtg atatgcctgt atctgttttt
aattttcatt cttcacttag 3900 gggaagtgaa atcgctcaga gatgagatcc
tttaattgaa aacgaagtgt 3950 aacggaatct agtgtctttc taatgtggta
aaattctcca tcaacatcac 4000 agtcagctgg cagctgaact tcagaatctc
acttacagca ggcgacacgg 4050 gggtacaccg atgggtcaca ctgggtctgg
gggctccctg gagctcctcc 4100 tgcgtgtggt ctggttagga gttgagttgt
ttgctccagg gttattctcc 4150 tcctcgagtc acagtcacac gaatacctgc
cttctctggc tttcctgcta 4200 tacacatatt cacatggcgc tcaagaagtt
aggctcatgg caacgtgtgt 4250 ctttctctgg acaactggcc cagtttacag
tgaaatggag aatttcaggt 4300 ctccacgtct gcccaggaaa gaacttcagc
tgactccacg gggatctgga 4350 aatccacgac caatcccgat cggctcttat
tagctccccg ctccacaaga 4400 cacctgtgct ttggaaatcc accaccaatc
ccgatcggct cttattagct 4450 ccccgctcca caagacacct gtgatctgga
aatctaccac caatcccgat 4500 cggctcttat tagctccccg ctccacaaga
cacctgtgac atcctccagg 4550 gccacaggag cacgtgctga ccagttttcc
cttccagttc ctgcacaaaa 4600 agtgtccaga gggctgtttg caaacactag
tgcactttgt agcttttcac 4650 cctctgtccc agggaatcta ggagagatga
ggcccgtcag agtcaagaga 4700 tgtcatcccc ccagggtctc caaggcattt
ccacactatt ggtggcacct 4750 ggaggacatg caccaaggct tgccagagcc
aacaggaagt gagcccagag 4800 catggcacat gagcatcacc cgctgatggt
ggcctgctgt gcctggtgcc 4850 aacaggggca tcccggccca tacccctcca
gacaggaagc atgggtttgc 4900 ccacagacct gtcgggtgct cctgtgagtg
gcctccagat gtctttgtgc 4950 ataggcacaa gtgggccagg gctggaggga
ggtgggaaac ctcatcatcc 5000 ggtgggccct gccaatctta acccagaacc
cttaggtatt cctggcagta 5050 gccatgacat tggagcacct tcctctccag
ccagaggctg acctgagggc 5100 cactgtcctc agatgacacc acccaggagc
accctaggtg aggggtgagg 5150 gcccccttat gtgaacctct tgcctcttcc
tttctcccat cagagtggtt 5200 ggatggagcc attggcctcc ttttcttcag
cgggcccttc aacctctctg 5250 caccatgttg tctggctgag gagctactag
aaaagctgag tggagtctcc 5300 tttccaacag gatgatgcat ttgctcaatt
ctcagggctg gaatgagccg 5350 gctggtcccc cagaaagctg gagtggggta
cagagttcag ttttcctctc 5400 tgtttacagc tccttgacag tcccacgccc
atctggagtg ggagctggga 5450 gtcagtgttg gagaagaaac aacaaaagcc
aattagaacc actattttta 5500 aaaagtgctt actgtgcaca gatactcttc
aagcactgga cgtggattct 5550 ctctctagcc ctcagcaccc ctgcggtagg
agtgccgcct ctacccactt 5600 gtgatggggt acagaggcac ttgctcttct
gcatggtgtt caataggctg 5650 ggagttttat ttatctcttc aaactttgta
caagagctca tggcttgtct 5700 tgggctttcg tcattaaacc aaaggaaatg
gaagccattc ccctgttgct 5750 ctccttagtc ttggtcatca gaacctcact
tggtaccata tagatcaaaa 5800 gctttgtaac cacaggaaaa aataaactct
tccatccctt aaagaataga 5850 atagtttgtc cctctcatgg gaattgggct
gtatgtatat tgttcttcct 5900 ccttagaatt tagagataca agagttctac
ttagaacttt tcatggacac 5950 aatttccaca acctttcaga tgctgatgta
gagctattgg gaaagaactt 6000 ccaaactcag gaagtttgca gagagcagac
agctagagat aactcgggac 6050 ccagagttgg tcgacagatg ttagatgtat
cctagctttt agctataaac 6100 cactcaaaga ttcagccccc agatcccaca
gtcagaactg aatctgcgtt 6150 gttgggaagc cagcagtggc cttgggaagg
aagccatggc tgtggttcag 6200 agagggtggg ctggcaagcc acttccgggg
aaaactcctt ccgccccagg 6250 tttcttcttc tcttaaggag agattattct
caccaacccg ctgccttcat 6300 gctgccttca aagctagatc atgtttgcct
tgcttagaga attactgcaa 6350 atcagcccca gtgcttggcg atgcatttac
agatttctag gccctcaggg 6400 ttttgtagag tgtgagccct ggtgggcagg
gttggggggt ctgtcttctg 6450 ctggatgctg cttgtaatcc atttgg 6476 51
11389 DNA Homo sapien 51 atggcgccgc cgccgccgcc cgtgctgccc
gtgctgctgc tcctggccgc 50 cgccgccgcc ctgccggcga tggggctgcg
agcggccgcc tgggagccgc 100 gcgtacccgg cgggacccgc gccttcgccc
tccggcccgg ctgtacctac 150 gcggtgggcg ccgcttgcac gccccgggcg
ccgcgggagc tgctggacgt 200 gggccgcgat gggcggctgg caggacgtcg
gcgcgtctcg ggcgcggggc 250 gcccgctgcc gctgcaagtc cgcttggtgg
cccgcagtgc cccgacggcg 300 ctgagccgcc gcctgcgggc gcgcacgcac
cttcccggct
gcggagcccg 350 tgcccggctc tgcggaaccg gtgcccggct ctgcggggcg
ctctgcttcc 400 ccgtccccgg cggctgcgcg gccgcgcagc attcggcgct
cgcagctccg 450 accaccttac ccgcctgccg ctgcccgccg cgccccaggc
cccgctgtcc 500 cggccgtccc atctgcctgc cgccgggcgg ctcggtccgc
ctgcgtctgc 550 tgtgcgccct gcggcgcgcg gctggcgccg tccgggtggg
actggcgctg 600 gaggccgcca ccgcggggac gccctccgcg tcgccatccc
catcgccgcc 650 cctgccgccg aacttgcccg aagcccgggc ggggccggcg
cgacgggccc 700 ggcggggcac gagcggcaga gggagcctga agtttccgat
gcccaactac 750 caggtggcgt tgtttgagaa cgaaccggcg ggcaccctca
tcctccagct 800 gcacgcgcac tacaccatcg agggcgagga ggagcgcgtg
agctattaca 850 tggaggggct gttcgacgag cgctcccggg gctacttccg
aatcgactct 900 gccacgggcg ccgtgagcac ggacagcgta ctggaccgcg
agaccaagga 950 gacgcacgtc ctcagggtga aagccgtgga ctacagtacg
ccgccgcgct 1000 cggccaccac ctacatcact gtcttggtca aagacaccaa
cgaccacagc 1050 ccggtcttcg agcagtcgga gtaccgcgag cgcgtgcggg
agaacctgga 1100 ggtgggctac gaggtgctga ccatccgcgc cagcgaccgc
gactcgccca 1150 tcaacgccaa cttgcgttac cgcgtgttgg ggggcgcgtg
ggacgtcttc 1200 cagctcaacg agagctctgg cgtggtgagc acacgggcgg
tgctggaccg 1250 ggaggaggcg gccgagtacc agctcctggt ggaggccaac
gaccaggggc 1300 gcaatccggg cccgctcagt gccacggcca ccgtgtacat
cgaggtggag 1350 gacgagaacg acaactaccc ccagttcagc gagcagaact
acgtggtcca 1400 ggtgcccgag gacgtggggc tcaacacggc tgtgctgcga
gtgcaggcca 1450 cggaccggga ccagggccag aacgcggcca ttcactacag
catcctcagc 1500 gggaacgtgg ccggccagtt ctacctgcac tcgctgagcg
ggatcctgga 1550 tgtgatcaac cccttggatt tcgaggatgt ccagaaatac
tcgctgagca 1600 ttaaggccca ggatgggggc cggcccccgc tcatcaattc
ttcaggggtg 1650 gtgtctgtgc aggtgctgga tgtcaacgac aacgagccta
tctttgtgag 1700 cagccccttc caggccacgg tgctggagaa tgtgcccctg
ggctaccccg 1750 tggtgcacat tcaggcggtg gacgcggact ctggagagaa
cgcccggctg 1800 cactatcgcc tggtggacac ggcctccacc tttctggggg
gcggcagcgc 1850 tgggcctaag aatcctgccc ccacccctga cttccccttc
cagatccaca 1900 acagctccgg ttggatcaca gtgtgtgccg agctggaccg
cgaggaggtg 1950 gagcactaca gcttcggggt ggaggcggtg gaccacggct
cgccccccat 2000 gagctcctcc accagcgtgt ccatcacggt gctggacgtg
aatgacaacg 2050 acccggtgtt cacgcagccc acctacgagc ttcgtctgaa
tgaggatgcg 2100 gccgtgggga gcagcgtgct gaccctgcag gcccgcgacc
gtgacgccaa 2150 cagtgtgatt acctaccagc tcacaggcgg caacacccgg
aaccgctttg 2200 cactcagcag ccagagaggg ggcggcctca tcaccctggc
gctacctctg 2250 gactacaagc aggagcagca gtacgtgctg gcggtgacag
catccgacgg 2300 cacacggtcg cacactgcgc atgtcctaat caacgtcact
gatgccaaca 2350 cccacaggcc tgtctttcag agctcccatt acacagtgag
tgtcagtgag 2400 gacaggcctg tgggcacctc cattgctacc ctcagtgcca
acgatgagga 2450 cacaggagag aatgcccgca tcacctacgt gattcaggac
cccgtgccgc 2500 agttccgcat tgaccccgac agtggcacca tgtacaccat
gatggagctg 2550 gactatgaga accaggtcgc ctacacgctg accatcatgg
cccaggacaa 2600 cggcatcccg cagaaatcag acaccaccac cctagagatc
ctcatcctcg 2650 atgccaatga caatgcaccc cagttcctgt gggatttcta
ccagggttcc 2700 atctttgagg atgctccacc ctcgaccagc atcctccagg
tctctgccac 2750 ggaccgggac tcaggtccca atgggcgtct gctgtacacc
ttccagggtg 2800 gggacgacgg cgatggggac ttctacatcg agcccacgtc
cggtgtgatt 2850 cgcacccagc gccggctgga ccgggagaat gtggccgtgt
acaacctttg 2900 ggctctggct gtggatcggg gcagtcccac tccccttagc
gcctcggtag 2950 aaatccaggt gaccatcttg gacattaatg acaatgcccc
catgtttgag 3000 aaggacgaac tggagctgtt tgttgaggag aacaacccag
tggggtcggt 3050 ggtggcaaag attcgtgcta acgaccctga tgaaggccct
aatgcccaga 3100 tcatgtatca gattgtggaa ggggacatgc ggcatttctt
ccagctggac 3150 ctgctcaacg gggacctgcg tgccatggtg gagctggact
ttgaggtccg 3200 gcgggagtat gtgctggtgg tgcaggccac gtcggctccg
ctggtgagcc 3250 gagccacggt gcacatcctt ctcgtggacc agaatgacaa
cccgcctgtg 3300 ctgcccgact tccagatcct cttcaacaac tatgtcacca
acaagtccaa 3350 cagtttcccc accggcgtga tcggctgcat cccggcccat
gaccccgacg 3400 tgtcagacag cctcaactac accttcgtgc agggcaacga
gctgcgcctg 3450 ttgctgctgg accccgccac gggcgaactg cagctcagcc
gcgacctgga 3500 caacaaccgg ccgctggagg cgctcatgga ggtgtctgtg
tctgatggca 3550 tccacagcgt cacggccttc tgcaccctgc gtgtcaccat
catcacggac 3600 gacatgctga ccaacagcat cactgtccgc ctggagaaca
tgtcccagga 3650 gaagttcctg tccccgctgc tggccctctt cgtggagggg
gtggccgccg 3700 tgctgtccac caccaaggac gacgtcttcg tcttcaacgt
ccagaacgac 3750 accgacgtca gctccaacat cctgaacgtg accttctcgg
cgctgctgcc 3800 tggcggcgtc cgcggccagt tcttcccgtc ggaggacctg
caggagcaga 3850 tctacctgaa tcggacgctg ctgaccacca tctccacgca
gcgcgtgctg 3900 cccttcgacg acaacatctg cctgcgcgag ccctgcgaga
actacatgaa 3950 gtgcgtgtcc gttctgcgat tcgacagctc cgcgcccttc
ctcagctcca 4000 ccaccgtgct cttccggccc atccacccca tcaacggcct
gcgctgccgc 4050 tgcccgcccg gcttcaccgg cgactactgc gagacggaga
tcgacctctg 4100 ctactccgac ccgtgcggcg ccaacggccg ctgccgcagc
cgcgagggcg 4150 gctacacctg cgagtgcttc gaggacttca ctggagagca
ctgtgaggtg 4200 gatgcccgct caggccgctg tgccaacggg gtgtgcaaga
acgggggcac 4250 ctgcgtgaac ctgctcatcg gcggcttcca ctgcgtgtgt
cctcctggcg 4300 agtatgagag gccctactgt gaggtgacca ccaggagctt
cccgccccag 4350 tccttcgtca ccttccgggg cctgagacag cgcttccact
tcaccatctc 4400 cctcacgttt gccactcagg aaaggaacgg cttgcttctc
tacaacggcc 4450 gcttcaatga gaagcacgac ttcatcgccc tggagatcgt
ggacgagcag 4500 gtgcagctca ccttctctgc aggcgagaca acaacgaccg
tggcaccgaa 4550 ggttcccagt ggtgtgagtg acgggcggtg gcactctgtg
caggtgcagt 4600 actacaacaa gcccaatatt ggccacctgg gcctgcccca
tgggccgtcc 4650 ggggaaaaga tggccgtggt gacagtggat gattgtgaca
caaccatggc 4700 tgtgcgcttt ggaaaggaca tcgggaacta cagctgcgct
gcccagggca 4750 ctcagaccgg ctccaagaag tccctggatc tgaccggccc
tctactcctg 4800 gggggtgtcc ccaacctgcc agaagacttc ccagtgcaca
accggcagtt 4850 cgtgggctgc atgcggaacc tgtcagtcga cggcaaaaat
gtggacatgg 4900 ccggattcat cgccaacaat ggcacccggg aaggctgcgc
tgctcggagg 4950 aacttctgcg atgggaggcg gtgtcagaat ggaggcacct
gtgtcaacag 5000 gtggaatatg tatctgtgtg agtgtccact ccgattcggc
gggaagaact 5050 gtgagcaagc catgcctcac ccccagctct tcagcggtga
gagcgtcgtg 5100 tcctggagtg acctgaacat catcatctct gtgccctggt
acctggggct 5150 catgttccgg acccggaagg aggacagcgt tctgatggag
gccaccagtg 5200 gtgggcccac cagctttcgc ctccagatcc tgaacaacta
cctccagttt 5250 gaggtgtccc acggcccctc cgatgtggag tccgtgatgc
tgtccgggtt 5300 gcgggtgacc gacggggagt ggcaccacct gctgatcgag
ctgaagaatg 5350 ttaaggagga cagtgagatg aagcacctgg tcaccatgac
cttggactat 5400 gggatggacc agaacaaggc agatatcggg ggcatgcttc
ccgggctgac 5450 ggtaaggagc gtggtggtcg gaggcgcctc tgaagacaag
gtctccgtgc 5500 gccgtggatt ccgaggctgc atgcagggag tgaggatggg
ggggacgccc 5550 accaacgtcg ccaccctgaa catgaacaac gcactcaagg
tcagggtgaa 5600 ggacggctgt gatgtggacg acccctgtac ctcgagcccc
tgtcccccca 5650 atagccgctg ccacgacgcc tgggaggact acagctgcgt
ctgtgacaaa 5700 gggtaccttg gaataaactg tgtggatgcc tgtcacctga
acccctgcga 5750 gaacatgggg gcctgcgtgc gctcccccgg ctccccgcag
ggctacgtgt 5800 gcgagtgtgg gcccagtcac tacgggccgt actgtgagaa
caaactcgac 5850 cttccgtgcc ccagaggctg gtgggggaac cccgtctgtg
gaccctgcca 5900 ctgtgccgtc agcaaaggct ttgatcccga ctgtaataag
accaacggcc 5950 agtgccaatg caaggagaat tactacaagc tcctagccca
ggacacctgt 6000 ctgccctgcg actgcttccc ccatggctcc cacagccgca
cttgcgacat 6050 ggccaccggg cagtgtgcct gcaagcccgg cgtcatcggc
cgccagtgca 6100 accgctgcga caacccgttt gccgaggtca ccacgctcgg
ctgtgaagtg 6150 atctacaatg gctgtcccaa agcatttgag gccggcatct
ggtggccaca 6200 gaccaagttc gggcagccgg ctgcggtgcc atgccctaag
ggatccgttg 6250 gaaatgcggt ccgacactgc agcggggaga agggctggct
gcccccagag 6300 ctctttaact gtaccaccat ctccttcgtg gacctcaggg
ccatgaatga 6350 gaagctgagc cgcaatgaga cgcaggtgga cggcgccagg
gccctgcagc 6400 tggtgagggc gctgcgcagt gctacacagc acacgggcac
gctctttggc 6450 aatgacgtgc gcacggccta ccagctgctg ggccacgtcc
ttcagcacga 6500 gagctggcag cagggcttcg acctggcagc cacgcaggac
gccgactttc 6550 acgaggacgt catccactcg ggcagcgccc tcctggcccc
agccaccagg 6600 gcggcgtggg agcagatcca gcggagcgag ggcggcacgg
cacagctgct 6650 ccggcgcctc gagggctact tcagcaacgt ggcacgcaac
gtgcggcgga 6700 cgtacctgcg gcccttcgtc atcgtcaccg ccaacatgat
tcttgctgtc 6750 gacatctttg acaagttcaa ctttacggga gccagggtcc
cgcgattcga 6800 caccatccat gaagagttcc ccagggagct ggagtcctcc
gtctccttcc 6850 cagccgactt cttcagacca cctgaagaaa aagaaggccc
cctgctgagg 6900 ccggctggcc ggaggaccac cccgcagacc acgcgcccgg
ggcctggcac 6950 cgagagggag gccccgatca gcaggcggag gcgacaccct
gatgacgctg 7000 gccagttcgc cgtcgctctg gtcatcattt accgcaccct
ggggcagctc 7050 ctgcccgagc gctacgaccc cgaccgtcgc agcctccggt
tgcctcaccg 7100 gcccatcatt aataccccga tggtgagcac gctggtgtac
agcgaggggg 7150 ctccgctccc gagacccctg gagaggcccg tcctggtgga
gttcgccctg 7200 ctggaggtgg aggagcgaac caagcctgtc tgcgtgttct
ggaaccactc 7250 cctggccgtt ggtgggacgg gagggtggtc tgcccggggc
tgcgagctcc 7300 tgtccaggaa ccggacacat gtcgcctgcc agtgcagcca
cacagccagc 7350 tttgcggtgc tcatggatat ctccaggcgt gagaacgggg
aggtcctgcc 7400 tctgaagatt gtcacctatg ccgctgtgtc cttgtcactg
gcagccctgc 7450 tggtggcctt cgtcctcctg agcctggtcc gcatgctgcg
ctccaacctg 7500 cacagcattc acaagcacct cgccgtggcg ctcttcctct
ctcagctggt 7550 gttcgtgatt gggatcaacc agacggaaaa cccgtttctg
tgcacagtgg 7600 ttgccatcct cctccactac atctacatga gcacctttgc
ctggaccctc 7650 gtggagagcc tgcatgtcta ccgcatgctg accgaggtgc
gcaacatcga 7700 cacggggccc atgcggttct actacgtcgt gggctggggc
atcccggcca 7750 ttgtcacagg actggcggtc ggcctggacc cccagggcta
cgggaacccc 7800 gacttctgct ggctgtcgct tcaagacacc ctgatttgga
gctttgcggg 7850 gcccatcgga gctgttataa tcatcaacac agtcacttct
gtcctatctg 7900 caaaggtttc ctgccaaaga aagcaccatt attatgggaa
aaaagggatc 7950 gtctccctgc tgaggaccgc attcctcctg ctgctgctca
tcagcgccac 8000 ctggctgctg gggctgctgg ctgtgaaccg cgatgcactg
agctttcact 8050 acctcttcgc catcttcagc ggcttacagg gccccttcgt
cctccttttc 8100 cactgcgtgc tcaaccagga ggtccggaag cacctgaagg
gcgtgctcgg 8150 cgggaggaag ctgcacctgg aggactccgc caccaccagg
gccaccctgc 8200 tgacgcgctc cctcaactgc aacaccacct tcggtgacgg
gcctgacatg 8250 ctgcgcacag acttgggcga gtccaccgcc tcgctggaca
gcatcgtcag 8300 ggatgaaggg atccagaagc tcggcgtgtc ctctgggctg
gtgaggggca 8350 gccacggaga gccagacgcg tccctcatgc ccaggagctg
caaggatccc 8400 cctggccacg attccgactc agatagcgag ctgtccctgg
atgagcagag 8450 cagctcttac gcctcctcac actcgtcaga cagcgaggac
gatggggtgg 8500 gagctgagga aaaatgggac ccggccaggg gcgccgtcca
cagcaccccc 8550 aaaggggacg ctgtggccaa ccacgttccg gccggctggc
ccgaccagag 8600 cctggctgag agtgacagtg aggaccccag cggcaagccc
cgcctgaagg 8650 tggagaccaa ggtcagcgtg gagctgcacc gcgaggagca
gggcagtcac 8700 cgtggagagt accccccgga ccaggagagc gggggcgcag
ccaggcttgc 8750 tagcagccag cccccagagc agaggaaagg catcttgaaa
aataaagtca 8800 cctacccgcc gccgctgacg ctgacggagc agacgctgaa
gggccggctc 8850 cgggagaagc tggccgactg tgagcagagc cccacatcct
cgcgcacgtc 8900 ttccctgggc tctggcggcc ccgactgcgc catcacagtc
aagagccctg 8950 ggagggagcc ggggcgtgac cacctcaacg gggtggccat
gaatgtgcgc 9000 actgggagcg cccaggccga tggctccgac tctgagaaac
cgtgaggcaa 9050 gcccgtcacc ccacacaggc tgcggcatca ccctcagacc
ttggagccca 9100 aggggccact gcccttgaag tggagtgggc ccagagtgtg
gcggtcccca 9150 tggtggcagc cccccgactg atcatccaga cacaaaggtc
ttggttctcc 9200 caggagctca gggcctgtca gacctggtga caagtgccaa
aggccacagg 9250 catgagggag gcgtggacca ctgggccagc accgctgagt
cctaagactg 9300 cagtcaaagc cagaactgag aggggacccc agactgggcc
cagaggctgg 9350 ccagagttca ggaacgccgg gcacagacca aagaccgcgg
tccagccccg 9400 cccaggcggg catctcatgg cagtgcggac ccgtggctgg
cagcccgggc 9450 agtcctttgc aaaggcaccc cttgtcttaa aatcacttcg
ctatgtggga 9500 aaggtggaga tacttttata tatttgtatg ggactctgag
gaggtgcaac 9550 ctgtatatat attgcattcg tgctgacttt gttatcccga
gagatccatg 9600 caatgatctc ttgctgtctt ctctgtcaag attgcacagt
tgtacttgaa 9650 tctggcatgt gttgacgaaa ctggtgcccc agcagatcaa
aggtgggaaa 9700 tacgtcagca gtggggctaa aaccaagcgg ctagaagccc
tacagctgcc 9750 ttcggccagg aagtgaggat ggtgtgggcc ctccccgccg
gccccctggg 9800 tccccagtgt tcgctgtgtg tgcgtttgtc ctctgctgcc
atctgccccg 9850 gctgtgtgaa ttcaagacag ggcagtgcag cactaggcag
gtgtgaggag 9900 ccctgctgag gtcactgtgg ggcacggttg ccacacggct
gtcatttttc 9950 acctggtcat tctgtgacca ccaccccctc ccctcaccgc
ctcccaggtg 10000 gcccgggagc tgcaggtggg gatggctttg tcctttgctc
ctgctccccg 10050 tgggacctgg gaccttaaag cgttgcaggt tcctgatttg
gacagaggtg 10100 tggggccttc caggccgtta catacctcct gccaattctc
taactctctg 10150 agactgcgag gatctccagg cagggttctc ccctctggag
tctgaccaat 10200 tacttcattt tgcttcaaat ggccaattgt gcagagggac
aaagccacag 10250 ccacactctt caacggttac caaactgttt ttggaaattc
acaccaaggt 10300 cgggcccact gcaggcagct ggcacagcgt ggcccgaggg
gctgtggaac 10350 gggtcccgga actgtcagac atgtttgatt ttagcgtttc
ctttgttctt 10400 caaatcaggt gcccaaataa gtgatcagca cagctgcttc
caaataggag 10450 aaaccataaa ataggatgaa aatcaagtaa aatgcaaaga
tgtccacact 10500 gttttaaact tgaccctgat gaaaatgtga gcactgttag
cagatgccta 10550 tgggagagga aaagcgtatc tgaaaatggt ccaggacagg
aggatgaaat 10600 gagatcccag agtcctcaca cctgaatgaa ttatacatgt
gccttaccag 10650 gtgagtggtc tttcgaagat aaaaaactct agtcccttta
aacgtttgcc 10700 cctggcgttt cctaagtacg aaaaggtttt taagtcttcg
aacagtctcc 10750 tttcatgact ttaacaggat tctgccccct gaggtgtaat
ttttttgttc 10800 tatttttttc cacgtactcc acagccaaca tcacgaggtg
taatttttaa 10850 tttgatcaga actgttacca aaaaacaact gtcagtttta
ttgagatggg 10900 aaaaatgtaa acctattttt attacttaag actttatggg
agagattaga 10950 cactggaggt ttttaacaga acgtgtattt attaatgttc
aaaacactgg 11000 aattacaaat gagaagagtc tacaataaat taagattttt
gaatttgtac 11050 ttctgcggtg ctggtttttc tccacaaaca cccccgcccc
tccccatgcc 11100 cagggtggcc gtggaaggga cggtttacgg acgtgcagct
gagctgtccg 11150 tgtcccatgc tccctcagcc agtggaacgt gccggaactt
tttgtccatt 11200 ccctagtagg cctgccacag cctagatggg cagtttttgt
ctttcaccaa 11250 atttgaggac tttttttttt tgccattatt tcttcagttt
tcttttcttg 11300 cactgatctt tctcctctcc ttctgtgact ccagtgactc
agacgttaga 11350 cctcttgatg ttttcccact ggtccctgag gctctgttc 11389
52 1107 DNA Homo Sapien unsure 170-208 unknown base 52 cggcctaagg
tagcgacggg actggccggg ggcggcagga cccgaaggcg 50 ctaggcggat
tcaccggatg ggagttgaat cgcgtcccgg tctttctagc 100 tgtgcccgga
aatcgggcgt gcgggcagct acagcagaga atcggacaag 150 gagggaagaa
agagatggtn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 200 nnnnnnnnga
agtgagtgca agaggagccg gcttagcatc taaactgatt 250 ctaccatcag
aaaagaggcc aaacttctat catcatggtg gatgtgaagt 300 gtctgagtga
ctgtaaattg cagaaccaac ttgagaagct tggattttca 350 cctggcccaa
tactaccttc caccagaaag ttgtatgaaa aaaagttagt 400 acagttgttg
gtctcacctc cctgtgcacc acctgtgatg aatggaccca 450 gagagctgga
tggagcgcag gacagtgatg acagcgaaga gcttaatatc 500 attttgcaag
gaaatatcat actctcaaca gaaaaaagca agaaactcaa 550 aaaatggcct
gaggcttcca ccactaaacg caaagctgta gatacctatt 600 gcttggatta
taagccttcc aagggaagaa ggtgggctgc aagagcacca 650 agcaccagaa
tcacatatgg gactatcacc aaagagagag actactgcgc 700 ggaagaccag
actatcgaga gctggagaga agaaggtttc ccagtgggct 750 tgaagcttgc
tgtgcttggt attttcatca ttgtggtgtt tgtctacctg 800 actgtggaaa
ataagtcgct gtttggttaa gtaatttagg agcaaagcaa 850 tgctccaagc
gaggcctcct gcttcaggaa agaaccaaaa cactaccctg 900 aagggccagc
ctagcctgca gccctccctt gcagggagcc ttcccttgca 950 ctgtgctgct
ctcacagatc ggtgtctggg ctcagccagg tggaaggaac 1000 ctgcctaacc
aggcacctgt gttaagagca tgatggttag gaaatccccc 1050 aagtcatgtc
aactctcatt aaaggtgctt ccatatttga gcaggcgtca 1100 aacaagg 1107 53
3946 DNA Homo Sapien 53 accgctccgg agcgggaggg gaggcttcgc ggaacgctct
cggcgccagg 50 actcgcgtgc aaagcccagg cccgggcggc cagaccaaga
gggaagaagc 100 acagaattcc tcaactccca gtgtgcccat gagtaagagc
aaatgctccg 150 tgggactcat gtcttccgtg gtggccccgg ctaaggagcc
caatgccgtg 200 ggcccgaagg aggtggagct catccttgtc aaggagcaga
acggagtgca 250 gctcaccagc tccaccctca ccaacccgcg
gcagagcccc gtggaggccc 300 aggatcggga gacctggggc aagaagatcg
actttctcct gtccgtcatt 350 ggctttgctg tggacctggc caacgtctgg
cggttcccct acctgtgcta 400 caaaaatggt ggcggtgcct tcctggtccc
ctacctgctc ttcatggtca 450 ttgctgggat gccacttttc tacatggagc
tggccctcgg ccagttcaac 500 agggaagggg ccgctggtgt ctggaagatc
tgccccatac tgaaaggtgt 550 gggcttcacg gtcatcctca tctcactgta
tgtcggcttc ttctacaacg 600 tcatcatcgc ctgggcgctg cactatctct
tctcctcctt caccacggag 650 ctcccctgga tccactgcaa caactcctgg
aacagcccca actgctcgga 700 tgcccatcct ggtgactcca gtggagacag
ctcgggcctc aacgacactt 750 ttgggaccac acctgctgcc gagtactttg
aacgtggcgt gctgcacctc 800 caccagagcc atggcatcga cgacctgggg
cctccgcggt ggcagctcac 850 agcctgcctg gtgctggtca tcgtgctgct
ctacttcagc ctctggaagg 900 gcgtgaagac ctcagggaag gtggtatgga
tcacagccac catgccatac 950 gtggtcctca ctgccctgct cctgcgtggg
gtcaccctcc ctggagccat 1000 agacggcatc agagcatacc tgagcgttga
cttctaccgg ctctgcgagg 1050 cgtctgtttg gattgacgcg gccacccagg
tgtgcttctc cctgggcgtg 1100 gggttcgggg tgctgatcgc cttctccagc
tacaacaagt tcaccaacaa 1150 ctgctacagg gacgcgattg tcaccacctc
catcaactcc ctgacgagct 1200 tctcctccgg cttcgtcgtc ttctccttcc
tggggtacat ggcacagaag 1250 cacagtgtgc ccatcgggga cgtggccaag
gacgggccag ggctgatctt 1300 catcatctac ccggaagcca tcgccacgct
ccctctgtcc tcagcctggg 1350 ccgtggtctt cttcatcatg ctgctcaccc
tgggtatcga cagcgccatg 1400 ggtggtatgg agtcagtgat caccgggctc
atcgatgagt tccagctgct 1450 gcacagacac cgtgagctct tcacgctctt
catcgtcctg gcgaccttcc 1500 tcctgtccct gttctgcgtc accaacggtg
gcatctacgt cttcacgctc 1550 ctggaccatt ttgcagccgg cacgtccatc
ctctttggag tgctcatcga 1600 agccatcgga gtggcctggt tctatggtgt
tgggcagttc agcgacgaca 1650 tccagcagat gaccgggcag cggcccagcc
tgtactggcg gctgtgctgg 1700 aagctggtca gcccctgctt tctcctgttc
gtggtcgtgg tcagcattgt 1750 gaccttcaga cccccccact acggagccta
catcttcccc gactgggcca 1800 acgcgctggg ctgggtcatc gccacatcct
ccatggccat ggtgcccatc 1850 tatgcggcct acaagttctg cagcctgcct
gggtcctttc gagagaaact 1900 ggcctacgcc attgcacccg agaaggaccg
tgagctggtg gacagagggg 1950 aggtgcgcca gttcacgctc cgccactggc
tcaaggtgta gagggagcag 2000 agacgaagac cccaggaagt catcctgcaa
tgggagagac acgaacaaac 2050 caaggaaatc taagtttcga gagaaaggag
ggcaacttct actcttcaac 2100 ctctactgaa aacacaaaca acaaagcaga
agactcctct cttctgactg 2150 tttacacctt tccgtgccgg gagcgcacct
cgccgtgtct tgtgttgctg 2200 taataacgac gtagatctgt gcagcgaggt
ccaccccgtt gttgtccctg 2250 cagggcagaa aaacgtctaa cttcatgctg
tctgtgtgag gctccctccc 2300 tccctgctcc ctgctcccgg ctctgaggct
gccccagggg cactgtgttc 2350 tcaggcgggg atcacgatcc ttgtagacgc
acctgctgag aatccccgtg 2400 ctcacagtag cttcctagac catttacttt
gcccatatta aaaagccaag 2450 tgtcctgctt ggtttagctg tgcagaaggt
gaaatggagg aaaccacaaa 2500 ttcatgcaaa gtcctttccc gatgcgtggc
tcccagcaga ggccgtaaat 2550 tgagcgttca gttgacacat tgcacacaca
gtctgttcag aggcattgga 2600 ggatgggggt cctggtatgt ctcaccagga
aattctgttt atgttcttgc 2650 agcagagaga aataaaactc cttgaaacca
gctcaggcta ctgccactca 2700 ggcagcctgt gggtccttgt ggtgtaggga
acggcctgag aggagcgtgt 2750 cctatccccg gacgcatgca gggcccccac
aggagcgtgt cctatccccg 2800 gacgcatgca gggcccccac aggagcatgt
cctatccctg gacgcatgca 2850 gggcccccac aggagcgtgt actaccccag
aacgcatgca gggcccccac 2900 aggagcgtgt actaccccag gacgcatgca
gggcccccac tggagcgtgt 2950 actaccccag gacgcatgca gggcccccac
aggagcgtgt cctatccccg 3000 gaccggacgc atgcagggcc cccacaggag
cgtgtactac cccaggacgc 3050 atgcagggcc cccacaggag cgtgtactac
cccaggatgc atgcagggcc 3100 cccacaggag cgtgtactac cccaggacgc
atgcagggcc cccatgcagg 3150 cagcctgcag accaacactc tgcctggcct
tgagccgtga cctccaggaa 3200 gggaccccac tggaatttta tttctctcag
gtgcgtgcca catcaataac 3250 aacagttttt atgtttgcga atggcttttt
aaaatcatat ttacctgtga 3300 atcaaaacaa attcaagaat gcagtatccg
cgagcctgct tgctgatatt 3350 gcagtttttg tttacaagaa taattagcaa
tactgagtga aggatgttgg 3400 ccaaaagctg ctttccatgg cacactgccc
tctgccactg acaggaaagt 3450 ggatgccata gtttgaattc atgcctcaag
tcggtgggcc tgcctacgtg 3500 ctgcccgagg gcaggggccg tgcagggcca
gtcatggctg tcccctgcaa 3550 gtggacgtgg gctccaggga ctggagtgta
atgctcggtg ggagccgtca 3600 gcctgtgaac tgccaggcag ctgcagttag
cacagaggat ggcttcccca 3650 ttgccttctg gggagggaca cagaggacgg
cttccccatc gccttctggc 3700 cgctgcagtc agcacagaga gcggcttccc
cattgccttc tggggaggga 3750 cacagaggac agtttcccca tcgccttctg
gttgttgaag acagcacaga 3800 gagcggcttc cccatcgcct tctggggagg
ggctccgtgt agcaacccag 3850 gtgttgtccg tgtctgttga ccaatctcta
ttcagcatcg tgtgggtccc 3900 taagcacaat aaaagacatc cacaatggaa
aaaaaaaaag gaattc 3946 54 2317 DNA Homo Sapien 54 cggacgcgtg
ggtgagcagg gacggtgcac cggacggcgg gatcgagcaa 50 atgggtctgg
ccatggagca cggagggtcc tacgctcggg cggggggcag 100 ctctcggggc
tgctggtatt acctgcgcta cttcttcctc ttcgtctccc 150 tcatccaatt
cctcatcatc ctggggctcg tgctcttcat ggtctatggc 200 aacgtgcacg
tgagcacaga gtccaacctg caggccaccg agcgccgagc 250 cgagggccta
tacagtcagc tcctagggct cacggcctcc cagtccaact 300 tgaccaagga
gctcaacttc accacccgcg ccaaggatgc catcatgcag 350 atgtggctga
atgctcgccg cgacctggac cgcatcaatg ccagcttccg 400 ccagtgccag
ggtgaccggg tcatctacac gaacaatcag aggtacatgg 450 ctgccatcat
cttgagtgag aagcaatgca gagatcaatt caaggacatg 500 aacaagagct
gcgatgcctt gctcttcatg ctgaatcaga aggtgaagac 550 gctggaggtg
gagatagcca aggagaagac catttgcact aaggataagg 600 aaagcgtgct
gctgaacaaa cgcgtggcgg aggaacagct ggttgaatgc 650 gtgaaaaccc
gggagctgca gcaccaagag cgccagctgg ccaaggagca 700 actgcaaaag
gtgcaagccc tctgcctgcc cctggacaag gacaagtttg 750 agatggacct
tcgtaacctg tggagggact ccattatccc acgcagcctg 800 gacaacctgg
gttacaacct ctaccatccc ctgggctcgg aattggcctc 850 catccgcaga
gcctgcgacc acatgcccag cctcatgagc tccaaggtgg 900 aggagctggc
ccggagcctc cgggcggata tcgaacgcgt ggcccgcgag 950 aactcagacc
tccaacgcca gaagctggaa gcccagcagg gcctgcgggc 1000 cagtcaggag
gcgaaacaga aggtggagaa ggaggctcag gcccgggagg 1050 ccaagctcca
agctgaatgc tcccggcaga cccagctagc gctggaggag 1100 aaggcggtgc
tgcggaagga acgagacaac ctggccaagg agctggaaga 1150 gaagaagagg
gaggcggagc agctcaggat ggagctggcc atcagaaact 1200 cagccctgga
cacctgcatc aagaccaagt cgcagccgat gatgccagtg 1250 tcaaggccca
tgggccctgt ccccaacccc cagcccatcg acccagctag 1300 cctggaggag
ttcaagagga agatcctgga gtcccagagg ccccctgcag 1350 gcatccctgt
agccccatcc agtggctgag gaggctccag gcctgaggac 1400 caagggatgg
cccgactcgg cggtttgcgg aggatgcagg gatatgctca 1450 cagcgcccga
cacaaccccc tcccgccgcc cccaaccacc cagggccacc 1500 atcagacaac
tccctgcatg caaaccccta gtaccctctc acacccgcac 1550 ccgcgcctca
cgatccctca cccagagcac acggccgcgg agatgacgtc 1600 acgcaagcaa
cggcgctgac gtcacatatc accgtggtga tggcgtcacg 1650 tggccatgta
gacgtcacga agagatatag cgatggcgtc gtgcagatgc 1700 agcacgtcgc
acacagacat ggggaacttg gcatgacgtc acaccgagat 1750 gcagcaacga
cgtcacgggc catgtcgacg tcacacatat taatgtcaca 1800 cagacgcggc
gatggcatca cacagacggt gatgatgtca cacacagaca 1850 cagtgacaac
acacaccatg acaacgacac ctatagatat ggcaccaaca 1900 tcacatgcac
gcatgccctt tcacacacac tttctaccca attctcacct 1950 agtgtcacgt
tcccccgacc ctggcacacg ggccaaggta cccacaggat 2000 cccatcccct
cccgcacagc cctgggcccc agcacctccc ctcctccagc 2050 ttcctggcct
cccagccact tcctcacccc cagtgcctgg acccggaggt 2100 gagaacagga
agccattcac ctccgctcct tgagcgtgag tgtttccagg 2150 accccctcgg
ggccctgagc cgggggtgag ggtcacctgt tgtcgggagg 2200 ggagccactc
cttctccccc aactcccagc cctgcctgtg gcccgttgaa 2250 atgttggtgg
cacttaataa atattagtaa atccttaaaa aaaaaaaaaa 2300 aaaaaaaaaa aaaaaaa
2317 55 756 DNA Homo Sapien 55 cggacttggc ttgttagaag gctgaaagat
gatggcagga atgaaaatcc 50 agcttgtatg catgctactc ctggctttca
gctcctggag tctgtgctca 100 gattcagaag aggaaatgaa agcattagaa
gcagatttct tgaccaatat 150 gcatacatca aagattagta aagcacatgt
tccctcttgg aagatgactc 200 tgctaaatgt ttgcagtctt gtaaataatt
tgaacagccc agctgaggaa 250 acaggagaag ttcatgaaga ggagcttgtt
gcaagaagga aacttcctac 300 tgctttagat ggctttagct tggaagcaat
gttgacaata taccagctcc 350 acaaaatctg tcacagcagg gcttttcaac
actgggagtt aatccaggaa 400 gatattcttg atactggaaa tgacaaaaat
ggaaaggaag aagtcataaa 450 gagaaaaatt ccttatattc tgaaacggca
gctgtatgag aataaaccca 500 gaagacccta catactcaaa agagattctt
actattactg agagaataaa 550 tcatttattt acatgtgatt gtgattcatc
atcccttaat taaatatcaa 600 attatatttg tgtgaaaatg tgacaaacac
acttatctgt ctcttctaca 650 attgtggttt attgaatgtg tttttctgca
ctaatagaaa ttagactaag 700 tgttttcaaa taaatctaaa tcttcaaaaa
aaaaaaaaaa aaatggggcc 750 gcaatt 756 56 3722 DNA Homo Sapien 56
cgcggggcgc ggagtcggcg gggcctcgcg ggacgcgggc agtgcggaga 50
ccgcggcgct gaggacgcgg gagccgggag cgcacgcgcg gggtggagtt 100
cagcctactc tttcttagat gtgaaaggaa aggaagatca tttcatgcct 150
tgttgataaa ggttcagact tctgctgatt cataaccatt tggctctgag 200
ctatgacaag agaggaaaca aaaagttaaa cttacaagcc tgccataagt 250
gagaagcaaa cttccttgat aacatgcttt tgcgaagtgc aggaaaatta 300
aatgtgggca ccaagaaaga ggatggtgag agtacagccc ccaccccccg 350
tccaaaggtc ttgcgttgta aatgccacca ccattgtcca gaagactcag 400
tcaacaatat ttgcagcaca gacggatatt gtttcacgat gatagaagag 450
gatgactctg ggttgcctgt ggtcacttct ggttgcctag gactagaagg 500
ctcagatttt cagtgtcggg acactcccat tcctcatcaa agaagatcaa 550
ttgaatgctg cacagaaagg aacgaatgta ataaagacct acaccctaca 600
ctgcctccat tgaaaaacag agattttgtt gatggaccta tacaccacag 650
ggctttactt atatctgtga ctgtctgtag tttgctcttg gtccttatca 700
tattattttg ttacttccgg tataaaagac aagaaaccag acctcgatac 750
agcattgggt tagaacagga tgaaacttac attcctcctg gagaatccct 800
gagagactta attgagcagt ctcagagctc aggaagtgga tcaggcctcc 850
ctctgctggt ccaaaggact atagctaagc agattcagat ggtgaaacag 900
attggaaaag gtcgctatgg ggaagtttgg atgggaaagt ggcgtggcga 950
aaaggtagct gtgaaagtgt tcttcaccac agaggaagcc agctggttca 1000
gagagacaga aatatatcag acagtgttga tgaggcatga aaacattttg 1050
ggtttcattg ctgcagatat caaagggaca gggtcctgga cccagttgta 1100
cctaatcaca gactatcatg aaaatggttc cctttatgat tatctgaagt 1150
ccaccaccct agacgctaaa tcaatgctga agttagccta ctcttctgtc 1200
agtggcttat gtcatttaca cacagaaatc tttagtactc aaggcaaacc 1250
agcaattgcc catcgagatc tgaaaagtaa aaacattctg gtgaagaaaa 1300
atggaacttg ctgtattgct gacctgggcc tggctgttaa atttattagt 1350
gatacaaatg aagttgacat accacctaac actcgagttg gcaccaaacg 1400
ctatatgcct ccagaagtgt tggacgagag cttgaacaga aatcacttcc 1450
agtcttacat catggctgac atgtatagtt ttggcctcat cctttgggag 1500
gttgctagga gatgtgtatc aggaggtata gtggaagaat accagcttcc 1550
ttatcatgac ctagtgccca gtgacccctc ttatgaggac atgagggaga 1600
ttgtgtgcat caagaagtta cgcccctcat tcccaaaccg gtggagcagt 1650
gatgagtgtc taaggcagat gggaaaactc atgacagaat gctgggctca 1700
caatcctgca tcaaggctga cagccctgcg ggttaagaaa acacttgcca 1750
aaatgtcaga gtcccaggac attaaactct gataggagag gaaaagtaag 1800
catctctgca gaaagccaac aggtactctt ctgtttgtgg gcagagcaaa 1850
agacatcaaa taagcatcca cagtacaagc cttgaacatc gtcctgcttc 1900
ccagtgggtt cagacctcac ctttcaggga gcgacctggg caaagacaga 1950
gaagctccca gaaggagaga ttgatccatg tctgtttgta ggacggagaa 2000
accgcttggg taacttgttc aagatatgat gcatgttgct ttctaagaaa 2050
gccctgtatt ttgtgattgc cttttttttt ttttaagatg ctttcatttt 2100
gccaaaataa aacagataat gtggatggtt taagggttat agtattatag 2150
tttaaataat aacaacaaaa ttcttcccag gaactctgct ggaaggtaaa 2200
ttaaaatact tgtttttcca ttggtaaaat attgttgcac tctgtgaacc 2250
aaaagacagt ctaagttgga ggacatagaa cggaactcat cttaaacata 2300
ctccccaccc cgtcttggcc tcctcagacc actttggcca tccctgcatt 2350
tggggccgct atggtaatgt gaatgcactg ggtacaaaca ccgcctgtct 2400
aggaccacat ttggaattcc tgcaggtggc cttttgcagc ttcaggcaat 2450
atggaacaaa tgaaggttta tgtgactcta atagaagtaa ttgttgatag 2500
gtgtttttca gatccacttc tgtttctgat tgagttaggc atctctttca 2550
tggtaaaacc cttttcatta aacacaaaaa aagctttttt tttttttttt 2600
tttttttttt ttttttaatg tgcagaggat tgacctgtgc atgcttttga 2650
tctctcattc aaaggatcaa tattaaataa aattgtcatg agctgtgttg 2700
aagacagggt gctttcaaat agaggtaatt tgctcttgtg ttgtaagagg 2750
aacatgtcaa caaagatagg aaatgagggt gatcgtgcag atggcttgta 2800
tcttatatat gcaaaggagc caatctcaga agcacaaaga aaaaagtgtg 2850
cataccttat tttgtacaga taaagatgat gtctttttgt tattgtctgt 2900
ctgttttgta tgtgtctgag ataagggata gagaggaaac atccgtcagg 2950
ctaatttaac tacattttat tttaaaaata gagaaacata acctctagat 3000
gggacagcag aggacagtta gtagaggcca caaactgtta tgggctgctg 3050
tgttttgttc taaaatcaat atggttggag catgtatatc ttaggtgatc 3100
atttcacatc ttaggaatgc ctactcattt tattttattc tagtgatgct 3150
caattcacta tttaatttat tatattttct cttctgtggc acttatacaa 3200
aatatctctt cacctactta gttctacagg gttttaactt tggagcaaca 3250
tgaataaaat catcgagaag gccaatattg tttagcaaca tgaatacaat 3300
acagtttaaa gttgtacaca tcctgctcaa ctttattcat atacatttcc 3350
tttctgtggt tttcttttgc ttcttagaaa ttctgttagt ggttagtaaa 3400
gaatttgaaa gtactttctc cttgctgttt tttttttttt ttaagacatt 3450
cctcccagaa tactccaggg ggcagtgttt tataacacat tttccccact 3500
gggtgattga aggatggagg atttttgaaa atttgacagc tacatgaaac 3550
atgagaaaac attttcctca cttctgaagt cggtttgcag ctggtaactt 3600
gttcatccag aaaacattct aaagcaatga gactttgtga gctgtgctta 3650
cagtttggga gaatcatgaa gattctttct atattttgca tttacttccc 3700
agtgcttcat agctgcattt tg 3722 57 837 PRT Homo Sapien 57 Met Leu Arg
Thr Ala Met Gly Leu Arg Ser Trp Leu Ala Ala Pro 1 5 10 15 Trp Gly
Ala Leu Pro Pro Arg Pro Pro Leu Leu Leu Leu Leu Leu 20 25 30 Leu
Leu Leu Leu Leu Gln Pro Pro Pro Pro Thr Trp Ala Leu Ser 35 40 45
Pro Arg Ile Ser Leu Pro Leu Gly Ser Glu Glu Arg Pro Phe Leu 50 55
60 Arg Phe Glu Ala Glu His Ile Ser Asn Tyr Thr Ala Leu Leu Leu 65
70 75 Ser Arg Asp Gly Arg Thr Leu Tyr Val Gly Ala Arg Glu Ala Leu
80 85 90 Phe Ala Leu Ser Ser Asn Leu Ser Phe Leu Pro Gly Gly Glu
Tyr 95 100 105 Gln Glu Leu Leu Trp Gly Ala Asp Ala Glu Lys Lys Gln
Gln Cys 110 115 120 Ser Phe Lys Gly Lys Asp Pro Gln Arg Asp Cys Gln
Asn Tyr Ile 125 130 135 Lys Ile Leu Leu Pro Leu Ser Gly Ser His Leu
Phe Thr Cys Gly 140 145 150 Thr Ala Ala Phe Ser Pro Met Cys Thr Tyr
Ile Asn Met Glu Asn 155 160 165 Phe Thr Leu Ala Arg Asp Glu Lys Gly
Asn Val Leu Leu Glu Asp 170 175 180 Gly Lys Gly Arg Cys Pro Phe Asp
Pro Asn Phe Lys Ser Thr Ala 185 190 195 Leu Val Val Asp Gly Glu Leu
Tyr Thr Gly Thr Val Ser Ser Phe 200 205 210 Gln Gly Asn Asp Pro Ala
Ile Ser Arg Ser Gln Ser Leu Arg Pro 215 220 225 Thr Lys Thr Glu Ser
Ser Leu Asn Trp Leu Gln Asp Pro Ala Phe 230 235 240 Val Ala Ser Ala
Tyr Ile Pro Glu Ser Leu Gly Ser Leu Gln Gly 245 250 255 Asp Asp Asp
Lys Ile Tyr Phe Phe Phe Ser Glu Thr Gly Gln Glu 260 265 270 Phe Glu
Phe Phe Glu Asn Thr Ile Val Ser Arg Ile Ala Arg Ile 275 280 285 Cys
Lys Gly Asp Glu Gly Gly Glu Arg Val Leu Gln Gln Arg Trp 290 295 300
Thr Ser Phe Leu Lys Ala Gln Leu Leu Cys Ser Arg Pro Asp Asp 305 310
315 Gly Phe Pro Phe Asn Val Leu Gln Asp Val Phe Thr Leu Ser Pro 320
325 330 Ser Pro Gln Asp Trp Arg Asp Thr Leu Phe Tyr Gly Val Phe Thr
335 340 345 Ser Gln
Trp His Arg Gly Thr Thr Glu Gly Ser Ala Val Cys Val 350 355 360 Phe
Thr Met Lys Asp Val Gln Arg Val Phe Ser Gly Leu Tyr Lys 365 370 375
Glu Val Asn Arg Glu Thr Gln Gln Trp Tyr Thr Val Thr His Pro 380 385
390 Val Pro Thr Pro Arg Pro Gly Ala Cys Ile Thr Asn Ser Ala Arg 395
400 405 Glu Arg Lys Ile Asn Ser Ser Leu Gln Leu Pro Asp Arg Val Leu
410 415 420 Asn Phe Leu Lys Asp His Phe Leu Met Asp Gly Gln Val Arg
Ser 425 430 435 Arg Met Leu Leu Leu Gln Pro Gln Ala Arg Tyr Gln Arg
Val Ala 440 445 450 Val His Arg Val Pro Gly Leu His His Thr Tyr Asp
Val Leu Phe 455 460 465 Leu Gly Thr Gly Asp Gly Arg Leu His Lys Ala
Val Ser Val Gly 470 475 480 Pro Arg Val His Ile Ile Glu Glu Leu Gln
Ile Phe Ser Ser Gly 485 490 495 Gln Pro Val Gln Asn Leu Leu Leu Asp
Thr His Arg Gly Leu Leu 500 505 510 Tyr Ala Ala Ser His Ser Gly Val
Val Gln Val Pro Met Ala Asn 515 520 525 Cys Ser Leu Tyr Arg Ser Cys
Gly Asp Cys Leu Leu Ala Arg Asp 530 535 540 Pro Tyr Cys Ala Trp Ser
Gly Ser Ser Cys Lys His Val Ser Leu 545 550 555 Tyr Gln Pro Gln Leu
Ala Thr Arg Pro Trp Ile Gln Asp Ile Glu 560 565 570 Gly Ala Ser Ala
Lys Asp Leu Cys Ser Ala Ser Ser Val Val Ser 575 580 585 Pro Ser Phe
Val Pro Thr Gly Glu Lys Pro Cys Glu Gln Val Gln 590 595 600 Phe Gln
Pro Asn Thr Val Asn Thr Leu Ala Cys Pro Leu Leu Ser 605 610 615 Asn
Leu Ala Thr Arg Leu Trp Leu Arg Asn Gly Ala Pro Val Asn 620 625 630
Ala Ser Ala Ser Cys His Val Leu Pro Thr Gly Asp Leu Leu Leu 635 640
645 Val Gly Thr Gln Gln Leu Gly Glu Phe Gln Cys Trp Ser Leu Glu 650
655 660 Glu Gly Phe Gln Gln Leu Val Ala Ser Tyr Cys Pro Glu Val Val
665 670 675 Glu Asp Gly Val Ala Asp Gln Thr Asp Glu Gly Gly Ser Val
Pro 680 685 690 Val Ile Ile Ser Thr Ser Arg Val Ser Ala Pro Ala Gly
Gly Lys 695 700 705 Ala Ser Trp Gly Ala Asp Arg Ser Tyr Trp Lys Glu
Phe Leu Val 710 715 720 Met Cys Thr Leu Phe Val Leu Ala Val Leu Leu
Pro Val Leu Phe 725 730 735 Leu Leu Tyr Arg His Arg Asn Ser Met Lys
Val Phe Leu Lys Gln 740 745 750 Gly Glu Cys Ala Ser Val His Pro Lys
Thr Cys Pro Val Val Leu 755 760 765 Pro Pro Glu Thr Arg Pro Leu Asn
Gly Leu Gly Pro Pro Ser Thr 770 775 780 Pro Leu Asp His Arg Gly Tyr
Gln Ser Leu Ser Asp Ser Pro Pro 785 790 795 Gly Ala Arg Val Phe Thr
Glu Ser Glu Lys Arg Pro Leu Ser Ile 800 805 810 Gln Asp Ser Phe Val
Glu Val Ser Pro Val Cys Pro Arg Pro Arg 815 820 825 Val Arg Leu Gly
Ser Glu Ile Arg Asp Ser Val Val 830 835 58 188 PRT Homo Sapien 58
Met Asp Cys Arg Lys Met Ala Arg Phe Ser Tyr Ser Val Ile Trp 1 5 10
15 Ile Met Ala Ile Ser Lys Val Phe Glu Leu Gly Leu Val Ala Gly 20
25 30 Leu Gly His Gln Glu Phe Ala Arg Pro Ser Arg Gly Tyr Leu Ala
35 40 45 Phe Arg Asp Asp Ser Ile Trp Pro Gln Glu Glu Pro Ala Ile
Arg 50 55 60 Pro Arg Ser Ser Gln Arg Val Pro Pro Met Gly Ile Gln
His Ser 65 70 75 Lys Glu Leu Asn Arg Thr Cys Cys Leu Asn Gly Gly
Thr Cys Met 80 85 90 Leu Gly Ser Phe Cys Ala Cys Pro Pro Ser Phe
Tyr Gly Arg Asn 95 100 105 Cys Glu His Asp Val Arg Lys Glu Asn Cys
Gly Ser Val Pro His 110 115 120 Asp Thr Trp Leu Pro Lys Lys Cys Ser
Leu Cys Lys Cys Trp His 125 130 135 Gly Gln Leu Arg Cys Phe Pro Gln
Ala Phe Leu Pro Gly Cys Asp 140 145 150 Gly Leu Val Met Asp Glu His
Leu Val Ala Ser Arg Thr Pro Glu 155 160 165 Leu Pro Pro Ser Ala Arg
Thr Thr Thr Phe Met Leu Val Gly Ile 170 175 180 Cys Leu Ser Ile Gln
Ser Tyr Tyr 185 59 80 PRT Homo Sapien 59 Met Ala Ala Arg Ala Leu
Cys Met Leu Gly Leu Val Leu Ala Leu 1 5 10 15 Leu Ser Ser Ser Ser
Ala Glu Glu Tyr Val Gly Leu Ser Ala Asn 20 25 30 Gln Cys Ala Val
Pro Ala Lys Asp Arg Val Asp Cys Gly Tyr Pro 35 40 45 His Val Thr
Pro Lys Glu Cys Asn Asn Arg Gly Cys Cys Phe Asp 50 55 60 Ser Arg
Ile Pro Gly Val Pro Trp Cys Phe Lys Pro Leu Gln Glu 65 70 75 Ala
Glu Cys Thr Phe 80 60 314 PRT Homo Sapien 60 Met Arg Ile Ala Val
Ile Cys Phe Cys Leu Leu Gly Ile Thr Cys 1 5 10 15 Ala Ile Pro Val
Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys 20 25 30 Gln Leu Tyr
Asn Lys Tyr Pro Asp Ala Val Ala Thr Trp Leu Asn 35 40 45 Pro Asp
Pro Ser Gln Lys Gln Asn Leu Leu Ala Pro Gln Asn Ala 50 55 60 Val
Ser Ser Glu Glu Thr Asn Asp Phe Lys Gln Glu Thr Leu Pro 65 70 75
Ser Lys Ser Asn Glu Ser His Asp His Met Asp Asp Met Asp Asp 80 85
90 Glu Asp Asp Asp Asp His Val Asp Ser Gln Asp Ser Ile Asp Ser 95
100 105 Asn Asp Ser Asp Asp Val Asp Asp Thr Asp Asp Ser His Gln Ser
110 115 120 Asp Glu Ser His His Ser Asp Glu Ser Asp Glu Leu Val Thr
Asp 125 130 135 Phe Pro Thr Asp Leu Pro Ala Thr Glu Val Phe Thr Pro
Val Val 140 145 150 Pro Thr Val Asp Thr Tyr Asp Gly Arg Gly Asp Ser
Val Val Tyr 155 160 165 Gly Leu Arg Ser Lys Ser Lys Lys Phe Arg Arg
Pro Asp Ile Gln 170 175 180 Tyr Pro Asp Ala Thr Asp Glu Asp Ile Thr
Ser His Met Glu Ser 185 190 195 Glu Glu Leu Asn Gly Ala Tyr Lys Ala
Ile Pro Val Ala Gln Asp 200 205 210 Leu Asn Ala Pro Ser Asp Trp Asp
Ser Arg Gly Lys Asp Ser Tyr 215 220 225 Glu Thr Ser Gln Leu Asp Asp
Gln Ser Ala Glu Thr His Ser His 230 235 240 Lys Gln Ser Arg Leu Tyr
Lys Arg Lys Ala Asn Asp Glu Ser Asn 245 250 255 Glu His Ser Asp Val
Ile Asp Ser Gln Glu Leu Ser Lys Val Ser 260 265 270 Arg Glu Phe His
Ser His Glu Phe His Ser His Glu Asp Met Leu 275 280 285 Val Val Asp
Pro Lys Ser Lys Glu Glu Asp Lys His Leu Lys Phe 290 295 300 Arg Ile
Ser His Glu Leu Asp Ser Ala Ser Ser Glu Val Asn 305 310 61 184 PRT
Homo Sapien 61 Met Ser Arg Thr Ala Tyr Thr Val Gly Ala Leu Leu Leu
Leu Leu 1 5 10 15 Gly Thr Leu Leu Pro Ala Ala Glu Gly Lys Lys Lys
Gly Ser Gln 20 25 30 Gly Ala Ile Pro Pro Pro Asp Lys Ala Gln His
Asn Asp Ser Glu 35 40 45 Gln Thr Gln Ser Pro Gln Gln Pro Gly Ser
Arg Asn Arg Gly Arg 50 55 60 Gly Gln Gly Arg Gly Thr Ala Met Pro
Gly Glu Glu Val Leu Glu 65 70 75 Ser Ser Gln Glu Ala Leu His Val
Thr Glu Arg Lys Tyr Leu Lys 80 85 90 Arg Asp Trp Cys Lys Thr Gln
Pro Leu Lys Gln Thr Ile His Glu 95 100 105 Glu Gly Cys Asn Ser Arg
Thr Ile Ile Asn Arg Phe Cys Tyr Gly 110 115 120 Gln Cys Asn Ser Phe
Tyr Ile Pro Arg His Ile Arg Lys Glu Glu 125 130 135 Gly Ser Phe Gln
Ser Cys Ser Phe Cys Lys Pro Lys Lys Phe Thr 140 145 150 Thr Met Met
Val Thr Leu Asn Cys Pro Glu Leu Gln Pro Pro Thr 155 160 165 Lys Lys
Lys Arg Val Thr Arg Val Lys Gln Cys Arg Cys Ile Ser 170 175 180 Ile
Asp Leu Asp 62 460 PRT Homo Sapien 62 Met Phe Leu Ala Thr Leu Tyr
Phe Ala Leu Pro Leu Leu Asp Leu 1 5 10 15 Leu Leu Ser Ala Glu Val
Ser Gly Gly Asp Arg Leu Asp Cys Val 20 25 30 Lys Ala Ser Asp Gln
Cys Leu Lys Glu Gln Ser Cys Ser Thr Lys 35 40 45 Tyr Arg Thr Leu
Arg Gln Cys Val Ala Gly Lys Glu Thr Asn Phe 50 55 60 Ser Leu Ala
Ser Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala 65 70 75 Met Glu
Ala Leu Lys Gln Lys Ser Leu Tyr Asn Cys Arg Cys Lys 80 85 90 Arg
Gly Met Lys Lys Glu Lys Asn Cys Leu Arg Ile Tyr Trp Ser 95 100 105
Met Tyr Gln Ser Leu Gln Gly Asn Asp Leu Leu Glu Asp Ser Pro 110 115
120 Tyr Glu Pro Val Asn Ser Arg Leu Ser Asp Ile Phe Arg Val Val 125
130 135 Pro Phe Ile Ser Val Glu His Ile Pro Lys Gly Asn Asn Cys Leu
140 145 150 Asp Ala Ala Lys Ala Cys Asn Leu Asp Asp Ile Cys Lys Lys
Tyr 155 160 165 Arg Ser Ala Tyr Ile Thr Pro Cys Thr Thr Ser Val Ser
Asn Asp 170 175 180 Val Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg
Gln Phe Phe 185 190 195 Asp Lys Val Pro Ala Lys His Ser Tyr Gly Met
Leu Phe Cys Ser 200 205 210 Cys Arg Asp Ile Ala Cys Thr Glu Arg Arg
Arg Gln Thr Ile Val 215 220 225 Pro Val Cys Ser Tyr Glu Glu Arg Glu
Lys Pro Asn Cys Leu Asn 230 235 240 Leu Gln Asp Ser Cys Lys Thr Asn
Tyr Ile Cys Arg Ser Arg Leu 245 250 255 Ala Asp Phe Phe Thr Asn Cys
Gln Pro Glu Ser Arg Ser Val Ser 260 265 270 Ser Cys Leu Lys Glu Asn
Tyr Ala Asp Cys Leu Leu Ala Tyr Ser 275 280 285 Gly Leu Ile Gly Thr
Val Met Thr Pro Asn Tyr Ile Asp Ser Ser 290 295 300 Ser Leu Ser Val
Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn 305 310 315 Asp Leu Glu
Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn 320 325 330 Thr Cys
Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp 335 340 345 Val
Thr Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr Thr Ala 350 355 360
Thr Thr Thr Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro 365 370
375 Ala Gly Ser Glu Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys 380
385 390 Ala Asn Leu Gln Ala Gln Lys Leu Lys Ser Asn Val Ser Gly Asn
395 400 405 Thr His Leu Cys Ile Ser Asn Gly Asn Tyr Glu Lys Glu Gly
Leu 410 415 420 Gly Ala Ser Ser His Ile Thr Thr Lys Ser Met Ala Ala
Pro Pro 425 430 435 Ser Cys Gly Leu Ser Pro Leu Leu Val Leu Val Val
Thr Ala Leu 440 445 450 Ser Thr Leu Leu Ser Leu Thr Glu Thr Ser 455
460 63 143 PRT Homo Sapien 63 Met Gln His Arg Gly Phe Leu Leu Leu
Thr Leu Leu Ala Leu Leu 1 5 10 15 Ala Leu Thr Ser Ala Val Ala Lys
Lys Lys Asp Lys Val Lys Lys 20 25 30 Gly Gly Pro Gly Ser Glu Cys
Ala Glu Trp Ala Trp Gly Pro Cys 35 40 45 Thr Pro Ser Ser Lys Asp
Cys Gly Val Gly Phe Arg Glu Gly Thr 50 55 60 Cys Gly Ala Gln Thr
Gln Arg Ile Arg Cys Arg Val Pro Cys Asn 65 70 75 Trp Lys Lys Glu
Phe Gly Ala Asp Cys Lys Tyr Lys Phe Glu Asn 80 85 90 Trp Gly Ala
Cys Asp Gly Gly Thr Gly Thr Lys Val Arg Gln Gly 95 100 105 Thr Leu
Lys Lys Ala Arg Tyr Asn Ala Gln Cys Gln Glu Thr Ile 110 115 120 Arg
Val Thr Lys Pro Cys Thr Pro Lys Thr Lys Ala Lys Ala Lys 125 130 135
Ala Lys Lys Gly Lys Gly Lys Asp 140 64 141 PRT Homo sapien 64 Met
Trp Val Leu Gly Ile Ala Ala Thr Phe Cys Gly Leu Phe Leu 1 5 10 15
Leu Pro Gly Phe Ala Leu Gln Ile Gln Cys Tyr Gln Cys Glu Glu 20 25
30 Phe Gln Leu Asn Asn Asp Cys Ser Ser Pro Glu Phe Ile Val Asn 35
40 45 Cys Thr Val Asn Val Gln Asp Met Cys Gln Lys Glu Val Met Glu
50 55 60 Gln Ser Ala Gly Ile Met Tyr Arg Lys Ser Cys Ala Ser Ser
Ala 65 70 75 Ala Cys Leu Ile Ala Ser Ala Gly Tyr Gln Ser Phe Cys
Ser Pro 80 85 90 Gly Lys Leu Asn Ser Val Cys Ile Ser Cys Cys Asn
Thr Pro Leu 95 100 105 Cys Asn Gly Pro Arg Pro Lys Lys Arg Gly Ser
Ser Ala Ser Ala 110 115 120 Leu Arg Pro Gly Leu Arg Thr Thr Ile Leu
Phe Leu Lys Leu Ala 125 130 135 Leu Phe Ser Ala His Cys 140 65 242
PRT Homo Sapien 65 Met Lys Asn Ile Gly Leu Val Met Glu Trp Glu Ile
Pro Glu Ile 1 5 10 15 Ile Cys Thr Cys Ala Lys Leu Arg Leu Pro Pro
Gln Ala Thr Phe 20 25 30 Gln Val Leu Arg Gly Asn Gly Ala Ser Val
Gly Thr Val Leu Met 35 40 45 Phe Arg Cys Pro Ser Asn His Gln Met
Val Gly Ser Gly Leu Leu 50 55 60 Thr Cys Thr Trp Lys Gly Ser Ile
Ala Glu Trp Ser Ser Gly Ser 65 70 75 Pro Val Cys Lys Leu Val Pro
Pro His Glu Thr Phe Gly Phe Lys 80 85 90 Val Ala Val Ile Ala Ser
Ile Val Ser Cys Ala Ile Ile Leu Leu 95 100 105 Met Ser Met Ala Phe
Leu Thr Cys Cys Leu Leu Lys Cys Val Lys 110 115 120 Lys Ser Lys Arg
Arg Arg Ser Asn Arg Ser Ala Gln Leu Trp Ser 125 130 135 Gln Leu Lys
Asp Glu Asp Leu Glu Thr Val Gln Ala Ala Tyr Leu 140 145 150 Gly Leu
Lys His Phe Asn Lys Pro Val Ser Gly Pro Ser Gln Ala 155 160 165 His
Asp Asn His Ser Phe Thr Thr Asp His Gly Glu Ser Thr Ser 170 175 180
Lys Leu Ala Ser Val Thr Arg Ser Val Asp Lys Asp Pro Gly Ile 185 190
195 Pro Arg Ala Leu Ser Leu Ser Gly Ser Ser Ser Ser Pro Gln Ala 200
205 210 Gln Val Met Val His Met Ala Asn Pro Arg Gln Pro Leu Pro Ala
215 220 225 Ser Gly Leu Ala Thr Gly Met Pro Gln Gln Pro Ala Ala Tyr
Ala 230 235 240 Leu Gly 66 672 PRT Homo sapien 66 Asp Cys Thr Gly
Asp Gly Pro Trp Gln Ser Asn Leu Ala Pro Ser 1 5 10 15 Gln Leu Glu
Tyr Tyr Ala Ser Ser Pro Asp Glu Lys Ala Leu Val 20 25 30 Glu Ala
Ala Ala Arg Ile Gly Ile Val Phe Ile Gly Asn Ser Glu
35 40 45 Glu Thr Met Glu Val Lys Thr Leu Gly Lys Leu Glu Arg Tyr
Lys 50 55 60 Leu Leu His Ile Leu Glu Phe Asp Ser Asp Arg Arg Arg
Met Ser 65 70 75 Val Ile Val Gln Ala Pro Ser Gly Glu Lys Leu Leu
Phe Ala Lys 80 85 90 Gly Ala Glu Ser Ser Ile Leu Pro Lys Cys Ile
Gly Gly Glu Ile 95 100 105 Glu Lys Thr Arg Ile His Val Asp Glu Phe
Ala Leu Lys Gly Leu 110 115 120 Arg Thr Leu Cys Ile Ala Tyr Arg Lys
Phe Thr Ser Lys Glu Tyr 125 130 135 Glu Glu Ile Asp Lys Arg Ile Phe
Glu Ala Arg Thr Ala Leu Gln 140 145 150 Gln Arg Glu Glu Lys Leu Ala
Ala Val Phe Gln Phe Ile Glu Lys 155 160 165 Asp Leu Ile Leu Leu Gly
Ala Thr Ala Val Glu Asp Arg Leu Gln 170 175 180 Asp Lys Val Arg Glu
Thr Ile Glu Ala Leu Arg Met Ala Gly Ile 185 190 195 Lys Val Trp Val
Leu Thr Gly Asp Lys His Glu Thr Ala Val Ser 200 205 210 Val Ser Leu
Ser Cys Gly His Phe His Arg Thr Met Asn Ile Leu 215 220 225 Glu Leu
Ile Asn Gln Lys Ser Asp Ser Glu Cys Ala Glu Gln Leu 230 235 240 Arg
Gln Leu Ala Arg Arg Ile Thr Glu Asp His Val Ile Gln His 245 250 255
Gly Leu Val Val Asp Gly Thr Ser Leu Ser Leu Ala Leu Arg Glu 260 265
270 His Glu Lys Leu Phe Met Glu Val Cys Arg Asn Cys Ser Ala Val 275
280 285 Leu Cys Cys Arg Met Ala Pro Leu Gln Lys Ala Lys Val Ile Arg
290 295 300 Leu Ile Lys Ile Ser Pro Glu Lys Pro Ile Thr Leu Ala Val
Gly 305 310 315 Asp Gly Ala Asn Asp Val Ser Met Ile Gln Glu Ala His
Val Gly 320 325 330 Ile Gly Ile Met Gly Lys Glu Gly Arg Gln Ala Ala
Arg Asn Ser 335 340 345 Asp Tyr Ala Ile Ala Arg Phe Lys Phe Leu Ser
Lys Leu Leu Phe 350 355 360 Val His Gly His Phe Tyr Tyr Ile Arg Ile
Ala Thr Leu Val Gln 365 370 375 Tyr Phe Phe Tyr Lys Asn Val Cys Phe
Ile Thr Pro Gln Phe Leu 380 385 390 Tyr Gln Phe Tyr Cys Leu Phe Ser
Gln Gln Thr Leu Tyr Asp Ser 395 400 405 Val Tyr Leu Thr Leu Tyr Asn
Ile Cys Phe Thr Ser Leu Pro Ile 410 415 420 Leu Ile Tyr Ser Leu Leu
Glu Gln His Val Asp Pro His Val Leu 425 430 435 Gln Asn Lys Pro Thr
Leu Tyr Arg Asp Ile Ser Lys Asn Arg Leu 440 445 450 Leu Ser Ile Lys
Thr Phe Leu Tyr Trp Thr Ile Leu Gly Phe Ser 455 460 465 His Ala Phe
Ile Phe Phe Phe Gly Ser Tyr Leu Leu Ile Gly Lys 470 475 480 Asp Thr
Ser Leu Leu Gly Asn Gly Gln Met Phe Gly Asn Trp Thr 485 490 495 Phe
Gly Thr Leu Val Phe Thr Val Met Val Ile Thr Val Thr Val 500 505 510
Lys Met Ala Leu Glu Thr His Phe Trp Thr Trp Ile Asn His Leu 515 520
525 Val Thr Trp Gly Ser Ile Ile Phe Tyr Phe Val Phe Ser Leu Phe 530
535 540 Tyr Gly Gly Ile Leu Trp Pro Phe Leu Gly Ser Gln Asn Met Tyr
545 550 555 Phe Val Phe Ile Gln Leu Leu Ser Ser Gly Ser Ala Trp Phe
Ala 560 565 570 Ile Ile Leu Met Val Val Thr Cys Leu Phe Leu Asp Ile
Ile Lys 575 580 585 Lys Val Phe Asp Arg His Leu His Pro Thr Ser Thr
Glu Lys Ala 590 595 600 Gln Leu Thr Glu Thr Asn Ala Gly Ile Lys Cys
Leu Asp Ser Met 605 610 615 Cys Cys Phe Pro Glu Gly Glu Ala Ala Cys
Ala Ser Val Gly Arg 620 625 630 Met Leu Glu Arg Val Ile Gly Arg Cys
Ser Pro Thr His Ile Ser 635 640 645 Arg Ser Trp Ser Ala Ser Asp Pro
Phe Tyr Thr Asn Asp Arg Ser 650 655 660 Ile Leu Thr Leu Ser Thr Met
Asp Ser Ser Thr Cys 665 670 67 877 PRT Homo Sapien 67 Met Trp Glu
Glu Glu Asp Ile Ala Ile Leu Phe Asn Lys Glu Pro 1 5 10 15 Gly Lys
Thr Glu Asn Ile Glu Asn Asn Leu Ser Ser Asn His Arg 20 25 30 Arg
Ser Cys Arg Arg Ser Glu Glu Ser Asp Asp Asp Leu Asp Phe 35 40 45
Asp Ile Gly Leu Glu Asn Thr Gly Gly Asp Pro Gln Ile Leu Arg 50 55
60 Phe Ile Ser Asp Phe Leu Ala Phe Leu Val Leu Tyr Asn Phe Ile 65
70 75 Ile Pro Ile Ser Leu Tyr Val Thr Val Glu Met Gln Lys Phe Leu
80 85 90 Gly Ser Phe Phe Ile Gly Trp Asp Leu Asp Leu Tyr His Glu
Glu 95 100 105 Ser Asp Gln Lys Ala Gln Val Asn Thr Ser Asp Leu Asn
Glu Glu 110 115 120 Leu Gly Gln Val Glu Tyr Val Phe Thr Asp Lys Thr
Gly Thr Leu 125 130 135 Thr Glu Asn Glu Met Gln Phe Arg Glu Cys Ser
Ile Asn Gly Met 140 145 150 Lys Tyr Gln Glu Ile Asn Gly Arg Leu Val
Pro Glu Gly Pro Thr 155 160 165 Pro Asp Ser Ser Glu Gly Asn Leu Ser
Tyr Leu Ser Ser Leu Ser 170 175 180 His Leu Asn Asn Leu Ser His Leu
Thr Thr Ser Ser Ser Phe Arg 185 190 195 Thr Ser Pro Glu Asn Glu Thr
Glu Leu Ile Lys Glu His Asp Leu 200 205 210 Phe Phe Lys Ala Val Ser
Leu Cys His Thr Val Gln Ile Ser Asn 215 220 225 Val Gln Thr Asp Cys
Thr Gly Asp Gly Pro Trp Gln Ser Asn Leu 230 235 240 Ala Pro Ser Gln
Leu Glu Tyr Tyr Ala Ser Ser Pro Asp Glu Lys 245 250 255 Ala Leu Val
Glu Ala Ala Ala Arg Tyr Lys Leu Leu His Ile Leu 260 265 270 Glu Phe
Asp Ser Asp Arg Arg Arg Met Ser Val Ile Val Gln Ala 275 280 285 Pro
Ser Gly Glu Lys Leu Leu Phe Ala Lys Gly Ala Glu Ser Ser 290 295 300
Ile Leu Pro Lys Cys Ile Gly Gly Glu Ile Glu Lys Thr Arg Ile 305 310
315 His Val Asp Glu Phe Ala Leu Lys Gly Leu Arg Thr Leu Cys Ile 320
325 330 Ala Tyr Arg Lys Phe Thr Ser Lys Glu Tyr Glu Glu Ile Asp Lys
335 340 345 Arg Ile Phe Glu Ala Arg Thr Ala Leu Gln Gln Arg Glu Glu
Lys 350 355 360 Leu Ala Ala Val Phe Gln Phe Ile Glu Lys Asp Leu Ile
Leu Leu 365 370 375 Gly Ala Thr Ala Val Glu Asp Arg Leu Gln Asp Lys
Val Arg Glu 380 385 390 Thr Ile Glu Ala Leu Arg Met Ala Gly Ile Lys
Val Trp Val Leu 395 400 405 Thr Gly Asp Lys His Glu Thr Ala Val Ser
Val Ser Leu Ser Cys 410 415 420 Gly His Phe His Arg Thr Met Asn Ile
Leu Glu Leu Ile Asn Gln 425 430 435 Lys Ser Asp Ser Glu Cys Ala Glu
Gln Leu Arg Gln Leu Ala Arg 440 445 450 Arg Ile Thr Glu Asp His Val
Ile Gln His Gly Leu Val Val Asp 455 460 465 Gly Thr Ser Leu Ser Leu
Ala Leu Arg Glu His Glu Lys Leu Phe 470 475 480 Met Glu Val Cys Arg
Asn Cys Ser Ala Val Leu Cys Cys Arg Met 485 490 495 Ala Pro Leu Gln
Lys Ala Lys Val Ile Arg Leu Ile Lys Ile Ser 500 505 510 Pro Glu Lys
Pro Ile Thr Leu Ala Val Gly Asp Gly Ala Asn Asp 515 520 525 Val Ser
Met Ile Gln Glu Ala His Val Gly Ile Gly Ile Met Gly 530 535 540 Lys
Glu Gly Arg Gln Ala Ala Arg Asn Ser Asp Tyr Ala Ile Ala 545 550 555
Arg Phe Lys Phe Leu Ser Lys Leu Leu Phe Val His Gly His Phe 560 565
570 Tyr Tyr Ile Arg Ile Ala Thr Leu Val Gln Tyr Phe Phe Tyr Lys 575
580 585 Asn Val Cys Phe Ile Thr Pro Gln Phe Leu Tyr Gln Phe Tyr Cys
590 595 600 Leu Phe Ser Gln Gln Thr Leu Tyr Asp Ser Val Tyr Leu Thr
Leu 605 610 615 Tyr Asn Ile Cys Phe Thr Ser Leu Pro Ile Leu Ile Tyr
Ser Leu 620 625 630 Leu Glu Gln His Val Asp Pro His Val Leu Gln Asn
Lys Pro Thr 635 640 645 Leu Tyr Arg Asp Ile Ser Lys Asn Arg Leu Leu
Ser Ile Lys Thr 650 655 660 Phe Leu Tyr Trp Thr Ile Leu Gly Phe Ser
His Ala Phe Ile Phe 665 670 675 Phe Phe Gly Ser Tyr Leu Leu Ile Gly
Lys Asp Thr Ser Leu Leu 680 685 690 Gly Asn Gly Gln Met Phe Gly Asn
Trp Thr Phe Gly Thr Leu Val 695 700 705 Phe Thr Val Met Val Ile Thr
Val Thr Val Lys Met Ala Leu Glu 710 715 720 Thr His Phe Trp Thr Trp
Ile Asn His Leu Val Thr Trp Gly Ser 725 730 735 Ile Ile Phe Tyr Phe
Val Phe Ser Leu Phe Tyr Gly Gly Ile Leu 740 745 750 Trp Pro Phe Leu
Gly Ser Gln Asn Met Tyr Phe Val Phe Ile Gln 755 760 765 Leu Leu Ser
Ser Gly Ser Ala Trp Phe Ala Ile Ile Leu Met Val 770 775 780 Val Thr
Cys Leu Phe Leu Asp Ile Ile Lys Lys Val Phe Asp Arg 785 790 795 His
Leu His Pro Thr Ser Thr Glu Lys Ala Gln Leu Thr Glu Thr 800 805 810
Asn Ala Gly Ile Lys Cys Leu Asp Ser Met Cys Cys Phe Pro Glu 815 820
825 Gly Glu Ala Ala Cys Ala Ser Val Gly Arg Met Leu Glu Arg Val 830
835 840 Ile Gly Arg Cys Ser Pro Thr His Ile Ser Arg Ser Trp Ser Ala
845 850 855 Ser Asp Pro Phe Tyr Thr Asn Asp Arg Ser Ile Leu Thr Leu
Ser 860 865 870 Thr Met Asp Ser Ser Thr Cys 875 68 63 PRT Homo
Sapien 68 Met Lys His Val Leu Asn Leu Tyr Leu Leu Gly Val Val Leu
Thr 1 5 10 15 Leu Leu Ser Ile Phe Val Arg Val Met Glu Ser Leu Glu
Gly Leu 20 25 30 Leu Glu Ser Pro Ser Pro Gly Thr Ser Trp Thr Thr
Arg Ser Gln 35 40 45 Leu Ala Asn Thr Glu Pro Thr Lys Gly Leu Pro
Asp His Pro Ser 50 55 60 Arg Ser Met 69 137 PRT Homo Sapien unsure
101, 136 unknown amino acid 69 Met Lys Thr Gly Leu Phe Phe Leu Cys
Leu Leu Gly Thr Ala Ala 1 5 10 15 Ala Ile Pro Thr Asn Ala Arg Leu
Leu Ser Asp His Ser Lys Pro 20 25 30 Thr Ala Glu Thr Val Ala Pro
Asp Asn Thr Ala Ile Pro Ser Leu 35 40 45 Arg Ala Glu Asp Glu Glu
Asn Glu Lys Glu Thr Ala Val Ser Thr 50 55 60 Glu Asp Asp Ser His
His Lys Ala Glu Lys Ser Ser Val Leu Lys 65 70 75 Ser Lys Glu Glu
Ser His Glu Gln Ser Ala Glu Gln Gly Lys Ser 80 85 90 Ser Ser Gln
Glu Leu Gly Leu Lys Asp Gln Xaa Asp Ser Asp Gly 95 100 105 Asp Leu
Ser Val Asn Leu Glu Tyr Ala Pro Thr Glu Gly Thr Leu 110 115 120 Asp
Ile Lys Glu Asp Met Ser Glu Pro Gln Glu Lys Asn Ser Gln 125 130 135
Xaa His 70 318 PRT Homo Sapien 70 Met Ala Pro Trp Ala Glu Ala Glu
His Ser Ala Leu Asn Pro Leu 1 5 10 15 Arg Ala Val Trp Leu Thr Leu
Thr Ala Ala Phe Leu Leu Thr Leu 20 25 30 Leu Leu Gln Leu Leu Pro
Pro Gly Leu Leu Pro Gly Cys Ala Ile 35 40 45 Phe Gln Asp Leu Ile
Arg Tyr Gly Lys Thr Lys Cys Gly Glu Pro 50 55 60 Ser Arg Pro Ala
Ala Cys Arg Ala Phe Asp Val Pro Lys Arg Tyr 65 70 75 Phe Ser His
Phe Tyr Ile Ile Ser Val Leu Trp Asn Gly Phe Leu 80 85 90 Leu Trp
Cys Leu Thr Gln Ser Leu Phe Leu Gly Ala Pro Phe Pro 95 100 105 Ser
Trp Leu His Gly Leu Leu Arg Ile Leu Gly Ala Ala Gln Phe 110 115 120
Gln Gly Gly Glu Leu Ala Leu Ser Ala Phe Leu Val Leu Val Phe 125 130
135 Leu Trp Leu His Ser Leu Arg Arg Leu Phe Glu Cys Leu Tyr Val 140
145 150 Ser Val Phe Ser Asn Val Met Ile His Val Val Gln Tyr Cys Phe
155 160 165 Gly Leu Val Tyr Tyr Val Leu Val Gly Leu Thr Val Leu Ser
Gln 170 175 180 Val Pro Met Asp Gly Arg Asn Ala Tyr Ile Thr Gly Lys
Asn Leu 185 190 195 Leu Met Gln Ala Arg Trp Phe His Ile Leu Gly Met
Met Met Phe 200 205 210 Ile Trp Ser Ser Ala His Gln Tyr Lys Cys His
Val Ile Leu Gly 215 220 225 Asn Leu Arg Lys Asn Lys Ala Gly Val Val
Ile His Cys Asn His 230 235 240 Arg Ile Pro Phe Gly Asp Trp Phe Glu
Tyr Val Ser Ser Pro Asn 245 250 255 Tyr Leu Ala Glu Leu Met Ile Tyr
Val Ser Met Ala Val Thr Phe 260 265 270 Gly Phe His Asn Leu Thr Trp
Trp Leu Val Val Thr Asn Val Phe 275 280 285 Phe Asn Gln Ala Leu Ser
Ala Phe Leu Ser His Gln Phe Tyr Lys 290 295 300 Ser Lys Phe Val Ser
Tyr Pro Lys His Arg Lys Ala Phe Leu Pro 305 310 315 Phe Leu Phe 71
426 PRT Homo sapien 71 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu
Ala Ser Cys Trp 1 5 10 15 Ile Ile Val Arg Ser Ser Pro Thr Pro Gly
Ser Glu Gly His Ser 20 25 30 Ala Ala Pro Asp Cys Pro Ser Cys Ala
Leu Ala Ala Leu Pro Lys 35 40 45 Asp Val Pro Asn Ser Gln Pro Glu
Met Val Glu Ala Val Lys Lys 50 55 60 His Ile Leu Asn Met Leu His
Leu Lys Lys Arg Pro Asp Val Thr 65 70 75 Gln Pro Val Pro Lys Ala
Ala Leu Leu Asn Ala Ile Arg Lys Leu 80 85 90 His Val Gly Lys Val
Gly Glu Asn Gly Tyr Val Glu Ile Glu Asp 95 100 105 Asp Ile Gly Arg
Arg Ala Glu Met Asn Glu Leu Met Glu Gln Thr 110 115 120 Ser Glu Ile
Ile Thr Phe Ala Glu Ser Gly Thr Ala Arg Lys Thr 125 130 135 Leu His
Phe Glu Ile Ser Lys Glu Gly Ser Asp Leu Ser Val Val 140 145 150 Glu
Arg Ala Glu Val Trp Leu Phe Leu Lys Val Pro Lys Ala Asn 155 160 165
Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe Gln Gln Gln Lys 170 175
180 His Pro Gln Gly Ser Leu Asp Thr Gly Glu Glu Ala Glu Glu Val 185
190 195 Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val
200 205 210 Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser
Ser 215 220 225 Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu
Asp Val 230 235 240 Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala
Ser Leu Val 245 250 255 Leu Leu
Gly Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys 260 265 270 Lys
Lys Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys 275 280 285
Glu Gln Ser His Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser 290 295
300 Glu Asp His Pro His Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp 305
310 315 Gly Lys Val Asn Ile Cys Cys Lys Lys Gln Phe Phe Val Ser Phe
320 325 330 Lys Asp Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro Ser Gly
Tyr 335 340 345 His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser His Ile
Ala Gly 350 355 360 Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr Val
Ile Asn His 365 370 375 Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn
Leu Lys Ser Cys 380 385 390 Cys Val Pro Thr Lys Leu Arg Pro Met Ser
Met Leu Tyr Tyr Asp 395 400 405 Asp Gly Gln Asn Ile Ile Lys Lys Asp
Ile Gln Asn Met Ile Val 410 415 420 Glu Glu Cys Gly Cys Ser 425 72
238 PRT Homo Sapien 72 Met Ala Ala Ala Pro Leu Leu Leu Leu Leu Leu
Leu Val Pro Val 1 5 10 15 Pro Leu Leu Pro Leu Leu Ala Gln Gly Pro
Gly Gly Ala Leu Gly 20 25 30 Asn Arg His Ala Val Tyr Trp Asn Ser
Ser Asn Gln His Leu Arg 35 40 45 Arg Glu Gly Tyr Thr Val Gln Val
Asn Val Asn Asp Tyr Leu Asp 50 55 60 Ile Tyr Cys Pro His Tyr Asn
Ser Ser Gly Val Gly Pro Gly Ala 65 70 75 Gly Pro Gly Pro Gly Gly
Gly Ala Glu Gln Tyr Val Leu Tyr Met 80 85 90 Val Ser Arg Asn Gly
Tyr Arg Thr Cys Asn Ala Ser Gln Gly Phe 95 100 105 Lys Arg Trp Glu
Cys Asn Arg Pro His Ala Pro His Ser Pro Ile 110 115 120 Lys Phe Ser
Glu Lys Phe Gln Arg Tyr Ser Ala Phe Ser Leu Gly 125 130 135 Tyr Glu
Phe His Ala Gly His Glu Tyr Tyr Tyr Ile Ser Thr Pro 140 145 150 Thr
His Asn Leu His Trp Lys Cys Leu Arg Met Lys Val Phe Val 155 160 165
Cys Cys Ala Ser Thr Ser His Ser Gly Glu Lys Pro Val Pro Thr 170 175
180 Leu Pro Gln Phe Thr Met Gly Pro Asn Val Lys Ile Asn Val Leu 185
190 195 Glu Asp Phe Glu Gly Glu Asn Pro Gln Val Pro Lys Leu Glu Lys
200 205 210 Ser Ile Ser Gly Thr Ser Pro Lys Arg Glu His Leu Pro Leu
Ala 215 220 225 Val Gly Ile Ala Phe Phe Leu Met Thr Phe Leu Ala Ser
230 235 73 541 PRT Homo Sapien 73 Met Gly His Ser Pro Pro Val Leu
Pro Leu Cys Ala Ser Val Ser 1 5 10 15 Leu Leu Gly Gly Leu Thr Phe
Gly Tyr Glu Leu Ala Val Ile Ser 20 25 30 Gly Ala Leu Leu Pro Leu
Gln Leu Asp Phe Gly Leu Ser Cys Leu 35 40 45 Glu Gln Glu Phe Leu
Val Gly Ser Leu Leu Leu Gly Ala Leu Leu 50 55 60 Ala Ser Leu Val
Gly Gly Phe Leu Ile Asp Cys Tyr Gly Arg Lys 65 70 75 Gln Ala Ile
Leu Gly Ser Asn Leu Val Leu Leu Ala Gly Ser Leu 80 85 90 Thr Leu
Gly Leu Ala Gly Ser Leu Ala Trp Leu Val Leu Gly Arg 95 100 105 Ala
Val Val Gly Phe Ala Ile Ser Leu Ser Ser Met Ala Cys Cys 110 115 120
Ile Tyr Val Ser Glu Leu Val Gly Pro Arg Gln Arg Gly Val Leu 125 130
135 Val Ser Leu Tyr Glu Ala Gly Ile Thr Val Gly Ile Leu Leu Ser 140
145 150 Tyr Ala Leu Asn Tyr Ala Leu Ala Gly Thr Pro Trp Gly Trp Arg
155 160 165 His Met Phe Gly Trp Ala Thr Ala Pro Ala Val Leu Gln Ser
Leu 170 175 180 Ser Leu Leu Phe Leu Pro Ala Gly Thr Asp Glu Thr Ala
Thr His 185 190 195 Lys Asp Leu Ile Pro Leu Gln Gly Gly Glu Ala Pro
Lys Leu Gly 200 205 210 Pro Gly Arg Pro Arg Tyr Ser Phe Leu Asp Leu
Phe Arg Ala Arg 215 220 225 Asp Asn Met Arg Gly Arg Thr Thr Val Gly
Leu Gly Leu Val Leu 230 235 240 Phe Gln Gln Leu Thr Gly Gln Pro Asn
Val Leu Cys Tyr Ala Ser 245 250 255 Thr Ile Phe Ser Ser Val Gly Phe
His Gly Gly Ser Ser Ala Val 260 265 270 Leu Ala Ser Val Gly Leu Gly
Ala Val Lys Val Ala Ala Thr Leu 275 280 285 Thr Ala Met Gly Leu Val
Asp Arg Ala Gly Arg Arg Ala Leu Leu 290 295 300 Leu Ala Gly Cys Ala
Leu Met Ala Leu Ser Val Ser Gly Ile Gly 305 310 315 Leu Val Ser Phe
Ala Val Pro Met Asp Ser Gly Pro Ser Cys Leu 320 325 330 Ala Val Pro
Asn Ala Thr Gly Gln Thr Gly Leu Pro Gly Asp Ser 335 340 345 Gly Leu
Leu Gln Asp Ser Ser Leu Pro Pro Ile Pro Arg Thr Asn 350 355 360 Glu
Asp Gln Arg Glu Pro Ile Leu Ser Thr Ala Lys Lys Thr Lys 365 370 375
Pro His Pro Arg Ser Gly Asp Pro Ser Ala Pro Pro Arg Leu Ala 380 385
390 Leu Ser Ser Ala Leu Pro Gly Pro Pro Leu Pro Ala Arg Gly His 395
400 405 Ala Leu Leu Arg Trp Thr Ala Leu Leu Cys Leu Met Val Phe Val
410 415 420 Ser Ala Phe Ser Phe Gly Phe Gly Pro Val Thr Trp Leu Val
Leu 425 430 435 Ser Glu Ile Tyr Pro Val Glu Ile Arg Gly Arg Ala Phe
Ala Phe 440 445 450 Cys Asn Ser Phe Asn Trp Ala Ala Asn Leu Phe Ile
Ser Leu Ser 455 460 465 Phe Leu Asp Leu Ile Gly Thr Ile Gly Leu Ser
Trp Thr Phe Leu 470 475 480 Leu Tyr Gly Leu Thr Ala Val Leu Gly Leu
Gly Phe Ile Tyr Leu 485 490 495 Phe Val Pro Glu Thr Lys Gly Gln Ser
Leu Ala Glu Ile Asp Gln 500 505 510 Gln Phe Gln Lys Arg Arg Phe Thr
Leu Ser Phe Gly His Arg Gln 515 520 525 Asn Ser Thr Gly Ile Pro Tyr
Ser Arg Ile Glu Ile Ser Ala Ala 530 535 540 Ser 74 1114 PRT Homo
Sapien 74 Met Ala Lys Ala Thr Ser Gly Ala Ala Gly Leu Arg Leu Leu
Leu 1 5 10 15 Leu Leu Leu Leu Pro Leu Leu Gly Lys Val Ala Leu Gly
Leu Tyr 20 25 30 Phe Ser Arg Asp Ala Tyr Trp Glu Lys Leu Tyr Val
Asp Gln Ala 35 40 45 Ala Gly Thr Pro Leu Leu Tyr Val His Ala Leu
Arg Asp Ala Pro 50 55 60 Glu Glu Val Pro Ser Phe Arg Leu Gly Gln
His Leu Tyr Gly Thr 65 70 75 Tyr Arg Thr Arg Leu His Glu Asn Asn
Trp Ile Cys Ile Gln Glu 80 85 90 Asp Thr Gly Leu Leu Tyr Leu Asn
Arg Ser Leu Asp His Ser Ser 95 100 105 Trp Glu Lys Leu Ser Val Arg
Asn Arg Gly Phe Pro Leu Leu Thr 110 115 120 Val Tyr Leu Lys Val Phe
Leu Ser Pro Thr Ser Leu Arg Glu Gly 125 130 135 Glu Cys Gln Trp Pro
Gly Cys Ala Arg Val Tyr Phe Ser Phe Phe 140 145 150 Asn Thr Ser Phe
Pro Ala Cys Ser Ser Leu Lys Pro Arg Glu Leu 155 160 165 Cys Phe Pro
Glu Thr Arg Pro Ser Phe Arg Ile Arg Glu Asn Arg 170 175 180 Pro Pro
Gly Thr Phe His Gln Phe Arg Leu Leu Pro Val Gln Phe 185 190 195 Leu
Cys Pro Asn Ile Ser Val Ala Tyr Arg Leu Leu Glu Gly Glu 200 205 210
Gly Leu Pro Phe Arg Cys Ala Pro Asp Ser Leu Glu Val Ser Thr 215 220
225 Arg Trp Ala Leu Asp Arg Glu Gln Arg Glu Lys Tyr Glu Leu Val 230
235 240 Ala Val Cys Thr Val His Ala Gly Ala Arg Glu Glu Val Val Met
245 250 255 Val Pro Phe Pro Val Thr Val Tyr Asp Glu Asp Asp Ser Ala
Pro 260 265 270 Thr Phe Pro Ala Gly Val Asp Thr Ala Ser Ala Val Val
Glu Phe 275 280 285 Lys Arg Lys Glu Asp Thr Val Val Ala Thr Leu Arg
Val Phe Asp 290 295 300 Ala Asp Val Val Pro Ala Ser Gly Glu Leu Val
Arg Arg Tyr Thr 305 310 315 Ser Thr Leu Leu Pro Gly Asp Thr Trp Ala
Gln Gln Thr Phe Arg 320 325 330 Val Glu His Trp Pro Asn Glu Thr Ser
Val Gln Ala Asn Gly Ser 335 340 345 Phe Val Arg Ala Thr Val His Asp
Tyr Arg Leu Val Leu Asn Arg 350 355 360 Asn Leu Ser Ile Ser Glu Asn
Arg Thr Met Gln Leu Ala Val Leu 365 370 375 Val Asn Asp Ser Asp Phe
Gln Gly Pro Gly Ala Gly Val Leu Leu 380 385 390 Leu His Phe Asn Val
Ser Val Leu Pro Val Ser Leu His Leu Pro 395 400 405 Ser Thr Tyr Ser
Leu Ser Val Ser Arg Arg Ala Arg Arg Phe Ala 410 415 420 Gln Ile Gly
Lys Val Cys Val Glu Asn Cys Gln Ala Phe Ser Gly 425 430 435 Ile Asn
Val Gln Tyr Lys Leu His Ser Ser Gly Ala Asn Cys Ser 440 445 450 Thr
Leu Gly Val Val Thr Ser Ala Glu Asp Thr Ser Gly Ile Leu 455 460 465
Phe Val Asn Asp Thr Lys Ala Leu Arg Arg Pro Lys Cys Ala Glu 470 475
480 Leu His Tyr Met Val Val Ala Thr Asp Gln Gln Thr Ser Arg Gln 485
490 495 Ala Gln Ala Gln Leu Leu Val Thr Val Glu Gly Ser Tyr Val Ala
500 505 510 Glu Glu Ala Gly Cys Pro Leu Ser Cys Ala Val Ser Lys Arg
Arg 515 520 525 Leu Glu Cys Glu Glu Cys Gly Gly Leu Gly Ser Pro Thr
Gly Arg 530 535 540 Cys Glu Trp Arg Gln Gly Asp Gly Lys Gly Ile Thr
Arg Asn Phe 545 550 555 Ser Thr Cys Ser Pro Ser Thr Lys Thr Cys Pro
Asp Gly His Cys 560 565 570 Asp Val Val Glu Thr Gln Asp Ile Asn Ile
Cys Pro Gln Asp Cys 575 580 585 Leu Arg Gly Ser Ile Val Gly Gly His
Glu Pro Gly Glu Pro Arg 590 595 600 Gly Ile Lys Ala Gly Tyr Gly Thr
Cys Asn Cys Phe Pro Glu Glu 605 610 615 Glu Lys Cys Phe Cys Glu Pro
Glu Asp Ile Gln Asp Pro Leu Cys 620 625 630 Asp Glu Leu Cys Arg Thr
Val Ile Ala Ala Ala Val Leu Phe Ser 635 640 645 Phe Ile Val Ser Val
Leu Leu Ser Ala Phe Cys Ile His Cys Tyr 650 655 660 His Lys Phe Ala
His Lys Pro Pro Ile Ser Ser Ala Glu Met Thr 665 670 675 Phe Arg Arg
Pro Ala Gln Ala Phe Pro Val Ser Tyr Ser Ser Ser 680 685 690 Gly Ala
Arg Arg Pro Ser Leu Asp Ser Met Glu Asn Gln Val Ser 695 700 705 Val
Asp Ala Phe Lys Ile Leu Glu Asp Pro Lys Trp Glu Phe Pro 710 715 720
Arg Lys Asn Leu Val Leu Gly Lys Thr Leu Gly Glu Gly Glu Phe 725 730
735 Gly Lys Val Val Lys Ala Thr Ala Phe His Leu Lys Gly Arg Ala 740
745 750 Gly Tyr Thr Thr Val Ala Val Lys Met Leu Lys Glu Asn Ala Ser
755 760 765 Pro Ser Glu Leu Arg Asp Leu Leu Ser Glu Phe Asn Val Leu
Lys 770 775 780 Gln Val Asn His Pro His Val Ile Lys Leu Tyr Gly Ala
Cys Ser 785 790 795 Gln Asp Gly Pro Leu Leu Leu Ile Val Glu Tyr Ala
Lys Tyr Gly 800 805 810 Ser Leu Arg Gly Phe Leu Arg Glu Ser Arg Lys
Val Gly Pro Gly 815 820 825 Tyr Leu Gly Ser Gly Gly Ser Arg Asn Ser
Ser Ser Leu Asp His 830 835 840 Pro Asp Glu Arg Ala Leu Thr Met Gly
Asp Leu Ile Ser Phe Ala 845 850 855 Trp Gln Ile Ser Gln Gly Met Gln
Tyr Leu Ala Glu Met Lys Leu 860 865 870 Val His Arg Asp Leu Ala Ala
Arg Asn Ile Leu Val Ala Glu Gly 875 880 885 Arg Lys Met Lys Ile Ser
Asp Phe Gly Leu Ser Arg Asp Val Tyr 890 895 900 Glu Glu Asp Ser Tyr
Val Lys Arg Ser Gln Gly Arg Ile Pro Val 905 910 915 Lys Trp Met Ala
Ile Glu Ser Leu Phe Asp His Ile Tyr Thr Thr 920 925 930 Gln Ser Asp
Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Val 935 940 945 Thr Leu
Gly Gly Asn Pro Tyr Pro Gly Ile Pro Pro Glu Arg Leu 950 955 960 Phe
Asn Leu Leu Lys Thr Gly His Arg Met Glu Arg Pro Asp Asn 965 970 975
Cys Ser Glu Glu Met Tyr Arg Leu Met Leu Gln Cys Trp Lys Gln 980 985
990 Glu Pro Asp Lys Arg Pro Val Phe Ala Asp Ile Ser Lys Asp Leu 995
1000 1005 Glu Lys Met Met Val Lys Arg Arg Asp Tyr Leu Asp Leu Ala
Ala 1010 1015 1020 Ser Thr Pro Ser Asp Ser Leu Ile Tyr Asp Asp Gly
Leu Ser Glu 1025 1030 1035 Glu Glu Thr Pro Leu Val Asp Cys Asn Asn
Ala Pro Leu Pro Arg 1040 1045 1050 Ala Leu Pro Ser Thr Trp Ile Glu
Asn Lys Leu Tyr Gly Met Ser 1055 1060 1065 Asp Pro Asn Trp Pro Gly
Glu Ser Pro Val Pro Leu Thr Arg Ala 1070 1075 1080 Asp Gly Thr Asn
Thr Gly Phe Pro Arg Tyr Pro Asn Asp Ser Val 1085 1090 1095 Tyr Ala
Asn Trp Met Leu Ser Pro Ser Ala Ala Lys Leu Met Asp 1100 1105 1110
Thr Phe Asp Ser 75 790 PRT Homo Sapien 75 Met Arg Thr Tyr Arg Tyr
Phe Leu Leu Leu Phe Trp Val Gly Gln 1 5 10 15 Pro Tyr Pro Thr Leu
Ser Thr Pro Leu Ser Lys Arg Thr Ser Gly 20 25 30 Phe Pro Ala Lys
Lys Arg Ala Leu Glu Leu Ser Gly Asn Ser Lys 35 40 45 Asn Glu Leu
Asn Arg Ser Lys Arg Ser Trp Met Trp Asn Gln Phe 50 55 60 Phe Leu
Leu Glu Glu Tyr Thr Gly Ser Asp Tyr Gln Tyr Val Gly 65 70 75 Lys
Leu His Ser Asp Gln Asp Arg Gly Asp Gly Ser Leu Lys Tyr 80 85 90
Ile Leu Ser Gly Asp Gly Ala Gly Asp Leu Phe Ile Ile Asn Glu 95 100
105 Asn Thr Gly Asp Ile Gln Ala Thr Lys Arg Leu Asp Arg Glu Glu 110
115 120 Lys Pro Val Tyr Ile Leu Arg Ala Gln Ala Ile Asn Arg Arg Thr
125 130 135 Gly Arg Pro Val Glu Pro Glu Ser Glu Phe Ile Ile Lys Ile
His 140 145 150 Asp Ile Asn Asp Asn Glu Pro Ile Phe Thr Lys Glu Val
Tyr Thr 155 160 165 Ala Thr Val Pro Glu Met Ser Asp Val Gly Thr Phe
Val Val Gln 170 175 180 Val Thr Ala Thr Asp Ala Asp Asp Pro Thr Tyr
Gly Asn Ser Ala 185 190 195 Lys Val Val Tyr Ser Ile Leu Gln Gly Gln
Pro Tyr Phe Ser Val 200 205 210 Glu Ser Glu Thr Gly Ile Ile Lys Thr
Ala Leu Leu Asn Met Asp 215 220 225 Arg Glu Asn Arg Glu Gln
Tyr Gln Val Val Ile Gln Ala Lys Asp 230 235 240 Met Gly Gly Gln Met
Gly Gly Leu Ser Gly Thr Thr Thr Val Asn 245 250 255 Ile Thr Leu Thr
Asp Val Asn Asp Asn Pro Pro Arg Phe Pro Gln 260 265 270 Ser Thr Tyr
Gln Phe Lys Thr Pro Glu Ser Ser Pro Pro Gly Thr 275 280 285 Pro Ile
Gly Arg Ile Lys Ala Ser Asp Ala Asp Val Gly Glu Asn 290 295 300 Ala
Glu Ile Glu Tyr Ser Ile Thr Asp Gly Glu Gly Leu Asp Met 305 310 315
Phe Asp Val Ile Thr Asp Gln Glu Thr Gln Glu Gly Ile Ile Thr 320 325
330 Val Lys Lys Leu Leu Asp Phe Glu Lys Lys Lys Val Tyr Thr Leu 335
340 345 Lys Val Glu Ala Ser Asn Pro Tyr Val Glu Pro Arg Phe Leu Tyr
350 355 360 Leu Gly Pro Phe Lys Asp Ser Ala Thr Val Arg Ile Val Val
Glu 365 370 375 Asp Val Asp Glu Pro Pro Val Phe Ser Lys Leu Ala Tyr
Ile Leu 380 385 390 Gln Ile Arg Glu Asp Ala Gln Ile Asn Thr Thr Ile
Gly Ser Val 395 400 405 Thr Ala Gln Asp Pro Asp Ala Ala Arg Asn Pro
Val Lys Tyr Ser 410 415 420 Val Asp Arg His Thr Asp Met Asp Arg Ile
Phe Asn Ile Asp Ser 425 430 435 Gly Asn Gly Ser Ile Phe Thr Ser Lys
Leu Leu Asp Arg Glu Thr 440 445 450 Leu Leu Trp His Asn Ile Thr Val
Ile Ala Thr Glu Ile Asn Asn 455 460 465 Pro Lys Gln Ser Ser Arg Val
Pro Leu Tyr Ile Lys Val Leu Asp 470 475 480 Val Asn Asp Asn Ala Pro
Glu Phe Ala Glu Phe Tyr Glu Thr Phe 485 490 495 Val Cys Glu Lys Ala
Lys Ala Asp Gln Leu Ile Gln Thr Leu His 500 505 510 Ala Val Asp Lys
Asp Asp Pro Tyr Ser Gly His Gln Phe Ser Phe 515 520 525 Ser Leu Ala
Pro Glu Ala Ala Ser Gly Ser Asn Phe Thr Ile Gln 530 535 540 Asp Asn
Lys Asp Asn Thr Ala Gly Ile Leu Thr Arg Lys Asn Gly 545 550 555 Tyr
Asn Arg His Glu Met Ser Thr Tyr Leu Leu Pro Val Val Ile 560 565 570
Ser Asp Asn Asp Tyr Pro Val Gln Ser Ser Thr Gly Thr Val Thr 575 580
585 Val Arg Val Cys Ala Cys Asp His His Gly Asn Met Gln Ser Cys 590
595 600 His Ala Glu Ala Leu Ile His Pro Thr Gly Leu Ser Thr Gly Ala
605 610 615 Leu Val Ala Ile Leu Leu Cys Ile Val Ile Leu Leu Val Thr
Val 620 625 630 Val Leu Phe Ala Ala Leu Arg Arg Gln Arg Lys Lys Glu
Pro Leu 635 640 645 Ile Ile Ser Lys Glu Asp Ile Arg Asp Asn Ile Val
Ser Tyr Asn 650 655 660 Asp Glu Gly Gly Gly Glu Glu Asp Thr Gln Ala
Phe Asp Ile Gly 665 670 675 Thr Leu Arg Asn Pro Glu Ala Ile Glu Asp
Asn Lys Leu Arg Arg 680 685 690 Asp Ile Val Pro Glu Ala Leu Phe Leu
Pro Arg Arg Thr Pro Thr 695 700 705 Ala Arg Asp Asn Thr Asp Val Arg
Asp Phe Ile Asn Gln Arg Leu 710 715 720 Lys Glu Asn Asp Thr Asp Pro
Thr Ala Pro Pro Tyr Asp Ser Leu 725 730 735 Ala Thr Tyr Ala Tyr Glu
Gly Thr Gly Ser Val Ala Asp Ser Leu 740 745 750 Ser Ser Leu Glu Ser
Val Thr Thr Asp Ala Asp Gln Asp Tyr Asp 755 760 765 Tyr Leu Ser Asp
Trp Gly Pro Arg Phe Lys Lys Leu Ala Asp Met 770 775 780 Tyr Gly Gly
Val Asp Ser Asp Lys Asp Ser 785 790 76 794 PRT Homo Sapien 76 Met
Leu Thr Arg Asn Cys Leu Ser Leu Leu Leu Trp Val Leu Phe 1 5 10 15
Asp Gly Gly Leu Leu Thr Pro Leu Gln Pro Gln Pro Gln Gln Thr 20 25
30 Leu Ala Thr Glu Pro Arg Glu Asn Val Ile His Leu Pro Gly Gln 35
40 45 Arg Ser His Phe Gln Arg Val Lys Arg Gly Trp Val Trp Asn Gln
50 55 60 Phe Phe Val Leu Glu Glu Tyr Val Gly Ser Glu Pro Gln Tyr
Val 65 70 75 Gly Lys Leu His Ser Asp Leu Asp Lys Gly Glu Gly Thr
Val Lys 80 85 90 Tyr Thr Leu Ser Gly Asp Gly Ala Gly Thr Val Phe
Thr Ile Asp 95 100 105 Glu Thr Thr Gly Asp Ile His Ala Ile Arg Ser
Leu Asp Arg Glu 110 115 120 Glu Lys Pro Phe Tyr Thr Leu Arg Ala Gln
Ala Val Asp Ile Glu 125 130 135 Thr Arg Lys Pro Leu Glu Pro Glu Ser
Glu Phe Ile Ile Lys Val 140 145 150 Gln Asp Ile Asn Asp Asn Glu Pro
Lys Phe Leu Asp Gly Pro Tyr 155 160 165 Val Ala Thr Val Pro Glu Met
Ser Pro Val Gly Ala Tyr Val Leu 170 175 180 Gln Val Lys Ala Thr Asp
Ala Asp Asp Pro Thr Tyr Gly Asn Ser 185 190 195 Ala Arg Val Val Tyr
Ser Ile Leu Gln Gly Gln Pro Tyr Phe Ser 200 205 210 Ile Asp Pro Lys
Thr Gly Val Ile Arg Thr Ala Leu Pro Asn Met 215 220 225 Asp Arg Glu
Val Lys Glu Gln Tyr Gln Val Leu Ile Gln Ala Lys 230 235 240 Asp Met
Gly Gly Gln Leu Gly Gly Leu Ala Gly Thr Thr Ile Val 245 250 255 Asn
Ile Thr Leu Thr Asp Val Asn Asp Asn Pro Pro Arg Phe Pro 260 265 270
Lys Ser Ile Phe His Leu Lys Val Pro Glu Ser Ser Pro Ile Gly 275 280
285 Ser Ala Ile Gly Arg Ile Arg Ala Val Asp Pro Asp Phe Gly Gln 290
295 300 Asn Ala Glu Ile Glu Tyr Asn Ile Val Pro Gly Asp Gly Gly Asn
305 310 315 Leu Phe Asp Ile Val Thr Asp Glu Asp Thr Gln Glu Gly Val
Ile 320 325 330 Lys Leu Lys Lys Pro Leu Asp Phe Glu Thr Lys Lys Ala
Tyr Thr 335 340 345 Phe Lys Val Glu Ala Ser Asn Leu His Leu Asp His
Arg Phe His 350 355 360 Ser Ala Gly Pro Phe Lys Asp Thr Ala Thr Val
Lys Ile Ser Val 365 370 375 Leu Asp Val Asp Glu Pro Pro Val Phe Ser
Lys Pro Leu Tyr Thr 380 385 390 Met Glu Val Tyr Glu Asp Thr Pro Val
Gly Thr Ile Ile Gly Ala 395 400 405 Val Thr Ala Gln Asp Leu Asp Val
Gly Ser Gly Ala Val Arg Tyr 410 415 420 Phe Ile Asp Trp Lys Ser Asp
Gly Asp Ser Tyr Phe Thr Ile Asp 425 430 435 Gly Asn Glu Gly Thr Ile
Ala Thr Asn Glu Leu Leu Asp Arg Glu 440 445 450 Ser Thr Ala Gln Tyr
Asn Phe Ser Ile Ile Ala Ser Lys Val Ser 455 460 465 Asn Pro Leu Leu
Thr Ser Lys Val Asn Ile Leu Ile Asn Val Leu 470 475 480 Asp Val Asn
Glu Phe Pro Pro Glu Ile Ser Val Pro Tyr Glu Thr 485 490 495 Ala Val
Cys Glu Asn Ala Lys Pro Gly Gln Ile Ile Gln Ile Val 500 505 510 Ser
Ala Ala Asp Arg Asp Leu Ser Pro Ala Gly Gln Gln Phe Ser 515 520 525
Phe Arg Leu Ser Pro Glu Ala Ala Ile Lys Pro Asn Phe Thr Val 530 535
540 Arg Asp Phe Arg Asn Asn Thr Ala Gly Ile Glu Thr Arg Arg Asn 545
550 555 Gly Tyr Ser Arg Arg Gln Gln Glu Leu Tyr Phe Leu Pro Val Val
560 565 570 Ile Glu Asp Ser Ser Tyr Pro Val Gln Ser Ser Thr Asn Thr
Met 575 580 585 Thr Ile Arg Val Cys Arg Cys Asp Ser Asp Gly Thr Ile
Leu Ser 590 595 600 Cys Asn Val Glu Ala Ile Phe Leu Pro Val Gly Leu
Ser Thr Gly 605 610 615 Ala Leu Ile Ala Ile Leu Leu Cys Ile Val Ile
Leu Leu Ala Ile 620 625 630 Val Val Leu Tyr Val Ala Leu Arg Arg Gln
Lys Lys Lys His Thr 635 640 645 Leu Met Thr Ser Lys Glu Asp Ile Arg
Asp Asn Val Ile His Tyr 650 655 660 Asp Asp Glu Gly Gly Gly Glu Glu
Asp Thr Gln Ala Phe Asp Ile 665 670 675 Gly Ala Leu Arg Asn Pro Lys
Val Ile Glu Glu Asn Lys Ile Arg 680 685 690 Arg Asp Ile Lys Pro Asp
Ser Leu Cys Leu Pro Arg Gln Arg Pro 695 700 705 Pro Met Glu Asp Asn
Thr Asp Ile Arg Asp Phe Ile His Gln Arg 710 715 720 Leu Gln Glu Asn
Asp Val Asp Pro Thr Ala Pro Pro Ile Asp Ser 725 730 735 Leu Ala Thr
Tyr Ala Tyr Glu Gly Ser Gly Ser Val Ala Glu Ser 740 745 750 Leu Ser
Ser Ile Asp Ser Leu Thr Thr Glu Ala Asp Gln Asp Tyr 755 760 765 Asp
Tyr Leu Thr Asp Trp Gly Pro Arg Phe Lys Val Leu Ala Asp 770 775 780
Met Phe Gly Glu Glu Glu Ser Tyr Asn Pro Asp Lys Val Thr 785 790 77
141 PRT Homo Sapien 77 Met Ala Arg Pro Leu Cys Thr Leu Leu Leu Leu
Met Ala Thr Leu 1 5 10 15 Ala Gly Ala Leu Ala Ser Ser Ser Lys Glu
Glu Asn Arg Ile Ile 20 25 30 Pro Gly Gly Ile Tyr Asp Ala Asp Leu
Asn Asp Glu Trp Val Gln 35 40 45 Arg Ala Leu His Phe Ala Ile Ser
Glu Tyr Asn Lys Ala Thr Glu 50 55 60 Asp Glu Tyr Tyr Arg Arg Pro
Leu Gln Val Leu Arg Ala Arg Glu 65 70 75 Gln Thr Phe Gly Gly Val
Asn Tyr Phe Phe Asp Val Glu Val Gly 80 85 90 Arg Thr Ile Cys Thr
Lys Ser Gln Pro Asn Leu Asp Thr Cys Ala 95 100 105 Phe His Glu Gln
Pro Glu Leu Gln Lys Lys Gln Leu Cys Ser Phe 110 115 120 Glu Ile Tyr
Glu Val Pro Trp Glu Asp Arg Met Ser Leu Val Asn 125 130 135 Ser Arg
Cys Gln Glu Ala 140 78 466 PRT Homo Sapien 78 Met Thr Thr Ser Pro
Ile Leu Gln Leu Leu Leu Arg Leu Ser Leu 1 5 10 15 Cys Gly Leu Leu
Leu Gln Arg Ala Glu Thr Gly Ser Lys Gly Gln 20 25 30 Thr Ala Gly
Glu Leu Tyr Gln Arg Trp Glu Arg Tyr Arg Arg Glu 35 40 45 Cys Gln
Glu Thr Leu Ala Ala Ala Glu Pro Pro Ser Gly Leu Ala 50 55 60 Cys
Asn Gly Ser Phe Asp Met Tyr Val Cys Trp Asp Tyr Ala Ala 65 70 75
Pro Asn Ala Thr Ala Arg Ala Ser Cys Pro Trp Tyr Leu Pro Trp 80 85
90 His His His Val Ala Ala Gly Phe Val Leu Arg Gln Cys Gly Ser 95
100 105 Asp Gly Gln Trp Gly Leu Trp Arg Asp His Thr Gln Cys Glu Asn
110 115 120 Pro Glu Lys Asn Glu Ala Phe Leu Asp Gln Arg Leu Ile Leu
Glu 125 130 135 Arg Leu Gln Val Met Tyr Thr Val Gly Tyr Ser Leu Ser
Leu Ala 140 145 150 Thr Leu Leu Leu Ala Leu Leu Ile Leu Ser Leu Phe
Arg Arg Leu 155 160 165 His Cys Thr Arg Asn Tyr Ile His Ile Asn Leu
Phe Thr Ser Phe 170 175 180 Met Leu Arg Ala Ala Ala Ile Leu Ser Arg
Asp Arg Leu Leu Pro 185 190 195 Arg Pro Gly Pro Tyr Leu Gly Asp Gln
Ala Leu Ala Leu Trp Asn 200 205 210 Gln Ala Leu Ala Ala Cys Arg Thr
Ala Gln Ile Val Thr Gln Tyr 215 220 225 Cys Val Gly Ala Asn Tyr Thr
Trp Leu Leu Val Glu Gly Val Tyr 230 235 240 Leu His Ser Leu Leu Val
Leu Val Gly Gly Ser Glu Glu Gly His 245 250 255 Phe Arg Tyr Tyr Leu
Leu Leu Gly Trp Gly Ala Pro Ala Leu Phe 260 265 270 Val Ile Pro Trp
Val Ile Val Arg Tyr Leu Tyr Glu Asn Thr Gln 275 280 285 Cys Trp Glu
Arg Asn Glu Val Lys Ala Ile Trp Trp Ile Ile Arg 290 295 300 Thr Pro
Ile Leu Met Thr Ile Leu Ile Asn Phe Leu Ile Phe Ile 305 310 315 Arg
Ile Leu Gly Ile Leu Leu Ser Lys Leu Arg Thr Arg Gln Met 320 325 330
Arg Cys Arg Asp Tyr Arg Leu Arg Leu Ala Arg Ser Thr Leu Thr 335 340
345 Leu Val Pro Leu Leu Gly Val His Glu Val Val Phe Ala Pro Val 350
355 360 Thr Glu Glu Gln Ala Arg Gly Ala Leu Arg Phe Ala Lys Leu Gly
365 370 375 Phe Glu Ile Phe Leu Ser Ser Phe Gln Gly Phe Leu Val Ser
Val 380 385 390 Leu Tyr Cys Phe Ile Asn Lys Glu Val Gln Ser Glu Ile
Arg Arg 395 400 405 Gly Trp His His Cys Arg Leu Arg Arg Ser Leu Gly
Glu Glu Gln 410 415 420 Arg Gln Leu Pro Glu Arg Ala Phe Arg Ala Leu
Pro Ser Gly Ser 425 430 435 Gly Pro Gly Glu Val Pro Thr Ser Arg Gly
Leu Ser Ser Gly Thr 440 445 450 Leu Pro Gly Pro Gly Asn Glu Ala Ser
Arg Glu Leu Glu Ser Tyr 455 460 465 Cys 79 506 PRT Homo Sapien 79
Met Leu Ser Lys Val Leu Pro Val Leu Leu Gly Ile Leu Leu Ile 1 5 10
15 Leu Gln Ser Arg Val Glu Gly Pro Gln Thr Glu Ser Lys Asn Glu 20
25 30 Ala Ser Ser Arg Asp Val Val Tyr Gly Pro Gln Pro Gln Pro Leu
35 40 45 Glu Asn Gln Leu Leu Ser Glu Glu Thr Lys Ser Thr Glu Thr
Glu 50 55 60 Thr Gly Ser Arg Val Gly Lys Leu Pro Glu Ala Ser Arg
Ile Leu 65 70 75 Asn Thr Ile Leu Ser Asn Tyr Asp His Lys Leu Arg
Pro Gly Ile 80 85 90 Gly Glu Lys Pro Thr Val Val Thr Val Glu Ile
Ala Val Asn Ser 95 100 105 Leu Gly Pro Leu Ser Ile Leu Asp Met Glu
Tyr Thr Ile Asp Ile 110 115 120 Ile Phe Ser Gln Thr Trp Tyr Asp Glu
Arg Leu Cys Tyr Asn Asp 125 130 135 Thr Phe Glu Ser Leu Val Leu Asn
Gly Asn Val Val Ser Gln Leu 140 145 150 Trp Ile Pro Asp Thr Phe Phe
Arg Asn Ser Lys Arg Thr His Glu 155 160 165 His Glu Ile Thr Met Pro
Asn Gln Met Val Arg Ile Tyr Lys Asp 170 175 180 Gly Lys Val Leu Tyr
Thr Ile Arg Met Thr Ile Asp Ala Gly Cys 185 190 195 Ser Leu His Met
Leu Arg Phe Pro Met Asp Ser His Ser Cys Pro 200 205 210 Leu Ser Phe
Ser Ser Phe Ser Tyr Pro Glu Asn Glu Met Ile Tyr 215 220 225 Lys Trp
Glu Asn Phe Lys Leu Glu Ile Asn Glu Lys Asn Ser Trp 230 235 240 Lys
Leu Phe Gln Phe Asp Phe Thr Gly Val Ser Asn Lys Thr Glu 245 250 255
Ile Ile Thr Thr Pro Val Gly Asp Phe Met Val Met Thr Ile Phe 260 265
270 Phe Asn Val Ser Arg Arg Phe Gly Tyr Val Ala Phe Gln Asn Tyr 275
280 285 Val Pro Ser Ser Val Thr Thr Met Leu Ser Trp Val Ser Phe Trp
290 295 300 Ile Lys Thr Glu Ser Ala Pro Ala Arg Thr Ser Leu Gly Ile
Thr 305 310 315 Ser Val Leu Thr Met Thr Thr Leu Gly Thr Phe Ser Arg
Lys Asn 320
325 330 Phe Pro Arg Val Ser Tyr Ile Thr Ala Leu Asp Phe Tyr Ile Ala
335 340 345 Ile Cys Phe Val Phe Cys Phe Cys Ala Leu Leu Glu Phe Ala
Val 350 355 360 Leu Asn Phe Leu Ile Tyr Asn Gln Thr Lys Ala His Ala
Ser Pro 365 370 375 Lys Leu Arg His Pro Arg Ile Asn Ser Arg Ala His
Ala Arg Thr 380 385 390 Arg Ala Arg Ser Arg Ala Cys Ala Arg Gln His
Gln Glu Ala Phe 395 400 405 Val Cys Gln Ile Val Thr Thr Glu Gly Ser
Asp Gly Glu Glu Arg 410 415 420 Pro Ser Cys Ser Ala Gln Gln Pro Pro
Ser Pro Gly Ser Pro Glu 425 430 435 Gly Pro Arg Ser Leu Cys Ser Lys
Leu Ala Cys Cys Glu Trp Cys 440 445 450 Lys Arg Phe Lys Lys Tyr Phe
Cys Met Val Pro Asp Cys Glu Gly 455 460 465 Ser Thr Trp Gln Gln Gly
Arg Leu Cys Ile His Val Tyr Arg Leu 470 475 480 Asp Asn Tyr Ser Arg
Val Val Phe Pro Val Thr Phe Phe Phe Phe 485 490 495 Asn Val Leu Tyr
Trp Leu Val Cys Leu Asn Leu 500 505 80 1212 PRT Homo Sapien 80 Met
Glu Pro Arg Pro Thr Ala Pro Ser Ser Gly Ala Pro Gly Leu 1 5 10 15
Ala Gly Val Gly Glu Thr Pro Ser Ala Ala Ala Leu Ala Ala Ala 20 25
30 Arg Val Glu Leu Pro Gly Thr Ala Val Pro Ser Val Pro Glu Asp 35
40 45 Ala Ala Pro Ala Ser Arg Asp Gly Gly Gly Val Arg Asp Glu Gly
50 55 60 Pro Ala Ala Ala Gly Asp Gly Leu Gly Arg Pro Leu Gly Pro
Thr 65 70 75 Pro Ser Gln Ser Arg Phe Gln Val Asp Leu Val Ser Glu
Asn Ala 80 85 90 Gly Arg Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala 95 100 105 Ala Ala Gly Ala Gly Ala Gly Ala Lys Gln Thr
Pro Ala Asp Gly 110 115 120 Glu Ala Ser Gly Glu Ser Glu Pro Ala Lys
Gly Ser Glu Glu Ala 125 130 135 Lys Gly Arg Phe Arg Val Asn Phe Val
Asp Pro Ala Ala Ser Ser 140 145 150 Ser Ala Glu Asp Ser Leu Ser Asp
Ala Ala Gly Val Gly Val Asp 155 160 165 Gly Pro Asn Val Ser Phe Gln
Asn Gly Gly Asp Thr Val Leu Ser 170 175 180 Glu Gly Ser Ser Leu His
Ser Gly Gly Gly Gly Gly Ser Gly His 185 190 195 His Gln His Tyr Tyr
Tyr Asp Thr His Thr Asn Thr Tyr Tyr Leu 200 205 210 Arg Thr Phe Gly
His Asn Thr Met Asp Ala Val Pro Arg Ile Asp 215 220 225 His Tyr Arg
His Thr Ala Ala Gln Leu Gly Glu Lys Leu Leu Arg 230 235 240 Pro Ser
Leu Ala Glu Leu His Asp Glu Leu Glu Lys Glu Pro Phe 245 250 255 Glu
Asp Gly Phe Ala Asn Gly Glu Glu Ser Thr Pro Thr Arg Asp 260 265 270
Ala Val Val Thr Tyr Thr Ala Glu Ser Lys Gly Val Val Lys Phe 275 280
285 Gly Trp Ile Lys Gly Val Leu Val Arg Cys Met Leu Asn Ile Trp 290
295 300 Gly Val Met Leu Phe Ile Arg Leu Ser Trp Ile Val Gly Gln Ala
305 310 315 Gly Ile Gly Leu Ser Val Leu Val Ile Met Met Ala Thr Val
Val 320 325 330 Thr Thr Ile Thr Gly Leu Ser Thr Ser Ala Ile Ala Thr
Asn Gly 335 340 345 Phe Val Arg Gly Gly Gly Ala Tyr Tyr Leu Ile Ser
Arg Ser Leu 350 355 360 Gly Pro Glu Phe Gly Gly Ala Ile Gly Leu Ile
Phe Ala Phe Ala 365 370 375 Asn Ala Val Ala Val Ala Met Tyr Val Val
Gly Phe Ala Glu Thr 380 385 390 Val Val Glu Leu Leu Lys Glu His Ser
Ile Leu Met Ile Asp Glu 395 400 405 Ile Asn Asp Ile Arg Ile Ile Gly
Ala Ile Thr Val Val Ile Leu 410 415 420 Leu Gly Ile Ser Val Ala Gly
Met Glu Trp Glu Ala Lys Ala Gln 425 430 435 Ile Val Leu Leu Val Ile
Leu Leu Leu Ala Ile Gly Asp Phe Val 440 445 450 Ile Gly Thr Phe Ile
Pro Leu Glu Ser Lys Lys Pro Lys Gly Phe 455 460 465 Phe Gly Tyr Lys
Ser Glu Ile Phe Asn Glu Asn Phe Gly Pro Asp 470 475 480 Phe Arg Glu
Glu Glu Thr Phe Phe Ser Val Phe Ala Ile Phe Phe 485 490 495 Pro Ala
Ala Thr Gly Ile Leu Ala Gly Ala Asn Ile Ser Gly Asp 500 505 510 Leu
Ala Asp Pro Gln Ser Ala Ile Pro Lys Gly Thr Leu Leu Ala 515 520 525
Ile Leu Ile Thr Thr Leu Val Tyr Val Gly Ile Ala Val Ser Val 530 535
540 Gly Ser Cys Val Val Arg Asp Ala Thr Gly Asn Val Asn Asp Thr 545
550 555 Ile Val Thr Glu Leu Thr Asn Cys Thr Ser Ala Ala Cys Lys Leu
560 565 570 Asn Phe Asp Phe Ser Ser Cys Glu Ser Ser Pro Cys Ser Tyr
Gly 575 580 585 Leu Met Asn Asn Phe Gln Val Met Ser Met Val Ser Gly
Phe Thr 590 595 600 Pro Leu Ile Ser Ala Gly Ile Phe Ser Ala Thr Leu
Ser Ser Ala 605 610 615 Leu Ala Ser Leu Val Ser Ala Pro Lys Ile Phe
Gln Ala Leu Cys 620 625 630 Lys Asp Asn Ile Tyr Pro Ala Phe Gln Met
Phe Ala Lys Gly Tyr 635 640 645 Gly Lys Asn Asn Glu Pro Leu Arg Gly
Tyr Ile Leu Thr Phe Leu 650 655 660 Ile Ala Leu Gly Phe Ile Leu Ile
Ala Glu Leu Asn Val Ile Ala 665 670 675 Pro Ile Ile Ser Asn Phe Phe
Leu Ala Ser Tyr Ala Leu Ile Asn 680 685 690 Phe Ser Val Phe His Ala
Ser Leu Ala Lys Ser Pro Gly Trp Arg 695 700 705 Pro Ala Phe Lys Tyr
Tyr Asn Met Trp Ile Ser Leu Leu Gly Ala 710 715 720 Ile Leu Cys Cys
Ile Val Met Phe Val Ile Asn Trp Trp Ala Ala 725 730 735 Leu Leu Thr
Tyr Val Ile Val Leu Gly Leu Tyr Ile Tyr Val Thr 740 745 750 Tyr Lys
Lys Pro Asp Val Asn Trp Gly Ser Ser Thr Gln Ala Leu 755 760 765 Thr
Tyr Leu Asn Ala Leu Gln His Ser Ile Arg Leu Ser Gly Val 770 775 780
Glu Asp His Val Lys Asn Phe Arg Pro Gln Cys Leu Val Met Thr 785 790
795 Gly Ala Pro Asn Ser Arg Pro Ala Leu Leu His Leu Val His Asp 800
805 810 Phe Thr Lys Asn Val Gly Leu Met Ile Cys Gly His Val His Met
815 820 825 Gly Pro Arg Arg Gln Ala Met Lys Glu Met Ser Ile Asp Gln
Ala 830 835 840 Lys Tyr Gln Arg Trp Leu Ile Lys Asn Lys Met Lys Ala
Phe Tyr 845 850 855 Ala Pro Val His Ala Asp Asp Leu Arg Glu Gly Ala
Gln Tyr Leu 860 865 870 Met Gln Ala Ala Gly Leu Gly Arg Met Lys Pro
Asn Thr Leu Val 875 880 885 Leu Gly Phe Lys Lys Asp Trp Leu Gln Ala
Asp Met Arg Asp Val 890 895 900 Asp Met Tyr Ile Asn Leu Phe His Asp
Ala Phe Asp Ile Gln Tyr 905 910 915 Gly Val Val Val Ile Arg Leu Lys
Glu Gly Leu Asp Ile Ser His 920 925 930 Leu Gln Gly Gln Glu Glu Leu
Leu Ser Ser Gln Glu Lys Ser Pro 935 940 945 Gly Thr Lys Asp Val Val
Val Ser Val Glu Tyr Ser Lys Lys Ser 950 955 960 Asp Leu Asp Thr Ser
Lys Pro Leu Ser Glu Lys Pro Ile Thr His 965 970 975 Lys Val Glu Glu
Glu Asp Gly Lys Thr Ala Thr Gln Pro Leu Leu 980 985 990 Lys Lys Glu
Ser Lys Gly Pro Ile Val Pro Leu Asn Val Ala Asp 995 1000 1005 Gln
Lys Leu Leu Glu Ala Ser Thr Gln Phe Gln Lys Lys Gln Gly 1010 1015
1020 Lys Asn Thr Ile Asp Val Trp Trp Leu Phe Asp Asp Gly Gly Leu
1025 1030 1035 Thr Leu Leu Ile Pro Tyr Leu Leu Thr Thr Lys Lys Lys
Trp Lys 1040 1045 1050 Asp Cys Lys Ile Arg Val Phe Ile Gly Gly Lys
Ile Asn Arg Ile 1055 1060 1065 Asp His Asp Arg Arg Ala Met Ala Thr
Leu Leu Ser Lys Phe Arg 1070 1075 1080 Ile Asp Phe Ser Asp Ile Met
Val Leu Gly Asp Ile Asn Thr Lys 1085 1090 1095 Pro Lys Lys Glu Asn
Ile Ile Ala Phe Glu Glu Ile Ile Glu Pro 1100 1105 1110 Tyr Arg Leu
His Glu Asp Asp Lys Glu Gln Asp Ile Ala Asp Lys 1115 1120 1125 Met
Lys Glu Asp Glu Pro Trp Arg Ile Thr Asp Asn Glu Leu Glu 1130 1135
1140 Leu Tyr Lys Thr Lys Thr Tyr Arg Gln Ile Arg Leu Asn Glu Leu
1145 1150 1155 Leu Lys Glu His Ser Ser Thr Ala Asn Ile Ile Val Met
Ser Leu 1160 1165 1170 Pro Val Ala Arg Lys Gly Ala Val Ser Ser Ala
Leu Tyr Met Ala 1175 1180 1185 Trp Leu Glu Ala Leu Ser Lys Asp Leu
Pro Pro Ile Leu Leu Val 1190 1195 1200 Arg Gly Asn His Gln Ser Val
Leu Thr Phe Tyr Ser 1205 1210 81 674 PRT Homo Sapien 81 Met Ala Thr
Ala Val Ser Arg Pro Cys Ala Gly Arg Ser Arg Asp 1 5 10 15 Ile Leu
Trp Arg Val Leu Gly Trp Arg Ile Val Ala Ser Ile Val 20 25 30 Trp
Ser Val Leu Phe Leu Pro Ile Cys Thr Thr Val Phe Ile Ile 35 40 45
Phe Ser Arg Ile Asp Leu Phe His Pro Ile Gln Trp Leu Ser Asp 50 55
60 Ser Phe Ser Asp Leu Tyr Ser Ser Tyr Val Ile Phe Tyr Phe Leu 65
70 75 Leu Leu Ser Val Val Ile Ile Ile Ile Ser Ile Phe Asn Val Glu
80 85 90 Phe Tyr Ala Val Val Pro Ser Ile Pro Cys Ser Arg Leu Ala
Leu 95 100 105 Ile Gly Lys Ile Ile His Pro Gln Gln Leu Met His Ser
Phe Ile 110 115 120 His Ala Ala Met Gly Met Val Met Ala Trp Cys Ala
Ala Val Ile 125 130 135 Thr Gln Gly Gln Tyr Ser Phe Leu Val Val Pro
Cys Thr Gly Thr 140 145 150 Asn Ser Phe Gly Ser Pro Ala Ala Gln Thr
Cys Leu Asn Glu Tyr 155 160 165 His Leu Phe Phe Leu Leu Thr Gly Ala
Phe Met Gly Tyr Ser Tyr 170 175 180 Ser Leu Leu Tyr Phe Val Asn Asn
Met Asn Tyr Leu Pro Phe Pro 185 190 195 Ile Ile Gln Gln Tyr Lys Phe
Leu Arg Phe Arg Arg Ser Leu Leu 200 205 210 Leu Leu Val Lys His Ser
Cys Val Glu Ser Leu Phe Leu Val Arg 215 220 225 Asn Phe Cys Ile Leu
Tyr Tyr Phe Leu Gly Tyr Ile Pro Lys Ala 230 235 240 Trp Ile Ser Thr
Ala Met Asn Leu His Ile Asp Glu Gln Val His 245 250 255 Arg Pro Leu
Asp Thr Val Ser Gly Leu Leu Asn Leu Ser Leu Leu 260 265 270 Tyr His
Val Trp Leu Cys Gly Val Phe Leu Leu Thr Thr Trp Tyr 275 280 285 Val
Ser Trp Ile Leu Phe Lys Ile Tyr Ala Thr Glu Ala His Val 290 295 300
Phe Pro Val Gln Pro Pro Phe Ala Glu Gly Ser Asp Glu Cys Leu 305 310
315 Pro Lys Val Leu Asn Ser Asn Pro Pro Pro Ile Ile Lys Tyr Leu 320
325 330 Ala Leu Gln Asp Leu Met Leu Leu Ser Gln Tyr Ser Pro Ser Arg
335 340 345 Arg Gln Glu Val Phe Ser Leu Ser Gln Pro Gly Gly His Pro
His 350 355 360 Asn Trp Thr Ala Ile Ser Arg Glu Cys Leu Asn Leu Leu
Asn Gly 365 370 375 Met Thr Gln Lys Leu Ile Leu Tyr Gln Glu Ala Ala
Ala Thr Asn 380 385 390 Gly Arg Val Ser Ser Ser Tyr Pro Val Glu Pro
Lys Lys Leu Asn 395 400 405 Ser Pro Glu Glu Thr Ala Phe Gln Thr Pro
Lys Ser Ser Gln Met 410 415 420 Pro Arg Pro Ser Val Pro Pro Leu Val
Lys Thr Ser Leu Phe Ser 425 430 435 Ser Lys Leu Ser Thr Pro Asp Val
Val Ser Pro Phe Gly Thr Pro 440 445 450 Phe Gly Ser Ser Val Met Asn
Arg Met Ala Gly Ile Phe Asp Val 455 460 465 Asn Thr Cys Tyr Gly Ser
Pro Gln Ser Pro Gln Leu Ile Arg Arg 470 475 480 Gly Pro Arg Leu Trp
Thr Ser Ala Ser Asp Gln Gln Met Thr Glu 485 490 495 Phe Ser Asn Pro
Ser Pro Ser Thr Ser Ile Ser Ala Glu Gly Lys 500 505 510 Thr Met Arg
Gln Pro Ser Val Ile Tyr Ser Trp Ile Gln Asn Lys 515 520 525 Arg Glu
Gln Ile Lys Asn Phe Leu Ser Lys Arg Val Leu Ile Met 530 535 540 Tyr
Phe Phe Ser Lys His Pro Glu Ala Ser Ile Gln Ala Val Phe 545 550 555
Ser Asp Ala Gln Met His Ile Trp Ala Leu Glu Gly Leu Ser His 560 565
570 Leu Val Ala Ala Ser Phe Thr Glu Asp Arg Phe Gly Val Val Gln 575
580 585 Thr Thr Leu Pro Ala Ile Leu Asn Thr Leu Leu Thr Leu Gln Glu
590 595 600 Ala Val Asp Lys Tyr Phe Lys Leu Pro His Ala Ser Ser Lys
Pro 605 610 615 Pro Arg Ile Ser Gly Ser Leu Val Asp Thr Ser Tyr Lys
Thr Leu 620 625 630 Arg Phe Ala Phe Arg Ala Ser Leu Lys Thr Ala Ile
Tyr Arg Ile 635 640 645 Thr Thr Thr Phe Gly Glu His Leu Asn Ala Val
Gln Ala Ser Ala 650 655 660 Glu His Gln Lys Arg Leu Gln Gln Phe Leu
Glu Phe Lys Glu 665 670 82 1321 PRT Homo Sapien 82 Met Gly Ala Pro
Phe Val Trp Ala Leu Gly Leu Leu Met Leu Gln 1 5 10 15 Met Leu Leu
Phe Val Ala Gly Glu Gln Gly Thr Gln Asp Ile Thr 20 25 30 Asp Ala
Ser Glu Arg Gly Leu His Met Gln Lys Leu Gly Ser Gly 35 40 45 Ser
Val Gln Ala Ala Leu Ala Glu Leu Val Ala Leu Pro Cys Leu 50 55 60
Phe Thr Leu Gln Pro Arg Pro Ser Ala Ala Arg Asp Ala Pro Arg 65 70
75 Ile Lys Trp Thr Lys Val Arg Thr Ala Ser Gly Gln Arg Gln Asp 80
85 90 Leu Pro Ile Leu Val Ala Lys Asp Asn Val Val Arg Val Ala Lys
95 100 105 Ser Trp Gln Gly Arg Val Ser Leu Pro Ser Tyr Pro Arg Arg
Arg 110 115 120 Ala Asn Ala Thr Leu Leu Leu Gly Pro Leu Arg Ala Ser
Asp Ser 125 130 135 Gly Leu Tyr Arg Cys Gln Val Val Arg Gly Ile Glu
Asp Glu Gln 140 145 150 Asp Leu Val Pro Leu Glu Val Thr Gly Val Val
Phe His Tyr Arg 155 160 165 Ser Ala Arg Asp Arg Tyr Ala Leu Thr Phe
Ala Glu Ala Gln Glu 170 175 180 Ala Cys Arg Leu Ser Ser Ala Ile Ile
Ala Ala Pro Arg His Leu 185 190 195 Gln Ala Ala Phe Glu Asp Gly Phe
Asp Asn Cys Asp Ala Gly Trp 200 205 210 Leu Ser Asp Arg Thr Val Arg
Tyr Pro Ile Thr Gln Ser Arg Pro 215 220 225 Gly Cys Tyr Gly Asp Arg
Ser
Ser Leu Pro Gly Val Arg Ser Tyr 230 235 240 Gly Arg Arg Asn Pro Gln
Glu Leu Tyr Asp Val Tyr Cys Phe Ala 245 250 255 Arg Glu Leu Gly Gly
Glu Val Phe Tyr Val Gly Pro Ala Arg Arg 260 265 270 Leu Thr Leu Ala
Gly Ala Arg Ala Gln Cys Arg Arg Gln Gly Ala 275 280 285 Ala Leu Ala
Ser Val Gly Gln Leu His Leu Ala Trp His Glu Gly 290 295 300 Leu Asp
Gln Cys Asp Pro Gly Trp Leu Ala Asp Gly Ser Val Arg 305 310 315 Tyr
Pro Ile Gln Thr Pro Arg Arg Arg Cys Gly Gly Pro Ala Pro 320 325 330
Gly Val Arg Thr Val Tyr Arg Phe Ala Asn Arg Thr Gly Phe Pro 335 340
345 Ser Pro Ala Glu Arg Phe Asp Ala Tyr Cys Phe Arg Ala His His 350
355 360 Pro Thr Ser Gln His Gly Asp Leu Glu Thr Pro Ser Ser Gly Asp
365 370 375 Glu Gly Glu Ile Leu Ser Ala Glu Gly Pro Pro Val Arg Glu
Leu 380 385 390 Glu Pro Thr Leu Glu Glu Glu Glu Val Val Thr Pro Asp
Phe Gln 395 400 405 Glu Pro Leu Val Ser Ser Gly Glu Glu Glu Thr Leu
Ile Leu Glu 410 415 420 Glu Lys Gln Glu Ser Gln Gln Thr Leu Ser Pro
Thr Pro Gly Asp 425 430 435 Pro Met Leu Ala Ser Trp Pro Thr Gly Glu
Val Trp Leu Ser Thr 440 445 450 Val Ala Pro Ser Pro Ser Asp Met Gly
Ala Gly Thr Ala Ala Ser 455 460 465 Ser His Thr Glu Val Ala Pro Thr
Asp Pro Met Pro Arg Arg Arg 470 475 480 Gly Arg Phe Lys Gly Leu Asn
Gly Arg Tyr Phe Gln Gln Gln Glu 485 490 495 Pro Glu Pro Gly Leu Gln
Gly Gly Met Glu Ala Ser Ala Gln Pro 500 505 510 Pro Thr Ser Glu Ala
Ala Val Asn Gln Met Glu Pro Pro Leu Ala 515 520 525 Met Ala Val Thr
Glu Met Leu Gly Ser Gly Gln Ser Arg Ser Pro 530 535 540 Trp Ala Asp
Leu Thr Asn Glu Val Asp Met Pro Gly Ala Gly Ser 545 550 555 Ala Gly
Gly Lys Ser Ser Pro Glu Pro Trp Leu Trp Pro Pro Thr 560 565 570 Met
Val Pro Pro Ser Ile Ser Gly His Ser Arg Ala Pro Val Leu 575 580 585
Glu Leu Glu Lys Ala Glu Gly Pro Ser Ala Arg Pro Ala Thr Pro 590 595
600 Asp Leu Phe Trp Ser Pro Leu Glu Ala Thr Val Ser Ala Pro Ser 605
610 615 Pro Ala Pro Trp Glu Ala Phe Pro Val Ala Thr Ser Pro Asp Leu
620 625 630 Pro Met Met Ala Met Leu Arg Gly Pro Lys Glu Trp Met Leu
Pro 635 640 645 His Pro Thr Pro Ile Ser Thr Glu Ala Asn Arg Val Glu
Ala His 650 655 660 Gly Glu Ala Thr Ala Thr Ala Pro Pro Ser Pro Ala
Ala Glu Thr 665 670 675 Lys Val Tyr Ser Leu Pro Leu Ser Leu Thr Pro
Thr Gly Gln Gly 680 685 690 Gly Glu Ala Met Pro Thr Thr Pro Glu Ser
Pro Arg Ala Asp Phe 695 700 705 Arg Glu Thr Gly Glu Thr Ser Pro Ala
Gln Val Asn Lys Ala Glu 710 715 720 His Ser Ser Ser Ser Pro Trp Pro
Ser Val Asn Arg Asn Val Ala 725 730 735 Val Gly Phe Val Pro Thr Glu
Thr Ala Thr Glu Pro Thr Gly Leu 740 745 750 Arg Gly Ile Pro Gly Ser
Glu Ser Gly Val Phe Asp Thr Ala Glu 755 760 765 Ser Pro Thr Ser Gly
Leu Gln Ala Thr Val Asp Glu Val Gln Asp 770 775 780 Pro Trp Pro Ser
Val Tyr Ser Lys Gly Leu Asp Ala Ser Ser Pro 785 790 795 Ser Ala Pro
Leu Gly Ser Pro Gly Val Phe Leu Val Pro Lys Val 800 805 810 Thr Pro
Asn Leu Glu Pro Trp Val Ala Thr Asp Glu Gly Pro Thr 815 820 825 Val
Asn Pro Met Asp Ser Thr Val Thr Pro Ala Pro Ser Asp Ala 830 835 840
Ser Gly Ile Trp Glu Pro Gly Ser Gln Val Phe Glu Glu Ala Glu 845 850
855 Ser Thr Thr Leu Ser Pro Gln Val Ala Leu Asp Thr Ser Ile Val 860
865 870 Thr Pro Leu Thr Thr Leu Glu Gln Gly Asp Lys Val Gly Val Pro
875 880 885 Ala Met Ser Thr Leu Gly Ser Ser Ser Ser Gln Pro His Pro
Glu 890 895 900 Pro Glu Asp Gln Val Glu Thr Gln Gly Thr Ser Gly Ala
Ser Val 905 910 915 Pro Pro His Gln Ser Ser Pro Leu Gly Lys Pro Ala
Val Pro Pro 920 925 930 Gly Thr Pro Thr Ala Ala Ser Val Gly Glu Ser
Ala Ser Val Ser 935 940 945 Ser Gly Glu Pro Thr Val Pro Trp Asp Pro
Ser Ser Thr Leu Leu 950 955 960 Pro Val Thr Leu Gly Ile Glu Asp Phe
Glu Leu Glu Val Leu Ala 965 970 975 Gly Ser Pro Gly Val Glu Ser Phe
Trp Glu Glu Val Ala Ser Gly 980 985 990 Glu Glu Pro Ala Leu Pro Gly
Thr Pro Met Asn Ala Gly Ala Glu 995 1000 1005 Glu Val His Ser Asp
Pro Cys Glu Asn Asn Pro Cys Leu His Gly 1010 1015 1020 Gly Thr Cys
Asn Ala Asn Gly Thr Met Tyr Gly Cys Ser Cys Asp 1025 1030 1035 Gln
Gly Phe Ala Gly Glu Asn Cys Glu Ile Asp Ile Asp Asp Cys 1040 1045
1050 Leu Cys Ser Pro Cys Glu Asn Gly Gly Thr Cys Ile Asp Glu Val
1055 1060 1065 Asn Gly Phe Val Cys Leu Cys Leu Pro Ser Tyr Gly Gly
Ser Phe 1070 1075 1080 Cys Glu Lys Asp Thr Glu Gly Cys Asp Arg Gly
Trp His Lys Phe 1085 1090 1095 Gln Gly His Cys Tyr Arg Tyr Phe Ala
His Arg Arg Ala Trp Glu 1100 1105 1110 Asp Ala Glu Lys Asp Cys Arg
Arg Arg Ser Gly His Leu Thr Ser 1115 1120 1125 Val His Ser Pro Glu
Glu His Ser Phe Ile Asn Ser Phe Gly His 1130 1135 1140 Glu Asn Thr
Trp Ile Gly Leu Asn Asp Arg Ile Val Glu Arg Asp 1145 1150 1155 Phe
Gln Trp Thr Asp Asn Thr Gly Leu Gln Phe Glu Asn Trp Arg 1160 1165
1170 Glu Asn Gln Pro Asp Asn Phe Phe Ala Gly Gly Glu Asp Cys Val
1175 1180 1185 Val Met Val Ala His Glu Ser Gly Arg Trp Asn Asp Val
Pro Cys 1190 1195 1200 Asn Tyr Asn Leu Pro Tyr Val Cys Lys Lys Gly
Thr Val Leu Cys 1205 1210 1215 Gly Pro Pro Pro Ala Val Glu Asn Ala
Ser Leu Ile Gly Ala Arg 1220 1225 1230 Lys Ala Lys Asn Asn Val His
Ala Thr Val Arg Tyr Gln Cys Asn 1235 1240 1245 Glu Gly Phe Ala Gln
His His Val Val Thr Ile Arg Cys Arg Ser 1250 1255 1260 Asn Gly Lys
Trp Asp Arg Pro Gln Ile Val Cys Thr Lys Pro Arg 1265 1270 1275 Arg
Ser His Arg Met Arg Gly His His His His His Gln His His 1280 1285
1290 His Gln His His His His Lys Ser Arg Lys Glu Arg Arg Lys His
1295 1300 1305 Lys Lys His Pro Thr Glu Asp Trp Glu Lys Asp Glu Gly
Asn Phe 1310 1315 1320 Cys 83 696 PRT Homo Sapien 83 Met Lys Phe
Ala Glu His Leu Ser Ala His Ile Thr Pro Glu Trp 1 5 10 15 Arg Lys
Gln Tyr Ile Gln Tyr Glu Ala Phe Lys Asp Met Leu Tyr 20 25 30 Ser
Ala Gln Asp Gln Ala Pro Ser Val Glu Val Thr Asp Glu Asp 35 40 45
Thr Val Lys Arg Tyr Phe Ala Lys Phe Glu Glu Lys Phe Phe Gln 50 55
60 Thr Cys Glu Lys Glu Leu Ala Lys Ile Asn Thr Phe Tyr Ser Glu 65
70 75 Lys Leu Ala Glu Ala Gln Arg Arg Phe Ala Thr Leu Gln Asn Glu
80 85 90 Leu Gln Ser Ser Leu Asp Ala Gln Lys Glu Ser Thr Gly Val
Thr 95 100 105 Thr Leu Arg Gln Arg Arg Lys Pro Val Phe His Leu Ser
His Glu 110 115 120 Glu Arg Val Gln His Arg Asn Ile Lys Asp Leu Lys
Leu Ala Phe 125 130 135 Ser Glu Phe Tyr Leu Ser Leu Ile Leu Leu Gln
Asn Tyr Gln Asn 140 145 150 Leu Asn Phe Thr Gly Phe Arg Lys Ile Leu
Lys Lys His Asp Lys 155 160 165 Ile Leu Glu Thr Ser Arg Gly Ala Asp
Trp Arg Val Ala His Val 170 175 180 Glu Val Ala Pro Phe Tyr Thr Cys
Lys Lys Ile Asn Gln Leu Ile 185 190 195 Ser Glu Thr Glu Ala Val Val
Thr Asn Glu Leu Glu Asp Gly Asp 200 205 210 Arg Gln Lys Ala Met Lys
Arg Leu Arg Val Pro Pro Leu Gly Ala 215 220 225 Ala Gln Pro Ala Pro
Ala Trp Thr Thr Phe Arg Val Gly Leu Phe 230 235 240 Cys Gly Ile Phe
Ile Val Leu Asn Ile Thr Leu Val Leu Ala Ala 245 250 255 Val Phe Lys
Leu Glu Thr Asp Arg Ser Ile Trp Pro Leu Ile Arg 260 265 270 Ile Tyr
Arg Gly Gly Phe Leu Leu Ile Glu Phe Leu Phe Leu Leu 275 280 285 Gly
Ile Asn Thr Tyr Gly Trp Arg Gln Ala Gly Val Asn His Val 290 295 300
Leu Ile Phe Glu Leu Asn Pro Arg Ser Asn Leu Ser His Gln His 305 310
315 Leu Phe Glu Ile Ala Gly Phe Leu Gly Ile Leu Trp Cys Leu Ser 320
325 330 Leu Leu Ala Cys Phe Phe Ala Pro Ile Ser Val Ile Pro Thr Tyr
335 340 345 Val Tyr Pro Leu Ala Leu Tyr Gly Phe Met Val Phe Phe Leu
Ile 350 355 360 Asn Pro Thr Lys Thr Phe Tyr Tyr Lys Ser Arg Phe Trp
Leu Leu 365 370 375 Lys Leu Leu Phe Arg Val Phe Thr Ala Pro Phe His
Lys Val Gly 380 385 390 Phe Ala Asp Phe Trp Leu Ala Asp Gln Leu Asn
Ser Leu Ser Val 395 400 405 Ile Leu Met Asp Leu Glu Tyr Met Ile Cys
Phe Tyr Ser Leu Glu 410 415 420 Leu Lys Trp Asp Glu Ser Lys Gly Leu
Leu Pro Asn Asn Ser Glu 425 430 435 Glu Ser Gly Ile Cys His Lys Tyr
Thr Tyr Gly Val Arg Ala Ile 440 445 450 Val Gln Cys Ile Pro Ala Trp
Leu Arg Phe Ile Gln Cys Leu Arg 455 460 465 Arg Tyr Arg Asp Thr Lys
Arg Ala Phe Pro His Leu Val Asn Ala 470 475 480 Gly Lys Tyr Ser Thr
Thr Phe Phe Met Val Ala Phe Ala Ala Leu 485 490 495 Tyr Ser Thr His
Lys Glu Arg Gly His Ser Asp Thr Met Val Phe 500 505 510 Phe Tyr Leu
Trp Ile Val Phe Tyr Ile Ile Ser Ser Cys Tyr Thr 515 520 525 Leu Ile
Trp Asp Leu Lys Met Asp Trp Gly Leu Phe Asp Lys Asn 530 535 540 Ala
Gly Glu Asn Thr Phe Leu Arg Glu Glu Ile Val Tyr Pro Gln 545 550 555
Lys Ala Tyr Tyr Tyr Cys Ala Ile Ile Glu Asp Val Ile Leu Arg 560 565
570 Phe Ala Trp Thr Ile Gln Ile Ser Ile Thr Ser Thr Thr Leu Leu 575
580 585 Pro His Ser Gly Asp Ile Ile Ala Thr Val Phe Ala Pro Leu Glu
590 595 600 Val Phe Arg Arg Phe Val Trp Asn Phe Phe Arg Leu Glu Asn
Glu 605 610 615 His Leu Asn Asn Cys Gly Glu Phe Arg Ala Val Arg Asp
Ile Ser 620 625 630 Val Ala Pro Leu Asn Ala Asp Asp Gln Thr Leu Leu
Glu Gln Met 635 640 645 Met Asp Gln Asp Asp Gly Val Arg Asn Arg Gln
Lys Asn Arg Ser 650 655 660 Trp Lys Tyr Asn Gln Ser Ile Ser Leu Arg
Arg Pro Arg Leu Ala 665 670 675 Ser Gln Ser Lys Ala Arg Asp Thr Lys
Val Leu Ile Glu Asp Thr 680 685 690 Asp Asp Glu Ala Asn Thr 695 84
696 PRT Homo Sapien 84 Met Lys Phe Ala Glu His Leu Ser Ala His Ile
Thr Pro Glu Trp 1 5 10 15 Arg Lys Gln Tyr Ile Gln Tyr Glu Ala Phe
Lys Asp Met Leu Tyr 20 25 30 Ser Ala Gln Asp Gln Ala Pro Ser Val
Glu Val Thr Asp Glu Asp 35 40 45 Thr Val Lys Arg Tyr Phe Ala Lys
Phe Glu Glu Lys Phe Phe Gln 50 55 60 Thr Cys Glu Lys Glu Leu Ala
Lys Ile Asn Thr Phe Tyr Ser Glu 65 70 75 Lys Leu Ala Glu Ala Gln
Arg Arg Phe Ala Thr Leu Gln Asn Glu 80 85 90 Leu Gln Ser Ser Leu
Asp Ala Gln Lys Glu Ser Thr Gly Val Thr 95 100 105 Thr Leu Arg Gln
Arg Arg Lys Pro Val Phe His Leu Ser His Glu 110 115 120 Glu Arg Val
Gln His Arg Asn Ile Lys Asp Leu Lys Leu Ala Phe 125 130 135 Ser Glu
Phe Tyr Leu Ser Leu Ile Leu Leu Gln Asn Tyr Gln Asn 140 145 150 Leu
Asn Phe Thr Gly Phe Arg Lys Ile Leu Lys Lys His Asp Lys 155 160 165
Ile Leu Glu Thr Ser Arg Gly Ala Asp Trp Arg Val Ala His Val 170 175
180 Glu Val Ala Pro Phe Tyr Thr Cys Lys Lys Ile Asn Gln Leu Ile 185
190 195 Ser Glu Thr Glu Ala Val Val Thr Asn Glu Leu Glu Asp Gly Asp
200 205 210 Arg Gln Lys Ala Met Lys Arg Leu Arg Val Pro Pro Leu Gly
Ala 215 220 225 Ala Gln Pro Ala Pro Ala Trp Thr Thr Phe Arg Val Gly
Leu Phe 230 235 240 Cys Gly Ile Phe Ile Val Leu Asn Ile Thr Leu Val
Leu Ala Ala 245 250 255 Val Phe Lys Leu Glu Thr Asp Arg Ser Ile Trp
Pro Leu Ile Arg 260 265 270 Ile Tyr Arg Gly Gly Phe Leu Leu Ile Glu
Phe Leu Phe Leu Leu 275 280 285 Gly Ile Asn Thr Tyr Gly Trp Arg Gln
Ala Gly Val Asn His Val 290 295 300 Leu Ile Phe Glu Leu Asn Pro Arg
Ser Asn Leu Ser His Gln His 305 310 315 Leu Phe Glu Ile Ala Gly Phe
Leu Gly Ile Leu Trp Cys Leu Ser 320 325 330 Leu Leu Ala Cys Phe Phe
Ala Pro Ile Ser Val Ile Pro Thr Tyr 335 340 345 Val Tyr Pro Leu Ala
Leu Tyr Gly Phe Met Val Phe Phe Leu Ile 350 355 360 Asn Pro Thr Lys
Thr Phe Tyr Tyr Lys Ser Arg Phe Trp Leu Leu 365 370 375 Lys Leu Leu
Phe Arg Val Phe Thr Ala Pro Phe His Lys Val Gly 380 385 390 Phe Ala
Asp Phe Trp Leu Ala Asp Gln Leu Asn Ser Leu Ser Val 395 400 405 Ile
Leu Met Asp Leu Glu Tyr Met Ile Cys Phe Tyr Ser Leu Glu 410 415 420
Leu Lys Trp Asp Glu Ser Lys Gly Leu Leu Pro Asn Asn Ser Glu 425 430
435 Glu Ser Gly Ile Cys His Lys Tyr Thr Tyr Gly Val Arg Ala Ile 440
445 450 Val Gln Cys Ile Pro Ala Trp Leu Arg Phe Ile Gln Cys Leu Arg
455 460 465 Arg Tyr Arg Asp Thr Lys Arg Ala Phe Pro His Leu Val Asn
Ala 470 475 480 Gly Lys Tyr Ser Thr Thr Phe Phe Met Val Thr Phe Ala
Ala Leu 485 490 495 Tyr Ser Thr His Lys Glu Arg Gly His Ser Asp Thr
Met Val Phe 500
505 510 Phe Tyr Leu Trp Ile Val Phe Tyr Ile Ile Ser Ser Cys Tyr Thr
515 520 525 Leu Ile Trp Asp Leu Lys Met Asp Trp Gly Leu Phe Asp Lys
Asn 530 535 540 Ala Gly Glu Asn Thr Phe Leu Arg Glu Glu Ile Val Tyr
Pro Gln 545 550 555 Lys Ala Tyr Tyr Tyr Cys Ala Ile Ile Glu Asp Val
Ile Leu Arg 560 565 570 Phe Ala Trp Thr Ile Gln Ile Ser Ile Thr Ser
Thr Thr Leu Leu 575 580 585 Pro His Ser Gly Asp Ile Ile Ala Thr Val
Phe Ala Pro Leu Glu 590 595 600 Val Phe Arg Arg Phe Val Trp Asn Phe
Phe Arg Leu Glu Asn Glu 605 610 615 His Leu Asn Asn Cys Gly Glu Phe
Arg Ala Val Arg Asp Ile Ser 620 625 630 Val Ala Pro Leu Asn Ala Asp
Asp Gln Thr Leu Leu Glu Gln Met 635 640 645 Met Asp Gln Asp Asp Gly
Val Arg Asn Arg Gln Lys Asn Arg Ser 650 655 660 Trp Lys Tyr Asn Gln
Ser Ile Ser Leu Arg Arg Pro Arg Leu Ala 665 670 675 Ser Gln Ser Lys
Ala Arg Asp Thr Lys Val Leu Ile Glu Asp Thr 680 685 690 Asp Asp Glu
Ala Asn Thr 695 85 635 PRT Homo Sapien 85 Met Ser Val Gly Val Ser
Thr Ser Ala Pro Leu Ser Pro Thr Ser 1 5 10 15 Gly Thr Ser Val Gly
Met Ser Thr Phe Ser Ile Met Asp Tyr Val 20 25 30 Val Phe Val Leu
Leu Leu Val Leu Ser Leu Ala Ile Gly Leu Tyr 35 40 45 His Ala Cys
Arg Gly Trp Gly Arg His Thr Val Gly Glu Leu Leu 50 55 60 Met Ala
Asp Arg Lys Met Gly Cys Leu Pro Val Ala Leu Ser Leu 65 70 75 Leu
Ala Thr Phe Gln Ser Ala Val Ala Ile Leu Gly Val Pro Ser 80 85 90
Glu Ile Tyr Arg Phe Gly Thr Gln Tyr Trp Phe Leu Gly Cys Cys 95 100
105 Tyr Phe Leu Gly Leu Leu Ile Pro Ala His Ile Phe Ile Pro Val 110
115 120 Phe Tyr Arg Leu His Leu Thr Ser Ala Tyr Glu Tyr Leu Glu Leu
125 130 135 Arg Phe Asn Lys Thr Val Arg Val Cys Gly Thr Val Thr Phe
Ile 140 145 150 Phe Gln Met Val Ile Tyr Met Gly Val Val Leu Tyr Ala
Pro Ser 155 160 165 Leu Ala Leu Asn Ala Val Thr Gly Phe Asp Leu Trp
Leu Ser Val 170 175 180 Leu Ala Leu Gly Ile Val Cys Thr Val Tyr Thr
Ala Leu Gly Gly 185 190 195 Leu Lys Ala Val Ile Trp Thr Asp Val Phe
Gln Thr Leu Val Met 200 205 210 Phe Leu Gly Gln Leu Ala Val Ile Ile
Val Gly Ser Ala Lys Val 215 220 225 Gly Gly Leu Gly Arg Val Trp Ala
Val Ala Ser Gln His Gly Arg 230 235 240 Ile Ser Gly Phe Glu Leu Asp
Pro Asp Pro Phe Val Arg His Thr 245 250 255 Phe Trp Thr Leu Ala Phe
Gly Gly Val Phe Met Met Leu Ser Leu 260 265 270 Tyr Gly Val Asn Gln
Ala Gln Val Gln Arg Tyr Leu Ser Ser Arg 275 280 285 Thr Glu Lys Ala
Ala Val Leu Ser Cys Tyr Ala Val Phe Pro Phe 290 295 300 Gln Gln Val
Ser Leu Cys Val Gly Cys Leu Ile Gly Leu Val Met 305 310 315 Phe Ala
Tyr Tyr Gln Glu Tyr Pro Met Ser Ile Gln Gln Ala Gln 320 325 330 Ala
Ala Pro Asp Gln Phe Val Leu Tyr Phe Val Met Asp Leu Leu 335 340 345
Lys Gly Leu Pro Gly Leu Pro Gly Leu Phe Ile Ala Cys Leu Phe 350 355
360 Ser Gly Ser Leu Ser Thr Ile Ser Ser Ala Phe Asn Ser Leu Ala 365
370 375 Thr Val Thr Met Glu Asp Leu Ile Arg Pro Trp Phe Pro Glu Phe
380 385 390 Ser Glu Ala Arg Ala Ile Met Leu Ser Arg Gly Leu Ala Phe
Gly 395 400 405 Tyr Gly Leu Leu Cys Leu Gly Met Ala Tyr Ile Ser Ser
Gln Met 410 415 420 Gly Pro Val Leu Gln Ala Ala Ile Ser Ile Phe Gly
Met Val Gly 425 430 435 Gly Pro Leu Leu Gly Leu Phe Cys Leu Gly Met
Phe Phe Pro Cys 440 445 450 Ala Asn Pro Pro Gly Ala Val Val Gly Leu
Leu Ala Gly Leu Val 455 460 465 Met Ala Phe Trp Ile Gly Ile Gly Ser
Ile Val Thr Ser Met Gly 470 475 480 Phe Ser Met Pro Pro Ser Pro Ser
Asn Gly Ser Ser Phe Ser Leu 485 490 495 Pro Thr Asn Leu Thr Val Ala
Thr Val Thr Thr Leu Met Pro Leu 500 505 510 Thr Thr Phe Ser Lys Pro
Thr Gly Leu Gln Arg Phe Tyr Ser Leu 515 520 525 Ser Tyr Leu Trp Tyr
Ser Ala His Asn Ser Thr Thr Val Ile Val 530 535 540 Val Gly Leu Ile
Val Ser Leu Leu Thr Gly Arg Met Arg Gly Arg 545 550 555 Ser Leu Asn
Pro Ala Thr Ile Tyr Pro Val Leu Pro Lys Leu Leu 560 565 570 Ser Leu
Leu Pro Leu Ser Cys Gln Lys Arg Leu His Cys Arg Ser 575 580 585 Tyr
Gly Gln Asp His Leu Asp Thr Gly Leu Phe Pro Glu Lys Pro 590 595 600
Arg Asn Gly Val Leu Gly Asp Ser Arg Asp Lys Glu Ala Met Ala 605 610
615 Leu Asp Gly Thr Ala Tyr Gln Gly Ser Ser Ser Thr Cys Ile Leu 620
625 630 Gln Glu Thr Ser Leu 635 86 351 PRT Homo Sapien 86 Met Ala
Leu Thr Gly Ala Ser Asp Pro Ser Ala Glu Ala Glu Ala 1 5 10 15 Asn
Gly Glu Lys Pro Phe Leu Leu Arg Ala Leu Gln Ile Ala Leu 20 25 30
Val Val Ser Leu Tyr Trp Val Thr Ser Ile Ser Met Val Phe Leu 35 40
45 Asn Lys Tyr Leu Leu Asp Ser Pro Ser Leu Arg Leu Asp Thr Pro 50
55 60 Ile Phe Val Thr Phe Tyr Gln Cys Leu Val Thr Thr Leu Leu Cys
65 70 75 Lys Gly Leu Ser Ala Leu Ala Ala Cys Cys Pro Gly Ala Val
Asp 80 85 90 Phe Pro Ser Leu Arg Leu Asp Leu Arg Val Ala Arg Ser
Val Leu 95 100 105 Pro Leu Ser Val Val Phe Ile Gly Met Ile Thr Phe
Asn Asn Leu 110 115 120 Cys Leu Lys Tyr Val Gly Val Ala Phe Tyr Asn
Val Gly Arg Ser 125 130 135 Leu Thr Thr Val Phe Asn Val Leu Leu Ser
Tyr Leu Leu Leu Lys 140 145 150 Gln Thr Thr Ser Phe Tyr Ala Leu Leu
Thr Cys Gly Ile Ile Ile 155 160 165 Gly Gly Phe Trp Leu Gly Val Asp
Gln Glu Gly Ala Glu Gly Thr 170 175 180 Leu Ser Trp Leu Gly Thr Val
Phe Gly Val Leu Ala Ser Leu Cys 185 190 195 Val Ser Leu Asn Ala Ile
Tyr Thr Thr Lys Val Leu Pro Ala Val 200 205 210 Asp Gly Ser Ile Trp
Arg Leu Thr Phe Tyr Asn Asn Val Asn Ala 215 220 225 Cys Ile Leu Phe
Leu Pro Leu Leu Leu Leu Leu Gly Glu Leu Gln 230 235 240 Ala Leu Arg
Asp Leu Ala Gln Leu Gly Ser Ala His Phe Trp Gly 245 250 255 Met Met
Thr Leu Gly Gly Leu Phe Gly Phe Ala Ile Gly Tyr Val 260 265 270 Thr
Gly Leu Gln Ile Lys Phe Thr Ser Pro Leu Thr His Asn Val 275 280 285
Ser Gly Thr Ala Lys Ala Cys Ala Gln Thr Val Leu Ala Val Leu 290 295
300 Tyr Tyr Glu Glu Thr Lys Ser Phe Leu Trp Trp Thr Ser Asn Met 305
310 315 Met Val Leu Gly Gly Ser Ser Ala Tyr Thr Trp Val Arg Gly Trp
320 325 330 Glu Met Lys Lys Thr Pro Glu Glu Pro Ser Pro Lys Asp Ser
Glu 335 340 345 Lys Ser Ala Met Gly Val 350 87 351 PRT Homo Sapien
87 Met Ala Leu Thr Gly Ala Ser Asp Pro Ser Ala Glu Ala Glu Ala 1 5
10 15 Asn Gly Glu Lys Pro Phe Leu Leu Arg Ala Leu Gln Ile Ala Leu
20 25 30 Val Val Ser Leu Tyr Trp Val Thr Ser Ile Ser Met Val Phe
Leu 35 40 45 Asn Lys Tyr Leu Leu Asp Ser Pro Ser Leu Arg Leu Asp
Thr Pro 50 55 60 Ile Phe Val Thr Phe Tyr Gln Cys Leu Val Thr Thr
Leu Leu Cys 65 70 75 Lys Gly Leu Ser Ala Leu Ala Ala Cys Cys Pro
Gly Ala Val Asp 80 85 90 Phe Pro Ser Leu Arg Leu Asp Leu Arg Val
Ala Arg Ser Val Leu 95 100 105 Pro Leu Ser Val Val Phe Ile Gly Met
Ile Thr Phe Asn Asn Leu 110 115 120 Cys Leu Lys Tyr Val Gly Val Ala
Phe Tyr Asn Val Gly Arg Ser 125 130 135 Leu Thr Thr Val Phe Asn Val
Leu Leu Ser Tyr Leu Leu Leu Lys 140 145 150 Gln Thr Thr Ser Phe Tyr
Ala Leu Leu Thr Cys Gly Ile Ile Ile 155 160 165 Gly Gly Phe Trp Leu
Gly Val Asp Gln Glu Gly Ala Glu Gly Thr 170 175 180 Leu Ser Trp Leu
Gly Thr Val Phe Gly Val Leu Ala Ser Leu Cys 185 190 195 Val Ser Leu
Asn Ala Ile Tyr Thr Thr Lys Val Leu Pro Ala Val 200 205 210 Asp Gly
Ser Ile Trp Arg Leu Thr Phe Tyr Asn Asn Val Asn Ala 215 220 225 Cys
Ile Leu Phe Leu Pro Leu Leu Leu Leu Leu Gly Glu Leu Gln 230 235 240
Ala Leu Arg Asp Phe Ala Gln Leu Gly Ser Ala His Phe Trp Gly 245 250
255 Met Met Thr Leu Gly Gly Leu Phe Gly Phe Ala Ile Gly Tyr Val 260
265 270 Thr Gly Leu Gln Ile Lys Phe Thr Ser Pro Leu Thr His Asn Val
275 280 285 Ser Gly Thr Ala Lys Ala Cys Ala Gln Thr Val Leu Ala Val
Leu 290 295 300 Tyr Tyr Glu Glu Thr Lys Ser Phe Leu Trp Trp Thr Ser
Asn Met 305 310 315 Met Val Leu Gly Gly Ser Ser Ala Tyr Thr Trp Val
Arg Gly Trp 320 325 330 Glu Met Lys Lys Thr Pro Glu Glu Pro Ser Pro
Lys Asp Ser Glu 335 340 345 Lys Ser Ala Met Gly Val 350 88 208 PRT
Homo Sapien 88 Met Gly Ser Cys Ser Gly Arg Cys Ala Leu Val Val Leu
Cys Ala 1 5 10 15 Phe Gln Leu Val Ala Ala Leu Glu Arg Gln Val Phe
Asp Phe Leu 20 25 30 Gly Tyr Gln Trp Ala Pro Ile Leu Ala Asn Phe
Val His Ile Ile 35 40 45 Ile Val Ile Leu Gly Leu Phe Gly Thr Ile
Gln Tyr Arg Leu Arg 50 55 60 Tyr Val Met Val Tyr Thr Leu Trp Ala
Ala Val Trp Val Thr Trp 65 70 75 Asn Val Phe Ile Ile Cys Phe Tyr
Leu Glu Val Gly Gly Leu Leu 80 85 90 Gln Asp Ser Glu Leu Leu Thr
Phe Ser Leu Ser Arg His Arg Ser 95 100 105 Trp Trp Arg Glu Arg Trp
Pro Gly Cys Leu His Glu Glu Val Pro 110 115 120 Ala Val Gly Leu Gly
Ala Pro His Gly Gln Ala Leu Val Ser Gly 125 130 135 Ala Gly Cys Ala
Leu Glu Pro Ser Tyr Val Glu Ala Leu His Ser 140 145 150 Gly Leu Gln
Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly Cys 155 160 165 Gln Val
Val Ser Val Phe Thr Glu Glu Glu Asp Ser Phe Asp Phe 170 175 180 Ile
Gly Gly Phe Asp Pro Phe Pro Leu Tyr His Val Asn Glu Lys 185 190 195
Pro Ser Ser Leu Leu Ser Lys Gln Val Tyr Leu Pro Ala 200 205 89 208
PRT Homo Sapien 89 Met Gly Ser Cys Ser Gly Arg Cys Ala Leu Val Val
Leu Cys Ala 1 5 10 15 Phe Gln Leu Val Ala Ala Leu Glu Arg Gln Val
Phe Asp Phe Leu 20 25 30 Gly Tyr Gln Trp Ala Pro Ile Leu Ala Asn
Phe Val His Ile Ile 35 40 45 Ile Val Ile Leu Gly Leu Phe Gly Thr
Ile Gln Tyr Arg Leu Arg 50 55 60 Tyr Val Met Val Tyr Thr Leu Trp
Ala Ala Val Trp Val Thr Trp 65 70 75 Asn Val Phe Ile Ile Cys Phe
Tyr Leu Glu Val Gly Gly Leu Leu 80 85 90 Lys Asp Ser Glu Leu Leu
Thr Phe Ser Leu Ser Arg His Arg Ser 95 100 105 Trp Trp Arg Glu Arg
Trp Pro Gly Cys Leu His Glu Glu Val Pro 110 115 120 Ala Val Gly Leu
Gly Ala Pro His Gly Gln Ala Leu Val Ser Gly 125 130 135 Ala Gly Cys
Ala Leu Glu Pro Ser Tyr Val Glu Ala Leu His Ser 140 145 150 Cys Leu
Gln Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly Cys 155 160 165 Gln
Val Val Ser Val Phe Thr Glu Glu Glu Asp Ser Phe Asp Phe 170 175 180
Ile Gly Gly Phe Asp Pro Phe Pro Leu Tyr His Val Asn Glu Lys 185 190
195 Pro Ser Ser Leu Leu Ser Lys Gln Val Tyr Leu Pro Ala 200 205 90
181 PRT Homo Sapien 90 Met Gly Ser Cys Ser Gly Arg Cys Ala Leu Val
Val Leu Cys Ala 1 5 10 15 Phe Gln Leu Val Ala Ala Leu Glu Arg Gln
Val Phe Asp Phe Leu 20 25 30 Gly Tyr Gln Trp Ala Pro Ile Leu Ala
Asn Phe Val His Ile Ile 35 40 45 Ile Val Ile Leu Gly Leu Phe Gly
Thr Ile Gln Tyr Arg Leu Arg 50 55 60 Tyr Val Met Val Tyr Thr Leu
Trp Ala Ala Val Trp Val Thr Trp 65 70 75 Asn Val Phe Ile Ile Cys
Phe Tyr Leu Glu Val Gly Gly Leu Leu 80 85 90 Lys Asp Ser Glu Leu
Leu Thr Phe Ser Leu Ser Arg His Arg Ser 95 100 105 Trp Trp Arg Glu
Arg Trp Pro Gly Cys Leu His Glu Glu Val Pro 110 115 120 Ala Val Gly
Leu Gly Ala Pro His Gly Gln Ala Leu Val Ser Gly 125 130 135 Ala Gly
Cys Ala Leu Glu Pro Ser Tyr Val Glu Ala Leu His Ser 140 145 150 Cys
Leu Gln Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly Cys 155 160 165
Gln Val Val Ser Val Phe Thr Glu Glu Glu Asp Ser Cys Leu Arg 170 175
180 Lys 91 181 PRT Homo Sapien 91 Met Gly Ser Cys Ser Gly Arg Cys
Ala Leu Val Val Leu Cys Ala 1 5 10 15 Phe Gln Leu Val Ala Ala Leu
Glu Arg Gln Val Phe Asp Phe Leu 20 25 30 Gly Tyr Gln Trp Ala Pro
Ile Leu Ala Asn Phe Val His Ile Ile 35 40 45 Ile Val Ile Leu Gly
Leu Phe Gly Thr Ile Gln Tyr Arg Leu Arg 50 55 60 Tyr Val Met Val
Tyr Thr Leu Trp Ala Ala Val Trp Val Thr Trp 65 70 75 Asn Val Phe
Ile Ile Cys Phe Tyr Leu Glu Val Gly Gly Leu Leu 80 85 90 Gln Asp
Ser Glu Leu Leu Thr Phe Ser Leu Ser Arg His Arg Ser 95 100 105 Trp
Trp Arg Glu Arg Trp Pro Gly Cys Leu His Glu Glu Val Pro 110 115 120
Ala Val Gly Leu Gly Ala Pro His Gly Gln Ala Leu Val Ser Gly 125 130
135 Ala Gly Cys Ala Leu Glu Pro Ser Tyr Val Glu Ala Leu His Ser 140
145 150 Gly Leu Gln Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly
Cys
155 160 165 Gln Val Val Ser Val Phe Thr Glu Glu Glu Asp Ser Cys Leu
Arg 170 175 180 Lys 92 382 PRT Homo Sapien 92 Met Ala Val Leu Phe
Leu Leu Leu Phe Leu Cys Gly Thr Pro Gln 1 5 10 15 Ala Ala Asp Asn
Met Gln Ala Ile Tyr Val Ala Leu Gly Glu Ala 20 25 30 Val Glu Leu
Pro Cys Pro Ser Pro Pro Thr Leu His Gly Asp Glu 35 40 45 His Leu
Ser Trp Phe Cys Ser Pro Ala Ala Gly Ser Phe Thr Thr 50 55 60 Leu
Val Ala Gln Val Gln Val Gly Arg Pro Ala Pro Asp Pro Gly 65 70 75
Lys Pro Gly Arg Glu Ser Arg Leu Arg Leu Leu Gly Asn Tyr Ser 80 85
90 Leu Trp Leu Glu Gly Ser Lys Glu Glu Asp Ala Gly Arg Tyr Trp 95
100 105 Cys Ala Val Leu Gly Gln His His Asn Tyr Gln Asn Trp Arg Val
110 115 120 Tyr Asp Val Leu Val Leu Lys Gly Ser Gln Leu Ser Ala Arg
Ala 125 130 135 Ala Asp Gly Ser Pro Cys Asn Val Leu Leu Cys Ser Val
Val Pro 140 145 150 Ser Arg Arg Met Asp Ser Val Thr Trp Gln Glu Gly
Lys Gly Pro 155 160 165 Val Arg Gly Arg Val Gln Ser Phe Trp Gly Ser
Glu Ala Ala Leu 170 175 180 Leu Leu Val Cys Pro Gly Glu Gly Leu Ser
Glu Pro Arg Ser Arg 185 190 195 Arg Pro Arg Ile Ile Arg Cys Leu Met
Thr His Asn Lys Gly Val 200 205 210 Ser Phe Ser Leu Ala Ala Ser Ile
Asp Ala Ser Pro Ala Leu Cys 215 220 225 Ala Pro Ser Thr Gly Trp Asp
Met Pro Trp Ile Leu Met Leu Leu 230 235 240 Leu Thr Met Gly Gln Gly
Val Val Ile Leu Ala Leu Ser Ile Val 245 250 255 Leu Trp Arg Gln Arg
Val Arg Gly Ala Pro Gly Arg Gly Asn Arg 260 265 270 Met Arg Cys Tyr
Asn Cys Gly Gly Ser Pro Ser Ser Ser Cys Lys 275 280 285 Glu Ala Val
Thr Thr Cys Gly Glu Gly Arg Pro Gln Pro Gly Leu 290 295 300 Glu Gln
Ile Lys Leu Pro Gly Asn Pro Pro Val Thr Leu Ile His 305 310 315 Gln
His Pro Ala Cys Val Ala Ala His His Cys Asn Gln Val Glu 320 325 330
Thr Glu Ser Val Gly Asp Val Thr Tyr Pro Ala His Arg Asp Cys 335 340
345 Tyr Leu Gly Asp Leu Cys Asn Ser Ala Val Ala Ser His Val Ala 350
355 360 Pro Ala Gly Ile Leu Ala Ala Ala Ala Thr Ala Leu Thr Cys Leu
365 370 375 Leu Pro Gly Leu Trp Ser Gly 380 93 783 PRT Homo Sapien
93 Met Ser Gly Gly His Gln Leu Gln Leu Ala Ala Leu Trp Pro Trp 1 5
10 15 Leu Leu Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu
20 25 30 Val Leu Ala Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln
Lys 35 40 45 Ala Val Ile Arg Val Ile Pro Leu Lys Met Asp Pro Thr
Gly Lys 50 55 60 Leu Asn Leu Thr Leu Glu Gly Val Phe Ala Gly Val
Ala Glu Ile 65 70 75 Thr Pro Ala Glu Gly Lys Leu Met Gln Ser His
Pro Leu Tyr Leu 80 85 90 Cys Asn Ala Ser Asp Asp Asp Asn Leu Glu
Pro Gly Phe Ile Ser 95 100 105 Ile Val Lys Leu Glu Ser Pro Arg Arg
Ala Pro Arg Pro Cys Leu 110 115 120 Ser Leu Ala Ser Lys Ala Arg Met
Ala Gly Glu Arg Gly Ala Ser 125 130 135 Ala Val Leu Phe Asp Ile Thr
Glu Asp Arg Ala Ala Ala Glu Gln 140 145 150 Leu Gln Gln Pro Leu Gly
Leu Thr Trp Pro Val Val Leu Ile Trp 155 160 165 Gly Asn Asp Ala Glu
Lys Leu Met Glu Phe Val Tyr Lys Asn Gln 170 175 180 Lys Ala His Val
Arg Ile Glu Leu Lys Glu Pro Pro Ala Trp Pro 185 190 195 Asp Tyr Asp
Val Trp Ile Leu Met Thr Val Val Gly Thr Ile Phe 200 205 210 Val Ile
Ile Leu Ala Ser Val Leu Arg Ile Arg Cys Arg Pro Arg 215 220 225 His
Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp Ala Ile 230 235 240
Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln Ala 245 250
255 Arg Gly Glu Trp Pro Asp Ser Gly Ser Ser Cys Ser Ser Ala Pro 260
265 270 Val Cys Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu
275 280 285 Arg Val Ile Ser Cys Leu His Glu Phe His Arg Asn Cys Val
Asp 290 295 300 Pro Trp Leu His Gln His Arg Thr Cys Pro Leu Cys Val
Phe Asn 305 310 315 Ile Thr Glu Gly Asp Ser Phe Ser Gln Ser Leu Gly
Pro Ser Arg 320 325 330 Ser Tyr Gln Glu Pro Gly Arg Arg Leu His Leu
Ile Arg Gln His 335 340 345 Pro Gly His Ala His Tyr His Leu Pro Ala
Ala Tyr Leu Leu Gly 350 355 360 Pro Ser Arg Ser Ala Val Ala Arg Pro
Pro Arg Pro Gly Pro Phe 365 370 375 Leu Pro Ser Gln Glu Pro Gly Met
Gly Pro Arg His His Arg Phe 380 385 390 Pro Arg Ala Ala His Pro Arg
Ala Pro Gly Glu Gln Gln Arg Leu 395 400 405 Ala Gly Ala Gln His Pro
Tyr Ala Gln Gly Trp Gly Met Ser His 410 415 420 Leu Gln Ser Thr Ser
Gln His Pro Ala Ala Cys Pro Val Pro Leu 425 430 435 Arg Arg Ala Arg
Pro Pro Asp Ser Ser Gly Ser Gly Glu Ser Tyr 440 445 450 Cys Thr Glu
Arg Ser Gly Tyr Leu Ala Asp Gly Pro Ala Ser Asp 455 460 465 Ser Ser
Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser Val Val 470 475 480 Asn
Cys Thr Asp Ile Ser Leu Gln Gly Val His Gly Ser Ser Ser 485 490 495
Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr 500 505
510 Cys Ser Pro Lys Gly Asp Pro Gln Arg Val Asp Met Gln Pro Ser 515
520 525 Val Thr Ser Arg Pro Arg Ser Leu Asp Ser Val Val Pro Thr Gly
530 535 540 Glu Thr Gln Val Ser Ser His Val His Tyr His Arg His Arg
His 545 550 555 His His Tyr Lys Lys Arg Phe Gln Trp His Gly Arg Lys
Pro Gly 560 565 570 Pro Glu Thr Gly Val Pro Gln Ser Arg Pro Pro Ile
Pro Arg Thr 575 580 585 Gln Pro Gln Pro Glu Pro Pro Ser Pro Asp Gln
Gln Val Thr Gly 590 595 600 Ser Asn Ser Ala Ala Pro Ser Gly Arg Leu
Ser Asn Pro Gln Cys 605 610 615 Pro Arg Ala Leu Pro Glu Pro Ala Pro
Gly Pro Val Asp Ala Ser 620 625 630 Ser Ile Cys Pro Ser Thr Ser Ser
Leu Phe Asn Leu Gln Lys Ser 635 640 645 Ser Leu Ser Ala Arg His Pro
Gln Arg Lys Arg Arg Gly Gly Pro 650 655 660 Ser Glu Pro Thr Pro Gly
Ser Arg Pro Gln Asp Ala Thr Val His 665 670 675 Pro Ala Cys Gln Ile
Phe Pro His Tyr Thr Pro Ser Val Ala Tyr 680 685 690 Pro Trp Ser Pro
Glu Ala His Pro Leu Ile Cys Gly Pro Pro Gly 695 700 705 Leu Asp Lys
Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser 710 715 720 Asn Ser
Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu 725 730 735 Glu
Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp 740 745 750
Thr Ala Glu Gly Arg Pro Cys Pro Tyr Pro His Cys Gln Val Leu 755 760
765 Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys Glu 770
775 780 Gln Ala Val 94 510 PRT Homo Sapien 94 Met Pro Leu Ser Leu
Gly Ala Glu Met Trp Gly Pro Glu Ala Trp 1 5 10 15 Leu Leu Leu Leu
Leu Leu Leu Ala Ser Phe Thr Gly Arg Cys Pro 20 25 30 Ala Gly Glu
Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly 35 40 45 Gln Asp
Ala Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu 50 55 60 Gln
Val Gly Gln Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly 65 70 75
Ala Gln Glu Leu Ala Leu Leu His Ser Lys Tyr Gly Leu His Val 80 85
90 Ser Pro Ala Tyr Glu Gly Arg Val Glu Gln Pro Pro Pro Pro Arg 95
100 105 Asn Pro Leu Asp Gly Ser Val Leu Leu Arg Asn Ala Val Gln Ala
110 115 120 Asp Glu Gly Glu Tyr Glu Cys Arg Val Ser Thr Phe Pro Ala
Gly 125 130 135 Ser Phe Gln Ala Arg Leu Arg Leu Arg Val Leu Val Pro
Pro Leu 140 145 150 Pro Ser Leu Asn Pro Gly Pro Ala Leu Glu Glu Gly
Gln Gly Leu 155 160 165 Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser
Pro Ala Pro Ser 170 175 180 Val Thr Trp Asp Thr Glu Val Lys Gly Thr
Thr Ser Ser Arg Ser 185 190 195 Phe Lys His Ser Arg Ser Ala Ala Val
Thr Ser Glu Phe His Leu 200 205 210 Val Pro Ser Arg Ser Met Asn Gly
Gln Pro Leu Thr Cys Val Val 215 220 225 Ser His Pro Gly Leu Leu Gln
Asp Gln Arg Ile Thr His Ile Leu 230 235 240 His Val Ser Phe Leu Ala
Glu Ala Ser Val Arg Gly Leu Glu Asp 245 250 255 Gln Asn Leu Trp His
Ile Gly Arg Glu Gly Ala Met Leu Lys Cys 260 265 270 Leu Ser Glu Gly
Gln Pro Pro Pro Ser Tyr Asn Trp Thr Arg Leu 275 280 285 Asp Gly Pro
Leu Pro Ser Gly Val Arg Val Asp Gly Asp Thr Leu 290 295 300 Gly Phe
Pro Pro Leu Thr Thr Glu His Ser Gly Ile Tyr Val Cys 305 310 315 His
Val Ser Asn Glu Phe Ser Ser Arg Asp Ser Gln Val Thr Val 320 325 330
Asp Val Leu Asp Pro Gln Glu Asp Ser Gly Lys Gln Val Asp Leu 335 340
345 Val Ser Ala Ser Val Val Val Val Gly Val Ile Ala Ala Leu Leu 350
355 360 Phe Cys Leu Leu Val Val Val Val Val Leu Met Ser Arg Tyr His
365 370 375 Arg Arg Lys Ala Gln Gln Met Thr Gln Lys Tyr Glu Glu Glu
Leu 380 385 390 Thr Leu Thr Arg Glu Asn Ser Ile Arg Arg Leu His Ser
His His 395 400 405 Thr Asp Pro Arg Ser Gln Pro Glu Glu Ser Val Gly
Leu Arg Ala 410 415 420 Glu Gly His Pro Asp Ser Leu Lys Asp Asn Ser
Ser Cys Ser Val 425 430 435 Met Ser Glu Glu Pro Glu Gly Arg Ser Tyr
Ser Thr Leu Thr Thr 440 445 450 Val Arg Glu Ile Glu Thr Gln Thr Glu
Leu Leu Ser Pro Gly Ser 455 460 465 Gly Arg Ala Glu Glu Glu Glu Asp
Gln Asp Glu Gly Ile Lys Gln 470 475 480 Ala Met Asn His Phe Val Gln
Glu Asn Gly Thr Leu Arg Ala Lys 485 490 495 Pro Thr Gly Asn Gly Ile
Tyr Ile Asn Gly Arg Gly His Leu Val 500 505 510 95 523 PRT Homo
Sapien 95 Met Thr Gln Asn Lys Leu Lys Leu Cys Ser Lys Ala Asn Val
Tyr 1 5 10 15 Thr Glu Val Pro Asp Gly Gly Trp Gly Trp Ala Val Ala
Val Ser 20 25 30 Phe Phe Phe Val Glu Val Phe Thr Tyr Gly Ile Ile
Lys Thr Phe 35 40 45 Gly Val Phe Phe Asn Asp Leu Met Asp Ser Phe
Asn Glu Ser Asn 50 55 60 Ser Arg Ile Ser Trp Ile Ile Ser Ile Cys
Val Phe Val Leu Thr 65 70 75 Phe Ser Ala Pro Leu Ala Thr Val Leu
Ser Asn Arg Phe Gly His 80 85 90 Arg Leu Val Val Met Leu Gly Gly
Leu Leu Val Ser Thr Gly Met 95 100 105 Val Ala Ala Ser Phe Ser Gln
Glu Val Ser His Met Tyr Val Ala 110 115 120 Ile Gly Ile Ile Ser Gly
Leu Gly Tyr Cys Phe Ser Phe Leu Pro 125 130 135 Thr Val Thr Ile Leu
Ser Gln Tyr Phe Gly Lys Arg Arg Ser Ile 140 145 150 Val Thr Ala Val
Ala Ser Thr Gly Glu Cys Phe Ala Val Phe Ala 155 160 165 Phe Ala Pro
Ala Ile Met Ala Leu Lys Glu Arg Ile Gly Trp Arg 170 175 180 Tyr Ser
Leu Leu Phe Val Gly Leu Leu Gln Leu Asn Ile Val Ile 185 190 195 Phe
Gly Ala Leu Leu Arg Pro Ile Ile Ile Arg Gly Pro Ala Ser 200 205 210
Pro Lys Ile Val Ile Gln Glu Asn Arg Lys Glu Ala Gln Tyr Met 215 220
225 Leu Glu Asn Glu Lys Thr Arg Thr Ser Ile Asp Ser Ile Asp Ser 230
235 240 Gly Val Glu Leu Thr Thr Ser Pro Lys Asn Val Pro Thr His Thr
245 250 255 Asn Leu Glu Leu Glu Pro Lys Ala Asp Met Gln Gln Val Leu
Val 260 265 270 Lys Thr Ser Pro Arg Pro Ser Glu Lys Lys Ala Pro Leu
Leu Asp 275 280 285 Phe Ser Ile Leu Lys Glu Lys Ser Phe Ile Cys Tyr
Ala Leu Phe 290 295 300 Gly Leu Phe Ala Thr Leu Gly Phe Phe Ala Pro
Ser Leu Tyr Ile 305 310 315 Ile Pro Leu Gly Ile Ser Leu Gly Ile Asp
Gln Asp Arg Ala Ala 320 325 330 Phe Leu Leu Ser Thr Met Ala Ile Ala
Glu Val Phe Gly Arg Ile 335 340 345 Gly Ala Gly Phe Val Leu Asn Arg
Glu Pro Ile Arg Lys Ile Tyr 350 355 360 Ile Glu Leu Ile Cys Val Ile
Leu Leu Thr Val Ser Leu Phe Ala 365 370 375 Phe Thr Phe Ala Thr Glu
Phe Trp Gly Leu Met Ser Cys Ser Ile 380 385 390 Phe Phe Gly Phe Met
Val Gly Thr Ile Gly Gly Leu Thr Phe His 395 400 405 Cys Leu Leu Lys
Met Met Ser Trp Ala Leu Gln Lys Met Ser Ser 410 415 420 Ala Ala Gly
Val Tyr Ile Phe Ile Gln Ser Ile Ala Gly Leu Ala 425 430 435 Gly Pro
Pro Leu Ala Gly Leu Leu Val Asp Gln Ser Lys Ile Tyr 440 445 450 Ser
Arg Ala Phe Tyr Ser Cys Ala Ala Gly Met Ala Leu Ala Ala 455 460 465
Val Cys Leu Ala Leu Val Arg Pro Cys Lys Met Gly Leu Cys Gln 470 475
480 Arg His His Ser Gly Glu Thr Lys Val Val Ser His Arg Gly Lys 485
490 495 Thr Leu Gln Asp Ile Pro Glu Asp Phe Leu Glu Met Asp Leu Ala
500 505 510 Lys Asn Glu His Arg Val His Val Gln Met Glu Pro Val 515
520 96 124 PRT Homo Sapien 96 Met Leu Leu Trp Val Ile Leu Leu Val
Leu Ala Pro Val Ser Gly 1 5 10 15 Gln Phe Ala Arg Thr Pro Arg Pro
Ile Ile Phe Leu Gln Pro Pro 20 25 30 Trp Thr Thr Val Phe Gln Gly
Glu Arg Val Thr Leu Thr Cys Lys 35 40 45 Gly Phe Arg Phe Tyr Ser
Pro Gln Lys Thr Lys Trp Tyr His Arg 50 55 60 Tyr Leu Gly Lys Glu
Ile Leu Arg Glu Thr Pro Asp Asn Ile
Leu 65 70 75 Glu Val Gln Glu Ser Gly Glu Tyr Arg Cys Gln Ala Gln
Gly Ser 80 85 90 Pro Leu Ser Ser Pro Val His Leu Asp Phe Ser Ser
Glu Met Gly 95 100 105 Phe Pro His Ala Ala Gln Ala Asn Val Glu Leu
Leu Gly Ser Ser 110 115 120 Asp Leu Leu Thr 97 977 PRT Homo Sapien
97 Met Leu Leu Trp Val Ile Leu Leu Val Leu Ala Pro Val Ser Gly 1 5
10 15 Gln Phe Ala Arg Thr Pro Arg Pro Ile Ile Phe Leu Gln Pro Pro
20 25 30 Trp Thr Thr Val Phe Gln Gly Glu Arg Val Thr Leu Thr Cys
Lys 35 40 45 Gly Phe Arg Phe Tyr Ser Pro Gln Lys Thr Lys Trp Tyr
His Arg 50 55 60 Tyr Leu Gly Lys Glu Ile Leu Arg Glu Thr Pro Asp
Asn Ile Leu 65 70 75 Glu Val Gln Glu Ser Gly Glu Tyr Arg Cys Gln
Ala Gln Gly Ser 80 85 90 Pro Leu Ser Ser Pro Val His Leu Asp Phe
Ser Ser Ala Ser Leu 95 100 105 Ile Leu Gln Ala Pro Leu Ser Val Phe
Glu Gly Asp Ser Val Val 110 115 120 Leu Arg Cys Arg Ala Lys Ala Glu
Val Thr Leu Asn Asn Thr Ile 125 130 135 Tyr Lys Asn Asp Asn Val Leu
Ala Phe Leu Asn Lys Arg Thr Asp 140 145 150 Phe His Ile Pro His Ala
Cys Leu Lys Asp Asn Gly Ala Tyr Arg 155 160 165 Cys Thr Gly Tyr Lys
Glu Ser Cys Cys Pro Val Ser Ser Asn Thr 170 175 180 Val Lys Ile Gln
Val Gln Glu Pro Phe Thr Arg Pro Val Leu Arg 185 190 195 Ala Ser Ser
Phe Gln Pro Ile Ser Gly Asn Pro Val Thr Leu Thr 200 205 210 Cys Glu
Thr Gln Leu Ser Leu Glu Arg Ser Asp Val Pro Leu Arg 215 220 225 Phe
Arg Phe Phe Arg Asp Asp Gln Thr Leu Gly Leu Gly Trp Ser 230 235 240
Leu Ser Pro Asn Phe Gln Ile Thr Ala Met Trp Ser Lys Asp Ser 245 250
255 Gly Phe Tyr Trp Cys Lys Ala Ala Thr Met Pro His Ser Val Ile 260
265 270 Ser Asp Ser Pro Arg Ser Trp Ile Gln Val Gln Ile Pro Ala Ser
275 280 285 His Pro Val Leu Thr Leu Ser Pro Glu Lys Ala Leu Asn Phe
Glu 290 295 300 Gly Thr Lys Val Thr Leu His Cys Glu Thr Gln Glu Asp
Ser Leu 305 310 315 Arg Thr Leu Tyr Arg Phe Tyr His Glu Gly Val Pro
Leu Arg His 320 325 330 Lys Ser Val Arg Cys Glu Arg Gly Ala Ser Ile
Ser Phe Ser Leu 335 340 345 Thr Thr Glu Asn Ser Gly Asn Tyr Tyr Cys
Thr Ala Asp Asn Gly 350 355 360 Leu Gly Ala Lys Pro Ser Lys Ala Val
Ser Leu Ser Val Thr Val 365 370 375 Pro Val Ser His Pro Val Leu Asn
Leu Ser Ser Pro Glu Asp Leu 380 385 390 Ile Phe Glu Gly Ala Lys Val
Thr Leu His Cys Glu Ala Gln Arg 395 400 405 Gly Ser Leu Pro Ile Leu
Tyr Gln Phe His His Glu Asp Ala Ala 410 415 420 Leu Glu Arg Arg Ser
Ala Asn Ser Ala Gly Gly Val Ala Ile Ser 425 430 435 Phe Ser Leu Thr
Ala Glu His Ser Gly Asn Tyr Tyr Cys Thr Ala 440 445 450 Asp Asn Gly
Phe Gly Pro Gln Arg Ser Lys Ala Val Ser Leu Ser 455 460 465 Ile Thr
Val Pro Val Ser His Pro Val Leu Thr Leu Ser Ser Ala 470 475 480 Glu
Ala Leu Thr Phe Glu Gly Ala Thr Val Thr Leu His Cys Glu 485 490 495
Val Gln Arg Gly Ser Pro Gln Ile Leu Tyr Gln Phe Tyr His Glu 500 505
510 Asp Met Pro Leu Trp Ser Ser Ser Thr Pro Ser Val Gly Arg Val 515
520 525 Ser Phe Ser Phe Ser Leu Thr Glu Gly His Ser Gly Asn Tyr Tyr
530 535 540 Cys Thr Ala Asp Asn Gly Phe Gly Pro Gln Arg Ser Glu Val
Val 545 550 555 Ser Leu Phe Val Thr Val Pro Val Ser Arg Pro Ile Leu
Thr Leu 560 565 570 Arg Val Pro Arg Ala Gln Ala Val Val Gly Asp Leu
Leu Glu Leu 575 580 585 His Cys Glu Ala Pro Arg Gly Ser Pro Pro Ile
Leu Tyr Trp Phe 590 595 600 Tyr His Glu Asp Val Thr Leu Gly Ser Ser
Ser Ala Pro Ser Gly 605 610 615 Gly Glu Ala Ser Phe Asn Leu Ser Leu
Thr Ala Glu His Ser Gly 620 625 630 Asn Tyr Ser Cys Glu Ala Asn Asn
Gly Leu Val Ala Gln His Ser 635 640 645 Asp Thr Ile Ser Leu Ser Val
Ile Val Pro Val Ser Arg Pro Ile 650 655 660 Leu Thr Phe Arg Ala Pro
Arg Ala Gln Ala Val Val Gly Asp Leu 665 670 675 Leu Glu Leu His Cys
Glu Ala Leu Arg Gly Ser Ser Pro Ile Leu 680 685 690 Tyr Trp Phe Tyr
His Glu Asp Val Thr Leu Gly Lys Ile Ser Ala 695 700 705 Pro Ser Gly
Gly Gly Ala Ser Phe Asn Leu Ser Leu Thr Thr Glu 710 715 720 His Ser
Gly Ile Tyr Ser Cys Glu Ala Asp Asn Gly Pro Glu Ala 725 730 735 Gln
Arg Ser Glu Met Val Thr Leu Lys Val Ala Val Pro Val Ser 740 745 750
Arg Pro Val Leu Thr Leu Arg Ala Pro Gly Thr His Ala Ala Val 755 760
765 Gly Asp Leu Leu Glu Leu His Cys Glu Ala Leu Arg Gly Ser Pro 770
775 780 Leu Ile Leu Tyr Arg Phe Phe His Glu Asp Val Thr Leu Gly Asn
785 790 795 Arg Ser Ser Pro Ser Gly Gly Ala Ser Leu Asn Leu Ser Leu
Thr 800 805 810 Ala Glu His Ser Gly Asn Tyr Ser Cys Glu Ala Asp Asn
Gly Leu 815 820 825 Gly Ala Gln Arg Ser Glu Thr Val Thr Leu Tyr Ile
Thr Gly Leu 830 835 840 Thr Ala Asn Arg Ser Gly Pro Phe Ala Thr Gly
Val Ala Gly Gly 845 850 855 Leu Leu Ser Ile Ala Gly Leu Ala Ala Gly
Ala Leu Leu Leu Tyr 860 865 870 Cys Trp Leu Ser Arg Lys Ala Gly Arg
Lys Pro Ala Ser Asp Pro 875 880 885 Ala Arg Ser Pro Pro Asp Ser Asp
Ser Gln Glu Pro Thr Tyr His 890 895 900 Asn Val Pro Ala Trp Glu Glu
Leu Gln Pro Val Tyr Thr Asn Ala 905 910 915 Asn Pro Arg Gly Glu Asn
Val Val Tyr Ser Glu Val Arg Ile Ile 920 925 930 Gln Glu Lys Lys Lys
His Ala Val Ala Ser Asp Pro Arg His Leu 935 940 945 Arg Asn Lys Gly
Ser Pro Ile Ile Tyr Ser Glu Val Lys Val Ala 950 955 960 Ser Thr Pro
Val Ser Gly Ser Leu Phe Leu Ala Ser Ser Ala Pro 965 970 975 His Arg
98 146 PRT Homo Sapien 98 Met Leu Leu Trp Cys Pro Pro Gln Cys Ala
Cys Ser Leu Gly Val 1 5 10 15 Phe Pro Ser Ala Pro Ser Pro Val Trp
Gly Thr Arg Arg Ser Cys 20 25 30 Glu Pro Ala Thr Arg Val Pro Glu
Val Trp Ile Leu Ser Pro Leu 35 40 45 Leu Arg His Gly Gly His Thr
Gln Thr Gln Asn His Thr Ala Ser 50 55 60 Pro Arg Ser Pro Val Met
Glu Ser Pro Lys Lys Lys Asn Gln Gln 65 70 75 Leu Lys Val Gly Ile
Leu His Leu Gly Ser Arg Gln Lys Lys Ile 80 85 90 Arg Ile Gln Leu
Arg Ser Gln Cys Ala Thr Trp Lys Val Ile Cys 95 100 105 Lys Ser Cys
Ile Ser Gln Thr Pro Gly Ile Asn Leu Asp Leu Gly 110 115 120 Ser Gly
Val Lys Val Lys Ile Ile Pro Lys Glu Glu His Cys Lys 125 130 135 Met
Pro Glu Ala Gly Glu Glu Gln Pro Gln Val 140 145 99 235 PRT Homo
Sapien 99 Met Arg Glu Leu Ala Ile Glu Ile Gly Val Arg Ala Leu Leu
Phe 1 5 10 15 Gly Val Phe Val Phe Thr Glu Phe Leu Asp Pro Phe Gln
Arg Val 20 25 30 Ile Gln Pro Glu Glu Ile Trp Leu Tyr Lys Asn Pro
Leu Val Gln 35 40 45 Ser Asp Asn Ile Pro Thr Arg Leu Met Phe Ala
Ile Ser Phe Leu 50 55 60 Thr Pro Leu Ala Val Ile Cys Val Val Lys
Ile Ile Arg Arg Thr 65 70 75 Asp Lys Thr Glu Ile Lys Glu Ala Phe
Leu Ala Val Ser Leu Ala 80 85 90 Leu Ala Leu Asn Gly Val Cys Thr
Asn Thr Ile Lys Leu Ile Val 95 100 105 Gly Arg Pro Arg Ala Asp Phe
Phe Tyr Arg Cys Phe Pro Asp Gly 110 115 120 Val Met Asn Ser Glu Met
His Cys Thr Gly Asp Pro Asp Leu Val 125 130 135 Ser Glu Gly Arg Lys
Ser Phe Pro Ser Ile His Ser Ser Phe Ala 140 145 150 Phe Ser Gly Leu
Gly Phe Thr Thr Phe Tyr Leu Ala Gly Lys Leu 155 160 165 His Cys Phe
Thr Glu Ser Gly Arg Gly Lys Ser Trp Arg Leu Cys 170 175 180 Ala Ala
Ile Leu Pro Leu Tyr Cys Ala Met Met Ile Ala Leu Ser 185 190 195 Arg
Met Cys Asp Tyr Lys His His Trp Gln Asp Ser Phe Val Gly 200 205 210
Gly Val Ile Ala Leu Ile Phe Ala Tyr Ile Cys Tyr Arg Gln His 215 220
225 Tyr Pro Pro Leu Gly Gln His Ser Leu Pro 230 235 100 252 PRT
Homo Sapien 100 Met Ala Glu Leu Glu Phe Val Gln Ile Ile Ile Ile Val
Val Val 1 5 10 15 Met Met Val Met Val Val Val Ile Thr Cys Leu Leu
Ser His Tyr 20 25 30 Lys Leu Ser Ala Arg Ser Phe Ile Ser Arg His
Ser Gln Gly Arg 35 40 45 Arg Arg Glu Asp Ala Leu Ser Ser Glu Gly
Cys Leu Trp Pro Ser 50 55 60 Glu Ser Thr Val Ser Gly Asn Gly Ile
Pro Glu Pro Gln Val Tyr 65 70 75 Ala Pro Pro Arg Pro Thr Asp Arg
Leu Ala Val Pro Pro Phe Ala 80 85 90 Gln Arg Glu Arg Phe His Arg
Phe Gln Pro Thr Tyr Pro Tyr Leu 95 100 105 Gln His Glu Ile Asp Leu
Pro Pro Thr Ile Ser Leu Ser Asp Gly 110 115 120 Glu Glu Pro Pro Pro
Tyr Gln Gly Pro Cys Thr Leu Gln Leu Arg 125 130 135 Asp Pro Glu Gln
Gln Leu Glu Leu Asn Arg Glu Ser Val Arg Ala 140 145 150 Pro Pro Asn
Arg Thr Ile Phe Asp Ser Asp Leu Met Asp Ser Ala 155 160 165 Arg Leu
Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser Gly Ile Ser 170 175 180 Ala
Thr Cys Tyr Gly Ser Gly Gly Arg Met Glu Gly Pro Pro Pro 185 190 195
Thr Tyr Ser Glu Val Ile Gly His Tyr Pro Gly Ser Ser Phe Gln 200 205
210 His Gln Gln Ser Ser Gly Pro Pro Ser Leu Leu Glu Gly Thr Arg 215
220 225 Leu His His Thr His Ile Ala Pro Leu Glu Ser Ala Ala Ile Trp
230 235 240 Ser Lys Glu Lys Asp Lys Gln Lys Gly His Pro Leu 245 250
101 252 PRT Homo Sapien 101 Met Ala Glu Leu Glu Phe Val Gln Ile Ile
Ile Ile Val Val Val 1 5 10 15 Met Met Val Met Val Val Val Ile Thr
Cys Leu Leu Ser His Tyr 20 25 30 Lys Leu Ser Ala Arg Ser Phe Ile
Ser Arg His Ser Gln Gly Arg 35 40 45 Arg Arg Glu Asp Ala Leu Ser
Ser Glu Gly Cys Leu Trp Pro Ser 50 55 60 Glu Ser Thr Val Ser Gly
Asn Gly Ile Pro Glu Pro Gln Val Tyr 65 70 75 Ala Pro Pro Arg Pro
Thr Asp Arg Leu Ala Val Pro Pro Phe Ala 80 85 90 Gln Arg Glu Arg
Phe His Arg Phe Gln Pro Thr Tyr Pro Tyr Leu 95 100 105 Gln His Glu
Ile Asp Leu Pro Pro Thr Ile Ser Leu Ser Asp Gly 110 115 120 Glu Glu
Pro Pro Pro Tyr Gln Gly Pro Cys Thr Leu Gln Leu Arg 125 130 135 Asp
Pro Glu Gln Gln Leu Glu Leu Asn Arg Glu Ser Val Arg Ala 140 145 150
Pro Pro Asn Arg Thr Ile Phe Asp Ser Asp Leu Met Asp Ser Ala 155 160
165 Arg Leu Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser Gly Ile Ser 170
175 180 Ala Thr Cys Tyr Gly Ser Gly Gly Arg Met Glu Gly Pro Pro Pro
185 190 195 Thr Tyr Ser Glu Val Ile Gly His Tyr Pro Gly Ser Ser Phe
Gln 200 205 210 His Gln Gln Ser Ser Gly Pro Pro Ser Leu Leu Glu Gly
Thr Arg 215 220 225 Leu His His Thr His Ile Ala Pro Leu Glu Ser Ala
Ala Ile Trp 230 235 240 Ser Lys Glu Lys Asp Lys Gln Lys Gly His Pro
Leu 245 250 102 465 PRT Homo Sapien 102 Met Gly Gly Ala Val Val Asp
Glu Gly Pro Thr Gly Val Lys Ala 1 5 10 15 Pro Asp Gly Gly Trp Gly
Trp Ala Val Leu Phe Gly Cys Phe Val 20 25 30 Ile Thr Gly Phe Ser
Tyr Ala Phe Pro Lys Ala Val Ser Val Phe 35 40 45 Phe Lys Glu Leu
Ile Gln Glu Phe Gly Ile Gly Tyr Ser Asp Thr 50 55 60 Ala Trp Ile
Ser Ser Ile Leu Leu Ala Met Leu Tyr Gly Thr Gly 65 70 75 Pro Leu
Cys Ser Val Cys Val Asn Arg Phe Gly Cys Arg Pro Val 80 85 90 Met
Leu Val Gly Gly Leu Phe Ala Ser Leu Gly Met Val Ala Ala 95 100 105
Ser Phe Cys Arg Ser Ile Ile Gln Val Tyr Leu Thr Thr Gly Val 110 115
120 Ile Thr Gly Leu Gly Leu Ala Leu Asn Phe Gln Pro Ser Leu Ile 125
130 135 Met Leu Asn Arg Tyr Phe Ser Lys Arg Arg Pro Met Ala Asn Gly
140 145 150 Leu Ala Ala Ala Gly Ser Pro Val Phe Leu Cys Ala Leu Ser
Pro 155 160 165 Leu Gly Gln Leu Leu Gln Asp Arg Tyr Gly Trp Arg Gly
Gly Phe 170 175 180 Leu Ile Leu Gly Gly Leu Leu Leu Asn Cys Cys Val
Cys Ala Ala 185 190 195 Leu Met Arg Pro Leu Val Val Thr Ala Gln Pro
Gly Ser Gly Pro 200 205 210 Pro Arg Pro Ser Arg Arg Leu Leu Asp Leu
Ser Val Phe Arg Asp 215 220 225 Arg Gly Phe Val Leu Tyr Ala Val Ala
Ala Ser Val Met Val Leu 230 235 240 Gly Leu Phe Val Pro Pro Val Phe
Val Val Ser Tyr Ala Lys Asp 245 250 255 Leu Gly Val Pro Asp Thr Lys
Ala Ala Phe Leu Leu Thr Ile Leu 260 265 270 Gly Phe Ile Asp Ile Phe
Ala Arg Pro Ala Ala Gly Phe Val Ala 275 280 285 Gly Leu Gly Lys Val
Arg Pro Tyr Ser Val Tyr Leu Phe Ser Phe 290 295 300 Ser Met Phe Phe
Asn Gly Leu Ala Asp Leu Ala Gly Ser Thr Ala 305 310 315 Gly Asp Tyr
Gly Gly Leu Val Val Phe Cys Ile Phe Phe Gly Ile 320 325 330 Ser Tyr
Gly Met Val Gly Ala Leu Gln Phe Glu Val Leu Met Ala 335 340 345 Ile
Val Gly Thr His Lys Phe Ser Ser Ala Ile Gly Leu Val Leu 350 355 360
Leu Met Glu Ala Val Ala Val Leu Val Gly Pro
Pro Ser Gly Gly 365 370 375 Lys Leu Leu Asp Ala Thr His Val Tyr Met
Tyr Val Phe Ile Leu 380 385 390 Ala Gly Ala Glu Val Leu Thr Ser Ser
Leu Ile Leu Leu Leu Gly 395 400 405 Asn Phe Phe Cys Ile Arg Lys Lys
Pro Lys Glu Pro Gln Pro Glu 410 415 420 Val Ala Ala Ala Glu Glu Glu
Lys Leu His Lys Pro Pro Ala Asp 425 430 435 Ser Gly Val Asp Leu Arg
Glu Val Glu His Phe Leu Lys Ala Glu 440 445 450 Pro Glu Lys Asn Gly
Glu Val Val His Thr Pro Glu Thr Ser Val 455 460 465 103 445 PRT
Homo Sapien 103 Met Ala Ala Pro Thr Pro Ala Arg Pro Val Leu Thr His
Leu Leu 1 5 10 15 Val Ala Leu Phe Gly Met Gly Ser Trp Ala Ala Val
Asn Gly Ile 20 25 30 Trp Val Glu Leu Pro Val Val Val Lys Glu Leu
Pro Glu Gly Trp 35 40 45 Ser Leu Pro Ser Tyr Val Ser Val Leu Val
Ala Leu Gly Asn Leu 50 55 60 Gly Leu Leu Val Val Thr Leu Trp Arg
Arg Leu Ala Pro Gly Lys 65 70 75 Asp Glu Gln Val Pro Ile Arg Val
Val Gln Val Leu Gly Met Val 80 85 90 Gly Thr Ala Leu Leu Ala Ser
Leu Trp His His Val Ala Pro Val 95 100 105 Ala Gly Gln Leu His Ser
Val Ala Phe Leu Ala Leu Ala Phe Val 110 115 120 Leu Ala Leu Ala Cys
Cys Ala Ser Asn Val Thr Phe Leu Pro Phe 125 130 135 Leu Ser His Leu
Pro Pro Arg Phe Leu Arg Ser Phe Phe Leu Gly 140 145 150 Gln Gly Leu
Ser Ala Leu Leu Pro Cys Val Leu Ala Leu Val Gln 155 160 165 Gly Val
Gly Arg Leu Glu Cys Pro Pro Ala Pro Ile Asn Gly Thr 170 175 180 Pro
Gly Pro Pro Leu Asp Phe Leu Glu Arg Phe Pro Ala Ser Thr 185 190 195
Phe Phe Trp Ala Leu Thr Ala Leu Leu Val Ala Ser Ala Ala Ala 200 205
210 Phe Gln Gly Leu Leu Leu Leu Leu Pro Pro Pro Pro Ser Val Pro 215
220 225 Thr Gly Glu Leu Gly Ser Gly Leu Gln Val Gly Ala Pro Gly Ala
230 235 240 Glu Glu Glu Val Glu Glu Ser Ser Pro Leu Gln Glu Pro Pro
Ser 245 250 255 Gln Ala Ala Gly Thr Thr Pro Gly Pro Asp Pro Lys Ala
Tyr Gln 260 265 270 Leu Leu Ser Ala Arg Ser Ala Cys Leu Leu Gly Leu
Leu Ala Ala 275 280 285 Thr Asn Ala Leu Thr Asn Gly Val Leu Pro Ala
Val Gln Ser Phe 290 295 300 Ser Cys Leu Pro Tyr Gly Arg Leu Ala Tyr
His Leu Ala Val Val 305 310 315 Leu Gly Ser Ala Ala Asn Pro Leu Ala
Cys Phe Leu Ala Met Gly 320 325 330 Val Leu Cys Arg Ser Leu Ala Gly
Leu Gly Gly Leu Ser Leu Leu 335 340 345 Gly Val Phe Cys Gly Gly Tyr
Leu Met Ala Leu Ala Val Leu Ser 350 355 360 Pro Cys Pro Pro Leu Val
Gly Thr Ser Ala Gly Val Val Leu Val 365 370 375 Val Leu Ser Trp Val
Leu Cys Leu Gly Val Phe Ser Tyr Val Lys 380 385 390 Val Ala Ala Ser
Ser Leu Leu His Gly Gly Gly Arg Pro Ala Leu 395 400 405 Leu Ala Ala
Gly Val Ala Ile Gln Val Gly Ser Leu Leu Gly Ala 410 415 420 Val Ala
Met Phe Pro Pro Thr Ser Ile Tyr His Val Phe His Ser 425 430 435 Arg
Lys Asp Cys Ala Asp Pro Cys Asp Ser 440 445 104 398 PRT Homo Sapien
104 Met His Thr Val Ala Thr Ser Gly Pro Asn Ala Ser Trp Gly Ala 1 5
10 15 Pro Ala Asn Ala Ser Gly Cys Pro Gly Cys Gly Ala Asn Ala Ser
20 25 30 Asp Gly Pro Val Pro Ser Pro Arg Ala Val Asp Ala Trp Leu
Val 35 40 45 Pro Leu Phe Phe Ala Ala Leu Met Leu Leu Gly Leu Val
Gly Asn 50 55 60 Ser Leu Val Ile Tyr Val Ile Cys Arg His Lys Pro
Met Arg Thr 65 70 75 Val Thr Asn Phe Tyr Ile Ala Asn Leu Ala Ala
Thr Asp Val Thr 80 85 90 Phe Leu Leu Cys Cys Val Pro Phe Thr Ala
Leu Leu Tyr Pro Leu 95 100 105 Pro Gly Trp Val Leu Gly Asp Phe Met
Cys Lys Phe Val Asn Tyr 110 115 120 Ile Gln Gln Val Ser Val Gln Ala
Thr Cys Ala Thr Leu Thr Ala 125 130 135 Met Ser Val Asp Arg Trp Tyr
Val Thr Val Phe Pro Leu Arg Ala 140 145 150 Leu His Arg Arg Thr Pro
Arg Leu Ala Leu Ala Val Ser Leu Ser 155 160 165 Ile Trp Val Gly Ser
Ala Ala Val Ser Ala Pro Val Leu Ala Leu 170 175 180 His Arg Leu Ser
Pro Gly Pro Arg Ala Tyr Cys Ser Glu Ala Phe 185 190 195 Pro Ser Arg
Ala Leu Glu Arg Ala Phe Ala Leu Tyr Asn Leu Leu 200 205 210 Ala Leu
Tyr Leu Leu Pro Leu Leu Ala Thr Cys Ala Cys Tyr Ala 215 220 225 Ala
Met Leu Arg His Leu Gly Arg Val Ala Val Arg Pro Ala Pro 230 235 240
Ala Asp Ser Ala Leu Gln Gly Gln Val Leu Ala Glu Arg Ala Gly 245 250
255 Ala Val Arg Ala Lys Val Ser Arg Leu Val Ala Ala Val Val Leu 260
265 270 Leu Phe Ala Ala Cys Trp Gly Pro Ile Gln Leu Phe Leu Val Leu
275 280 285 Gln Ala Leu Gly Pro Ala Gly Ser Trp His Pro Arg Ser Tyr
Ala 290 295 300 Ala Tyr Ala Leu Lys Thr Trp Ala His Cys Met Ser Tyr
Ser Asn 305 310 315 Ser Ala Leu Asn Pro Leu Leu Tyr Ala Phe Leu Gly
Ser His Phe 320 325 330 Arg Gln Ala Phe Arg Arg Val Cys Pro Cys Ala
Pro Arg Arg Pro 335 340 345 Arg Arg Pro Arg Arg Pro Gly Pro Ser Asp
Pro Ala Ala Pro His 350 355 360 Ala Glu Leu His Arg Leu Gly Ser His
Pro Ala Pro Ala Arg Ala 365 370 375 Gln Lys Pro Gly Ser Ser Gly Leu
Ala Ala Arg Gly Leu Cys Val 380 385 390 Leu Gly Glu Asp Asn Ala Pro
Leu 395 105 359 PRT Homo Sapien 105 Met Ser Met Asn Asn Ser Lys Gln
Leu Val Ser Pro Ala Ala Ala 1 5 10 15 Leu Leu Ser Asn Thr Thr Cys
Gln Thr Glu Asn Arg Leu Ser Val 20 25 30 Phe Phe Ser Val Ile Phe
Met Thr Val Gly Ile Leu Ser Asn Ser 35 40 45 Leu Ala Ile Ala Ile
Leu Met Lys Ala Tyr Gln Arg Phe Arg Gln 50 55 60 Lys Ser Lys Ala
Ser Phe Leu Leu Leu Ala Ser Gly Leu Val Ile 65 70 75 Thr Asp Phe
Phe Gly His Leu Ile Asn Gly Ala Ile Ala Val Phe 80 85 90 Val Tyr
Ala Ser Asp Lys Glu Trp Ile Arg Phe Asp Gln Ser Asn 95 100 105 Val
Leu Cys Ser Ile Phe Gly Ile Cys Met Val Phe Ser Gly Leu 110 115 120
Cys Pro Leu Leu Leu Gly Ser Val Met Ala Ile Glu Arg Cys Ile 125 130
135 Gly Val Thr Lys Pro Ile Phe His Ser Thr Lys Ile Thr Ser Lys 140
145 150 His Val Lys Met Met Leu Ser Gly Val Cys Leu Phe Ala Val Phe
155 160 165 Ile Ala Leu Leu Pro Ile Leu Gly His Arg Asp Tyr Lys Ile
Gln 170 175 180 Ala Ser Arg Thr Trp Cys Phe Tyr Asn Thr Glu Asp Ile
Lys Asp 185 190 195 Trp Glu Asp Arg Phe Tyr Leu Leu Leu Phe Ser Phe
Leu Gly Leu 200 205 210 Leu Ala Leu Gly Val Ser Leu Leu Cys Asn Ala
Ile Thr Gly Ile 215 220 225 Thr Leu Leu Arg Val Lys Phe Lys Ser Gln
Gln His Arg Gln Gly 230 235 240 Arg Ser His His Leu Glu Met Val Ile
Gln Leu Leu Ala Ile Met 245 250 255 Cys Val Ser Cys Ile Cys Trp Ser
Pro Phe Leu Val Thr Met Ala 260 265 270 Asn Ile Gly Ile Asn Gly Asn
His Ser Leu Glu Thr Cys Glu Thr 275 280 285 Thr Leu Phe Ala Leu Arg
Met Ala Thr Trp Asn Gln Ile Leu Asp 290 295 300 Pro Trp Val Tyr Ile
Leu Leu Arg Lys Ala Val Leu Lys Asn Leu 305 310 315 Tyr Lys Leu Ala
Ser Gln Cys Cys Gly Val His Val Ile Ser Leu 320 325 330 His Ile Trp
Glu Leu Ser Ser Ile Lys Asn Ser Leu Lys Val Ala 335 340 345 Ala Ile
Ser Glu Ser Pro Val Ala Glu Lys Ser Ala Ser Thr 350 355 106 819 PRT
Homo Sapien 106 Met Ser Arg Met Ser Arg His Pro Asp Lys Asp Leu Ala
Gln Gly 1 5 10 15 Pro Phe Asn Thr Cys Cys Gly Cys Thr Leu Met Ala
Ser Pro Ala 20 25 30 Asn Leu Pro Pro Asn Thr Gln Ala Ala Ala Glu
Arg Ala Leu Ser 35 40 45 Gln Ser Arg Trp Lys Arg Val Gln Val Pro
Ala Pro Ala Ser Leu 50 55 60 Ser Pro Phe Pro Leu Ala Met Ala Ser
Val Ala Phe Trp Ile Ser 65 70 75 Ile Leu Ile Gly Cys Glu Glu Gln
Thr Leu Cys Arg Gly Trp Arg 80 85 90 Ser Pro Val Gly Asp Gly Cys
Ala His Val Pro Pro Gln Glu Arg 95 100 105 Ala Thr Ala Glu Ala Asp
Pro Pro Gly Arg Cys Ser Thr Ser Thr 110 115 120 Ala Ser Ser Thr Ile
Cys Gly Leu Trp His Leu Ser Pro Arg Leu 125 130 135 Gln Leu Leu Pro
Pro Leu His Ser Arg Gln Gly Glu Glu Ser Gly 140 145 150 Lys Thr Glu
Lys Val Leu Leu Trp Gly Arg Glu Gly Leu His Val 155 160 165 Trp Lys
Pro Gly Val Leu Gln Pro Asp Val His Gly Thr Ser Asn 170 175 180 Leu
Gly Asn Cys Ser Phe Leu His Gly Leu Val Thr Ala Pro Ser 185 190 195
Cys Pro Arg Arg Ala Gly Ala Glu Leu Leu Asn Ser Leu Gly Ser 200 205
210 Gln Phe Ala Ile Ser Leu Phe Glu Val Gln Ser Gly Thr Glu Pro 215
220 225 Ser Ile Thr Gly Val Ala Thr Ser Gly Gln Cys Arg Ala Met Pro
230 235 240 Leu Lys His Tyr Leu Leu Leu Leu Val Gly Cys Gln Ala Trp
Gly 245 250 255 Ala Gly Leu Ala Tyr His Gly Cys Pro Ser Glu Cys Thr
Cys Ser 260 265 270 Arg Ala Ser Gln Val Glu Cys Thr Gly Ala Arg Ile
Val Ala Val 275 280 285 Pro Thr Pro Leu Pro Trp Asn Ala Met Ser Leu
Gln Ile Leu Asn 290 295 300 Thr His Ile Thr Glu Leu Asn Glu Ser Pro
Phe Leu Asn Ile Ser 305 310 315 Ala Leu Ile Ala Leu Arg Ile Glu Lys
Asn Glu Leu Ser Arg Ile 320 325 330 Thr Pro Gly Ala Phe Arg Asn Leu
Gly Ser Leu Arg Tyr Leu Ser 335 340 345 Leu Ala Asn Asn Lys Leu Gln
Val Leu Pro Ile Gly Leu Phe Gln 350 355 360 Gly Leu Asp Ser Leu Glu
Ser Leu Leu Leu Ser Ser Asn Gln Leu 365 370 375 Leu Gln Ile Gln Pro
Ala His Phe Ser Gln Cys Ser Asn Leu Lys 380 385 390 Glu Leu Gln Leu
His Gly Asn His Leu Glu Tyr Ile Pro Asp Gly 395 400 405 Ala Phe Asp
His Leu Val Gly Leu Thr Lys Leu Asn Leu Gly Lys 410 415 420 Asn Ser
Leu Thr His Ile Ser Pro Arg Val Phe Gln His Leu Gly 425 430 435 Asn
Leu Gln Val Leu Arg Leu Tyr Glu Asn Arg Leu Thr Asp Ile 440 445 450
Pro Met Gly Thr Phe Asp Gly Leu Val Asn Leu Gln Glu Leu Ala 455 460
465 Leu Gln Gln Asn Gln Ile Gly Leu Leu Ser Pro Gly Leu Phe His 470
475 480 Asn Asn His Asn Leu Gln Arg Leu Tyr Leu Ser Asn Asn His Ile
485 490 495 Ser Gln Leu Pro Pro Ser Ile Phe Met Gln Leu Pro Gln Leu
Asn 500 505 510 Arg Leu Thr Leu Phe Gly Asn Ser Leu Lys Glu Leu Ser
Leu Gly 515 520 525 Ile Phe Gly Pro Met Pro Asn Leu Arg Glu Leu Trp
Leu Tyr Asp 530 535 540 Asn His Ile Ser Ser Leu Pro Asp Asn Val Phe
Ser Asn Leu Arg 545 550 555 Gln Leu Gln Val Leu Ile Leu Ser Arg Asn
Gln Ile Ser Phe Ile 560 565 570 Ser Pro Gly Ala Phe Asn Gly Leu Thr
Glu Leu Arg Glu Leu Ser 575 580 585 Leu His Thr Asn Ala Leu Gln Asp
Leu Asp Gly Asn Val Phe Arg 590 595 600 Met Leu Ala Asn Leu Gln Asn
Ile Ser Leu Gln Asn Asn Arg Leu 605 610 615 Arg Gln Leu Pro Gly Asn
Ile Phe Ala Asn Val Asn Gly Leu Met 620 625 630 Ala Ile Gln Leu Gln
Asn Asn Gln Leu Glu Asn Leu Pro Leu Gly 635 640 645 Ile Phe Asp His
Leu Gly Lys Leu Cys Glu Leu Arg Leu Tyr Asp 650 655 660 Asn Pro Trp
Arg Cys Asp Ser Asp Ile Leu Pro Leu Arg Asn Trp 665 670 675 Leu Leu
Leu Asn Gln Pro Arg Leu Gly Thr Asp Thr Val Pro Val 680 685 690 Cys
Phe Ser Pro Ala Asn Val Arg Gly Gln Ser Leu Ile Ile Ile 695 700 705
Asn Val Asn Val Ala Val Pro Ser Val His Val Pro Glu Val Pro 710 715
720 Ser Tyr Pro Glu Thr Pro Trp Tyr Pro Asp Thr Pro Ser Tyr Pro 725
730 735 Asp Thr Thr Ser Val Ser Ser Thr Thr Glu Leu Thr Ser Pro Val
740 745 750 Glu Asp Tyr Thr Asp Leu Thr Thr Ile Gln Val Thr Asp Asp
Arg 755 760 765 Ser Val Trp Gly Met Thr His Ala His Ser Gly Leu Ala
Ile Ala 770 775 780 Ala Ile Val Ile Gly Ile Val Ala Leu Ala Cys Ser
Leu Ala Ala 785 790 795 Cys Val Gly Cys Cys Cys Cys Lys Lys Arg Ser
Gln Ala Val Leu 800 805 810 Met Gln Met Lys Ala Pro Asn Glu Cys 815
107 3014 PRT Homo Sapien 107 Met Ala Pro Pro Pro Pro Pro Val Leu
Pro Val Leu Leu Leu Leu 1 5 10 15 Ala Ala Ala Ala Ala Leu Pro Ala
Met Gly Leu Arg Ala Ala Ala 20 25 30 Trp Glu Pro Arg Val Pro Gly
Gly Thr Arg Ala Phe Ala Leu Arg 35 40 45 Pro Gly Cys Thr Tyr Ala
Val Gly Ala Ala Cys Thr Pro Arg Ala 50 55 60 Pro Arg Glu Leu Leu
Asp Val Gly Arg Asp Gly Arg Leu Ala Gly 65 70 75 Arg Arg Arg Val
Ser Gly Ala Gly Arg Pro Leu Pro Leu Gln Val 80 85 90 Arg Leu Val
Ala Arg Ser Ala Pro Thr Ala Leu Ser Arg Arg Leu 95 100 105 Arg Ala
Arg Thr His Leu Pro Gly Cys Gly Ala Arg Ala Arg Leu 110 115 120 Cys
Gly Thr Gly Ala Arg Leu Cys Gly Ala Leu Cys Phe Pro Val 125 130 135
Pro Gly Gly Cys Ala Ala Ala Gln His Ser Ala Leu Ala Ala Pro 140 145
150 Thr Thr Leu Pro Ala Cys Arg Cys Pro Pro Arg Pro Arg Pro Arg 155
160 165 Cys Pro Gly Arg
Pro Ile Cys Leu Pro Pro Gly Gly Ser Val Arg 170 175 180 Leu Arg Leu
Leu Cys Ala Leu Arg Arg Ala Ala Gly Ala Val Arg 185 190 195 Val Gly
Leu Ala Leu Glu Ala Ala Thr Ala Gly Thr Pro Ser Ala 200 205 210 Ser
Pro Ser Pro Ser Pro Pro Leu Pro Pro Asn Leu Pro Glu Ala 215 220 225
Arg Ala Gly Pro Ala Arg Arg Ala Arg Arg Gly Thr Ser Gly Arg 230 235
240 Gly Ser Leu Lys Phe Pro Met Pro Asn Tyr Gln Val Ala Leu Phe 245
250 255 Glu Asn Glu Pro Ala Gly Thr Leu Ile Leu Gln Leu His Ala His
260 265 270 Tyr Thr Ile Glu Gly Glu Glu Glu Arg Val Ser Tyr Tyr Met
Glu 275 280 285 Gly Leu Phe Asp Glu Arg Ser Arg Gly Tyr Phe Arg Ile
Asp Ser 290 295 300 Ala Thr Gly Ala Val Ser Thr Asp Ser Val Leu Asp
Arg Glu Thr 305 310 315 Lys Glu Thr His Val Leu Arg Val Lys Ala Val
Asp Tyr Ser Thr 320 325 330 Pro Pro Arg Ser Ala Thr Thr Tyr Ile Thr
Val Leu Val Lys Asp 335 340 345 Thr Asn Asp His Ser Pro Val Phe Glu
Gln Ser Glu Tyr Arg Glu 350 355 360 Arg Val Arg Glu Asn Leu Glu Val
Gly Tyr Glu Val Leu Thr Ile 365 370 375 Arg Ala Ser Asp Arg Asp Ser
Pro Ile Asn Ala Asn Leu Arg Tyr 380 385 390 Arg Val Leu Gly Gly Ala
Trp Asp Val Phe Gln Leu Asn Glu Ser 395 400 405 Ser Gly Val Val Ser
Thr Arg Ala Val Leu Asp Arg Glu Glu Ala 410 415 420 Ala Glu Tyr Gln
Leu Leu Val Glu Ala Asn Asp Gln Gly Arg Asn 425 430 435 Pro Gly Pro
Leu Ser Ala Thr Ala Thr Val Tyr Ile Glu Val Glu 440 445 450 Asp Glu
Asn Asp Asn Tyr Pro Gln Phe Ser Glu Gln Asn Tyr Val 455 460 465 Val
Gln Val Pro Glu Asp Val Gly Leu Asn Thr Ala Val Leu Arg 470 475 480
Val Gln Ala Thr Asp Arg Asp Gln Gly Gln Asn Ala Ala Ile His 485 490
495 Tyr Ser Ile Leu Ser Gly Asn Val Ala Gly Gln Phe Tyr Leu His 500
505 510 Ser Leu Ser Gly Ile Leu Asp Val Ile Asn Pro Leu Asp Phe Glu
515 520 525 Asp Val Gln Lys Tyr Ser Leu Ser Ile Lys Ala Gln Asp Gly
Gly 530 535 540 Arg Pro Pro Leu Ile Asn Ser Ser Gly Val Val Ser Val
Gln Val 545 550 555 Leu Asp Val Asn Asp Asn Glu Pro Ile Phe Val Ser
Ser Pro Phe 560 565 570 Gln Ala Thr Val Leu Glu Asn Val Pro Leu Gly
Tyr Pro Val Val 575 580 585 His Ile Gln Ala Val Asp Ala Asp Ser Gly
Glu Asn Ala Arg Leu 590 595 600 His Tyr Arg Leu Val Asp Thr Ala Ser
Thr Phe Leu Gly Gly Gly 605 610 615 Ser Ala Gly Pro Lys Asn Pro Ala
Pro Thr Pro Asp Phe Pro Phe 620 625 630 Gln Ile His Asn Ser Ser Gly
Trp Ile Thr Val Cys Ala Glu Leu 635 640 645 Asp Arg Glu Glu Val Glu
His Tyr Ser Phe Gly Val Glu Ala Val 650 655 660 Asp His Gly Ser Pro
Pro Met Ser Ser Ser Thr Ser Val Ser Ile 665 670 675 Thr Val Leu Asp
Val Asn Asp Asn Asp Pro Val Phe Thr Gln Pro 680 685 690 Thr Tyr Glu
Leu Arg Leu Asn Glu Asp Ala Ala Val Gly Ser Ser 695 700 705 Val Leu
Thr Leu Gln Ala Arg Asp Arg Asp Ala Asn Ser Val Ile 710 715 720 Thr
Tyr Gln Leu Thr Gly Gly Asn Thr Arg Asn Arg Phe Ala Leu 725 730 735
Ser Ser Gln Arg Gly Gly Gly Leu Ile Thr Leu Ala Leu Pro Leu 740 745
750 Asp Tyr Lys Gln Glu Gln Gln Tyr Val Leu Ala Val Thr Ala Ser 755
760 765 Asp Gly Thr Arg Ser His Thr Ala His Val Leu Ile Asn Val Thr
770 775 780 Asp Ala Asn Thr His Arg Pro Val Phe Gln Ser Ser His Tyr
Thr 785 790 795 Val Ser Val Ser Glu Asp Arg Pro Val Gly Thr Ser Ile
Ala Thr 800 805 810 Leu Ser Ala Asn Asp Glu Asp Thr Gly Glu Asn Ala
Arg Ile Thr 815 820 825 Tyr Val Ile Gln Asp Pro Val Pro Gln Phe Arg
Ile Asp Pro Asp 830 835 840 Ser Gly Thr Met Tyr Thr Met Met Glu Leu
Asp Tyr Glu Asn Gln 845 850 855 Val Ala Tyr Thr Leu Thr Ile Met Ala
Gln Asp Asn Gly Ile Pro 860 865 870 Gln Lys Ser Asp Thr Thr Thr Leu
Glu Ile Leu Ile Leu Asp Ala 875 880 885 Asn Asp Asn Ala Pro Gln Phe
Leu Trp Asp Phe Tyr Gln Gly Ser 890 895 900 Ile Phe Glu Asp Ala Pro
Pro Ser Thr Ser Ile Leu Gln Val Ser 905 910 915 Ala Thr Asp Arg Asp
Ser Gly Pro Asn Gly Arg Leu Leu Tyr Thr 920 925 930 Phe Gln Gly Gly
Asp Asp Gly Asp Gly Asp Phe Tyr Ile Glu Pro 935 940 945 Thr Ser Gly
Val Ile Arg Thr Gln Arg Arg Leu Asp Arg Glu Asn 950 955 960 Val Ala
Val Tyr Asn Leu Trp Ala Leu Ala Val Asp Arg Gly Ser 965 970 975 Pro
Thr Pro Leu Ser Ala Ser Val Glu Ile Gln Val Thr Ile Leu 980 985 990
Asp Ile Asn Asp Asn Ala Pro Met Phe Glu Lys Asp Glu Leu Glu 995
1000 1005 Leu Phe Val Glu Glu Asn Asn Pro Val Gly Ser Val Val Ala
Lys 1010 1015 1020 Ile Arg Ala Asn Asp Pro Asp Glu Gly Pro Asn Ala
Gln Ile Met 1025 1030 1035 Tyr Gln Ile Val Glu Gly Asp Met Arg His
Phe Phe Gln Leu Asp 1040 1045 1050 Leu Leu Asn Gly Asp Leu Arg Ala
Met Val Glu Leu Asp Phe Glu 1055 1060 1065 Val Arg Arg Glu Tyr Val
Leu Val Val Gln Ala Thr Ser Ala Pro 1070 1075 1080 Leu Val Ser Arg
Ala Thr Val His Ile Leu Leu Val Asp Gln Asn 1085 1090 1095 Asp Asn
Pro Pro Val Leu Pro Asp Phe Gln Ile Leu Phe Asn Asn 1100 1105 1110
Tyr Val Thr Asn Lys Ser Asn Ser Phe Pro Thr Gly Val Ile Gly 1115
1120 1125 Cys Ile Pro Ala His Asp Pro Asp Val Ser Asp Ser Leu Asn
Tyr 1130 1135 1140 Thr Phe Val Gln Gly Asn Glu Leu Arg Leu Leu Leu
Leu Asp Pro 1145 1150 1155 Ala Thr Gly Glu Leu Gln Leu Ser Arg Asp
Leu Asp Asn Asn Arg 1160 1165 1170 Pro Leu Glu Ala Leu Met Glu Val
Ser Val Ser Asp Gly Ile His 1175 1180 1185 Ser Val Thr Ala Phe Cys
Thr Leu Arg Val Thr Ile Ile Thr Asp 1190 1195 1200 Asp Met Leu Thr
Asn Ser Ile Thr Val Arg Leu Glu Asn Met Ser 1205 1210 1215 Gln Glu
Lys Phe Leu Ser Pro Leu Leu Ala Leu Phe Val Glu Gly 1220 1225 1230
Val Ala Ala Val Leu Ser Thr Thr Lys Asp Asp Val Phe Val Phe 1235
1240 1245 Asn Val Gln Asn Asp Thr Asp Val Ser Ser Asn Ile Leu Asn
Val 1250 1255 1260 Thr Phe Ser Ala Leu Leu Pro Gly Gly Val Arg Gly
Gln Phe Phe 1265 1270 1275 Pro Ser Glu Asp Leu Gln Glu Gln Ile Tyr
Leu Asn Arg Thr Leu 1280 1285 1290 Leu Thr Thr Ile Ser Thr Gln Arg
Val Leu Pro Phe Asp Asp Asn 1295 1300 1305 Ile Cys Leu Arg Glu Pro
Cys Glu Asn Tyr Met Lys Cys Val Ser 1310 1315 1320 Val Leu Arg Phe
Asp Ser Ser Ala Pro Phe Leu Ser Ser Thr Thr 1325 1330 1335 Val Leu
Phe Arg Pro Ile His Pro Ile Asn Gly Leu Arg Cys Arg 1340 1345 1350
Cys Pro Pro Gly Phe Thr Gly Asp Tyr Cys Glu Thr Glu Ile Asp 1355
1360 1365 Leu Cys Tyr Ser Asp Pro Cys Gly Ala Asn Gly Arg Cys Arg
Ser 1370 1375 1380 Arg Glu Gly Gly Tyr Thr Cys Glu Cys Phe Glu Asp
Phe Thr Gly 1385 1390 1395 Glu His Cys Glu Val Asp Ala Arg Ser Gly
Arg Cys Ala Asn Gly 1400 1405 1410 Val Cys Lys Asn Gly Gly Thr Cys
Val Asn Leu Leu Ile Gly Gly 1415 1420 1425 Phe His Cys Val Cys Pro
Pro Gly Glu Tyr Glu Arg Pro Tyr Cys 1430 1435 1440 Glu Val Thr Thr
Arg Ser Phe Pro Pro Gln Ser Phe Val Thr Phe 1445 1450 1455 Arg Gly
Leu Arg Gln Arg Phe His Phe Thr Ile Ser Leu Thr Phe 1460 1465 1470
Ala Thr Gln Glu Arg Asn Gly Leu Leu Leu Tyr Asn Gly Arg Phe 1475
1480 1485 Asn Glu Lys His Asp Phe Ile Ala Leu Glu Ile Val Asp Glu
Gln 1490 1495 1500 Val Gln Leu Thr Phe Ser Ala Gly Glu Thr Thr Thr
Thr Val Ala 1505 1510 1515 Pro Lys Val Pro Ser Gly Val Ser Asp Gly
Arg Trp His Ser Val 1520 1525 1530 Gln Val Gln Tyr Tyr Asn Lys Pro
Asn Ile Gly His Leu Gly Leu 1535 1540 1545 Pro His Gly Pro Ser Gly
Glu Lys Met Ala Val Val Thr Val Asp 1550 1555 1560 Asp Cys Asp Thr
Thr Met Ala Val Arg Phe Gly Lys Asp Ile Gly 1565 1570 1575 Asn Tyr
Ser Cys Ala Ala Gln Gly Thr Gln Thr Gly Ser Lys Lys 1580 1585 1590
Ser Leu Asp Leu Thr Gly Pro Leu Leu Leu Gly Gly Val Pro Asn 1595
1600 1605 Leu Pro Glu Asp Phe Pro Val His Asn Arg Gln Phe Val Gly
Cys 1610 1615 1620 Met Arg Asn Leu Ser Val Asp Gly Lys Asn Val Asp
Met Ala Gly 1625 1630 1635 Phe Ile Ala Asn Asn Gly Thr Arg Glu Gly
Cys Ala Ala Arg Arg 1640 1645 1650 Asn Phe Cys Asp Gly Arg Arg Cys
Gln Asn Gly Gly Thr Cys Val 1655 1660 1665 Asn Arg Trp Asn Met Tyr
Leu Cys Glu Cys Pro Leu Arg Phe Gly 1670 1675 1680 Gly Lys Asn Cys
Glu Gln Ala Met Pro His Pro Gln Leu Phe Ser 1685 1690 1695 Gly Glu
Ser Val Val Ser Trp Ser Asp Leu Asn Ile Ile Ile Ser 1700 1705 1710
Val Pro Trp Tyr Leu Gly Leu Met Phe Arg Thr Arg Lys Glu Asp 1715
1720 1725 Ser Val Leu Met Glu Ala Thr Ser Gly Gly Pro Thr Ser Phe
Arg 1730 1735 1740 Leu Gln Ile Leu Asn Asn Tyr Leu Gln Phe Glu Val
Ser His Gly 1745 1750 1755 Pro Ser Asp Val Glu Ser Val Met Leu Ser
Gly Leu Arg Val Thr 1760 1765 1770 Asp Gly Glu Trp His His Leu Leu
Ile Glu Leu Lys Asn Val Lys 1775 1780 1785 Glu Asp Ser Glu Met Lys
His Leu Val Thr Met Thr Leu Asp Tyr 1790 1795 1800 Gly Met Asp Gln
Asn Lys Ala Asp Ile Gly Gly Met Leu Pro Gly 1805 1810 1815 Leu Thr
Val Arg Ser Val Val Val Gly Gly Ala Ser Glu Asp Lys 1820 1825 1830
Val Ser Val Arg Arg Gly Phe Arg Gly Cys Met Gln Gly Val Arg 1835
1840 1845 Met Gly Gly Thr Pro Thr Asn Val Ala Thr Leu Asn Met Asn
Asn 1850 1855 1860 Ala Leu Lys Val Arg Val Lys Asp Gly Cys Asp Val
Asp Asp Pro 1865 1870 1875 Cys Thr Ser Ser Pro Cys Pro Pro Asn Ser
Arg Cys His Asp Ala 1880 1885 1890 Trp Glu Asp Tyr Ser Cys Val Cys
Asp Lys Gly Tyr Leu Gly Ile 1895 1900 1905 Asn Cys Val Asp Ala Cys
His Leu Asn Pro Cys Glu Asn Met Gly 1910 1915 1920 Ala Cys Val Arg
Ser Pro Gly Ser Pro Gln Gly Tyr Val Cys Glu 1925 1930 1935 Cys Gly
Pro Ser His Tyr Gly Pro Tyr Cys Glu Asn Lys Leu Asp 1940 1945 1950
Leu Pro Cys Pro Arg Gly Trp Trp Gly Asn Pro Val Cys Gly Pro 1955
1960 1965 Cys His Cys Ala Val Ser Lys Gly Phe Asp Pro Asp Cys Asn
Lys 1970 1975 1980 Thr Asn Gly Gln Cys Gln Cys Lys Glu Asn Tyr Tyr
Lys Leu Leu 1985 1990 1995 Ala Gln Asp Thr Cys Leu Pro Cys Asp Cys
Phe Pro His Gly Ser 2000 2005 2010 His Ser Arg Thr Cys Asp Met Ala
Thr Gly Gln Cys Ala Cys Lys 2015 2020 2025 Pro Gly Val Ile Gly Arg
Gln Cys Asn Arg Cys Asp Asn Pro Phe 2030 2035 2040 Ala Glu Val Thr
Thr Leu Gly Cys Glu Val Ile Tyr Asn Gly Cys 2045 2050 2055 Pro Lys
Ala Phe Glu Ala Gly Ile Trp Trp Pro Gln Thr Lys Phe 2060 2065 2070
Gly Gln Pro Ala Ala Val Pro Cys Pro Lys Gly Ser Val Gly Asn 2075
2080 2085 Ala Val Arg His Cys Ser Gly Glu Lys Gly Trp Leu Pro Pro
Glu 2090 2095 2100 Leu Phe Asn Cys Thr Thr Ile Ser Phe Val Asp Leu
Arg Ala Met 2105 2110 2115 Asn Glu Lys Leu Ser Arg Asn Glu Thr Gln
Val Asp Gly Ala Arg 2120 2125 2130 Ala Leu Gln Leu Val Arg Ala Leu
Arg Ser Ala Thr Gln His Thr 2135 2140 2145 Gly Thr Leu Phe Gly Asn
Asp Val Arg Thr Ala Tyr Gln Leu Leu 2150 2155 2160 Gly His Val Leu
Gln His Glu Ser Trp Gln Gln Gly Phe Asp Leu 2165 2170 2175 Ala Ala
Thr Gln Asp Ala Asp Phe His Glu Asp Val Ile His Ser 2180 2185 2190
Gly Ser Ala Leu Leu Ala Pro Ala Thr Arg Ala Ala Trp Glu Gln 2195
2200 2205 Ile Gln Arg Ser Glu Gly Gly Thr Ala Gln Leu Leu Arg Arg
Leu 2210 2215 2220 Glu Gly Tyr Phe Ser Asn Val Ala Arg Asn Val Arg
Arg Thr Tyr 2225 2230 2235 Leu Arg Pro Phe Val Ile Val Thr Ala Asn
Met Ile Leu Ala Val 2240 2245 2250 Asp Ile Phe Asp Lys Phe Asn Phe
Thr Gly Ala Arg Val Pro Arg 2255 2260 2265 Phe Asp Thr Ile His Glu
Glu Phe Pro Arg Glu Leu Glu Ser Ser 2270 2275 2280 Val Ser Phe Pro
Ala Asp Phe Phe Arg Pro Pro Glu Glu Lys Glu 2285 2290 2295 Gly Pro
Leu Leu Arg Pro Ala Gly Arg Arg Thr Thr Pro Gln Thr 2300 2305 2310
Thr Arg Pro Gly Pro Gly Thr Glu Arg Glu Ala Pro Ile Ser Arg 2315
2320 2325 Arg Arg Arg His Pro Asp Asp Ala Gly Gln Phe Ala Val Ala
Leu 2330 2335 2340 Val Ile Ile Tyr Arg Thr Leu Gly Gln Leu Leu Pro
Glu Arg Tyr 2345 2350 2355 Asp Pro Asp Arg Arg Ser Leu Arg Leu Pro
His Arg Pro Ile Ile 2360 2365 2370 Asn Thr Pro Met Val Ser Thr Leu
Val Tyr Ser Glu Gly Ala Pro 2375 2380 2385 Leu Pro Arg Pro Leu Glu
Arg Pro Val Leu Val Glu Phe Ala Leu 2390 2395 2400 Leu Glu Val Glu
Glu Arg Thr Lys Pro Val Cys Val Phe Trp Asn 2405 2410 2415 His Ser
Leu Ala Val Gly Gly Thr Gly Gly Trp Ser Ala Arg Gly 2420 2425 2430
Cys Glu Leu Leu Ser Arg Asn Arg Thr His Val Ala Cys Gln Cys 2435
2440 2445 Ser His Thr Ala Ser Phe Ala Val Leu Met Asp Ile Ser Arg
Arg 2450 2455 2460 Glu Asn Gly Glu Val Leu Pro Leu Lys Ile Val Thr
Tyr Ala Ala
2465 2470 2475 Val Ser Leu Ser Leu Ala Ala Leu Leu Val Ala Phe Val
Leu Leu 2480 2485 2490 Ser Leu Val Arg Met Leu Arg Ser Asn Leu His
Ser Ile His Lys 2495 2500 2505 His Leu Ala Val Ala Leu Phe Leu Ser
Gln Leu Val Phe Val Ile 2510 2515 2520 Gly Ile Asn Gln Thr Glu Asn
Pro Phe Leu Cys Thr Val Val Ala 2525 2530 2535 Ile Leu Leu His Tyr
Ile Tyr Met Ser Thr Phe Ala Trp Thr Leu 2540 2545 2550 Val Glu Ser
Leu His Val Tyr Arg Met Leu Thr Glu Val Arg Asn 2555 2560 2565 Ile
Asp Thr Gly Pro Met Arg Phe Tyr Tyr Val Val Gly Trp Gly 2570 2575
2580 Ile Pro Ala Ile Val Thr Gly Leu Ala Val Gly Leu Asp Pro Gln
2585 2590 2595 Gly Tyr Gly Asn Pro Asp Phe Cys Trp Leu Ser Leu Gln
Asp Thr 2600 2605 2610 Leu Ile Trp Ser Phe Ala Gly Pro Ile Gly Ala
Val Ile Ile Ile 2615 2620 2625 Asn Thr Val Thr Ser Val Leu Ser Ala
Lys Val Ser Cys Gln Arg 2630 2635 2640 Lys His His Tyr Tyr Gly Lys
Lys Gly Ile Val Ser Leu Leu Arg 2645 2650 2655 Thr Ala Phe Leu Leu
Leu Leu Leu Ile Ser Ala Thr Trp Leu Leu 2660 2665 2670 Gly Leu Leu
Ala Val Asn Arg Asp Ala Leu Ser Phe His Tyr Leu 2675 2680 2685 Phe
Ala Ile Phe Ser Gly Leu Gln Gly Pro Phe Val Leu Leu Phe 2690 2695
2700 His Cys Val Leu Asn Gln Glu Val Arg Lys His Leu Lys Gly Val
2705 2710 2715 Leu Gly Gly Arg Lys Leu His Leu Glu Asp Ser Ala Thr
Thr Arg 2720 2725 2730 Ala Thr Leu Leu Thr Arg Ser Leu Asn Cys Asn
Thr Thr Phe Gly 2735 2740 2745 Asp Gly Pro Asp Met Leu Arg Thr Asp
Leu Gly Glu Ser Thr Ala 2750 2755 2760 Ser Leu Asp Ser Ile Val Arg
Asp Glu Gly Ile Gln Lys Leu Gly 2765 2770 2775 Val Ser Ser Gly Leu
Val Arg Gly Ser His Gly Glu Pro Asp Ala 2780 2785 2790 Ser Leu Met
Pro Arg Ser Cys Lys Asp Pro Pro Gly His Asp Ser 2795 2800 2805 Asp
Ser Asp Ser Glu Leu Ser Leu Asp Glu Gln Ser Ser Ser Tyr 2810 2815
2820 Ala Ser Ser His Ser Ser Asp Ser Glu Asp Asp Gly Val Gly Ala
2825 2830 2835 Glu Glu Lys Trp Asp Pro Ala Arg Gly Ala Val His Ser
Thr Pro 2840 2845 2850 Lys Gly Asp Ala Val Ala Asn His Val Pro Ala
Gly Trp Pro Asp 2855 2860 2865 Gln Ser Leu Ala Glu Ser Asp Ser Glu
Asp Pro Ser Gly Lys Pro 2870 2875 2880 Arg Leu Lys Val Glu Thr Lys
Val Ser Val Glu Leu His Arg Glu 2885 2890 2895 Glu Gln Gly Ser His
Arg Gly Glu Tyr Pro Pro Asp Gln Glu Ser 2900 2905 2910 Gly Gly Ala
Ala Arg Leu Ala Ser Ser Gln Pro Pro Glu Gln Arg 2915 2920 2925 Lys
Gly Ile Leu Lys Asn Lys Val Thr Tyr Pro Pro Pro Leu Thr 2930 2935
2940 Leu Thr Glu Gln Thr Leu Lys Gly Arg Leu Arg Glu Lys Leu Ala
2945 2950 2955 Asp Cys Glu Gln Ser Pro Thr Ser Ser Arg Thr Ser Ser
Leu Gly 2960 2965 2970 Ser Gly Gly Pro Asp Cys Ala Ile Thr Val Lys
Ser Pro Gly Arg 2975 2980 2985 Glu Pro Gly Arg Asp His Leu Asn Gly
Val Ala Met Asn Val Arg 2990 2995 3000 Thr Gly Ser Ala Gln Ala Asp
Gly Ser Asp Ser Glu Lys Pro 3005 3010 108 181 PRT Homo Sapien 108
Met Val Asp Val Lys Cys Leu Ser Asp Cys Lys Leu Gln Asn Gln 1 5 10
15 Leu Glu Lys Leu Gly Phe Ser Pro Gly Pro Ile Leu Pro Ser Thr 20
25 30 Arg Lys Leu Tyr Glu Lys Lys Leu Val Gln Leu Leu Val Ser Pro
35 40 45 Pro Cys Ala Pro Pro Val Met Asn Gly Pro Arg Glu Leu Asp
Gly 50 55 60 Ala Gln Asp Ser Asp Asp Ser Glu Glu Leu Asn Ile Ile
Leu Gln 65 70 75 Gly Asn Ile Ile Leu Ser Thr Glu Lys Ser Lys Lys
Leu Lys Lys 80 85 90 Trp Pro Glu Ala Ser Thr Thr Lys Arg Lys Ala
Val Asp Thr Tyr 95 100 105 Cys Leu Asp Tyr Lys Pro Ser Lys Gly Arg
Arg Trp Ala Ala Arg 110 115 120 Ala Pro Ser Thr Arg Ile Thr Tyr Gly
Thr Ile Thr Lys Glu Arg 125 130 135 Asp Tyr Cys Ala Glu Asp Gln Thr
Ile Glu Ser Trp Arg Glu Glu 140 145 150 Gly Phe Pro Val Gly Leu Lys
Leu Ala Val Leu Gly Ile Phe Ile 155 160 165 Ile Val Val Phe Val Tyr
Leu Thr Val Glu Asn Lys Ser Leu Phe 170 175 180 Gly 109 620 PRT
Homo Sapien 109 Met Ser Lys Ser Lys Cys Ser Val Gly Leu Met Ser Ser
Val Val 1 5 10 15 Ala Pro Ala Lys Glu Pro Asn Ala Val Gly Pro Lys
Glu Val Glu 20 25 30 Leu Ile Leu Val Lys Glu Gln Asn Gly Val Gln
Leu Thr Ser Ser 35 40 45 Thr Leu Thr Asn Pro Arg Gln Ser Pro Val
Glu Ala Gln Asp Arg 50 55 60 Glu Thr Trp Gly Lys Lys Ile Asp Phe
Leu Leu Ser Val Ile Gly 65 70 75 Phe Ala Val Asp Leu Ala Asn Val
Trp Arg Phe Pro Tyr Leu Cys 80 85 90 Tyr Lys Asn Gly Gly Gly Ala
Phe Leu Val Pro Tyr Leu Leu Phe 95 100 105 Met Val Ile Ala Gly Met
Pro Leu Phe Tyr Met Glu Leu Ala Leu 110 115 120 Gly Gln Phe Asn Arg
Glu Gly Ala Ala Gly Val Trp Lys Ile Cys 125 130 135 Pro Ile Leu Lys
Gly Val Gly Phe Thr Val Ile Leu Ile Ser Leu 140 145 150 Tyr Val Gly
Phe Phe Tyr Asn Val Ile Ile Ala Trp Ala Leu His 155 160 165 Tyr Leu
Phe Ser Ser Phe Thr Thr Glu Leu Pro Trp Ile His Cys 170 175 180 Asn
Asn Ser Trp Asn Ser Pro Asn Cys Ser Asp Ala His Pro Gly 185 190 195
Asp Ser Ser Gly Asp Ser Ser Gly Leu Asn Asp Thr Phe Gly Thr 200 205
210 Thr Pro Ala Ala Glu Tyr Phe Glu Arg Gly Val Leu His Leu His 215
220 225 Gln Ser His Gly Ile Asp Asp Leu Gly Pro Pro Arg Trp Gln Leu
230 235 240 Thr Ala Cys Leu Val Leu Val Ile Val Leu Leu Tyr Phe Ser
Leu 245 250 255 Trp Lys Gly Val Lys Thr Ser Gly Lys Val Val Trp Ile
Thr Ala 260 265 270 Thr Met Pro Tyr Val Val Leu Thr Ala Leu Leu Leu
Arg Gly Val 275 280 285 Thr Leu Pro Gly Ala Ile Asp Gly Ile Arg Ala
Tyr Leu Ser Val 290 295 300 Asp Phe Tyr Arg Leu Cys Glu Ala Ser Val
Trp Ile Asp Ala Ala 305 310 315 Thr Gln Val Cys Phe Ser Leu Gly Val
Gly Phe Gly Val Leu Ile 320 325 330 Ala Phe Ser Ser Tyr Asn Lys Phe
Thr Asn Asn Cys Tyr Arg Asp 335 340 345 Ala Ile Val Thr Thr Ser Ile
Asn Ser Leu Thr Ser Phe Ser Ser 350 355 360 Gly Phe Val Val Phe Ser
Phe Leu Gly Tyr Met Ala Gln Lys His 365 370 375 Ser Val Pro Ile Gly
Asp Val Ala Lys Asp Gly Pro Gly Leu Ile 380 385 390 Phe Ile Ile Tyr
Pro Glu Ala Ile Ala Thr Leu Pro Leu Ser Ser 395 400 405 Ala Trp Ala
Val Val Phe Phe Ile Met Leu Leu Thr Leu Gly Ile 410 415 420 Asp Ser
Ala Met Gly Gly Met Glu Ser Val Ile Thr Gly Leu Ile 425 430 435 Asp
Glu Phe Gln Leu Leu His Arg His Arg Glu Leu Phe Thr Leu 440 445 450
Phe Ile Val Leu Ala Thr Phe Leu Leu Ser Leu Phe Cys Val Thr 455 460
465 Asn Gly Gly Ile Tyr Val Phe Thr Leu Leu Asp His Phe Ala Ala 470
475 480 Gly Thr Ser Ile Leu Phe Gly Val Leu Ile Glu Ala Ile Gly Val
485 490 495 Ala Trp Phe Tyr Gly Val Gly Gln Phe Ser Asp Asp Ile Gln
Gln 500 505 510 Met Thr Gly Gln Arg Pro Ser Leu Tyr Trp Arg Leu Cys
Trp Lys 515 520 525 Leu Val Ser Pro Cys Phe Leu Leu Phe Val Val Val
Val Ser Ile 530 535 540 Val Thr Phe Arg Pro Pro His Tyr Gly Ala Tyr
Ile Phe Pro Asp 545 550 555 Trp Ala Asn Ala Leu Gly Trp Val Ile Ala
Thr Ser Ser Met Ala 560 565 570 Met Val Pro Ile Tyr Ala Ala Tyr Lys
Phe Cys Ser Leu Pro Gly 575 580 585 Ser Phe Arg Glu Lys Leu Ala Tyr
Ala Ile Ala Pro Glu Lys Asp 590 595 600 Arg Glu Leu Val Asp Arg Gly
Glu Val Arg Gln Phe Thr Leu Arg 605 610 615 His Trp Leu Lys Val 620
110 442 PRT Homo Sapien 110 Met Gly Leu Ala Met Glu His Gly Gly Ser
Tyr Ala Arg Ala Gly 1 5 10 15 Gly Ser Ser Arg Gly Cys Trp Tyr Tyr
Leu Arg Tyr Phe Phe Leu 20 25 30 Phe Val Ser Leu Ile Gln Phe Leu
Ile Ile Leu Gly Leu Val Leu 35 40 45 Phe Met Val Tyr Gly Asn Val
His Val Ser Thr Glu Ser Asn Leu 50 55 60 Gln Ala Thr Glu Arg Arg
Ala Glu Gly Leu Tyr Ser Gln Leu Leu 65 70 75 Gly Leu Thr Ala Ser
Gln Ser Asn Leu Thr Lys Glu Leu Asn Phe 80 85 90 Thr Thr Arg Ala
Lys Asp Ala Ile Met Gln Met Trp Leu Asn Ala 95 100 105 Arg Arg Asp
Leu Asp Arg Ile Asn Ala Ser Phe Arg Gln Cys Gln 110 115 120 Gly Asp
Arg Val Ile Tyr Thr Asn Asn Gln Arg Tyr Met Ala Ala 125 130 135 Ile
Ile Leu Ser Glu Lys Gln Cys Arg Asp Gln Phe Lys Asp Met 140 145 150
Asn Lys Ser Cys Asp Ala Leu Leu Phe Met Leu Asn Gln Lys Val 155 160
165 Lys Thr Leu Glu Val Glu Ile Ala Lys Glu Lys Thr Ile Cys Thr 170
175 180 Lys Asp Lys Glu Ser Val Leu Leu Asn Lys Arg Val Ala Glu Glu
185 190 195 Gln Leu Val Glu Cys Val Lys Thr Arg Glu Leu Gln His Gln
Glu 200 205 210 Arg Gln Leu Ala Lys Glu Gln Leu Gln Lys Val Gln Ala
Leu Cys 215 220 225 Leu Pro Leu Asp Lys Asp Lys Phe Glu Met Asp Leu
Arg Asn Leu 230 235 240 Trp Arg Asp Ser Ile Ile Pro Arg Ser Leu Asp
Asn Leu Gly Tyr 245 250 255 Asn Leu Tyr His Pro Leu Gly Ser Glu Leu
Ala Ser Ile Arg Arg 260 265 270 Ala Cys Asp His Met Pro Ser Leu Met
Ser Ser Lys Val Glu Glu 275 280 285 Leu Ala Arg Ser Leu Arg Ala Asp
Ile Glu Arg Val Ala Arg Glu 290 295 300 Asn Ser Asp Leu Gln Arg Gln
Lys Leu Glu Ala Gln Gln Gly Leu 305 310 315 Arg Ala Ser Gln Glu Ala
Lys Gln Lys Val Glu Lys Glu Ala Gln 320 325 330 Ala Arg Glu Ala Lys
Leu Gln Ala Glu Cys Ser Arg Gln Thr Gln 335 340 345 Leu Ala Leu Glu
Glu Lys Ala Val Leu Arg Lys Glu Arg Asp Asn 350 355 360 Leu Ala Lys
Glu Leu Glu Glu Lys Lys Arg Glu Ala Glu Gln Leu 365 370 375 Arg Met
Glu Leu Ala Ile Arg Asn Ser Ala Leu Asp Thr Cys Ile 380 385 390 Lys
Thr Lys Ser Gln Pro Met Met Pro Val Ser Arg Pro Met Gly 395 400 405
Pro Val Pro Asn Pro Gln Pro Ile Asp Pro Ala Ser Leu Glu Glu 410 415
420 Phe Lys Arg Lys Ile Leu Glu Ser Gln Arg Pro Pro Ala Gly Ile 425
430 435 Pro Val Ala Pro Ser Ser Gly 440 111 170 PRT Homo Sapien 111
Met Met Ala Gly Met Lys Ile Gln Leu Val Cys Met Leu Leu Leu 1 5 10
15 Ala Phe Ser Ser Trp Ser Leu Cys Ser Asp Ser Glu Glu Glu Met 20
25 30 Lys Ala Leu Glu Ala Asp Phe Leu Thr Asn Met His Thr Ser Lys
35 40 45 Ile Ser Lys Ala His Val Pro Ser Trp Lys Met Thr Leu Leu
Asn 50 55 60 Val Cys Ser Leu Val Asn Asn Leu Asn Ser Pro Ala Glu
Glu Thr 65 70 75 Gly Glu Val His Glu Glu Glu Leu Val Ala Arg Arg
Lys Leu Pro 80 85 90 Thr Ala Leu Asp Gly Phe Ser Leu Glu Ala Met
Leu Thr Ile Tyr 95 100 105 Gln Leu His Lys Ile Cys His Ser Arg Ala
Phe Gln His Trp Glu 110 115 120 Leu Ile Gln Glu Asp Ile Leu Asp Thr
Gly Asn Asp Lys Asn Gly 125 130 135 Lys Glu Glu Val Ile Lys Arg Lys
Ile Pro Tyr Ile Leu Lys Arg 140 145 150 Gln Leu Tyr Glu Asn Lys Pro
Arg Arg Pro Tyr Ile Leu Lys Arg 155 160 165 Asp Ser Tyr Tyr Tyr 170
112 502 PRT Homo Sapien 112 Met Leu Leu Arg Ser Ala Gly Lys Leu Asn
Val Gly Thr Lys Lys 1 5 10 15 Glu Asp Gly Glu Ser Thr Ala Pro Thr
Pro Arg Pro Lys Val Leu 20 25 30 Arg Cys Lys Cys His His His Cys
Pro Glu Asp Ser Val Asn Asn 35 40 45 Ile Cys Ser Thr Asp Gly Tyr
Cys Phe Thr Met Ile Glu Glu Asp 50 55 60 Asp Ser Gly Leu Pro Val
Val Thr Ser Gly Cys Leu Gly Leu Glu 65 70 75 Gly Ser Asp Phe Gln
Cys Arg Asp Thr Pro Ile Pro His Gln Arg 80 85 90 Arg Ser Ile Glu
Cys Cys Thr Glu Arg Asn Glu Cys Asn Lys Asp 95 100 105 Leu His Pro
Thr Leu Pro Pro Leu Lys Asn Arg Asp Phe Val Asp 110 115 120 Gly Pro
Ile His His Arg Ala Leu Leu Ile Ser Val Thr Val Cys 125 130 135 Ser
Leu Leu Leu Val Leu Ile Ile Leu Phe Cys Tyr Phe Arg Tyr 140 145 150
Lys Arg Gln Glu Thr Arg Pro Arg Tyr Ser Ile Gly Leu Glu Gln 155 160
165 Asp Glu Thr Tyr Ile Pro Pro Gly Glu Ser Leu Arg Asp Leu Ile 170
175 180 Glu Gln Ser Gln Ser Ser Gly Ser Gly Ser Gly Leu Pro Leu Leu
185 190 195 Val Gln Arg Thr Ile Ala Lys Gln Ile Gln Met Val Lys Gln
Ile 200 205 210 Gly Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp
Arg Gly 215 220 225 Glu Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu
Glu Ala Ser 230 235 240 Trp Phe Arg Glu Thr Glu Ile Tyr Gln Thr Val
Leu Met Arg His 245 250 255 Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp
Ile Lys Gly Thr Gly 260 265 270 Ser Trp Thr Gln Leu Tyr Leu Ile Thr
Asp Tyr His Glu Asn Gly 275 280 285 Ser Leu Tyr Asp Tyr Leu Lys Ser
Thr Thr Leu Asp Ala Lys Ser 290 295 300 Met Leu Lys Leu Ala Tyr Ser
Ser Val Ser Gly Leu Cys His Leu 305 310 315 His Thr Glu Ile Phe Ser
Thr Gln Gly Lys Pro Ala Ile Ala His 320
325 330 Arg Asp Leu Lys Ser Lys Asn Ile Leu Val Lys Lys Asn Gly Thr
335 340 345 Cys Cys Ile Ala Asp Leu Gly Leu Ala Val Lys Phe Ile Ser
Asp 350 355 360 Thr Asn Glu Val Asp Ile Pro Pro Asn Thr Arg Val Gly
Thr Lys 365 370 375 Arg Tyr Met Pro Pro Glu Val Leu Asp Glu Ser Leu
Asn Arg Asn 380 385 390 His Phe Gln Ser Tyr Ile Met Ala Asp Met Tyr
Ser Phe Gly Leu 395 400 405 Ile Leu Trp Glu Val Ala Arg Arg Cys Val
Ser Gly Gly Ile Val 410 415 420 Glu Glu Tyr Gln Leu Pro Tyr His Asp
Leu Val Pro Ser Asp Pro 425 430 435 Ser Tyr Glu Asp Met Arg Glu Ile
Val Cys Ile Lys Lys Leu Arg 440 445 450 Pro Ser Phe Pro Asn Arg Trp
Ser Ser Asp Glu Cys Leu Arg Gln 455 460 465 Met Gly Lys Leu Met Thr
Glu Cys Trp Ala His Asn Pro Ala Ser 470 475 480 Arg Leu Thr Ala Leu
Arg Val Lys Lys Thr Leu Ala Lys Met Ser 485 490 495 Glu Ser Gln Asp
Ile Lys Leu 500 113 2403 DNA Homo Sapien 113 ttgaagtgca ttgctgcagc
tggtagcatg agtggtggcc accacctgca 50 gctggctgcc ctctggccct
ggctgctgat ggctaccctg caggcaggct 100 ttggacgcac aggactggta
ctggcagcag cggtggagtc tgaaagatca 150 gcagaacaga aagctgttat
cagagtgatc cccttgaaaa tggaccccac 200 aggaaaactg aatctcactt
tggaaggtgt gtttgctggt gttgctgaaa 250 taactccagc agaaggaaaa
ttaatgcagt cccacccgct gtacctgtgc 300 aatgccagtg atgacgacaa
tctggagcct ggattcatca gcatcgtcaa 350 gctggagagt cctcgacggg
ccccccaccc ctgcctgtca ctggctagca 400 aggctcggat ggcgggtgag
cgaggagcca gtgctgtcct ctttgacatc 450 actgaggatc gagctgctgc
tgagcagctg cagcagccgc tggggctgac 500 ctggccagtg gtgttgatct
ggggtaatga cgctgagaag ctgatggagt 550 ttgtgtacaa gaaccaaaag
gcccatgtga ggattgagct gaaggagccc 600 ccggcctggc cagattatga
tgtgtggatc ctaatgacag tggtgggcac 650 catctttgtg atcatcctgg
cttcggtgct gcgcatccgg tgccgccccc 700 gccacagcag gccggatccg
cttcagcaga gaacagcctg ggccatcagc 750 cagctggcca ccaggaggta
ccaggccagc tgcaggcagg cccggggtga 800 gtggccagac tcagggagca
gctgcagctc agcccctgtg tgtgccatct 850 gtctggagga gttctctgag
gggcaggagc tacgggtcat ttcctgcctc 900 catgagttcc atcgtaactg
tgtggacccc tggttacatc agcatcggac 950 ttgccccctc tgcatgttca
acatcacaga gggagattca ttttcccagt 1000 ccctgggacc ctctcgatct
taccaagaac caggtcgaag actccacctc 1050 attcgccagc atcccggcca
tgcccactac cacctccctg ctgcctacct 1100 gttgggccct tcccggagtg
cagtggctcg gcccccacga cctggtccct 1150 tcctgccatc ccaggagcca
ggcatgggcc ctcggcatca ccgcttcccc 1200 agagctacac atccccgggc
tccaggagag cagcagcgcc tggcaggagc 1250 ccagcacccc tatgcacaag
gctggggact gagccacctc caatccacct 1300 cacagcaccc tgctgcttgc
ccagtgcccc tacgccgggc caggccccct 1350 gacagcagtg gatctggaga
aagctattgc acagaacgca gtgggtacct 1400 ggcagatggg ccagccagtg
actccagctc agggccctgt catggctctt 1450 ccagtgactc tgtggtcaac
tgcacggaca tcagcctaca gggggtccat 1500 ggcagcagtt ctactttctg
cagctcccta agcagtgact ttgaccccct 1550 agtgtactgc agccctaaag
gggatcccca gcgagtggac atgcagccta 1600 gtgtgacctc tcggcctcgt
tccttggact cggtggtgcc cacaggggaa 1650 acccaggttt ccagccatgt
ccactaccac cgccaccggc accaccacta 1700 caaaaagcgg ttccagtggc
atggcaggaa gcctggccca gaaaccggag 1750 tcccccagtc caggcctcct
attcctcgga cacagcccca gccagagcca 1800 ccttctcctg atcagcaagt
caccagatcc aactcagcag ccccttcggg 1850 gcggctctct aacccacagt
gccccagggc cctccctgag ccagcccctg 1900 gcccagttga cgcctccagc
atctgcccca gtaccagcag tctgttcaac 1950 ttgcaaaaat ccagcctctc
tgcccgacac ccacagagga aaaggcgggg 2000 gggtccctcc gagcccaccc
ctggctctcg gccccaggat gcaactgtgc 2050 acccagcttg ccagattttt
ccccattaca cccccagtgt ggcatatcct 2100 tggtccccag aggcacaccc
cttgatctgt ggacctccag gcctggacaa 2150 gaggctgcta ccagaaaccc
caggcccctg ttactcaaat tcacagccag 2200 tgtggttgtg cctgactcct
cgccagcccc tggaaccaca tccacctggg 2250 gaggggcctt ctgaatggag
ttctgacacc gcagagggca ggccatgccc 2300 ttatccgcac tgccaggtgc
tgtcggccca gcctggctca gaggaggaac 2350 tcgaggagct gtgtgaacag
gctgtgtgag atgttcaggc ctagctccaa 2400 cca 2403 114 783 PRT Homo
Sapien 114 Met Ser Gly Gly His His Leu Gln Leu Ala Ala Leu Trp Pro
Trp 1 5 10 15 Leu Leu Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr
Gly Leu 20 25 30 Val Leu Ala Ala Ala Val Glu Ser Glu Arg Ser Ala
Glu Gln Lys 35 40 45 Ala Val Ile Arg Val Ile Pro Leu Lys Met Asp
Pro Thr Gly Lys 50 55 60 Leu Asn Leu Thr Leu Glu Gly Val Phe Ala
Gly Val Ala Glu Ile 65 70 75 Thr Pro Ala Glu Gly Lys Leu Met Gln
Ser His Pro Leu Tyr Leu 80 85 90 Cys Asn Ala Ser Asp Asp Asp Asn
Leu Glu Pro Gly Phe Ile Ser 95 100 105 Ile Val Lys Leu Glu Ser Pro
Arg Arg Ala Pro His Pro Cys Leu 110 115 120 Ser Leu Ala Ser Lys Ala
Arg Met Ala Gly Glu Arg Gly Ala Ser 125 130 135 Ala Val Leu Phe Asp
Ile Thr Glu Asp Arg Ala Ala Ala Glu Gln 140 145 150 Leu Gln Gln Pro
Leu Gly Leu Thr Trp Pro Val Val Leu Ile Trp 155 160 165 Gly Asn Asp
Ala Glu Lys Leu Met Glu Phe Val Tyr Lys Asn Gln 170 175 180 Lys Ala
His Val Arg Ile Glu Leu Lys Glu Pro Pro Ala Trp Pro 185 190 195 Asp
Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly Thr Ile Phe 200 205 210
Val Ile Ile Leu Ala Ser Val Leu Arg Ile Arg Cys Arg Pro Arg 215 220
225 His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp Ala Ile 230
235 240 Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln Ala
245 250 255 Arg Gly Glu Trp Pro Asp Ser Gly Ser Ser Cys Ser Ser Ala
Pro 260 265 270 Val Cys Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln
Glu Leu 275 280 285 Arg Val Ile Ser Cys Leu His Glu Phe His Arg Asn
Cys Val Asp 290 295 300 Pro Trp Leu His Gln His Arg Thr Cys Pro Leu
Cys Met Phe Asn 305 310 315 Ile Thr Glu Gly Asp Ser Phe Ser Gln Ser
Leu Gly Pro Ser Arg 320 325 330 Ser Tyr Gln Glu Pro Gly Arg Arg Leu
His Leu Ile Arg Gln His 335 340 345 Pro Gly His Ala His Tyr His Leu
Pro Ala Ala Tyr Leu Leu Gly 350 355 360 Pro Ser Arg Ser Ala Val Ala
Arg Pro Pro Arg Pro Gly Pro Phe 365 370 375 Leu Pro Ser Gln Glu Pro
Gly Met Gly Pro Arg His His Arg Phe 380 385 390 Pro Arg Ala Thr His
Pro Arg Ala Pro Gly Glu Gln Gln Arg Leu 395 400 405 Ala Gly Ala Gln
His Pro Tyr Ala Gln Gly Trp Gly Leu Ser His 410 415 420 Leu Gln Ser
Thr Ser Gln His Pro Ala Ala Cys Pro Val Pro Leu 425 430 435 Arg Arg
Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly Glu Ser Tyr 440 445 450 Cys
Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro Ala Ser Asp 455 460 465
Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser Val Val 470 475
480 Asn Cys Thr Asp Ile Ser Leu Gln Gly Val His Gly Ser Ser Ser 485
490 495 Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr
500 505 510 Cys Ser Pro Lys Gly Asp Pro Gln Arg Val Asp Met Gln Pro
Ser 515 520 525 Val Thr Ser Arg Pro Arg Ser Leu Asp Ser Val Val Pro
Thr Gly 530 535 540 Glu Thr Gln Val Ser Ser His Val His Tyr His Arg
His Arg His 545 550 555 His His Tyr Lys Lys Arg Phe Gln Trp His Gly
Arg Lys Pro Gly 560 565 570 Pro Glu Thr Gly Val Pro Gln Ser Arg Pro
Pro Ile Pro Arg Thr 575 580 585 Gln Pro Gln Pro Glu Pro Pro Ser Pro
Asp Gln Gln Val Thr Arg 590 595 600 Ser Asn Ser Ala Ala Pro Ser Gly
Arg Leu Ser Asn Pro Gln Cys 605 610 615 Pro Arg Ala Leu Pro Glu Pro
Ala Pro Gly Pro Val Asp Ala Ser 620 625 630 Ser Ile Cys Pro Ser Thr
Ser Ser Leu Phe Asn Leu Gln Lys Ser 635 640 645 Ser Leu Ser Ala Arg
His Pro Gln Arg Lys Arg Arg Gly Gly Pro 650 655 660 Ser Glu Pro Thr
Pro Gly Ser Arg Pro Gln Asp Ala Thr Val His 665 670 675 Pro Ala Cys
Gln Ile Phe Pro His Tyr Thr Pro Ser Val Ala Tyr 680 685 690 Pro Trp
Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro Pro Gly 695 700 705 Leu
Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser 710 715 720
Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu 725 730
735 Glu Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp 740
745 750 Thr Ala Glu Gly Arg Pro Cys Pro Tyr Pro His Cys Gln Val Leu
755 760 765 Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys
Glu 770 775 780 Gln Ala Val 115 2407 DNA Homo Sapien 115 ccctttgaag
tgcattgctg cagctggtag catgagtggt ggccaccagc 50 tgcagctggc
tgccctctgg ccctggctgc tgatggctac cctgcaggca 100 ggctttggac
gcacaggact ggtactggca gcagcggtgg agtctgaaag 150 atcagcagaa
cagaaagctg ttatcagagt gatccccttg aaaatggacc 200 ccacaggaaa
actgaatctc actttggaag gtgtgtttgc tggtgttgct 250 gaaataactc
cagcagaagg aaaattaatg cagtcccacc cgctgtacct 300 gtgcaatgcc
agtgatgacg acaatctgga gcctggattc atcagcatcg 350 tcaagctgga
gagtcctcga cgggcccccc gcccctgcct gtcactggct 400 agcaaggctc
ggatggcggg tgagcgagga gccagtgctg tcctctttga 450 catcactgag
gatcgagctg ctgctgagca gctgcagcag ccgctggggc 500 tgacctggcc
agtggtgttg atctggggta atgacgctga gaagctgatg 550 gagtttgtgt
acaagaacca aaaggcccat gtgaggattg agctgaagga 600 gcccccggcc
tggccagatt atgatgtgtg gatcctaatg acagtggtgg 650 gcaccatctt
tgtgatcatc ctggcttcgg tgctgcgcat ccagtgccgc 700 ccccgccaca
gcaggccgga tccgcttcag cagagaacag cctgggccat 750 cagccagctg
gccaccagga ggtaccaggc cagctgcagg caggcccggg 800 gtgagtggcc
agactcaggg agcagctgca gctcagcccc tgtgtgtgcc 850 atctgtctgg
aggagttctc tgaggggcag gagctacggg tcatttcctg 900 cctccatgag
ttccatcgta actgtgtgga cccctggtta catcagcatc 950 ggacttgccc
cctctgcatg ttcaacatca cagagggaga ttcattttcc 1000 cagtccctgg
gaccctctcg atcttaccaa gaaccaggtc gaagactcca 1050 cctcattcgc
cagcatcccg gccatgccca ctaccacctc cctgctgcct 1100 acctgttggg
cccttcccgg agtgcagtgg ctcggccccc acgacctggt 1150 cccttcctgc
catcccagga gccaggcatg ggccctcggc atcaccgctt 1200 ccccagagct
gcacatcccc gggctccagg agagcagcag cgcctggcag 1250 gagcccagca
cccctatgca caaggctggg gactgagcca cctccaatcc 1300 acctcacagc
accctgctgc ttgcccagtg cccctacgcc gggccaggcc 1350 ccctgacagc
agtggatctg gagaaagcta ttgcacagaa cgcagtgggt 1400 acctggcaga
tgggccagcc agtgactcca gctcagggcc ctgtcatggc 1450 tcttccagtg
actctgtggt caactgcacg gacatcagcc tacagggggt 1500 ccatggcagc
agttctactt tctgcagctc cctaagcagt gactttgacc 1550 ccctagtgta
ctgcagccct aaaggggatc cccagcgagt ggacatgcag 1600 cctagtgtga
cctctcggcc tcgttccttg gactcggtgg tgcccacagg 1650 ggaaacccag
gtttccagcc atgtccacta ccaccgccac cggcaccacc 1700 actacaaaaa
gcggttccag tggcatggca ggaagcctgg cccagaaacc 1750 ggagtccccc
agtccaggcc tcctattcct cggacacagc cccagccaga 1800 gccaccttct
cctgatcagc aagtcaccag atccaactca gcagcccctt 1850 cggggcggct
ctctaaccca cagtgcccca gggccctccc tgagccagcc 1900 cctggcccag
ttgacgcctc cagcatctgc cccagtacca gcagtctgtt 1950 caacttgcaa
aaatccagcc tctctgcccg acacccacag aggaaaaggc 2000 gggggggtcc
ctccgagccc acccctggct ctcggcccca ggatgcaact 2050 gtgcacccag
cttgccagat ttttccccat tacaccccca gtgtggcata 2100 tccttggtcc
ccagaggcac accccttgat ctgtggacct ccaggcctgg 2150 acaagaggct
gctaccagaa accccaggcc cctgttactc aaattcacag 2200 ccagtgtggt
tgtgcctgac tcctcgccag cccctggaac cacatccacc 2250 tggggagggg
ccttctgaat ggagttctga caccgcagag ggcaggccat 2300 gcccttgtcc
gcactgccag gtgctgtcgg cccagcctgg ctcagaggag 2350 gaactcgagg
agctgtgtga acaggctgtg tgagatgttc aggcctagct 2400 ccaacca 2407 116
783 PRT Homo Sapien 116 Met Ser Gly Gly His Gln Leu Gln Leu Ala Ala
Leu Trp Pro Trp 1 5 10 15 Leu Leu Met Ala Thr Leu Gln Ala Gly Phe
Gly Arg Thr Gly Leu 20 25 30 Val Leu Ala Ala Ala Val Glu Ser Glu
Arg Ser Ala Glu Gln Lys 35 40 45 Ala Val Ile Arg Val Ile Pro Leu
Lys Met Asp Pro Thr Gly Lys 50 55 60 Leu Asn Leu Thr Leu Glu Gly
Val Phe Ala Gly Val Ala Glu Ile 65 70 75 Thr Pro Ala Glu Gly Lys
Leu Met Gln Ser His Pro Leu Tyr Leu 80 85 90 Cys Asn Ala Ser Asp
Asp Asp Asn Leu Glu Pro Gly Phe Ile Ser 95 100 105 Ile Val Lys Leu
Glu Ser Pro Arg Arg Ala Pro Arg Pro Cys Leu 110 115 120 Ser Leu Ala
Ser Lys Ala Arg Met Ala Gly Glu Arg Gly Ala Ser 125 130 135 Ala Val
Leu Phe Asp Ile Thr Glu Asp Arg Ala Ala Ala Glu Gln 140 145 150 Leu
Gln Gln Pro Leu Gly Leu Thr Trp Pro Val Val Leu Ile Trp 155 160 165
Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val Tyr Lys Asn Gln 170 175
180 Lys Ala His Val Arg Ile Glu Leu Lys Glu Pro Pro Ala Trp Pro 185
190 195 Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly Thr Ile Phe
200 205 210 Val Ile Ile Leu Ala Ser Val Leu Arg Ile Gln Cys Arg Pro
Arg 215 220 225 His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp
Ala Ile 230 235 240 Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys
Arg Gln Ala 245 250 255 Arg Gly Glu Trp Pro Asp Ser Gly Ser Ser Cys
Ser Ser Ala Pro 260 265 270 Val Cys Ala Ile Cys Leu Glu Glu Phe Ser
Glu Gly Gln Glu Leu 275 280 285 Arg Val Ile Ser Cys Leu His Glu Phe
His Arg Asn Cys Val Asp 290 295 300 Pro Trp Leu His Gln His Arg Thr
Cys Pro Leu Cys Met Phe Asn 305 310 315 Ile Thr Glu Gly Asp Ser Phe
Ser Gln Ser Leu Gly Pro Ser Arg 320 325 330 Ser Tyr Gln Glu Pro Gly
Arg Arg Leu His Leu Ile Arg Gln His 335 340 345 Pro Gly His Ala His
Tyr His Leu Pro Ala Ala Tyr Leu Leu Gly 350 355 360 Pro Ser Arg Ser
Ala Val Ala Arg Pro Pro Arg Pro Gly Pro Phe 365 370 375 Leu Pro Ser
Gln Glu Pro Gly Met Gly Pro Arg His His Arg Phe 380 385 390 Pro Arg
Ala Ala His Pro Arg Ala Pro Gly Glu Gln Gln Arg Leu 395 400 405 Ala
Gly Ala Gln His Pro Tyr Ala Gln Gly Trp Gly Leu Ser His 410 415 420
Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro Val Pro Leu 425
430 435 Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly Glu Ser Tyr
440 445 450 Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro Ala Ser
Asp 455 460 465 Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser
Val Val 470 475 480 Asn Cys Thr Asp Ile Ser Leu Gln Gly Val His Gly
Ser Ser Ser 485 490 495 Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp
Pro Leu Val Tyr 500 505 510 Cys Ser Pro Lys Gly Asp Pro Gln Arg Val
Asp Met Gln Pro Ser 515 520 525 Val Thr Ser Arg Pro Arg Ser Leu Asp
Ser Val Val Pro Thr Gly 530 535 540 Glu Thr Gln Val Ser Ser His Val
His Tyr His Arg His Arg His 545 550 555 His His Tyr Lys Lys Arg Phe
Gln Trp His Gly Arg Lys Pro Gly 560 565 570 Pro Glu Thr Gly Val Pro
Gln Ser Arg Pro Pro Ile Pro Arg Thr 575 580 585 Gln Pro Gln Pro Glu
Pro Pro Ser Pro Asp Gln Gln Val Thr Arg 590 595 600 Ser Asn Ser Ala
Ala Pro Ser Gly Arg Leu Ser Asn Pro Gln Cys 605 610 615 Pro Arg Ala
Leu Pro Glu Pro Ala Pro Gly Pro Val Asp Ala Ser 620 625 630 Ser Ile
Cys Pro Ser Thr Ser Ser Leu Phe Asn Leu Gln Lys Ser 635 640 645 Ser
Leu Ser Ala Arg His Pro Gln Arg Lys Arg Arg Gly Gly Pro 650 655 660
Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala Thr Val His 665 670
675 Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro Ser Val Ala Tyr 680
685 690 Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro Pro Gly
695 700 705 Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr
Ser 710 715 720 Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln
Pro Leu 725 730 735 Glu Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp
Ser Ser Asp 740 745 750 Thr Ala Glu Gly Arg Pro Cys Pro Cys Pro His
Cys Gln Val Leu 755 760 765 Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu
Glu Glu Leu Cys Glu 770 775 780 Gln Ala Val 117 2403 DNA Homo
Sapien 117 ttgaagtgca ttgctgcagc tggtagcatg agtggtggcc accacctgca
50 gctggctgcc ctctggccct ggctgctgat ggctaccctg caggcaggct 100
ttggacgcac aggactggta ctggcagcag cggtggagtc tgaaagatca 150
gcagaacaga aagctgttat cagagtgatc cccttgaaaa tggaccccac 200
aggaaaactg aatctcactt tggaaggtgt gtttgctggt gttgctgaaa 250
taactccagc agaaggaaaa ttaatgcagt cccacccgct gtacctgtgc 300
aatgccagtg atgacgacaa tctggagcct ggattcatca gcatcgtcaa 350
gctggagagt cctcgacggg ccccccaccc ctgcctgtca ctggctagca 400
aggctcggat ggcgggtgag cgaggagcca gtgctgtcct ctttgacatc 450
actgaggatc gagctgctgc tgagcagctg cagcagccgc tggggctgac 500
ctggccagtg gtgttgatct ggggtaatga cgctgagaag ctgatggagt 550
ttgtgtacaa gaaccaaaag gcccatgtga ggattgagct gaaggagccc 600
ccggcctggc cagattatga tgtgtggatc ctaatgacag tggtgggcac 650
catctttgtg atcatcctgg cttcggtgct gcgcatccgg tgccgccccc 700
gccacagcag gccggatccg cttcagcaga gaacagcctg ggccatcagc 750
cagctggcca ccaggaggta ccaggccagc tgcaggcagg cccggggtga 800
gtggccagac tcagggagca gctgcagctc agcccctgtg tgtgccatct 850
gtctggagga gttctctgag gggcaggagc tacgggtcat ttcctgcctc 900
catgagttcc atcgtaactg tgtggacccc tggttacatc agcatcggac 950
ttgccccctc tgcatgttca acatcacaga gggagattca ttttcccagt 1000
ccctgggacc ctctcgatct taccaagaac caggtcgaag actccacctc 1050
attcgccagc atcccggcca tgcccactac cacctccctg ctgcctacct 1100
gttgggccct tcccggagtg cagtggctcg gcccccacga cctggtccct 1150
tcctgccatc ccaggagcca ggcatgggcc ctcggcatca ccgcttcccc 1200
agagctgcac atccccgggc tccaggagag cagcagcgcc tggcaggagc 1250
ccagcacccc tatgcacaag gctggggaat gagccacctc caatccacct 1300
cacagcaccc tgctgcttgc ccagtgcccc tacgccgggc caggccccct 1350
gacagcagtg gatctggaga aagctattgc acagaacgca gtgggtacct 1400
ggcagatggg ccagccagtg actccagctc agggccctgt catggctctt 1450
ccagtgactc tgtggtcaac tgcacggaca tcagcctaca gggggtccat 1500
ggcagcagtt ctactttctg cagctcccta agcagtgact ttgaccccct 1550
agtgtactgc agccctaaag gggatcccca gcgagtggac atgcagccta 1600
gtgtgacctc tcggcctcgt tccttggact cggtggtgcc cacaggggaa 1650
acccaggttt ccagccatgt ccactaccac cgccaccggc accaccacta 1700
caaaaagcgg ttccagtggc atggcaggaa gcctggccca gaaaccggag 1750
tcccccagtc caggcctcct attcctcgga cacagcccca gccagagcca 1800
ccttctcctg atcagcaagt caccagatcc aactcagcag ccccttcggg 1850
gcggctctct aacccacagt gccccagggc cctccctgag ccagcccctg 1900
gcccagttga cgcctccagc atctgcccca gtaccagcag tctgttcaac 1950
ttgcaaaaat ccagcctctc tgcccgacac ccacagagga aaaggcgggg 2000
gggtccctcc gagcccaccc ctggctctcg gccccaggat gcaactgtgc 2050
acccagcttg ccagattttt ccccattaca cccccagtgt ggcatatcct 2100
tggtccccag aggcacaccc cttgatctgt ggacctccag gcctggacaa 2150
gaggctgcta ccagaaaccc caggcccctg ttactcaaat tcacagccag 2200
tgtggttgtg cctgactcct cgccagcccc tggaaccaca tccacctggg 2250
gaggggcctt ctgaatggag ttctgacacc gcagagggca ggccatgccc 2300
ttatccgcac tgccaggtgc tgtcggccca gcctggctca gaggaggaac 2350
tcgaggagct gtgtgaacag gctgtgtgag atgttcaggc ctagctccaa 2400 cca
2403 118 783 PRT Homo Sapien 118 Met Ser Gly Gly His His Leu Gln
Leu Ala Ala Leu Trp Pro Trp 1 5 10 15 Leu Leu Met Ala Thr Leu Gln
Ala Gly Phe Gly Arg Thr Gly Leu 20 25 30 Val Leu Ala Ala Ala Val
Glu Ser Glu Arg Ser Ala Glu Gln Lys 35 40 45 Ala Val Ile Arg Val
Ile Pro Leu Lys Met Asp Pro Thr Gly Lys 50 55 60 Leu Asn Leu Thr
Leu Glu Gly Val Phe Ala Gly Val Ala Glu Ile 65 70 75 Thr Pro Ala
Glu Gly Lys Leu Met Gln Ser His Pro Leu Tyr Leu 80 85 90 Cys Asn
Ala Ser Asp Asp Asp Asn Leu Glu Pro Gly Phe Ile Ser 95 100 105 Ile
Val Lys Leu Glu Ser Pro Arg Arg Ala Pro His Pro Cys Leu 110 115 120
Ser Leu Ala Ser Lys Ala Arg Met Ala Gly Glu Arg Gly Ala Ser 125 130
135 Ala Val Leu Phe Asp Ile Thr Glu Asp Arg Ala Ala Ala Glu Gln 140
145 150 Leu Gln Gln Pro Leu Gly Leu Thr Trp Pro Val Val Leu Ile Trp
155 160 165 Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val Tyr Lys Asn
Gln 170 175 180 Lys Ala His Val Arg Ile Glu Leu Lys Glu Pro Pro Ala
Trp Pro 185 190 195 Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly
Thr Ile Phe 200 205 210 Val Ile Ile Leu Ala Ser Val Leu Arg Ile Arg
Cys Arg Pro Arg 215 220 225 His Ser Arg Pro Asp Pro Leu Gln Gln Arg
Thr Ala Trp Ala Ile 230 235 240 Ser Gln Leu Ala Thr Arg Arg Tyr Gln
Ala Ser Cys Arg Gln Ala 245 250 255 Arg Gly Glu Trp Pro Asp Ser Gly
Ser Ser Cys Ser Ser Ala Pro 260 265 270 Val Cys Ala Ile Cys Leu Glu
Glu Phe Ser Glu Gly Gln Glu Leu 275 280 285 Arg Val Ile Ser Cys Leu
His Glu Phe His Arg Asn Cys Val Asp 290 295 300 Pro Trp Leu His Gln
His Arg Thr Cys Pro Leu Cys Met Phe Asn 305 310 315 Ile Thr Glu Gly
Asp Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg 320 325 330 Ser Tyr Gln
Glu Pro Gly Arg Arg Leu His Leu Ile Arg Gln His 335 340 345 Pro Gly
His Ala His Tyr His Leu Pro Ala Ala Tyr Leu Leu Gly 350 355 360 Pro
Ser Arg Ser Ala Val Ala Arg Pro Pro Arg Pro Gly Pro Phe 365 370 375
Leu Pro Ser Gln Glu Pro Gly Met Gly Pro Arg His His Arg Phe 380 385
390 Pro Arg Ala Ala His Pro Arg Ala Pro Gly Glu Gln Gln Arg Leu 395
400 405 Ala Gly Ala Gln His Pro Tyr Ala Gln Gly Trp Gly Met Ser His
410 415 420 Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro Val Pro
Leu 425 430 435 Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly Glu
Ser Tyr 440 445 450 Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro
Ala Ser Asp 455 460 465 Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser
Asp Ser Val Val 470 475 480 Asn Cys Thr Asp Ile Ser Leu Gln Gly Val
His Gly Ser Ser Ser 485 490 495 Thr Phe Cys Ser Ser Leu Ser Ser Asp
Phe Asp Pro Leu Val Tyr 500 505 510 Cys Ser Pro Lys Gly Asp Pro Gln
Arg Val Asp Met Gln Pro Ser 515 520 525 Val Thr Ser Arg Pro Arg Ser
Leu Asp Ser Val Val Pro Thr Gly 530 535 540 Glu Thr Gln Val Ser Ser
His Val His Tyr His Arg His Arg His 545 550 555 His His Tyr Lys Lys
Arg Phe Gln Trp His Gly Arg Lys Pro Gly 560 565 570 Pro Glu Thr Gly
Val Pro Gln Ser Arg Pro Pro Ile Pro Arg Thr 575 580 585 Gln Pro Gln
Pro Glu Pro Pro Ser Pro Asp Gln Gln Val Thr Arg 590 595 600 Ser Asn
Ser Ala Ala Pro Ser Gly Arg Leu Ser Asn Pro Gln Cys 605 610 615 Pro
Arg Ala Leu Pro Glu Pro Ala Pro Gly Pro Val Asp Ala Ser 620 625 630
Ser Ile Cys Pro Ser Thr Ser Ser Leu Phe Asn Leu Gln Lys Ser 635 640
645 Ser Leu Ser Ala Arg His Pro Gln Arg Lys Arg Arg Gly Gly Pro 650
655 660 Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala Thr Val His
665 670 675 Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro Ser Val Ala
Tyr 680 685 690 Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro
Pro Gly 695 700 705 Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly Pro
Cys Tyr Ser 710 715 720 Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro
Arg Gln Pro Leu 725 730 735 Glu Pro His Pro Pro Gly Glu Gly Pro Ser
Glu Trp Ser Ser Asp 740 745 750 Thr Ala Glu Gly Arg Pro Cys Pro Tyr
Pro His Cys Gln Val Leu 755 760 765 Ser Ala Gln Pro Gly Ser Glu Glu
Glu Leu Glu Glu Leu Cys Glu 770 775 780 Gln Ala Val 119 4839 DNA
Homo Sapien 119 ggaaagctag cggcagaggc tcagccccgg cggcagcgcg
cgccccgctg 50 ccagcccatt ttccggacgc cacccgcggg cactgccgac
gcccccgggg 100 ctgccgaggg gaggccgggg gggcgcagcg gagcgcggtc
ccgcgcactg 150 agccccgcgg cgccccggga acttggcggc gacccgagcc
cggcgagccg 200 gggcgcgcct cccccgccgc gcgcctcctg catgcggggc
cccagctccg 250 ggcgccggcc ggagcccccc ccggccgccc ccgagccccc
cgcgccccgc 300 gccgcgccgc cgcgccgtcc atgcaccgct tgatgggggt
caacagcacc 350 gccgccgccg ccgccgggca gcccaatgtc tcctgcacgt
gcaactgcaa 400 acgctctttg ttccagagca tggagatcac ggagctggag
tttgttcaga 450 tcatcatcat cgtggtggtg atgatggtga tggtggtggt
gatcacgtgc 500 ctgctgagcc actacaagct gtctgcacgg tccttcatca
gccggcacag 550 ccaggggcgg aggagagaag atgccctgtc ctcagaagga
tgcctgtggc 600 cctcggagag cacagtgtca ggcaacggaa tcccagagcc
gcaggtctac 650 gccccgcctc ggcccaccga ccgcctggcc gtgccgccct
tcgcccagcg 700 ggagcgcttc caccgcttcc agcccaccta tccgtacctg
cagcacgaga 750 tcgacctgcc acccaccatc tcgctgtcag acggggagga
gcccccaccc 800 taccagggcc cctgcaccct ccagcttcgg gaccccgagc
agcagctgga 850 actgaaccgg gagtcggtgc gcgcaccccc aaacagaacc
atcttcgaca 900 gtgacctgat ggatagtgcc aggctgggcg gcccctgccc
ccccagcagt 950 aactcgggca tcagcgccac gtgctacggc agcggcgggc
gcatggaggg 1000 gccgccgccc acctacagcg aggtcatcgg ccactacccg
gggtcctcct 1050 tccagcacca gcagagcagt gggccgccct ccttgctgga
ggggacccgg 1100 ctccaccaca cacacatcgc gcccctagag agcgcagcca
tctggagcaa 1150 agagaaggat aaacagaaag gacaccctct ctagggtccc
caggggggcc 1200 gggctggggc tgcgtaggtg aaaaggcaga acactccgcg
cttcttagaa 1250 gaggagtgag aggaaggcgg ggggcgcagc aacgcatcgt
gtggccctcc 1300 cctcccacct ccctgtgtat aaatatttac atgtgatgtc
tggtctgaat 1350 gcacaagcta agagagcttg caaaaaaaaa aagaaaaaag
aaaaaaaaaa 1400 accacgtttc tttgttgagc tgtgtcttga aggcaaaaga
aaaaaaattt 1450 ctacagtagt ctttcttgtt tctagttgag ctgcgtgcgt
gaatgcttat 1500 tttcttttgt ttatgataat ttcacttaac tttaaagaca
tatttgcaca 1550 aaacctttgt ttaaagatct gcaatattat atatataaat
atatataaga 1600 taagagaaac tgtatgtgcg agggcaggag tatttttgta
ttagaagagg 1650 cctattaaaa aaaaaagttg ttttctgaac tagaagagga
aaaaaatggc 1700 aatttttgag tgccaagtca gaaagtgtgt attaccttgt
aaagaaaaaa 1750 attacaaagc aggggtttag agttatttat ataaatgttg
agattttgca 1800 ctatttttta atataaatat gtcagtgctt gcttgatgga
aacttctctt 1850 gtgtctgttg agactttaag ggagaaatgt cggaatttca
gagtcgcctg 1900 acggcagagg gtgagccccc gtggagtctg cagagaggcc
ttggccagga 1950 gcggcgggct ttcccgaggg gccactgtcc ctgcagagtg
gatgcttctg 2000 cctagtgaca ggttatcacc acgttatata ttccctaccg
aaggagacac 2050 cttttccccc ctgacccaga acagccttta aatcacaagc
aaaataggaa 2100 agttaaccac ggaggcaccg agttccaggt agtggttttg
cctttcccaa 2150 aaatgaaaat aaactgttac cgaaggaatt agtttttcct
cttctttttt 2200 ccaactgtga aggtccccgt ggggtggagc atggtgcccc
tcacaagccg 2250 cagcggctgg tgcccgggct accagggaca tgccagaggg
ctcgatgact 2300 tgtctctgca gggcgctttg gtggttgttc agctggctaa
aggttcaccg 2350 gtgaaggcag gtgcggtaac tgccgcactg gaccctagga
agccccaggt 2400 attcgcaatc tgacctcctc ctgtctgttt cccttcacgg
atcaattctc 2450 acttaagagg ccaataaaca acccaacatg aaaaggtgac
aagcctgggt 2500 ttctcccagg ataggtgaaa gggttaaaat gagtaaagca
gttgagcaaa 2550 caccaacccg agcttcgggc gcagaattct tcaccttctc
ttcccctttc 2600 catctccttt ccccgcggaa acaacgcttc ccttctggtg
tgtctgttga 2650 tctgtgtttt catttacatc tctcttagac tccgctcttg
ttctccaggt 2700 tttcaccaga tagatttggg gttggcggga cctgctggtg
acgtgcaggt 2750 gaaggacagg aaggggcatg tgagcgtaaa tagaggtgac
cagaggagag 2800 catgaggggt ggggctttgg gacccaccgg ggccagtggc
tggagcttga 2850 cgtctttcct ccccatgggg gtgggagggc ccccagctgg
aagagcagac 2900 tcccagctgc taccccctcc cttcccatgg gagtggcttt
ccattttggg 2950 cagaatgctg actagtagac taacataaaa gatataaaag
gcaataacta 3000 ttgtttgtga gcaacttttt tataacttcc aaaacaaaaa
cctgagcaca 3050 gttttgaagt tctagccact cgagctcatg catgtgaaac
gtgtgcttta 3100 cgaaggtggc agctgacaga cgtgggctct gcatgccgcc
agcctagtag 3150 aaagttctcg ttcattggca acagcagaac ctgcctctcc
gtgaagtcgt 3200 cagcctaaaa tttgtttctc tcttgaagag gattctttga
aaaggtcctg 3250 cagagaaatc agtacaggtt atcccgaaag gtacaaggac
gcacttgtaa 3300 agatgattaa aacgtatctt tcctttatgt gacgcgtctc
tagtgcctta 3350 ctgaagaagc agtgacactc ccgtcgctcg gtgaggacgt
tcccggacag 3400 tgcctcactc acctgggact ggtatcccct cccagggtcc
accaagggct 3450 cctgcttttc agacacccca tcatcctcgc gcgtcctcac
cctgtctcta 3500 ccagggaggt gcctagcttg gtgaggttac tcctgctcct
ccaacctttt 3550 tttgccaagg tttgtacacg actcccatct aggctgaaaa
cctagaagtg 3600 gaccttgtgt gtgtgcatgg tgtcagccca aagccaggct
gagacagtcc 3650 tcatatcctc ttgagccaaa ctgtttgggt ctcgttgctt
catggtatgg 3700 tctggatttg tgggaatggc tttgcgtgag aaaggggagg
agagtggttg 3750 ctgccctcag ccggcttgag gacagagcct gtccctctca
tgacaactca 3800 gtgttgaagc ccagtgtcct cagcttcatg tccagtggat
ggcagaagtt 3850 catggggtag tggcctctca
aaggctgggc gcatcccaag acagccagca 3900 ggttgtctct ggaaacgacc
agagttaagc tctcggcttc tctgctgagg 3950 gtgcaccctt tcctctagat
ggtagttgtc acgttatctt tgaaaactct 4000 tggactgctc ctgaggaggc
cctcttttcc agtaggaagt tagatggggg 4050 ttctcagaag tggctgattg
gaaggggaca agcttcgttt caggggtctg 4100 ccgttccatc ctggttcaga
gaaggccgag cgtggctttc tctagccttg 4150 tcactgtctc cctgcctgtc
aatcaccacc tttcctccag aggaggaaaa 4200 ttatctcccc tgcaaagccc
ggttctacac agatttcaca aattgtgcta 4250 agaaccgtcc gtgttctcag
aaagcccagt gtttttgcaa agaatgaaaa 4300 gggaccccat atgtagcaaa
aatcagggct gggggagagc cgggttcatt 4350 ccctgtcctc attggtcgtc
cctatgaatt gtacgtttca gagaaatttt 4400 ttttcctatg tgcaacacga
agcttccaga accataaaat atcccgtcga 4450 taaggaaaga aaatgtcgtt
gttgttgttt ttctggaaac tgcttgaaat 4500 cttgctgtac tatagagctc
agaaggacac agcccgtcct cccctgcctg 4550 cctgattcca tggctgttgt
gctgattcca atgctttcac gttggttcct 4600 ggcgtgggaa ctgctctcct
ttgcagcccc atttcccaag ctctgttcaa 4650 gttaaactta tgtaagcttt
ccgtggcatg cggggcgcgc acccacgtcc 4700 ccgctgcgta agactctgta
tttggatgcc aatccacagg cctgaagaaa 4750 ctgcttgttg tgtatcagta
atcattagtg gcaatgatga cattctgaaa 4800 agctgcaata cttatacaat
aaattttaca attctttgg 4839 120 287 PRT Homo Sapien 120 Met His Arg
Leu Met Gly Val Asn Ser Thr Ala Ala Ala Ala Ala 1 5 10 15 Gly Gln
Pro Asn Val Ser Cys Thr Cys Asn Cys Lys Arg Ser Leu 20 25 30 Phe
Gln Ser Met Glu Ile Thr Glu Leu Glu Phe Val Gln Ile Ile 35 40 45
Ile Ile Val Val Val Met Met Val Met Val Val Val Ile Thr Cys 50 55
60 Leu Leu Ser His Tyr Lys Leu Ser Ala Arg Ser Phe Ile Ser Arg 65
70 75 His Ser Gln Gly Arg Arg Arg Glu Asp Ala Leu Ser Ser Glu Gly
80 85 90 Cys Leu Trp Pro Ser Glu Ser Thr Val Ser Gly Asn Gly Ile
Pro 95 100 105 Glu Pro Gln Val Tyr Ala Pro Pro Arg Pro Thr Asp Arg
Leu Ala 110 115 120 Val Pro Pro Phe Ala Gln Arg Glu Arg Phe His Arg
Phe Gln Pro 125 130 135 Thr Tyr Pro Tyr Leu Gln His Glu Ile Asp Leu
Pro Pro Thr Ile 140 145 150 Ser Leu Ser Asp Gly Glu Glu Pro Pro Pro
Tyr Gln Gly Pro Cys 155 160 165 Thr Leu Gln Leu Arg Asp Pro Glu Gln
Gln Leu Glu Leu Asn Arg 170 175 180 Glu Ser Val Arg Ala Pro Pro Asn
Arg Thr Ile Phe Asp Ser Asp 185 190 195 Leu Met Asp Ser Ala Arg Leu
Gly Gly Pro Cys Pro Pro Ser Ser 200 205 210 Asn Ser Gly Ile Ser Ala
Thr Cys Tyr Gly Ser Gly Gly Arg Met 215 220 225 Glu Gly Pro Pro Pro
Thr Tyr Ser Glu Val Ile Gly His Tyr Pro 230 235 240 Gly Ser Ser Phe
Gln His Gln Gln Ser Ser Gly Pro Pro Ser Leu 245 250 255 Leu Glu Gly
Thr Arg Leu His His Thr His Ile Ala Pro Leu Glu 260 265 270 Ser Ala
Ala Ile Trp Ser Lys Glu Lys Asp Lys Gln Lys Gly His 275 280 285 Pro
Leu
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