U.S. patent application number 15/488734 was filed with the patent office on 2017-08-03 for cells useful for immuno-based botulinum toxin serotype a activity assays.
The applicant listed for this patent is Allergan, Inc.. Invention is credited to Ester Fernandez-Salas, D. Dianne Hodges, Birgitte P.S. Jacky, Joanne Wang, Hong Zhu.
Application Number | 20170218334 15/488734 |
Document ID | / |
Family ID | 42288894 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170218334 |
Kind Code |
A1 |
Zhu; Hong ; et al. |
August 3, 2017 |
Cells Useful for Immuno-Based Botulinum Toxin Serotype A Activity
Assays
Abstract
The present specification discloses clonal cell lines
susceptible to BoNT/A intoxication, methods of producing such
clonal cell lines, and methods of detecting Botulinum toxin
serotype A activity using such clonal cell lines.
Inventors: |
Zhu; Hong; (San Diego,
CA) ; Wang; Joanne; (Irvine, CA) ; Jacky;
Birgitte P.S.; (Orange, CA) ; Hodges; D. Dianne;
(Tustin, CA) ; Fernandez-Salas; Ester; (Ann Arbor,
MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Allergan, Inc. |
Irvine |
CA |
US |
|
|
Family ID: |
42288894 |
Appl. No.: |
15/488734 |
Filed: |
April 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13339248 |
Dec 28, 2011 |
9625447 |
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15488734 |
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12722801 |
Mar 12, 2010 |
8361789 |
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13339248 |
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61160199 |
Mar 13, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5014 20130101;
C12N 5/0693 20130101; C12N 5/0618 20130101 |
International
Class: |
C12N 5/079 20060101
C12N005/079; G01N 33/50 20060101 G01N033/50; C12N 5/09 20060101
C12N005/09 |
Claims
1. An established single cell derived clonal cell line comprising
cells susceptible to intoxication by botulinum toxin type A
(BoNT/A), wherein the cells exhibit at least a 1.5-fold increase in
mRNA expression levels after differentiation in the genes ADAMTS9,
ATAD2, CDC45L, CNTN1, Cyclin A, Cyclin E, E2F1, E2F2, E2F7, FGFR2,
MYBL1, MYBL2, NDC80, NDN, ORC1L, PLS3, RAD54L, RBL1, RRM2, S1PR3,
SPC24, SPC25, TFDP1, TFPI2, TK1, TTK, TYMS, TYK and ZWINT relative
to the mRNA expression levels of each of these genes in cells from
the parental SiMa cell line DSMZ ACC 164; and, wherein the cells
exhibit at least a 1.5-fold decrease in mRNA expression levels
after differentiation in the genes CDKN2A and CNTN2 as compared to
the mRNA expression levels of each of these genes in cells from the
parental SiMa cell line DSMZ ACC 164; and, wherein the clonal cell
line comprises cells susceptible to BoNT/A intoxication by about
100 pM or less of a BoNT/A.
2. The established clonal cell line of claim 1, wherein the cells
exhibit at least a 2.0-fold difference in gene expression levels of
at least one gene as compared to the expression levels of the gene
in cells from the parental SiMa cell line DSMZ ACC 164.
3. The established clonal cell line of claim 1, wherein the cells
exhibit at least a 3.0-fold difference in gene expression levels of
at least one gene as compared to the expression levels of the gene
in cells from the parental SiMa cell line DSMZ ACC 164.
4. The established clonal cell line of claim 1, wherein the cells
exhibit at least a 4.0-fold difference in gene expression levels of
at least one gene as compared to the expression levels of the gene
in cells from the parental SiMa cell line DSMZ ACC 164.
5. The established clonal cell line of claim 1, wherein the cell
from an established clonal cell line exhibits about 100 pM or less
of a BoNT/A for at least 5 or more cell passages.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/339,248, filed Dec. 28, 2011 which is a divisional of
U.S. patent application Ser. No. 12/722,801, filed Mar. 12, 2010,
which claims priority pursuant to 35 U.S.C. .sctn.119(e) to U.S.
Provisional Patent Application No. 61/160,199 filed Mar. 13, 2009,
each of which are incorporated entirely by reference.
[0002] The sequences disclosed in the present specification are
contained in the Sequence Listing submitted with the present
specification which is hereby incorporated by reference in its
entirety.
[0003] The ability of Clostridial toxins, such as, e.g., Botulinum
neurotoxins (BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E,
BoNT/F and BoNT/G, and Tetanus neurotoxin (TeNT), to inhibit
neuronal transmission are being exploited in a wide variety of
therapeutic and cosmetic applications, see e.g., William J. Lipham,
Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc.,
2004). Clostridial toxins commercially available as pharmaceutical
compositions include, BoNT/A preparations, such as, e.g.,
BOTOX.RTM. (Allergan, Inc., Irvine, Calif.),
DYSPORT.RTM./RELOXIN.RTM., (Ipsen Ltd., Slough, England),
PURTOX.RTM. (Mentor Corp., Santa Barbara, Calif.), XEOMIN.RTM.
(Merz Pharmaceuticals, GmbH., Frankfurt, Germany), NEURONOX.RTM.
(Medy-Tox, Inc., Ochang-myeon, South Korea), BTX-A (Biogen-tech
Ltd., University, Yantai, Shandong, China); and BoNT/B
preparations, such as, e.g., MYOBLOC.RTM./NEUROBLOC.RTM. (Solstice
Neurosciences, Inc., South San Francisco, Calif.). As an example,
BOTOX.RTM. is currently approved in one or more countries for the
following indications: achalasia, adult spasticity, anal fissure,
back pain, blepharospasm, bruxism, cervical dystonia, essential
tremor, glabellar lines or hyperkinetic facial lines, headache,
hemifacial spasm, hyperactivity of bladder, hyperhidrosis, juvenile
cerebral palsy, multiple sclerosis, myoclonic disorders, nasal
labial lines, spasmodic dysphonia, strabismus and VII nerve
disorder.
[0004] At present the mouse LD.sub.50 bioassay, a lethality test,
remains the "gold standard" test used by all pharmaceutical
manufacturers to express the potency of their preparations. S. S.
Arnon et al., JAMA 285: 1059-1070 (2001). In fact, the units on the
pharmaceutical preparations' labels are mouse LD.sub.50 units and
the number of animals needed to produce statistically useful
LD.sub.50 data is large. The advantage of the mouse LD.sub.50
bioassay is that it measures all the steps necessary for botulinum
toxin uptake (e.g., toxin binding to a cell surface receptor,
internalization of the toxin-receptor complex, light chain
translocation into the cytoplasm, light chain cleavage of
substrate), instead of merely determining the activity for only
part of this intoxication process, such as, e.g., in vitro assays
that only measure light chain enzymatic activity. Unfortunately,
the mouse LD.sub.50 bioassay suffers from many drawbacks including
high operational cost due to the large numbers of laboratory
animals required, a lack of specificity since all BoNT serotypes
will cause the same measurable end-point, and the potential for
inaccuracy unless large animal groups are used. In addition, animal
rights groups have exerted pressure on regulatory agencies in the
United States (FDA/NICEATM/ICCVAM) and Europe (MHRA and EDQM), and
on pharmaceutical companies manufacturing botulinum neurotoxin
products to reduce animal testing and more importantly replace the
mouse LD.sub.50 bioassay for product release. The regulatory
agencies are engaging pharmaceutical companies to apply the three
"Rs" principle to the potency testing of botulinum neurotoxins:
Reduce, Refine, Replace. D. Straughan, Progress in Applying the
Three Rs to the Potency Testing of Botulinum Toxin Type A, Altern.
Lab. Anim. 34(3): 305-313 (2006). In recent years, several steps
have been already taken to reduce and refine the mouse LD.sub.50
bioassay in order to standardize the protocol and produce more
consistent data using fewer animals per assay.
[0005] Thus, a simple, reliable, validated and governmental agency
acceptable botulinum toxin activity assay that can evaluate the
integrity of all the steps necessary in botulinum toxin uptake
would be of significant value because such a non-animal based assay
would alleviate the need for animal testing and all the
disadvantages, costs and ethical concerns associated with this type
of animal-based assay. Companion patent application Ester
Fernandez-Salas, et al., Immuno-Based Botulinum Toxin Serotype A
Activity Assays, U.S. patent application Ser. No. 12/403,531,
provides novel compositions, cells, and methods for assaying the
activity of a BoNT/A useful for various industries, such as, e.g.,
the pharmaceutical and food industries. Such compositions, cells,
and methods do not use live animals or tissues taken from live
animals, but can evaluate all the steps necessary for neurotoxin
action, namely, binding and cellular uptake of toxin, translocation
into the cell cytosol, and protease activity.
[0006] One of the necessary components for the methods disclosed in
U.S. patent application Ser. No. 12/403,531 is the use of cells
from an established cell line that are susceptible to low levels of
BoNT/A intoxication because the amount of BoNT/A contained within a
commercially-available pharmaceutical composition is very low. For
example, approximately 4-5 ng of a BoNT/A complex is contained in
the pharmaceutical composition sold as BOTOX.RTM.. Thus, there is a
need to identify and create established cell lines comprising cells
that are susceptible to BoNT/A intoxication when only very low
levels of the neurotoxin is present in the sample. The present
specification provides novel cells and cell compositions. that are
susceptible to BoNT/A intoxication when only very low amounts of
BoNT/A is present and thus allow assaying of commercially-available
pharmaceutical composition comprising BoNT/A.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIGS. 1A and 1B show a schematic of the current paradigm of
neurotransmitter release and Clostridial toxin intoxication in a
central and peripheral neuron. FIG. 1A shows a schematic for the
neurotransmitter release mechanism of a central and peripheral
neuron. The release process can be described as comprising two
steps: 1) vesicle docking, where the vesicle-bound SNARE protein of
a vesicle containing neurotransmitter molecules associates with the
membrane-bound SNARE proteins located at the plasma membrane; and
2) neurotransmitter release, where the vesicle fuses with the
plasma membrane and the neurotransmitter molecules are exocytosed.
FIG. 1B shows a schematic of the intoxication mechanism for tetanus
and botulinum toxin activity in a central and peripheral neuron.
This intoxication process can be described as comprising four
steps: 1) receptor binding, where Clostridial toxin binding to a
Clostridial receptor complex and initiates the intoxication
process; 2) complex internalization, where after toxin binding, a
vesicle containing a toxin/receptor system complex is endocytosed
into the cell; 3) light chain translocation, where multiple events
are thought to occur, including changes in the internal pH of the
vesicle, formation of a channel pore comprising the H.sub.N domain
of Clostridial toxin heavy chain, separation of the Clostridial
toxin light chain from the heavy chain, and release of the light
chain and 4) enzymatic target modification, where the light chain
of Clostridial toxin proteolytically cleaves its target SNARE
substrates, such as, e.g., SNAP-25, VAMP or Syntaxin, thereby
preventing vesicle docking and neurotransmitter release.
[0008] FIGS. 2A, 2B, 2C, 2D, and 2E show volcano plots of genes
that are more than 4-fold (log.sub.2(4)=2) up or down regulated in
BB10 and H1 cell lines as compared to the 2D6 cell line. FIG. 2A
shows the genes that are up regulated in the BB10 cell line as
compared to the 2D6 cell line. FIG. 2B shows the genes that are up
regulated in the H1 cell line as compared to the 2D6 cell line.
FIG. 2C shows the genes that are up regulated in the BB10 cell line
as compared to the parental SiMa cell line (PA). FIG. 2D shows the
genes that are up regulated in the H1 cell line as compared to the
parental SiMa cell line (PA). FIG. 2E shows the genes that are up
regulated in the parental SiMa cell line (PA) as compared to the
2D6 cell line. Each dot represents a probe set. The dashed line
marks the 95% confidence interval, p.ltoreq.0.05. Most of the genes
that are over expressed in BB10 and H1 compared to 2D6 (A and B)
are also over or under-expressed in BB10 compared to PA (C), in H1
compared to PA (D), and in PA compared to 2D6 (E), however a number
of them are less than 4-fold over-expressed and/or do not pass the
95% confidence interval.
[0009] FIGS. 3A and 3B show normalized BIAcore SPR curves of 7.8 nM
of the antibodies 2E2A6, 1D3B8, 3C1A5 and 2C9B10 and commercial
MC-6050 and MC-6053. FIG. 3A shows the normalized data for the
on-rate of each antibody. FIG. 3B shows the normalized data for the
off-rate of each antibody.
DETAILED DESCRIPTION
[0010] The present specification provides novel cells and cell
compositions that are susceptible to BoNT/A intoxication to allow
assaying of BoNT/A and BoNT/A compositions such as, e.g.,
commercially-available pharmaceutical composition comprising
BoNT/A. The cell and cell compositions disclosed in the present
specification are useful to conduct methods that can detect
picomolar quantities of BoNT/A in a sample, such as the methods
disclosed in, e.g., Ester Fernandez-Salas, et al., Immuno-Based
Botulinum Toxin Serotype A Activity Assays, U.S. patent application
Ser. No. 12/403,531, which is hereby incorporated by reference in
its entirety. The cells and cell compositions, and their use in
methods of detecting BoNT/A activity reduce the need for animal
toxicity studies, yet serve to analyze the multiple steps of BoNT/A
intoxication, namely, binding and cellular uptake of toxin,
translocation into the cell cytosol, and protease activity. As
discussed further below, the novel cells and cell compositions of
the present specification can be used in assays that analyze crude
and bulk samples as well as highly purified di-chain toxins and
formulated toxin products and further are amenable to automated
high throughput assay formats.
[0011] Thus, aspects of the present specification provide a cell
composition comprising cells from a clonal cell line that are
susceptible to BoNT/A intoxication.
[0012] Other aspects of the present specification provide a method
of detecting BoNT/A activity using the clonal cell lines disclosed
in the present specification.
[0013] Clostridia toxins produced by Clostridium botulinum,
Clostridium tetani, Clostridium baratii and Clostridium butyricum
are the most widely used in therapeutic and cosmetic treatments of
humans and other mammals. Strains of C. botulinum produce seven
antigenically-distinct serotypes of botulinum toxins (BoNTs), which
have been identified by investigating botulism outbreaks in man
(BoNT/A, BoNT/B, BoNT/E and BoNT/F), animals (BoNT/C1 and BoNT/D),
or isolated from soil (BoNT/G). While all seven botulinum toxin
serotypes have similar structure and biological properties, each
also displays heterogeneous characteristics, such as, e.g.,
different pharmacological properties. In contrast, tetanus toxin
(TeNT) is produced by a uniform group of C. tetani. Two other
species of Clostridia, C. baratii and C. butyricum, also produce
toxins similar to BoNT/F and BoNT/E, respectively.
[0014] Clostridial toxins are each translated as a single chain
polypeptide of approximately 150 kDa that is subsequently cleaved
by proteolytic scission within a disulfide loop by a
naturally-occurring protease, such as, e.g., an endogenous
Clostridial toxin protease or a naturally-occurring protease
produced in the environment. This posttranslational processing
yields a di-chain molecule comprising an approximately 50 kDa light
chain (LC) and an approximately 100 kDa heavy chain (HC) held
together by a single disulfide bond and noncovalent interactions.
Each mature di-chain molecule comprises three functionally distinct
domains: 1) an enzymatic domain located in the LC that includes a
metalloprotease region containing a zinc-dependent endopeptidase
activity which specifically targets core components of the
neurotransmitter release apparatus; 2) a translocation domain
contained within the amino-terminal half of the HC (H.sub.N) that
facilitates release of the LC from intracellular vesicles into the
cytoplasm of the target cell; and 3) a binding domain found within
the carboxyl-terminal half of the HC (H.sub.C) that determines the
binding activity and binding specificity of the toxin to the
receptor complex located at the surface of the target cell.
[0015] The binding, translocation and enzymatic activity of these
three functional domains are all necessary for toxicity. While all
details of this process are not yet precisely known, the overall
cellular intoxication mechanism whereby Clostridial toxins enter a
neuron and inhibit neurotransmitter release is similar, regardless
of serotype or subtype. Although the applicants have no wish to be
limited by the following description, the intoxication mechanism
can be described as comprising at least four steps: 1) receptor
binding, 2) complex internalization, 3) light chain translocation,
and 4) enzymatic target modification (FIG. 1). The process is
initiated when the H.sub.C binding domain of a Clostridial toxin
binds to a toxin-specific receptor system located on the plasma
membrane surface of a target cell. The binding specificity of a
receptor complex is thought to be achieved, in part, by specific
combinations of gangliosides and protein receptors that appear to
distinctly comprise each Clostridial toxin receptor complex. Once
bound, the toxin/receptor complexes are internalized by endocytosis
and the internalized vesicles are sorted to specific intracellular
routes. The translocation step appears to be triggered by the
acidification of the vesicle compartment. This process seems to
initiate important pH-dependent structural rearrangements that
increase hydrophobicity, promote pore formation, and facilitate
separation of the heavy and light chains of the toxin. Once
separated, the light chain endopeptidase of the toxin is released
from the intracellular vesicle into the cytosol where it appears to
specifically target core components of the neurotransmitter release
apparatus. These core proteins, vesicle-associated membrane protein
(VAMP)/synaptobrevin, synaptosomal-associated protein of 25 kDa
(SNAP-25) and Syntaxin, are necessary for synaptic vesicle docking
and fusion at the nerve terminal and constitute members of the
soluble N-ethylmaleimide-sensitive factor-attachment
protein-receptor (SNARE) family. BoNT/A and BoNT/E cleave SNAP-25
in the carboxyl terminal region, releasing a nine or twenty six
amino acid fragment, respectively, and BoNT/C1 also cleaves SNAP-25
near the carboxyl terminus releasing an eight amino acid fragment.
The botulinum serotypes BoNT/B, BoNT/D, BoNT/F and BoNT/G, and
tetanus toxin, act on the conserved central portion of VAMP, and
release the amino terminal portion of VAMP into the cytosol.
BoNT/C1 cleaves syntaxin at a single site near the cytosolic
membrane surface. The selective proteolysis of synaptic SNAREs
accounts for the block of neurotransmitter release caused by
Clostridial toxins in vivo. The SNARE protein targets of
Clostridial toxins are common to exocytosis in a variety of
non-neuronal types; in these cells, as in neurons, light chain
peptidase activity inhibits exocytosis, see, e.g., Yann Humeau et
al., How Botulinum and Tetanus Neurotoxins Block Neurotransmitter
Release, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al.,
Botulinum and Tetanus Neurotoxins: Structure, Function and
Therapeutic Utility, 27(11) Trends Biochem. Sci. 552-558. (2002);
Giovanna Lalli et al., The Journey of Tetanus and Botulinum
Neurotoxins in Neurons, 11(9) Trends Microbiol. 431-437,
(2003).
[0016] Aspects of the present disclosure comprise, in part, a cell
from an established cell line. As used herein, the term "cell"
refers to any eukaryotic cell susceptible to BoNT/A intoxication by
a BoNT/A or any eukaryotic cell that can uptake a BoNT/A. The term
cell encompasses cells from a variety of organisms, such as, e.g.,
murine, rat, porcine, bovine, equine, primate and human cells; from
a variety of cell types such as, e.g., neuronal and non-neuronal;
and can be isolated from or part of a heterogeneous cell
population, tissue or organism. As used herein, the term
"established cell line" is synonymous with "immortal cell line," or
"transformed cell line" and refers to a cell culture of cells
selected for indefinite propagation from a cell population derived
from an organism, tissue, or organ source. By definition, an
established cell line excludes a cell culture of primary cells. As
used herein, the term "primary cells" are cells harvested directly
from fresh tissues or organs and do not have the potential to
propagate indefinitely. An established cell line can comprise a
heterogeneous population of cells or a uniform population of cells.
An established cell line derived from a single cell is referred to
as a clonal cell line. An established cell line can be one whose
cells endogenously express all component necessary for the cells to
undergo the overall cellular mechanism whereby a BoNT/A
proteolytically cleaves a SNAP-25 substrate and encompasses the
binding of a BoNT/A to a BoNT/A receptor, the internalization of
the neurotoxin/receptor complex, the translocation of the BoNT/A
light chain from an intracellular vesicle into the cytoplasm and
the proteolytic cleavage of a SNAP-25. Alternatively, an
established cell line can be one whose cells have had introduced
from an exogenous source at least one component necessary for the
cells to undergo the overall cellular mechanism whereby a BoNT/A
proteolytically cleaves a SNAP-25 substrate and encompasses the
binding of a BoNT/A to a BoNT/A receptor, the internalization of
the neurotoxin/receptor complex, the translocation of the BoNT/A
light chain from an intracellular vesicle into the cytoplasm and
the proteolytic cleavage of a SNAP-25. Also referred to as a
genetically-engineered cell line, cells from such an established
cell line may, e.g., express an exogenous FGFR2, an exogenous
FGFR3, an exogenous SV2, an exogenous SNAP-25, or any combination
thereof. As used herein, the term "established clonal cell line" or
"clonal cell line" refers to a cell culture of cells selected from
a heterogenous population of cell types comprising an established
cell line, which can also be referred to as a parental cell line.
Thus, as a non-limiting example, the cells from the A6, A7, A9,
A10, A11, B5, B10, C5, C11, C12, D7, D11, E11, F10, H1, H3, H8,
H10, 1E3, 2B9, 2E4, 3B8, 3D5, 3G10, 4B5, 4C8, 5F3, AC9, AF4, BB3,
BB10, BE3, BF8, CC11, CD6, CE6, CG8, CG10, DC4, DD10, DE7, YF5, and
H1 1.4 clonal cell lines were selected from the heterogenous
population of cell types comprising the SiMa cell line (or parental
SiMa cell line) DSMZ ACC 164.
[0017] Aspects of the present disclosure comprise, in part, a cell
from an established clonal cell line susceptible to BoNT/A
intoxication. As used herein, the terms "cell(s) susceptible to
BoNT/A intoxication," "cell(s) susceptible to BoNT/A intoxication
by a BoNT/A," or "cell(s) from an established clonal cell line
susceptible to BoNT/A intoxication by a BoNT/A" refer to cell(s)
that can undergo the overall cellular mechanism whereby a BoNT/A
proteolytically cleaves a SNAP-25 substrate and encompasses the
binding of a BoNT/A to a BoNT/A receptor, the internalization of
the neurotoxin/receptor complex, the translocation of the BoNT/A
light chain from an intracellular vesicle into the cytoplasm and
the proteolytic cleavage of a SNAP-25. By definition, cell(s)
susceptible to BoNT/A intoxication must express, or be engineered
to express, at least one BoNT/A receptor and at least one SNAP-25
substrate. Non-limiting examples of a cell or cells from an
established clonal cell line susceptible to BoNT/A intoxication,
include A6, A7, A9, A10, A11, B5, B10, C5, C11, C12, D7, D11, E11,
F10, H1, H3, H8, H10, 1E3, 2B9, 2D2, 2D6, 2E4, 3B8, 3D5, 3G10, 4B5,
4C8, 4D3, 5C10, 5F3, AC9, AF4, BB3, BB10, BE3, BF8, CC11, CD6, CE6,
CG8, CG10, DC4, DD10, DE7, DF5, YB7, YF5, and H1 1.4 clonal cell
lines. As used herein, the terms "cell(s) that can uptake BoNT/A"
or "cell(s) comprising an established clonal cell line that can
uptake BoNT/A" refer to cells that can undergo the overall cellular
mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25
substrate and encompasses the binding of a BoNT/A to a BoNT/A
receptor, the internalization of the neurotoxin/receptor complex,
the translocation of the BoNT/A light chain from an intracellular
vesicle into the cytoplasm and the proteolytic cleavage of a
SNAP-25. By definition, cell(s) that can uptake BoNT/A must
express, or be engineered to express, at least one BoNT/A receptor
and at least one SNAP-25 substrate. Non-limiting examples of a cell
or cells from an established clonal cell line that can uptake
BoNT/A, include A6, A7, A9, A10, A11, B5, B10, C5, C11, C12, D7,
D11, E11, F10, H1, H3, H8, H10, 1E3, 2B9, 2D2, 2D6, 2E4, 3B8, 3D5,
3G10, 4B5, 4C8, 4D3, 5C10, 5F3, AC9, AF4, BB3, BB10, BE3, BF8,
CC11, CD6, CE6, CG8, CG10, DC4, DD10, DE7, DF5, YB7, YF5, and H1
1.4 clonal cell lines. Cell lines comprising cell(s) from an
established clonal cell line susceptible to BoNT/A intoxication or
cell(s) that can uptake BoNT/A are referred to as a "responder cell
line."
[0018] Aspects of the present disclosure comprise, in part, cells
from an established clonal cell line more susceptible to BoNT/A
intoxication. As used herein, the terms "cell(s) more susceptible
to BoNT/A intoxication," "cell(s) more susceptible to BoNT/A
intoxication by a BoNT/A," or "cell(s) from an established clonal
cell line more susceptible to BoNT/A intoxication by a BoNT/A"
refer to cell(s) that undergo the overall cellular mechanism
whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate to a
greater degree as compared to a cell(s) comprising a parental SiMa
cell line, such as, e.g., parental SiMa cell line DSMZ ACC 164.
Non-limiting examples of a cell or cells from an established clonal
cell line more susceptible to BoNT/A intoxication, include a cell
or cells from a A10, H1, 2E4, 3B8, 3D5, 5F3, AF4, BB3, BB10, DC4,
or H1 1.4 clonal cell lines. As used herein, the terms "cell(s)
that uptake BoNT/A more" or "cell(s) comprising an established
clonal cell line that uptake BoNT/A more" refer to cells that
undergo the overall cellular mechanism whereby a BoNT/A
proteolytically cleaves a SNAP-25 substrate to a greater degree as
compared to a cell(s) comprising a parental SiMa cell line, such
as, e.g., parental SiMa cell line DSMZ ACC 164. Non-limiting
examples of a cell or cells from an established clonal cell line
that can uptake BoNT/A more, include a cell or cells from a A10,
H1, 2E4, 3B8, 3D5, 5F3, AF4, BB3, BB10, DC4, or H1 1.4 clonal cell
lines. Cell lines comprising cell(s) from an established clonal
cell line more susceptible to BoNT/A intoxication or cell(s) that
can uptake BoNT/A more are referred to as a "high responder cell
line."
[0019] Aspects of the present disclosure comprise, in part, cells
from an established clonal cell line less susceptible to BoNT/A
intoxication. As used herein, the terms "cell(s) less susceptible
to BoNT/A intoxication," "cell(s) less susceptible to BoNT/A
intoxication by a BoNT/A," or "cell(s) from an established clonal
cell line less susceptible to BoNT/A intoxication by a BoNT/A"
refer to cell(s) that undergo the overall cellular mechanism
whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate to a
lesser degree as compared to a cell(s) comprising a parental SiMa
cell line, such as, e.g., parental SiMa cell line DSMZ ACC 164.
Non-limiting examples of a cell or cells from an established clonal
cell line less susceptible to BoNT/A intoxication, include A7, B10,
D11, H10, 2D2, 2D6, 4D3, 5C10, DF5, and YB7 clonal cell lines. As
used herein, the terms "cell(s) that uptake BoNT/A less" or
"cell(s) comprising an established clonal cell line that uptake
BoNT/A less" refer to cells that undergo the overall cellular
mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25
substrate to a lesser degree as compared to a cell(s) comprising a
parental SiMa cell line, such as, e.g., parental SiMa cell line
DSMZ ACC 164. Non-limiting examples of a cell or cells from an
established clonal cell line that can uptake BoNT/A less, include
A7, B10, D11, H10, 2D2, 2D6, 4D3, 5C10, DF5, and YB7 clonal cell
lines. Cell lines comprising cell(s) from an established clonal
cell line less susceptible to BoNT/A intoxication or cell(s) that
can uptake BoNT/A less are referred to as a "low responder cell
line."
[0020] Aspects of the present disclosure comprise, in part, a cell
from an established clonal cell line not susceptible to BoNT/A
intoxication. As used herein, the terms "cell(s) not susceptible to
BoNT/A intoxication," "cell(s) not susceptible to BoNT/A
intoxication by a BoNT/A," or "cell(s) from an established clonal
cell line not susceptible to BoNT/A intoxication by a BoNT/A" refer
to cell(s) that cannot undergo the overall cellular mechanism
whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate. As
used herein, the terms "cell(s) that cannot uptake BoNT/A" or
"cell(s) comprising an established clonal cell line that cannot
uptake BoNT/A" refer to cells that cannot undergo the overall
cellular mechanism whereby a BoNT/A proteolytically cleaves a
SNAP-25 substrate. Cell lines comprising cell(s) from an
established clonal cell line not susceptible to BoNT/A intoxication
or cell(s) that cannot uptake BoNT/A are referred to as a
"non-responder cell line."
[0021] Thus in an embodiment, cells from an established clonal cell
line are susceptible to BoNT/A intoxication. In aspects of this
embodiment, cells from an established clonal cell line are
susceptible to BoNT/A intoxication by, e.g., about 500 pM or less,
about 400 pM or less, about 300 pM or less, about 200 pM or less,
or about 100 pM or less of a BoNT/A. In other aspects of this
embodiment, cells from an established clonal cell line are
susceptible to BoNT/A intoxication by, e.g., about 90 pM or less,
about 80 pM or less, about 70 pM or less, about 60 pM or less,
about 50 pM or less, about 40 pM or less, about 30 pM or less,
about 20 pM or less, or about 10 pM or less of a BoNT/A. In still
other aspects, cells from an established clonal cell line are
susceptible to BoNT/A intoxication by, e.g., about 9 pM or less,
about 8 pM or less, about 7 pM or less, about 6 pM or less, about 5
pM or less, about 4 pM or less, about 3 pM or less, about 2 pM or
less, or about 1 pM or less of a BoNT/A. In yet other aspects,
cells from an established clonal cell line are susceptible to
BoNT/A intoxication by, e.g., about 0.9 pM or less, about 0.8 pM or
less, about 0.7 pM or less, about 0.6 pM or less, about 0.5 pM or
less, about 0.4 pM or less, about 0.3 pM or less, about 0.2 pM, or
about 0.1 pM or less of a BoNT/A. As used herein, the term "about"
when qualifying a value of a stated item, number, percentage, or
term refers to a range of plus or minus ten percent of the value of
the stated item, percentage, parameter, or term.
[0022] In another embodiment, cells comprising an established
clonal cell line can uptake a BoNT/A. In aspects of this
embodiment, cells comprising an established clonal cell line can
uptake, e.g., about 500 pM or less, about 400 pM or less, about 300
pM or less, about 200 pM or less, or about 100 pM or less of a
BoNT/A. In other aspects of this embodiment, cells comprising an
established clonal cell line possess the ability to uptake about 90
pM or less, about 80 pM or less, about 70 pM or less, about 60 pM
or less, about 50 pM or less, about 40 pM or less, about 30 pM or
less, about 20 pM or less, or about 10 pM or less of a BoNT/A. In
still other aspects, cells comprising an established clonal cell
line possess the ability to uptake about 9 pM or less, about 8 pM
or less, about 7 pM or less, about 6 pM or less, about 5 pM or
less, about 4 pM or less, about 3 pM or less, about 2 pM or less,
or about 1 pM or less of a BoNT/A. In yet other aspects, cells
comprising an established clonal cell line possess the ability to
uptake about 0.9 pM or less, about 0.8 pM or less, about 0.7 pM or
less, about 0.6 pM or less, about 0.5 pM or less, about 0.4 pM or
less, about 0.3 pM or less, about 0.2 pM or less, or about 0.1 pM
or less of a BoNT/A. In other aspects, cells comprising an
established clonal cell line can uptake BoNT/A from, e.g., about
0.01 pM to about 100 pM, about 0.01 pM to about 75 pM, about 0.01
pM to about 50 pM, about 0.01 pM to about 25 pM, about 0.01 pM to
about 20 pM, about 0.01 pM to about 15 pM, about 0.01 pM to about
10 pM, about 0.01 pM to about 5 pM, about 0.001 pM to about 100 pM,
about 0.001 pM to about 75 pM, about 0.001 pM to about 50 pM, about
0.001 pM to about 25 pM, about 0.001 pM to about 20 pM, about 0.001
pM to about 15 pM, about 0.001 pM to about 10 pM, or about 0.001 pM
to about 5 pM of BoNT/A.
[0023] In another embodiment, cells from an established clonal cell
line are not susceptible to BoNT/A intoxication. In aspects of this
embodiment, cells from an established clonal cell line are not
susceptible to BoNT/A intoxication by, e.g., about 500 pM or less,
about 400 pM or less, about 300 pM or less, about 200 pM or less,
or about 100 pM or less of a BoNT/A. In other aspects of this
embodiment, cells from an established clonal cell line are
susceptible to BoNT/A intoxication by, e.g., about 90 pM or less,
about 80 pM or less, about 70 pM or less, about 60 pM or less,
about 50 pM or less, about 40 pM or less, about 30 pM or less,
about 20 pM or less A, or about 10 pM or less of a BoNT/A. In still
other aspects, cells from an established clonal cell line are not
susceptible to BoNT/A intoxication by, e.g., about 9 pM or less,
about 8 pM or less, about 7 pM or less, about 6 pM or less, about 5
pM or less, about 4 pM or less, about 3 pM or less, about 2 pM or
less, or about 1 pM or less of a BoNT/A. In yet other aspects,
cells from an established clonal cell line are not susceptible to
BoNT/A intoxication by, e.g., about 0.9 pM or less, about 0.8 pM or
less, about 0.7 pM or less, about 0.6 pM or less, about 0.5 pM or
less, about 0.4 pM or less, about 0.3 pM or less, about 0.2 pM, or
about 0.1 pM or less of a BoNT/A. In other aspects, cells from an
established clonal cell line are not susceptible to BoNT/A
intoxication from, e.g., about 0.01 pM to about 100 pM, about 0.01
pM to about 75 pM, about 0.01 pM to about 50 pM, about 0.01 pM to
about 25 pM, about 0.01 pM to about 20 pM, about 0.01 pM to about
15 pM, about 0.01 pM to about 10 pM, about 0.01 pM to about 5 pM,
about 0.001 pM to about 100 pM, about 0.001 pM to about 75 pM,
about 0.001 pM to about 50 pM, about 0.001 pM to about 25 pM, about
0.001 pM to about 20 pM, about 0.001 pM to about 15 pM, about 0.001
pM to about 10 pM, or about 0.001 pM to about 5 pM of BoNT/A.
[0024] Aspects of the present disclosure comprise, in part, cells
from an established clonal cell line susceptible to BoNT/A
intoxication that are more stable than cells from a parental SiMa
cell line, such as, e.g., parental SiMa cell line DSMZ ACC 164. As
used herein, the term "stable" refers to cells from an established
clonal cell line for a particular passage number that exhibit a
relative EC.sub.50, sensitivity, efficacy, well-defined upper
asymptote, and/or a well-defined dose-response curve for BoNT/A
activity that is similar to the values for relative EC.sub.50,
sensitivity, efficacy, well-defined upper asymptote, and/or a
well-defined dose-response curve exhibited by cells from a parental
SiMa cell line, such as, e.g., parental SiMa cell line DSMZ ACC
164, at the same or similar passage number, where the same assay
conditions and the same BoNT/A (or molecule) are used in both
assays.
[0025] Thus in an embodiment, cells from an established clonal cell
line are more stable as compared to a parental SiMa cell line. In
an aspect of this embodiment, cells from an established clonal cell
line are more stable as compared to the parental SiMa cell line
DSMZ ACC 164. In other aspects of this embodiment, cells from an
established clonal cell line are more stable for, e.g., at least 5
more passages, at least 10 more passages, at least 15 more
passages, at least 20 more passages, at least 25 more passages, or
at least 30 more passages, as compared to a parental SiMa cell
line. In yet other aspects of this embodiment, cells from an
established clonal cell line are more stable for, e.g., at least 5
more passages, at least 10 more passages, at least 15 more
passages, at least 20 more passages, at least 25 more passages, or
at least 30 more passages, as compared to a parental SiMa cell line
DSMZ ACC 164.
[0026] Aspects of the present disclosure comprise, in part, cells
from an established clonal cell line susceptible to BoNT/A
intoxication that are stable over a plurality of cell passages. As
used herein, the term "stable" refers to cells from an established
clonal cell line for a particular passage number that exhibit a
relative EC.sub.50, sensitivity, efficacy, well-defined upper
asymptote, and/or a well-defined dose-response curve for BoNT/A
activity that is similar to the values for relative EC.sub.50,
sensitivity, efficacy, well-defined upper asymptote, and/or a
well-defined dose-response curve exhibited by cells from the same
established clonal cell line, but from a prior passage or passages,
where the same assay conditions and the same BoNT/A (or molecule)
are used in both assays.
[0027] Cells from an established cell line disclosed in the present
specification can exhibit consistent sensitivity to BoNT/A activity
over a plurality of cell passages. As used herein, the term
"sensitivity to BoNT/A activity" refers to the lowest dose that an
assay can measure consistently above the signal detected by a
non-treatment control or background signal.
[0028] Thus, in an embodiment, cells from the established clonal
cell line exhibit a sensitivity for BoNT/A activity for any given
passages that is e.g., 100 pM or less, about 80 pM or less, about
70 pM or less, about 60 pM or less, about 50 pM or less, about 40
pM or less, about 30 pM or less, about 20 pM or less, about 10 pM
or less, about 1 pM or less, about 0.9 pM or less, about 0.8 pM or
less, about 0.7 pM or less, about 0.6 pM or less, about 0.5 pM or
less, about 0.4 pM or less, about 0.3 pM or less, about 0.2 pM or
less, or about 0.1 pM or less of a BoNT/A. In aspects of this
embodiment, cells from the established clonal cell line exhibit a
sensitivity for BoNT/A activity for any given passages that is,
e.g., about 0.01 pM to about 100 pM, about 0.01 pM to about 75 pM,
about 0.01 pM to about 50 pM, about 0.01 pM to about 25 pM, about
0.01 pM to about 20 pM, about 0.01 pM to about 15 pM, about 0.01 pM
to about 10 pM, about 0.01 pM to about 5 pM, about 0.001 pM to
about 100 pM, about 0.001 pM to about 75 pM, about 0.001 pM to
about 50 pM, about 0.001 pM to about 25 pM, about 0.001 pM to about
20 pM, about 0.001 pM to about 15 pM, about 0.001 pM to about 10
pM, or about 0.001 pM to about 5 pM of BoNT/A.
[0029] In another embodiment, cells from the established clonal
cell line exhibit a sensitivity for BoNT/A activity that is about
100 pM or less, about 75 pM or less, about 50 pM or less, about 25
pM or less, less about 20 pM or less, about 15 pM or less, about 10
pM or less, or about 1 pM or less for, e.g., 5 or more cell
passages, 10 or more cell passages, 15 or more cell passages, 20 or
more cell passages, 25 or more cell passages, 30 or more cell
passages, 35 or more cell passages, 40 or more cell passages, 45 or
more cell passages, or 50 or more cell passages. In other aspects
of this embodiment, cells from the established clonal cell line
exhibit a sensitivity for BoNT/A activity that is about 100 pM or
less, about 75 pM or less, about 50 pM or less, about 25 pM or
less, less about 20 pM or less, about 15 pM or less, about 10 pM or
less, or about 1 pM or less for, e.g., about 15 to about 60
passages, about 20 to about 60 passages, about 25 to about 60
passages, about 30 to about 60 passages, about 35 to about 60
passages, about 40 to about 60 passages, about 45 to about 60
passages, about 50 to about 60 passages, about 15 to about 50
passages, about 20 to about 50 passages, about 25 to about 50
passages, about 30 to about 50 passages, about 35 to about 50
passages, about 40 to about 50 passages, about 15 to about 40
passages, about 20 to about 40 passages, about 25 to about 40
passages, or about 30 to about 40 passages.
[0030] Cells from an established cell line disclosed in the present
specification can exhibit a consistent relative efficacy of BoNT/A
uptake, BoNT/A activity, or BoNT/A intoxication over a plurality of
cell passages. As used herein, the term "relative efficacy" refers
to how well the upper asymptote for the BoNT/A activity detected in
the current assay run compares to the upper asymptote for the
BoNT/A activity detected in a reference standard, a reference
molecule, or a reference passage number used on that assay. As used
herein, the term "signal to noise ratio for the upper asymptote"
refers to the signal detected in an assay at the upper limit of
detection divided by the signal detected by a non-treatment control
or background signal. The upper limit of detection is the highest
dose that an assay can measure consistently before saturation of
the signal occurs.
[0031] Thus, in an embodiment, cells from an established cell line
disclosed in the present specification can exhibit a well defined
upper asymptote over a plurality of cell passages and maintain a
signal to noise ratio that is consistent and adequate for the
assay. In aspects of this embodiment, cells from an established
cell line disclosed in the present specification must have a signal
to noise ratio for the upper asymptote for BoNT/A activity of,
e.g., at least 3:1, at least 4:1, at least 5:1, at least 6:1, at
least 7:1, at least 8:1, at least 9:1, at least 10:1, at least
15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1,
at least 40:1, at least 45:1, at least 50:1, at least 60:1, at
least 70:1, at least 80:1, at least 90:1, or at least 100:1, at
least 150:1, at least 200:1, at least 250:1, at least 300:1, at
least 350:1, at least 400:1, at least 450:1, at least 500:1, at
least 550:1, or at least 600:1, over, e.g., 5 or more cell
passages, 10 or more cell passages, 15 or more cell passages, 20 or
more cell passages, 25 or more cell passages, 30 or more cell
passages, 35 or more cell passages, 40 or more cell passages, 45 or
more cell passages, or 50 or more cell passages. In other aspects
of this embodiment, cells from an established cell line disclosed
in the present specification must have a signal to noise ratio for
the upper asymptote for BoNT/A activity of, e.g., at least 3:1, at
least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1,
at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least
25:1, at least 30:1, at least 35:1, at least 40:1, at least 45:1,
at least 50:1, at least 60:1, at least 70:1, at least 80:1, at
least 90:1, or at least 100:1, at least 150:1, at least 200:1, at
least 250:1, at least 300:1, at least 350:1, at least 400:1, at
least 450:1, at least 500:1, at least 550:1, or at least 600:1,
over, e.g., about 15 to about 60 passages, about 20 to about 60
passages, about 25 to about 60 passages, about 30 to about 60
passages, about 35 to about 60 passages, about 40 to about 60
passages, about 45 to about 60 passages, about 50 to about 60
passages, about 15 to about 50 passages, about 20 to about 50
passages, about 25 to about 50 passages, about 30 to about 50
passages, about 35 to about 50 passages, about 40 to about 50
passages, about 15 to about 40 passages, about 20 to about 40
passages, about 25 to about 40 passages, or about 30 to about 40
passages.
[0032] Cells from an established cell line disclosed in the present
specification can exhibit a well defined dose-response curve for
BoNT/A activity over a plurality of cell passages. As used herein,
the term "dose-response curve" refers to the how well the raw data
fits the statistical model of choice for that assay. As a
non-limiting example, a sigmoidal curve with a four parameter
logistics fit is a dose-response curve for an enzymatic activity
assay, such as, e.g., a potency assay. As another non-limiting
example, a ligand binding with one site saturation fit is a
dose-response curve for a ligand/antibody binding assay.
[0033] Thus, in an embodiment, cells from an established cell line
disclosed in the present specification exhibit a well defined
dose-response curve for BoNT/A activity over a plurality of cell
passages. In aspects of this embodiment, cells from an established
cell line disclosed in the present specification exhibit a well
defined dose-response curve for BoNT/A activity over, e.g., 5 or
more cell passages, 10 or more cell passages, 15 or more cell
passages, 20 or more cell passages, 25 or more cell passages, 30 or
more cell passages, 35 or more cell passages, 40 or more cell
passages, 45 or more cell passages, or 50 or more cell passages. In
other aspects of this embodiment, cells from an established cell
line disclosed in the present specification exhibit a well defined
dose-response curve for BoNT/A activity over, e.g., about 15 to
about 60 passages, about 20 to about 60 passages, about 25 to about
60 passages, about 30 to about 60 passages, about 35 to about 60
passages, about 40 to about 60 passages, about 45 to about 60
passages, about 50 to about 60 passages, about 15 to about 50
passages, about 20 to about 50 passages, about 25 to about 50
passages, about 30 to about 50 passages, about 35 to about 50
passages, about 40 to about 50 passages, about 15 to about 40
passages, about 20 to about 40 passages, about 25 to about 40
passages, or about 30 to about 40 passages.
[0034] Cells from an established cell line disclosed in the present
specification can exhibit a consistent relative EC.sub.50 value for
BoNT/A activity over a plurality of cell passages. As used herein,
the term "relative EC.sub.50" or "relative EC.sub.50 value" refers
to an EC.sub.50 value of BoNT/A activity that is normalized against
the EC.sub.50 calculated for a reference standard, a reference
molecule, or a reference passage number used on that assay.
[0035] Thus, in an embodiment, cells from an established clonal
cell line exhibit a consistent relative EC.sub.50 for BoNT/A
activity over a plurality of cell passages. In aspects of this
embodiment, cells from an established clonal cell line exhibit a
consistent relative EC.sub.50 for BoNT/A activity that is, e.g.,
about .+-.10%, about .+-.20%, about .+-.30%, about .+-.40%, about
.+-.50%, about 60%, about 70%, or about .+-.75%, the relative
EC.sub.50 for BoNT/A activity over, e.g., 5 or more cell passages,
10 or more cell passages, 15 or more cell passages, 20 or more cell
passages, 25 or more cell passages, 30 or more cell passages, 35 or
more cell passages, 40 or more cell passages, 45 or more cell
passages, or 50 or more cell passages. In other aspects of this
embodiment, cells from an established clonal cell line exhibit a
relative EC.sub.50 for BoNT/A activity that is, e.g., about .+-.10%
to about 75%, about .+-.10% to about 70%, about .+-.10% to about
60%, about .+-.10% to about 50%, about .+-.10% to about 40%, about
.+-.10% to about 30%, or about .+-.10% to about 20% the relative
EC.sub.50 for BoNT/A activity over, e.g., 5 or more cell passages,
10 or more cell passages, 15 or more cell passages, 20 or more cell
passages, 25 or more cell passages, 30 or more cell passages, 35 or
more cell passages, 40 or more cell passages, 45 or more cell
passages, or 50 or more cell passages.
[0036] Aspects of the present disclosure comprise, in part, cells
from an established clonal cell line susceptible to BoNT/A
intoxication that exhibit at least a 1.5-fold difference in gene
expression levels of one or more genes listed in Tables 5, 6, 7, or
8 as compared to the expression levels of these genes in cells from
the 2D6 cell line. In other aspects of this embodiment, cells from
an established clonal cell line susceptible to BoNT/A intoxication
exhibit a difference in gene expression levels of one or more genes
listed in Tables 5, 6, 7, or 8 of, e.g., at least a 1.5-fold, at
least a 2.0-fold, at least a 2.5-fold, at least a 3.0-fold, at
least a 3.5-fold, at least a 4.0-fold, at least a 4.5-fold, at
least a 5.0-fold, at least a 5.5-fold, at least a 6.0-fold, at
least a 7.0-fold, or at least a 8.0-fold as compared to the
expression levels of these genes in cells from the 2D6 cell line.
In other aspects of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit a
difference in gene expression levels of, e.g., 2 or more genes, 3
or more genes, 4 or more genes, 5 or more genes, 6 or more genes, 7
or more genes, 8 or more genes, 9 or more genes, 10 or more genes,
20 or more genes, 30 or more genes, 40 or more genes, 50 or more
genes, 60 or more genes, 70 or more genes, 80 or more genes, 90 or
more genes, or 100 or more genes listed in Tables 5, 6, 7, or 8 as
compared to the expression levels of these genes in cells from the
2D6 cell line. In yet other aspects, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit a
difference in gene expression levels of, e.g., about 5 genes to
about 100 genes, about 10 genes to about 100 genes, about 15 genes
to about 100 genes, about 20 genes to about 100 genes, about 25
genes to about 100 genes, about 5 genes to about 75 genes, about 10
genes to about 75 genes, about 15 genes to about 75 genes, about 20
genes to about 75 genes, about 25 genes to about 75 genes, about 5
genes to about 50 genes, about 10 genes to about 50 genes, about 15
genes to about 50 genes, about 20 genes to about 50 genes, or about
25 genes to about 50 genes listed in Tables 5, 6, 7, or 8 as
compared to the expression levels of these genes in cells from the
2D6 cell line. The log ratio in Tables 5, 6, 7, and 8 represent
log.sub.2 values where 0.585 is log.sub.2(1.5) which is a 1.5-fold
difference, 1 is log.sub.2(2) which is a 2-fold difference, 1.584
is log.sub.2(3) which is a 3-fold difference, 2 is log.sub.2(4)
which is a 4-fold difference, 2.321 is log.sub.2(5) which is a
5-fold difference, 2.584 is log.sub.2(6) which is a 6-fold
difference, 2.807 is log.sub.2(7) which is a 7-fold difference, 3
is log.sub.2(8) which is a 8-fold difference, 3.169 is log.sub.2(9)
which is a 9-fold difference, and 3.321 is log.sub.2(10) which is a
10-fold difference.
[0037] In an embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes listed in
Tables 5 or 7 as compared to the expression levels of these genes
in cells from the 2D6 cell line. In aspects of this embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit an increase in gene expression levels of one
or more genes listed in Tables 5 or 7 of, e.g., at least a
2.0-fold, at least a 2.5-fold, at least a 3.0-fold, at least a
3.5-fold, at least a 4.0-fold, at least a 4.5-fold, at least a
5.0-fold, at least a 5.5-fold, at least a 6.0-fold, at least a
7.0-fold, or at least a 8.0-fold as compared to the expression
levels of these genes in cells from the 2D6 cell line. In other
aspects of this embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit an increase in gene
expression levels of, e.g., 2 or more genes, 3 or more genes, 4 or
more genes, 5 or more genes, 6 or more genes, 7 or more genes, 8 or
more genes, 9 or more genes, 10 or more genes, 20 or more genes, 30
or more genes, 40 or more genes, 50 or more genes, 60 or more
genes, 70 or more genes, 80 or more genes, 90 or more genes, or 100
or more genes listed in Tables 5 or 7 as compared to the expression
levels of these genes in cells from the 2D6 cell line. In yet other
aspects, cells from an established clonal cell line susceptible to
BoNT/A intoxication exhibit an increase in gene expression levels
of, e.g., about 5 genes to about 100 genes, about 10 genes to about
100 genes, about 15 genes to about 100 genes, about 20 genes to
about 100 genes, about 25 genes to about 100 genes, about 5 genes
to about 75 genes, about 10 genes to about 75 genes, about 15 genes
to about 75 genes, about 20 genes to about 75 genes, about 25 genes
to about 75 genes, about 5 genes to about 50 genes, about 10 genes
to about 50 genes, about 15 genes to about 50 genes, about 20 genes
to about 50 genes, or about 25 genes to about 50 genes listed in
Tables 5 or 7 as compared to the expression levels of these genes
in cells from the 2D6 cell line.
[0038] In an embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes selected
from A2BP1, ACO1, ACOT7, ACOT9, ACTL6A, ACTL6B, ADRBK2, AGPS, AIDA,
AKAP13, ALCAM, ALDH7A1, AMOTL1, ANAPC7, ANKRD13A, ANKRD54, ANKS1B,
ANLN, ANP32E, ANXA6, APAF1, APBB2, APOO, ARF3, ARHGAP11A, ARHGAP24,
ARHGAP24, ARHGAP6, ARHGEF3, ARHGEF6, ARL13B, ARL6, ARL6IP1, ASCL1,
ASF1A, ASF1B, ASXL3, ATAD2, ATP6V1A, AURKA, AURKB, BARD1, BASP1,
BAX, BCL11A, BCR, BNC2, BOK, BRCA1, BRCA2, BRD4, BRI3BP, BRIP1,
BTBD3, BTG3, BUB1, BUB1B, BUB3, BVES, C10ORF58, C10ORF78, C11ORF75,
C12ORF48, C12ORF49, C14ORF106, C14ORF143, C15ORF23, C15ORF42,
C18ORF54, C1ORF112, C1ORF183, C1ORF43, C1QL1, C20ORF108, C20ORF20,
C20ORF7, C20ORF72, C22ORF28, C22ORF29, C22ORF39, C3ORF14, C3ORF70,
C4ORF46, C4ORF49, C5ORF32, C6ORF115, C9ORF100, CARHSP1, CBLB,
CBWD1, CBX5, CCDC109B, CCDC117, CCDC15, CCDC21, CCDC3, CCDC34,
CCDC52, CCDC86, CCDC99, CCNB1, CCNE2, CCNF, CCNYL1, CD24, CD47,
CD9, CDC2, CDC25B, CDC42EP4, CDC45L, CDC5L, CDC6, CDCA2, CDCA4,
CDCA5, CDCA7, CDCA8, CDH2, CDK2, CDK2AP1, CDK6, CDKAL1, CDKN2C,
CDKN2D, CDO1, CDS2, CECR5, CELSR3, CENPF, CENPH), CENPI, CENPJ,
CENPK, CENPL, CENPM, CENPN, CENPO, CEP135, CEP152, CEP55, CEP78,
CEP97, CHAF1A, CHD6, CHEK1, CHEK2, CHMP4B, CHRNA3, CHRNA7, CHST15,
CIT, CKAP2, CKAP2L, CKLF, CKS1B, CLDND1, CLSTN2, CMTM7, CNN3,
CNOT4, CNP, CNTN1, CNTN4, COBLL1, COQ3, CPNE4, CPT1A, CPVL,
CRISPLD1, CRTAC1, CRYBG3, CRYZ, CSE1L, CSRP2, CSRP2BP, CSTF1,
CTDSPL2, CTNNBL1, CTSL2, CUGBP2, CXCR4, CYTSA, DACT1, DAZ1, DBF4B,
DBH, DCLRE1B, DCPS, DDAH2, DDT, DEK, DENR, DEPDC1, DEPDC1B, DERA,
DGCR14, DGKE, DHX15, DHX35, DIAPH1, DIAPH3, DKFZP434L187, DLEU2,
DLGAPS, DLL3, DLX6, DNA2, DNM3, DNMT3B, DOCK10, DOK4, DOK5, DPF1,
DPYD, DPYSL3, DRAM1, DRG1, DSCC1, DSN1, DTL, DTNBP1, DTYMK, DVL2,
DYNLT3, DYRK4, E2F1, E2F2, E2F7, E2F8, EAF2, EBF1, ECT2, EFNA5,
EFNB2, ELAVL4, ELOVL7, EME1, EMILIN2, EMILIN3, EML1, ENC1, EPB41L5,
EPOR, ERCC6L, ESCO2, ESF1, ESPL1, ETS1, ETV1, EXO1, EXOC5, EXOSC6,
EXPH5, EZH2, FAM101B, FAM105A, FAM110A, FAM114A1, FAM118A, FAM120C,
FAM129B, FAM13A, FAM162B, FAM181B, FAM19A4, FAM19A5, FAM54A,
FAM64A, FAM7A3, FAM83D, FANCG, FANCI, FANCL, FANCM, FARP1, FAT1,
FBLN1, FBN1, FBP1, FBXO43, FBXO5, FEN1, FGFR2, FH, FIGNL1, FKBP5,
FNDC5, FOXD1, FOXN3, FRMD6, FRZB, FSTL1, FZD2, FZD5, FZD8, G2E3,
GABBR2, GAP43, GAP43, GAS2L3, GATM, GEMIN4, GFPT2, GFRA1, GFRA2,
GGCX, GINS1, GINS3, GJC1, GLDC, GMNN, GNAI1, GNASAS, GNB4, GNG11,
GNG12, GNG4, GPAM, GPN3, GPR125, GPR161, GPSM2, GPX3, GRP, GSG2,
GSS, GSTCD, GTSE1, GTSF1, GXYLT1, H1F0, H2AFX, HAT1, HAUS6, HDAC8,
HEG1, HES6, HGF, HIC2, HMG4L, HMGB1, HMGB2, HMGB3, HMGXB4, HMMR,
HOOK3, HPS4, HRH3, HS3ST2, HS6ST2, HSPB11, HTATSF1, IDH2, IFI27L1,
IQGAP1, ITGA6, ITGAV, ITGB5, ITPRIP, JARID2, KCNG3, KCNJ8, KCNN1,
KCTD12, KDELC2, KHDRBS3, KIAA0101, KIAA0406, KIAA1211, KIAA1524,
KIF11, KIF14, KIF16B, KIF18A, KIF23, KIFC1, KLF7, KLHL13, KLHL5,
KNTC1, KPNA2, LBH, LGR5, LHFPL2, LIFR, LIG3, LMF2, LMNB1,
LOC100127983, LOC100128844, LOC100288551, LOC340109, LOC344595,
LOC440288, LOC642597, LOC728052, LPAR1, LRFN2, LRRC1, LSM4, LUM,
MAB21L1, MAB21L2, MAGEH1, MAN1A1, MAN2A1, MAOA, MAP3K13, MAPK11,
MAPK12, MAPKAPK3, MASTL, MBD2, MCAM, MCM10, MCM3, MCM4, MCM5, MCM6,
MCM7, MCM8, MDC1, MDM1, MED20, MEIS1, MEIS2, MELK, MEST, MFAP2,
MGAT5B, MINA, MKI67, MKL1, MLF1IP, MMD, MNS1, MPHOSPH9, MPP5,
MPPED2, MRC2, MRPL35, MRPL49, MRS2, MSN, MTF2, MTHFD1, MTUS1, MVK,
MYBL1, MYBL2, MYD88, MYO1B, MYO6, MYST1, NAAA, NAGA, NANP, NARG1,
NASP, NAT11, NAT13, NCAPD3, NCAPG, NCAPG2, NCAPH, NCAPH2, NDN,
NEDD1, NEDD9, NEIL3, NELL2, NFIB, NOC4L, NOS1, NR3C1, NRG3, NRM,
NRXN1, NSMCE4A, NTAN1, NUCKS1, NUDT1, NUF2, NUP107, NUP37, NUP43,
NUP50, NUP93, NUSAP1, NXPH1, NXT2, ODZ3, ODZ4, OIP5, ORC1L, ORC6L,
OSBPL3, PAICS, PANK2, PARP3, PASK, PBK, PCDH17, PCDH8, PCGF5, PCNA,
PDCL, PDE5A, PDLIM5, PDRG1, PEG3, PELI2, PELO, PEX13, PEX26,
PFKFB3, PGAM5, PGM2, PHF19, PHF20, PHF21B, PHF5A, PHLPP1, PHYHIPL,
PI4KA, PIP4K2A, PIR, PKMYT1, PLCL2, PLD5, PLEKHF2, PLK2, PLK4,
PLS3, PLXNA2, PLXNA4, PLXNB2, PM20D2, POLA1, POLA2, POLE, POLE2,
POLQ, POSTN, PPAP2B, PPAT, PPIF, PPIL5, PPM1F, PPP1R13B, PPP1R3C,
PPP2R2B, PPP3CB, PRIM1, PRIM2, PRLHR, PRPS1, PRSS12, PSMA7,
PSMC3IP, PSMD5, PSMD9, PSRC1, PTER, PTGES2, PTGFRN, PTGR1, PTPRE,
PTPRG, PTPRK, PTPRM, PTTG1, PVRL3, PXMP4, PXN, RAB35, RAB3B, RAD18,
RAD51, RAD51C, RALY, RAN, RANBP1, RANGAP1, RASEF, RBBP7, RBBP9,
RBL1, RBPMS, RBX1, RECQL4, REEP1, RELB, RELL1, REXO2, RFC1, RFC2,
RFC3, RFC4, RFC5, RGMA, RGS5, RILPL2, RIMBP2, RMI1, RNASEH2A,
RNF182, RNF26, RNF34, RPA1, RPP30, RRM1, RRM2, RRP7A, RSRC1, S1PR3,
SALL4, SAP30, SASS6, SBF2, SCD5, SCML1, SCML2, SDC1, SEC23A,
SEC61A2, SELM, SEMA6A, SEMA6D, SESN3, SEZ6L, SFI1, SFRP1, SGOL2,
SH3D19, SHCBP1, SHMT1, SHOX2, SIM1, SIVA1, SKA1, SKA2, SKA3, SKP2,
SLC25A10, SLC26A2, SLC2A4RG, SLC2A8, SLC43A3, SLC44A5, SLC7A2,
SLC8A1, SLCO3A1, SLIT3, SLITRK5, SLK, SLMO2, SMC1A, SMC2, SMC4,
SMC6, SMO, SMYD5, SNAI2, SNAP29, SNCAIP, SNRPD3, SNX18, SNX5, SORD,
SOX4, SPAG6, SPARC, SPATS2L, SPC24, SPC25, SPOCK1, SPSB4, SRGAP1,
SRRD, SRRM4, SSH2, ST8SIA1, ST8SIA2, ST8SIA4, STARD4, STIL, STK17A,
STMN3, STOM, STON2, STRA6, SUSD5, SUV39H1, SVIP, SYN2, SYNE2, SYNM,
SYT17, SYTL4, TBC1D1, TBX3, TCF19, TCF7L2, TCFL5, TDRKH, TEAD3,
TESC, TFDP1, TFDP2, TFPI2, TH1L, THOC4, TIFA, TIMP3, TLE3, TLE4,
TMCC3, TMEM107, TMEM132C, TMEM151B, TMEM170B, TMEM178, TMEM194A,
TMEM38B, TMEM48, TMEM56, TMEM98, TMPO, TMSB15B, TNFAIP8, TOB2,
TOMM34, TPBG, TPTE, TPX2, TRIM29, TRIM36, TRIM68, TRIM9, TRIP13,
TROAP, TSHZ3, TSPAN14, TSPAN4, TSPAN5, TTC28, TTC9, TUBA1B, TUBB,
TWIST1, TXNRD1, TYMS, TYRO3, UBE2C, UBE2L3, UBE2V1, UBE3B, UCK1,
UCP2, UFD1L, UHRF1, UNC119B, UNG, UQCC, USP1, USP48, UTP18, VAPB,
VAV3, VPS29, VSTM2L, WDHD1, WDR51A, WDR53, WDR62, WDR67, WEE1,
WEE1, WHSC1, WRAP53, WWC3, XRCC4, XRCC6, ZAK, ZFHX4, ZFP82, ZGPAT,
ZNF215, ZNF217, ZNF238, ZNF253, ZNF280B, ZNF367, ZNF43, ZNF443,
ZNF503, ZNF521, ZNF560, ZNF608, ZNF626, ZNF681, ZNF71, ZNF823,
ZNF85, ZNF92, ZNF93, and/or ZWINT, as compared to the expression
levels of these genes in cells from the 2D6 cell line.
[0039] In an aspect of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold increase in gene expression levels of one or more
genes selected from A2BP1, ACOT9, ALCAM, ANLN, ARHGAP24, ARHGAP6,
ARHGEF3, ARHGEF6, ASCL1, ASF1B, ATAD2, AURKA, AURKB, BARD1, BASP1,
BNC2, BRCA2, BRIP1, BTG3, BUB1, BUB1B, BVES, C11ORF75, C12ORF48,
C14ORF106, C15ORF42, C18ORF54, C1ORF112, C3ORF70, C4ORF49,
CCDC109B, CCDC3, CCNB1, CCNE2, CD9, CDC2, CDC25B, CDC45L, CDC6,
CDCA2, CDCA5, CDCA8, CDK2, CDKN2D, CENPF, CENPI, CENPL, CENPN,
CEP55, CHEK1, CHEK2, CHRNA7, CIT, CKAP2L, CLSTN2, CNTN1, CNTN4,
CPVL, CRYBG3, CSRP2, CTSL2, CUGBP2, CXCR4, DAZ1, DEPDC1, DEPDC1B,
DIAPH3, DLGAPS, DNA2, DOK5, DPYD, DPYSL3, DSCC1, DSN1, DTL, DYNLT3,
E2F1, E2F2, E2F7, E2F8, ECT2, EFNB2, ELOVL7, EME1, EMILIN2,
EMILIN3, EML1, ENC1, ERCC6L, ESCO2, ESPL1, ETV1, EXO1, EXPH5,
FAM101B, FAM114A1, FAM54A, FAM64A, FAM7A3, FAM83D, FANCI, FAT1,
FBLN1, FBP1, FBXO43, FGFR2, FNDC5, FOXD1, FRMD6, FRZB, FZD5,
GAS2L3, GFRA2, GINS1, GINS3, GNAI1, GNB4, GNG11, GNG12, GPSM2, GRP,
GTSE1, GTSF1, HGF, HMMR, HS3ST2, ITGA6, ITPRIP, KCNG3, KCTD12,
KDELC2, KIAA0101, KIAA1524, KIF11, KIF14, KIF18A, KIF23, KIFC1,
LOC340109, LOC642597, LOC728052, LOC728052, LPAR1, LUM, MAB21L1,
MAB21L2, MAOA, MCM10, MCM5, MELK, MINA, MKI67, MLF1IP, MPPED2,
MRC2, MSN, MYBL1, MYBL2, MYO6, NCAPG, NCAPH, NDN, NEDD9, NEIL3,
NR3C1, NRXN1, NUF2, NUSAP1, OIP5, ORC1L, OSBPL3, PBK, PCGF5, PDE5A,
PEG3, PELO, PFKFB3, PHLPP1, PLD5, PLK2, PLK4, PLS3, POLA2, POLE2,
POLQ, PPAP2B, PPP1R3C, PRLHR, PRSS12, PSRC1, PTGR1, PTPRE, PTPRK,
PTPRM, PTTG1, PVRL3, RAD51, RBL1, RBPMS, RELL1, RFC5, RGMA, RGS5,
RNF182, RRM2, S1PR3, SGOL2, SHCBP1, SHOX2, SIM1, SKA1, SLC43A3,
SLC44A5, SLC7A2, SLITRK5, SMC2, SMC6, SPAG6, SPARC, SPC24, SPC25,
ST8SIA4, STK17A, SUSD5, SYN2, SYNM, SYT17, TCF19, TESC, TFDP2,
TFPI2, TIMP3, TLE3, TMEM132C, TMEM178, TNFAIP8, TPBG, TPTE, TPX2,
TRIM29, TRIM36, TRIM68, TROAP, TWIST1, TYMS, UBE2C, UBE3B, UHRF1,
WDHD1, WDR51A, WDR67, WEE1, ZFP82, ZNF367, ZNF521, and/or ZWINT, as
compared to the expression levels of these genes in cells from the
2D6 cell line.
[0040] In another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold increase in gene expression levels of
one or more genes selected from A2BP1, ALCAM, ANLN, ARHGAP24,
ARHGAP6, ASCL1, ASF1B, ATAD2, AURKA, AURKB, BARD1, BASP1, BNC2,
BRIP1, BUB1, BVES, C11ORF75, C12ORF48, C14ORF106, C18ORF54,
C3ORF70, CCDC109B, CCDC3, CD9, CDC2, CDC45L, CDCA2, CDCA8, CDK2,
CENPL, CEP55, CHEK2, CHRNA7, CKAP2L, CNTN1, CRYBG3, CUGBP2, CXCR4,
DAZ1, DEPDC1, DIAPH3, DLGAPS, DOK5, DPYD, DTL, DYNLT3, E2F1, E2F7,
E2F8, EFNB2, ELOVL7, EMILIN2, EML1, ENC1, ESPL1, ETV1, EXO1, EXPH5,
FAM54A, FAM64A, FAM7A3, FAM83D, FANCI, FAT1, FBLN1, FBXO43, FGFR2,
FZD5, GNAI1, GNB4, GNG11, GNG12, GPSM2, GRP, GTSE1, GTSF1, HGF,
HMMR, ITGA6, ITPRIP, KCTD12, KDELC2, KIF11, KIF14, KIF18A, KIF23,
KIFC1, LOC340109, LOC728052, LPAR1, LUM, MAB21L1, MAB21L2, MAOA,
MCM10, MELK, MINA, MKI67, MSN, MYBL1, NCAPH, NDN, NEDD9, NEIL3,
NRXN1, NUF2, NUSAP1, OIP5, ORC1L, OSBPL3, PBK, PCGF5, PEG3, PEG3,
PHLPP1, PLD5, PLK2, PLK4, PLS3, POLE2, PPAP2B, PPP1R3C, PRLHR,
PRSS12, PTPRE, PTPRK, PTPRM, RBPMS, RELL1, RGS5, RNF182, RRM2,
S1PR3, SGOL2, SHCBP1, SHOX2, SIM1, SLC43A3, SLC44A5, SLC7A2,
SLITRK5, SMC6, SPAG6, SPARC, SPC24, SPC25, ST8SIA4, STK17A, SYT17,
TFPI2, TIMP3, TMEM132C, TMEM178, TPBG, TPTE, TPX2, TRIM36, TWIST1,
UBE3B, and/or ZNF367, as compared to the expression levels of these
genes in cells from the 2D6 cell line.
[0041] In yet another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold increase in gene expression levels of
one or more genes selected from A2BP1, ARHGAP24, ARHGAP6, ASCL1,
ASF1B, ATAD2, AURKA, BARD1, BASP1, BNC2, BUB1, BVES, C11ORF75,
CCDC3, CHEK2, CHRNA7, CKAP2L, CNTN1, CRYBG3, CUGBP2, CUGBP2, CXCR4,
DAZ1, DEPDC1, DIAPH3, DLGAPS, DOK5, DPYD, DTL, DYNLT3, E2F8, EFNB2,
ELOVL7, ENC1, EXPH5, FAM7A3, FANCI, FAT1, FBLN1, FGFR2, GNAI1,
GNB4, GNG11, GNG12, GRP, GTSE1, GTSF1, HMMR, ITGA6, ITPRIP, KCTD12,
KDELC2, LOC728052, LPAR1, LUM, MAB21L1, MAB21L2, MAOA, MCM10, MELK,
MINA, MKI67, MSN, MYBL1, NDN, NEDD9, NRXN1, OSBPL3, PEG3, PLK2,
PLS3, PRLHR, PRSS12, PTPRK, PTPRM, RBPMS, RGS5, RNF182, RRM2,
SGOL2, SIM1, SLC43A3, SLC44A5, SLC7A2, SMC6, SPAG6, SPARC, STK17A,
TFPI2, TIMP3, TMEM132C, TMEM178, and/or TPTE, as compared to the
expression levels of these genes in cells from the 2D6 cell
line.
[0042] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes selected
from ADAMTS9, ATAD2, C11ORF82, CDC45L, CNTN1, CNTN4, Cyclin A,
Cyclin E, E2F1, E2F2, E2F7, ELOVL7, EME1, FGFR, FGFR2, KIAA1524,
MELK, MYBL1, MYBL2, NDC80, NDN, ORC1L, PLS3, PRIMA1, RAD54L, RBL1,
RBPMS, RRM2, S1PR3, SCLY, SLCIA3, SPC24, SPC25, ST8SIA4, TFDP1,
TFP12, TK1, TMEM35, TTK, TWIST1, TYMS, TYK, and/or ZWINT, as
compared to the expression levels of these genes in cells from the
2D6 cell line.
[0043] In yet another embodiment, cells from an established clonal
cell line susceptible to BoNT/A intoxication exhibit at least a
1.5-fold increase in gene expression levels of one or more genes
selected from AURKB, BIRC5, BRCA1, BRCA2, BRIP1, BUB1B, CD9,
DLGAP3, DYNLT3, ENC1, FBLN1, FOXM1, GB.gamma., GNAI1, GNG11, GNG12,
GPSM2, GUCY1B3, HGF, ITGA6, JNK, KCNJ5, KIF18A, KITLG, MMD, MSN,
MYRIP, NEK2, NR3C1, NXPH1, OSBPL3, PKMYT1, PTPRM, RAD51, RAD51AP1,
SLC7A2, SLC43A3, SMC6, SNAI2, SNCAIP, SSH2, STK17A, SYNPO2, TOP2A,
TPTE, TRAF4, TSPAN, TSPAN4, UBE3, UBE3B, and/or VAV3, as compared
to the expression levels of these genes in cells from the 2D6 cell
line. In still another embodiment, cells from an established clonal
cell line susceptible to BoNT/A intoxication exhibit at least a
1.5-fold increase in gene expression levels of one or more genes
selected from ALCAM, AURKA, CHEK, CIT, CSRP2, E2F, ECT2, EFNB2,
ERK, ESPL1, GNAI, GPR161, HMMR, KIF4A, KIF14, KIF15, KIF22, KIF23,
KIFC1, LPAR1, MK167, OIP5, PHLPP, PP1/PP2A, PPP1R3C, PRC1, PTTG1,
RACGAP, RB, RGS5, SDC2, and/or TPX2, as compared to the expression
levels of these genes in cells from the 2D6 cell line.
[0044] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes selected
from ASCL1, HES6, MAPK, NMU, PEG3, PTPRK, PRLHR, PTPRK, SGOL2,
SPARC, and/or ZNF217, as compared to the expression levels of these
genes in cells from the 2D6 cell line. In yet another embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit at least a 1.5-fold increase in gene
expression levels of one or more genes selected from ASF1B, BASP1,
CHAF1A, NCAPH, PBK, PRAME, SMC2, UHRF1, and/or VRK1, as compared to
the expression levels of these genes in cells from the 2D6 cell
line. In still another embodiment, cells from an established clonal
cell line susceptible to BoNT/A intoxication exhibit at least a
1.5-fold increase in gene expression levels of one or more genes
selected from C14ORF106, CEP72, KIF20A, PCNA, PEX13, PFC5, POLQ,
SPAG5, SYTL4, TROAP, and/or WDR51A, as compared to the expression
levels of these genes in cells from the 2D6 cell line. In a further
embodiment, cells from an established clonal cell line susceptible
to BoNT/A intoxication exhibit at least a 1.5-fold increase in gene
expression levels of one or more genes selected from ANLN,
ARHGAP24, ASPM, BUB1, CCDC99, CEP55, CKAP2, DRAM, E2F8, PLXNA2,
SLC16A10, UBE2C, UBE2S, and/or WDHD1, as compared to the expression
levels of these genes in cells from the 2D6 cell line.
[0045] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes selected
from ARHGEF3, CTSL2, DIAPH3, FBP1, KIF2C, KIF11, PFKFB3, and/or
PLK4, as compared to the expression levels of these genes in cells
from the 2D6 cell line. In yet another embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold increase in gene expression levels of
one or more genes selected from ARHGEF3, CTSL2, DIAPH3, FBP1,
KIF2C, KIF11, PFKFB3, and/or PLK4, as compared to the expression
levels of these genes in cells from the 2D6 cell line. In still
another embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes selected
from FNDC5, HSPC159, MAB21L2, SLITRK5, SYN2, and/or ZNF367, as
compared to the expression levels of these genes in cells from the
2D6 cell line. In a further another embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold increase in gene expression levels of
one or more genes selected from EXO1, KCTD12, MYO6, PHEBL1, SHCBP1,
TPBG, and/or TUBB6, as compared to the expression levels of these
genes in cells from the 2D6 cell line. In yet another embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit at least a 1.5-fold increase in gene
expression levels of one or more genes selected from BTG3, GABRA5,
TR1P10, and/or ZNF521, as compared to the expression levels of
these genes in cells from the 2D6 cell line. In still another
embodiment, cells from an established clonal cell line susceptible
to BoNT/A intoxication exhibit at least a 1.5-fold increase in gene
expression levels of one or more genes selected from BNC2, DPYD,
EMILIN2, PPIL5, and/or TACC3, as compared to the expression levels
of these genes in cells from the 2D6 cell line.
[0046] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
difference in gene expression levels of one or more genes listed in
Tables 14 or 16 as compared to the expression levels of these genes
in cells from the 2D6 cell line. In an aspect of this embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit at least a 1.5-fold increase in gene
expression levels of one or more genes selected from ACOT9,
ADAMTS9, ARHGAP24, ARHGEF3, ASCL1, BARD1, BASP1, C3ORF70, C11ORF75,
CCDC109B, CD9, CDCA7L, CDK2, CENPL, CLSTN2, CNTN1, CSRP2, CTSL2,
CUGBP2, DEPDC1, DIAPH3, DOK5, DPYD, DYNLT3, EMILIN2, ETV1, FAM101B,
FBLN1, FGFR2, FNDC5, GNAI1, GNB4, GNG11, GNG12, GPR177, GTSE1, HGF,
KITLG, LPAR1, MAB21L2, MAOA, MCM10, MINA, MSN, MYO6, MYRIP, PAG1,
PEG3, PLK2, POLA2, PPP1R3C, PRLHR, PRSS12, PTGR1, PTPRK, PVRL3,
RAB32, RBPMS, SDC2, SGOL2, SLC43A3, SLC7A2, SMC2, SMC6, SPARC,
SPC25, ST8SIA4, TCF7L1, TFPI2, TMEM35, TMEM178, TNFAIP8, TPTE,
TRIP10, TWIST1, and/or ZNF521, as compared to the expression levels
of these genes in cells from the 2D6 cell line. In another aspect
of this embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes selected
from ADAMTS9, ARHGAP24, ASCL1, BARD1, BASP1, BVES, C11ORF75,
CDCA7L, CNTN1, CUGBP2, DOK5, DPYD, DYNLT3, FBLN1, FGFR2, GNAI1,
GNB4, GNG11, GNG12, GTSE1, GTSF1, ITPRIP, KDELC2, LOC728052, LPAR1,
MAB21L2, MAOA, MINA, MSN, PEG3, PLK2, PRLHR, PRSS12, PTPRK, RBPMS,
RNF182, SGOL2, SLC43A3, SLC44A5, SLC7A2, SMC6, SPARC, TFPI2,
TMEM178, and/or TPTE, as compared to the expression levels of these
genes in cells from the 2D6 cell line. In yet another aspect of
this embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes selected
from ADAMTS9, ASCL1, BASP1, DOK5, DPYD, GNB4, GNG11, GTSF1, MAOA,
MINA, MSN, PEG3, PLK2, PRSS12, RNF182, SLC44A5, SPARC, TFPI2,
and/or TPTE, as compared to the expression levels of these genes in
cells from the 2D6 cell line.
[0047] In yet another embodiment, cells from an established clonal
cell line susceptible to BoNT/A intoxication exhibit at least a
1.5-fold increase in gene expression levels of one or more genes
selected from ADAMTS9, ARHGAP24, ARHGEF3, ASCL1, BARD1, CD9, CDK2,
CSRP2, CTSL2, DIAPH3, DOK5, DYNLT3, EMILIN2, ETV1, FBLN1, FGFR2,
GNAI1, GNB4, GNG11, GNG12, HGF, KITLG, LPAR1, MCM10, MSN, PAG1,
PEG3, PLK2, POLA2, PPP1R3C, PTPRK, RAB32, SDC2, SLC43A3, SLC7A2,
SMC6, SPARC, SPC25, ST8SIA4, TCF7L1, TFPI2, TMEM35, TNFAIP8, TPTE,
TRIP10, and/or TWIST1, as compared to the expression levels of
these genes in cells from the 2D6 cell line. In an aspect of this
embodiment, cells from an established clonal cell line susceptible
to BoNT/A intoxication exhibit at least a 1.5-fold increase in gene
expression levels of one or more genes selected from ADAMTS9,
ARHGAP24, ASCL1, BARD1, DOK5, DYNLT3, FBLN1, FGFR2, GNAI1, GNB4,
GNG11, GNG12, LPAR1, MSN, PEG3, PLK2, PTPRK, SLC43A3, SLC7A2, SMC6,
SPARC, TFPI2, and/or TPTE, as compared to the expression levels of
these genes in cells from the 2D6 cell line. In another aspect of
this embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes selected
from ADAMTS9, ASCL1, DOK5, GNB4, GNG11, MSN, PEG3, PLK2, SPARC,
TFPI2, and/or TPTE, as compared to the expression levels of these
genes in cells from the 2D6 cell line.
[0048] In still another embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold increase in gene expression levels of one or more
genes selected from ACOT9, BASP1, C11ORF75, CCDC109B, CDCA7L,
CLSTN2, CNTN1, CUGBP2, DEPDC1, DPYD, FAM101B, FNDC5, GTSE1, MAOA,
MINA, MYO6, MYRIP, PLK2, PRLHR, PVRL3, RBPMS, SGOL2, SMC2, TFP12,
TMEM178, and/or ZNF521, as compared to the expression levels of
these genes in cells from the 2D6 cell line. In an aspect of this
embodiment, cells from an established clonal cell line susceptible
to BoNT/A intoxication exhibit at least a 1.5-fold increase in gene
expression levels of one or more genes selected from BASP1,
C11ORF75, CDCA7L, CNTN1, CUGBP2, DPYD, GTSE1, MAOA, MINA, PLK2,
PRLHR, RBPMS, SGOL2, TFPI2, and/or TMEM178, as compared to the
expression levels of these genes in cells from the 2D6 cell line.
In another aspect of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold increase in gene expression levels of one or more
genes selected from BASP1, DPYD, MAOA, MINA, PLK2, and/or TFPI2, as
compared to the expression levels of these genes in cells from the
2D6 cell line.
[0049] In a further embodiment, cells from an established clonal
cell line susceptible to BoNT/A intoxication exhibit at least a
1.5-fold increase in gene expression levels of one or more genes
selected from C3ORF70, MAB21L2, PRSS12, CENPL, GPR177, and/or
PTGR1, as compared to the expression levels of these genes in cells
from the 2D6 cell line. In an aspect of this embodiment, cells from
an established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold increase in gene expression levels of
one or more genes selected from MAB21L2 and/or PRSS12, as compared
to the expression levels of these genes in cells from the 2D6 cell
line.
[0050] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
decrease in gene expression levels of one or more genes listed in
Tables 6 or 8 as compared to the expression levels of these genes
in cells from a 2D6 cell line. In other aspects of this embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit a decrease in gene expression levels of one or
more genes listed in Tables 6 or 8 of, e.g., at least a 1.5-fold,
at least a 2.0-fold, at least a 2.5-fold, at least a 3.0-fold, at
least a 3.5-fold, at least a 4.0-fold, at least a 4.5-fold, at
least a 5.0-fold, at least a 5.5-fold, at least a 6.0-fold, at
least a 7.0-fold, or at least a 8.0-fold as compared to the
expression levels of these genes in cells from a 2D6 cell line. In
other aspects of this embodiment, cells from an established clonal
cell line susceptible to BoNT/A intoxication exhibit a decrease in
gene expression levels of, e.g., 2 or more genes, 3 or more genes,
4 or more genes, 5 or more genes, 6 or more genes, 7 or more genes,
8 or more genes, 9 or more genes, 10 or more genes, 20 or more
genes, 30 or more genes, 40 or more genes, 50 or more genes, 60 or
more genes, 70 or more genes, 80 or more genes, 90 or more genes,
or 100 or more genes listed in Tables 6 or 8 as compared to the
expression levels of these genes in cells from the 2D6 cell line.
In yet other aspects of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit an
decrease in gene expression levels of, e.g., about 5 genes to about
100 genes, about 10 genes to about 100 genes, about 15 genes to
about 100 genes, about 20 genes to about 100 genes, about 25 genes
to about 100 genes, about 5 genes to about 75 genes, about 10 genes
to about 75 genes, about 15 genes to about 75 genes, about 20 genes
to about 75 genes, about 25 genes to about 75 genes, about 5 genes
to about 50 genes, about 10 genes to about 50 genes, about 15 genes
to about 50 genes, about 20 genes to about 50 genes, or about 25
genes to about 50 genes listed in Tables 6 or 8 as compared to the
expression levels of these genes in cells from the 2D6 cell
line.
[0051] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
decrease in gene expression levels of one or more genes selected
from AATK, ABCA2, ABCC5, ABCC8, ABR, ABTB1, ACOT1, ACRBP, ACRV1,
ACYP2, ADARB1, ADCY1, ADM, AFF1, AGAP4, AGPAT3, AGXT2L2, AHI1,
AKAP9, AKIRIN1, AKT2, AMH, ANK2, ANK3, ANKHD1, ANKRD17, ANKRD50,
ANKS1A, AQP1, ARF1, ARHGAP1, ARHGAP23, ARL17A, ASAM, ASPHD1,
ATP2B3, ATP2B3, ATP6V0A1, ATP8B2, ATP9A, BACE1, BACH2, BLZF1, BPTF,
BRUNOL4, BRUNOL5, BRWD1, BRWD2, BTBD18, BTN3A3, BZRAP1, C14ORF159,
C15ORF24, C15ORF57, C16ORF52, C17ORF28, C1ORF21, C1ORF21, C1ORF50,
C21ORF57, C21ORF59, C21ORF66, C2CD2, C2CD4A, C2ORF60, C2ORF68,
C3ORF23, C4ORF41, C5ORF42, C6ORF154, C6ORF52, C7ORF28B, C7ORF54,
C9ORF150, C9ORF68, CACNA1D, CALY, CAMK2B, CAMK2N2, CAP2, CAPN2,
CARTPT, CBS, CCDC104, CCDC50, CCDC76, CD151, CD163L1, CD302,
CDC42EP1, CDH10, CDH12, CDKN1C, CDKN2A, CDS1, CFC1, CHD5, CLCN3,
CLDN12, CLIP4, CLMN, CNGA3, CNNM1, CNOT6L, COL6A1, COPA, CPNE8,
CRKRS, CRTC1, CRYGS, CSTB, CTSK, CUL4A, CYGB, CYLD, CYP2E1, CYP3A5,
CYTL1, D4S234E, DCLK1, DCTD, DEPDC6, DHX36, DIP2A, DIS3,
DKFZP547G183, DNAJC22, DTNA, DTWD1, DUSP16, DUSP5, DVL3, EFNB3,
EIF3C, ELFN2, ENOSF1, ERAP1, ERBB2, ERBB4, ERMAP, ETFDH, EXOSC6,
FADS3, FAM120AOS, FAM150B, FAM165B, FAM184B, FAM30A, FAM46A,
FAM66C, FAT4, FBRSL1, FBXW7, FGD5, FGF13, FLJ33630, FNBP1, FOXO6,
FUBP3, GAL, GAL, GLT25D2, GNAS, GOLGA8A, GPR123, GPX7, GRIA2,
GRIP2, GRM5, GUCY1A3, H2AFY, HCG 1776018, HCG 2022304, HCN4,
HEATR1, HELQ, HERC4, HERPUD2, HEXDC, HGSNAT, HIST1H2AC, HIST1H2AE,
HIST1H2BD, HIST1H2BK, HIST1H3I, HIST2H2BE, HIVEP3, HNRNPM, HNRNPR,
HPCAL1, HPCAL4, HSPD1, IER3, IL10RB, IL17B, INTU, JMY, KAT2B,
KATNB1, KBTBD11, KCNMA1, KCNQ2, KCNS2, KCTD13, KHDC1, KIAA0125,
KIAA1370, KIAA1598, KIF1A, KIF5C, KIF5C, KISS1R, KLHDC1, LCORL,
LGALS3BP, LOC100130097, LOC100130360, LOC100130522, LOC100272228,
LOC284408, LOC399491, LOC401320, LOC641298, LOC642852, LOC90110,
LOC94431, LONRF2, LRP2BP, LRRC37A2, LRRFIP1, LTBP3, LYPLAL1, LYRM5,
MAP3K5, MAP7, MAP9, MCM3AP, MCM3APAS, MCTP1, MEOX2, METTL3, MFSD4,
MGAT4A, MLXIP, MRPL1, MRPS33, MST1, MUC20, MXD1, MZF1, NAMPT,
NAP1L3, NBPF1, NBPF10, NCOA7, NCRNA00171, NCRNA00182, NDRG1,
NDUFA4L2, NDUFV3, NEBL, NELF, NHEDC2, NHEG1, NIPAL2, NIPAL3, NLRX1,
NOL3, NSMAF, NUDT19, NUP153, NUP54, OLFM3, OR7D2, OSBP, PABPC1L,
PABPC4, PACRGL, PAM, PAPPA, PAQR6, PARP12, PCBD2, PCNXL2, PDCD6,
PDE4C, PDE9A, PDGFRB, PDIA2, PGAP1, PHF17, PHKA1, PHKA2, PHLDA2,
PIGH, PION, PKD1, PLA2G4C, PLCB4, PLEKHH1, PLP2, PLRG1, PLXNC1,
PNCK, PNMA3, POFUT2, POGK, PPAPDC1A, PPAPDC1B, PPID, PPIE, PPM1K,
PPP1R2, PPP2R2C, PPT1, PRKACB, PRPH, PSMB7, PTCD1, PTGER2, PTGS1,
PTN, PTPRD, PTPRN, RAB11FIP3, RAB6B, RAF1, RGAG4, RGS11, RGS8,
RHBDL1, RHOQ, RHOU, RLF, RNASET2, RNF13, RNF149, RNF165, RNF207,
RNF41, RPL37, RSL1D1, RUNDC3A, S100A6, SCMH1, SCN2A, SERP1, SETX,
SFRS18, SFXN3, SGK3, SH3BP5, SH3GL2, SH3YL1, SHC2, SHC4, SIGIRR,
SIK1, SLC12A7, SLC1A2, SLC1A6, SLC22A17, SLC35F3, SLC38A5, SLCO1A2,
SMAGP, SMAP2, SNCA, SOBP, SORCS1, SORL1, SPINT2, SPIRE2, SPOCK2,
SRR, ST8SIA3, STAR, STEAP3, STOX2, STX3, SYNJ1, SYT13, SYT5, TAF10,
TANC2, TCEA1, THBS2, THSD4, TIMP1, TM2D1, TMEM111, TMEM151A,
TMEM184C, TMEM41B, TMEM43, TMEM5, TMEM59L, TMIE, TNFRSF25,
TNFRSF25, TOX4, TP53BP1, TPM3, TRA2A, TRIM33, TRIM73, TRIMS, TRIT1,
TSR1, TTC17, TTC3, TTC39C, TUSC3, TXLNB, U2AF1, UBE2D3, UBE2G2,
UBN2, UBXN6, UCN, UGP2, UNC80, UNQ1887, USP36, USPL1, VN1R1,
VPS13C, VPS53, VPS8, WDFY3, WDR27, WDR85, WSB1, YJEFN3, YLPM1,
ZDHHC11, ZER1, ZG16B, ZNF275, ZNF440, ZNF573, ZNF641, ZNF662,
ZNF785, and/or ZNF814, as compared to the expression levels of
these genes in cells from the 2D6 cell line.
[0052] In an aspect of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold decrease in gene expression levels of one or more
genes selected from ACOT1, ADARB1, ADM, ANK2, AQP1, ASAM, C2CD4A,
C3ORF23, CACNA1D, CAMK2B, CAP2, CARTPT, CD163L1, CDC42EP1, CDH10,
CDKN1C, CDKN2A, CFC1, CLMN, CPNE8, CYGB, CYP2E1, CYTL1, DNAJC22,
DUSP5, ENOSF1, ERAP1, FGF13, FOXO6, GAL, GNAS, GPX7, GRIA2, GRM5,
HCG 1776018, HIST1H2AC, HIST1H2BD, HIST1H2BK, HIST2H2BE, IL17B,
KIAA0125, KIAA1598, KISS1R, LRP2BP, MEOX2, NCRNA00182, NDRG1,
NDUFA4L2, NIPAL2, NUDT19, PDE4C, PHKA2, PHLDA2, PTGER2, RGS11,
S100A6, SCN2A, SHC4, SIGIRR, SLC1A2, SLC1A6, SLC35F3, SLC38A5,
SORCS1, SYT13, SYT5, THBS2, TIMP1, and/or TMEM111, as compared to
the expression levels of these genes in cells from the 2D6 cell
line.
[0053] In another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold decrease in gene expression levels of
one or more genes selected from ACOT1, ADARB1, ANK2, AQP1, C3ORF23,
CAP2, CARTPT, CD163L1, CDC42EP1, CDKN2A, CFC1, CPNE8, CYGB, CYP2E1,
CYTL1, FGF13, FOXO6, GNAS, GRIA2, GRM5, HIST1H2AC, IL17B, KIAA0125,
KIAA1598, KISS1R, MEOX2, NDRG1, NDUFA4L2, NIPAL2, PDE4C, PHKA2,
RGS11, SCN2A, SLC1A2, SLC35F3, SLC38A5, SYT13, and/or THBS2, as
compared to the expression levels of these genes in cells from the
2D6 cell line.
[0054] In yet another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold decrease in gene expression levels of
one or more genes selected from AQP1, CAP2, CARTPT, CD163L1,
CDC42EP1, CFC1, CPNE8, CYGB, CYP2E1, CYTL1, FGF13, GNAS, GRIA2,
GRM5, HIST1H2AC, IL17B, KIAA0125, MEOX2, NDUFA4L2, PDE4C, PHKA2,
RGS11, SLC1A2, SLC35F3, SLC38A5, and/or SYT13, as compared to the
expression levels of these genes in cells from the 2D6 cell
line.
[0055] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
decrease in gene expression levels of one or more genes selected
from ANXA2, AQP1, ARHGAP9, CDH10, CDKN2A, CHPT1, CNTN2, ERAP1,
and/or RGS11, as compared to the expression levels of these genes
in cells from the 2D6 cell line. In yet another embodiment, cells
from an established clonal cell line susceptible to BoNT/A
intoxication exhibit at least a 1.5-fold decrease in gene
expression levels of one or more genes selected from ABCC8, AELIM3,
CAP2, IL17B, MEF2A, NEEBL, PHC, S100A6, SLC1A6, SMAD1, SMAD5,
SMAD8, SYT13, and/or SYTL1, as compared to the expression levels of
these genes in cells from the 2D6 cell line. In still another
embodiment, cells from an established clonal cell line susceptible
to BoNT/A intoxication exhibit at least a 1.5-fold decrease in gene
expression levels of one or more genes selected from CSTB, GPCR,
GRIM5, KISSR, SCN2A, SLC1A2, and/or THBS2, as compared to the
expression levels of these genes in cells from the 2D6 cell
line.
[0056] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
decrease in gene expression levels of one or more genes selected
from ADARB1, ADM, PTPPH, and/or SLCO1A2, as compared to the
expression levels of these genes in cells from the 2D6 cell line.
In yet another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
decrease in gene expression levels of one or more genes selected
from CNGA3, HIST1H3E, and/or PTGS1, as compared to the expression
levels of these genes in cells from the 2D6 cell line. In still
another embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
decrease in gene expression levels of one or more genes selected
from HIST1H2BD and/or OSCAR as compared to the expression levels of
these genes in cells from the 2D6 cell line. In a further
embodiment, cells from an established clonal cell line susceptible
to BoNT/A intoxication exhibit at least a 1.5-fold decrease in gene
expression levels of one or more genes selected from CALY as
compared to the expression levels of these genes in cells from the
2D6 cell line.
[0057] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
decrease in gene expression levels of one or more genes selected
from COL5A1 and/or MICAL2 as compared to the expression levels of
these genes in cells from the 2D6 cell line. In yet another
embodiment, cells from an established clonal cell line susceptible
to BoNT/A intoxication exhibit at least a 1.5-fold decrease in gene
expression levels of one or more genes selected from OLFML2A and/or
SIGIRR as compared to the expression levels of these genes in cells
from the 2D6 cell line. In still another embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold decrease in gene expression levels of
one or more genes selected from HPCAL1 and/or LPAR5 as compared to
the expression levels of these genes in cells from the 2D6 cell
line. In a further embodiment, cells from an established clonal
cell line susceptible to BoNT/A intoxication exhibit at least a
1.5-fold decrease in gene expression levels of one or more genes
selected from HTR1E and/or SORCS1 as compared to the expression
levels of these genes in cells from the 2D6 cell line.
[0058] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
decrease in gene expression levels of one or more genes selected
from ANK2, CPNE8, CREB5, IL17B, KIAA0125, LOC100289109, LOC144571,
NPAS4, SLC1A2, SORCS1, THBS2, and/or ZNF814, as compared to the
expression levels of these genes in cells from the 2D6 cell
line.
[0059] Aspects of the present disclosure comprise, in part, cells
from an established clonal cell line susceptible to BoNT/A
intoxication that exhibit at least a 1.5-fold difference in gene
expression levels of one or more genes listed in Tables 9, 10, 11,
or 12 as compared to the expression levels of these genes in cells
from a parental SiMa cell line. In aspects of this embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit at least a 1.5-fold difference in gene
expression levels of one or more genes listed in Tables 9, 10, 11,
or 12 as compared to the expression levels of these genes in cells
from the parental SiMa cell line DSMZ ACC 164. In other aspects of
this embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit a difference in gene
expression levels of one or more genes listed in Tables 9, 10, 11,
or 12 of, e.g., at least a 1.5-fold, at least a 2.0-fold, at least
a 2.5-fold, at least a 3.0-fold, at least a 3.5-fold, at least a
4.0-fold, at least a 4.5-fold, at least a 5.0-fold, at least a
5.5-fold, at least a 6.0-fold, at least a 7.0-fold, or at least a
8.0-fold as compared to the expression levels of these genes in
cells from the parental SiMa cell line DSMZ ACC 164. In yet other
aspects of this embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit a difference in
gene expression levels of, e.g., 2 or more genes, 3 or more genes,
4 or more genes, 5 or more genes, 6 or more genes, 7 or more genes,
8 or more genes, 9 or more genes, 10 or more genes, 20 or more
genes, 30 or more genes, 40 or more genes, 50 or more genes, 60 or
more genes, 70 or more genes, 80 or more genes, 90 or more genes,
or 100 or more genes listed in Tables 9, 10, 11, or 12 as compared
to the expression levels of these genes in cells from the parental
SiMa cell line DSMZ ACC 164. In still other aspects of this
embodiment, cells from an established clonal cell line susceptible
to BoNT/A intoxication exhibit a difference in gene expression
levels of, e.g., about 5 genes to about 100 genes, about 10 genes
to about 100 genes, about 15 genes to about 100 genes, about 20
genes to about 100 genes, about 25 genes to about 100 genes, about
5 genes to about 75 genes, about 10 genes to about 75 genes, about
15 genes to about 75 genes, about 20 genes to about 75 genes, about
25 genes to about 75 genes, about 5 genes to about 50 genes, about
10 genes to about 50 genes, about 15 genes to about 50 genes, about
20 genes to about 50 genes, or about 25 genes to about 50 genes
listed in Tables 9, 10, 11, or 12 as compared to the expression
levels of these genes in cells from the parental SiMa cell line
DSMZ ACC 164. The log ratio in Tables 9, 10, 11, or 12 represent
log.sub.2 values where 0.585 is log.sub.2(1.5) which is a 1.5-fold
difference, 1 is log.sub.2(2) which is a 2-fold difference, 1.584
is log.sub.2(3) which is a 3-fold difference, 2 is log.sub.2(4)
which is a 4-fold difference, 2.321 is log.sub.2(5) which is a
5-fold difference, 2.584 is log.sub.2(6) which is a 6-fold
difference, 2.807 is log.sub.2(7) which is a 7-fold difference, 3
is log.sub.2(8) which is a 8-fold difference, 3.169 is log.sub.2(9)
which is a 9-fold difference, and 3.321 is log.sub.2(10) which is a
10-fold difference.
[0060] In an embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
difference in gene expression levels of one or more genes selected
from ACOT9, ADAMTS9, ARHGAP24, ARHGEF3, ASCL1, BARD1, BASP1,
C3ORF70, C11ORF75, CCDC109B, CD9, CDCA7L, CDK2, CENPL, CLSTN2,
CNTN1, CSRP2, CTSL2, CUGBP2, DEPDC1, DIAPH3, DOK5, DPYD, DYNLT3,
EMILIN2, ETV1, FAM101B, FBLN1, FGFR2, FNDC5, GNAI1, GNB4, GNG11,
GNG12, GPR177, GTSE1, HGF, KITLG, LPAR1, MAB21L2, MAOA, MCM10,
MINA, MSN, MYO6, MYRIP, PAG1, PEG3, PLK2, POLA2, PPP1R3C, PRLHR,
PRSS12, PTGR1, PTPRK, PVRL3, RAB32, RBPMS, SDC2, SGOL2, SLC43A3,
SLC7A2, SMC2, SMC6, SPARC, SPC25, ST8SIA4, TCF7L1, TFPI2, TMEM35,
TMEM178, TNFAIP8, TPTE, TRIP10, TWIST1, and/or ZNF521, as compared
to the expression levels of these genes in cells from the parental
SiMa cell line DSMZ ACC 164. In another aspect of this embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit at least a 1.5-fold difference in gene
expression levels of one or more genes selected from ADAMTS9,
ARHGAP24, ASCL1, BARD1, BASP1, BVES, C11ORF75, CDCA7L, CNTN1,
CUGBP2, DOK5, DPYD, DYNLT3, FBLN1, FGFR2, GNAI1, GNB4, GNG11,
GNG12, GTSE1, GTSF1, ITPRIP, KDELC2, LOC728052, LPAR1, MAB21L2,
MAOA, MINA, MSN, PEG3, PLK2, PRLHR, PRSS12, PTPRK, RBPMS, RNF182,
SGOL2, SLC43A3, SLC44A5, SLC7A2, SMC6, SPARC, TFPI2, TMEM178,
and/or TPTE, as compared to the expression levels of these genes in
cells from the parental SiMa cell line DSMZ ACC 164. In yet another
aspect of this embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
difference in gene expression levels of one or more genes selected
from ADAMTS9, ASCL1, BASP1, DOK5, DPYD, GNB4, GNG11, GTSF1, MAOA,
MINA, MSN, PEG3, PLK2, PRSS12, RNF182, SLC44A5, SPARC, TFPI2,
and/or TPTE, as compared to the expression levels of these genes in
cells from the parental SiMa cell line DSMZ ACC 164.
[0061] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes listed in
Tables 9 or 11 as compared to the expression levels of these genes
in cells from a parental SiMa cell line DSMZ ACC 164. In other
aspects of this embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit an increase in gene
expression levels of one or more genes listed in Tables 9 or 11 of,
e.g., at least a 1.5-fold, at least a 2.0-fold, at least a
2.5-fold, at least a 3.0-fold, at least a 3.5-fold, at least a
4.0-fold, at least a 4.5-fold, at least a 5.0-fold, at least a
5.5-fold, at least a 6.0-fold, at least a 7.0-fold, or at least a
8.0-fold as compared to the expression levels of these genes in
cells from a parental SiMa cell line DSMZ ACC 164. In other aspects
of this embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit an increase in gene
expression levels of, e.g., 2 or more genes, 3 or more genes, 4 or
more genes, 5 or more genes, 6 or more genes, 7 or more genes, 8 or
more genes, 9 or more genes, 10 or more genes, 20 or more genes, 30
or more genes, 40 or more genes, 50 or more genes, 60 or more
genes, 70 or more genes, 80 or more genes, 90 or more genes, or 100
or more genes listed in Tables 9 or 11 as compared to the
expression levels of these genes in cells from the parental SiMa
cell line DSMZ ACC 164. In yet other aspects of this embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit an increase in gene expression levels of,
e.g., about 5 genes to about 100 genes, about 10 genes to about 100
genes, about 15 genes to about 100 genes, about 20 genes to about
100 genes, about 25 genes to about 100 genes, about 5 genes to
about 75 genes, about 10 genes to about 75 genes, about 15 genes to
about 75 genes, about 20 genes to about 75 genes, about 25 genes to
about 75 genes, about 5 genes to about 50 genes, about 10 genes to
about 50 genes, about 15 genes to about 50 genes, about 20 genes to
about 50 genes, or about 25 genes to about 50 genes listed in
Tables 9 or 11 as compared to the expression levels of these genes
in cells from the parental SiMa cell line DSMZ ACC 164.
[0062] In an embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes selected
from A2BP1, ADRM1, AFF4, AGAP4, ALCAM, ANAPC7, ANKRD13D, ANKRD28,
API5, ARF1, ARHGAP6, ARHGEF10, ARHGEF6, ARL17A, ASCL1, ASXL1,
ASXL3, ATF7IP, B3GALNT2, BASP1, BLCAP, BNC2, C10ORF58, C12ORF49,
C12ORF51, C1ORF43, C20ORF117, C20ORF7, CADM1, CADPS, CALCB, CAPN1,
CAPRIN2, CBLB, CBLN2, CBWD1, CCDC150, CCDC3, CD9, CDC25B, CFDP1,
CHRNA7, CIAPIN1, CLASP2, CNOT7, CPVL, CRYBG3, CSE1L, CTTNBP2NL,
CUGBP2, CXCR4, DACT1, DAZ1, DBH, DCAF8, DENR, DHX35, DKFZP434L187,
DLEU2, DLL1, DLL3, DYNC2H1, EAF2, EFNA5, EIF3B, EIF3C, ELOVL7,
EML1, EML4, ENC1, EVL, EXOSC6, EXPH5, EZH2, FAM178A, FAM181B,
FAM19A4, FAM7A3, FLJ10213, FUBP3, FUS, FZD5, GAP43, GFM1, GFRA2,
GGA2, GNAI1, GNAS, GNB4, GNG11, GOLGA4, GPR125, GRM8, GRP, GSPT1,
GSS, GSTCD, GULP1, HAUS2, HEG1, HNRNPL, HNRNPM, HOXA7, HOXD4, ID2,
IDH3B, IL1A, INSM1, IREB2, ITGA6, ITGB5, KCTD12, KDELC2, KHDRBS3,
KIAA0907, KIAA1267, KIF16B, KLC1, KLHL13, LBH, LMO4, LOC100128844,
LOC340109, LOC641298, LOC647190, LOC728052, LOC728153, LRPPRC,
LRRFIP2, LSM3, LUM, MAB21L1, MAB21L2, MAN2A1, MAOA, MARS, MDN1,
MED13L, MED22, MGC24103, MINA, MKLN1, MLEC, MMD, MORF4L2, MPZL1,
MSH6, MSI2, MSN, MTMR9, MYO1B, MYO6, N4BP2, NAAA, NDUFS8, NEDD9,
NFIB, NKTR, NLN, NOS1, NR2C1, NUFIP2, NUPL1, OSBPL3, PAPD4, PCBP2,
PCDH9, PCGF5, PCNX, PDLIM5, PDZRN3, PELI2, PFKFB3, PGP, PHF20,
PLK2, PLXNA2, PLXNA4, PM20D2, POLE, POLQ, PPP2R2A, PPP2R3C, PRDX2,
PSMA7, PSME4, PTGFRN, PTK2, PTPN1, PTPRE, PTPRG, QKI, RAB12, RAB35,
RAB3GAP2, RAD23B, RAF1, RALGAPA1, RAN, RASEF, RBL1, RDH11, RELL1,
REPS1, RGS5, RIMBP2, RNF182, RNF34, RNPEP, ROBO2, RPAIN, RPL35A,
RPRD1A, S1PR3, SALL4, SBNO1, SCARB2, SDHA, SEMA6A, SEZ6L, SF1,
SFRS8, SIM1, SIRT2, SKIL, SLC20A1, SLC44A5, SLC7A2, SLCO3A1,
SLITRK5, SMAD4, SMARCC2, SMC3, SNRPB2, SNRPN, SNX5, SOX2, SPAG6,
SPAG9, SPATS2L, SPON1, SR140, SSBP2, ST8SIA1, STMN3, STRA6,
SYNCRIP, SYTL3, TAF15, TCF7L2, TDG, TERT, TFPI2, TGFBR1, TH1L,
THOC4, TIMP3, TLE3, TMEM132C, TMEM178, TMEM181, TNRC6A, TPBG,
TRA2A, TRIM29, TRIM36, TSHZ3, TXNRD1, U2AF1, UBE2O, UBE2V1, UBE2Z,
UBXN2A, UCHL1, USP25, USP32, USP34, VPS29, XPO1, XPO7, ZFAND6,
ZFHX4, ZFYVE16, ZGPAT, ZNF217, ZNF451, ZNF503, and/or ZNF664, as
compared to the expression levels of these genes in cells from the
parental SiMa cell line DSMZ ACC 164.
[0063] In an aspect of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold increase in gene expression levels of one or more
genes selected from ALCAM, ARHGAP6, ARHGEF6, ASCL1, BNC2, CBLN2,
CCDC150, CCDC3, CHRNA7, CRYBG3, CUGBP2, CXCR4, DAZ1, DKFZP434L187,
DLL1, EFNA5, ELOVL7, EML1, EML4, ENC1, EXPH5, FAM7A3, FZD5, GFRA2,
GNAI1, GNB4, GNG11, GRP, 102, ITGA6, KCTD12, LOC100128844,
LOC340109, LOC728052, LUM, MAB21L1, MAB21L2, MAOA, MGC24103, MSN,
NEDD9, NFIB, OSBPL3, PAPD4, PCDH9, PDZRN3, PLK2, POLQ, PTGFRN,
PTPRE, PTPRG, RAD23B, RGS5, RNF182, ROBO2, SIM1, SLC20A1, SLC44A5,
SLITRK5, SPAG6, SPAG9, SR140, TFPI2, TIMP3, TMEM132C, TPBG, TRIM29,
TRIM36, and/or ZFAND6, as compared to the expression levels of
these genes in cells from the parental SiMa cell line DSMZ ACC
164.
[0064] In another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold increase in gene expression levels of
one or more genes selected from ALCAM, ARHGAP6, ARHGEF6, CBLN2,
CCDC3, CHRNA7, CRYBG3, CUGBP2, CXCR4, DAZ1, ELOVL7, EML1, EXPH5,
FAM7A3, GNB4, GNG11, GRP, ITGA6, KCTD12, LOC340109, LUM, MAB21L1,
MAB21L2, MGC24103, PCDH9, PLK2, POLQ, PTPRE, RGS5, RNF182, ROBO2,
SIM1, SLC44A5, SLITRK5, SPAG6, TIMP3, and/or TMEM1320, as compared
to the expression levels of these genes in cells from the parental
SiMa cell line DSMZ ACC 164.
[0065] In yet another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold increase in gene expression levels of
one or more genes selected from CBLN2, CCDC3, CHRNA7, CRYBG3,
CXCR4, DAZ1, EXPH5, FAM7A3, GNB4, GRP, KCTD12, LUM, MGC24103,
PCDH9, PLK2, POLQ, PTPRE, RGS5, ROBO2, SIM1, SLITRK5, TIMP3, and/or
TMEM132C, as compared to the expression levels of these genes in
cells from the parental SiMa cell line DSMZ ACC 164.
[0066] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes listed in
Tables 14 or 16 as compared to the expression levels of these genes
in cells from the parental SiMa cell line DSMZ ACC 164. In aspects
of this embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes listed in
Tables 14 or 16 as compared to the expression levels of these genes
in cells from a parental SiMa cell line DSMZ ACC 164. In other
aspects of this embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit an increase in gene
expression levels of one or more genes listed in Tables 14 or 16
of, e.g., at least a 1.5-fold, at least a 2.0-fold, at least a
2.5-fold, at least a 3.0-fold, at least a 3.5-fold, at least a
4.0-fold, at least a 4.5-fold, at least a 5.0-fold, at least a
5.5-fold, at least a 6.0-fold, at least a 7.0-fold, or at least a
8.0-fold as compared to the expression levels of these genes in
cells from a parental SiMa cell line DSMZ ACC 164. In other aspects
of this embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit an increase in gene
expression levels of, e.g., 2 or more genes, 3 or more genes, 4 or
more genes, 5 or more genes, 6 or more genes, 7 or more genes, 8 or
more genes, 9 or more genes, 10 or more genes, 20 or more genes, 30
or more genes, 40 or more genes, 50 or more genes, 60 or more
genes, 70 or more genes, 80 or more genes, 90 or more genes, or 100
or more genes listed in Tables 14 or 16 as compared to the
expression levels of these genes in cells from the parental SiMa
cell line DSMZ ACC 164. In yet other aspects of this embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit an increase in gene expression levels of,
e.g., about 5 genes to about 100 genes, about 10 genes to about 100
genes, about 15 genes to about 100 genes, about 20 genes to about
100 genes, about 25 genes to about 100 genes, about 5 genes to
about 75 genes, about 10 genes to about 75 genes, about 15 genes to
about 75 genes, about 20 genes to about 75 genes, about 25 genes to
about 75 genes, about 5 genes to about 50 genes, about 10 genes to
about 50 genes, about 15 genes to about 50 genes, about 20 genes to
about 50 genes, or about 25 genes to about 50 genes listed in
Tables 14 or 16 as compared to the expression levels of these genes
in cells from the parental SiMa cell line DSMZ ACC 164.
[0067] In aspects of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold increase in gene expression levels of one or more
genes selected from ACOT9, ADAMTS9, ARHGAP24, ARHGEF3, ASCL1,
BARD1, BASP1, C3ORF70, C11ORF75, CCDC109B, CD9, CDCA7L, CDK2,
CENPL, CLSTN2, CNTN1, CSRP2, CTSL2, CUGBP2, DEPDC1, DIAPH3, DOK5,
DPYD, DYNLT3, EMILIN2, ETV1, FAM101B, FBLN1, FGFR2, FNDC5, GNAI1,
GNB4, GNG11, GNG12, GPR177, GTSE1, HGF, KITLG, LPAR1, MAB21L2,
MAOA, MCM10, MINA, MSN, MYO6, MYRIP, PAG1, PEG3, PLK2, POLA2,
PPP1R3C, PRLHR, PRSS12, PTGR1, PTPRK, PVRL3, RAB32, RBPMS, SDC2,
SGOL2, SLC43A3, SLC7A2, SMC2, SMC6, SPARC, SPC25, ST8SIA4, TCF7L1,
TFPI2, TMEM35, TMEM178, TNFAIP8, TPTE, TRIP10, TWIST1, and/or
ZNF521, as compared to the expression levels of these genes in
cells from the parental SiMa cell line DSMZ ACC 164. In another
aspect of this embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes selected
from ADAMTS9, ARHGAP24, ASCL1, BARD1, BASP1, BVES, C11ORF75,
CDCA7L, CNTN1, CUGBP2, DOK5, DPYD, DYNLT3, FBLN1, FGFR2, GNAI1,
GNB4, GNG11, GNG12, GTSE1, GTSF1, ITPRIP, KDELC2, LOC728052, LPAR1,
MAB21L2, MAOA, MINA, MSN, PEG3, PLK2, PRLHR, PRSS12, PTPRK, RBPMS,
RNF182, SGOL2, SLC43A3, SLC44A5, SLC7A2, SMC6, SPARC, TFPI2,
TMEM178, and/or TPTE, as compared to the expression levels of these
genes in cells from the parental SiMa cell line DSMZ ACC 164. In
yet another aspect of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold increase in gene expression levels of one or more
genes selected from ADAMTS9, ASCL1, BASP1, DOK5, DPYD, GNB4, GNG11,
GTSF1, MAOA, MINA, MSN, PEG3, PLK2, PRSS12, RNF182, SLC44A5, SPARC,
TFPI2, and/or TPTE, as compared to the expression levels of these
genes in cells from the parental SiMa cell line DSMZ ACC 164.
[0068] In yet another embodiment, cells from an established clonal
cell line susceptible to BoNT/A intoxication exhibit at least a
1.5-fold increase in gene expression levels of one or more genes
selected from ADAMTS9, ARHGAP24, ARHGEF3, ASCL1, BARD1, CD9, CDK2,
CSRP2, CTSL2, DIAPH3, DOK5, DYNLT3, EMILIN2, ETV1, FBLN1, FGFR2,
GNAI1, GNB4, GNG11, GNG12, HGF, KITLG, LPAR1, MCM10, MSN, PAG1,
PEG3, PLK2, POLA2, PPP1R3C, PTPRK, RAB32, SDC2, SLC43A3, SLC7A2,
SMC6, SPARC, SPC25, ST8SIA4, TCF7L1, TFPI2, TMEM35, TNFAIP8, TPTE,
TRIP10, and/or TWIST1, as compared to the expression levels of
these genes in cells from the parental SiMa cell line DSMZ ACC 164.
In an aspect of this embodiment, cells from an established clonal
cell line susceptible to BoNT/A intoxication exhibit at least a
1.5-fold increase in gene expression levels of one or more genes
selected from ADAMTS9, ARHGAP24, ASCL1, BARD1, DOK5, DYNLT3, FBLN1,
FGFR2, GNAI1, GNB4, GNG11, GNG12, LPAR1, MSN, PEG3, PLK2, PTPRK,
SLC43A3, SLC7A2, SMC6, SPARC, TFPI2, and/or TPTE, as compared to
the expression levels of these genes in cells from the parental
SiMa cell line DSMZ ACC 164. In another aspect of this embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit at least a 1.5-fold increase in gene
expression levels of one or more genes selected from ADAMTS9,
ASCL1, DOK5, GNB4, GNG11, MSN, PEG3, PLK2, SPARC, TFPI2, and/or
TPTE, as compared to the expression levels of these genes in cells
from the parental SiMa cell line DSMZ ACC 164.
[0069] In still another embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold increase in gene expression levels of one or more
genes selected from ACOT9, BASP1, C11ORF75, CCDC109B, CDCA7L,
CLSTN2, CNTN1, CUGBP2, DEPDC1, DPYD, FAM101B, FNDC5, GTSE1, MAOA,
MINA, MYO6, MYRIP, PLK2, PRLHR, PVRL3, RBPMS, SGOL2, SMC2, TFP12,
TMEM178, and/or ZNF521, as compared to the expression level of
these genes in cells from the parental SiMa cell line DSMZ ACC 164.
In an aspect of this embodiment, cells from an established clonal
cell line susceptible to BoNT/A intoxication exhibit at least a
1.5-fold increase in gene expression levels of one or more genes
selected from BASP1, C11ORF75, CDCA7L, CNTN1, CUGBP2, DPYD, GTSE1,
MAOA, MINA, PLK2, PRLHR, RBPMS, SGOL2, TFPI2, and/or TMEM178, as
compared to the expression levels of these genes in cells from the
parental SiMa cell line DSMZ ACC 164. In another aspect of this
embodiment, cells from an established clonal cell line susceptible
to BoNT/A intoxication exhibit at least a 1.5-fold increase in gene
expression levels of one or more genes selected from BASP1, DPYD,
MAOA, MINA, PLK2, and/or TFPI2, as compared to the expression
levels of these genes in cells from the parental SiMa cell line
DSMZ ACC 164.
[0070] In a further embodiment, cells from an established clonal
cell line susceptible to BoNT/A intoxication exhibit at least a
1.5-fold increase in gene expression levels of one or more genes
selected from C3ORF70, MAB21L2, PRSS12, CENPL, GPR177, and/or
PTGR1, as compared to the expression levels of these genes in cells
from the parental SiMa cell line DSMZ ACC 164. In an aspect of this
embodiment, cells from an established clonal cell line susceptible
to BoNT/A intoxication exhibit at least a 1.5-fold increase in gene
expression levels of one or more genes selected from MAB21 L2
and/or PRSS12, as compared to the expression levels of these genes
in cells from the parental SiMa cell line DSMZ ACC 164.
[0071] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
decrease in gene expression levels of one or more genes listed in
Tables 10 or 12 as compared to the expression levels of these genes
in cells from a parental SiMa cell line. In aspects of this
embodiment, cells from an established clonal cell line susceptible
to BoNT/A intoxication exhibit at least a 1.5-fold decrease in gene
expression levels of one or more genes listed in Tables 10 or 12 as
compared to the expression levels of these genes in cells from the
parental SiMa cell line DSMZ ACC 164. In other aspects of this
embodiment, cells from an established clonal cell line susceptible
to BoNT/A intoxication exhibit a decrease in gene expression levels
of one or more genes listed in Tables 10 or 12 of, e.g., at least a
1.5-fold, at least a 2.0-fold, at least a 2.5-fold, at least a
3.0-fold, at least a 3.5-fold, at least a 4.0-fold, at least a
4.5-fold, at least a 5.0-fold, at least a 5.5-fold, at least a
6.0-fold, at least a 7.0-fold, or at least a 8.0-fold as compared
to the expression levels of these genes in cells from the parental
SiMa cell line DSMZ ACC 164. In other aspects cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit a decrease in gene expression levels of, e.g., 2 or more
genes, 3 or more genes, 4 or more genes, 5 or more genes, 6 or more
genes, 7 or more genes, 8 or more genes, 9 or more genes, 10 or
more genes, 20 or more genes, 30 or more genes, 40 or more genes,
50 or more genes, 60 or more genes, 70 or more genes, 80 or more
genes, 90 or more genes, or 100 or more genes listed in Tables 10
or 12 as compared to the expression levels of these genes in cells
from the parental SiMa cell line DSMZ ACC 164. In yet other aspects
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit an decrease in gene expression levels of,
e.g., about 5 genes to about 100 genes, about 10 genes to about 100
genes, about 15 genes to about 100 genes, about 20 genes to about
100 genes, about 25 genes to about 100 genes, about 5 genes to
about 75 genes, about 10 genes to about 75 genes, about 15 genes to
about 75 genes, about 20 genes to about 75 genes, about 25 genes to
about 75 genes, about 5 genes to about 50 genes, about 10 genes to
about 50 genes, about 15 genes to about 50 genes, about 20 genes to
about 50 genes, or about 25 genes to about 50 genes listed in
Tables 10 or 12 as compared to the expression levels of these genes
in cells from the parental SiMa cell line DSMZ ACC 164.
[0072] In an embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
decrease in gene expression levels of one or more genes selected
from 1-Mar, ABCG1, ABTB1, ACOT1, ACSL1, ACVR1, ACVR2A, ACYP2,
ADAMTS1, ADAMTS3, ADAMTSL1, ADARB1, ADCY1, ADD2, ADO, AFF3, AKAP12,
ALDH1A2, ALKBH1, ALLC, AMN1, ANKH, ANKRA2, ANKRD50, ANXA5, AP3B2,
ARMC10, ARMCX5, ASAM, ASB13, ASPHD1, ATG5, ATL3, ATP2B3, ATP2C1,
ATRNL1, B3GALTL, BACE1, BACE2, BBS10, BBS7, BCAP29, BCL2, BCORL1,
BRUNOL4, BRUNOL6, BRWD1, BTN3A3, C10ORF10, C10ORF104, C11ORF57,
C11ORF70, C14ORF167, C15ORF39, C16ORF52, C17ORF69, C18ORF18,
C1GALT1C1, C1ORF21, C1ORF25, C1ORF97, C1RL, C21ORF57, C2CD2,
C2CD4A, C2ORF67, C2ORF68, C3ORF23, C4ORF12, C4ORF39, C4ORF41,
C5ORF42, C6ORF120, C9ORF150, CADM2, CAMK2D, CAMK2N1, CAMK2N2, CAP2,
CAPN2, CARTPT, CCDC126, CCDC40, CCDC50, CONY, CD248, CD302, CDC2L6,
CDC37L1, CDC42EP3, CDH12, CDKN1C, CDKN2A, CDS1, CFC1, CHMP1B,
CLCN3, CLDN12, CLMN, CLN8, CNGA3, CNNM1, CNOT6L, COL27A1, COL5A1,
COL6A1, COL6A2, COL6A3, COX18, COX5B, CRYGD, CRYZL1, CTPS2, CTSC,
CYB5R2, CYGB, CYLD, CYP2E1, CYP2U1, CYTL1, CYYR1, D4S234E, DAB1,
DBT, DCAF10, DCLK1, DCTD, DDAH1, DHRSX, DISP1, DKFZP434I0714, DKK1,
DNAJC12, DNAJC24, DNASE1L1, DNER, DOPEY2, DRAM2, DSCAM, DSCR3,
DTX3L, DUSP16, DUSP22, ECEL1, ECHDC3, EEF1D, EEF2K, EFEMP2, EFNB3,
EMID1, ENOX2, ERAP1, ERBB2, ERBB4, ETFDH, ETS2, EYA1, FAM13C,
FAM162B, FAM165B, FAM172A, FAM175A, FAM190A, FAM26F, FAM46A,
FAM49A, FAM71E1, FAM76A, FAM85A, FBXL5, FBXW7, FCRLB, FGD5, FGF1,
FGF13, FGF19, FGF3, FGF7, FIP1L1, FKTN, FLJ10038, FLJ35220,
FLJ35390, FLJ37798, FLJ39051, FOXO6, FSTL1, FUCA2, GAL, GART, GAS5,
GDPD5, GLCE, GLI2, GLIS1, GLIS3, GLT25D2, GNA14, GNAS, GPR123,
GPX7, GRM5, GTF2H5, GUCY1A3, H2AFJ, HCG 1776018, HDAC4, HEBP2,
HELQ, HERPUD2, HHLA3, HIST1H2AC, HIST1H2BD, HIST1H2BK, HIST2H2BE,
HMGCLL1, HNRNPR, HPCAL1, HPDL, HPS3, HSBP1L1, HSPA1A, HTATIP2,
ICAM2, IFNAR1, IGFBP5, IGFBP7, IGSF5, IL10RB, IL13RA2, IL17D,
IL20RA, IL7, IMMP2L, INSR, IRS1, ITGB1BP1, JMY, JRKL, KAT2B,
KBTBD11, KCMF1, KCNMA1, KCNQ2, KCNQ5, KCTD18, KDM1A, KDSR, KHDC1,
KIAA1109, KIAA1324, KIAA1598, KIAA1804, KIRREL3, KLHDC1, KRCC1,
LGALS3, LGALS3BP, LIFR, LINGO1, LIPT1, LMAN2L, LMCD1, LOC100129195,
LOC100129884, LOC100130522, LOC100130856, LOC100132167,
LOC100216479, LOC100272217, LOC100287039, LOC151146, LOC153682,
LOC220930, LOC254128, LOC255167, LOC283588, LOC285286, LOC285550,
LOC285878, LOC286052, LOC339290, LOC401321, LOC645513, LOC80154,
LOC90246, LOC93622, LOC94431, LPAR3, LPHN2, LRCH2, LRP2BP, LYRM1,
LYRM5, MAGI2, MANSC1, MAP3K13, MAP3K5, MAP9, MAPKAP1, MBD5, MBLAC2,
MBNL1, MCTP1, MED6, MEGF11, MEOX2, MET, METT5D1, MFSD4, MFSD6,
MFSD9, MGAT4A, MORC3, MREG, MRPS18C, MRPS33, MST1, MTMR3, MTUS2,
MXRA7, N4BP3, NAP1L3, NCAM1, NCAM2, NCOA7, NCRNA00081, NCRNA00117,
NCRNA00171, NDUFA4L2, NDUFV3, NEAT1, NEBL, NEIL2, NETO1, NFATC4,
NHEDC2, NIPAL3, NLRX1, NNAT, NOTCH4, NPW, NPY, NR1H3, NR2F1, NT5E,
NUDT19, NUDT6, OGFRL1, OLFM3, OMA1, OPRM1, OPTN, P4HTM, PABPC4L,
PABPC5, PAK1, PAM, PAPOLG, PAPPA, PBX1, PCDHB10, PCDHB16, PCNX,
PCNXL2, PDCD2, PDE4DIP, PDGFRB, PEX12, PEX6, PHKA1, PHLDB3, PIBF1,
PID1, PIGH, PIGP, PIK3CB, PIWIL2, PKNOX1, PKNOX2, PLA2G12A, PLCB1,
PLEKHA2, PLEKHA3, PLIN2, PLRG1, POLR3GL, POU6F1, PPAPDC1A, PPOX,
PPP1R14A, PPP2R5C, PPP3CA, PRAME, PRKACB, PRMT2, PRPH, PSD3, PSTK,
PTGER2, PTGES, PTN, PTP4A3, PTPRD, PTPRN2, PTPRR, QPCT, RAB4A,
RAB6B, RAC2, RAI2, RCAN1, RCC1, RDH13, RFPL1S, RG9MTD2, RGAG4,
RHBDD1, RHBDF2, RHOU, RNF13, RNF41, RNLS, RPL31, RPL37, RPRD1A,
RRN3, RSL1D1, RSPH3, SAP30, SAV1, SCG5, SCN5A, SERTAD4, SGMS1,
SH3BGR, SH3GL2, SH3KBP1, SH3YL1, SIAE, SIGIRR, SIK1, SIK3, SIX2,
SLC12A7, SLC16A14, SLC22A17, SLC22A5, SLC25A12, SLC25A4, SLC35F3,
SLC6A15, SLIT1, SMARCA2, SMEK2, SNAP91, SNCA, SOCS5, SORCS2,
SPATA17, SPATA7, SPIN3, SPINT1, SPINT2, SPOCK2, SPRED1, SSPN,
ST8SIA3, STAC2, STAR, STEAP3, STOX2, STX12, STXBP5L, SUCLG2, SYNJ1,
SYNPR, SYT13, TAF12, TAF1B, TBC1D12, TBC1D15, TCEAL2, TEX264,
THAP2, TM2D1, TMCC1, TMEM182, TMEM1840, TMEM196, TMEM45B, TMEM5,
TMEM59L, TMEM65, TMIE, TNC, TNFRSF10D, TNFSF4, TOX, TRAPPC9, TRHDE,
TRIM61, TRIM69, TSPAN7, TSPAN9, TTC23, TTC39C, TUSC3, TXLNB,
UBE2D3, UBE2W, UCN, UNC5A, UTP23, VPS37A, VSTM2A, WASF3, WDFY3,
WDTC1, XPR1, XYLT1, ZBTB41, ZC3H12B, ZNF148, ZNF185, ZNF22, ZNF23,
ZNF25, ZNF250, ZNF280D, ZNF285A, ZNF295, ZNF346, ZNF528, ZNF585A,
ZNF610, ZNF641, ZNF662, ZNF677, and/or ZNF862, as compared to the
expression levels of these genes in cells from the parental SiMa
cell line DSMZ ACC 164.
[0073] In an aspect of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold decrease in gene expression levels of one or more
genes selected from ACOT1, ADARB1, ASAM, BACE2, C11ORF70, C2CD4A,
C3ORF23, CADM2, CAP2, CARTPT, CDKN1C, CFC1, CNGA3, COL5A1, CYGB,
CYP2E1, CYTL1, DSCAM, ECEL1, ECHDC3, FAM26F, FAM49A, FGF1, FGF13,
FGF19, FLJ39051, FUCA2, GAL, GPX7, GRM5, HCG 1776018, HHLA3,
HIST1H2AC, HIST1H2BD, HSPA1A, HTATIP2, ICAM2, IGFBP5, IGFBP7,
IL13RA2, KCNMA1, KCNQ5, KIAA1598, KRCC1, LMCD1, LOC100216479,
LOC254128, LOC339290, LPAR3, MEGF11, MEOX2, NCAM2, NDUFA4L2, NEAT1,
NNAT, NPW, NPY, PABPC4L, PAPPA, PID1, PPAPDC1A, PRAME, PRKACB,
PTGER2, PTN, PTP4A3, RAC2, SLC35F3, SYT13, TCEAL2, THAP2, TMIE,
TNFRSF10D, TRHDE, TXLNB, and/or ZNF662, as compared to the
expression levels of these genes in cells from the parental SiMa
cell line DSMZ ACC 164.
[0074] In another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold decrease in gene expression levels of
one or more genes selected from ACOT1, ASAM, BACE2, C11ORF70,
C2CD4A, C3ORF23, CADM2, CAP2, CARTPT, CFC1, CNGA3, CYGB, CYP2E1,
CYTL1, DSCAM, ECEL1, ECHDC3, FGF1, FGF13, FUCA2, GAL, GPX7, HSPA1A,
HTATIP2, ICAM2, IGFBP5, IGFBP7, KIAA1598, KRCC1, LMCD1,
LOC100216479, LOC254128, LOC339290, LPAR3, MEOX2, NDUFA4L2, NEAT1,
NNAT, NPW, NPY, PAPPA, PID1, PPAPDC1A, PRAME, PTN, SLC35F3, SYT13,
TCEAL2, TMIE, TNFRSF10D, TRHDE, and/or ZNF662, as compared to the
expression levels of these genes in cells from the parental SiMa
cell line DSMZ ACC 164.
[0075] In yet another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold decrease in gene expression levels of
one or more genes selected from C11ORF70, CADM2, CAP2, CARTPT,
CNGA3, CYGB, CYP2E1, CYTL1, DSCAM, FGF13, FUCA2, GAL, HSPA1A,
HTATIP2, ICAM2, IGFBP5, IGFBP7, LMCD1, LOC100216479, MEOX2,
NDUFA4L2, NEAT1, NNAT, NPW, PID1, PRAME, SLC35F3, SYT13, TCEAL2,
TMIE, TNFRSF10D, and/or ZNF662, as compared to the expression
levels of these genes in cells from the parental SiMa cell line
DSMZ ACC 164.
[0076] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes listed in
Tables 5 or 7 as compared to the expression levels of these genes
in cells from the 2D6 cell line, and at least a 1.5-fold increase
in gene expression levels of one or more genes listed in Tables 9
or 11 as compared to the expression levels of these genes in cells
from a parental SiMa cell line. In aspects of this embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit at least a 1.5-fold increase in gene
expression levels of one or more genes listed in Tables 5 or 7 as
compared to the expression levels of these genes in cells from the
2D6 cell line, and at least a 1.5-fold increase in gene expression
levels of one or more genes listed in Tables 9 or 11 as compared to
the expression levels of these genes in cells from the parental
SiMa cell line DSMZ ACC 164. In other aspects of this embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit an increase in gene expression levels of one
or more genes listed in Tables 5 or 7 of, e.g., at least a
1.5-fold, at least a 2.0-fold, at least a 2.5-fold, at least a
3.0-fold, at least a 3.5-fold, at least a 4.0-fold, at least a
4.5-fold, at least a 5.0-fold, at least a 5.5-fold, at least a
6.0-fold, at least 7.0-fold, or at least 8.0-fold as compared to
the expression levels of these genes in cells from the 2D6 cell
line, and exhibit an increase in gene expression levels of one or
more genes listed in Tables 9 or 11 of, e.g., at least a 1.5-fold,
at least a 2.0-fold, at least a 2.5-fold, at least a 3.0-fold, at
least a 3.5-fold, at least a 4.0-fold, at least a 4.5-fold, at
least a 5.0-fold, at least a 5.5-fold, at least a 6.0-fold, at
least 7.0-fold, or at least 8.0-fold as compared to the expression
levels of these genes in cells from the parental SiMa cell line
DSMZ ACC 164. In other aspects of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit an increase in gene expression levels of, e.g., 2 or more
genes, 3 or more genes, 4 or more genes, 5 or more genes, 6 or more
genes, 7 or more genes, 8 or more genes, 9 or more genes, 10 or
more genes, 20 or more genes, 30 or more genes, 40 or more genes,
50 or more genes, 60 or more genes, 70 or more genes, 80 or more
genes, 90 or more genes, or 100 or more genes listed in Tables 5 or
7 as compared to the expression levels of these genes in cells from
the 2D6 cell line, and listed in Tables 9 or 11 as compared to the
expression levels of these genes in cells from the parental SiMa
cell line DSMZ ACC 164. In yet other aspects cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit an increase in gene expression levels of, e.g., about 5
genes to about 100 genes, about 10 genes to about 100 genes, about
15 genes to about 100 genes, about 20 genes to about 100 genes,
about 25 genes to about 100 genes, about 5 genes to about 75 genes,
about 10 genes to about 75 genes, about 15 genes to about 75 genes,
about 20 genes to about 75 genes, about 25 genes to about 75 genes,
about 5 genes to about 50 genes, about 10 genes to about 50 genes,
about 15 genes to about 50 genes, about 20 genes to about 50 genes,
or about 25 genes to about 50 genes listed in Tables 5 or 7 as
compared to the expression levels of these genes in cells from the
2D6 cell line, and listed in Tables 9 or 11 as compared to the
expression levels of these genes in cells from the parental SiMa
cell line DSMZ ACC 164.
[0077] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes selected
from A2BP1, ALCAM, ANAPC7, ARF1, ARHGAP6, ARHGEF6, ARL17A, ASCL1,
ASXL3, BASP1, BNC2, C10ORF58, C12ORF49, C1ORF43, C20ORF7, CBLB,
CBWD1, CCDC3, CD9, CDC25B, CHRNA7, CPVL, CRYBG3, CSE1L, CUGBP2,
CXCR4, DACT1, DAZ1, DBH, DHX35, DKFZP434L187, DLEU2, DLL3, EAF2,
EFNA5, EIF3C, ELOVL7, EML1, ENC1, EXOSC6, EXPH5, EZH2, FAM181B,
FAM19A4, FAM7A3, FUBP3, FZD5, GAP43, GFRA2, GNAI1, GNB4, GNG11,
GPR125, GRP, GSS, ITGA6, ITGA6, ITGB5, KCTD12, KDELC2, KHDRBS3,
KIF16B, KLHL13, LBH, LOC100128844, LOC340109, LOC641298, LOC728052,
LUM, MAB21L1, MAN2A1, MAOA, MINA, MMD, MSN, MYO1B, MYO6, NAAA,
NEDD9, NOS1, OSBPL3, PCGF5, PELI2, PFKFB3, PHF20, PLK2, PLS3,
PLXNA2, PLXNA4, PM20D2, POLE, PSMA7, PTGFRN, PTPRE, PTPRG, RAB35,
RAF1, RAN, RASEF, RBL1, RELL1, RGS5, RIMBP2, RNF182, RNF34, S1PR3,
SALL4, SEMA6A, SEZ6L, SIM1, SLC44A5, SLC7A2, SLCO3A1, SLITRK5,
SPAG6, SPATS2L, ST8SIA1, STMN3, STRA6, TCF7L2, TFPI2, TH1L, THOC4,
TIMP3, TLE3, TMEM132C, TMEM178, TPBG, TRA2A, TRIM29, TRIM36, TSHZ3,
TXNRD1, U2AF1, UBE2V1, VPS29, ZFHX4, ZGPAT, ZNF217, and/or ZNF503,
as compared to the expression levels of these genes in cells from
both the 2D6 cell line and the parental SiMa cell line DSMZ ACC
164.
[0078] In an aspect of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold increase in gene expression levels of one or more
genes selected from ALCAM, ARHGAP6, ARHGEF6, ASCL1, BNC2, CCDC3,
CHRNA7, CRYBG3, CUGBP2, CXCR4, DAZ1, EFNA5, ELOVL7, EML1, ENC1,
EXPH5, FAM7A3, FZD5, GFRA2, GNAI1, GNB4, GNG11, GRP, ITGA6, KCTD12,
LOC100128844, LOC340109, LOC728052, LUM, MAB21L1, MAB21L2, MAOA,
MSN, NEDD9, OSBPL3, PLK2, PTGFRN, PTPRE, RGS5, RNF182, SIM1,
SLC44A5, SLITRK5, SPAG6, TFPI2, TIMP3, TMEM132C, TPBG, TRIM29,
and/or TRIM36, as compared to the expression levels of these genes
in cells from both the 2D6 cell line and the parental SiMa cell
line DSMZ ACC 164.
[0079] In another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold increase in gene expression levels of
one or more genes selected from ALCAM, ARHGAP6, ARHGEF6, CCDC3,
CHRNA7, CRYBG3, CUGBP2, CXCR4, DAZ1, ELOVL7, EML1, EXPH5, FAM7A3,
GNB4, GNG11, GRP, ITGA6, KCTD12, LOC340109, LUM, MAB21L1, MAB21L2,
PLK2, PTPRE, RGS5, RNF182, SIM1, SLC44A5, SLITRK5, SPAG6, TIMP3,
and/or TMEM1320, as compared to the expression levels of these
genes in cells from both the 2D6 cell line and the parental SiMa
cell line DSMZ ACC 164.
[0080] In yet another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold increase in gene expression levels of
one or more genes selected from CCDC3, CHRNA7, CRYBG3, CXCR4, DAZ1,
EXPH5, FAM7A3, GNB4, GRP, KCTD12, LUM, PLK2, PTPRE, RGS5, SIM1,
SLITRK5, TIMP3, and/or TMEM1320, as compared to the expression
levels of these genes in cells from both the 2D6 cell line and the
parental SiMa cell line DSMZ ACC 164.
[0081] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes selected
from A2BP1, ALCAM, ARF1, ARL17A, ASCL1, BASP1, CD9, CUGBP2, EAF2,
EIF3C, FAM181B, FUBP3, GNAI1, GNB4, GNG11, KDELC2, KHDRBS3, KLHL13,
LOC641298, LOC728052, MAB21L2, MAOA, MINA, MSN, MYO6, PLK2, PLS3,
RAF1, RELL1, RNF182, SEZ6L, SEZ6L, SIM1, SLC44A5, SLC7A2, SLCO3A1,
SLITRK5, SPATS2L, TCF7L2, TFPI2, TMEM178, TRA2A, U2AF1, and/or
ZNF217, as compared to the expression levels of these genes in
cells from both the 2D6 cell line and the parental SiMa cell line
DSMZ ACC 164.
[0082] In an aspect of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold increase in gene expression levels of one or more
genes selected from A2BP1, ALCAM, ARF1, ASCL1, BASP1, CD9, CUGBP2,
EIF3C, FUBP3, GNAI1, GNB4, GNG11, KDELC2, KHDRBS3, LOC641298,
LOC728052, MAB21L2, MAOA, MINA, MSN, MYO6, PLK2, PLS3, RELL1,
RNF182, SIM1, SLC44A5, SLC7A2, SLCO3A1, SLITRK5, TCF7L2, TFPI2,
TMEM178, TRA2A, and/or ZNF217, as compared to the expression levels
of these genes in cells from both the 2D6 cell line and the
parental SiMa cell line DSMZ ACC 164.
[0083] In another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold increase in gene expression levels of
one or more genes selected from ASCL1, CUGBP2, GNAI1, GNB4, GNG11,
LOC728052, MAB21L2, MAOA, MSN, PLK2, RNF182, SIM1, SLC44A5,
SLITRK5, and/or TFPI2, as compared to the expression levels of
these genes in cells from both the 2D6 cell line and the parental
SiMa cell line DSMZ ACC 164.
[0084] In yet another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold increase in gene expression levels of
one or more genes selected from GNB4, GNG11, PLK2, RNF182, SIM1,
SLC44A5, and/or SLITRK5, as compared to the expression levels of
these genes in cells from both the 2D6 cell line and the parental
SiMa cell line DSMZ ACC 164.
[0085] In still another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold increase in gene expression levels of
one or more genes selected from GNB4, PLK2, SIM1, and/or SLITRK5,
as compared to the expression levels of these genes in cells from
both the 2D6 cell line and the parental SiMa cell line DSMZ ACC
164.
[0086] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
decrease in gene expression levels of one or more genes listed in
Tables 6 or 8 as compared to the expression levels of these genes
in cells from the 2D6 cell line, and at least a 1.5-fold decrease
in gene expression levels of one or more genes listed in Tables 10
or 12 as compared to the expression levels of these genes in cells
from a parental SiMa cell line. In aspects of this embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit at least a 1.5-fold decrease in gene
expression levels of one or more genes listed in Tables 6 or 8 as
compared to the expression levels of these genes in cells from the
2D6 cell line, and at least a 1.5-fold decrease in gene expression
levels of one or more genes listed in Tables 10 or 12 as compared
to the expression levels of these genes in cells from the parental
SiMa cell line DSMZ ACC 164. In other aspects of this embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit a decrease in gene expression levels of one or
more genes listed in Tables 6 or 8 of, e.g., at least a 1.5-fold,
at least a 2.0-fold, at least a 2.5-fold, at least a 3.0-fold, at
least a 3.5-fold, at least a 4.0-fold, at least a 4.5-fold, at
least a 5.0-fold, at least a 5.5-fold, at least a 6.0-fold, at
least 7.0-fold, or at least 8.0-fold as compared to the expression
levels of these genes in cells from the 2D6 cell line, and a
decrease in gene expression levels of one or more genes listed in
Tables 10 or 12 of, e.g., at least a 1.5-fold, at least a 2.0-fold,
at least a 2.5-fold, at least a 3.0-fold, at least a 3.5-fold, at
least a 4.0-fold, at least a 4.5-fold, at least a 5.0-fold, at
least a 5.5-fold, at least a 6.0-fold, at least 7.0-fold, or at
least 8.0-fold as compared to the expression levels of these genes
in cells from the parental SiMa cell line DSMZ ACC 164. In other
aspects of this embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit a decrease in gene
expression levels of, e.g., 2 or more genes, 3 or more genes, 4 or
more genes, 5 or more genes, 6 or more genes, 7 or more genes, 8 or
more genes, 9 or more genes, 10 or more genes, 20 or more genes, 30
or more genes, 40 or more genes, 50 or more genes, 60 or more
genes, 70 or more genes, 80 or more genes, 90 or more genes, or 100
or more genes listed in Tables 6 or 8 as compared to the expression
levels of these genes in cells from the 2D6 cell line, and listed
in Tables 10 or 12 as compared to the expression levels of these
genes in cells from the parental SiMa cell line DSMZ ACC 164. In
yet other aspects cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit a decrease in gene
expression levels of, e.g., about 5 genes to about 100 genes, about
10 genes to about 100 genes, about 15 genes to about 100 genes,
about 20 genes to about 100 genes, about 25 genes to about 100
genes, about 5 genes to about 75 genes, about 10 genes to about 75
genes, about 15 genes to about 75 genes, about 20 genes to about 75
genes, about 25 genes to about 75 genes, about 5 genes to about 50
genes, about 10 genes to about 50 genes, about 15 genes to about 50
genes, about 20 genes to about 50 genes, or about 25 genes to about
50 genes listed in Tables 6 or 8 as compared to the expression
levels of these genes in cells from the 2D6 cell line, and listed
in Tables 10 or 12 as compared to the expression levels of these
genes in cells from the parental SiMa cell line DSMZ ACC 164.
[0087] In an embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
decrease in gene expression levels of one or more genes selected
from ABTB1, ACOT1, ACYP2, ADARB1, ADCY1, ANKRD50, ASAM, ASPHD1,
ATP2B3, BACE1, BRUNOL4, BRWD1, BTN3A3, C16ORF52, C1ORF21, C21ORF57,
C2CD2, C2CD4A, C3ORF23, C9ORF150, CAMK2N2, CAP2, CAPN2, CARTPT,
CD302, CDH12, CDKN2A, CDS1, CFC1, CLCN3, CLDN12, CLMN, CNGA3,
CNNM1, CNOT6L, COL6A1, CYGB, CYLD, CYP2E1, CYTL1, D4S234E, DCLK1,
DCTD, DUSP16, EFNB3, ERAP1, ERBB2, ERBB4, ETFDH, FAM162B, FAM165B,
FAM46A, FBXW7, FGD5, FGF13, FOXO6, FSTL1, GAL, GLT25D2, GNAS,
GPR123, GPX7, GRM5, GUCY1A3, HCG 1776018, HELQ, HERPUD2, HIST1H2AC,
HIST1H2BD, HIST1H2BK, HIST2H2BE, HNRNPR, HPCAL1, IL10RB, JMY,
KAT2B, KBTBD11, KCNMA1, KCNQ2, KHDC1, KIAA1598, KLHDC1, LGALS3BP,
LIFR, LOC100130522, LOC94431, LRP2BP, LYRM5, MAP3K5, MAP9, MCTP1,
MEOX2, MFSD4, MGAT4A, MRPS33, MST1, NAP1L3, NCOA7, NCRNA00171,
NDUFA4L2, NDUFV3, NEBL, NHEDC2, NIPAL3, NLRX1, NUDT19, OLFM3, PAM,
PAPPA, PCNXL2, PDGFRB, PHKA1, PIGH, PLRG1, PLRG1, PPAPDC1A, PRKACB,
PRPH, PTGER2, PTN, PTPRD, RAB6B, RGAG4, RHOU, RNF13, RNF41, RSL1D1,
SH3GL2, SH3YL1, SIGIRR, SIK1, SLC12A7, SLC22A17, SLC35F3, SNCA,
SPINT2, SPOCK2, ST8SIA3, STAR, STEAP3, STOX2, SYNJ1, SYT13, TM2D1,
TMEM184C, TMEM5, TMEM59L, TMIE, TTC39C, TUSC3, TXLNB, UCN, WDFY3,
WDFY3, ZNF641, and/or ZNF662, as compared to the expression levels
of these genes in cells from both the 2D6 cell line and the
parental SiMa cell line DSMZ ACC 164.
[0088] In an aspect of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold decrease in gene expression levels of one or more
genes selected from ACOT1, ADARB1, ASAM, C2CD4A, C3ORF23, CAP2,
CARTPT, CFC1, CNGA3, CYGB, CYP2E1, CYTL1, FGF13, GAL, GPX7, GRM5,
HCG 1776018, HIST1H2AC, HIST1H2BD, KCNMA1, KIAA1598, MEOX2,
NDUFA4L2, PPAPDC1A, PRKACB, PTGER2, PTN, SLC35F3, SYT13, TMIE,
TXLNB, and/or ZNF662, as compared to the expression levels of these
genes in cells from both the 2D6 cell line and the parental SiMa
cell line DSMZ ACC 164.
[0089] In another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold decrease in gene expression levels of
one or more genes selected from ACOT1, ASAM, C2CD4A, C3ORF23, CAP2,
CARTPT, CFC1, CNGA3, CYGB, CYP2E1, CYTL1, FGF13, GAL, GPX7,
KIAA1598, MEOX2, NDUFA4L2, PPAPDC1A, PTN, SLC35F3, SYT13, TMIE,
and/or ZNF662, as compared to the expression levels of these genes
in cells from both the 2D6 cell line and the parental SiMa cell
line DSMZ ACC 164.
[0090] In yet another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold decrease in gene expression levels of
one or more genes selected from CAP2, CARTPT, CNGA3, CYGB, CYP2E1,
CYTL1, FGF13, GAL, MEOX2, NDUFA4L2, SLC35F3, SYT13, TMIE, and/or
ZNF662, as compared to the expression levels of these genes in
cells from both the 2D6 cell line and the parental SiMa cell line
DSMZ ACC 164.
[0091] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
decrease in gene expression levels of one or more genes selected
from CAP2, CDKN2A, CNGA3, FAM162B, FGF13, FOXO6, FSTL1, KCNMA1,
KHDC1, LIFR, MCTP1, MEOX2, PPAPDC1A, PTN, SYT13, TMIE, and/or
ZNF662, as compared to the expression levels of these genes in
cells from both the 2D6 cell line and the parental SiMa cell line
DSMZ ACC 164.
[0092] In an aspect of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold decrease in gene expression levels of one or more
genes selected from CAP2, CDKN2A, CNGA3, FGF13, FOXO6, FSTL1,
KCNMA1, KHDC1, MCTP1, MEOX2, PPAPDC1A, PTN, SYT13, TMIE, and/or
ZNF662, as compared to the expression levels of these genes in
cells from both the 2D6 cell line and the parental SiMa cell line
DSMZ ACC 164.
[0093] In another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold decrease in gene expression levels of
one or more genes selected from CNGA3, FGF13, KCNMA1, MEOX2,
PPAPDC1A, PTN, SYT13, TMIE, and/or ZNF662, as compared to the
expression levels of these genes in cells from both the 2D6 cell
line and the parental SiMa cell line DSMZ ACC 164.
[0094] In yet another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold decrease in gene expression levels of
one or more genes selected from CNGA3, FGF13, MEOX2, PPAPDC1A, PTN,
SYT13, TMIE, and/or ZNF662, as compared to the expression levels of
these genes in cells from both the 2D6 cell line and the parental
SiMa cell line DSMZ ACC 164.
[0095] In still another aspect of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold decrease in gene expression levels of
one or more genes selected from CNGA3, FGF13, MEOX2, SYT13, TMIE,
and/or ZNF662, as compared to the expression levels of these genes
in cells from both the 2D6 cell line and the parental SiMa cell
line DSMZ ACC 164.
[0096] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
difference in gene expression levels of one or more genes listed in
Tables 14 or 16 as compared to the expression levels of these genes
in cells from both the 2D6 cell line and the parental SiMa cell
line DSMZ ACC 164. In aspects of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit at least a 1.5-fold difference in gene expression levels of
one or more genes listed in Tables 14 or 16 as compared to the
expression levels of these genes in cells from both the 2D6 cell
line and the parental SiMa cell line DSMZ ACC 164. In other aspects
of this embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit a difference in gene
expression levels of one or more genes listed in Tables 14 or 16
of, e.g., at least a 1.5-fold, at least a 2.0-fold, at least a
2.5-fold, at least a 3.0-fold, at least a 3.5-fold, at least a
4.0-fold, at least a 4.5-fold, at least a 5.0-fold, at least a
5.5-fold, at least a 6.0-fold, at least a 7.0-fold, or at least a
8.0-fold as compared to the expression levels of these genes in
cells from both the 2D6 cell line and the parental SiMa cell line
DSMZ ACC 164. In other aspects of this embodiment, cells from an
established clonal cell line susceptible to BoNT/A intoxication
exhibit an increase in gene expression levels of, e.g., 2 or more
genes, 3 or more genes, 4 or more genes, 5 or more genes, 6 or more
genes, 7 or more genes, 8 or more genes, 9 or more genes, 10 or
more genes, 20 or more genes, 30 or more genes, 40 or more genes,
50 or more genes, 60 or more genes, 70 or more genes, 80 or more
genes, 90 or more genes, or 100 or more genes listed in Tables 14
or 16 as compared to the expression levels of these genes in cells
from both the 2D6 cell line and the parental SiMa cell line DSMZ
ACC 164. In yet other aspects cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit an increase in gene
expression levels of, e.g., about 5 genes to about 100 genes, about
10 genes to about 100 genes, about 15 genes to about 100 genes,
about 20 genes to about 100 genes, about 25 genes to about 100
genes, about 5 genes to about 75 genes, about 10 genes to about 75
genes, about 15 genes to about 75 genes, about 20 genes to about 75
genes, about 25 genes to about 75 genes, about 5 genes to about 50
genes, about 10 genes to about 50 genes, about 15 genes to about 50
genes, about 20 genes to about 50 genes, or about 25 genes to about
50 genes listed in Tables 14 or 16 as compared to the expression
levels of these genes in cells from both the 2D6 cell line and the
parental SiMa cell line DSMZ ACC 164.
[0097] In an embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
difference in gene expression levels of one or more genes selected
from ACOT9, ADAMTS9, ARHGAP24, ARHGEF3, ASCL1, BARD1, BASP1,
C3ORF70, C11ORF75, CCDC109B, CD9, CDCA7L, CDK2, CENPL, CLSTN2,
CNTN1, CSRP2, CTSL2, CUGBP2, DEPDC1, DIAPH3, DOK5, DPYD, DYNLT3,
EMILIN2, ETV1, FAM101B, FBLN1, FGFR2, FNDC5, GNAI1, GNB4, GNG11,
GNG12, GPR177, GTSE1, HGF, KITLG, LPAR1, MAB21L2, MAOA, MCM10,
MINA, MSN, MYO6, MYRIP, PAG1, PEG3, PLK2, POLA2, PPP1R3C, PRLHR,
PRSS12, PTGR1, PTPRK, PVRL3, RAB32, RBPMS, SDC2, SGOL2, SLC43A3,
SLC7A2, SMC2, SMC6, SPARC, SPC25, ST8SIA4, TCF7L1, TFPI2, TMEM35,
TMEM178, TNFAIP8, TPTE, TRIP10, TWIST1, and/or ZNF521, as compared
to the expression levels of these genes in cells from both the 2D6
cell line and the parental SiMa cell line DSMZ ACC 164. In another
aspect of this embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
difference in gene expression levels of one or more genes selected
from ADAMTS9, ARHGAP24, ASCL1, BARD1, BASP1, BVES, C11ORF75,
CDCA7L, CNTN1, CUGBP2, DOK5, DPYD, DYNLT3, FBLN1, FGFR2, GNAI1,
GNB4, GNG11, GNG12, GTSE1, GTSF1, ITPRIP, KDELC2, LOC728052, LPAR1,
MAB21L2, MAOA, MINA, MSN, PEG3, PLK2, PRLHR, PRSS12, PTPRK, RBPMS,
RNF182, SGOL2, SLC43A3, SLC44A5, SLC7A2, SMC6, SPARC, TFPI2,
TMEM178, and/or TPTE, as compared to the expression levels of these
genes in cells from both the 2D6 cell line and the parental SiMa
cell line DSMZ ACC 164. In yet another aspect of this embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit at least a 1.5-fold difference in gene
expression levels of one or more genes selected from ADAMTS9,
ASCL1, BASP1, DOK5, DPYD, GNB4, GNG11, GTSF1, MAOA, MINA, MSN,
PEG3, PLK2, PRSS12, RNF182, SLC44A5, SPARC, TFPI2, and/or TPTE, as
compared to the expression levels of these genes in cells from both
the 2D6 cell line and the parental SiMa cell line DSMZ ACC 164.
[0098] In another embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes selected
from ADAMTS9, ARHGAP24, ARHGEF3, ASCL1, BARD1, CD9, CDK2, CSRP2,
CTSL2, DIAPH3, DOK5, DYNLT3, EMILIN2, ETV1, FBLN1, FGFR2, GNAI1,
GNB4, GNG11, GNG12, HGF, KITLG, LPAR1, MCM10, MSN, PAG1, PEG3,
PLK2, POLA2, PPP1R3C, PTPRK, RAB32, SDC2, SLC43A3, SLC7A2, SMC6,
SPARC, SPC25, ST8SIA4, TCF7L1, TFPI2, TMEM35, TNFAIP8, TPTE,
TRIP10, and/or TWIST1, as compared to the expression levels of
these genes in cells from both the 2D6 cell line and the parental
SiMa cell line DSMZ ACC 164. In an aspect of this embodiment, cells
from an established clonal cell line susceptible to BoNT/A
intoxication exhibit at least a 1.5-fold increase in gene
expression levels of one or more genes selected from ADAMTS9,
ARHGAP24, ASCL1, BARD1, DOK5, DYNLT3, FBLN1, FGFR2, GNAI1, GNB4,
GNG11, GNG12, LPAR1, MSN, PEG3, PLK2, PTPRK, SLC43A3, SLC7A2, SMC6,
SPARC, TFPI2, and/or TPTE, as compared to the expression levels of
these genes in cells from both the 2D6 cell line and the parental
SiMa cell line DSMZ ACC 164. In another aspect of this embodiment,
cells from an established clonal cell line susceptible to BoNT/A
intoxication exhibit at least a 1.5-fold increase in gene
expression levels of one or more genes selected from ADAMTS9,
ASCL1, DOK5, GNB4, GNG11, MSN, PEG3, PLK2, SPARC, TFPI2, and/or
TPTE, as compared to the expression levels of these genes in cells
from both the 2D6 cell line and the parental SiMa cell line DSMZ
ACC 164.
[0099] In yet another embodiment, cells from an established clonal
cell line susceptible to BoNT/A intoxication exhibit at least a
1.5-fold increase in gene expression levels of one or more genes
selected from ACOT9, BASP1, C11ORF75, CCDC109B, CDCA7L, CLSTN2,
CNTN1, CUGBP2, DEPDC1, DPYD, FAM101B, FNDC5, GTSE1, MAOA, MINA,
MYO6, MYRIP, PLK2, PRLHR, PVRL3, RBPMS, SGOL2, SMC2, TFP12,
TMEM178, and/or ZNF521, as compared to the expression levels of
these genes in cells from both the 2D6 cell line and the parental
SiMa cell line DSMZ ACC 164. In an aspect of this embodiment, cells
from an established clonal cell line susceptible to BoNT/A
intoxication exhibit at least a 1.5-fold increase in gene
expression levels of one or more genes selected from BASP1,
C11ORF75, CDCA7L, CNTN1, CUGBP2, DPYD, GTSE1, MAOA, MINA, PLK2,
PRLHR, RBPMS, SGOL2, TFPI2, and/or TMEM178, as compared to the
expression levels of these genes in cells from both the 2D6 cell
line and the parental SiMa cell line DSMZ ACC 164. In another
aspect of this embodiment, cells from an established clonal cell
line susceptible to BoNT/A intoxication exhibit at least a 1.5-fold
increase in gene expression levels of one or more genes selected
from BASP1, DPYD, MAOA, MINA, PLK2, and/or TFPI2, as compared to
the expression levels of these genes in cells from both the 2D6
cell line and the parental SiMa cell line DSMZ ACC 164.
[0100] In a still another embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold increase in gene expression levels of one or more
genes selected from C3ORF70, MAB21L2, PRSS12, CENPL, GPR177, and/or
PTGR1, as compared to the expression levels of these genes in cells
from both the 2D6 cell line and the parental SiMa cell line DSMZ
ACC 164. In an aspect of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit at
least a 1.5-fold increase in gene expression levels of one or more
genes selected from MAB21L2 and/or PRSS12, as compared to the
expression levels of these genes in cells from both the 2D6 cell
line and the parental SiMa cell line DSMZ ACC 164.
[0101] Aspects of the present disclosure comprise, in part, cells
from an established clonal cell line susceptible to BoNT/A
intoxication that exhibit an equivalent or lower EC.sub.50 for
BoNT/A activity relative to the EC.sub.50 for BoNT/A activity of
cells from a parental SiMa cell line. In an aspect of this
embodiment, cells from an established clonal cell line susceptible
to BoNT/A intoxication exhibit an equivalent or lower EC.sub.50 for
BoNT/A activity relative to the EC.sub.50 for BoNT/A activity of
cells from the parental SiMa cell line DSMZ ACC 164. In an aspect
of this embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit an equivalent EC.sub.50
for BoNT/A activity relative to the EC.sub.50 for BoNT/A activity
of cells from the parental SiMa cell line DSMZ ACC 164. In aspects
of this embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit a lower EC.sub.50 for
BoNT/A activity of, e.g., at least 10%, at least 20%, at least 30%,
at least 40%, at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, or at least 100% relative to the EC.sub.50 for
BoNT/A activity of cells from the parental SiMa cell line DSMZ ACC
164. In other aspects of this embodiment, cells from an established
clonal cell line susceptible to BoNT/A intoxication exhibit a lower
EC.sub.50 for BoNT/A activity of, e.g., at least 110%, at least
120%, at least 130%, at least 140%, at least 150%, at least 160%,
at least 170%, at least 180%, at least 190%, or at least 200%
relative to the EC.sub.50 for BoNT/A activity of cells from the
parental SiMa cell line DSMZ ACC 164. In yet other aspects of this
embodiment, cells from an established clonal cell line susceptible
to BoNT/A intoxication exhibit a lower EC.sub.50 for BoNT/A
activity of, e.g., at least 225%, at least 250%, at least 275%, at
least 300%, at least 325%, at least 350%, at least 375%, at least
400%, at least 425%, at least 450%, at least 475%, or at least 500%
relative to the EC.sub.50 for BoNT/A activity of cells from the
parental SiMa cell line DSMZ ACC 164. In still other aspects of
this embodiment, cells from an established clonal cell line
susceptible to BoNT/A intoxication exhibit an EC.sub.50 for BoNT/A
activity of, e.g., 10 pM or less, 9 pM or less, 8 pM or less, 7 pM
or less, 6 pM or less, 5 pM or less, 4 pM or less, 3.0 pM or less,
2.9 pM or less, 2.8 pM or less, 2.7 pM or less, 2.6 pM or less, 2.5
pM or less, 2.4 pM or less, 2.3 pM or less, 2.2 pM or less, 2.1 pM
or less, 2.0 pM or less, 1.9 pM or less, 1.8 pM or less, 1.7 pM or
less, 1.6 pM or less, 1.5 pM or less, 1.4 pM or less, 1.3 pM or
less, 1.2 pM or less, 1.1 pM or less, 1.0 pM or less, 0.9 pM or
less, 0.8 pM or less, 0.7 pM or less, 0.6 pM or less, 0.5 pM or
less, 0.4 pM or less, 0.3 pM or less, 0.2 pM or less, or 0.1 pM or
less.
[0102] Aspects of the present disclosure comprise, in part, a
BoNT/A. As used herein, the term "BoNT/A" is synonymous with
"botulinum neurotoxin serotype A" or "botulinum neurotoxin type A"
and refers to both a naturally-occurring BoNT/A or a non-naturally
occurring BoNT/As thereof, and includes BoNT/A complex comprising
the about 150 kDa BoNT/A neurotoxin and associated non-toxin
associated proteins (NAPs), as well as the about 150 kDa BoNT/A
neurotoxin alone. Non-limiting examples of BoNT/A complexes
include, e.g., the 900-kDa BoNT/A complex, the 500-kDa BoNT/A
complex, the 300-kDa BoNT/A complex. Non-limiting examples of the
about 150 kDa BoNT/A neurotoxin include, e.g., SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4.
[0103] As used herein, the term "naturally occurring BoNT/A" refers
to any BoNT/A produced by a naturally-occurring process, including,
without limitation, BoNT/A isoforms produced from a
post-translational modification, an alternatively-spliced
transcript, or a spontaneous mutation, and BoNT/A subtypes, such
as, e.g., a BoNT/A1 subtype, BoNT/A2 subtype, BoNT/A3 subtype,
BoNT/A4 subtype, and BoNT/A5 subtype. A naturally occurring BoNT/A
includes, without limitation, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 3, SEQ ID NO: 4, or one that substitutes, deletes or adds,
e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or
more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30
or more, 40 or more, 50 or more, or 100 amino acids from SEQ ID NO:
1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Commercially
available pharmaceutical compositions of a naturally-occurring
BoNT/A includes, without limitation, BOTOX.RTM. (Allergan, Inc.,
Irvine, Calif.), DYSPORT.RTM./RELOXIN.RTM., (Ipsen Ltd., Slough,
England), PURTOX.RTM. (Mentor Corp., Santa Barbara, Calif.),
XEOMIN.RTM. (Merz Pharmaceuticals, GmbH., Frankfurt, Germany),
NEURONOX.RTM. (Medy-Tox, Inc., Ochang-myeon, South Korea),
BTX-A.
[0104] As used herein, the term "non-naturally occurring BoNT/A"
refers to any BoNT/A whose structure was modified with the aid of
human manipulation, including, without limitation, a BoNT/A with an
altered amino acid sequence produced by genetic engineering using
random mutagenesis or rational design and a BoNT/A produced by in
vitro chemical synthesis. Non-limiting examples of non-naturally
occurring BoNT/As are described in, e.g., Steward, L. E. et al.,
Post-translational Modifications and Clostridial Neurotoxins, U.S.
Pat. No. 7,223,577; Dolly, J. O. et al., Activatable Clostridial
Toxins, U.S. Pat. No. 7,419,676; Steward, L. E. et al., Clostridial
Neurotoxin Compositions and Modified Clostridial Neurotoxins, US
2004/0220386; Steward, L. E. et al., Modified Clostridial Toxins
With Enhanced Targeting Capabilities For Endogenous Clostridial
Toxin Receptor Systems, U.S. Patent Publication No. 2008/0096248;
Steward, L. E. et al., Modified Clostridial Toxins With Altered
Targeting Capabilities For Clostridial Toxin Target Cells, U.S.
Patent Publication No. 2008/0161543; Steward, L. E. et al.,
Modified Clostridial Toxins With Enhanced Translocation
Capabilities and Altered Targeting Activity For Clostridial Toxin
Target Cells, U.S. Patent Publication No. 2008/0241881, each of
which is hereby incorporated by reference in its entirety.
[0105] Thus in an embodiment, the BoNT/A activity being detected is
from a naturally occurring BoNT/A. In aspects of this embodiment,
the BoNT/A activity being detected is from a BoNT/A isoform or a
BoNT/A subtype. In aspects of this embodiment, the BoNT/A activity
being detected is from the BoNT/A of SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO: 3, or SEQ ID NO: 4. In other aspects of this embodiment,
the BoNT/A activity being detected is from a BoNT/A having, e.g.,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, or at least 95% amino acid identity with SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In other aspects of this
embodiment, the BoNT/A activity being detected is from BOTOX.RTM.,
DYSPORT.RTM./RELOXIN.RTM., PURTOX.RTM., XEOMIN.RTM., NEURONOX.RTM.,
or BTX-A.
[0106] In another embodiment, the BoNT/A activity being detected is
from a non-naturally occurring BoNT/A. In other aspects of this
embodiment, the BoNT/A activity being detected is from a
non-naturally occurring BoNT/A variant having, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% amino acid identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:
3, or SEQ ID NO: 4. In other aspects of this embodiment, the BoNT/A
activity being detected is from a non-naturally occurring BoNT/A
variant having, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5
or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20
or more, 30 or more, 40 or more, 50 or more, or 100 or more
non-contiguous amino acid substitutions, deletions, or additions
relative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:
4. In yet other aspects of this embodiment, the BoNT/A activity
being detected is from a non-naturally occurring BoNT/A variant
having, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or
more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or
more, 30 or more, 40 or more, 50 or more, or 100 or more contiguous
amino acid substitutions, deletions, or additions relative to SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
[0107] Aspects of the present disclosure comprise, in part, a
SNAP-25. As used herein, the term "SNAP-25" refers to a
naturally-occurring SNAP-25 or a non-naturally occurring SNAP-25
which is preferentially cleaved by a BoNT/A. As used herein, the
term "preferentially cleaved" refers to that the cleavage rate of a
BoNT/A substrate by a BoNT/A that is at least one order of
magnitude higher than the cleavage rate of any other substrate by
BoNT/A. In aspects of this embodiment, the cleavage rate of a
BoNT/A substrate by a BoNT/A that is at least two orders of
magnitude higher, at least three orders of magnitude higher, at
least four orders of magnitude higher, or at least five orders of
magnitude higher than the cleavage rate of any other substrate by
BoNT/A.
[0108] As used herein, the term "naturally occurring SNAP-25"
refers to any SNAP-25 produced by a naturally-occurring process,
including, without limitation, SNAP-25 isoforms produced from a
post-translational modification, an alternatively-spliced
transcript, or a spontaneous mutation, and SNAP-25 subtypes. A
naturally occurring SNAP-25 includes, without limitation, SEQ ID
NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ
ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:
14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ
ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:
23, or SEQ ID NO: 24, or one that substitutes, deletes or adds,
e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or
more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30
or more, 40 or more, 50 or more, or 100 or more amino acids from
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ
ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:
18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ
ID NO: 23, or SEQ ID NO: 24.
[0109] As used herein, the term "non-naturally occurring SNAP-25"
refers to any SNAP-25 whose structure was modified with the aid of
human manipulation, including, without limitation, a SNAP-25
produced by genetic engineering using random mutagenesis or
rational design and a SNAP-25 produced by in vitro chemical
synthesis. Non-limiting examples of non-naturally occurring
SNAP-25s are described in, e.g., Steward, L. E. et al., FRET
Protease Assays for Clostridial Toxins, U.S. Pat. No. 7,332,567;
Fernandez-Salas et al., Lipohilic Dye-based FRET Assays for
Clostridial Toxin Activity, U.S. Patent Publication 2008/0160561,
each of which is hereby incorporated by reference in its entirety.
A non-naturally occurring SNAP-25 may substitute, delete or add,
e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or
more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30
or more, 40 or more, 50 or more, or 100 or more amino acids from
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ
ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:
18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ
ID NO: 23, or SEQ ID NO: 24.
[0110] Thus in an embodiment, a SNAP-25 is a naturally occurring
SNAP-25. In aspects of this embodiment, the SNAP-25 is a SNAP-25
isoform or a SNAP-25 subtype. In aspects of this embodiment, the
naturally occurring SNAP-25 is the naturally occurring SNAP-25 of
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ
ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:
18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ
ID NO: 23, or SEQ ID NO: 24. In other aspects of this embodiment,
the SNAP-25 is a naturally occurring SNAP-25 having, e.g., at least
70% amino acid identity, at least 75%, at least 80%, at least 85%,
at least 90%, or at least 95% amino acid identity with SEQ ID NO:
5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,
SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID
NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,
or SEQ ID NO: 24.
[0111] In another embodiment, a SNAP-25 is a non-naturally
occurring SNAP-25. In other aspects of this embodiment, the SNAP-25
is a non-naturally occurring SNAP-25 having, e.g., at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, or at least
95% amino acid identity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID
NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,
SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID
NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In other
aspects of this embodiment, the SNAP-25 is a non-naturally
occurring SNAP-25 having, e.g., 1 or more, 2 or more, 3 or more, 4
or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10
or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or
more non-contiguous amino acid substitutions, deletions, or
additions relative to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ
ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:
12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ
ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:
21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In yet other
aspects of this embodiment, the SNAP-25 is a non-naturally
occurring SNAP-25 having, e.g., 1 or more, 2 or more, 3 or more, 4
or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10
or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or
more contiguous amino acid substitutions, deletions, or additions
relative to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,
SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID
NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,
SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID
NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.
[0112] A SNAP-25 can be an endogenous SNAP-25 or an exogenous
SNAP-25. As used herein, the term "endogenous SNAP-25" refers to a
SNAP-25 naturally present in the cell because it is naturally
encoded within the cell's genome, such that the cell inherently
expresses the SNAP-25 without the need of an external source of
SNAP-25 or an external source of genetic material encoding a
SNAP-25. The expression of an endogenous SNAP-25 may be with or
without environmental stimulation such as, e.g., cell
differentiation. By definition, an endogenous SNAP-25 can only be a
naturally-occurring SNAP-25 or variants thereof. For example, the
following established cell lines express an endogenous SNAP-25:
BE(2)-M17, Kelly, LA1-55n, N1E-115, N4TG3, N18, Neuro-2a, NG108-15,
PC12, SH-SY5Y, SiMa, and SK-N-BE(2)-C.
[0113] As used herein, the term "exogenous SNAP-25" refers to a
SNAP-25 expressed in a cell through the introduction of an external
source of SNAP-25 or an external source of genetic material
encoding a SNAP-25 by human manipulation. The expression of an
exogenous SNAP-25 may be with or without environmental stimulation
such as, e.g., cell differentiation. As a non-limiting example,
cells from an established clonal cell line can express an exogenous
SNAP-25 by transient or stably transfection of a SNAP-25 coding
sequence. As another non-limiting example, cells from an
established clonal cell line can express an exogenous SNAP-25 by
protein transfection of a SNAP-25. An exogenous SNAP-25 can be a
naturally-occurring SNAP-25 or variants thereof, or a non-naturally
occurring SNAP-25 or variants thereof.
[0114] Thus in an embodiment, cells from an established clonal cell
line express an endogenous SNAP-25. In aspects of this embodiment,
the endogenous SNAP-25 expressed by cells from an established
clonal cell line is a naturally-occurring SNAP-25. In other aspects
of this embodiment, the endogenous SNAP-25 expressed by cells from
an established clonal cell line is SEQ ID NO: 5, SEQ ID NO: 6, SEQ
ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,
SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In
yet other aspects of this embodiment, the endogenous SNAP-25
expressed by cells from an established clonal cell line is a
naturally occurring SNAP-25, such as, e.g., a SNAP-25 isoform or a
SNAP-25 subtype. In other aspects of this embodiment, the
endogenous SNAP-25 expressed by cells from an established clonal
cell line is a naturally occurring SNAP-25 having, e.g., at least
70% amino acid identity, at least 75%, at least 80%, at least 85%,
at least 90%, or at least 95% amino acid identity with SEQ ID NO:
5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,
SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID
NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,
or SEQ ID NO: 24.
[0115] In another embodiment, cells from an established clonal cell
line are transiently or stably engineered to express an exogenous
SNAP-25. In an aspect of this embodiment, cells from an established
clonal cell line are transiently or stably engineered to express a
naturally-occurring SNAP-25. In other aspects of this embodiment,
cells from an established clonal cell line are transiently or
stably engineered to express the naturally-occurring SNAP-25 of SEQ
ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,
SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID
NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,
SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID
NO: 23, or SEQ ID NO: 24. In yet other aspects of this embodiment,
cells from an established clonal cell line are transiently or
stably engineered to express a naturally occurring SNAP-25, such
as, e.g., a SNAP-25 isoform or a SNAP-25 subtype. In still other
aspects of this embodiment, cells from an established clonal cell
line are transiently or stably engineered to express a naturally
occurring SNAP-25 having, e.g., at least 70% amino acid identity,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% amino acid identity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID
NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,
SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID
NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.
[0116] In another aspect of the embodiment, cells from an
established clonal cell line are transiently or stably engineered
to express a non-naturally occurring SNAP-25. In other aspects of
this embodiment, cells from an established clonal cell line are
transiently or stably engineered to express a non-naturally
occurring SNAP-25 having, e.g., at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, or at least 95% amino acid
identity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ
ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:
17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ
ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In other aspects of
this embodiment, cells from an established clonal cell line are
transiently or stably engineered to express a non-naturally
occurring SNAP-25 having, e.g., 1 or more, 2 or more, 3 or more, 4
or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10
or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or
more non-contiguous amino acid substitutions, deletions, or
additions relative to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ
ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:
12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ
ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:
21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In yet other
aspects of this embodiment, cells from an established clonal cell
line are transiently or stably engineered to express a
non-naturally occurring SNAP-25 having, e.g., 1 or more, 2 or more,
3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9
or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or
more, or 100 or more contiguous amino acid substitutions,
deletions, or additions relative to SEQ ID NO: 5, SEQ ID NO: 6, SEQ
ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,
SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.
[0117] Assays that detect the cleavage of a BoNT/A substrate after
exposure to a BoNT/A can be used to assess whether a cell is
expressing an endogenous or an exogenous SNAP-25. In these assays,
generation of a SNAP-25 cleavage-product would be detected in cells
expressing a SNAP-25 after BoNT/A treatment. Non-limiting examples
of specific Western blot analysis, as well as well-characterized
reagents, conditions and protocols are readily available from
commercial vendors that include, without limitation, Amersham
Biosciences, Piscataway, N.J.; Bio-Rad Laboratories, Hercules,
Calif.; Pierce Biotechnology, Inc., Rockford, Ill.; Promega
Corporation, Madison, Wis., and Stratagene, Inc., La Jolla, Calif.
It is understood that these and similar assays for SNAP-25 cleavage
can be useful in identifying cells expressing an endogenous or an
exogenous SNAP-25.
[0118] As non-limiting examples, Western blot analysis using an
antibody that recognizes BoNT/A SNAP-25-cleaved product or both the
cleaved and uncleaved forms of SNAP-25 can be used to assay for
uptake of BoNT/A. Examples of .alpha.-SNAP-25 antibodies useful for
these assays include, without limitation, .alpha.-SNAP-25 mouse
monoclonal antibody SMI-81 (Sternberger Monoclonals Inc.,
Lutherville, Md.), mouse .alpha.-SNAP-25 monoclonal antibody CI
71.1 (Synaptic Systems, Goettingen, Germany), .alpha.-SNAP-25 mouse
monoclonal antibody CI 71.2 (Synaptic Systems, Goettingen,
Germany), .alpha.-SNAP-25 mouse monoclonal antibody SP12 (Abcam,
Cambridge, Mass.), .alpha.-SNAP-25 rabbit polyclonal antiserum
(Synaptic Systems, Goettingen, Germany), .alpha.-SNAP-25 rabbit
polyclonal antiserum (Abeam, Cambridge, Mass.), and .alpha.-SNAP-25
rabbit polyclonal antiserum S9684 (Sigma, St Louis, Mo.).
[0119] Aspects of the present disclosure comprise, in part, a
BoNT/A receptor. As used herein, the term "BoNT/A receptor" refers
to either a naturally-occurring BoNT/A receptor or a non-naturally
occurring BoNT/A receptor which preferentially interacts with
BoNT/A in a manner that elicits a BoNT/A intoxication response. As
used herein, the term "preferentially interacts" refers to that the
equilibrium dissociation constant (KD) of BoNT/A for a BoNT/A
receptor is at least one order of magnitude less than that of
BoNT/A for any other receptor. The equilibrium dissociation
constant, a specific type of equilibrium constant that measures the
propensity of an BoNT/A-BoNT/A receptor complex to separate
(dissociate) reversibly into its component molecules, namely the
BoNT/A and the BoNT/A receptor complex. The disassociation constant
is defined as KD=[L][R]/[C], where [L] equals the molar
concentration of BoNT/A, [R] is the molar concentration of a BoNT/A
receptor, and [C] is the molar concentration of the BoNT/A-BoNT/A
receptor complex, and where all concentrations are of such
components when the system is at equilibrium. The smaller the
dissociation constant, the more tightly bound the BoNT/A is to its
receptor, or the higher the binding affinity between BoNT/A and
BoNT/A receptor. In aspects of this embodiment, the disassociation
constant of BoNT/A for a BoNT/A receptor is at least two orders of
magnitude less, at least three orders of magnitude less, at least
four orders of magnitude less, or at least five orders of magnitude
less than that of BoNT/A for any other receptor. In other aspects
of this embodiment, the binding affinity of a BoNT/A that
preferentially interacts with a BoNT/A receptor can have an
equilibrium disassociation constant of, e.g., of 500 nM or less,
400 nM or less, 300 nM or less, 200 nM, or less 100 nM or less. In
other aspects of this embodiment, the binding affinity of a BoNT/A
that preferentially interacts with a BoNT/A receptor can have an
equilibrium disassociation constant of, e.g., of 90 nM or less, 80
nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or
less, 30 nM or less, 20 nM, or less 10 nM or less. As used herein,
the term "elicits a BoNT/A intoxication response" refers to the
ability of a BoNT/A receptor to interact with a BoNT/A to form a
neurotoxin/receptor complex and the subsequent internalization of
that complex into the cell cytoplasm.
[0120] As used herein, the term "naturally occurring BoNT/A
receptor" refers to any BoNT/A receptor produced by a
naturally-occurring process, including, without limitation, BoNT/A
receptor isoforms produced from a post-translational modification,
an alternatively-spliced transcript, or a spontaneous mutation, and
BoNT/A receptor subtypes. A naturally occurring BoNT/A receptor
includes, without limitation, a fibroblast growth factor receptor 2
(FGFR2), a fibroblast growth factor receptor 3 (FGFR3), a synaptic
vesicle glycoprotein 2 (SV2), and a complex ganglioside like GT1b,
such as those described in Ester Fernandez-Salas, et al., Botulinum
Toxin Screening Assays, U.S. Patent Publication 2008/0003240; Ester
Fernandez-Salas, et al., Botulinum Toxin Screening Assays, U.S.
Patent Publication 2008/0182799; Min Dong et al., SV2 is the
Protein Receptor for Botulinum Neurotoxin A, Science (2006); S.
Mahrhold et al, The Synaptic Vesicle Protein 2C Mediates the Uptake
of Botulinum Neurotoxin A into Phrenic Nerves, 580(8) FEBS Lett.
2011-2014 (2006), each of which is hereby incorporated by reference
in its entirety. A naturally occurring FGFR2 includes, without
limitation, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO:
62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ
ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70, or one
that substitutes, deletes or adds, e.g., 1 or more, 2 or more, 3 or
more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or
more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more,
or 100 or more amino acids from SEQ ID NO: 59, SEQ ID NO: 60, SEQ
ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO:
65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and
SEQ ID NO: 70. A naturally occurring FGFR3 includes, without
limitation, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, or one
that substitutes, deletes or adds, e.g., 1 or more, 2 or more, 3 or
more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or
more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more,
or 100 or more amino acids from SEQ ID NO: 25, SEQ ID NO: 26, and
SEQ ID NO: 27. A naturally occurring SV2 includes, without
limitation, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID
NO: 31, or one that substitutes, deletes or adds, e.g., 1 or more,
2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8
or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more,
50 or more, or 100 or more amino acids from SEQ ID NO: 28, SEQ ID
NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31.
[0121] As used herein, the term "non-naturally occurring BoNT/A
receptor variant" refers to any BoNT/A receptor produced with the
aid of human manipulation or design, including, without limitation,
a BoNT/A receptor produced by genetic engineering using random
mutagenesis or rational design and a BoNT/A receptor produced by
chemical synthesis. Non-limiting examples of non-naturally
occurring BoNT/A variants include, e.g., conservative BoNT/A
receptor variants, non-conservative BoNT/A receptor variants,
BoNT/A receptor chimeric variants and active BoNT/A receptor
fragments.
[0122] As used herein, the term "non-naturally occurring BoNT/A
receptor" refers to any BoNT/A receptor whose structure was
modified with the aid of human manipulation, including, without
limitation, a BoNT/A receptor produced by genetic engineering using
random mutagenesis or rational design and a BoNT/A receptor
produced by in vitro chemical synthesis. Non-limiting examples of
non-naturally occurring BoNT/A receptors are described in, e.g.,
Ester Fernandez-Salas, et al., Botulinum Toxin Screening Assays,
U.S. Patent Publication 2008/0003240; Ester Fernandez-Salas, et
al., Botulinum Toxin Screening Assays, U.S. Patent Publication
2008/0182799, each of which is hereby incorporated by reference in
its entirety. A non-naturally occurring BoNT/A receptor may
substitute, delete or add, e.g., 1 or more, 2 or more, 3 or more, 4
or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10
or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or
more amino acids from SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,
SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID
NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63,
SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID
NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70.
[0123] Thus in an embodiment, a BoNT/A receptor is a naturally
occurring BoNT/A receptor such as, e.g., FGFR2, FGFR3 or SV2. In
aspects of this embodiment, the BoNT/A receptor is a BoNT/A
receptor isoform or a BoNT/A receptor subtype. In aspects of this
embodiment, the naturally occurring BoNT/A receptor is the
naturally occurring BoNT/A receptor of SEQ ID NO: 25, SEQ ID NO:
26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ
ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO:
62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ
ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In other
aspects of this embodiment, the BoNT/A receptor is a naturally
occurring BoNT/A receptor having, e.g., at least 70% amino acid
identity, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% amino acid identity with SEQ ID NO: 25, SEQ ID NO:
26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ
ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO:
62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ
ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70.
[0124] In another embodiment, a BoNT/A receptor is a non-naturally
occurring BoNT/A receptor, such as, e.g., a genetically-engineered
FGFR2, a genetically-engineered FGFR3, or a genetically-engineered
SV2. In other aspects of this embodiment, the BoNT/A receptor is a
non-naturally occurring BoNT/A receptor having, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% amino acid identity with SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID
NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,
SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID
NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67,
SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In other aspects of
this embodiment, the BoNT/A receptor is a non-naturally occurring
BoNT/A receptor having, e.g., 1 or more, 2 or more, 3 or more, 4 or
more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or
more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or
more non-contiguous amino acid substitutions, deletions, or
additions relative to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,
SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID
NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63,
SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID
NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In yet other aspects of
this embodiment, the BoNT/A receptor is a non-naturally occurring
BoNT/A receptor having, e.g., 1 or more, 2 or more, 3 or more, 4 or
more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or
more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or
more contiguous amino acid substitutions, deletions, or additions
relative to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:
28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ
ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:
64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ
ID NO: 69, or SEQ ID NO: 70.
[0125] A BoNT/A receptor can be an endogenous BoNT/A receptor or an
exogenous BoNT/A receptor. As used herein, the term "endogenous
BoNT/A receptor" refers to a BoNT/A receptor naturally present in
the cell because it is naturally encoded within the cell's genome,
such that the cell inherently expresses the BoNT/A receptor without
the need an external source of BoNT/A receptor or an external
source of genetic material encoding a BoNT/A receptor. Expression
of an endogenous BoNT/A receptor may be with or without
environmental stimulation such as e.g., cell differentiation or
promoter activation. For example, the following established clonal
cell lines express at least one endogenous BoNT/A receptor:
BE(2)-M17, Kelly, LA1-55n, N1E-115, N4TG3, N18, Neuro-2a, NG108-15,
PC12, SH-SY5Y, SiMa, and SK-N-BE(2)-C. An endogenous BoNT/A
receptor can only be a naturally-occurring BoNT/A receptor or
naturally-occurring variants thereof.
[0126] As used herein, the term "exogenous BoNT/A receptor" refers
to a BoNT/A receptor expressed in a cell through the introduction
of an external source of BoNT/A receptor or an external source of
genetic material encoding a BoNT/A receptor by human manipulation.
The expression of an exogenous BoNT/A receptor may be with or
without environmental stimulation such as, e.g., cell
differentiation or promoter activation. As a non-limiting example,
cells from an established clonal cell line can express one or more
exogenous BoNT/A receptors by transient or stably transfection of a
polynucleotide molecule encoding a BoNT/A receptor, such as, e.g.,
a FGFR2, a FGFR3, or a SV2. As another non-limiting example, cells
from an established clonal cell line can express one or more
exogenous BoNT/A receptors by protein transfection of the BoNT/A
receptors, such as, e.g., a FGFR2, a FGFR3, or a SV2. An exogenous
BoNT/A receptor can be a naturally-occurring BoNT/A receptor or
naturally occurring variants thereof, or non-naturally occurring
BoNT/A receptor or non-naturally occurring variants thereof.
[0127] Thus in an embodiment, cells from an established clonal cell
line express an endogenous BoNT/A receptor. In aspects of this
embodiment, the endogenous BoNT/A receptor expressed by cells from
an established clonal cell line is a naturally-occurring BoNT/A
receptor. In other aspects of this embodiment, the endogenous
BoNT/A receptor expressed by cells from an established clonal cell
line is SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,
SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID
NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64,
SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID
NO: 69, or SEQ ID NO: 70. In yet aspects of this embodiment, the
endogenous BoNT/A receptor expressed by cells from an established
clonal cell line is a naturally occurring BoNT/A receptor, such as,
e.g., a BoNT/A receptor isoform or a BoNT/A receptor subtype. In
other aspects of this embodiment, the endogenous BoNT/A receptor
expressed by cells from an established clonal cell line is a
naturally occurring BoNT/A receptor having, e.g., at least 70%
amino acid identity, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% amino acid identity with SEQ ID NO: 25,
SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID
NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61,
SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID
NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO:
70.
[0128] In another embodiment, cells from an established clonal cell
line are transiently or stably engineered to express an exogenous
BoNT/A receptor. In an aspect of this embodiment, cells from an
established clonal cell line are transiently or stably engineered
to express a naturally-occurring BoNT/A receptor. In other aspects
of this embodiment, cells from an established clonal cell line are
transiently or stably engineered to express the naturally-occurring
BoNT/A receptor of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ
ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:
59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ
ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO:
68, SEQ ID NO: 69, or SEQ ID NO: 70. In yet other aspects of this
embodiment, cells from an established clonal cell line are
transiently or stably engineered to express a naturally occurring
BoNT/A receptor, such as, e.g., a BoNT/A receptor isoform or a
BoNT/A receptor subtype. In still other aspects of this embodiment,
cells from an established clonal cell line are transiently or
stably engineered to express a naturally occurring BoNT/A receptor
having, e.g., at least 70% amino acid identity, at least 75%, at
least 80%, at least 85%, at least 90%, or at least 95% amino acid
identity with SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID
NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59,
SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID
NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68,
SEQ ID NO: 69, or SEQ ID NO: 70.
[0129] In another aspect of the embodiment, cells from an
established clonal cell line are transiently or stably engineered
to express a non-naturally occurring BoNT/A receptor. In other
aspects of this embodiment, cells from an established clonal cell
line are transiently or stably engineered to express a
non-naturally occurring BoNT/A receptor having, e.g., at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% amino acid identity with SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID
NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,
SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID
NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67,
SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In other aspects of
this embodiment, cells from an established clonal cell line are
transiently or stably engineered to express a non-naturally
occurring BoNT/A receptor having, e.g., 1 or more, 2 or more, 3 or
more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or
more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more,
or 100 or more non-contiguous amino acid substitutions, deletions,
or additions relative to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:
27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ
ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO:
63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ
ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In yet other aspects of
this embodiment, cells from an established clonal cell line are
transiently or stably engineered to express a non-naturally
occurring BoNT/A receptor having, e.g., 1 or more, 2 or more, 3 or
more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or
more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more,
or 100 or more contiguous amino acid substitutions, deletions, or
additions relative to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,
SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID
NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63,
SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID
NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70
[0130] In another embodiment, cells from an established clonal cell
line are transiently or stably engineered to express an exogenous
FGFR2, an exogenous FGFR3, an exogenous SV2, or any combination
thereof. In aspects of this embodiment, cells from an established
clonal cell line are transiently or stably engineered to express a
naturally-occurring FGFR2, a naturally-occurring FGFR3, a
naturally-occurring SV2, or any combination thereof. In yet other
aspects of this embodiment, cells from an established clonal cell
line are transiently or stably engineered to express a
non-naturally-occurring FGFR2, a non-naturally-occurring FGFR3, a
non-naturally-occurring SV2, or any combination thereof. In still
other aspects of this embodiment, cells from an established clonal
cell line are transiently or stably engineered to express either a
naturally-occurring FGFR2 or a non-naturally-occurring FGFR2, a
naturally-occurring FGFR3 or a non-naturally-occurring FGFR3, a
naturally-occurring SV2 or a non-naturally-occurring SV2, or any
combination thereof.
[0131] Cells that express one or more endogenous or exogenous
BoNT/A receptors can be identified by routine methods including
direct and indirect assays for toxin uptake. Assays that determine
BoNT/A binding or uptake properties can be used to assess whether a
cell is expressing a BoNT/A receptor. Such assays include, without
limitation, cross-linking assays using labeled BoNT/A, such as,
e.g., [.sup.125I] BoNT/A, see, e.g., Noriko Yokosawa et al.,
Binding of Clostridium botulinum type C neurotoxin to different
neuroblastoma cell lines, 57(1) Infect. Immun. 272-277 (1989);
Noriko Yokosawa et al., Binding of botulinum type Cl, D and E
neurotoxins to neuronal cell lines and synaptosomes, 29(2) Toxicon
261-264 (1991); and Tei-ichi Nishiki et al., Identification of
protein receptor for Clostridium botulinum type B neurotoxin in rat
brain synaptosomes, 269(14) J. Biol. Chem. 10498-10503 (1994).
Other non-limiting assays include immunocytochemical assays that
detect BoNT/A binding using labeled or unlabeled antibodies, see,
e.g., Atsushi Nishikawa et al., The receptor and transporter for
internalization of Clostridium botulinum type C progenitor toxin
into HT-29 cells, 319(2) Biochem. Biophys. Res. Commun. 327-333
(2004) and immunoprecipitation assays, see, e.g., Yukako Fujinaga
et al., Molecular characterization of binding subcomponents of
Clostridium botulinum type C progenitor toxin for intestinal
epithelial cells and erythrocytes, 150(Pt 5) Microbiology 1529-1538
(2004), that detect bound toxin using labeled or unlabeled
antibodies. Antibodies useful for these assays include, without
limitation, antibodies selected against BoNT/A, antibodies selected
against a BoNT/A receptor, such as, e.g., FGFR2, FGFR3, or SV2,
and/or antibodies selected against a ganglioside, such as, e.g.,
GD1a, GD1b, GD3, GQ1b, or GT1b. If the antibody is labeled, the
binding of the molecule can be detected by various means, including
Western blot analysis, direct microscopic observation of the
cellular location of the antibody, measurement of cell or
substrate-bound antibody following a wash step, flow cytometry,
electrophoresis or capillary electrophoresis, employing techniques
well-known to those of skill in the art. If the antibody is
unlabeled, one may employ a labeled secondary antibody for indirect
detection of the bound molecule, and detection can proceed as for a
labeled antibody. It is understood that these and similar assays
that determine BoNT/A uptake properties or characteristics can be
useful in identifying cells expressing endogenous or exogenous
BoNT/A receptors.
[0132] Assays that monitor the release of a molecule after exposure
to BoNT/A can also be used to assess whether a cell is expressing
one or more endogenous or exogenous BoNT/A receptors. In these
assays, inhibition of the molecule's release would occur in cells
expressing a BoNT/A receptor after BoNT/A treatment. Well known
assays include methods that measure inhibition of radio-labeled
catecholamine release from neurons, such as, e.g., [3H]
noradrenaline or [3H] dopamine release, see e.g., A Fassio et al.,
Evidence for calcium-dependent vesicular transmitter release
insensitive to tetanus toxin and botulinum toxin type F, 90(3)
Neuroscience 893-902 (1999); and Sara Stigliani et al., The
sensitivity of catecholamine release to botulinum toxin C1 and E
suggests selective targeting of vesicles set into the readily
releasable pool, 85(2) J. Neurochem. 409-421 (2003), or measures
catecholamine release using a fluorometric procedure, see, e.g.,
Anton de Paiva et al., A role for the interchain disulfide or its
participating thiols in the internalization of botulinum neurotoxin
A revealed by a toxin derivative that binds to ecto-acceptors and
inhibits transmitter release intracellularly, 268(28) J. Biol.
Chem. 20838-20844 (1993); Gary W. Lawrence et al., Distinct
exocytotic responses of intact and permeabilised chromaffin cells
after cleavage of the 25-kDa synaptosomal-associated protein
(SNAP-25) or synaptobrevin by botulinum toxin A or B, 236(3) Eur.
J. Biochem. 877-886 (1996); and Patrick Foran et al., Botulinum
neurotoxin C1 cleaves both syntaxin and SNAP-25 in intact and
permeabilized chromaffin cells: correlation with its blockade of
catecholamine release, 35(8) Biochemistry 2630-2636 (1996). Other
non-limiting examples include assays that measure inhibition of
hormone release from endocrine cells, such as, e.g., anterior
pituitary cells or ovarian cells. It is understood that these and
similar assays for molecule release can be useful in identifying
cells expressing endogenous or exogenous or BoNT/A receptors.
[0133] Assays that detect the cleavage of a BoNT/A substrate after
exposure to a BoNT/A can also be used to assess whether a cell is
expressing one or more endogenous or exogenous BoNT/A receptors. In
these assays, generation of a BoNT/A substrate cleavage-product, or
disappearance of the intact BoNT/A substrate, would be detected in
cells expressing a BoNT/A receptor after BoNT/A treatment.
Non-limiting examples of specific Western blot analysis, as well as
well-characterized reagents, conditions and protocols are readily
available from commercial vendors that include, without limitation,
Amersham Biosciences, Piscataway, N.J.; Bio-Rad Laboratories,
Hercules, Calif.; Pierce Biotechnology, Inc., Rockford, Ill.;
Promega Corporation, Madison, Wis., and Stratagene, Inc., La Jolla,
Calif. It is understood that these and similar assays for BoNT/A
substrate cleavage can be useful in identifying cells expressing
endogenous or exogenous BoNT/A receptors.
[0134] As non-limiting examples, Western blot analysis using an
antibody that recognizes BoNT/A SNAP-25-cleaved product or both the
cleaved and uncleaved forms of SNAP-25 can be used to assay for
uptake of BoNT/A. Examples of .alpha.-SNAP-25 antibodies useful for
these assays include, without limitation, SMI-81 .alpha.-SNAP-25
mouse monoclonal antibody (Sternberger Monoclonals Inc.,
Lutherville, Md.), CI 71.1 mouse .alpha.-SNAP-25 monoclonal
antibody (Synaptic Systems, Goettingen, Germany), CI 71.2
.alpha.-SNAP-25 mouse monoclonal antibody (Synaptic Systems,
Goettingen, Germany), SP12 .alpha.-SNAP-25 mouse monoclonal
antibody (Abcam, Cambridge, Mass.), .alpha.-SNAP-25 rabbit
polyclonal antiserum (Synaptic Systems, Goettingen, Germany),
.alpha.-SNAP-25 rabbit polyclonal antiserum S9684 (Sigma, St.
Louis, Mo.), and .alpha.-SNAP-25 rabbit polyclonal antiserum
(Abcam, Cambridge, Mass.).
[0135] Aspects of the present disclosure provide cells that through
genetic manipulation or recombinant engineering are made to express
an exogenous SNAP-25 and/or one or more exogenous BoNT/A receptors.
Cells useful to express an exogenous SNAP-25 and/or one or more
exogenous BoNT/A receptors through genetic manipulation or
recombinant engineering include neuronal cells and non-neuronal
cells that may or may not express an endogenous SNAP-25 and/or one
or more endogenous BoNT/A receptors. It is further understood that
such genetically manipulated or recombinantly engineered cells may
express an exogenous SNAP-25 and one or more exogenous BoNT/A
receptors under control of a constitutive, tissue-specific,
cell-specific or inducible promoter element, enhancer element or
both. It is understood that any cell is useful as long as the cell
can be genetically manipulated or recombinantly engineered to
expresses an exogenous SNAP-25 and/or one or more exogenous BoNT/A
receptors and is capable of undergoing BoNT/A intoxication.
[0136] Methods useful for introducing into a cell an exogenous
polynucleotide molecule encoding a component necessary for the
cells to undergo the overall cellular mechanism whereby a BoNT/A
proteolytically cleaves a SNAP-25 substrate, such as, e.g., a
SNAP-25, a FGFR2, a FGFR3, or a SV2, include, without limitation,
chemical-mediated delivery methods, such as, e.g., calcium
phosphate-mediated, diethyl-aminoethyl (DEAE) dextran-mediated,
lipid-mediated, polyethyleneimine (PEI)-mediated,
polylysine-mediated and polybrene-mediated; physical-mediated
delivery methods, such as, e.g., biolistic particle delivery,
microinjection, protoplast fusion and electroporation; and
viral-mediated delivery methods, such as, e.g., retroviral-mediated
transfection, see e.g., Introducing Cloned Genes into Cultured
Mammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds.,
Molecular Cloning A Laboratory Manual, Vol. 3, 3.sup.rd ed. 2001);
Alessia Colosimo et al., Transfer and Expression of Foreign Genes
in Mammalian Cells, 29(2) Biotechniques 314-318, 320-322, 324
(2000); Philip Washbourne & A. Kimberley McAllister, Techniques
for Gene Transfer into Neurons, 12(5) Curr. Opin. Neurobiol.
566-573 (2002); and Current Protocols in Molecular Biology, John
Wiley and Sons, pp 9.16.4-9.16.11 (2000), each of which is
incorporated by reference in its entirety. One skilled in the art
understands that selection of a specific method to introduce a
polynucleotide molecule into a cell will depend, in part, on
whether the cell will transiently or stably contain a component
necessary for the cells to undergo the overall cellular mechanism
whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate.
Non-limiting examples of polynucleotide molecule encoding a
component necessary for the cells to undergo the overall cellular
mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25
substrate as follows: FGFR2 polynucleotide molecule of SEQ ID NO:
130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO:
134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, or SEQ ID NO:
138; FGFR3 polynucleotide molecule of SEQ ID NO: 139, SEQ ID NO:
140, or SEQ ID NO: 141; SV2 polynucleotide molecule of SEQ ID NO:
142, SEQ ID NO: 143, or SEQ ID NO: 144; and SNAP-25 polynucleotide
molecule of SEQ ID NO: 145, or SEQ ID NO: 146.
[0137] Chemical-mediated delivery methods are well-known to a
person of ordinary skill in the art and are described in, e.g.,
Martin Jordan & Florian Worm, Transfection of Adherent and
Suspended Cells by Calcium Phosphate, 33(2) Methods 136-143 (2004);
Chun Zhang et al., Polyethylenimine Strategies for Plasmid Delivery
to Brain-Derived Cells, 33(2) Methods 144-150 (2004), each of which
is hereby incorporated by reference in its entirety. Such
chemical-mediated delivery methods can be prepared by standard
procedures and are commercially available, see, e.g., CellPhect
Transfection Kit (Amersham Biosciences, Piscataway, N.J.);
Mammalian Transfection Kit, Calcium phosphate and DEAE Dextran,
(Stratagene, Inc., La Jolla, Calif.); Lipofectamine.TM.
Transfection Reagent (Invitrogen, Inc., Carlsbad, Calif.); ExGen
500 Transfection kit (Fermentas, Inc., Hanover, Md.), and SuperFect
and Effectene Transfection Kits (Qiagen, Inc., Valencia,
Calif.).
[0138] Physical-mediated delivery methods are well-known to a
person of ordinary skill in the art and are described in, e.g.,
Jeike E. Biewenga et al., Plasmid-Mediated Gene Transfer in Neurons
using the Biolistics Technique, 71(1) J. Neurosci. Methods. 67-75
(1997); John O'Brien & Sarah C. R. Lummis, Biolistic and
Diolistic Transfection: Using the Gene Gun to Deliver DNA and
Lipophilic Dyes into Mammalian Cells, 33(2) Methods 121-125 (2004);
M. Golzio et al., In Vitro and In Vivo Electric Field-Mediated
Permeabilization, Gene Transfer, and Expression, 33(2) Methods
126-135 (2004); and Oliver Gresch et al., New Non-Viral Method for
Gene Transfer into Primary Cells, 33(2) Methods 151-163 (2004),
each of which is hereby incorporated by reference in its
entirety.
[0139] Viral-mediated delivery methods are well-known to a person
of ordinary skill in the art and are described in, e.g., Chooi M.
Lai et al., Adenovirus and Adeno-Associated Virus Vectors, 21(12)
DNA Cell Biol. 895-913 (2002); Ilya Frolov et al., Alphavirus-Based
Expression Vectors: Strategies and Applications, 93(21) Proc. Natl.
Acad. Sci. U.S.A. 11371-11377 (1996); Roland Wolkowicz et al.,
Lentiviral Vectors for the Delivery of DNA into Mammalian Cells,
246 Methods Mol. Biol. 391-411 (2004); A. Huser & C. Hofmann,
Baculovirus Vectors: Novel Mammalian Cell Gene-Delivery Vehicles
and Their Applications, 3(1) Am. J. Pharmacogenomics 53-63 (2003);
Tiziana Tonini et al., Transient Production of Retro viral-and
Lentiviral-Based Vectors for the Transduction of Mammalian Cells,
285 Methods Mol. Biol. 141-148 (2004); Manfred Gossen & Hermann
Bujard, Tight Control of Gene Expression in Eukaryotic Cells by
Tetracycline-Responsive Promoters, U.S. Pat. No. 5,464,758; Hermann
Bujard & Manfred Gossen, Methods for Regulating Gene
Expression, U.S. Pat. No. 5,814,618; David S. Hogness,
Polynucleotides Encoding Insect Steroid Hormone Receptor
Polypeptides and Cells Transformed With Same, U.S. Pat. No.
5,514,578; David S. Hogness, Polynucleotide Encoding Insect
Ecdysone Receptor, U.S. Pat. No. 6,245,531; Elisabetta Vegeto et
al., Progesterone Receptor Having C. Terminal Hormone Binding
Domain Truncations, U.S. Pat. No. 5,364,791; Elisabetta Vegeto et
al., Mutated Steroid Hormone Receptors, Methods for Their Use and
Molecular Switch for Gene Therapy, U.S. Pat. No. 5,874,534, each of
which is hereby incorporated by reference in its entirety. Such
viral-mediated delivery methods can be prepared by standard
procedures and are commercially available, see, e.g., VIRAPOWER.TM.
Adenoviral Expression System (Invitrogen, Inc., Carlsbad, Calif.)
and VIRAPOWER.TM. Adenoviral Expression System Instruction Manual
25-0543 version A, Invitrogen, Inc., (Jul. 15, 2002); and
ADEASY.TM. Adenoviral Vector System (Stratagene, Inc., La Jolla,
Calif.) and ADEASY.TM. Adenoviral Vector System Instruction Manual
064004f, Stratagene, Inc. Furthermore, such viral delivery systems
can be prepared by standard methods and are commercially available,
see, e.g., BD.TM. Tet-Off and Tet-On Gene Expression Systems (BD
Biosciences-Clonetech, Palo Alto, Calif.) and BD.TM. Tet-Off and
Tet-On Gene Expression Systems User Manual, PT3001-1, BD
Biosciences Clonetech, (Mar. 14, 2003), GeneSwitch.TM. System
(Invitrogen, Inc., Carlsbad, Calif.) and GENESWITCH.TM. System A
Mifepristone-Regulated Expression System for Mammalian Cells
version D, 25-0313, Invitrogen, Inc., (Nov. 4, 2002); VIRAPOWER.TM.
Lentiviral Expression System (Invitrogen, Inc., Carlsbad, Calif.)
and VIRAPOWER.TM. Lentiviral Expression System Instruction Manual
25-0501 version E, Invitrogen, Inc., (Dec. 8, 2003); and COMPLETE
CONTROL.RTM. Retroviral Inducible Mammalian Expression System
(Stratagene, La Jolla, Calif.) and COMPLETE CONTROL.RTM. Retroviral
Inducible Mammalian Expression System Instruction Manual,
064005e.
[0140] As mentioned above, an exogenous component necessary for the
cells to undergo the overall cellular mechanism whereby a BoNT/A
proteolytically cleaves a SNAP-25 substrate, such as, e.g., a
SNAP-25, a FGFR2, a FGFR3, or a SV2 disclosed in the present
specification can be introduced into a cell. Any and all methods
useful for introducing such an exogenous component with a delivery
agent into a cell population can be useful with the proviso that
this method introduce the exogenous component disclosed in the
present specification in at least 50% of the cells within a given
cell population. Thus, aspects of this embodiment can include a
cell population in which, e.g., at least 50%, at least 60%, at
least 70%, at least 80%, or at least 90% of the given cell
population contains an exogenous component necessary for the cells
to undergo the overall cellular mechanism whereby a BoNT/A
proteolytically cleaves a SNAP-25 substrate, such as, e.g., a
SNAP-25, a FGFR2, a FGFR3, or a SV2 disclosed in the present
specification. As used herein, the term "delivery agent" refers to
any molecule that enables or enhances internalization of a
covalently-linked, non-covalently-linked or in any other manner
associated with a polypeptide into a cell. Thus, the term "delivery
agent" encompasses, without limitation, proteins, peptides,
peptidomimetics, small molecules, polynucleotide molecules,
liposomes, lipids, viruses, retroviruses and cells that, without
limitation, transport a covalently or non-covalently linked
molecule to the cell membrane, cell cytoplasm or nucleus. It
further is understood that the term "delivery agent" encompasses
molecules that are internalized by any mechanism, including
delivery agents which function via receptor mediated endocytosis
and those which are independent of receptor mediated
endocytosis.
[0141] A delivery agent can also be an agent that enables or
enhances cellular uptake of a covalently linked component, like
FGFR2, FGFR3, SV2, or SNAP-25, such as, e.g., by chemical
conjugation or by genetically produced fusion proteins. Methods
that covalently link delivery agents and methods of using such
agents are described in, e.g., Steven F. Dowdy, Protein
Transduction System and Methods of Use Thereof, International
Publication No WO 00/34308; Gerard Chassaing & Alain
Prochiantz, Peptides which can be Used as Vectors for the
Intracellular Addressing of Active Molecules, U.S. Pat. No.
6,080,724; Alan Frankel et al., Fusion Protein Comprising
TAT-derived Transport Moiert, U.S. Pat. No. 5,674,980; Alan Frankel
et al., TAT-derived Transport Polypeptide Conjugates, U.S. Pat. No.
5,747,641; Alan Frankel et al., TAT-derived Transport Polypeptides
and Fusion Proteins, U.S. Pat. No. 5,804,604; Peter F. J. O'Hare et
al., Use of Transport Proteins, U.S. Pat. No. 6,734,167; Yao-Zhong
Lin & Jack J. Hawiger, Method for Importing Biologically Active
Molecules into Cells, U.S. Pat. No. 5,807,746; Yao-Zhong Lin &
Jack J. Hawiger, Method for Importing Biologically Active Molecules
into Cells, U.S. Pat. No. 6,043,339; Yao-Zhong Lin et al., Sequence
and Method for Genetic Engineering of Proteins with Cell Membrane
Translocating Activity, U.S. Pat. No. 6,248,558; Yao-Zhong Lin et
al., Sequence and Method for Genetic Engineering of Proteins with
Cell Membrane Translocating Activity, U.S. Pat. No. 6,432,680; Jack
J. Hawiger et al., Method for Importing Biologically Active
Molecules into Cells, U.S. Pat. No. 6,495,518; Yao-Zhong Lin et
al., Sequence and Method for Genetic Engineering of Proteins with
Cell Membrane Translocating Activity, U.S. Pat. No. 6,780,843;
Jonathan B. Rothbard & Paul A Wender, Method and Composition
for Enhancing Transport Across Biological Membranes, U.S. Pat. No.
6,306,993; Jonathan B. Rothbard & Paul A Wender, Method and
Composition for Enhancing Transport Across Biological Membranes,
U.S. Pat. No. 6,495,663; and Pamela B. Davis et al., Fusion
Proteins for Protein Delivery, U.S. Pat. No. 6,287,817, each of
which is incorporated by reference in its entirety.
[0142] A delivery agent can also be an agent that enables or
enhances cellular uptake of a non-covalently associated component,
like FGFR2, FGFR3, SV2c, or SNAP-25. Methods that function in the
absence of covalent linkage and methods of using such agents are
described in, e.g., Gilles Divita et al, Peptide-Mediated
Transfection Agents and Methods of Use, U.S. Pat. No. 6,841,535;
Philip L Feigner and Olivier Zelphati, Intracellular Protein
Delivery Compositions and Methods of Use, U.S. Patent Publication
No. 2003/0008813; and Michael Karas, Intracellular Delivery of
Small Molecules, Proteins and Nucleic Acids, U.S. Patent
Publication 2004/0209797, each of which is incorporated by
reference in its entirety. Such peptide delivery agents can be
prepared and used by standard methods and are commercially
available, see, e.g. the CHARIOT.TM. Reagent (Active Motif,
Carlsbad, Calif.); BIO-PORTER.RTM. Reagent (Gene Therapy Systems,
Inc., San Diego, Calif.), BIO TREK.TM. Protein Delivery Reagent
(Stratagene, La Jolla, Calif.), and PRO-JECT.TM. Protein
Transfection Reagent (Pierce Biotechnology Inc., Rockford,
Ill.).
[0143] Aspects of the present disclosure can also be described as
follows: [0144] 1. An established clonal cell line comprising a
cell susceptible to intoxication by BoNT/A. [0145] 2. The
established clonal cell line of 1, wherein the clonal cell line is
selected from a parental SiMa cell line. [0146] 3. The established
clonal cell line of 1 or 2, wherein the cell is susceptible to
BoNT/A intoxication by about 100 pM or less, by about 50 pM or
less, by about 10 pM or less, by about 5 pM or less, by about 1 pM
or less, by about 0.5 pM or less, or by about 0.1 pM or less of a
BoNT/A. [0147] 4. The established clonal cell line of 1 or 2,
wherein susceptibility to BoNT/A intoxication is measured before
differentiation of the cell. [0148] 5. The established clonal cell
line of 2, wherein in the undifferentiated state, the cell exhibits
at least a 1.5-fold increase in gene expression of one or more
genes relative to the expression of the one or more genes in a cell
from a 2D6 cell line, the genes being taken from Table 5, or
wherein the cell exhibits at least a 1.5-fold decrease in gene
expression of one or more genes relative to the expression of the
one or more genes in a cell from a 2D6 cell line, the genes being
taken from Table 6. [0149] 6. The established clonal cell line of
2, wherein in the differentiated state, the cell exhibits at least
a 1.5-fold increase in gene expression of one or more genes
relative to the expression of the one or more genes in a cell from
a 2D6 cell line, the genes being taken from Table 9, or wherein the
cell exhibits at least a 1.5-fold decrease in gene expression of
one or more genes relative to the expression of the one or more
genes in a cell from a 2D6 cell line, the genes being taken from
Table 10. [0150] 7. The established clonal cell line of 2, wherein
in the undifferentiated state, the cell exhibits at least a
1.5-fold increase in gene expression of one or more genes relative
to the expression of the one or more genes in a cell from a
parental SiMa cell line, the genes being taken from Table 7, or
wherein the cell exhibits at least a 1.5-fold decrease in gene
expression of one or more genes relative to the expression of the
one or more genes in a cell from a parental SiMa cell line, the
genes being taken from Table 8. [0151] 8. The established clonal
cell line of 2, wherein in the differentiated state, the cell
exhibits at least a 1.5-fold increase in gene expression of one or
more genes relative to the expression of the one or more genes in a
cell from a parental SiMa cell line, the genes being taken from
Table 11, or wherein the cell exhibits at least a 1.5-fold decrease
in gene expression of one or more genes relative to the expression
of the one or more genes in a cell from a parental SiMa cell line,
the genes being taken from Table 12. [0152] 9. The established
clonal cell line of 1 or 2, wherein the cell exhibits a sensitivity
for BoNT/A activity that is 100 pM or less or about 25 pM for about
5 or more cell passages, 10 or more cell passages, 15 or more cell
passages, 20 or more cell passages, 25 or more cell passages, 30 or
more cell passages, 35 or more cell passages, 40 or more cell
passages, 45 or more cell passages, 50 or more cell passage, 55 or
more cell passage, or 60 or more cell passage. [0153] 10. The
established clonal cell line of 1 or 2, wherein the cell from an
established clonal cell line exhibits a well defined signal to
noise ratio for the upper asymptote for BoNT/A activity of at least
3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at
least 8:1, at least 9:1, at least 10:1, over, e.g., 5 or more cell
passages, 10 or more cell passages, 15 or more cell passages, 20 or
more cell passages, 25 or more cell passages, 30 or more cell
passages, 35 or more cell passages, 40 or more cell passages, 45 or
more cell passages, 50 or more cell passage, 55 or more cell
passage, or 60 or more cell passage. [0154] 11. An established
clonal cell line comprising a cell susceptible to intoxication by
BoNT/A produced from a parental SiMa cell line, wherein the cell is
susceptible to BoNT/A intoxication by about 100 pM or less, by
about 50 pM or less, by about 10 pM or less, by about 5 pM or less,
by about 1 pM or less, by about 0.5 pM or less, or by about 0.1 pM
or less of a BoNT/A. [0155] 12. An established clonal cell line
comprising cells susceptible to intoxication by BoNT/A, wherein the
clonal cell line is selected from a parental SiMa cell line; and
wherein the clonal cell line comprises cells susceptible to BoNT/A
intoxication by about 100 pM or less of a BoNT/A. [0156] 13. The
established clonal cell line of 12, wherein the parental SiMa cell
line is the parental SiMa cell line DSMZ ACC 164. [0157] 14. The
established clonal cell line of 12, wherein the cell exhibits a
sensitivity for BoNT/A activity that is 100 pM or less or about 25
pM for about 5 or more cell passages, 10 or more cell passages, 15
or more cell passages, 20 or more cell passages, 25 or more cell
passages, 30 or more cell passages, 35 or more cell passages, 40 or
more cell passages, 45 or more cell passages, 50 or more cell
passage, 55 or more cell passage, or 60 or more cell passage.
[0158] 15. The established clonal cell line of 12, wherein the cell
from an established clonal cell line exhibits a well defined signal
to noise ratio for the upper asymptote for BoNT/A activity of at
least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1,
at least 8:1, at least 9:1, at least 10:1, over, e.g., 5 or more
cell passages, 10 or more cell passages, 15 or more cell passages,
20 or more cell passages, 25 or more cell passages, 30 or more cell
passages, 35 or more cell passages, 40 or more cell passages, 45 or
more cell passages, 50 or more cell passage, 55 or more cell
passage, or 60 or more cell passage. [0159] 16. The established
clonal cell line of 12, wherein susceptibility to BoNT/A
intoxication is measured before differentiation of the cell or
after differentiation of the cell. [0160] 17. An established clonal
cell line comprising cells susceptible to intoxication by BoNT/A,
wherein the cells exhibits at least a 1.5-fold difference in gene
expression levels of three or more genes as compared to the
expression levels of these genes in cells from the 2D6 cell line;
wherein the gene expression levels are selected from the group
ADAMTS9, ASCL1, BASP1, DOK5, DPYD, GNB4, GNG11, GTSF1, MAOA, MINA,
MSN, PEG3, PLK2, PRSS12, RNF182, SLC44A5, SPARC, TFPI2, and TPTE;
and wherein the clonal cell line comprises cells susceptible to
BoNT/A intoxication by about 100 pM or less of a BoNT/A. [0161] 18.
The established clonal cell line of 17, wherein the cells exhibits
at least a 2.0-fold difference in gene expression levels of one or
more genes as compared to the expression levels of these genes in
cells from the 2D6 cell line, at least a 3.0-fold difference in
gene expression levels of one or more genes as compared to the
expression levels of these genes in cells from the 2D6 cell line,
or at least a 4.0-fold difference in gene expression levels of one
or more genes as compared to the expression levels of these genes
in cells from the 2D6 cell line. [0162] 19. The established clonal
cell line of 17, wherein the gene expression levels are measured
before differentiation of the cell or after differentiation of the
cell. [0163] 20. The established clonal cell line of 17, wherein
the cell exhibits a sensitivity for BoNT/A activity that is 100 pM
or less or about 25 pM for about 5 or more cell passages, 10 or
more cell passages, 15 or more cell passages, 20 or more cell
passages, 25 or more cell passages, 30 or more cell passages, 35 or
more cell passages, 40 or more cell passages, 45 or more cell
passages, 50 or more cell passage, 55 or more cell passage, or 60
or more cell passage. [0164] 21. The established clonal cell line
of 17, wherein the cell from an established clonal cell line
exhibits a well defined signal to noise ratio for the upper
asymptote for BoNT/A activity of at least 3:1, at least 4:1, at
least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1,
at least 10:1, over, e.g., 5 or more cell passages, 10 or more cell
passages, 15 or more cell passages, 20 or more cell passages, 25 or
more cell passages, 30 or more cell passages, 35 or more cell
passages, 40 or more cell passages, 45 or more cell passages, 50 or
more cell passage, 55 or more cell passage, or 60 or more cell
passage.
EXAMPLES
Example I
Screening for Cell Lines Comprising Cells Susceptible to BoNT/A
Intoxication
[0165] The following example illustrates how to identify clonal
cells from a parental established cell line that are susceptible to
BoNT/A intoxication or have neurotoxin uptake capacity.
1. Isolation of Clonal Cell Lines.
[0166] Companion patent application Ester Fernandez-Salas, et al.,
Immuno-Based Botulinum Toxin Serotype A Activity Assays, U.S.
patent application Ser. No. 12/403,531 identified several
established cell lines useful for conducting the disclosed
immuno-based methods of detecting BoNT/A activity, including, e.g.,
LA1-55n (ECACC 06041203), N18 (ECACC 88112301), Neuro-2a (ATCC
CCL-131), PC12 (ATCC CRL-1721), SH-SY5Y (ATCC CRL-2266), and SiMa
(DSMZ ACC 164). During characterization of the SiMa cell line, it
was discovered that the cells comprising this established cell line
comprised at least five different cellular phenotypes. To determine
whether any one of these phenotypically-distinct cell types was
responsible for the susceptibility of this cell line to BoNT/A
intoxication, two different limited-dilution screens were conducted
to obtain single colony isolates for each phenotypically-distinct
cell type.
[0167] In both screens, a suitable density of cells from a SiMa
stock were grown in RPMI 1640 medium, having 10% fetal bovine
serum, 1% Penicillin-Streptomycin, 2 mM L-Glutamine, contained in a
T175 Collagen IV coated flask. After the second passage, the cells
were trypsin-treated to produce a cell suspension and the cell
concentration was determined. About 4.0.times.10.sup.6 cells from
this cell suspension was transferred into a 50 mL tube and the
cells were dissociated into single cells by repeated vigorous
expulsion through an 18.5 gauge needle using a 10 mL syringe. Cells
from this disassociated single-cell suspension were then diluted to
a concentration of 0.2.times.10.sup.6 cells/mL by adding 15 mL of
fresh growth medium, and 2.5 .mu.L of this dilution was added to 50
mL of fresh growth medium to obtain a concentration of 10 cells/mL.
From this final dilution stock, 100 .mu.L of growth medium was
added to each well of a 96-well Collagen IV coated plates (final
average density of one cell/well), and the cells were grown
undisturbed in a 37.degree. C. incubator under 5% carbon dioxide
for four weeks. A total of nineteen 96-well plates were setup for
analysis. Plates were microscopically examined periodically to
assess colony growth. After four weeks, each well was
microscopically examined to identify growing colonies, and for each
growing colony identified, 100 .mu.L of fresh growth medium was
added to each well and the cells were grown undisturbed in a
37.degree. C. incubator under 5% carbon dioxide for two weeks.
After two additional weeks of growth, the growing single colonies
were trypsin-treated and transferred to a new 96-well plate for
continued growth. Once colonies grew to about 1,000 cells, based on
visual inspection, the cells were trypsin-treated and each
cell-suspension was transferred into a new well from a 24-well
Collagen IV-coated plate. The cells were grown in a 37.degree. C.
incubator under 5% carbon dioxide with fresh growth medium being
replenished every 2-3 days, if needed. The cells were grown until
the culture reached approximately 60% confluence or greater, at
which point the cells were trypsin-treated and each cell-suspension
was transferred into a 25 cm.sup.2 Collagen IV-coated flask. The
cells were grown in a 37.degree. C. incubator under 5% carbon
dioxide with fresh growth medium being replenished every 2-3 days,
if needed. Once the cells in the flask reached 70-80% confluence,
they were frozen and stored in liquid nitrogen until the clonal
cell lines were tested to determine their susceptibility to BoNT/A
intoxication. Of the 1,824 colony isolates initially setup from
both screens, 130 clonal cell lines were selected based on
viability and growth criteria and expanded for subsequent screening
procedures.
2. Primary Screen for BoNT/A Intoxication Susceptibility of Cells
from a Clonal Cell Line Using a BoNT/A Complex.
[0168] One way to determine whether cells from a clonal cell line
were susceptible to BoNT/A intoxication was to conduct a primary
screen using a CELLTITER-GLO.RTM. luminescent cell viability assay
(Promega, Madison, Wis.) for normalizing the cell number in each
well and an immuno-based method for determining BoNT/A activity as
described in Ester Fernandez-Salas, et al., Immuno-Based Botulinum
Toxin Serotype A Activity Assays, U.S. patent application Ser. No.
12/403,531, which is hereby incorporated by reference in its
entirety. Two separate 96-well plates were set up, one for the
BoNT/A activity assay and the other for the cell viability
assay.
[0169] To conduct the BoNT/A activity assay, a lysate was prepared
from cells treated with BoNT/A by plating cells from each isolated
clonal cell line into a well from a poly-D-lysine 96-well plate (BD
Biosciences, Bedford, Mass.). The wells contained 0.1 mL of a
serum-free medium comprising Minimum Essential Medium, 2 mM
GlutaMAX.TM. I with Earle's salts, 1.times.B27 supplement,
1.times.N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mM
HEPES and 25 .mu.g/mL GT1b (302-011-M005, Alexis Biochemicals, San
Diego, Calif.). These cells were incubated in a 37.degree. C.
incubator under 5% carbon dioxide until the cells differentiated,
as assessed by standard and routine morphological criteria, such as
growth arrest and neurite extension (approximately 3 days). The
media from the differentiated cells was aspirated from each well
and replaced with fresh media containing 1 nM of a BoNT/A complex.
After a 24 hrs incubation, the cells were washed by aspirating the
growth media and rinsing each well with 200 .mu.L of 1.times.PBS.
To harvest the cells, 1.times.PBS was aspirated, the cells lysed by
adding 30 .mu.l of Lysis Buffer comprising 20 mM Tris-HCl (pH 7.5),
150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 to each well,
and the plate incubated on a shaker rotating at 500 rpm for 30
minutes at 4.degree. C. The plate was centrifuged at 4000 rpm for
20 minutes at 4.degree. C. to pellet cellular debris and the
supernatant was transferred to a capture antibody coated 96-well
plate to perform the detection step.
[0170] To prepare an .alpha.-SNAP-25 capture antibody solution, the
.alpha.-SNAP-25 mouse monoclonal antibody contained in the ascites
from hybridoma cell line 2E2A6 (Example VII) was purified using a
standard Protein A purification protocol.
[0171] To prepare an .alpha.-SNAP-25 detection antibody solution,
.alpha.-SNAP-25 rabbit polyclonal antibody S9684 (Sigma, St. Louis,
Mo.) was conjugated to
Ruthenium(II)-tris-bipyridine-(4-methysulfonate) NHS ester labeling
reagent (Meso Scale Discovery, Gaithersburg, Md.) according to the
manufacturer's instructions (Meso Scale Discovery, Gaithersburg,
Md.). The conjugation reaction was performed by adding 30 .mu.L of
distilled water reconstituted MSD SULFO-TAG.TM. stock solution to
200 .mu.L of 2 mg/mL .alpha.-SNAP-25 polyclonal antibodies and
incubating the reaction at room temperature for 2 hours in the
dark. The labeled antibodies were purified using a standard spin
column protocol and the protein concentration determined using a
standard colorimetric protein assay. The absorbance of the
.alpha.-SNAP-25 antibody/MSD SULFO-TAG.TM. conjugate was measured
at 455 nm using a spectrophotometer to determine the concentration
in moles per liter. The detection antibody solution was stored at
4.degree. C. until needed.
[0172] To prepare an .alpha.-SNAP-25 solid phase support comprising
an .alpha.-SNAP-25 capture antibody, approximately 5 .mu.L of the
appropriate .alpha.-SNAP-25 monoclonal antibody solution (20
.mu.g/mL in 1.times.PBS) is added to each well of a 96-well MSD
High Bind plate and the solution is allowed to air dry in a
biological safety cabinet for 2-3 hours in order to liquid
evaporate the solution. The capture antibody-bound wells were
sealed and stored at 4.degree. C. until needed.
[0173] To detect the presence of a cleaved SNAP-25 product by ECL
sandwich ELISA, the wells from an .alpha.-SNAP-25 solid phase
support were then blocked by adding 150 .mu.L of Blocking Buffer
comprising 2% Amersham Blocking Reagent (GE Life Sciences,
Piscataway, N.J.) and 10% goat serum (VWR, West Chester, Pa.) at
room temperature for 2 hours. The Blocking Buffer was aspirated, 25
.mu.L of a lysate from cells treated with BoNT/A was added to each
well and the plates were incubated at 4.degree. C. for overnight.
Plate wells were washed three times by aspirating the cell lysate
and rinsing each well three times with 200 .mu.L 1.times.PBS, 0.1%
TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After
washing, 25 .mu.l of 5 .mu.g/mL .alpha.-SNAP-25 detection antibody
solution comprising 2% Amersham Blocking Reagent in 1.times.PBS,
0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate) was
added to each well, sealed, and incubated at room temperature for 1
hour with shaking. After .alpha.-SNAP-25 detection antibody
incubation, the wells were washed three times with 200 .mu.L
1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan
monolaureate). After washing 150 .mu.L of 1.times. Read Buffer
(Meso Scale Discovery, Gaithersburg, Md.) was added to each well
and the plates were read using a SECTOR.TM. Imager 6000 Image
Reader (Meso Scale Discovery, Gaithersburg, Md.). The raw data was
collected using the ECL imager.
[0174] To conduct the luminescent cell viability assay, cells from
each isolated clonal cell line were plated and differentiated as
described above. The media from the differentiated cells was
aspirated from each well and replaced with 100 .mu.l of the reagent
mixture comprising the CELLTITER-GLO.RTM. substrate and
CELLTITER-GLO.RTM. buffer according to the manufacturer's
instructions. The plate was then protected from light and mixed for
two minutes on an orbital shaker. The solution from the 96-well
cell plate with clear bottom was transferred to a white luminometer
plate and after 10 minutes incubation was read on the
luminometer.
[0175] These results indicated that the cell viability readings
ranged from 9,838 to 10,423,188 and BoNT/A activity signals ranged
from 84 to 27,242 (Table 1). In addition, a ratio of BoNT/A
activity over cell viability was calculated to normalize the data,
although there was not always a 1:1 ratio of viability and
activity. Seven clonal cell lines exhibited activity/viability
ratios of about 0.2.times.10.sup.-2 or greater: H1, A7, A11, F10,
A9, A10, and C11 (Table 1). Nine clonal cell lines exhibited
activity/viability ratios of about 0.125.times.10.sup.-2 to about
0.150.times.10.sup.-2: H3, H8, E11, H10, A6, C5, C12, D11, and B5
(Table 1). Two cell lines exhibited activity/viability ratios of
about 0.100.times.10.sup.-2: D7 and B10 (Table 1). Based on these
activity/viability ratios in conjunction with the cell viability
and BoNT/A activity signal measurements, these 18 clonal cell lines
were selected for further testing by conducting a secondary screen
using a BoNT/A activity assay in order to generate a dose-response
curve to BoNT/A.
TABLE-US-00001 TABLE 1 Primary Screen for Clonal Cell Lines
Susceptible to BoNT/A Intoxication Using Viability and Activity
Signals Cell Viability BoNT/A Reading Activity Activity/Viability
Well (RLU) Signal (RLU) Ratio A1 2634417 2425 0.092 .times.
10.sup.-2 B1 731808 198 0.027 .times. 10.sup.-2 C1 4258160 1641
0.039 .times. 10.sup.-2 D1 6554606 4885 0.075 .times. 10.sup.-2 E1
2374483 897 0.038 .times. 10.sup.-2 F1 2801444 1842 0.066 .times.
10.sup.-2 G1 5094510 4579 0.090 .times. 10.sup.-2 H1 3808699 10861
0.285 .times. 10.sup.-2 A2 9593106 7090 0.074 .times. 10.sup.-2 B2
5374966 5673 0.106 .times. 10.sup.-2 C2 4170158 2972 0.071 .times.
10.sup.-2 D2 31283 88 0.281 .times. 10.sup.-2 E2 5706902 3743 0.066
.times. 10.sup.-2 F2 4884798 4568 0.094 .times. 10.sup.-2 G2
3552045 2190 0.062 .times. 10.sup.-2 H2 3743690 3421 0.091 .times.
10.sup.-2 A3 15372 89 0.579 .times. 10.sup.-2 B3 5381672 1551 0.029
.times. 10.sup.-2 C3 4270534 3959 0.093 .times. 10.sup.-2 D3 48702
97 0.199 .times. 10.sup.-2 E3 4707349 2671 0.057 .times. 10.sup.-2
F3 3053132 1539 0.050 .times. 10.sup.-2 G3 5518292 4195 0.076
.times. 10.sup.-2 H3 4156632 6339 0.153 .times. 10.sup.-2 A4 19793
89 0.450 .times. 10.sup.-2 B4 6224766 4299 0.069 .times. 10.sup.-2
C4 3212859 3202 0.100 .times. 10.sup.-2 D4 4376180 2367 0.054
.times. 10.sup.-2 E4 3931793 3975 0.101 .times. 10.sup.-2 F4
1150134 526 0.046 .times. 10.sup.-2 G4 19023 94 0.494 .times.
10.sup.-2 H4 3368425 2645 0.079 .times. 10.sup.-2 A5 8231689 7809
0.095 .times. 10.sup.-2 B5 5332004 6545 0.123 .times. 10.sup.-2 C5
3869441 5042 0.130 .times. 10.sup.-2 D5 2554300 1161 0.045 .times.
10.sup.-2 E5 3781403 764 0.020 .times. 10.sup.-2 F5 4608801 3720
0.081 .times. 10.sup.-2 G5 7105403 7330 0.103 .times. 10.sup.-2 H5
5585607 7544 0.135 .times. 10.sup.-2 A6 7698708 10152 0.132 .times.
10.sup.-2 B6 4706560 3196 0.068 .times. 10.sup.-2 C6 5866251 6145
0.105 .times. 10.sup.-2 D6 2285787 768 0.034 .times. 10.sup.-2 E6
2471979 1038 0.042 .times. 10.sup.-2 F6 10958 91 0.830 .times.
10.sup.-2 G6 19590 91 0.465 .times. 10.sup.-2 H6 2057687 2564 0.125
.times. 10.sup.-2 A7 10132753 27242 0.269 .times. 10.sup.-2 B7
2977371 1642 0.055 .times. 10.sup.-2 C7 5746577 4878 0.085 .times.
10.sup.-2 D7 5861338 6044 0.103 .times. 10.sup.-2 E7 2819910 1399
0.050 .times. 10.sup.-2 F7 23523 84 0.357 .times. 10.sup.-2 G7
6232344 4031 0.065 .times. 10.sup.-2 H7 4916756 2759 0.056 .times.
10.sup.-2 A8 5945833 5611 0.094 .times. 10.sup.-2 B8 4591002 2128
0.046 .times. 10.sup.-2 C8 5648241 8758 0.155 .times. 10.sup.-2 D8
4207887 3360 0.080 .times. 10.sup.-2 E8 5894699 3844 0.065 .times.
10.sup.-2 F8 5585487 4651 0.083 .times. 10.sup.-2 G8 4758800 4545
0.096 .times. 10.sup.-2 H8 5439137 8017 0.147 .times. 10.sup.-2 A9
10423188 20706 0.199 .times. 10.sup.-2 B9 6493893 4444 0.068
.times. 10.sup.-2 C9 5742047 5788 0.101 .times. 10.sup.-2 D9
3424405 1848 0.054 .times. 10.sup.-2 E9 5771943 8064 0.140 .times.
10.sup.-2 F9 24524 88 0.359 .times. 10.sup.-2 G9 7291510 6674 0.092
.times. 10.sup.-2 H9 4169883 6048 0.145 .times. 10.sup.-2 A10
9050811 17617 0.195 .times. 10.sup.-2 B10 5590113 5175 0.093
.times. 10.sup.-2 C10 2325807 1713 0.074 .times. 10.sup.-2 D10
2644116 2589 0.098 .times. 10.sup.-2 E10 4210945 2666 0.063 .times.
10.sup.-2 F10 2932749 6390 0.218 .times. 10.sup.-2 G10 3980127 3114
0.078 .times. 10.sup.-2 H10 4519035 6187 0.137 .times. 10.sup.-2
A11 9870025 24690 0.250 .times. 10.sup.-2 B11 4973892 2235 0.045
.times. 10.sup.-2 C11 3326448 6405 0.193 .times. 10.sup.-2 D11
5747961 7169 0.125 .times. 10.sup.-2 E11 5831546 8432 0.145 .times.
10.sup.-2 F11 5291803 4317 0.082 .times. 10.sup.-2 G11 3499587 2790
0.080 .times. 10.sup.-2 H11 3225750 4518 0.140 .times. 10.sup.-2
A12 9838 89 0.905 .times. 10.sup.-2 B12 3728293 2016 0.054 .times.
10.sup.-2 C12 4086862 5308 0.130 .times. 10.sup.-2 D12 5044526 3860
0.077 .times. 10.sup.-2 E12 4787913 2719 0.057 .times. 10.sup.-2
F12 5101695 2585 0.051 .times. 10.sup.-2 G12 2887390 1622 0.056
.times. 10.sup.-2 H12 2829307 5105 0.180 .times. 10.sup.-2
[0176] To conduct BoNT/A activity assay in order to generate a
dose-response curve to BoNT/A, cells from each isolated clonal cell
line were seeded in a poly-D-lysine 96-well plate and
differentiated as described above. Of the top 18 clonal cell lines
selected for further testing, only 8 remained viable, the remaining
10 clonal cell lines failed to regrow and were lost. The media from
the differentiated cells was aspirated from each well and replaced
with fresh media containing either 0 (untreated sample), 0.03 pM,
0.1 pM, 0.3 pM, 0.9 pM, 2.8 pM, 8.3 pM, and 25 pM of a BoNT/A
complex. After a 24 hrs treatment, the cells were washed, incubated
for an additional two days in serum-free medium without toxin to
allow for the cleavage of the SNAP-25 substrate, and harvested as
described above. The supernatant was transferred to a capture
antibody coated 96-well plate to perform the detection step.
[0177] The .alpha.-SNAP-25 capture antibody solution, the
.alpha.-SNAP-25 detection antibody solution, and the
.alpha.-SNAP-25 solid phase support were prepared as described
above. Detection of the presence of cleaved SNAP-25 product by ECL
sandwich ELISA analysis was performed as described above. The raw
data obtained from the ECL imager was then transferred to SigmaPlot
v. 9.0 and a 4-parameter logistics fit was used to define the
dose-response curves. There were no constraints used for the
4-parameter logistic function when plotting the data. Graphical
reports were generated using the following analysis: R2
(correlation coefficient), a (Max for data set), b (hillslope), and
X0.+-.SE (EC.sub.50 value.+-.standard error).
[0178] These results indicate that of the 8 clonal cell lines
tested, only two were more susceptible to BoNT/A intoxication when
compared to cells comprising the parental SiMa cell line (Table 2).
For example, cells comprising the parental SiMa cell line exhibited
an EC.sub.50 activity for BoNT/A of 2.20 pM, whereas cells
comprising the H1 and A10 cell lines exhibited an EC.sub.50
activity for BoNT/A of 1.76 pM or less. Conversely, cells
comprising the H10 and D11 cell line exhibited an EC.sub.50
activity for BoNT/A that was higher than that observed for cells
comprising the parental SiMa cell line, but less than 5.0 pM. It
should be noted that any clonal cell line exhibiting an EC.sub.50
activity for BoNT/A that is 5.0 pM or less (100 U/mL or less) is
useful for an immuno-based method for determining BoNT/A activity
described in Ester Fernandez-Salas, et al., Immuno-Based Botulinum
Toxin Serotype A Activity Assays, U.S. patent application Ser. No.
12/403,531.
TABLE-US-00002 TABLE 2 Secondary Screen for Clonal Cell Lines
Susceptible to BoNT/A Intoxication BoNT/A Activity Signal.sub.MIN
Clone EC.sub.50 (pM) (RLU) Signal.sub.MAX (RLU) SiMa 2.20 123
47,611 H1 0.77 193 125,252 H3 ND ND ND B5 >25.0 115 22,277 C5 ND
ND ND A6 ND ND ND A7 5.59 153 100,412 D7 ND ND ND H8 ND ND ND A9 ND
ND ND A10 1.76 207 113,029 B10 6.35 147 101,935 F10 >25.0 111
32,663 H10 2.80 171 120,975 A11 ND ND ND C11 ND ND ND D11 4.4 1.84
105,527 E11 ND ND ND C12 ND ND ND ND = Not determined.
3. Primary Screen for BoNT/A Intoxication Susceptibility of Cells
from a Clonal Cell Line Using a Formulated BoNT/A Pharmaceutical
Product.
[0179] Another way to determine whether cells from a clonal cell
line were susceptibility to BoNT/A intoxication was to conduct a
primary screen by generating a dose-response curve to BoNT/A and
calculate the EC.sub.50 value using an immuno-based method for
determining BoNT/A activity as described in Ester Fernandez-Salas,
et al., Immuno-Based Botulinum Toxin Serotype A Activity Assays,
U.S. patent application Ser. No. 12/403,531, which is hereby
incorporated by reference in its entirety.
[0180] Initially six of 35 clonal cell line isolates were examined
using a full dose response to BoNT/A. To prepare a lysate from
cells treated with BoNT/A, cells from six clonal cell line were
seeded in a poly-D-lysine 96-well plate and differentiated as
described above in Section 2 except that the serum-free medium
comprised 60 .mu.g/mL GT1b. The clonal cell lines used were 3D8,
YB8, 1D4, 2D6, 1E11, 2F5. The H1 and H10 clonal cell lines
identified above, along with the parental SiMa cell line, were used
as references to identify cell lines highly susceptible to BoNT/A
intoxication. The media from the differentiated cells was aspirated
from each well and replaced with fresh media containing either 0
(untreated sample), 0.98 U/mL, 1.9 U/mL, 3.91 U/mL, 7.81 U/mL, 15.6
U/mL, 31.3 U/mL, 62.5 U/mL, 125 U/mL, 250 U/mL, or 500 U/mL of a
formulated BoNT/A pharmaceutical product. After a 24 hrs treatment,
the cells were washed, incubated for an additional two days in
serum-free medium without toxin to allow for the cleavage of the
SNAP-25 substrate. To harvest the cells, 1.times.PBS was aspirated,
the cells lysed by adding 30 .mu.l of Lysis Buffer comprising 20 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton
X-100 to each well, and the plate incubated on a shaker rotating at
500 rpm for 30 minutes at 4.degree. C. The plate was centrifuged at
4000 rpm for 20 minutes at 4.degree. C. to pellet cellular debris.
The protein concentration was determined using standard methods.
The supernatant was transferred to a capture antibody coated
96-well plate to perform the detection step of the immuno-based
method.
[0181] The .alpha.-SNAP-25 capture antibody solution, the
.alpha.-SNAP-25 detection antibody solution, and the
.alpha.-SNAP-25 solid phase support were prepared as described
above in Section 2. Detection of the presence of SNAP-25 cleavage
product by ECL sandwich ELISA analysis was performed, collected
data was analyzed and the EC.sub.50 calculated as described above
in Section 2, except that SigmaPlot v. 10 was used.
[0182] These results show that clonal cell line H1 comprises cells
that are more susceptible to BoNT/A than the parental SIMa cell
line (Table 3). In addition, results indicate that although clonal
cell lines H10, 1D4, 1E11, 2D6, 2F5, 3D8, and YB8 all comprise
cells that are less susceptible to BoNT/A than the parental SiMa
cell line, all have an EC.sub.50 of BoNT/A activity that is below
30 U/mL (Table 3). It should be noted that any clonal cell line
exhibiting an EC.sub.50 activity for BoNT/A that is 100 U/mL or
less (5.0 pM or less) is useful for an immuno-based method for
determining BoNT/A activity described in Ester Fernandez-Salas, et
al., Immuno-Based Botulinum Toxin Serotype A Activity Assays, U.S.
patent application Ser. No. 12/403,531.
TABLE-US-00003 TABLE 3 Primary Screen for Clonal Cell Lines
Susceptible to BoNT/A Intoxication BoNT/A Activity Clone EC.sub.50
(U/mL) Signal.sub.MIN (RLU) Signal.sub.MAX (RLU) SiMa 10.8 .+-. 1.0
10,519 253,484 H1 10.2 .+-. 0.6 13,996 276,982 H10 22.2 .+-. 2.0
5,379 296,168 1D4 22.4 .+-. 0.8 6,064 239,096 1E11 14.2 .+-. 1.7
8,698 276,323 2D6 27.3 .+-. 1.8 3,347 179,454 2F5 15.8 .+-. 0.9
7,007 265,461 3D8 13.4 .+-. 0.6 11,658 266,279 YB8 19.7 .+-. 1.3
8,477 293,559
[0183] This initial primary screen using a dose-response curve with
the 10 different BoNT/A concentrations identified three
concentrations that were useful for a shortened assay using 3
different BoNT/A concentrations, with one concentration being
slightly higher than the lower asymptote (8 U/mL), one
concentration near the EC.sub.50 (25 U/mL), and one concentration
representing about 80% of the upper asymptote (80 U/mL). In these
subsequent primary screens the remaining 29 clonal cell line
isolates were tested as described above except that the cells were
treated with either 0 (untreated sample), 8 U/mL, 25 U/mL, or 80
U/mL of a formulated BoNT/A pharmaceutical product. The clonal cell
lines tested were 1E3, 2B9, 2D2, 2E4, 3B8, 3D5, 3G10, 4B5, 4C8,
4D3, 5C10, 5F3, AC9, AF4, BB3, BB10, BE3, BF8, CC11, CD6, CE6, CG8,
CG10, DC4, DD10, DE7, DF5, YB7, and YF5. In addition, the parental
SiMa was used as positive control.
[0184] These results show that clonal cell lines 2E4, 3B8, 3D5,
5F3, AF4, BB3, BB10, and DC4 all comprise cells that are more
susceptible to BoNT/A than the parental SiMa cell line (Table 4).
In addition, these results indicate that clonal cell lines 1E3,
2B9, 3G10, 4B5, 4C8, AC9, BE3, BF8, CC11, CD6, CE6, CG8, CG10,
DD10, DE7, and YF5 all comprise cells that are of similar
susceptibility to BoNT/A to that of the parental SiMa cell line
(Table 4). Lastly, these results reveal that clonal cell lines 2D2,
4D3, 5C10, DF5, and YB7 all comprise cells that are less
susceptible to BoNT/A than the parental SiMa cell line (Table
4).
TABLE-US-00004 TABLE 4 Primary Screen for Clonal Cell Lines
Susceptible to BoNT/A Intoxication 8 U/mL BoNT/A 25 U/mL BoNT/A 80
U/mL BoNT/A Activity Signal Fold Activity Signal Fold Activity
Signal Fold Clone (RLU) Difference (RLU) Difference (RLU)
Difference SiMa 75811/80562* -- 138952/175685* -- 173979/225768* --
1E3 71040 0.9 128771 0.9 155107 0.9 2B9 66962 0.9 135006 1.0 166871
1.0 2D2 60224* 0.8 136947* 0.8 199786* 0.9 2E4 103680 1.4 217980
1.6 270463 1.6 3B8 123521 1.6 227367 1.6 249218 1.4 3D5 113532 1.5
201683 1.5 270578 1.6 3G10 83049 1.1 130930 0.9 175540 1.0 4B5
74344 1.0 137604 1.0 177363 1.0 4C8 87047 1.1 144395 1.0 185115 1.1
4D3 38063 0.5 80180 0.6 101724 0.6 5C10 16024* 0.2 49030 0.3
116396* 0.6 5F3 98263* 1.7 182902* 1.4 212275* 1.5 AC9 60016* 1.0
127030* 1.0 180384* 1.4 AF4 115460 1.5 179901 1.3 190867 1.1 BB3
74668 1.3 120966 1.4 170177 1.4 BB10 152748 2.0 232406 1.7 266555
1.5 BE3 100849 1.0 199487 0.9 236415 1.0 BF8 55763* 1.0 111746* 1.0
153074* 1.3 CC11 54421* 1.0 123576* 1.0 155337* 1.2 CD6 74970 1.0
151862 1.1 183377 1.1 CE6 70019 0.9 146292 1.1 208184 1.2 CG8
81790* 1.0 159098* 1.0 189209* 1.3 CG10 69503 0.9 141079 1.0 185801
1.1 DC4 112085 1.5 163319 1.2 169352 1.0 DD10 116325 1.5 205445 1.5
223473 1.3 DE7 60994* 0.9 131237* 0.9 174487* 0.9 DF5 34,165* 0.5
104,696* 0.7 179,314* 0.9 YB7 50433 0.7 103707 0.7 143423 0.8 YF5
65848 0.9 122487 0.9 146263 0.4 *Two separate screens were done. In
the first, the signal detected for the SiMa cell line was 75811 RLU
and this value was used to determine the fold difference for clonal
cell lines tested in this screen (numbers with no asterisk). For
the second screen, the signal detected for the SiMa cell line was
80562 RLU and this value was used to determine the fold difference
for clonal cell lines tested in this screen (numbers with
asterisk).
Example II
Stability Analysis of Clonal Cell Lines
[0185] The following example illustrates how to characterize the
stability of cells from an established clonal cell line that are
susceptible to BoNT/A intoxication or have neurotoxin uptake
capacity.
[0186] During characterization of the SiMa cell line, it was
determined that the parental SiMa cell line became unstable in
terms of a significant loss of sensitivity to BoNT/A. For example,
prior to passage 13, cells comprising the parental SiMa cell line
routinely exhibited an EC.sub.50 for BoNT/A activity that was under
5.0 pM. However, after passages about 14 to about 20 passages,
cells comprising the parental SiMa cell exhibited an EC.sub.50 for
BoNT/A activity that was routinely over 25.0 pM. One reason for
this dramatic reduction in the EC.sub.50 value may be because the
SiMa cell line is heterogeneous in nature since it comprises at
least five different cell types. Over time, one of these cell types
could overtake the culture to the exclusion of the other four. If
the overtaking cell type was less susceptible to BoNT/A
intoxication, or not susceptible at all, then as this cell type
became the dominant one in the cell line the EC.sub.50 for BoNT/A
activity in this overtaken SiMa cell line would decrease. Thus,
because the established clonal cell lines were derived from only
one of these cell types, then it follows that the clonal cell line
would exhibit greater stability in terms of maintaining an
EC.sub.50 for BoNT/A activity that was routinely under 5.0 pM.
[0187] To determine whether the cells from the established clonal
cell lines showed increased stability relative to the cells
comprising the parental SIMa cell line, the EC.sub.50 for BoNT/A
activity was determined for clonal cell lines BB10, H1, H10, and
the parental SiMa cell line at different passage numbers using an
immuno-based method for determining BoNT/A activity. The media from
the differentiated cells was aspirated from each well and replaced
with fresh media containing either 0 (untreated sample), 0.98 U/mL,
1.9 U/mL, 3.91 U/mL, 7.81 U/mL, 15.6 U/mL, 31.3 U/mL, 62.5 U/mL,
125 U/mL, 250 U/mL, or 500 U/mL of a formulated BoNT/A
pharmaceutical product. After a 24 hrs treatment, the cells were
washed, incubated for an additional two days in serum-free medium
without toxin to allow for the cleavage of the SNAP-25 substrate,
and harvested as described above. The supernatant was transferred
to a capture antibody coated 96-well plate to perform the detection
step.
[0188] Similarly, the preparation of the .alpha.-SNAP-25 capture
antibody solution, .alpha.-SNAP-25 detection antibody solution, and
the solid phase support comprising an .alpha.-SNAP-25 capture
antibody were as described in Section 3. Lastly, detection of the
presence of SNAP-25 cleavage product by ECL sandwich ELISA was
performed, collected data was analyzed, and the EC.sub.50
calculated as described above in Section 2, except that PLA v. 2
was used for the 4-parameter logistics fit.
[0189] The results from this stability experiments indicated that
BB10, H1 and H10 cell lines all exhibited increase stability as
compared to the parental SiMa cell line. For example, where cells
from the parental SiMa cell line exhibited an EC.sub.50 for BoNT/A
activity that was over 500 U/mL (or 25 pM) after passages about 14
to about 20 passages, cells comprising the BB10, H1 and H10 cell
lines all exhibited an EC.sub.50 for BoNT/A activity that was below
10 U/mL (or 0.5 pM) after passage about 30.
Example III
Gene Expression Profile of Cell Lines Comprising Cells Susceptible
to BoNT/A Intoxication
[0190] The following example illustrates how to characterize cells
from an established clonal cell line that are susceptible to BoNT/A
intoxication or have neurotoxin uptake capacity by gene expression
profiling.
[0191] To determine the changes in genome-wide gene transcription
levels in cells susceptible to BoNT/A intoxication, gene expression
analysis was performed on cells from H1, BB10, 2D6 and the parental
SiMa cell line, in both differentiated and undifferentiated states
using GENECHIP.RTM. U133 Plus 2 microarray technology (Affymetrix,
Inc., Santa Clara, Calif.) according to the manufacturer's
instructions, see, e.g., GENECHIP.RTM. Expression Analysis
Technical Manual, 702232, Rev. 2 (2006) which is hereby
incorporated by reference in its entirety.
[0192] To prepare differentiated cells for these cell lines, a
suitable density of cells from the H1, BB10, 2D6, and SiMa cell
lines were seeded in separate T175 tissue culture flasks containing
50 mL of a serum-free medium comprising Enhanced Minimum Essential
Medium, 10 mM HEPES, 1 mM sodium pyruvate, 0.1 mM Non-Essential
Amino Acids, 1.times.B27 supplement, 1.times.N2 supplement, and 60
.mu.g/mL GT1b (302-011-M005, Alexis Biochemicals, San Diego,
Calif.). These cells were incubated in a 37.degree. C. incubator
under 5% carbon dioxide until the cells differentiated, as assessed
by standard and routine morphological criteria, such as growth
arrest and neurite extension, and attained 80% confluence
(approximately 3 days). The cells were then washed with 50 mL of
1.times.PBS, trypsin-treated for 5 min at room temperature, and
then 15 mL of growth medium was added to the flask to inactivate
the trypsin. The cell suspension was transferred to a 50 mL tube
and centrifuge at 200 RCF for 5 min at room temperature to pellet
the cells. The cell pellet was washed once in 50 mL of 1.times.PBS,
centrifuge at 200 RCF for 5 min at room temperature to pellet the
cells, and the supernatant was removed. The cell pellet was quick
frozen on dry ice and stored at -80.degree. C. until needed.
[0193] To prepare undifferentiated cells for these cell lines, a
suitable density of cells from the H1, BB10, 2D6, and SiMa cell
lines were seeded in separate T175 tissue culture flasks containing
50 mL of growth medium comprising RPMI 1640, 10% fetal bovine
serum, 10 mM HEPES, 1 mM sodium pyruvate, 0.1 mM Non-Essential
Amino Acids, and 60 .mu.g/mL GT1b. These cells were incubated in a
37.degree. C. incubator under 5% carbon dioxide until the cells
attained 80% confluence (approximately 3 days). The cells were then
processed as described above and the cell pellet was quick frozen
on dry ice and stored at -80.degree. C. until needed.
[0194] To prepare total RNA, cells for each cell line were lysed
and total RNA isolated and purified using a RNA isolation kit
(QIAGEN, Valencia, Calif.) according to the manufacturer's
protocols. DNA was digested using an on-column DNase I procedure
(QIAGEN, Valencia, Calif.). RNA was quantified
photospectrometrically at 260 nm and purity assessed by the
A260A280 ratio using a spectrophotometer. RNA integrity was
assessed using a Bioanalyser 2100 (Agilent Technologies, Inc.,
Santa Clara, Calif.). Extracted total RNA aliquots were snap-frozen
in liquid nitrogen and stored at -80.degree. C.
[0195] To prepare cDNA probes for the gene microarray chips, about
1-15 .mu.g was reversed transcribed using a T7-Oligo(dT) promoter
primer in the first strand cDNA synthesis reaction. Following RNase
H-mediated second strand cDNA synthesis, the double-stranded cDNA
was purified and served as a template for subsequent in vitro
transcription reaction. The in vitro transcription reaction was
carried out in the presence of T7 RNA polymerase and a biotinylated
nucleotide analog/ribonucleotide mix for complementary RNA (cRNA)
amplification and biotin labeling. The biotinylated cRNA targets
were then cleaned up, fragmented, and hybridized to microchip
expression arrays for 17 hours at 65.degree. C. according to the
manufacturer's instructions, see, e.g., GENECHIP.RTM. Expression
Analysis Technical Manual, 702232, Rev. 2 (2006) which is hereby
incorporated by reference in its entirety. To identify substantial
treatment-related up- or down-regulated gene expression differences
compared to control cell lines, greater than 1.5-fold differences
(log.sub.2 0.58) were considered significant, and only those genes
that displayed consistent changes in expression in the triplicate
analysis were considered (Tables 5-12).
[0196] Expression profiling for cell lines H1, BB10 and 2D6
analyzed in the undifferentiated state revealed that 1,323 genes
were identified as having their expression levels increase by
1.5-fold (log.sub.2 0.58) or more in the H1 and BB10 cell lines as
compared to the gene expression levels obtained from the 2D6 cell
line (Table 5). Of these, 686 genes exhibited an increase in
expression levels in the H1 and BB10 cell lines by 2-fold
(log.sub.2 1.0) or more as compared to the expression level for
these genes determined from the 2D6 cell line; and 231 genes
exhibited an increase in expression levels in the H1 and BB10 cell
lines by 4-fold (log.sub.2 2.0) or more as compared to the
expression level for these genes determined from the 2D6 cell line.
With respect to decreased expression, 1,438 genes were identified
as having their expression levels decreased by 1.5-fold (log.sub.2
0.58) or more in the H1 and BB10 cell lines as compared to the gene
expression levels obtained from the 2D6 cell line (Table 6). Of
these, 668 genes exhibited a decrease in expression levels in the
H1 and BB10 cell lines by 2-fold (log.sub.2 1.0) or more as
compared to the expression level for these genes determined from
the 2D6 cell line; and 107 genes exhibited a decrease in expression
levels in the H1 and BB10 cell lines by 4-fold (log.sub.2 2.0) or
more as compared to the expression level for these genes determined
from the 2D6 cell line.
[0197] Expression profiling for cell lines H1, BB10 and 2D6
analyzed in the differentiated state revealed that 2,556 genes were
identified as having their expression levels increased by 1.5-fold
(log.sub.2 0.58) or more in the H1 and BB10 cell lines as compared
to the gene expression levels obtained from the 2D6 cell line
(Table 7). Of these, 1,634 genes exhibited an increase in
expression levels in the H1 and BB10 cell lines by 2-fold
(log.sub.2 1.0) or more as compared to the expression level for
these genes determined from the 2D6 cell line; and 696 genes
exhibited an increase in expression levels in the H1 and BB10 cell
lines by 4-fold (log.sub.2 2.0) or more as compared to the
expression level for these genes determined from the 2D6 cell line.
With respect to decreased expression, 2,087 genes were identified
as having their expression levels decreased by 1.5-fold (log.sub.2
0.58) or more in the H1 and BB10 cell lines as compared to the gene
expression levels obtained from the 2D6 cell line (Table 8). Of
these, 1,215 genes exhibited a decrease in expression levels in the
H1 and BB10 cell lines by 2-fold (log.sub.2 1.0) or more as
compared to the expression level for these genes determined from
the 2D6 cell line; and 280 genes exhibited a decrease in expression
levels in the H1 and BB10 cell lines by 4-fold (log.sub.2 2.0) or
more as compared to the expression level for these genes determined
from the 2D6 cell line.
[0198] Expression profiling for cell lines H1, BB10 and SiMa
analyzed in the undifferentiated state revealed that 1,232 genes
were identified as having their expression levels increase by
1.5-fold (log.sub.2 0.58) or more in the H1 and BB10 cell lines as
compared to the gene expression levels obtained from the SiMa
parental cell line (Table 9). Of these, 764 genes exhibited an
increase in expression levels in the H1 and BB10 cell lines by
2-fold (log.sub.2 1.0) or more as compared to the expression level
for these genes determined from the SiMa parental cell line; and
228 genes exhibited an increase in expression levels in H1 and BB10
cell lines by 4-fold (log.sub.2 2.0) or more as compared to the
expression level for these genes determined from the SiMa parental
cell line. With respect to decreased expression, 1,265 genes were
identified as having their expression levels decreased by 1.5-fold
(log.sub.2 0.58) or more in the H1 and BB10 cell lines as compared
to the gene expression levels obtained from the SiMa parental cell
line (Table 10). Of these, 648 genes exhibited a decrease in
expression levels in the H1 and BB10 cell lines by 2-fold
(log.sub.2 1.0) or more as compared to the expression level for
these genes determined from the SiMa parental cell line; and 189
genes exhibited a decrease in expression levels in H1 and BB10 cell
lines by 4-fold (log.sub.2 2.0) or more as compared to the
expression level for these genes determined from the SiMa parental
cell line.
[0199] Expression profiling for cell lines H1, BB10 and SiMa
analyzed in the differentiated state revealed that 756 genes were
identified as having their expression levels increase by 1.5-fold
(log.sub.2 0.58) or more in the H1 and BB10 cell lines as compared
to the gene expression levels obtained from the SiMa parental cell
line (Table 11). Of these, 472 genes exhibited an increase in
expression levels in the H1 and BB10 cell lines by 2-fold
(log.sub.2 1.0) or more as compared to the expression level for
these genes determined from the SiMa parental cell line; and 150
genes exhibited an increase in expression levels in H1 and BB10
cell lines by 4-fold (log.sub.2 2.0) or more as compared to the
expression level for these genes determined from the SiMa parental
cell line. With respect to decreased expression, 912 genes were
identified as having their expression levels decreased by 1.5-fold
(log.sub.2 0.58) or more in the H1 and BB10 cell lines as compared
to the gene expression levels obtained from the SiMa parental cell
line (Table 12). Of these, 411 genes exhibited a decrease in
expression levels in the H1 and BB10 cell lines by 2-fold
(log.sub.2 1.0) or more as compared to the expression level for
these genes determined from the SiMa parental cell line; and 108
genes exhibited a decrease in expression levels in H1 and BB10 cell
lines by 4-fold (log.sub.2 2.0) or more as compared to the
expression level for these genes determined from the SiMa parental
cell line.
Example IV
Pathway Analysis of Gene Expression Profiles from Clonal Cell
Lines
[0200] The following example illustrates how to characterize gene
expression profiles from clonal cells from an established clonal
cell line that are susceptible to BoNT/A intoxication or have
neurotoxin uptake capacity in order to identify biological networks
or pathways associated with BoNT/A intoxication.
[0201] An Ingenuity Pathway Analysis (IPA) Core Analysis (Ingenuity
Systems Inc., Redwood City, Calif.) was performed to characterize
the RNA expression phenotype of two independently derived BoNT/A
very sensitive single clone cell lines, BB10 and H1, compared to a
related but much less BoNT/A sensitive single clone cell line, 2D6.
The analysis was done to identify the specific gene expression
phenotype of a BoNT/A sensitive cell line. The result can also be
used to predict and potentially identify proteins that function to
enhance BoNT/A uptake.
[0202] An Ingenuity Pathway Analysis (IPA) Core Analysis was
performed that identified biologically relevant networks to BoNT/A
intoxication based on connectivity among the genes from an IPA
library and the genes exhibiting significant changes as determined
from the expression profiling discussed above. For this analysis,
all genes that exhibited an increased or decrease in expression
levels in the H1 and BB10 cell lines by 4-fold (log.sub.2 2.0) or
more as compared to the expression level for these genes determined
from the 2D6 cell line were used, in conjunction with, selected
genes with known or suspected relevance to BoNT/A intoxication,
hereafter referred to as the Profile genes. The significance of the
association to a given network was measured and ranked by the ratio
of the number of Profile genes that mapped to the specific network
divided by the total number of genes that map to that network.
Fishers exact test was used to calculate p-scores, which represent
the association between the Profile genes and the network, and
based on the p-scores the genes from the Profile genes were used to
rank the networks. The p-scores were derived from p-values. If
there are n genes in the network and f of them are Profile genes,
then the p-value is the probability of finding for more Profile
genes in a set of n genes randomly selected from the Global
Molecular Network. Since significant p-values are quite low (e.g.,
1.times.10.sup.-8), a p-score was defined as the component of the
p-value; p-score=-log 10 (p-value). The identified networks were
overlaid with differentially expressed genes which had more than
1.5-fold differential expression and a p-value .ltoreq.0.001 either
before or after differentiation.
[0203] Using IPA Core Analysis the differentially expressed Profile
genes were connected to 26 Networks. Nineteen of these included 10
or more Profile genes (Table 13). Among these nineteen networks
three overall networks groups, named A, B and C were identified
(Table 13). Within the networks in these three groups there were
overlapping genes and the expression for more than three genes was
increased after differentiation. Group A genes include the FGFR2
receptor which has been shown to function in the BoNT/A
intoxication process as a receptor for BoNT/A and are as follows:
genes exhibiting a 1.5-fold or more decrease in expression levels
in the H1 and BB10 cell lines as compared to their expression in
cells from the 2D6 cell line, ANXA2, AQP1, ARHGAP9, CDH10, CDKN2A,
CHPT1, CNTN2, ERAP1, and RGS11; genes exhibiting a 1.5-fold or more
increase in expression levels in the H1 and BB10 cell lines as
compared to their expression in cells from the 2D6 cell line,
ADAMTS9, ATAD2, C11ORF82, CDC45L, CNTN1, CNTN4, Cyclin A, Cyclin E,
E2F1, E2F2, E2F7, ELOVL7, EME1, FGFR, FGFR2, KIAA1524, MELK, MYBL1,
MYBL2, NDC80, NDN, ORC1L, PLS3, PRIMA1, RAD54L, RBL1, RBPMS, RRM2,
S1PR3, SCLY, SLCIA3, SPC24, SPC25, ST8SIA4, TFDP1, TFP12, TK1,
TMEM35, TTK, TWIST1, TYMS, TYK, and ZWINT.
[0204] Group B genes include the EGFR, a receptor which is
regulated by NGF, which has been shown to increase BoNT/A uptake in
cell culture experiments and are as follows: genes exhibiting a
1.5-fold or more decrease in expression levels in the H1 and BB10
cell lines as compared to their expression in cells from the 2D6
cell line, ABCC8, AELIM3, CAP2, IL17B, MEF2A, NEEBL, PHC, S100A6,
SLC1A6, SMAD1, SMAD5, SMAD8, SYT13, and SYTL1; genes exhibiting a
1.5-fold or more increase in expression levels in the H1 and BB10
cell lines as compared to their expression in cells from the 2D6
cell line, AURKB, BIRC5, BRCA1, BRCA2, BRIP1, BUB1B, CD9, DLGAP3,
DYNLT3, ENC1, FBLN1, FOXM1, G.beta..gamma., GNAI1, GNG11, GNG12,
GPSM2, GUCY1B3, HGF, ITGA6, JNK, KCNJ5, KIF18A, KITLG, MMD, MSN,
MYRIP, NEK2, NR3C1, NXPH1, OSBPL3, PKMYT1, PTPRM, RAD51, RAD51AP1,
SLC7A2, SLC43A3, SMC6, SNAI2, SNCAIP, SSH2, STK17A, SYNPO2, TOP2A,
TPTE, TRAF4, TSPAN, TSPAN4, UBE3, UBE3B, and VAV3.
[0205] Group C genes include a number of microtubule motor proteins
of the kinesin family that may function in the intracellular
trafficking of BoNT/A and are as follows: genes exhibiting a
1.5-fold or more decrease in expression levels in the H1 and BB10
cell lines as compared to their expression in cells from the 2D6
cell line, CSTB, GPCR, GRIM5, KISSR, SCN2A, SLC1A2, and THBS2;
genes exhibiting a 1.5-fold or more increase in expression levels
in the H1 and BB10 cell lines as compared to their expression in
cells from the 2D6 cell line, ALCAM, AURKA, CHEK, CIT, CSRP2, E2F,
ECT2, EFNB2, ERK, ESPL1, GNAI, GPR161, HMMR, KIF4A, KIF14, KIF15,
KIF22, KIF23, KIFC1, LPAR1, MK167, OIP5, PHLPP, PP1/PP2A, PPP1R3C,
PRC1, PTTG1, RACGAP, RB, RGS5, SDC2, and TPX2.
[0206] The remaining networks identified did not share common genes
between any other network. For Network 3, genes exhibiting a
1.5-fold or more decrease in expression levels in the H1 and BB10
cell lines as compared to their expression in cells from the 2D6
cell line, ADARB1, ADM, PTPPH, and SLCO1A2; genes exhibiting a
1.5-fold or more increase in expression levels in the H1 and BB10
cell lines as compared to their expression in cells from the 2D6
cell line, ASCL1, HES6, MAPK, NMU, PEG3, PTPRK, PRLHR, PTPRK,
SGOL2, SPARC, and ZNF217. For Network 6, genes exhibiting a
1.5-fold or more decrease in expression levels in the H1 and BB10
cell lines as compared to their expression in cells from the 2D6
cell line, CNGA3, HIST1H3E and PTGS1; genes exhibiting a 1.5-fold
or more increase in expression levels in the H1 and BB10 cell lines
as compared to their expression in cells from the 2D6 cell line,
ASF1B, BASP1, CHAF1A, NCAPH, PBK, PRAME, SMC2, UHRF1, and VRK1. For
Network 9, genes exhibiting a 1.5-fold or more decrease in
expression levels in the H1 and BB10 cell lines as compared to
their expression in cells from the 2D6 cell line, HIST1H2BD and
OSCAR; genes exhibiting a 1.5-fold or more increase in expression
levels in the H1 and BB10 cell lines as compared to their
expression in cells from the 2D6 cell line, C14ORF106, CEP72,
KIF20A, PCNA, PEX13, PFC5, POLQ, SPAG5, SYTL4, TROAP, and WDR51A.
For Network 12, genes exhibiting a 1.5-fold or more increase in
expression levels in the H1 and BB10 cell lines as compared to
their expression in cells from the 2D6 cell line, ANLN, ARHGAP24,
ASPM, BUB1, CCDC99, CEP55, CKAP2, DRAM, E2F8, PLXNA2, SLC16A10,
UBE2C, UBE2S, and WDHD1. For Network 13, genes exhibiting a
1.5-fold or more decrease in expression levels in the H1 and BB10
cell lines as compared to their expression in cells from the 2D6
cell line, CALY; genes exhibiting a 1.5-fold or more increase in
expression levels in the H1 and BB10 cell lines as compared to
their expression in cells from the 2D6 cell line, ARHGEF3, CTSL2,
DIAPH3, FBP1, KIF2C, KIF11, PFKFB3, and PLK4. For Network 14, genes
exhibiting a 1.5-fold or more decrease in expression levels in the
H1 and BB10 cell lines as compared to their expression in cells
from the 2D6 cell line, COL5A1 and MICAL2; genes exhibiting a
1.5-fold or more increase in expression levels in the H1 and BB10
cell lines as compared to their expression in cells from the 2D6
cell line, FNDC5, HSPC159, MAB21L2, SLITRK5, SYN2, and ZNF367. For
Network 15, genes exhibiting a 1.5-fold or more decrease in
expression levels in the H1 and BB10 cell lines as compared to
their expression in cells from the 2D6 cell line, OLFML2A and
SIGIRR; genes exhibiting a 1.5-fold or more increase in expression
levels in the H1 and BB10 cell lines as compared to their
expression in cells from the 2D6 cell line, EXO1, KCTD12, MYO6,
PHEBL1, SHCBP1, TPBG, and TUBB6. For Network 17, genes exhibiting a
1.5-fold or more decrease in expression levels in the H1 and BB10
cell lines as compared to their expression in cells from the 2D6
cell line, HPCAL1 and LPAR5; genes exhibiting a 1.5-fold or more
increase in expression levels in the H1 and BB10 cell lines as
compared to their expression in cells from the 2D6 cell line, BTG3,
GABRA5, TR1P10, and ZNF521. For Network 19, genes exhibiting a
1.5-fold or more decrease in expression levels in the H1 and BB10
cell lines as compared to their expression in cells from the 2D6
cell line, HTR1E and SORCS1; genes exhibiting a 1.5-fold or more
increase in expression levels in the H1 and BB10 cell lines as
compared to their expression in cells from the 2D6 cell line, BNC2,
DPYD, EMILIN2, PPIL5, and TACC3.
TABLE-US-00005 TABLE 13 IPA Core Analysis of Profile Genes Profile
Expression Expression Differentiated Proteins in Gene
Undifferentiated State State Group Network Network pvalue Number
Decreased Increased Decreased Increased A 1 ADAMTS9, 51 30 CDKN2A
ADAMTS9, CDKN2A, ADAMTS9, ATAD2, CNTN1, CNTN2, ATAD2, CDC45L, HDAC,
NDN, CDC45L, CDKN2A, PLS3, CNTN1, CNTN1, S1PR3, TFPI2 Cyclin A,
CNTN2, Cyclin Cyclin E, A, Cyclin E, E2F1, E2F2, DIRAS3, E2F1,
E2F7, E2F2, E2F7, FGFR2, FGFR2, HDAC, MYBL1, MYBL1, MYBL2, MYBL2,
NDC80, NDC80, NDN, NDN, NFkB, ORC1L, ORC1L, PLS3, PLS3, RAD54L,
RAD54L, RBL1, RRM2, RBL1, S1PR3, RRM2, SPC24, S1PR3, SPC25, SPC24,
TFDP1, TFPI2, SPC25, TK1, TFDP1, TNFRSF10D, TFPI2, TK1, TTK, TYMS,
TTK, TYMS, TYK, ZWINT TYK, ZWINT C 2 ALCAM, ANK2, 46 28 ANK2,
ALCAM, KISSR, ALCAM, ANK3, AURKA, ANK3, DOK5, ERK, SCN2A AURKA,
DOK5, E2F, PHKA2, GFRA2, E2F, ERK, ERK, ESPL1, PPP2R2C, PP1/PP2A,
ESPL1, GDF15, SCN2A PP2A, HMMR, GFRA2, PPP1R3C, KIF15, HMMR, ITGA9,
PPP2R2B KIF22, KIF15, KIF22, MKI67, OIP5, KISS1R, PHLPP, KPNA,
KPNB, PP1/PP2A, LGI1, MAFB, PPP1R3C, MAP2K1, PTTG1, RB, MAP2K2,
TPX2 MKI67, NRG3, OIP5, PHKA2, PHLPP, POSTN, PP1/PP2A, PP2A,
PPP1R3C, PPP2R2B, PPP2R2C, PTTG1, RAF1, RB, SCN2A, TPX2 -- 3
ADARB1, 46 28 ADARB1, ASCL1, ADARB1, ASCL1, ADM, AKAP12, GRIP2,
DLL1, FRZB, ADM, HES6, ALP, ASCL1, SLCO1A2 MAPK, PTPPH, MAPK, NMU,
BHLHB2, CRH, PEG3, PLAT, SLCO1A2 PEG3, DLL1, FRZB, PRLHR, PRLHR,
FSH, GRIA2, SMAD6, PTPRK, GRIP2, hCG, SPARC, SGOL2, HES6, ZNF217
SPARC, HTATIP2, ZNF217 IL13RA2, MAPK, NMU, PDGF BB, PDLIM3, PEG3,
PLAT, PPFIA4, PRLHR, PTPRH, PTPRK, SGOL2, SLCO1A2, SMAD6, SPARC,
STC1, TEAD4, TGFB, VEGF, ZNF217 C 4 AKT, ACTN1, 43 27 CSTB, GNAI,
CSTB, CIT, CSRP2, ACTN2, GPCR, GPR161, GPCR, ECT2, ACTN3, ATF5,
GRIM5, LPAR1, GRIM5, EFNB2, ATM/ATR, SLC1A2 RGS5 SLC1A2, CHEK,
GNAI, BDKRB2, THBS2 GPR161, CHEK1, KIF4A, CHEK2, CIT, KIF14, CSRP2,
CSTB, KIF23, CTSH, ECT2, KIFC1, EFNB2, LPAR1, ELMO1, GNAI, PRC1,
GABBR2, RACGAP, GPCR, RGS5, GPR161, SDC2, GRM5, ICAM2, KIF4A,
KIF14, KIF23, KIFC1, LPAR1, MMP, PDGFA PDGFB, PRC1, RAC, RAC2,
RACGAP1, RGS5, SDC2, SLC1A2, THBS2 B 5 ABLIM3, F- 38 25 SYT13, CD9,
CXCR4, AELIM3, CD9, ENC1, ACTF, G-ACT, S100A6 ENC1, G.beta..gamma.,
S100A6, G.beta..gamma., GNAI1, CD9, CXCR4, GNAI1, SYT13, GNG11,
DTNA, ENC1, GNG11, SYTL1 GNG12, FNBP1, G.beta..gamma., GNG12,
GPSM2, GNAI1, ITGA6, JNK, ITGA6, JNK, GNG11, MSN, MYRIP, KCNJ4,
GNG12, STK17A MSN, GPSM2, ITG, MYRIP, ITA5, ITGA6, NXPH1, JNK,
KCNJ4, STK17A, MLC, MSN, TRAF4, MYRIP, NOX, TSPAN, NET1, NRXN1,
TSPAN4, NXPH1, PI3K, VAV3 RHO, S100A6, STK17A, SYT13, SYTL1, TRAF4,
TSPAN, TSPAN3, TSPAN4, VAV3 -- 6 ADCY, ADCY1, 36 24 CAMK2B BASP1,
CNGA3, ASF1B, AP1, ASF1B, HRH3 HIST1H3E, BASP1, ATP2B4, PTGS1
CHAF1A, BASP1, NCAPH, BCL11B, CAM, PBK, CAMK2B, PRAME, CHAF1A,
SMC2, CNGA3, UHRF1, CYP2E1, VRK1 DACH1, HIST1H3E, HISH3, HRH3,
HSP90, IL1, IL12, INS, INFA, ISG20, KCNN2, LDL, MMP14, NCAPH, PBK,
PDGF, PIAS1, PRAME, PTGS1, SEC31A, SMC2, UHRF1, VRK1 B 7 BARD1, 32
24 CAP2, NR3C1, CAP2, BRCA1, BRCA1, CNA, SMC6, MEF2A, BRCA2, BRCA2,
PHLDA2, SNCAIP, PKC(s), BRIP1, BRIP1, PKC(s) TCF7L2 SLC1A6 BUB1B,
BUB1B, CNA, DLGAP3, CAP2, CK2, GUCY1B3, DLGAP5, FAT, NEK2, GUCY1B3,
KITLG, HSP70, KITLG, NR3C1, MEF2A, NEK2, RAD51, NFAT, NR3C1,
RAD51AP1, NF1, PHLDA2, SMC6, PKC(s), SNAI2, RAD51, SNCAIP,
RAD51AP1, UBE3, TOP2, UBE3B SAMD4A, SLC1A6, SMC6, SNAI2, SNCAIP,
STAT5a, STAT5b, TCF7L2, UBE3, UBE3B, UBN, XPO1 B 8 CBP, AURKB, 30
21 KCNMA1, ERK1, ERK2, NDRG1, AURKB, BIRC5, NDRG1, MAOA, P38 SMAD1,
BIRC5, CACNA1D, RAS MAPK, SMAD5, ERK1, ERK2, CBP/p300, RGMA SMAD8
FBLN1, CD3, CREB, FOXM1, CREB5, DBH, HGF, MAOA, EGR1, ERK1, P38
MAPK, ERK2, FBLN1, PKMYT1, FBN1, FOXM1, SYNPO2, HGF, INCENP, TOP2A
KCNMA1, LGALS1, MAOA, MEK, NDRG1, NID1, P38 MAPK, PKA, PKMYT1,
PRKAC, PRKACB, RAF, RAP1, RAS, RGMA, RSK, SMAD1, SMAD5, SMAD8,
SYNPO2, TOP2A -- 9 C14ORF106, 19 15 -- PCGF5 HIST1H2BD, C14ORF106,
CDKN1A, OSCAR CEP72, CEP72, DHX8, KIF20A, FKBPL, PCNA, HIST1H2BD,
PEX13, HNF4A, PFC5, KIF20A, POLQ, KRT18, SPAG5, MTHFS, SYTL4,
OSCAR, TROAP, PCGF5, PCNA, WDR51A PELO, PEX13, PFC5, POLD4, POLK,
POLL, POLM, POLQ, POLS, R3HDM1, RASL12, RFC5, SLC33A1, SPAG5,
STAT4, SULT1A1, SULT1C2, SYTL4, TCEAL3, TROAP, VEZT, WDR51A, ZBTB16
A 10 ARHGAP9, 18 15 CHPT1 CNTN4, ARHGAP9, CNTN4, CDH10, CDS1,
ELOVL7, CDH10 ELOVL7, CDS2, CHPT1, HAND1, EME1, CNTN4, ODS2, SOX2,
FGFR, ELOVL7, TWIST1 KIAA1524, EME1, FGFR, PRIMA1, GAS6, HAND1,
RBPMS, IFI202B, ISL1, ST8SIA4, KIAA1524, TWIST1 KRT74, MIRN31,
MUS81, MYF6, MYOD1, NFYB, OTX1, PRIMA1, QKI, RBM9,
RBPMS, RORA, SHOX2, SLC39A8, SOX2, SOX15, ST8SIA4, STAT3, TWIST1,
VPS39, ZFHX3 B 11 ALDH3A2, 18 15 PHLDA2 BAMBI, -- DYNLT3, B3GALT2,
CUGBP2, KIF18A, BAMBI, CNN2, DYNLT3, PTPRM, CTNNAL1, SLC7A2,
SLC7A2, CTNNB1, SLC43A3, SLC43A3, CTSC, STK17A, SSH2, CUGBP2, TPTE
STK17A, DYNLT3, IL13, TPTE KIF18A, LIN7C, MFI2, MPP5, MRC2, NDST1,
PDGF- CC, PHLDA2, PLAU, PPM1J, PPP1CA, PTPN14, PTPRM, SLC16A3,
SLC26A2, SLC43A3, SLC7A2, SSH2, STK17A, TAX1BP3, TCF7L1, TGFB1,
TPTE, TSPAN8 -- 12 AHR, AHRR, 17 15 HS3ST1 PDZRN3, -- ANLN, ANLN,
APH1B, PVRL3 ARHGAP24, ARHGAP24, ASPM, ASPM, BUB1, BUB1, CCDC99,
CCDC99, CEP55, CEP55, CKAP2, CKAP2, DRAM, E2F8, DRAM, HS3ST1, E2F8,
JPH1, PLXNA2, MIRN124-1, SLC16A10, NMT2, UBE2C, PDZRN3, UBE2S,
PLXNA2, WDHD1 PMM1, PPP1R13L, PVRL3, RFFL, S100A2, SLC16A10, TP53,
UBE2A, UBE2C, UBE2D2, UBE2S, UBE2V1, UBE2V2, UBL3, VPS37C, WDHD1 --
13 ARHGEF3, 17 14 CALY ARHGEF3, CALY ARHGEF3, CALY, CSH2, GSK3B,
CTSL2, CTSL2, RMP22 DIAPH3, DIAPH3, FBP1, ECEL1, FBP1, KIF2C,
FFAR2, KIF11, FRAT2, PFKFB3, GSK3B, PLK4 HSD17B4, IL6, IL19, KIF11,
KIF2C, KNG1, LARGE, LEFTY2, LGR5, LIF, LPAR3, MPZ, MSLN, ORM2,
PFKFB3, PLK4, PMP22, PRPH, PRSS8, PTGER3, SCN7A, SCNN1B, SLC4A11,
TAL1 -- 14 AGT, 15 13 CPNE3 CADPS, COL5A1, FNDC5, AGTRAP, Beta
FNDC5, MICAL2 HSPC159, ARK, CADPS, MAB21L2, MAB21L2, CD160,
SLITRK5, SLITRK5, COL5A1, ST8SIA2 SYN2, CPNE8, ZNF367 FNDC5,
HSPC159, INA, ITGA7, LIMA1, LPP, MAB21L2, MAPK1, MERTK, MICAL2,
MIRN294, MIRN185, MIRN352, NTN1, PLCG1, PLCG2, PTGFR, RASGEF1B,
RBM4B, RSU1, SKAP1, SLITRK5, SPRED1, ST8SIA2, SYN2, TGFB3, TNS1,
ZNF367 -- 15 ACT1, BAG5, 15 13 -- KCTD12, OLFML2A, EXO1, CPVL,
EXO1, TPBG SIGIRR KCTD12, GIPC1, GJC1, MYO6, GPR37, PHEBL1, HSPA5,
SHCBP1, HSPA1A, TPBG, HSPBP1, TUBB6 IkBKB, KCTD12, MAP3K3, MAP3K14,
MEKK3/NIK, MIRN30B, MYO6, OLFML2A, PELI3, RHEBL1, RNF126, RPL10A,
RPN1, SEC16A, SHC1, SHCBP1, SIGIRR, TJP3, TPBG, TRAF6, TRAF2-TRAF5,
TRAF2-TRAF5, TRAF6, TUBB6, TUBB2B B 16 ABCC8, 15 13 IL17B DACT1,
ABCC8, MMD, BACE2, PCDH8 IL17B, OSBPL3, CLASP1, NEEBL SYNPO2
COLEC12, CXCR7, DACT1, ENTPD5, FGD6, FMO1, GSTT1, IL17B, KRT34,
KYNU, LAMP3, LMCD1, MIRN101B, MMD, NEBL, NFRKB, OSBPL3, PCDH8,
PEMT, RASAL2, SAMD4A, ST3GAL3, SYNGR3, SYNPO2, TNF, TNNC1, TXN2,
YWHAG, ZNF267, ZYX -- 17 ARPP-19, 14 13 HPCAL1 PPAP2B HPCAL1, BTG3,
BTG3, LPAR5 GABRA5, CBFA2T2, TRIP10, CBLN1, ZNF521 CNGA2, EBF1,
EGF, EGFR, GABA, GABRA5, GABRB3, GABRD, GABRR1, HDAC9, HPCAL1, HTT,
IDS, INSL3, LPAR5, OPRL1, PCDH9, PDE10A, PDE11A, PDE1A, PDE1B,
PDE2A, PDE4C, PDE6H, PPAP2B, PPP1R1B, ROBO2, SH3D2C1, TRIP10,
ZNF521 A 18 ANXA2, AQP1, 14 12 AQP1, MINA, ANXA2, C11ORF82,
C11ORF82, CDKN2A PDE5A CDKN2A, MELK, MINA, CCNI, ERAP1, PDE5A,
CDKN2A, RGS11 SCLY, DEFB104A, SLC1A3, DEFB4, TMEM35 EGLN3, ERAP1,
FAM129A, GNAO1, IFNG, IL1B, IL1F7, INCA, LOC729687, MELK, MINA,
MIRN210, MPA2L, MYC, NNAT, PDE5A, RGS11, RNASE7, RT1- B, SCLY,
SCUBE1, SLC11A1, SLC14A1, SLC1A3, ST18, TSH, TMEM35, TREM3 -- 19
BIRC8, BNC2, 10 10 -- EMILIN2, HTR1E, BNC2, CASP5, PLXNA4 SORCS1
DPYD, CASP10, EMILIN2, CASP14, PPIL5, CASP, CBLN2, TACC3 DEDD2,
DNASE1L1, DPYD, DSG3, EIF4E, EMILIN2, HTR1D, HTR1E, HTR1F, HTR3A,
IFT57, KHDC1, MIRN20A, MTCH1, NGFR, PLXNA4, PPIL5, RB1,
RP11-257K9.7, SEMA6D, SERPINA3K,
SORCS1, STK25, SYT5, TACC3, TERT -- 20 EGFL7, GFI1B 2 1 -- -- -- --
-- 21 AGRN, 2 1 -- -- -- -- PRSS12 -- 22 KCNB1, 2 1 -- -- -- --
KCNG3 -- 23 NUP37, 2 1 -- -- -- -- NUP43 -- 24 HS6ST1, 2 1 -- -- --
-- HS6ST2 -- 25 FUCA1, 2 1 -- -- -- -- FUCA2 -- 26 IQGAP, 2 1 -- --
-- -- IQGAP3, MIRN339
Example V
Pathway Analysis of Gene Expression Profiles from Clonal Cell
Lines
[0207] The following example illustrates how to characterize gene
expression profiles from clonal cells from an established clonal
cell line that are susceptible to BoNT/A intoxication or have
neurotoxin uptake capacity in order to identify biological networks
or pathways associated with BoNT/A intoxication.
[0208] Computer analysis was performed to characterize the RNA
expression phenotype of two independently derived single clone cell
lines, BB10 and H1, very sensitive to BoNT/A activity and a single
clone cell line, 2D6, that was less sensitive BoNT/A activity. The
results are useful in identifying clonal cell lines selected from
the SiMa parental cell line that are useful to practice the methods
disclosed in the present specification.
[0209] To identify the specific gene expression phenotype
associated with a BoNT/A sensitive cell line, the data was analyzed
using JMP Genomic (SAS Institute Inc., Cary, N.C.). To perform a
JMP Genomics analysis, the RNA expression data was imported to JMP
Genomics by creating two SAS files, designated "data" and
"Experiment design." A basic expression workflow was performed,
including a variance analysis and one-way analysis of variance
(one-way ANOVA) to identify differences in mean expression values.
The variance analysis was used to identify differences between the
cell lines and the differentiation state of each cell line
(differentiated or non-differentiated). The expression values of
all four cell lines were compared pair wise, independent of
differentiation and genes that were .gtoreq.4-fold over-expressed
in the H1 cell line as compared to the 2D6 cell line, in the BB10
cell line as compared to the 2D6 cell line, and in SiMa parental
cell line (PA) compared to the 2D6 cell line. The variance analysis
treats all factors in the study as random effects, to find out what
their contribution is to the proportion of variance explained
(total variance=100%). About half of the variance, 46.9% was
assigned to differences among the different cell types, while 26%
was assigned to differences before and after differentiation. Only
4.6% of the variance was assigned to differences among
differentiated or non-differentiated cells types, meaning that the
genes that were differentially expressed after differentiation were
similar for all cell types examined, suggesting that the difference
in BoNT/A sensitivity among the cell lines is independent of
differentiation state. As such, the focus was placed on genes that
are different among the different cells types irrespective of
differentiation.
[0210] Based on the results from the variance analysis, the
expression values of all cell lines were compared pair wise,
independent of differentiation (FIG. 2). Only gene probes that were
.gtoreq.4-fold over-expressed in the H1 cell line as compared to
the 2D6 cell line, and in the BB10 cell line as compared to the 2D6
cell line are shown. The gene probes were plotted as expression
value (Log.sub.e) on the x-axis and p-value (-log.sub.10(p-value))
on the y-axis. The red dashed line marks the 95% confidence
interval. The genes probes that were .gtoreq.4-fold over-expressed
in the H1 cell line as compared to the 2D6 cell line (FIG. 2A), and
the BB10 cell line as compared to the 2D6 cell line (FIG. 2B), were
also to a large extend among the genes that were over-expressed in
the BB10 cell line as compared to the SiMa parental cell line (PA,
FIG. 2C), in the H1 cell line as compared to the SiMa parental cell
line (FIG. 2D), and in the SiMa parental cell line as compared to
the 2D6 cell line (FIG. 2E). These data suggest that BoNT/A
sensitivity was related to a gradual increase in expression of
these genes. Based on this finding, the number of gene probes was
further narrowed down to 119 by including only gene probes that
were also over-expressed in the SiMa parental cell line compared to
the 2D6 cell line, rationalizing that gene probes that were
differential expressed across one more cell line would be even more
likely to be important for BoNT/A sensitivity. As such, the JMP
Genomic analysis resulted in the identification of 119 gene probes,
designated the JMP probe set.
[0211] The JMP probe set was exported to Ingenuity Pathway Analysis
(IPA) Core Analysis (Ingenuity Systems Inc., Redwood City, Calif.)
to further characterize the JMP probe set. Using this analysis,
biologically relevant networks to BoNT/A intoxication were
identified based on connectivity among the genes from an IPA
library and the gene probes contained in the JMP probe set. The
significance of the association to a given network was measured and
ranked by the ratio of the number of gene probes from the JMP probe
set that mapped to the specific network divided by the total number
of genes that map to that network. Fisher's exact test was used to
calculate p-scores, and based on the p-scores the genes from the
JMP probe set were used to rank the networks. The p-scores were
derived from p-values. If there are n genes in the network and f of
them are gene probes from the JMP probe set, then the p-value is
the probability of finding for more gene probes from the JMP probe
set in a set of n genes randomly selected from the Global Molecular
Network. Since significant p-values are quite low (e.g.,
1.times.10.sup.-8), a p-score was defined as the component of the
p-value; p-score=-log 10 (p-value). The identified networks were
overlaid with differentially expressed genes which had more than
1.5-fold differential expression and a p-value .ltoreq.0.001 either
before or after differentiation. Using IPA Core Analysis, 111 of
the 119 gene probes from the JMP probe set were mapped to networks.
The eight unmapped probes most likely represent either a probe with
an outdated nomenclature or one representing a gene without a known
gene product. The 111 gene probes corresponded to 79 genes that
were greater than 4.0-fold differentially expressed in the H1 and
BB10 cell lines when compared to the 2D6 cell line and that were
also greater than 4.0-fold differentially expressed in the SiMa
parental cell lines when compared to the 2D6 cell line, when the
p-value cut off was set to 0.05 (Table 14).
TABLE-US-00006 TABLE 14 Genes Identified using IPA Core Analysis
Gene Log Gene Name Symbol Probe ID. No. Location Type Ratio
Achaete-scute complex homolog 1 ASCL1 209985_s_at Nucleus
Transcription 4.469 regulator Achaete-scute complex homolog 1 ASCL1
209988_s_at Nucleus Transcription 2.774 regulator Achaete-scute
complex homolog 1 ASCL1 213768_s_at Nucleus Transcription 2.955
regulator Acyl-CoA thioesterase 9 ACOT9 221641_s_at Cytoplasm
Enzyme 2.251 ADAM metallopeptidase with ADAMTS9 226814_at
Extracellular Peptidase 4.034 thrombospondin type 1 motif, 9 Space
Ankyrin 2, neuronal ANK2 216195_at Plasma Other -2.673 Membrane
Ankyrin 2, neuronal ANK2 232606_at Plasma Other -2.621 Membrane
Blood vessel epicardial BVES 228783_at Plasma Other 3.174 substance
Membrane Brain abundant, membrane BASP1 202391_at Plasma Other
4.549 attached signal protein 1 Membrane BRCA1 associated RING
domain 1 BARD1 205345_at Nucleus Transcription 3.44 regulator
Calsyntenin 2 CLSTN2 219414_at Plasma Transporter 2.024 Membrane
cAMP responsive element CREB5 232555_at Nucleus Transcription -2.67
binding protein 5 Cathepsin L2 CTSL2 210074_at Cytoplasm Peptidase
2.119 CD9 molecule CD9 201005_at Plasma Other 2.887 Membrane Cell
division cycle associated 7- CDCA7L 225081_s_at Nucleus Other 3.737
like Centromere protein L CENPL 1554271_a_at Unknown Other 2.582
Chromosome 11 open reading C11ORF75 219806_s_at Unknown Other 3.914
frame 75 Chromosome 3 open reading C3ORF70 242447_at Unknown Other
2.705 frame 70 Coiled-coil domain containing CCDC109B 218802_at
Unknown Other 2.881 109B Contactin 1 CNTN1 211203_s_at Plasma
Enzyme 3.684 Membrane Contactin 1 CNTN1 227202_at Plasma Enzyme
3.184 Membrane Contactin 1 CNTN1 227209_at Plasma Enzyme 3.188
Membrane Copine VIII CPNE8 228365_at Unknown Other -3.355 Copine
VIII CPNE8 241706_at Unknown Other -2.736 CUG triplet repeat, RNA
binding CUGBP2 202157_s_at Nucleus Other 3.588 protein 2
Cyclin-dependent kinase 2 CDK2 204252_at Nucleus Kinase 2.744
Cysteine and glycine-rich protein 2 CSRP2 207030_s_at Nucleus Other
2.487 Cysteine and glycine-rich protein 2 CSRP2 211126_s_at Nucleus
Other 2.109 DEP domain containing 1 DEPDC1 220295_x_at Unknown
Other 2.795 Diaphanous homolog 3 DIAPH3 232596_at Cytoplasm Enzyme
2.434 Dihydropyrimidine DPYD 204646_at Cytoplasm Enzyme 4.248
dehydrogenase Docking protein 5 DOK5 214844_s_at Plasma Other 4.56
Membrane Dynein, light chain, Tctex-type 3 DYNLT3 203303_at
Cytoplasm Other 3.449 Elastin microfibril interfacer 2 EMILIN2
224374_s_at Extracellular Other 2.603 Space Ets variant 1 ETV1
206501_x_at Nucleus Transcription 2.578 regulator Ets variant 1
ETV1 217053_x_at Nucleus Transcription 2.499 regulator Ets variant
1 ETV1 217061_s_at Nucleus Transcription 2.557 regulator Family
with sequence similarity FAM101B 226876_at Unknown Other 2.399 101,
member B Fibroblast growth factor receptor 2 FGFR2 203638_s_at
Plasma Kinase 2.306 Membrane Fibroblast growth factor receptor 2
FGFR2 203639_s_at Plasma Kinase 3.239 Membrane Fibroblast growth
factor receptor 2 FGFR2 208228_s_at Plasma Kinase 2.151 Membrane
Fibronectin type III domain FNDC5 226096_at Unknown Other 2.054
containing 5 Fibulin 1 FBLN1 201787_at Extracellular Other 2.801
Space Fibulin 1 FBLN1 202994_s_at Extracellular Other 2.378 Space
Fibulin 1 FBLN1 202995_s_at Extracellular Other 3.733 Space G
protein-coupled receptor 177 GPR177 221958_s_at Unknown Other 2.389
G protein-coupled receptor 177 GPR177 228950_s_at Unknown Other
2.622 G-2 and S-phase expressed 1 GTSE1 204317_at Cytoplasm Other
3.334 Gametocyte specific factor 1 GTSF1 227711_at Unknown Other
6.407 Guanine nucleotide binding GNAI1 227692_at Plasma Enzyme
3.123 protein (G protein), alpha Membrane inhibiting activity
polypeptide 1 Guanine nucleotide binding GNB4 225710_at Plasma
Enzyme 6.066 protein (G protein), beta Membrane polypeptide 4
Guanine nucleotide binding GNG11 204115_at Plasma Enzyme 4.251
protein (G protein), gamma 11 Membrane Guanine nucleotide binding
GNG12 212294_at Plasma Enzyme 2.315 protein (G protein), gamma 12
Membrane Guanine nucleotide binding GNG12 222834_s_at Plasma Enzyme
3.403 protein (G protein), gamma 12 Membrane Hepatocyte growth
factor HGF 209960_at Extracellular Growth 2.939 (hepapoietin A)
Space factor Hypothetical protein LOC100289109 216189_at Unknown
Other -3.256 LOC100289109 Hypothetical protein LOC144571 LOC144571
1564139_at Unknown Other -2.394 Inositol 1,4,5-triphosphate ITPRIP
225582_at Unknown Other 3.522 receptor interacting protein
Interleukin 17B IL17B 220273_at Extracellular Cytokine -4.735 Space
KDEL (Lys-Asp-Glu-Leu) KDELC2 225128_at Unknown Other 3.003
containing 2 KIAA0125 KIAA0125 206478_at Unknown Other -8.436 KIT
ligand KITLG 226534_at Extracellular Growth 2.59 Space factor
LY6/PLAUR domain containing 6 LYPD6 227764_at Extracellular Other
2.64 Space Lysophosphatidic acid receptor 1 LPAR1 204036_at Plasma
Receptor 3.151 Membrane MAB-21-like 2 MAB21L2 210303_at Unknown
Other 3.817 Minichromosome maintenance MCM10 223570_at Nucleus
Other 2.249 complex component 10 Moesin MSN 200600_at Plasma Other
5.842 Membrane Monoamine oxidase A MAOA 204388_s_at Cytoplasm
Enzyme 4.029 Monoamine oxidase A MAOA 204389_at Cytoplasm Enzyme
3.355 Monoamine oxidase A MAOA 212741_at Cytoplasm Enzyme 4.157 MYC
induced nuclear antigen MINA 213188_s_at Nucleus Other 4.019 MYC
induced nuclear antigen MINA 213189_at Nucleus Other 4.636 Myosin
VI MYO6 203216_s_at Cytoplasm Other 2.406 Myosin VIIA and Rab
interacting MYRIP 214156_at Cytoplasm Other 2.805 protein Neuronal
PAS domain protein 4 NPAS4 1554299_at Nucleus Transcription -2.515
regulator Paternally expressed 3 PEG3 209242_at Nucleus Kinase
5.896 Paternally expressed 3 PEG3 209243_s_at Nucleus Kinase 4.556
Phosphoprotein associated with PAG1 225626_at Plasma Other 2.193
glycosphingolipid microdomains 1 Membrane Poliovirus
receptor-related 3 PVRL3 213325_at Plasma Other 2.325 Membrane
Polo-like kinase 2 PLK2 201939_at Nucleus Kinase 4.93 Polymerase
(DNA directed), POLA2 204441_s_at Nucleus Enzyme 2.198 alpha 2 (70
kD subunit) Prolactin releasing hormone PRLHR 231805_at Plasma
Receptor 3.19 receptor Membrane Prostaglandin reductase 1 PTGR1
228824_s_at Cytoplasm Enzyme 2.398 Prostaglandin reductase 1 PTGR1
231897_at Cytoplasm Enzyme 2.33 Protease, serine, 12 PRSS12
205515_at Extracellular Peptidase 5.146 (neurotrypsin, motopsin)
Space Protein phosphatase 1, regulatory PPP1R3C 204284_at Cytoplasm
Phosphatase 2.827 (inhibitor) subunit 3C Protein tyrosine
phosphatase, PTPRK 203038_at Plasma Phosphatase 3.451 receptor
type, K Membrane RAB32, member RAS oncogene RAB32 204214_s_at
Cytoplasm Other 2.192 family RELT-like 1 RELL1 226430_at Unknown
Other 2.88 Rho GTPase activating protein 24 ARHGAP24 223422_s_at
Cytoplasm Other 3.043 Rho guanine nucleotide ARHGEF3 218501_at
Cytoplasm Other 2.473 exchange factor (GEF) 3 Ring finger protein
182 RNF182 230720_at Unknown Other 4.746 RNA binding protein with
multiple RBPMS 209487_at Unknown Other 2.329 splicing RNA binding
protein with multiple RBPMS 209488_s_at Unknown Other 3.292
splicing Secreted protein, acidic, cysteine- SPARC 200665_s_at
Extracellular Other 4.919 rich (osteonectin) Space Secreted
protein, acidic, cysteine- SPARC 212667_at Extracellular Other 4.58
rich (osteonectin) Space Shugoshin-like 2 SGOL2 230165_at Nucleus
Other 3.108 Similar to hCG2031213 LOC728052 1558795_at Unknown
Other 3.402 Solute carrier family 1 (glial high SLC1A2 225491_at
Plasma Transporter -4.354 affinity glutamate transporter), Membrane
member 2 Solute carrier family 43, member 3 SLC43A3 210692_s_at
Extracellular Other 3.126 Space Solute carrier family 43, member 3
SLC43A3 213113_s_at Extracellular Other 2.804 Space Solute carrier
family 44, member 5 SLC44A5 1569112_at Unknown Other 3.923 Solute
carrier family 44, member 5 SLC44A5 235763_at Unknown Other 4.439
Solute carrier family 7 (cationic SLC7A2 225516_at Plasma
Transporter 3.23 amino acid transporter, y+ Membrane system),
member 2 Sortilin-related VPS10 domain SORCS1 1556891_at Plasma
Transporter -2.424 containing receptor 1 Membrane SPC25, NDC80
kinetochore SPC25 209891_at Unknown Other 2.634 complex component,
homolog ST8 alpha-N-acetyl-neuraminide ST8SIA4 230836_at Cytoplasm
Enzyme 2.705 alpha-2,8-sialyltransferase 4 Structural maintenance
of SMC2 204240_s_at Nucleus Transporter 2.442 chromosomes 2
Structural maintenance of SMC6 218781_at Nucleus Other 3.327
chromosomes 6 Structural maintenance of SMC6 236535_at Nucleus
Other 3.44 chromosomes 6 Syndecan 2 SDC2 212154_at Plasma Other
2.367 Membrane Syndecan 2 SDC2 212158_at Plasma Other 2.098
Membrane Thrombospondin 2 THBS2 203083_at Extracellular Other -2.81
Space Thyroid hormone receptor TRIP10 202734_at Cytoplasm Other
2.663 interactor 10 Tissue factor pathway inhibitor 2 TFPI2
209278_s_at Extracellular Other 4.666 Space Transcription factor
7-like 1 (T- TCF7L1 221016_s_at Nucleus Transcription 2.835 cell
specific, HMG-box) regulator Transmembrane phosphatase TPTE
220205_at Plasma Phosphatase 6.04 with tensin homology Membrane
Transmembrane protein 178 TMEM178 229302_at Unknown Other 3.194
Transmembrane protein 35 TMEM35 219685_at Unknown Other 2.879 Tumor
necrosis factor, alpha- TNFAIP8 210260_s_at Cytoplasm Other 2.389
induced protein 8 Twist homolog 1 TWIST1 213943_at Nucleus
Transcription 2.573 regulator Zinc finger protein 521 ZNF521
226676_at Nucleus Other 2.382 Zinc finger protein 521 ZNF521
226677_at Nucleus Other 2.943 Zinc finger protein 814 ZNF814
1556204_a_at Unknown Other -2.505 The log ratio represent log.sub.2
values where 0.585 is log.sub.2(1.5) which is a 1.5-fold
difference, 1 is log.sub.2(2) which is a 2-fold difference, 1.584
is log.sub.2(3) which is a 3-fold difference, 2 is log.sub.2(4)
which is a 4-fold difference, 2.321 is log.sub.2(5) which is a
5-fold difference, 2.584 is log.sub.2(6) which is a 6-fold
difference, 2.807 is log.sub.2(7) which is a 7-fold difference, 3
is log.sub.2(8) which is a 8-fold difference, 3.169 is log.sub.2(9)
which is a 9-fold difference, and 3.321 is log.sub.2(10) which is a
10-fold difference.
[0212] Using IPA Core Analysis, 73 of the 79 genes were mapped to 5
different protein networks (Table 15). Based on overlapping gene
mapping, 73 of the 79 genes that were up-regulated could be
associated with two major protein network groups, named A and B
(Table 15). Networks 1, 2 and 5 belong to Group A, and Networks 3
and 4 belong to Group B. Similarly, all down-regulated genes were
all associated with one network, named C (Table 15).
[0213] Group A genes include the FGFR2 receptor which has been
shown to function in the BoNT/A intoxication process as a receptor
for BoNT/A and are as follows: genes exhibiting a 1.5-fold or more
increase in expression levels in the H1 and BB10 cell lines as
compared to their expression in cells from the 2D6 cell line,
ADAMTS9, ARHGAP24, ARHGEF3, ASCL1, BARD1, CD9, CDK2, CSRP2, CTSL2,
DIAPH3, DOK5, DYNLT3, EMILIN2, ETV1, FBLN1, FGFR2, GNAI1, GNB4,
GNG11, GNG12, HGF, KITLG, LPAR1, MCM10, MSN, PAG1, PEG3, PLK2,
POLA2, PPP1R3C, PTPRK, RAB32, SDC2, SLC43A3, SLC7A2, SMC6, SPARC,
SPC25, ST8SIA4, TCF7L1, TFPI2, TMEM35, TNFAIP8, TPTE, TRIP10, and
TWIST1.
[0214] Group B genes are as follows: genes exhibiting a 1.5-fold or
more increase in expression levels in the H1 and BB10 cell lines as
compared to their expression in cells from the 2D6 cell line,
ACOT9, BASP1, C11ORF75, CCDC109B, CDCA7L, CLSTN2, CNTN1, CUGBP2,
DEPDC1, DPYD, FAM101B, FNDC5, GTSE1, MAOA, MINA, MYO6, MYRIP, PLK2,
PRLHR, PVRL3, RBPMS, SGOL2, SMC2, TFP12, TMEM178, and ZNF521.
[0215] Group C genes are as follows: genes exhibiting a 1.5-fold or
more decrease in expression levels in the H1 and BB10 cell lines as
compared to their expression in cells from the 2D6 cell line, ANK2,
CPNE8, CREB5, IL17B, KIAA0125, LOC100289109, LOC144571, NPAS4,
SLC1A2, SORCS1, THBS2, and/or ZNF814.
[0216] The remaining networks identified did not share common genes
between any other network. Individually, however, genes exhibiting
a 1.5-fold or more increase in expression levels in the H1 and BB10
cell lines as compared to their expression in cells from the 2D6
cell line were as follows: C3ORF70 (Network 6), MAB21L2 (Network
7), PRSS12 (Network 8), CENPL (Network 9), GPR177 (Network 10), and
PTGR1 (Network 11).
TABLE-US-00007 TABLE 15 IPA Core Analysis of Genes JMP Gene
Expression Profile Network Group Proteins in Network pvalue Number
Increased Decreased 1 A ADAMTS9, AKT, ASCL1, CDK2, 48 22 ADAMTS9,
-- CSRP2, CTSL2, DOK5, E2F, ASCL1, CDK2, FBLN1, FSH, GNAI1, GNG11,
CSRP2, CTSL2, hCG, HGF, KITLG, LPAR1, DOK5, FBLN1, MAPK, MCM10,
MEK, MSN, GNAI1, GNG11, NF.kappa.B, P38, MAPK, PAG1, PDGFBB, HGF,
KITLG, PEG3, PLK2, RAC, RAS, LPAR1, SDC2, SPARC, TFPI2, TGF.beta.,
MCM10, MSN, TNFAIP8, TRIP10, VEGF PAG1, PEG3, PLK2, SDC2, SPARC,
TFPI2, TNFAIP8, TRIP10 2 A CASP3, Caspase 3/7, CCL6, 35 17 DIAPH3,
-- DIAPH3, DYNLT3, EMILIN2, DYNLT3, EPM2A, ETV1, EYA2, FAM3B,
EMILIN2, ETV1, FASTK, GNAI1, GNB4, GNG10, GNAI1, GNB4, GNG11,
GNG12, GSTM2, HK1, GNG11, IGFBP3, IL13, PLCE1, POLA2, GNG12,
PPP1R3C, PTPRK, PTPRZ1, POLA2, RAB32, SLC43A3, SLC7A2, PPP1R3C,
SRC, ST8SIA4, TNF, TPTE, PTPRK, RAB32, TWIST1 SLC43A3, SLC7A2,
ST8SIA4, TPTE, TWIST1 3 B ACOT9, AR, ATXN7, BASP1, 26 14 ACOT9,
BASP1, -- CCNE2, CCNK, CLSTN2, CLSTN2, CNTN1, CUGBP2, DGKA, CNTN1,
FNDC5, GTSE1, IL4, MAOA, CUGBP2, MSH2, NOVA1, NF1, PICK1, FNDC5,
PLK2, PNRC1, POM121, GTSE1, MAOA, PRLHR, PTP4A1, PTP4A2, PLK2,
PRLHR, PVRL1, PVRL3, PVRL4, PVRL3, RBPMS, SERPINB5, SMARCA4, RBPMS,
TFP12, SPN, TFP12, TP53, ZNF521 ZNF521 4 B ARHGAP17, AXL, C11ORF75,
24 13 C11ORF75, -- CAPN6, CCDC109B, CDCA7L, CCDC109B, DEPDC1, DPYD,
EIF4E, CDCA7L, FAM101B, GRB2, HNRNPF, DEPDC1, HTATIP2, MAOA, MINA,
DPYD, MIR124, MIRLET7A1, MYC, FAM101B, MYO6, MYRIP, NCAPD3, NF2,
MAOA, MINA, PPP2R5A, PPP2R5D, MYO6, MYRIP, PPP2R5E, RPS16, RPS23,
SGOL2, SMC2, SGOL2, SLC25A12, SMC2, TMEM178 SMC4, TMEM178, TUSC2,
TXNIP, ZBTB16 5 A ARHGAP24, ARHGEF, 17 10 ARHGAP24, -- ARHGEF3,
BARD1, CD9, CD53, ARHGEF3, CTNNB1, DUSP3, ERK, ERM, BARD1, CD9,
FGF9, FGF18, FGF21, FGF23, FGFR2, LPAR1, FGFR2, IL1, IL6, IL17RD,
SMC6, SPC25, Integrin.alpha.6.beta.1, KLB, LEF/TCF, TCF7L1, LPAR1,
MIRN297-2, PI3K, RAS TMEM35 homolog, RNAPOLII, SLC26A2, SMC6,
SPC25, SREBF1, TACC1, TCF7L1, TMEM35, TPT1, WISP1 6 -- C3ORF70,
MIR31 2 1 C3ORF70 -- 7 -- MAB21L2, MIRN294 2 1 MAB21L2 -- 8 --
AGPN, PRSS12 2 1 PRSS12 -- 9 -- CENPL, MIRN340 2 1 CENPL -- 10 --
GPR177, MIRN324 2 1 GPR177 -- 11 -- ADH, PTGR1 2 1 PTGR1 -- 12 C
ANK2, ATP2A2, BDNF, CEBPG, 22 8 -- ANK2, CPNE8, CPNE8, CREB5, GRM3,
GRM5, CREB5, IL17B, IGFBP2, IL17B, ITPR, ITPR1, KIAA0125, K+,
L1CAM, MIRN330, LOC100289109, Neurotrophin, NFE2L1, NGFR,
LOC144571, NME1, NPAS4, Pro-inflammatory NPAS4, Cytokine, PSEN1,
SCN2A, SLC1A2, SCN3A, SCNN1B, SLC1A2, SORCS1, SLC8A1, SORCS1,
SORT1, THBS2, ZNF814 TGFA, THBS2, TNF, TNFAIP6
[0217] Computer analysis was performed as in Example V, except that
the list of probes reflects genes that were greater than 1.5-fold
differential expressed in the H1 and the BB10 cell lines when
compared to both the 2D6 and SiMa parental cell lines. When the
p-value cut off was set to 0.05, this analysis identified 439 gene
probes that could be classified to 369 genes (Table 16).
TABLE-US-00008 TABLE 16 Genes Identified using IPA Core Analysis
Gene Log Gene Name Symbol Probe ID. No. Location Type Ratio
6-phosphofructo-2- PFKFB3 202464_s_at Cytoplasm kinase 1.811
kinase/fructose-2,6- biphosphatase 3 achaete-scute complex homolog
1 ASCL1 209985_s_at Nucleus transcription 2.429 regulator
achaete-scute complex homolog 1 ASCL1 209987_s_at Nucleus
transcription 2.097 regulator achaete-scute complex homolog 1 ASCL1
209988_s_at Nucleus transcription 1.446 regulator achaete-scute
complex homolog 1 ASCL1 213768_s_at Nucleus transcription 1.578
regulator activated leukocyte cell adhesion ALCAM 1569362_at Plasma
other 2.612 molecule Membrane activated leukocyte cell adhesion
ALCAM 201951_at Plasma other 1.774 molecule Membrane activated
leukocyte cell adhesion ALCAM 201952_at Plasma other 1.521 molecule
Membrane acyl-CoA thioesterase 1 ACOT1 202982_s_at Cytoplasm enzyme
-2.875 acylphosphatase 2, muscle type ACYP2 206833_s_at unknown
enzyme -1.15 additional sex combs like 3 ASXL3 233536_at unknown
other 1.572 adenosine deaminase, RNA- ADARB1 203865_s_at Nucleus
enzyme -2.474 specific, B1 adenylate cyclase 1 ADCY1 213245_at
Plasma enzyme -1.935 Membrane adipocyte-specific adhesion ASAM
228082_at Plasma other -2.802 molecule Membrane ADP-ribosylation
factor 1 ARF1 1565651_at Cytoplasm enzyme 1.063 ADP-ribosylation
factor-like 17A ARL17A 243899_at unknown other 0.741 anaphase
promoting complex ANAPC7 225554_s_at unknown other 0.679 subunit 7
ankyrin repeat and BTB (POZ) ABTB1 229164_s_at Cytoplasm
translation -0.656 domain containing 1 regulator ankyrin repeat
domain 50 ANKRD50 225731_at unknown other -0.654 ankyrin repeat
domain 50 ANKRD50 225735_at unknown other -1.114 aspartate
beta-hydroxylase ASPHD1 214993_at unknown other -0.763 domain
containing 1 ataxin 2-binding protein 1 A2BP1 1553422_s_at
Cytoplasm other 1.453 ataxin 2-binding protein 1 A2BP1 221217_s_at
Cytoplasm other 1.497 ATPase, Ca++ transporting, ATP2B3 207026_s_at
Plasma transporter -1.248 plasma membrane 3 Membrane ATPase, Ca++
transporting, ATP2B3 242036_x_at Plasma transporter -1.777 plasma
membrane 3 Membrane basonuclin 2 BNC2 220272_at Nucleus other 2.466
basonuclin 2 BNC2 230722_at Nucleus other 1.641
.beta..gamma.crystallin domain containing 3 CRYBG3 214030_at
unknown other 4.74 .beta.-site APP-cleaving enzyme 1 BACE1
217904_s_at Cytoplasm peptidase -1.57 brain abundant, membrane
BASP1 202391_at Plasma other 1.146 attached signal protein 1
Membrane bromodomain and WD repeat BRWD1 225446_at Nucleus
transcription -1.018 domain containing 1 regulator bruno-like 4,
RNA binding BRUNOL4 238966_at Nucleus translation -0.81 protein
regulator butyrophilin, subfamily 3, BTN3A3 38241_at unknown other
-1.108 member A3 C2 calcium-dependent domain C2CD2 212875_s_at
unknown other -1.27 containing 2 C2 calcium-dependent domain C2CD4A
241031_at unknown other -2.615 containing 4A cadherin 12, type 2
(N-cadherin CDH12 207149_at Plasma other -1.408 2) Membrane
calcium/calmodulin-dependent CAMK2N2 230706_s_at Nucleus other
-0.692 protein kinase II inhibitor 2 calmin (calponin-like, CLMN
213839_at Cytoplasm other -1.911 transmembrane) calpain 2, (m/II)
large subunit CAPN2 208683_at Cytoplasm peptidase -1.429 CAP,
adenylate cyclase- CAP2 212551_at Plasma other -1.974 associated
protein, 2 Membrane CAP, adenylate cyclase- CAP2 212554_at Plasma
other -3.68 associated protein, 2 Membrane carboxypeptidase,
vitellogenic- CPVL 208146_s_at unknown peptidase 1.708 like CART
prepropeptide CARTPT 206339_at Extracellular other -4.676 Space
Cas-Br-M (murine) ecotropic CBLB 209682_at Nucleus other 1.032
retroviral transforming sequence b CCR4-NOT transcription CNOT6L
227119_at Cytoplasm other -0.785 complex, subunit 6-like CD302
molecule CD302 203799_at Plasma receptor -1.518 Membrane CD9
molecule CD9 201005_at Plasma other 1.415 Membrane
CDP-diacylglycerol synthase CDS1 226185_at Cytoplasm enzyme -1.74
(phosphatidate cytidylyltransferase) 1 cell division cycle 25
homolog B CDC25B 201853_s_at Nucleus phosphatase 1.453 (S. pombe)
chemokine (C--X--C motif) CXCR4 209201_x_at Plasma receptor 4.135
receptor 4 Membrane chemokine (C--X--C motif) CXCR4 211919_s_at
Plasma receptor 3.957 receptor 4 Membrane chemokine (C--X--C motif)
CXCR4 217028_at Plasma receptor 3.176 receptor 4 Membrane chloride
channel 3 CLCN3 201733_at Plasma ion channel -0.908 Membrane
chloride channel 3 CLCN3 201734_at Plasma ion channel -0.987
Membrane cholinergic receptor, nicotinic, CHRNA7 210123_s_at Plasma
receptor 3.098 alpha 7 Membrane chromosome 1 open reading C1ORF21
221272_s_at unknown other -0.74 frame 21 chromosome 1 open reading
C1ORF21 223127_s_at unknown other -0.748 frame 21 chromosome 1 open
reading C1ORF43 1555225_at unknown other 1.604 frame 43 chromosome
10 open reading C10ORF58 224435_at Extracellular other 0.918 frame
58 Space chromosome 12 open reading C12ORF49 218867_s_at unknown
other 0.692 frame 49 chromosome 16 open reading C16ORF52 230721_at
unknown other -0.87 frame 52 chromosome 20 open reading C20ORF7
227160_s_at unknown other 0.624 frame 7 chromosome 21 open reading
C21ORF57 227421_at unknown other -1.086 frame 57 chromosome 21 open
reading C21ORF57 239208_s_at unknown other -0.657 frame 57
chromosome 3 open reading C3ORF23 1555905_a_at Cytoplasm other
-2.797 frame 23 chromosome 3 open reading C3ORF23 1555906_s_at
Cytoplasm other -1.502 frame 23 chromosome 3 open reading C3ORF23
241666_at Cytoplasm other -1.017 frame 23 chromosome 9 open reading
C9ORF150 227443_at unknown other -1.773 frame 150 claudin 12 CLDN12
223249_at Plasma other -0.766 Membrane COBW domain containing 1
CBWD1 226193_x_at unknown other 0.849 coiled-coil domain containing
3 CCDC3 223316_at Cytoplasm other 3.357 collagen, type VI, alpha 1
COL6A1 213428_s_at Extracellular other -1.638 Space cripto, FRL-1,
cryptic family 1 CFC1 223753_s_at Extracellular other -2.834 Space
cripto, FRL-1, cryptic family 1 CFC1 236724_at Extracellular other
-1.636 Space CSE1 chromosome segregation CSE1L 210765_at Nucleus
transporter 1.581 1-like CUG triplet repeat, RNA binding CUGBP2
202156_s_at Nucleus other 1.318 protein 2 CUG triplet repeat, RNA
binding CUGBP2 202157_s_at Nucleus other 2.156 protein 2 CUG
triplet repeat, RNA binding CUGBP2 202158_s_at Nucleus other 2.539
protein 2 cyclic nucleotide gated channel CNGA3 207261_at Plasma
ion channel -3.182 alpha 3 Membrane cyclin M1 CNNM1 220166_at
Plasma other -1.214 Membrane cyclin-dependent kinase inhibitor
CDKN2A 209644_x_at Nucleus transcription -1.277 2A (melanoma, p16,
inhibits regulator CDK4) cylindromatosis (turban tumor CYLD
213295_at Nucleus transcription -0.815 syndrome) regulator
cytochrome P450, family 2, CYP2E1 1431_at Cytoplasm enzyme -4.466
subfamily E, polypeptide 1 cytochrome P450, family 2, CYP2E1
209975_at Cytoplasm enzyme -3.946 subfamily E, polypeptide 1
cytochrome P450, family 2, CYP2E1 209976_s_at Cytoplasm enzyme
-3.429 subfamily E, polypeptide 1 cytoglobin CYGB 1553572_a_at
Cytoplasm transporter -4.921 cytokine-like 1 CYTL1 219837_s_at
Extracellular cytokine -6.33 Space dapper, antagonist of beta-
DACT1 219179_at Cytoplasm other 1.682 catenin, homolog 1 dCMP
deaminase DCTD 201572_x_at unknown enzyme -0.649 DEAH
(Asp-Glu-Ala-His) box DHX35 218579_s_at unknown enzyme 0.735
polypeptide 35 deleted in azoospermia 1 DAZ1 207909_x_at Nucleus
translation 6.92 regulator deleted in azoospermia 1 DAZ1
207912_s_at Nucleus translation 5.558 regulator deleted in
azoospermia 1 DAZ1 208281_x_at Nucleus translation 5.258 regulator
deleted in azoospermia 1 DAZ1 208282_x_at Nucleus translation 7.008
regulator deleted in azoospermia 1 DAZ1 216351_x_at Nucleus
translation 3.596 regulator deleted in azoospermia 1 DAZ1
216922_x_at Nucleus translation 5.185 regulator deleted in
lymphocytic leukemia DLEU2 216870_x_at unknown other 1.233 2
(non-protein coding) delta-like 3 (Drosophila) DLL3 219537_x_at
Extracellular other 0.779 Space DNA segment on chromosome 4 D4S234E
209569_x_at Cytoplasm other -1.247 (unique) 234 expressed sequence
DNA segment on chromosome 4 D4S234E 209570_s_at Cytoplasm other
-1.488 (unique) 234 expressed sequence DNA segment on chromosome 4
D4S234E 213533_at Cytoplasm other -1.507 (unique) 234 expressed
sequence dopamine beta-hydroxylase DBH 206450_at Cytoplasm enzyme
1.311 (dopamine beta- monooxygenase) doublecortin-like kinase 1
DCLK1 215303_at Cytoplasm kinase -1.62 dual specificity phosphatase
16 DUSP16 224832_at Nucleus phosphatase -0.918 echinoderm
microtubule EML1 204797_s_at Cytoplasm other 2.668 associated
protein like 1 ectodermal-neural cortex (with ENC1 201340_s_at
Nucleus peptidase 2.445 BTB-like domain) ectodermal-neural cortex
(with ENC1 201341_at Nucleus peptidase 1.91 BTB-like domain)
electron-transferring-flavoprotein ETFDH 33494_at Cytoplasm enzyme
-0.807 dehydrogenase ELL associated factor 2 EAF2 219551_at Nucleus
transcription 0.693 regulator ELOVL family member 7, ELOVL7
227180_at unknown other 2.644 elongation of long chain fatty acids
endoplasmic reticulum ERAP1 214012_at Extracellular peptidase
-1.885 aminopeptidase 1 Space enhancer of zeste homolog 2 EZH2
203358_s_at Nucleus transcription 0.94 regulator ephrin-A5 EFNA5
214036_at Plasma kinase 2.041 Membrane ephrin-A5 EFNA5 227955_s_at
Plasma kinase 1.84 Membrane ephrin-A5 EFNA5 233814_at Plasma kinase
1.805 Membrane ephrin-B3 EFNB3 205031_at Plasma kinase -0.816
Membrane eukaryotic translation initiation EIF3C 236700_at
Cytoplasm translation 1.378 factor 3, subunit C regulator exophilin
5 EXPH5 214734_at unknown other 3.973 exosome component 6 EXOSC6
231916_at Nucleus other 1.057 family with sequence similarity
FAM162B 228875_at unknown other -0.743 162, member B family with
sequence similarity FAM165B 228239_at Plasma other -0.822 165,
member B Membrane family with sequence similarity FAM181B 231430_at
unknown other 0.794 181, member B family with sequence similarity
FAM19A4 242348_at Extracellular other
1.282 19 (chemokine (C-C motif)-like), Space member A4 family with
sequence similarity FAM46A 224973_at unknown other -1.509 46,
member A family with sequence similarity 7, FAM7A3 243356_at
unknown other 3.495 member A3 far upstream element (FUSE) FUBP3
239193_at Nucleus transcription 1.192 binding protein 3 regulator
F-box and WD repeat domain FBXW7 229419_at Nucleus transcription
-0.921 containing 7 regulator fibroblast growth factor 13 FGF13
205110_s_at Extracellular growth factor -3.522 Space
follistatin-like 1 FSTL1 208782_at Extracellular other -1.275 Space
forkhead box O6 FOXO6 239657_x_at Nucleus other -1.512 frizzled
homolog 5 FZD5 221245_s_at Plasma receptor 2.347 Membrane FYVE,
RhoGEF and PH domain FGD5 226985_at Cytoplasm other -1.952
containing 5 G protein-coupled receptor 123 GPR123 239221_at Plasma
receptor -1.302 Membrane G protein-coupled receptor 125 GPR125
210473_s_at Plasma receptor 0.714 Membrane galanin prepropeptide
GAL 207466_at Extracellular other -1.434 Space galanin
prepropeptide GAL 214240_at Extracellular other -3.128 Space
gastrin-releasing peptide GRP 206326_at Extracellular growth factor
4.698 Space GDNF family receptor alpha 2 GFRA2 205721_at Plasma
receptor 1.847 Membrane GDNF family receptor alpha 2 GFRA2
205722_s_at Plasma receptor 2.265 Membrane glutamate receptor,
metabotropic 5 GRM5 214217_at Plasma receptor -2.231 Membrane
glutathione peroxidase 7 GPX7 213170_at Cytoplasm enzyme -2.692
glutathione synthetase GSS 211630_s_at Cytoplasm enzyme 0.599
glycosyltransferase 25 domain GLT25D2 209883_at unknown other
-1.223 containing 2 GNAS complex locus GNAS 214157_at Plasma enzyme
-1.794 Membrane growth associated protein 43 GAP43 204471_at Plasma
other 0.869 Membrane growth associated protein 43 GAP43 216963_s_at
Plasma other 1.151 Membrane guanine nucleotide binding GNAI1
209576_at Plasma enzyme 2.225 protein (G protein), alpha Membrane
inhibiting activity polypeptide 1 guanine nucleotide binding GNAI1
227692_at Plasma enzyme 2.111 protein (G protein), alpha Membrane
inhibiting activity polypeptide 1 guanine nucleotide binding GNB4
225710_at Plasma enzyme 4.695 protein (G protein), beta Membrane
polypeptide 4 guanine nucleotide binding GNG11 204115_at Plasma
enzyme 2.587 protein (G protein), gamma 11 Membrane guanylate
cyclase 1, soluble, GUCY1A3 221942_s_at Cytoplasm enzyme -1.522
alpha 3 guanylate cyclase 1, soluble, GUCY1A3 229530_at Cytoplasm
enzyme -1.696 alpha 3 helicase, POLQ-like HELQ 228736_at Nucleus
enzyme -1.28 HERPUD family member 2 HERPUD2 236170_x_at unknown
other -0.897 heterogeneous nuclear HNRNPR 208765_s_at Nucleus other
-0.64 ribonucleoprotein R hippocalcin-like 1 HPCAL1 205462_s_at
Cytoplasm other -1.551 hippocalcin-like 1 HPCAL1 212552_at
Cytoplasm other -1.387 histone cluster 1, H2ac HIST1H2AC
215071_s_at Nucleus other -2.462 histone cluster 1, H2bd HIST1H2BD
209911_x_at Nucleus other -2.036 histone cluster 1, H2bk HIST1H2BK
209806_at Nucleus other -1.575 histone cluster 2, H2be HIST2H2BE
202708_s_at Nucleus other -1.735 hypothetical LOC100130522
LOC100130522 230477_at unknown other -0.928 hypothetical LOC26082
DKFZP434L187 230861_at unknown other 0.968 hypothetical protein
LOC100128844 229110_at unknown other 2.106 LOC100128844
hypothetical protein LOC643401 LOC340109 1557765_at unknown other
2.848 integrin, alpha 6 ITGA6 201656_at Plasma other 2.822 Membrane
integrin, alpha 6 ITGA6 215177_s_at Plasma other 2.024 Membrane
integrin, beta 5 ITGB5 201125_s_at Plasma other 0.913 Membrane
interleukin 10 receptor, beta IL10RB 209575_at Plasma transmembrane
-1.444 Membrane receptor junction mediating and JMY 226352_at
Nucleus transcription -0.744 regulatory protein, p53 cofactor
regulator K(lysine) acetyltransferase 2B KAT2B 203845_at Nucleus
transcription -0.731 regulator KDEL (Lys-Asp-Glu-Leu) KDELC2
225128_at unknown other 1.085 containing 2 kelch domain containing
1 KLHDC1 1552733_at unknown other -0.952 kelch repeat and BTB (POZ)
KBTBD11 204301_at unknown other -0.733 domain containing 11
kelch-like 13 (Drosophila) KLHL13 227875_at unknown other 0.902 KH
domain containing, RNA KHDRBS3 209781_s_at Nucleus other 1.082
binding, signal transduction associated 3 KH homology domain
containing 1 KHDC1 230055_at unknown other -1.656 KIAA1598 KIAA1598
221802_s_at unknown other -2.811 kinesin family member 16B KIF16B
232083_at Cytoplasm other 1.688 lectin, galactoside-binding,
LGALS3BP 200923_at Plasma transmembrane -1.417 soluble, 3 binding
protein Membrane receptor leukemia inhibitory factor LIFR 225575_at
Plasma transmembrane -0.941 receptor alpha Membrane receptor limb
bud and heart development LBH 221011_s_at Nucleus transcription
0.788 homolog (mouse) regulator LRP2 binding protein LRP2BP
207797_s_at unknown other -1.365 lumican LUM 201744_s_at
Extracellular other 5.638 Space LYR motif containing 5 LYRM5
225469_at unknown other -0.93 mab-21-like 1 MAB21L1 206163_at
unknown other 2.722 mab-21-like 2 MAB21L2 210302_s_at unknown other
2.871 mab-21-like 2 MAB21L2 210303_at unknown other 2.204
macrophage stimulating 1 MST1 216320_x_at Extracellular growth
factor -1.012 (hepatocyte growth factor-like) Space major
facilitator superfamily MFSD4 229254_at unknown other -1.752 domain
containing 4 mannosidase, alpha, class 2A, MAN2A1 226538_at
Cytoplasm enzyme 0.864 member 1 mannosyl (alpha-1,3-)- MGAT4A
226039_at unknown enzyme -0.904 glycoprotein beta-1,4-N-
acetylglucosaminyltransferase, isozyme A mesenchyme homeobox 2
MEOX2 206201_s_at Nucleus transcription -4.213 regulator
microtubule-associated protein 9 MAP9 220145_at unknown other -0.7
microtubule-associated protein 9 MAP9 228423_at unknown other
-1.108 microtubule-associated protein 9 MAP9 235550_at unknown
other -0.863 mitochondrial ribosomal protein MRPS33 218654_s_at
Cytoplasm other -0.609 S33 mitogen-activated protein kinase MAP3K5
203836_s_at Cytoplasm kinase -1.159 kinase kinase 5 moesin MSN
200600_at Plasma other 2.082 Membrane monoamine oxidase A MAOA
204388_s_at Cytoplasm enzyme 2.15 monoamine oxidase A MAOA
204389_at Cytoplasm enzyme 1.88 monoamine oxidase A MAOA 212741_at
Cytoplasm enzyme 2.048 monocyte to macrophage MMD 244523_at Plasma
other 1.045 differentiation-associated Membrane multiple C2
domains, MCTP1 220122_at unknown other -1.597 transmembrane 1 MYC
induced nuclear antigen MINA 213188_s_at Nucleus other 1.391 MYC
induced nuclear antigen MINA 213189_at Nucleus other 1.641 myosin
IB MYO1B 212365_at Cytoplasm other 0.71 myosin VI MYO6 203216_s_at
Cytoplasm other 1.104 myosin VI MYO6 210480_s_at Cytoplasm other
1.41 Na+/H+ exchanger domain NHEDC2 1564746_at Plasma other -1.462
containing 2 Membrane N-acylethanolamine acid NAAA 214765_s_at
Cytoplasm enzyme 0.911 amidase NADH dehydrogenase NDUFA4L2
218484_at unknown enzyme -3.507 (ubiquinone) 1 alpha subcomplex,
4-like 2 NADH dehydrogenase NDUFV3 226616_s_at Cytoplasm enzyme
-0.875 (ubiquinone) flavoprotein 3, 10 kDa nebulette NEBL 203961_at
Cytoplasm other -1.359 neural precursor cell expressed, NEDD9
202150_s_at Nucleus other 2.41 developmentally down-regulated 9
NIPA-like domain containing 3 NIPAL3 225876_at unknown other -1.203
nitric oxide synthase 1 NOS1 239132_at Cytoplasm enzyme 1.138
(neuronal) nitric oxide synthase 1 NOS1 240911_at Cytoplasm enzyme
1.187 (neuronal) NLR family member X1 NLRX1 219680_at unknown other
-1.541 non-protein coding RNA 171 NCRNA00171 215985_at unknown
other -0.692 nuclear receptor coactivator 7 NCOA7 225344_at Nucleus
other -0.842 nucleosome assembly protein 1- NAP1L3 204749_at
Nucleus other -1.53 like 3 nudix (nucleoside diphosphate NUDT19
235384_at Cytoplasm other -1.685 linked moiety X)-type motif 19
olfactomedin 3 OLFM3 1554524_a_at Cytoplasm other -0.927 oxysterol
binding protein-like 3 OSBPL3 209626_s_at Cytoplasm other 2.083
pecanex-like 2 PCNXL2 1554256_a_at unknown other -0.728 pellino
homolog 2 PELI2 219132_at Cytoplasm other 0.732 peptidase M20
domain PM20D2 225421_at unknown other 1.6 containing 2
peptidylglycine alpha-amidating PAM 202336_s_at Plasma enzyme
-0.861 monooxygenase Membrane peripherin PRPH 213847_at Plasma
other -1.417 Membrane PHD finger protein 20 PHF20 235389_at Nucleus
other 0.596 phosphatidic acid phosphatase PPAPDC1A 236044_at
unknown phosphatase -2.621 type 2 domain containing 1A
phosphatidylinositol glycan PIGH 209625_at Cytoplasm enzyme -0.597
anchor biosynthesis, class H phosphoinositide-interacting
HCG1776018 232887_at unknown other -2.019 regulator of transient
receptor potential channels phosphorylase kinase, alpha 1 PHKA1
229876_at Cytoplasm kinase -1.2 (muscle) plastin 3 (T isoform) PLS3
201215_at Cytoplasm other 1.068 platelet-derived growth factor
PDGFRB 202273_at Plasma kinase -1.373 receptor, beta polypeptide
Membrane pleiotrophin PTN 209466_x_at Extracellular growth factor
-1.426 Space pleiotrophin PTN 211737_x_at Extracellular growth
factor -2.929 Space pleiotropic regulator 1 PLRG1 225194_at Nucleus
transcription -0.701 regulator pleiotropic regulator 1 PLRG1
227246_at Nucleus transcription -1.228 regulator plexin A2 PLXNA2
213030_s_at Plasma other 0.946 Membrane plexin A4 PLXNA4 232317_at
Plasma receptor 1.368 Membrane polo-like kinase 2 PLK2 201939_at
Nucleus kinase 3.606 polycomb group ring finger 5 PCGF5 227935_s_at
unknown other 0.791 polymerase (DNA directed), POLE 216026_s_at
Nucleus enzyme 1.053 epsilon potassium channel KCTD12 212188_at
unknown ion channel 3.396 tetramerisation domain containing 12
potassium channel KCTD12 212192_at unknown ion channel 4.806
tetramerisation domain containing 12 potassium large conductance
KCNMA1 221584_s_at Plasma ion channel -2.389 calcium-activated
channel, Membrane subfamily M, alpha member 1 potassium
voltage-gated KCNQ2 205737_at Plasma ion channel -1.246 channel,
KQT-like subfamily, Membrane member 2 pregnancy-associated plasma
PAPPA 201982_s_at Extracellular peptidase -1.672 protein A,
pappalysin 1 Space prostaglandin E receptor 2 PTGER2 206631_at
Plasma receptor -2.328 (subtype EP2), 53 kDa Membrane prostaglandin
F2 receptor PTGFRN 224937_at Plasma other 2.301 negative regulator
Membrane proteasome (prosome, PSMA7 216088_s_at Cytoplasm peptidase
0.658 macropain) subunit, alpha type, 7 protein kinase, cAMP-
PRKACB 235780_at Cytoplasm kinase -2.17 dependent, catalytic, beta
protein tyrosine phosphatase, PTPRD 214043_at Plasma phosphatase
-1.16 receptor type, D Membrane protein tyrosine phosphatase, PTPRE
221840_at Plasma phosphatase 3.044 receptor type, E Membrane
protein tyrosine phosphatase, PTPRG 204944_at Plasma phosphatase
1.237 receptor type, G Membrane RAB35, member RAS oncogene RAB35
205461_at Cytoplasm enzyme 0.737 family RAB6B, member RAS oncogene
RAB6B 225259_at Cytoplasm enzyme -1.229 family
Rac/Cdc42 guanine nucleotide ARHGEF6 209539_at Cytoplasm other
2.719 exchange factor (GEF) 6 RAN, member RAS oncogene RAN
200750_s_at Nucleus enzyme 0.59 family RAS and EF-hand domain RASEF
1553986_at unknown other 1.393 containing ras homolog gene family,
RHOU 223168_at Cytoplasm enzyme -1.154 member U regulator of
G-protein signaling 5 RGS5 1555725_a_at Plasma other 4.038 Membrane
regulator of G-protein signaling 5 RGS5 209070_s_at Plasma other
3.729 Membrane regulator of G-protein signaling 5 RGS5 209071_s_at
Plasma other 3.468 Membrane regulator of G-protein signaling 5 RGS5
218353_at Plasma other 3.65 Membrane RELT-like 1 RELL1 226430_at
unknown other 1.214 retinoblastoma-like 1 (p107) RBL1 1559307_s_at
Nucleus other 0.959 retrotransposon gag domain RGAG4 227823_at
unknown other -1.775 containing 4 Rho GTPase activating protein 6
ARHGAP6 206167_s_at Cytoplasm other 2.809 ribosomal L1 domain
containing 1 RSL1D1 213750_at Cytoplasm other -0.816 RIMS binding
protein 2 RIMBP2 238817_at Plasma other 1.679 Membrane ring finger
protein 13 RNF13 201780_s_at Nucleus other -0.707 ring finger
protein 182 RNF182 230720_at unknown other 2.696 ring finger
protein 34 RNF34 219035_s_at Cytoplasm enzyme 0.85 ring finger
protein 41 RNF41 201962_s_at Cytoplasm other -0.591 RNA polymerase
I transcription LOC94431 216908_x_at unknown other -0.674 factor
homolog pseudogene 1 sal-like 4 SALL4 229661_at Nucleus other 1.586
salt-inducible kinase 1 SIK1 208078_s_at Cytoplasm kinase -1.403
seizure related 6 homolog SEZ6L 207873_x_at Plasma other 0.866
(mouse)-like Membrane seizure related 6 homolog SEZ6L 211894_x_at
Plasma other 0.797 (mouse)-like Membrane sema domain, transmembrane
SEMA6A 220454_s_at Plasma other 1.356 domain (TM), and cytoplasmic
Membrane domain, (semaphorin) 6A serine peptidase inhibitor, Kunitz
SPINT2 210715_s_at Extracellular other -1.889 type, 2 Space SH3
domain containing, Ysc84- SH3YL1 204019_s_at unknown other -0.859
like 1 SH3-domain GRB2-like 2 SH3GL2 205751_at Plasma enzyme -1.605
Membrane similar to hCG2031213 LOC728052 1558795_at unknown other
2.239 similar to hCG2031213 LOC728052 1558796_a_at unknown other
1.887 single immunoglobulin and toll- SIGIRR 52940_at Plasma
receptor -1.42 interleukin 1 receptor (TIR) Membrane domain
single-minded homolog 1 SIM1 1556300_s_at Nucleus transcription
6.36 regulator single-minded homolog 1 SIM1 206876_at Nucleus
transcription 5.737 regulator SLIT and NTRK-like family, SLITRK5
214930_at unknown other 4.094 member 5 SMG1 homolog, LOC641298
244766_at unknown other 1.065 phosphatidylinositol 3-kinase-
related kinase pseudogene solute carrier family 12 SLC12A7
218066_at Plasma transporter -1.258 (potassium/chloride Membrane
transporters), member 7 solute carrier family 22, member SLC22A17
218675_at Plasma transporter -0.724 17 Membrane solute carrier
family 35, member SLC35F3 229065_at unknown other -3.048 F3 solute
carrier family 44, member 5 SLC44A5 1569112_at unknown other 2.738
solute carrier family 44, member 5 SLC44A5 235763_at unknown other
1.845 solute carrier family 7 (cationic SLC7A2 225516_at Plasma
transporter 1.892 amino acid transporter, y+ Membrane system),
member 2 solute carrier organic anion SLCO3A1 219229_at Plasma
transporter 1.622 transporter family, member 3A1 Membrane solute
carrier organic anion SLCO3A1 227367_at Plasma transporter 1.045
transporter family, member 3A1 Membrane sparc/osteonectin, cwcv and
SPOCK2 202524_s_at Extracellular other -0.83 kazal-like domains
proteoglycan Space (testican) 2 sperm associated antigen 6 SPAG6
210032_s_at Cytoplasm other 1.687 sperm associated antigen 6 SPAG6
210033_s_at Cytoplasm other 2.774 spermatogenesis associated,
SPATS2L 222154_s_at unknown other 0.907 serine-rich 2-like
sphingosine-1-phosphate S1PR3 228176_at Plasma receptor 1.9
receptor 3 Membrane ST8 alpha-N-acetyl-neuraminide ST8SIA1
210073_at Cytoplasm enzyme 1.114 alpha-2,8-sialyltransferase 1 ST8
alpha-N-acetyl-neuraminide ST8SIA3 230262_at Cytoplasm enzyme
-1.039 alpha-2,8-sialyltransferase 3 stathmin-like 3 STMN3
222557_at Nucleus other 0.788 STEAP family member 3 STEAP3
218424_s_at Cytoplasm transporter -1.827 steroidogenic acute
regulatory STAR 204548_at Cytoplasm transporter -1.093 protein
stimulated by retinoic acid gene STRA6 1569334_at Plasma other
1.359 6 homolog Membrane stimulated by retinoic acid gene STRA6
1569335_a_at Plasma other 1.167 6 homolog Membrane storkhead box 2
STOX2 226822_at unknown other -0.854 synaptojanin 1 SYNJ1 212990_at
Cytoplasm phosphatase -0.624 synaptotagmin XIII SYT13 226086_at
unknown transporter -4.754 synuclein, alpha (non A4 SNCA 236081_at
Cytoplasm other -0.935 component of amyloid precursor) taxilin beta
TXLNB 227834_at Cytoplasm other -2.226 teashirt zinc finger
homeobox 3 TSHZ3 223392_s_at Nucleus transcription 1.472 regulator
teashirt zinc finger homeobox 3 TSHZ3 223393_s_at Nucleus
transcription 0.962 regulator tetratricopeptide repeat domain
TTC39C 238480_at unknown other -0.824 39C TH1-like TH1L 220607_x_at
Nucleus other 1.027 TH1-like TH1L 225006_x_at Nucleus other 0.884
TH1-like TH1L 225261_x_at Nucleus other 0.883 TH1-like TH1L
225865_x_at Nucleus other 0.904 thioredoxin reductase 1 TXNRD1
201266_at Cytoplasm enzyme 1.032 THO complex 4 THOC4 226319_s_at
Nucleus transcription 0.996 regulator TIMP metallopeptidase
inhibitor 3 TIMP3 201147_s_at Extracellular other 5.151 Space TIMP
metallopeptidase inhibitor 3 TIMP3 201148_s_at Extracellular other
3.637 Space TIMP metallopeptidase inhibitor 3 TIMP3 201149_s_at
Extracellular other 5.037 Space TIMP metallopeptidase inhibitor 3
TIMP3 201150_s_at Extracellular other 4.312 Space tissue factor
pathway inhibitor 2 TFPI2 209277_at Extracellular other 1.592 Space
tissue factor pathway inhibitor 2 TFPI2 209278_s_at Extracellular
other 2.326 Space TM2 domain containing 1 TM2D1 213882_at Plasma
receptor -0.769 Membrane transcription factor 7-like 2 (T- TCF7L2
212761_at Nucleus transcription 1.005 cell specific, HMG-box)
regulator transcription factor 7-like 2 (T- TCF7L2 212762_s_at
Nucleus transcription 1.224 cell specific, HMG-box) regulator
transcription factor 7-like 2 (T- TCF7L2 216035_x_at Nucleus
transcription 1.072 cell specific, HMG-box) regulator transcription
factor 7-like 2 (T- TCF7L2 216037_x_at Nucleus transcription 0.81
cell specific, HMG-box) regulator transcription factor 7-like 2 (T-
TCF7L2 216511_s_at Nucleus transcription 1.003 cell specific,
HMG-box) regulator transducin-like enhancer of split 3 TLE3
212770_at Nucleus other 0.976 transformer 2 alpha homolog TRA2A
213593_s_at Nucleus other 1.12 transmembrane inner ear TMIE
1553601_a_at unknown other -3.334 transmembrane protein 132C
TMEM132C 232313_at unknown other 4.02 transmembrane protein 178
TMEM178 229302_at unknown other 1.697 transmembrane protein 184C
TMEM184C 219074_at unknown other -0.779 transmembrane protein 5
TMEM5 204808_s_at Plasma other -1.074 Membrane transmembrane
protein 59-like TMEM59L 219005_at Cytoplasm other -0.845 tripartite
motif-containing 29 TRIM29 211002_s_at Cytoplasm transcription
2.005 regulator tripartite motif-containing 36 TRIM36 219736_at
Cytoplasm other 2.221 trophoblast glycoprotein TPBG 203476_at
Plasma other 2.158 Membrane tumor suppressor candidate 3 TUSC3
232770_at Extracellular enzyme -1.407 Space U2 small nuclear RNA
auxiliary U2AF1 232141_at Nucleus other 0.776 factor 1
ubiquitin-conjugating enzyme E2 UBE2V1 201003_x_at Nucleus
transcription 0.663 variant 1 regulator urocortin UCN 206072_at
Extracellular other -0.961 Space vacuolar protein sorting 29 VPS29
223026_s_at Cytoplasm transporter 0.805 homolog v-erb-a
erythroblastic leukemia ERBB4 206794_at Plasma kinase -1.537 viral
oncogene homolog 4 Membrane v-erb-a erythroblastic leukemia ERBB4
214053_at Plasma kinase -1.708 viral oncogene homolog 4 Membrane
v-erb-b2 erythroblastic leukemia ERBB2 216836_s_at Plasma kinase
-0.698 viral oncogene homolog 2, Membrane neuro/glioblastoma
derived oncogene homolog v-raf-1 murine leukemia viral RAF1
1557675_at Cytoplasm kinase 0.696 oncogene homolog 1 WD repeat and
FYVE domain WDFY3 212602_at Cytoplasm enzyme -0.626 containing 3 WD
repeat and FYVE domain WDFY3 212606_at Cytoplasm enzyme -0.785
containing 3 zinc finger homeobox 4 ZFHX4 219779_at unknown other
1.36 zinc finger homeobox 4 ZFHX4 241700_at unknown other 1.234
zinc finger protein 217 ZNF217 203739_at Nucleus transcription
1.096 regulator zinc finger protein 503 ZNF503 227195_at Nucleus
other 1.373 zinc finger protein 641 ZNF641 226509_at unknown other
-1.427 zinc finger protein 662 ZNF662 228538_at unknown other
-3.352 zinc finger, CCCH-type with G ZGPAT 221848_at unknown other
0.991 patch domain
[0218] Computer analysis was performed as in Example V, except that
the list of probes reflects genes that were greater than 1.5-fold
differential expressed in the H1 cell line and the BB10 cell line
when compared to both the 2D6 and SiMa parental cell lines, and
also a greater than 1.5-fold differential expressed in the SiMa
parental cell line when compared to the 2D6 cell line. When the
p-value cut off was set to 0.05, this analysis identified 94 gene
probes that could be classified to 70 genes (Table 17).
TABLE-US-00009 TABLE 17 Genes Identified using IPA Core Analysis
Gene Log Gene Name Symbol Probe ID. No. Location Type Ratio
achaete-scute complex ASCL1 209985_s_at Nucleus transcription 2.429
homolog 1 regulator achaete-scute complex ASCL1 209988_s_at Nucleus
transcription 1.446 homolog 1 regulator achaete-scute complex ASCL1
213768_s_at Nucleus transcription 1.578 homolog 1 regulator
activated leukocyte cell ALCAM 201952_at Plasma other 1.521
adhesion molecule Membrane ADP-ribosylation factor 1 ARF1
1565651_at Cytoplasm enzyme 1.063 ADP-ribosylation factor-like
ARL17A 243899_at unknown other 0.741 17A ataxin 2-binding protein 1
A2BP1 1553422_s_at Cytoplasm other 1.453 ataxin 2-binding protein 1
A2BP1 221217_s_at Cytoplasm other 1.497 brain abundant, membrane
BASP1 202391_at Plasma other 1.146 attached signal protein 1
Membrane CAP, adenylate cyclase- CAP2 212551_at Plasma other -1.974
associated protein, 2 Membrane CD9 molecule CD9 201005_at Plasma
other 1.415 Membrane CUG triplet repeat, RNA CUGBP2 202157_s_at
Nucleus other 2.156 binding protein 2 cyclic nucleotide gated CNGA3
207261_at Plasma ion channel -3.182 channel alpha 3 Membrane
cyclin-dependent kinase CDKN2A 209644_x_at Nucleus transcription
-1.277 inhibitor 2A (melanoma, p16, regulator inhibits CDK4) ELL
associated factor 2 EAF2 219551_at Nucleus transcription 0.693
regulator eukaryotic translation initiation EIF3C 236700_at
Cytoplasm translation 1.378 factor 3, subunit C regulator family
with sequence FAM162B 228875_at unknown other -0.743 similarity
162, member B family with sequence FAM181B 231430_at unknown other
0.794 similarity 181, member B far upstream element (FUSE) FUBP3
239193_at Nucleus transcription 1.192 binding protein 3 regulator
fibroblast growth factor 13 FGF13 205110_s_at Extracellular growth
factor -3.522 Space follistatin-like 1 FSTL1 208782_at
Extracellular other -1.275 Space forkhead box O6 FOXO6 239657_x_at
Nucleus other -1.512 guanine nucleotide binding GNAI1 227692_at
Plasma enzyme 2.111 protein (G protein), alpha Membrane inhibiting
activity polypeptide 1 guanine nucleotide binding GNB4 225710_at
Plasma enzyme 4.695 protein (G protein), beta Membrane polypeptide
4 guanine nucleotide binding GNG11 204115_at Plasma enzyme 2.587
protein (G protein), gamma 11 Membrane KDEL (Lys-Asp-Glu-Leu)
KDELC2 225128_at unknown other 1.085 containing 2 kelch-like 13
KLHL13 227875_at unknown other 0.902 KH domain containing, RNA
KHDRBS3 209781_s_at Nucleus other 1.082 binding, signal
transduction associated 3 KH homology domain KHDC1 230055_at
unknown other -1.656 containing 1 leukemia inhibitory factor LIFR
225575_at Plasma transmembrane -0.941 receptor alpha Membrane
receptor mab-21-like 2 MAB21L2 210303_at unknown other 2.204
mesenchyme homeobox 2 MEOX2 206201_s_at Nucleus transcription
-4.213 regulator moesin MSN 200600_at Plasma other 2.082 Membrane
monoamine oxidase A MAOA 204388_s_at Cytoplasm enzyme 2.15
monoamine oxidase A MAOA 204389_at Cytoplasm enzyme 1.88 monoamine
oxidase A MAOA 212741_at Cytoplasm enzyme 2.048 multiple C2
domains, MCTP1 220122_at unknown other -1.597 transmembrane 1 MYC
induced nuclear antigen MINA 213188_s_at Nucleus other 1.391 MYC
induced nuclear antigen MINA 213189_at Nucleus other 1.641 myosin
VI MYO6 203216_s_at Cytoplasm other 1.104 phosphatidic acid
PPAPDC1A 236044_at unknown phosphatase -2.621 phosphatase type 2
domain containing 1A plastin 3 (T isoform) PLS3 201215_at Cytoplasm
other 1.068 pleiotrophin PTN 209466_x_at Extracellular growth
factor -1.426 Space pleiotrophin PTN 211737_x_at Extracellular
growth factor -2.929 Space polo-like kinase 2 PLK2 201939_at
Nucleus kinase 3.606 (Drosophila) potassium large conductance
KCNMA1 221584_s_at Plasma ion channel -2.389 calcium-activated
channel, Membrane subfamily M, alpha member 1 RELT-like 1 RELL1
226430_at unknown other 1.214 ring finger protein 182 RNF182
230720_at unknown other 2.696 seizure related 6 homolog-like SEZ6L
207873_x_at Plasma other 0.866 Membrane seizure related 6 homolog
SEZ6L 211894_x_at Plasma other 0.797 (mouse)-like Membrane similar
to hCG2031213 LOC728052 1558795_at unknown other 2.239
single-minded homolog 1 SIM1 1556300_s_at Nucleus transcription
6.36 (Drosophila) regulator single-minded homolog 1 SIM1 206876_at
Nucleus transcription 5.737 (Drosophila) regulator SLIT and
NTRK-like family, SLITRK5 214930_at unknown other 4.094 member 5
SMG1 homolog, LOC641298 244766_at unknown other 1.065
phosphatidylinositol 3-kinase- related kinase pseudogene solute
carrier family 44, SLC44A5 1569112_at unknown other 2.738 member 5
solute carrier family 44, SLC44A5 235763_at unknown other 1.845
member 5 solute carrier family 7 SLC7A2 225516_at Plasma
transporter 1.892 (cationic amino acid Membrane transporter, y+
system), member 2 solute carrier organic anion SLCO3A1 219229_at
Plasma transporter 1.622 transporter family, member Membrane 3A1
spermatogenesis associated, SPATS2L 222154_s_at unknown other 0.907
serine-rich 2-like synaptotagmin XIII SYT13 226086_at unknown
transporter -4.754 tissue factor pathway inhibitor 2 TFPI2
209278_s_at Extracellular other 2.326 Space transcription factor
7-like 2 (T- TCF7L2 212761_at Nucleus transcription 1.005 cell
specific, HMG-box) regulator transformer 2 alpha homolog TRA2A
213593_s_at Nucleus other 1.12 transmembrane inner ear TMIE
1553601_a_at unknown other -3.334 transmembrane protein 178 TMEM178
229302_at unknown other 1.697 U2 small nuclear RNA U2AF1 232141_at
Nucleus other 0.776 auxiliary factor 1 v-raf-1 murine leukemia
viral RAF1 1557675_at Cytoplasm kinase 0.696 oncogene homolog 1
zinc finger protein 217 ZNF217 203739_at Nucleus transcription
1.096 regulator zinc finger protein 662 ZNF662 228538_at unknown
other -3.352 The log ratio represent log.sub.2 values where 0.585
is log.sub.2(1.5) which is a 1.5-fold difference, 1 is log.sub.2(2)
which is a 2-fold difference, 1.584 is log.sub.2(3) which is a
3-fold difference, 2 is log.sub.2(4) which is a 4-fold difference,
2.321 is log.sub.2(5) which is a 5-fold difference, 2.584 is
log.sub.2(6) which is a 6-fold difference, 2.807 is log.sub.2(7)
which is a 7-fold difference, 3 is log.sub.2(8) which is a 8-fold
difference, 3.169 is log.sub.2(9) which is a 9-fold difference, and
3.321 is log.sub.2(10) which is a 10-fold difference.
Example VI
Immuno-Based Method to Detect Picomolar Amounts of BoNT/A
[0219] The following example illustrates how to perform
immuno-based methods of detecting BoNT/A activity that can detect
picomolar amounts of the BoNT/A pharmaceutical product, such as,
e.g., BOTOX.RTM. DYSPORT.RTM./RELOXIN.RTM., PURTOX.RTM.,
XEOMIN.RTM., NEURONOX.RTM., or BTX-A.
1. Immuno-Based Method of Detecting BoNT/A Using ECL Sandwich
ELISA.
[0220] To prepare a lysate from cells treated with a BoNT/A,
approximately 150,000 cells from an established clonal cell line
was plated into the wells of 96-well tissue culture poly-D-lysine
plates containing 100 .mu.L of a serum-free medium containing
Minimum Essential Medium, 2 mM GlutaMAX.TM. I with Earle's salts,
1.times.B27 supplement, 1.times.N2 supplement, 0.1 mM Non-Essential
Amino Acids, 10 mM HEPES and 60 .mu.g/mL GT1b. The cell lines used
were BB10, H1, and SiMa. These cells were incubated in a 37.degree.
C. incubator under 5% carbon dioxide until the cells
differentiated, as assessed by standard and routine morphological
criteria, such as growth arrest and neurite extension
(approximately 3 days). The media from the differentiated cells was
aspirated from each well and replaced with fresh media containing
one of the following: 1) SiMa cell line, 0 (untreated), 0.98 U/mL,
1.9 U/mL, 3.91 U/mL, 7.81 U/mL, 15.6 U/mL, 31.3 U/mL, 62.5 U/mL,
125 U/mL, 250 U/mL, or 500 U/mL of a BoNT/A pharmaceutical product
reconstituted in a sodium chloride free medium; 2) BB10, 0
(untreated), 0.49 U/mL, 0.98 U/mL, 1.9 U/mL, 3.91 U/mL, 7.81 U/mL,
15.6 U/mL, 31.3 U/mL, 62.5 U/mL, 125 U/mL, 250 U/mL; 3) H1, 0
(untreated), 0.3 U/mL, 1 U/mL, 3 U/mL, 9 U/mL, 28 U/mL, 83 U/mL,
and 250 U/mL. Because the BoNT/A pharmaceutical product contains
sodium chloride, its addition to the culture medium resulted in a
hypertonic media that was detrimental to cell viability. To
circumvent the hypertonicity issue, 200 .mu.L of MEM media made
without sodium chloride was used to reconstitute the BoNT/A
pharmaceutical product giving a final concentration of 25 pM BoNT/A
(500 U/mL). The matrix was kept constant for all concentrations
along the dose-response curve by adding sodium chloride in the
media used to make the dilutions match the amount of excipients
present at the highest concentration used (25 pM or 500 U/mL).
After a 24 hr treatment, the cells were washed, and incubated for
an additional two days without toxin. To harvest the cells,
1.times.PBS was aspirated, the cells lysed by adding 75 .mu.L of
Lysis Buffer comprising 50 mM HEPES, 150 mM NaCl, 1.5 mM
MgCl.sub.2, 1 mM EGTA, 1% Triton X-100 to each well, and the plate
incubated on a shaker rotating at 500 rpm for 30 minutes at
4.degree. C. The plate was centrifuged at 4000 rpm for 20 minutes
at 4.degree. C. to pellet cellular debris and the supernatant was
transferred to a capture antibody coated 96-well plate to perform
the detection step.
[0221] To prepare the .alpha.-SNAP-25 capture antibody solution,
the .alpha.-SNAP-25 monoclonal antibody contained in the ascites
from hybridoma cell line 2E2A6 (Example VII) was purified using a
standard Protein A purification protocol. To prepare the
.alpha.-SNAP-25 detection antibody solution, .alpha.-SNAP-25 rabbit
polyclonal antibody S9684 (Sigma, St. Louis, Mo.) was conjugated to
Ruthenium(II)-tris-bipyridine-(4-methysulfonate) NHS ester labeling
reagent (Meso Scale Discovery, Gaithersburg, Md.) according to the
manufacturer's instructions (Meso Scale Discovery, Gaithersburg,
Md.). The conjugation reaction was performed by adding 30 .mu.L of
distilled water reconstituted MSD SULFO-TAG.TM. stock solution to
200 .mu.L of 2 mg/mL .alpha.-SNAP-25 polyclonal antibodies and
incubating the reaction at room temperature for 2 hours in the
dark. The labeled antibodies were purified using a standard spin
column protocol and the protein concentration determined using a
standard colorimetric protein assay. The absorbance of the
.alpha.-SNAP-25 antibody/MSD SULFO-TAG.TM. conjugate was measured
at 455 nm using a spectrophotometer to determine the concentration
in moles per liter. The detection antibody solution was stored at
4.degree. C. until needed.
[0222] To prepare the solid phase support comprising the capture
antibody that is specific for a SNAP-25 cleaved product,
approximately 1 .mu.L of .alpha.-SNAP-25 monoclonal antibody 2E2A6
solution (45 .mu.g/mL in 1.times.PBS, 750 .mu.g/mL of BSA) was
added to each well of a 96-well MSD High Bind plate (Meso Scale
Discovery, Gaithersburg, Md.) and the solution is allowed to air
dry in a biological safety cabinet for 2-3 hours in order to liquid
evaporate the solution, and then the plates were sealed and stored
at 4.degree. C. until needed. The dried capture antibody-bound
wells were then blocked by adding 150 .mu.L of Blocking Buffer
comprising 1.times.PBS, 0.05% Tween-20, 2% ECL Blocking reagent (GE
Healthcare-Amersham), and 1% goat serum (Rockland Immunochemicals,
Gilbertsville, Pa.) at room temperature for 2 hours with rotation
at 600 rpm.
[0223] To detect the presence of a SNAP-25 cleavage product by ECL
sandwich ELISA analysis, the Blocking Buffer from plates was
aspirated, 25 .mu.L of a lysate from cells treated with BoNT/A was
added to each well and the plates were incubated at 4.degree. C.
for overnight. Plate wells were washed three times by aspirating
the cell lysate and rinsing each well three times with 200 .mu.L
1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan
monolaureate). After washing, 25 .mu.l of 5 .mu.g/mL
.alpha.-SNAP-25 detection antibody solution comprising 2% Amersham
Blocking Reagent in 1.times.PBS, 0.1% TWEEN-20.RTM.
(polyoxyethylene (20) sorbitan monolaureate) was added to each
well, the plate was sealed, and the sealed plate was incubated at
room temperature for 1 hour with shaking. After .alpha.-SNAP-25
detection antibody incubation, the wells were washed three times
with 250 .mu.L 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene
(20) sorbitan monolaureate). After washing 150 .mu.L of 1.times.
Read Buffer (Meso Scale Discovery, Gaithersburg, Md.) was added to
each well and the plates were read using a SECTOR.TM. Imager 6000
Image Reader (Meso Scale Discovery, Gaithersburg, Md.). The raw
data was then transferred to SigmaPlot v 10.0 and a 4-parameter
logistics fit was used to define the dose-response curves. There
were no constraints used for the 4-parameter logistic function when
plotting the data. Graphical reports were generated using the
following analysis: R2 (correlation coefficient), a (Max for data
set), b (hillslope), and X0.+-.SE (EC.sub.50 value.+-.standard
error).
[0224] These results indicated that on average the cells comprising
the BB10 and H1 clonal cell lines were more susceptible to BoNT/A
intoxication as compared to cells comprising the parental SiMa cell
line. Cells from a BB10 cell line exhibited an EC.sub.50 for BoNT/A
activity of 7 U/mL, cells from a H1 cell line exhibited an
EC.sub.50 for BoNT/A activity of 8 U/mL, and cells from the
parental SiMa cell line exhibited an EC.sub.50 for BoNT/A activity
of 13 U/mL. This method can also be performed in a multiplex
fashion as described in Ester Fernandez-Salas, et al., Immuno-Based
Botulinum Toxin Serotype A Activity Assays, U.S. patent application
Ser. No. 12/403,531, which is hereby incorporated by reference in
its entirety.
Example VII
Transfection of BoNT/A Receptor DNA into Sensitive Clonal Cell
Lines Further Improves Sensitivity of the Clonal Cell Line for
BoNT/A
[0225] The following example illustrates how to introduce a
polynucleotide molecule encoding a BoNT/A receptor into cells from
a clonal cell line to further improve susceptibility to BoNT/A
intoxication or improve BoNT/A uptake capacity.
[0226] To introduce an exogenous BoNT/A receptor into cells
comprising an established clonal cell line, a mammalian expression
construct comprising a polynucleotide molecule of SEQ ID NO: 139
encoding the FGFR3 of SEQ ID NO: 25, was transfected into cells
from a H1 clonal cell line by a cationic lipid method. A suitable
density (about 5.times.10.sup.6 cells) of cells from an established
clonal cell line are plated in a 100 mm tissue culture dish
containing 5 mL of complete culture media and grown in a 37.degree.
C. incubator under 5% carbon dioxide until the cells reached a
density appropriate for transfection. A 3 mL transfection solution
is prepared by adding 1.5 mL of OPTI-MEM.RTM. Reduced Serum Medium
containing 60 .mu.L of LIPOFECTAMINE.RTM. 2000 (Invitrogen,
Carlsbad, Calif.) incubated at room temperature for 5 minutes to
1.5 mL of OPTI-MEM.RTM. Reduced Serum Medium containing 24 .mu.g of
an expression construct encoding the FGFR3 of SEQ ID NO: 25. This
transfection mixture is incubated at room temperature for
approximately 30 minutes. The complete media on the cells is
replaced with the 3 mL transfection solution and the cells are
incubated in a 37.degree. C. incubator under 5% carbon dioxide for
approximately 8 hours. Transfection media is replaced with 3 mL of
fresh complete culture media and the cells are incubated in a
37.degree. C. incubator under 5% carbon dioxide for approximately
24 hours. Media is replaced with 3 mL of fresh complete culture
media containing approximately 1 mM G418 (Invitrogen, Carlsbad,
Calif.) for selection of transfected cells. Cells are incubated in
a 37.degree. C. incubator under 5% carbon dioxide for approximately
1 week, the old media is replaced with fresh complete culture media
containing 0.5 mM G418 and the cells are plated onto one 96-well
collagen IV coated plate at a plating density of 0.5 cells per
well. Cell growth and viability was monitored by microscopic
examination and after about 3 weeks single clones appeared in about
20% of the wells. These clones were grown for an additional 2 weeks
and then transferred to duplicate 24-well collagen IV coated
plates, one to maintain the clonal cell line and the other to test
for BoNT/A susceptibility.
[0227] To determine the susceptibility of the cells comprising the
clonal cell lines overexpressing a BoNT/A receptor, a Western blot
analysis was done to determine sensitivity and an ECL sandwich
ELISA was done to determine an EC.sub.50 value. A total of eight
clones were tested for the FGFR3 stably transfected H1 cells.
[0228] To prepare a cell lysate for the Western blot analysis,
cells from the clonal cell line were plated into two wells in a
poly-D-lysine coated 96-well plate in serum-free medium containing
Minimum Essential Medium, 2 mM GLUTAMAX.TM. I with Earle's salts,
1.times.B27 supplement, 1.times.N2 supplement, 0.1 mM Non-Essential
Amino Acids, 10 mM HEPES and 25 .mu.g/mL GT1b (Alexis Biochemicals,
Lausen, Switzerland). These cells were incubated in a 37.degree. C.
incubator under 5% carbon dioxide until the cells differentiated,
as assessed by standard and routine morphological criteria, such as
growth arrest and neurite extension (approximately 3 days). The
media from the differentiated cells was aspirated from each well
and replaced with fresh media containing either 0 (untreated), 0.5
nM or 0.25 nM of a BoNT/A complex. After a 6 hr treatment, the
cells were washed, and incubated for an additional 18 hr without
toxin. After the incubation, the cells were washed by aspirating
the growth media and rinsing each well with 200 .mu.l of
1.times.PBS. To harvest the cells, the 1.times.PBS was aspirated,
the cells were lysed by adding 40 .mu.L of 2.times.SDS Loading
Buffer and the plate was swirled for an even coating of Loading
Buffer. The lysate was transferred to a new 96-well PCR plate
(Axygen, Union City, Calif.) and the plate was heated to 95.degree.
C. for 5 min to complete cell lysis and denature proteins.
[0229] To detect for the presence of both uncleaved SNAP-25
substrate and cleaved SNAP-25 products, an aliquot from each
harvested sample was analyzed by Western blot. In this analysis, a
12 .mu.L aliquot of the harvested sample was separated by MOPS
polyacrylamide gel electrophoresis using NUPAGE.RTM. Novex 12%
Bis-Tris precast polyacrylamide gels (Invitrogen Inc., Carlsbad,
Calif.) under denaturing, reducing conditions. Separated proteins
were transferred from the gel onto nitrocellulose membranes
(Bio-Rad Laboratories, Hercules, Calif.) by Western blotting using
a "Transfer+4 cassettes & Cooler" transfer chamber (GE
Healthcare, Piscataway, N.J.). Membranes were blocked by incubating
at room temperature for 1 hr with gentle agitation in a solution
containing Tris-Buffered Saline (TBS) (25 mM
2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid
(Tris-HCl) (pH 7.4), 137 mM sodium chloride, 2.7 mM potassium
chloride), 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan
monolaureate), 2% Bovine Serum Albumin (BSA), 5% nonfat dry milk.
Blocked membranes were incubated at 4.degree. C. for overnight in
TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan
monolaureate), 2% BSA, and 5% nonfat dry milk containing a 1:5,000
dilution of .alpha.-SNAP-25 rabbit polyclonal antiserum S9684
(Sigma, St. Louis, Mo.) as the primary antibody. The
.alpha.-SNAP-25 rabbit polyclonal antibodies can detect both the
uncleaved SNAP-25 substrate and the SNAP-25 cleavage product,
allowing for the assessment of overall SNAP-25 expression in each
cell line and the percent of SNAP-25 cleaved after BoNT/A treatment
as a parameter to assess the amount of BoNT/A uptake. Primary
antibody probed blots were washed three times for 5 minutes each
time in TBS, TWEEN-20.RTM. (polyoxyethylene (20) sorbitan
monolaureate). Washed membranes were incubated at room temperature
for 2 hours in TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20)
sorbitan monolaureate), 2% BSA, and 5% nonfat dry milk containing a
1:10,000 dilution of goat polyclonal anti-rabbit immunoglobulin G,
heavy and light chains (IgG, H+L) antibody conjugated to
horseradish peroxidase (Invitrogen, Inc., Carlsbad, Calif.) as a
secondary antibody. Secondary antibody-probed blots were washed
five times for 5 minutes each time in TBS, 0.1% TWEEN-20.RTM.
(polyoxyethylene (20) sorbitan monolaureate). Signal detection of
the labeled SNAP-25 products were visualized using the ECL Plus.TM.
Western Blot Detection System (GE Healthcare, Amersham Biosciences,
Piscataway, N.J.) and the membrane was imaged and the percent of
cleaved SNAP-25 products quantified with a Typhoon 9410 Variable
Mode Imager and Imager Analysis software (GE Healthcare, Amersham
Biosciences, Piscataway, N.J.). The choice of pixel size (100 to
200 pixels) and PMT voltage settings (350 to 600, normally 400)
depended on the individual blot. The volumes of the bands
corresponding to either intact and cleaved SNAP-25 product were
quantified using Image Quant TL and the total amount of SNAP-25 and
the percentage of SNAP-25 cleaved were calculated based on the
intensity of these two SNAP-25 bands.
[0230] Clone H1 1.4 had a high percentage of SNAP-25 cleavage and
high levels of total SNAP25 and was selected for further testing.
The selected clone was first grown on a 10 cm.sup.2 dish and then
in T175 flasks to ensure that enough cells were produced for both
ECL sandwich ELISA assay and for freezing stocks. Frozen stocks
were made with 1.times.10.sup.6 cells and 1 mL of Recovery Cell
Culture Freezing Media (GIBCO-Invitrogen, #12648-010 Freezing
media).
[0231] To prepare a cell lysate for the ECL sandwich ELISA,
approximately 50,000 cells from H1 clonal cell line and from H1 1.4
clonal cell line overexpressing FGFR3 were plated and
differentiated as described above. The media from the
differentiated cells was aspirated from each well and replaced with
fresh media containing either (untreated), 0.27 pM, 0.82 pM, 2.47
pM, 7.4 pM, 22.2 pM, 66.6 pM, or 200 pM of a BoNT/A complex of a
BoNT/A complex. After a 6 hr treatment, the cells were washed, and
incubated overnight without toxin. After a 24 hr treatment, the
cells were washed by aspirating the BoNT/A containing media and
rinsing each well with 200 .mu.L of 1.times.PBS. Washed cells were
harvested by lysing in freshly prepared Triton X-100 Lysis Buffer
(150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1%
Triton X-100 plus protease inhibitors) at 4.degree. C. for 30
minutes with constant agitation. Lysed cells were centrifuged at
4000 rpm for 20 min at 4.degree. C. to pellet debris.
[0232] The .alpha.-SNAP-25 capture antibody solution, the
.alpha.-SNAP-25 detection antibody solution, and the solid phase
support comprising the capture antibody that is specific for a
SNAP-25 cleaved product were prepared as described in Example V. To
detect the presence of a cleaved SNAP-25 product by ECL sandwich
ELISA, the plates were processed, and the data collected and
analyzed, and the EC.sub.50 calculated as described in Example VI.
The EC.sub.50 for H1 1.4 cells was estimated to about 1.7 pM,
compared to an EC.sub.50 about 8.7 pM for H1 cells indicating that
the newly generated H1 1.4 cell line is about 5 times more
sensitive than the H1 cell line for BoNT/A uptake (average of two
independent experiments with similar results). The maximum signal
for both cell lines was identical indicating similar efficacy
towards SNAP25 cleavage. The increase in sensitivity is especially
prominent at lower concentrations of BoNT/A, where the H1 1.4 cells
are many times more sensitive to BoNT/A compared to H1 cells
producing a very robust signal over background at the 0.27 pM
concentration. Over-expression of FGFR3 was verified in a Western
blot using .alpha.-FGFR3 antibodies sc-123 (Santa Cruz
Biotechnologies, Santa Cruz, Calif.).
Example VIII
Development of .alpha.-SNAP-25 Monoclonal Antibodies that
Selectively Bind a SNAP-25 Epitope Having a Free Carboxyl-Terminus
at the P.sub.1 Residue of the BoNT/A Cleavage Site Scissile
Bond
[0233] The following example illustrates how to make
.alpha.-SNAP-25 monoclonal antibodies that can selectively bind to
a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue
of the BoNT/A cleavage site scissile bond.
1. Generation of .alpha.-SNAP-25 Monoclonal Antibodies.
[0234] .alpha.-SNAP-25 monoclonal antibodies with higher binding
affinity for a SNAP-25 cleavage product and/or .alpha.-SNAP-25
antibodies with a higher binding specificity for a SNAP-25 cleavage
product were developed. In addition, because a permanent, stable
and renewable source of .alpha.-SNAP-25 antibodies is preferable
for an FDA-approved assay, screens to isolate monoclonal antibodies
were undertaken. To develop monoclonal .alpha.-SNAP-25 antibodies
the 13-residue peptide CDSNKTRIDEANQ.sub.COOH (SEQ ID NO: 38) was
designed as a SNAP-25 antigen having a carboxyl-terminus at the
P.sub.1 residue of the BoNT/A cleavage site scissile bond. This
peptide comprises a flexible linker region and a N-terminal
Cysteine residue for conjugation to KLH and amino acids 186-197 of
human SNAP-25 (SEQ ID NO: 5) with a carboxylated C-terminal
glutamine (SEQ ID NO: 38). The generation of monoclonal antibodies
to well-chosen, unique peptide sequences provides control over
epitope specificity, allowing the identification of a particular
subpopulation of protein among a pool of closely related isoforms.
Blast searches revealed that this peptide has high homology only to
SNAP-25 and almost no possible cross-reactivity with other proteins
in neuronal cells. The sequence was also carefully scrutinized by
utilizing computer algorithms to determine hydropathy index,
protein surface probability, regions of flexibility, and favorable
secondary structure, followed by proper orientation and
presentation of the chosen peptide sequence. The peptide was
synthesized and conjugated to Keyhole Limpet Hemocyanin (KLH) to
increase immunogenicity. Six Balb/c mice were immunized with this
peptide, and after three immunizations in about eight weeks, the
mice were bled for testing. The blood was allowed to clot by
incubating at 4.degree. C. for 60 minutes. The clotted blood was
centrifuged at 10,000.times.g at 4.degree. C. for 10 minutes to
pellet the cellular debris. The resulting serum sample was
dispensed into 50 .mu.L aliquots and stored at -20.degree. C. until
needed.
[0235] A similar strategy based on other SNAP-25 antigens disclosed
in the present specification is used to develop .alpha.-SNAP-25
monoclonal antibodies that can selectively bind to a SNAP-25 having
a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage
site scissile bond. For example, the SNAP-25 antigen of SEQ ID NO:
45 can be conjugated to KLH instead of the SNAP-25 antigen of SEQ
ID NO: 38. As another example, the amino acids 186-197 of human
SNAP-25 from the SNAP-25 antigen of SEQ ID NO: 38 can be replaced
with SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35,
SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID
NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 147, or SEQ ID NO:
148.
2. Screening for the Presence of .alpha.-SNAP-25 Monoclonal
Antibodies.
[0236] To determine the presence of an .alpha.-SNAP-25 monoclonal
antibody that can selectively bind to a SNAP-25 antigen having a
carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage
site scissile bond, comparative ELISA and cell-based cleavage assay
were performed using the extracted mouse serum. For comparative
ELISA, two fusion proteins were constructed: BIRA-HIS
TAG.RTM.-SNAP-25.sub.134-197 of SEQ ID NO: 48 and the BIRA-HIS
TAG.RTM.-SNAP-25.sub.134-206 of SEQ ID NO: 49. BIRA-HIS
TAG.RTM.-SNAP-25.sub.134-197 comprised a naturally-biotinylated 16
amino acid BirA peptide of SEQ ID NO: 50 amino-terminally linked to
a SNAP-25 peptide comprising amino acids 134-197 of SEQ ID NO: 5.
BIRA-HIS TAG.RTM.-SNAP-25.sub.134-206 comprised a
naturally-biotinylated 16 amino acid BirA peptide of SEQ ID NO: 50
amino-terminally linked to a SNAP-25 peptide comprising amino acids
134-206 of SEQ ID NO: 5. These two substrates were suspended in
1.times.PBS at a concentration of 10 .mu.g/mL BIRA-HIS
TAG.RTM.-SNAP-25.sub.134-197 and the BIRA-HIS
TAG.RTM.-SNAP-25.sub.134-206. The BIRA-HIS
TAG.RTM.-SNAP-25.sub.134-197 and the BIRA-HIS
TAG.RTM.-SNAP-25.sub.134-206 were coated onto separate plates by
adding approximately 100 .mu.l of the appropriate Substrate
Solution and incubating the plates at room temperature for one
hour. Washed plates were incubated at 37.degree. C. for one hour in
0.5% BSA in 1.times.TBS containing a 1:10 to 1:100 dilution of an
antibody-containing serum derived from one of the six immunized
mice (Mouse 1, Mouse 2, Mouse 3, Mouse 4, Mouse 5, and Mouse 6).
Primary antibody probed plates were washed four times for 5 minutes
each time in 200 .mu.l TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene
(20) sorbitan monolaureate). Washed plates were incubated at
37.degree. C. for 1 hour in 1.times.TBS containing a 1:10,000
dilution of goat polyclonal anti-mouse IgG antibody conjugated to
Horseradish peroxidase (Pierce Biotechnology, Rockford, Ill.) as a
secondary antibody. Secondary antibody-probed plates were washed
four times in 200 .mu.l TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene
(20) sorbitan monolaureate). Chromogenic detection of the labeled
SNAP-25 products were visualized by chromogenic detection using
ImmunoPure TMB substrate kit (Pierce Biotechnology, Rockford,
Ill.). The development of a yellow color in the BIRA-HIS
TAG.RTM.-SNAP-25.sub.134-197 coated plates, but not the BIRA-HIS
TAG.RTM.-SNAP-25.sub.134-206 coated plates, indicated that the
.alpha.-SNAP-25 antibody preferentially recognized the
SNAP-25.sub.197 cleavage product. The resulted indicated that of
the six mice used for immunization, three mice (Mouse 2, Mouse 3,
and Mouse 4) had higher titers and more specificity towards a
SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue
of the BoNT/A cleavage site scissile bond.
[0237] These results were confirmed using an ELISA light chain
activity assay. A 96-well Reacti-Bind Streptavidin coated plates
(Pierce Biotechnology, Rockford, Ill.) were prepared by adding
approximately 100 .mu.l of the following Substrate Solution: Rows
A-C were coated with 100 .mu.L of BIRA-HIS
TAG.RTM.-SNAP-25.sub.134-197 at twelve different concentrations;
Rows D-H were coated with 100 .mu.L of BIRA-HIS
TAG.RTM.-SNAP-25.sub.134-206 at 10 .mu.g/mL. The plates were washed
by aspirating the Substrate Solution and rinsing each well three
times with 200 .mu.l TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20)
sorbitan monolaureate). Dilutions of BoNT/A were pre-reduced at
37.degree. C. for 20 minutes in BoNT/A Incubation Buffer (50 mM
HEPES, pH 7.4, 1% fetal bovine serum, 10 pM ZnCl.sub.2, 10 mM
dithiothreitol) and 100 .mu.l of the pre-reduced BoNT/A was added
to the substrate-coated plates and incubated at 37.degree. C. for
90 minutes. BoNT/A treated plates were washed by aspirating the
BoNT/A Incubation Buffer and rinsing each plate three times with
200 .mu.l TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan
monolaureate). Washed plates were incubated at 37.degree. C. for
one hour in 0.5% BSA in 1.times.TBS containing a 1:10 to 1:100
dilution of the antibody-containing serum being tested. Primary
antibody probed plates were washed four times for 5 minutes each
time in 200 .mu.l TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20)
sorbitan monolaureate). Washed plates were incubated at 37.degree.
C. for 1 hour in 1.times.TBS containing a 1:10,000 dilution of goat
polyclonal anti-mouse IgG antibody conjugated to Horseradish
peroxidase (Pierce Biotechnology, Rockford, Ill.) as a secondary
antibody. Secondary antibody-probed plates were washed four times
in 200 .mu.l TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan
monolaureate). Chromogenic detection of the labeled SNAP-25
products were visualized by chromogenic detection using ImmunoPure
TMB substrate kit (Pierce Biotechnology, Rockford, Ill.). The
development of a yellow color, which correlated with the presence
of the SNAP-25.sub.197 cleavage product was detected in BoNT/A
treated samples, but not untreated controls, using
antibody-containing serum derived from all six immunized mice
(Mouse 1, Mouse 2, Mouse 3, Mouse 4, Mouse 5, and Mouse 6). Thus,
the comparative ELISA analysis indicated that of the mice used for
immunization, three mice (Mouse 2, Mouse 3, and Mouse 4) had higher
titers and more specificity towards a SNAP-25 antigen having a
carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage
site scissile bond.
[0238] For cell-based cleavage assay, a suitable density of PC12
cells were plated into 60 mm.sup.2 tissue culture plates containing
3 mL of an appropriate serum medium (Table 1). The cells were grown
in a 37.degree. C. incubator under 5% carbon dioxide until cells
reached the appropriate density. A 500 .mu.L transfection solution
was prepared by adding 250 .mu.L of OPTI-MEM.RTM. Reduced Serum
Medium containing 15 .mu.L of LIPOFECTAINE.RTM. 2000 (Invitrogen
Inc., Carlsbad, Calif.) incubated at room temperature for 5 minutes
to 250 .mu.L of OPTI-MEM.RTM. Reduced Serum Medium containing 10
.mu.g of a pQBI-25/GFP-BoNT/A-LC expression construct (SEQ ID NO:
51). The pQBI-25/GFP-BoNT/A-LC expression construct comprises a
pQBI-25 expression vector (Qbiogene Inc., Carlsbad, Calif.) whose
promoter elements are functionally linked to a polynucleotide
encoding the GFP-BoNT/A light chain of SEQ ID NO: 52. This
transfection mixture was incubated at room temperature for
approximately 20 minutes. The media was replaced with fresh
unsupplemented media and the 500 .mu.L transfection solution was
added to the cells. The cells were then incubated in a 37.degree.
C. incubator under 5% carbon dioxide for approximately 6 to 18
hours. The cells were washed and harvested as described in Example
II. To detect for the presence of the cleaved SNAP-25.sub.197
product, an aliquot from each harvested sample was analyzed by
Western blot as described in Example II, except that the primary
antibody used was a 1:1,000 dilution of the antibody-containing
serum and the secondary antibody used was a 1:20,000 of mouse
.alpha.-IgG Horseradish Peroxidase (Pierce Biotechnology, Rockford,
Ill.). A single band corresponding to the SNAP-25.sub.197 cleavage
product was detected in BoNT/A treated samples, but not untreated
controls, using antibody-containing serum derived from three mice
(Mouse 2, Mouse 3, and Mouse 4). Thus, the cell-based cleavage
assay indicated that of the mice used for immunization, three mice
(Mouse 2, Mouse 3, and Mouse 4) had higher titers and more
specificity towards a SNAP-25 antigen having a carboxyl-terminus at
the P.sub.1 residue of the BoNT/A cleavage site scissile bond.
3. Production of Hybridomas.
[0239] To make hybridomas producing .alpha.-SNAP-25 monoclonal
antibodies that can selectively bind to a SNAP-25 antigen having a
carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage
site scissile bond, the spleen from Mouse 2 was harvested three
days subsequent to a final "booster" immunization and the spleen
cells were fused with myeloma cells P3-X63 Ag8.653 using standard
hybridoma protocols. These cells were plated into five 96-well
plates and hybrids were selected using HAT medium. Within 8-14 days
after fusion, the first screening of the approximately 480 parent
clones was carried out using comparative ELISA with the BIRA-HIS
TAG.RTM.-SNAP-25.sub.134-197 and the BIRA-HIS
TAG.RTM.-SNAP-25.sub.134-206 peptides coated in two separate
plates. The comparative ELISA provided a quick screen method to
identify hybridomas producing antibodies specific for the cleaved
SNAP-25.sub.197. The top 18 clones were subjected to further
screening using the cell-based cleavage assay described above and
immunostaining of LC/A transfected cells. (Table 18).
TABLE-US-00010 TABLE 18 Analysis of Supernatants Containing
.alpha.-SNAP-25 Monoclonal Antibody Comparative ELISA Cell-Based
Assay Clone OD SNAP-25.sub.197 OD SNAP-25.sub.206 Ratio.sub.197/206
Ratio.sub.206/197 SNAP-25.sub.197 SNAP-25.sub.206 1D3 1.805 0.225
8.02 0.13 +++ - 1F12 0.365 0.093 3.92 0.25 - - 1G10 0.590 0.137
4.31 0.23 ++ - 1H1 0.335 0.121 2.77 0.36 - - 1H8 0.310 0.302 1.03
0.97 + - 2C9 0.139 0.274 0.51 1.97 - - 2E2 0.892 0.036 24.78 0.04
++ - 2E4 0.228 0.069 3.30 0.30 + - 2F11 1.095 1.781 0.61 1.63 - -
3C1 1.268 0.053 23.92 0.04 ++ - 3C3 0.809 0.052 15.56 0.06 ++ - 3E1
0.086 0.155 0.55 1.80 0 - 3E8 2.048 0.053 38.64 0.03 +++ - 3G2
0.053 0.158 0.34 2.98 - - 4D1 0.106 0.218 0.49 2.06 - - 4G6 0.061
0.159 0.38 2.61 - - 5A5 0.251 0.106 2.37 0.42 + - 5F11 0.243 0.061
3.98 0.25 - -
[0240] Clones 1D3, 1G10, 2E2, 3C1, 3C3, and 3E8 were further cloned
by limiting dilution because the conditioned media produced by
these clones comprised .alpha.-SNAP-25 antibodies with a
preferential binding specificity having a ratio.sub.197/206 of at
least 4:1 for the SNAP-25.sub.197 cleavage product relative to the
SNAP-25.sub.206 uncleaved substrate and detected the
SNAP-25.sub.197-cleavage product using the cell-based cleavage
assay and the immunostaining of PC12 cells transfected with
GFP-LC/A. Similarly clones 2C9, 2F11, 3G2, 4D1 and 4G6 were further
cloned by limiting dilution because the conditioned media produced
by these clones comprised .alpha.-SNAP-25 antibodies with a
preferential binding specificity having a ratio.sub.2061107 of at
least 1.5:1 for the SNAP-25.sub.206 uncleaved substrate relative to
the SNAP-25.sub.197 cleavage product and detected the
SNAP-25.sub.206-uncleaved substrate using the cell-based cleavage
assay. These single-cell derived clones were screened again using
comparative ELISA, cell-based cleavage, and immunostaining to
confirm their affinity and specificity, and the antibodies were
isotyped using standard procedures. Ascites were produced from
clones 1D3B8 (IgM.k), 1G10A12 (IgG3.k), 2C9B10 (IgG3.k), 2E2A6
(IgG3.k), 2F11B6 (IgM.k), 3C1A5 (IgG2a.k), and 3C3E2 (IgG2a.k).
Clone 3E8 stopped producing antibodies during the cloning process
and could not be further evaluated.
4. Evaluation of Binding Specificity of .alpha.-SNAP-25 Monoclonal
Antibodies.
[0241] To evaluate binding specificity of an .alpha.-SNAP-25
monoclonal antibody that can selectively bind to a SNAP-25 antigen
having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A
cleavage site scissile bond, ascites from clones 1D3B8, 1G10A12,
2C9B10, 2E2A6, 2F11B6, 3C1A5, and 3C3E2 were used to detect SNAP-25
cleavage product using the cell-based activity assay,
immunocytochemistry and immunoprecipitation.
[0242] For the cell-based activity assay, binding specificity was
determined by analyzing the ability of .alpha.-SNAP-25
antibody-containing ascites to detect the uncleaved SNAP-25.sub.206
substrate and the cleaved SNAP-25.sub.197 product by Western blot
analysis. A suitable density of PC12 cells were plated into 60
mm.sup.2 tissue culture plates containing 3 mL of an appropriate
serum medium, grown in a 37.degree. C. incubator under 5% carbon
dioxide until an appropriate cell density was reached, and
transfected with the either a transfection solution lacking the
pQBI-25/GFP-BoNT/A-LC expression construct (untransfected cells) or
a transfection solution containing the pQBI-25/GFP-BoNT/A-LC
expression construct (transfected cells) as described above. The
cells were washed and harvested as described in Example I. To
detect for the presence of both the uncleaved SNAP-25.sub.206
substrate and the cleaved SNAP-25.sub.197 product, an aliquot from
each harvested sample was analyzed by Western blot as described in
Example I, except that the primary antibody used was a 1:100
dilution of the .alpha.-SNAP-25 monoclonal antibody-containing
ascites and the secondary antibody used was a 1:20,000 of
.alpha.-mouse IgG conjugated to Horseradish Peroxidase (Pierce
Biotechnology, Rockford, Ill.). In addition, three commercially
available mouse .alpha.-SNAP-25 monoclonal antibodies were tested.
SMI-81 (Sternberger Monoclonals Inc., Lutherville, Md.), an
.alpha.-SNAP-25 antibody the manufacturer indicates detects both
the uncleaved SNAP-25.sub.206 substrate and the cleaved
SNAP-25.sub.197 product, was used at a 15,000 dilution according to
the manufacturer's recommendations. MC-6050 (Research &
Diagnostic Antibodies, Las Vegas, Nev.), an .alpha.-SNAP-25
antibody the manufacturer indicates detects both the uncleaved
SNAP-25.sub.206 substrate and the cleaved SNAP-25.sub.197 product,
was used at a 1:100 dilution according to the manufacturer's
recommendations. MC-6053 (Research & Diagnostic Antibodies, Las
Vegas, Nev.), an .alpha.-SNAP-25 antibody the manufacturer
indicates detects only the cleaved SNAP-25.sub.197 product, was
used at a 1:100 dilution according to the manufacturer's
recommendations.
[0243] Table 19 indicates the .alpha.-SNAP-25 antibody-containing
ascites that detected only the SNAP-25.sub.197 cleavage product.
The cell-based cleavage assay indicated that ascites produced from
clones 1D3B8, 2C9B10, 2E2A6, 3C1A5, and 3C3E2 synthesize an
.alpha.-SNAP-25 monoclonal antibody having high binding specificity
for the SNAP-25.sub.197 cleavage product that allows for the
selective recognition of this cleavage product relative to the
SNAP-25.sub.206 uncleaved substrate. Commercial antibody SMI-81
detected the SNAP-25.sub.206 uncleaved substrate, but only poorly
recognized the SNAP-25.sub.197 cleavage product (Table 19).
Surprisingly, commercial antibody MC-6050 only detected the
SNAP-25.sub.206 uncleaved substrate, and failed to recognize the
SNAP-25.sub.197 cleavage product (Table 19). Even more
surprisingly, commercial antibody MC-6050 only detected the
SNAP-25.sub.206 uncleaved substrate, and failed to recognize the
SNAP-25.sub.197 cleavage product, even though the manufacturer
advertises that this antibody selectively detects the
SNAP-25.sub.197 cleavage product (Table 19). Thus, this analysis
indicates that while 1D3B8, 2C9B10, 2E2A6, 3C1A5, and 3C3E2 exhibit
suitable selectivity for the SNAP-25.sub.197 cleavage product,
1G10A12 and 2F11B6 do not. In addition, commercial antibodies
SMI-81, MC-6050 and MC-6053 all are unsuitable for the immuno-based
methods disclosed in the present application because all failed to
selectivity detect the SNAP-25.sub.197 cleavage product.
[0244] For immunocytochemistry analysis, binding specificity was
determined by analyzing the ability of .alpha.-SNAP-25
antibody-containing ascites to detect the uncleaved SNAP-25.sub.206
substrate and the cleaved SNAP-25.sub.197 product by
immunostaining. See e.g., Ester Fernandez-Salas et al., Plasma
Membrane Localization Signals in the Light Chain of Botulinum
Neurotoxin, Proc. Natl. Acad. Sci., U.S.A. 101(9): 3208-3213
(2004). A suitable density of PC12 cells were plated, grown, and
transfected with either a transfection solution lacking the
pQBI-25/GFP-BoNT/A-LC expression construct (untransfected cells) or
a transfection solution containing the pQBI-25/GFP-BoNT/A-LC
expression construct (transfected cells) as described above. The
cells were washed in 1.times.PBS and fixed in 5 mL of PAF at room
temperature for 30 minutes. Fixed cells were washed in phosphate
buffered saline, incubated in 5 mL of 0.5% Triton.RTM. X-100
(polyethylene glycol octylphenol ether) in 1.times.PBS, washed in
1.times.PBS, and permeabilized in 5 mL of methanol at -20.degree.
C. for six minutes. Permeabilized cells were blocked in 5 mL of 100
mM glycine at room temperature for 30 minutes, washed in
1.times.PBS, and blocked in 5 mL of 0.5% BSA in 1.times.PBS at room
temperature for 30 minutes. Blocked cells were washed in
1.times.PBS and incubated at room temperature for two hours in 0.5%
BSA in 1.times.PBS containing a 1:10 dilution of an ascites from a
clonal hybridoma cell line being tested. Primary antibody probed
cells were washed three times for 5 minutes each time in
1.times.PBS. Washed cells were incubated at room temperature for 2
hours in 1.times.PBS containing a 1:200 dilution of goat polyclonal
anti-mouse immunoglobulin G, heavy and light chains (IgG, H+L)
antibody conjugated to ALEXA.RTM. FLUOR 568 (Invitrogen Inc.,
Carlsbad, Calif.) as a secondary antibody. Secondary
antibody-probed cells were washed three times for 5 minutes each
time in 1.times.PBS. Washed cells were prepared for microscopic
examination by mounting in VECTASHIELD.RTM. Mounting Media (Vector
Laboratories, Burlingame, Calif.) and coverslipped. Images of
signal detection were obtained with a Leica confocal microscope
using appropriate laser settings. Table 19 indicates that the
.alpha.-SNAP-25 antibody-containing ascites that specifically
detected the SNAP-25.sub.197-cleavage product. The
immunocytochemistry analysis indicated that ascites produced from
clones 1D3B8, 2C9B10, 2E2A6, 3C1A5, and 3C3E2 synthesize an
.alpha.-SNAP-25 monoclonal antibody having high binding specificity
for the SNAP-25.sub.197 cleavage product that allows for the
preferential recognition of this cleavage product relative to the
SNAP-25.sub.206 uncleaved substrate.
[0245] For immunoprecipitation analysis, binding specificity was
determined by analyzing the ability of Protein A (HiTrap.TM.
Protein A HP Columns, GE Healthcare, Amersham, Piscataway, N.J.),
purified .alpha.-SNAP-25 monoclonal antibodies to precipitate the
uncleaved SNAP-25.sub.206 substrate and the cleaved SNAP-25.sub.197
product. See e.g., Chapter 8 Storing and Purifying Antibodies, pp.
309-311, Harlow & Lane, supra, 1998a. A suitable density of
PC12 cells were plated, grown, and transfected with either a
transfection solution containing a pQBI-25/GFP expression construct
(control cells; SEQ ID NO: 53) or a transfection solution
containing the pQBI-25/GFP-BoNT/A-LC expression construct
(experimental cells) as described above. The pQBI-25/GFP expression
construct comprises an expression vector whose promoter elements
are functionally linked to a polynucleotide encoding GFP of SEQ ID
NO: 54. After an overnight incubation, the cells were washed by
aspirating the growth media and rinsing each well with 200 .mu.L
1.times.PBS. To harvest the cells, the PBS was aspirated, the cells
were lysed by adding an Immunoprecipitation Lysis Buffer comprising
50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl.sub.2, 1 mM EGDT, 10%
glycerol, 1% Triton.RTM. X-100 (polyethylene glycol octylphenol
ether) and a 1.times. COMPLETE.TM. Protease inhibitor cocktail
(Roche Applied Biosciences, Indianapolis, Ind.) and incubating at
4.degree. C. for one hour. The lysed cells were centrifuged at
3,000.times.g at 4.degree. C. for 10 minutes to remove cellular
debris and the supernatant transferred to a clean tube and diluted
to a protein concentration of approximately 1 mg/mL. Approximately
5 .mu.g of purified monoclonal antibody was added to 0.5 mL of
diluted supernatant and incubated at 4.degree. C. for two hours.
After primary antibody incubation, approximately 50 .mu.l of
immobilized Protein G (Pierce Biotechnology, Rockford, Ill.) was
added to the diluted supernatant and incubated at 4.degree. C. for
one hour. The incubated supernatant was washed three times for 30
minutes each time by adding 0.5 mL of Immunoprecipitation Lysis
Buffer, centrifuging at 300.times.g at 4.degree. C. for one minute
to pellet the immobilized Protein G, and decanting the supernatant.
After washing, the pellet was resuspended in 30 .mu.L of
1.times.SDS Loading Buffer and the sample was heated to 95.degree.
C. for 5 minutes. To detect for the presence of both the uncleaved
SNAP-25.sub.206 substrate and the cleaved SNAP-25.sub.197 product,
an aliquot from each harvested sample was analyzed by Western blot
as described in Example I, except that the primary antibody used
was a 1:1,000 dilution of the .alpha.-SNAP-25 polyclonal antibody
serum (see Example IV) and the secondary antibody used was a
1:20,000 of rabbit .alpha.-IgG Horseradish Peroxidase (Pierce
Biotechnology, Rockford, Ill.). Table 19 indicates the
.alpha.-SNAP-25 antibody-containing ascites that specifically
pulled down the SNAP-25.sub.197-cleavage product by
immunoprecipitation analysis. The immunoprecipitation analysis
indicated that ascites produced from clones 2E2A6 and 3C1A5
synthesize an .alpha.-SNAP-25 monoclonal antibody having high
binding specificity for the SNAP-25.sub.197 cleavage product that
allows for the preferential recognition of this cleavage product
relative to the SNAP-25.sub.206 uncleaved substrate.
TABLE-US-00011 TABLE 19 Analysis of Clone Ascites Containing
.alpha.-SNAP-25 Monoclonal Antibody Cell-Based Assay
Immunocytochemistry Immunoprecipitation Clone SNAP-25.sub.197
SNAP-25.sub.206 SNAP-25.sub.197 SNAP-25.sub.206 SNAP-25.sub.197
SNAP-25.sub.206 1D3B8 ++ - ++ - Not Tested Not Tested 1G10A12 ++ ++
Not Tested Not Tested Not Tested Not Tested 2C9B10 ++ - ++ - Not
Tested Not Tested 2E2A6 ++ - ++ - ++ - 2F11B6 + + + + Not Tested
Not Tested 3C1A5 ++ - ++ - ++ - 3C3E2 + - Not Tested Not Tested Not
Tested Not Tested MC-6050 - + Not Tested Not Tested Not Tested Not
Tested MC-6053 - + Not Tested Not Tested Not Tested Not Tested
SMI-81 -/+ ++ Not Tested Not Tested Not Tested Not Tested
5. Evaluation of Binding Affinity of .alpha.-SNAP-25 Monoclonal
Antibodies.
[0246] To determine the binding affinity of an .alpha.-SNAP-25
monoclonal antibody showing high binding specificity for either the
SNAP-25.sub.197 cleavage product or the SNAP-25.sub.206 uncleaved
substrate, binding affinity assays were performed on a BIACORE 3000
instrument using carboxymethyl dextran (CM5) sensor chips (BIAcore,
Inc., Piscataway, N.J.). Runs were conducted at 25.degree. C. with
HBS-EP buffer comprising 10 mM HEPES (pH 7.4), 150 mM sodium
chloride, 3 mM EDTA, 0.005% (v/v) surfactant P20 at a flow rate of
10 .mu.l/min. SNAP-25 peptides comprising amino acids 134-197 of
SEQ ID NO: 5 (SNAP-25.sub.134-197) or amino acids 134-206 of SEQ ID
NO: 5 (SNAP-25.sub.134-206) were covalently attached to the surface
of the CM5 sensor chips using standard amine coupling. Briefly, the
CM5 chips were activated by a 7 minute injection of a mixture of
0.2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 0.05 M
N-hydroxysuccimide; the SNAP-25 peptides were then injected in 10
mM sodium acetate (pH 4.0) for 20 min at a flow rate of 10
.mu.L/min; and unreacted succimide esters were blocked by a 7-min
injection of 1 M ethanolamine hydrochloride, pH 8.5. The
immobilized amount of SNAP-25.sub.134-197 or SNAP-25 on the chip
was reflected by a 100-150 increase in response units (about
0.10-0.15 ng/mm.sup.2). Antibody samples comprising either ascites
or purified monoclonal antibodies produced from clones 1D3B8,
2C9B10, 2E2A6, 3C1A5, and 3C3E2, as well as, commercially available
.alpha.-SNAP-25 antibodies were passed over the surface of the CM5
chips allowing an association time of 10 min and a dissociation
time of 20 min. The surfaces were regenerated between runs by a 1
minute injection of 10 mM glycine-HCl (pH 2.5) at a flow rate of 15
.mu.L/min. Sensorgram curves were fitted to a 1:1 kinetic binding
model with the BIAevaluation 3.0 software.
[0247] The results indicate that both 2E2A6 and 3C1A5 were highly
specific for cleaved SNAP-25.sub.197 product over SNAP-25 uncleaved
substrate (Table 20). When compared to the binding affinities of
MC-6050 and MC-6053, 1D3B6 had an approximately 10-fold higher
equilibrium disassociation constant for the SNAP-25 cleavage
product relative to these commercial antibodies (Table 20).
Interestingly, 2E2A6 had only a slightly lower equilibrium
disassociation constant for the SNAP-25 cleavage product relative
to these commercial antibodies (0.405 nM versus 0.497 and 0.508)
(Table 20). As neither of these commercial .alpha.-SNAP-25
antibodies selectively recognized the SNAP-25 cleavage product
(Table 19), an equilibrium disassociation constant lower than about
0.5 nM appears, in part, critical to achieve such selectivity.
Similarly, when compared to the binding affinities of MC-6050 and
MC-6053, 2E2A6 had an about at least one-fold slower off
rate/dissociation constant (6.74.times.10.sup.-5 versus
8.82.times.10.sup.-4 s.sup.-1 and 1.18.times.10.sup.-3 s.sup.-1)
(Table 20). This further suggests that an off rate/dissociation
constant lower than about 8.82.times.10.sup.-4 appears, in part,
critical to achieve selective binding for the SNAP-25 cleavage
product. This result is consistent with 1 D3B8, which had an off
rate/dissociation constant of 5.78.times.10.sup.-5 s.sup.-1 (Table
20).
TABLE-US-00012 TABLE 20 Analysis of Binding Affinity
.alpha.-SNAP-25 Monoclonal Antibodies SPR 1D3B8 2E2A6 Parameter
SNAP-25.sub.197 SNAP-25.sub.206.sup.a SNAP-25.sub.197
SNAP-25.sub.206.sup.b Ka 1.06 .times. 10.sup.6 -- 1.70 .times.
10.sup.6 -- (M.sup.-1 s.sup.-1) (1.66 .times. 10.sup.5).sup. (--)
Kd (s.sup.-1) 5.78 .times. 10.sup.-5 -- 1.53 .times. 10.sup.-4 --
(6.74 .times. 10.sup.-5) (--) KD (nM) 0.050 -- 0.090 -- (0.405)
(--) SPR 3C1A5 2C9B10 Parameter SNAP-25.sub.197
SNAP-25.sub.206.sup.c SNAP-25.sub.197 SNAP-25.sub.206.sup.d Ka 2.17
.times. 10.sup.5 -- 1.15 .times. 10.sup.4 -- (M.sup.-1 s.sup.-1) Kd
(s.sup.-1) 2.88 .times. 10.sup.-4 -- 3.11 .times. 10.sup.-4 -- KD
(nM) 1.33 -- 27.1 -- SPR MC-6050 MC-6053 Parameter SNAP-25.sub.197
SNAP-25.sub.206 SNAP-25.sub.197 SNAP-25.sub.206 Ka 1.78 .times.
10.sup.6 3.06 .times. 10.sup.2 2.32 .times. 10.sup.6 1.06 .times.
10.sup.2 (M.sup.-1 s.sup.-1) Kd (s.sup.-1) 8.82 .times. 10.sup.-4
6.07 .times. 10.sup.-3 1.18 .times. 10.sup.-3 2.56 .times.
10.sup.-5 KD (nM) 0.497 19,800 0.508 240 .sup.aNo binding was
observed when up to 125 nM of .alpha.-SNAP-25 monoclonal antibody
1D3B8 was passed over the surface of the CM5 sensor chip after a 10
minute association time. .sup.bNo binding was observed when up to
10 .mu.M of .alpha.-SNAP-25 monoclonal antibody 2E2A6 was passed
over the surface of the CM5 sensor chip after a 10 minute
association time. .sup.cNo binding was observed when up to 100 nM
of .alpha.-SNAP-25 monoclonal antibody 3C1A5 was passed over the
surface of the CM5 sensor chip after a 10 minute association time.
.sup.dNo binding was observed when up to 100 nM of .alpha.-SNAP-25
monoclonal antibody 2C9B10 was passed over the surface of the CM5
sensor chip after a 10 minute association time.
[0248] To compare the six different antibodies, the on-rate (ka)
and off-rate (kd) for each was normalized using a program from the
BIA evaluation 4.1 software. For comparison of the on-rates, the
data were first individually trimmed by deleting the re-generation
portion and the injection spikes, and then normalized to a 0 to 100
scale. For comparison of the off-rate, the data were normalized to
the injection stop/top point. This analysis showed that 2C9B10 had
a much slower on-rate than the other antibodies (FIG. 3A), and that
MC-6053 has a much faster off-rate (dissociation) that the other
antibodies (FIG. 3B). The fast off-rate of MC-6053 indicates that
this antibody will not perform well in the methods disclosed in the
present specification because this antibody will have difficulty
staying bound to the substrate antigen during the washing
steps.
6. Sequencing of the Epitope from Isolated .alpha.-SNAP-25
Monoclonal Antibodies.
[0249] To determine the epitope of an isolated .alpha.-SNAP-25
monoclonal antibody that can selectively bind to a SNAP-25 antigen
having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A
cleavage site scissile bond, the polynucleotide molecule encoding
the variable heavy (V.sub.H) and variable light (V.sub.L) chains of
the .alpha.-SNAP-25 monoclonal antibody produced by hybridomas
1D3B8, 2C9B10, 2E2A6, 3C1A5 and 3C3E2 were sequenced. mRNA was
extracted and purified from each hybridoma using standard protocols
and reversed transcribed into cDNA using either an oligo dT
anti-sense primer or a gene-specific (murine IgG1 CH and kappa CL)
anti-sense primer. Specific murine and human constant domain
primers were used to amplify the cDNA by PCR after cDNA production
to determine the isotype of the antibody. Degenerate V.sub.H and
V.sub.L primers were used to amplify the variable domains from the
cDNA. For 5'RACE, a homopolymeric dCTP tail was added to the 3' end
of the cDNA. The heavy and light chains were then amplified with an
oligo dG sense primer and a gene specific (CH/KC) anti-sense
primer. PCR products included the sequence of the signal peptide,
variable domains and constant domains up to the anti-sense primer.
The PCR products were gel purified to remove small fragments, and
cloned into a blunt or TA vector for sequencing. Five independent
clones for each chain were sequenced and alignments of V.sub.H and
VL chains and consensus sequences were determined. Methods used to
determine the V.sub.H and V.sub.L amino acid sequences are
described in, e.g., Roger A. Sabbadini, et al., Novel Bioactive
Lipid Derivatives and Methods of Making and Using Same, U.S. Patent
Publication 2007/0281320; and Peter Amersdorfer, et al., Molecular
Characterization of Murine Humoral Immune Response to Botulinum
Neurotoxin Type A Binding Domain as Assessed by Using Phage
Antibody Libraries, 65(9) Infect. Immun. 3743-3752, each of which
is hereby incorporated by reference in its entirety. In addition,
commercial services are available to sequence the variable heavy
(V.sub.H) and variable light (V.sub.L) chains of an antibody and
identify the CDR regions, see, e.g., Fusion Antibodies Ltd.,
Northern Ireland.
[0250] The polynucleotide sequence comprising the V.sub.H and
V.sub.L chains of the .alpha.-SNAP-25 monoclonal antibody produced
by the hybridomas disclosed in the present specification is as
follows: 1 D3B8 V.sub.H (SEQ ID NO: 71), 2C9B10 V.sub.H (SEQ ID NO:
73), 2E2A6 V.sub.H (SEQ ID NO: 75), 3C1A5 V.sub.H (SEQ ID NO: 77),
3C3E2 V.sub.H variant 1 (SEQ ID NO: 79), 3C3E2 V.sub.H variant 2
(SEQ ID NO: 81), 3C3E2 V.sub.H variant 3 (SEQ ID NO: 149), 1D3B8
V.sub.L (SEQ ID NO: 83), 2C9B10 V.sub.L (SEQ ID NO: 85), 2E2A6
V.sub.L (SEQ ID NO: 87), 3C1A5 V.sub.L (SEQ ID NO: 89), and 3C3E2
V.sub.L (SEQ ID NO: 91). The amino acid sequence comprising the
V.sub.H and V.sub.L chains of the .alpha.-SNAP-25 monoclonal
antibody produced by the hybridomas disclosed in the present
specification is as follows: 1D3B8 V.sub.H (SEQ ID NO: 72), 2C9B10
V.sub.H (SEQ ID NO: 74), 2E2A6 V.sub.H (SEQ ID NO: 76), 3C1A5
V.sub.H (SEQ ID NO: 78), 3C3E2 V.sub.H variant 1 (SEQ ID NO: 80),
3C3E2 V.sub.H variant 2 (SEQ ID NO: 82); 3C3E2 V.sub.H variant 2
(SEQ ID NO: 150), 1D3B8 V.sub.L (SEQ ID NO: 84), 2C9B10 V.sub.L
(SEQ ID NO: 86), 2E2A6 V.sub.L (SEQ ID NO: 88), 3C1A5 V.sub.L (SEQ
ID NO: 90), and 3C3E2 V.sub.L (SEQ ID NO: 92). The amino acid
sequences comprising the V.sub.H and V.sub.L CDR domains of the
.alpha.-SNAP-25 monoclonal antibody produced by the hybridomas
1D3B8, 2C9B10, 2E2A6, 3C1A5, and 3C3E2 are given in Table 21.
TABLE-US-00013 TABLE 21 CDR Sequences of V.sub.H and V.sub.L
domains from .alpha.-SNAP-25 Monoclonal Antibodies CDR Sequence
Identified In SEQ ID NO: V.sub.H CDR 1 TFTDHSIH 2E2A6 93 2C9B10
3C1A5 V.sub.H CDR 1 TFTNYVIH 3C3E2 94 V.sub.H CDR 1 IFTDHALH 1D3B8
95 V.sub.H CDR 2 YIFPGNGNIEYNDKFKG 2E2A6 96 V.sub.H CDR 2
YLFPGNGNFEYNEKFKG 2C9B10 97 3C1A5 V.sub.H CDR 2 YINPYNDGSKYNEKFKG
3C3E2 98 V.sub.H CDR 2 YIFPGNGNIEYNEKFKG 1D3B8 99 V.sub.H CDR 3
KRMGY 2E2A6 100 3C1A5 V.sub.H CDR 3 KKMDY 2C9B10 101 1D3B8 V.sub.H
CDR 3 ARMDY 3C3E2var1 102 V.sub.H CDR 3 ARMGY 3C3E2var2 151 V.sub.H
CDR 3 ARHLANTYYYFDY 3C3E2var3 152 V.sub.L CDR 1 RSSQSIVHSNGNTYLE
1D3B8 103 V.sub.L CDR 1 RTTENIYSYFV 2C9B10 104 V.sub.L CDR 1
KSSQSLLYTNGKTYLT 2E2A6 105 V.sub.L CDR 1 KSSQSLLNTNGKTYLT 3C1A5 106
V.sub.L CDR 1 RASQNIGNYLH 3C3E2 107 V.sub.L CDR 2 KVSNRFS 1D3B8 108
V.sub.L CDR 2 NAKSLAE 2C9B10 109 V.sub.L CDR 2 LVSELDS 2E2A6 110
V.sub.L CDR 2 LVSKLDS 3C1A5 111 V.sub.L CDR 2 YASQSIS 3C3E2 112
V.sub.L CDR 3 FQGSHVPPT 1D3B8 113 V.sub.L CDR 3 QHHYGTPYT 2C9B10
114 V.sub.L CDR 3 LQSAHFPFT 2E2A6 115 V.sub.L CDR 3 LQSSHFPFT 3C1A5
116 V.sub.L CDR 3 QQSDTWPLT 3C3E2 117
[0251] Non-limiting examples of amino acid sequences comprising
V.sub.H CDR domain variants of the .alpha.-SNAP-25 monoclonal
antibody produced by the hybridomas disclosed in the present
specification include V.sub.H CDR1 variant SEQ ID NO: 118 for
1D3B8; V.sub.H CDR1 variant SEQ ID NO: 119 for 2C9B10, 2E2A6 and
3C1A5 V.sub.H; V.sub.H CDR1 variant SEQ ID NO: 120 for 3C1A5
V.sub.H and 3C3E2 variant 3; V.sub.H CDR2 variant SEQ ID NO: 121
for 1D3B8 and 2E2A6; V.sub.H CDR2 variant SEQ ID NO: 122 for 2C9B10
and 3C1A5 V.sub.H; V.sub.H CDR2 variant SEQ ID NO: 123 for 3C1A5
V.sub.H and 3C3E2 variant 3; V.sub.H CDR3 variant MDY for 1D3B8 and
2C9B10; V.sub.H CDR3 variant MGY for 2E2A6 and 3C1A5 V.sub.H; and
V.sub.H CDR3 variant SEQ ID NO: 124 for 3C1A5 V.sub.H and 3C3E2
variant 3. Non-limiting examples of amino acid sequences comprising
V.sub.L CDR domain variants of the .alpha.-SNAP-25 monoclonal
antibody produced by the hybridomas disclosed in the present
specification include V.sub.L CDR1 variant SEQ ID NO: 125 for
1D3B8; V.sub.L CDR1 variant SEQ ID NO: 126 for 2C9B10; V.sub.L CDR1
variant SEQ ID NO: 127 for 2E2A6; V.sub.L CDR1 variant SEQ ID NO:
128 for 3C1A5; V.sub.L CDR1 variant SEQ ID NO: 129 for 3C3E2;
V.sub.L CDR2 variant KVS for 1D3B8; V.sub.L CDR2 variant NAK for
2C9B10; V.sub.L CDR2 variant LVS for 2E2A6; V.sub.L CDR2 variant
YAT for 3C1A5; and V.sub.L CDR2 variant YAS for 3C3E2.
Example IX
Development of .alpha.-SNAP-25 Polyclonal Antibodies that
Selectively Bind a SNAP-25 Epitope Having a Free Carboxyl-Terminus
at the P.sub.1 Residue of the BoNT/A Cleavage Site Scissile
Bond
[0252] The following example illustrates how to make
.alpha.-SNAP-25 polyclonal antibodies that can selectively bind to
a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue
of the BoNT/A cleavage site scissile bond.
[0253] To develop .alpha.-SNAP-25 polyclonal antibodies that bind
an epitope comprising a carboxyl-terminus at the P1 residue from
the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage
product, the 10-residue peptide CGGGRIDEANQ (SEQ ID NO: 46) was
designed as a SNAP-25 cleavage product antigen. This peptide
comprising a N-terminal Cysteine residue for conjugation to KLH, a
G-spacer flexible spacer (GGG) linked to amino acids 191-197 of
human SNAP-25 (SEQ ID NO: 5) and has a carboxylated C-terminal
glutamine. Blast searches revealed that this peptide has high
homology only to SNAP-25 and almost no possible cross-reactivity
with other proteins in neuronal cells. The sequence was also
carefully scrutinized by utilizing computer algorithms to determine
hydropathy index, protein surface probability, regions of
flexibility, and favorable secondary structure, followed by proper
orientation and presentation of the chosen peptide sequence. The
peptide was synthesized and conjugated to Keyhole Limpet Hemocyanin
(KLH) to increase immunogenicity. Before the animals were
immunized, naive rabbits were first screened against cell lysates
from candidate cell lines in a Western blot in order to identify
animals that had no immunoreactivity to the proteins present in the
cell lysates. Two pre-screened rabbits were immunized with this
peptide, and after three immunizations in about eight weeks, the
rabbits were bled for testing. The blood was allowed to clot by
incubating at 4.degree. C. for 60 minutes. The clotted blood was
centrifuged at 10,000.times.g at 4.degree. C. for 10 minutes to
pellet the cellular debris. The resulting serum sample was
dispensed into 50 .mu.L aliquots and stored at -20.degree. C. until
needed.
[0254] A similar strategy based on other SNAP-25 antigens disclosed
in the present specification is used to develop .alpha.-SNAP-25
polyclonal antibodies that bind an epitope comprising a
carboxyl-terminus at the P1 residue from the BoNT/A cleavage site
scissile bond from a SNAP-25 cleavage product. For example, the
SNAP-25 antigen of SEQ ID NO: 47 can be conjugated to KLH instead
of the SNAP-25 antigen of SEQ ID NO: 46. As another example, the
amino acids 191-197 of human SNAP-25 from the SNAP-25 antigen of
SEQ ID NO: 38 can be replaced with SEQ ID NO: 33, SEQ ID NO: 34,
SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID
NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO:
44.
2. Screening for the Presence of .alpha.-SNAP-25 Polyclonal
Antibodies.
[0255] To determine the presence of .alpha.-SNAP-25 polyclonal
antibodies that can selectively bind to a SNAP-25 antigen having a
carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage
site scissile bond, comparative ELISA and cell-based cleavage
assays were performed using the extracted rabbit serum as described
in Example VIII. The serum from both rabbits contained
.alpha.-SNAP-25 polyclonal antibodies that can selectively bind to
a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue
of the BoNT/A cleavage site scissile bond. The .alpha.-SNAP-25
rabbit polyclonal antibodies were designated as NTP 22 and NTP
23.
3. Purification of .alpha.-SNAP-25 Polyclonal Antibodies.
[0256] To purify .alpha.-SNAP-25 polyclonal antibodies that can
selectively bind to a SNAP-25 antigen having a carboxyl-terminus at
the P.sub.1 residue of the BoNT/A cleavage site scissile bond, NTP
22 and NTP 23 antibodies from rabbit serum were purified using
affinity columns containing the SNAP-25 antigen of SEQ ID NO:
46.
4. Evaluation of Binding Specificity of .alpha.-SNAP-25 Polyclonal
Antibodies.
[0257] To evaluate binding specificity of an .alpha.-SNAP-25
polyclonal antibody that can selectively bind to a SNAP-25 antigen
having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A
cleavage site scissile bond, purified NTP 22 and NTP 23
.alpha.-SNAP-25 polyclonal antibodies were used to detect cleavage
product using the cell-based activity assay, immunnocytochemistry
and immunoprecipitation as described in Example VIII. The
cell-based cleavage assay, immunocytochemistry analysis and
immunoprecipitation analysis all indicated that NTP 22 and NTP 23
.alpha.-SNAP-25 polyclonal antibodies did not cross-react with
uncleaved SNAP-25. Thus both NTP 22 and NTP 23 have high binding
specificity for the SNAP-25.sub.197 cleavage product that allows
for the preferential recognition of this cleavage product relative
to the SNAP-25.sub.206 uncleaved substrate. Affinity for the
antigens can be determined using SPR in the BiAcore as described in
Example VIII.
[0258] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as molecular weight,
reaction conditions, and so forth used in the specification and
claims are to be understood as being modified in all instances by
the term "about." Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the specification and
attached claims are approximations that may vary depending upon the
desired properties sought to be obtained by the present invention.
At the very least, and not as an attempt to limit the application
of the doctrine of equivalents to the scope of the claims, each
numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary
rounding techniques. Notwithstanding that the numerical ranges and
parameters setting forth the broad scope of the invention are
approximations, the numerical values set forth in the specific
examples are reported as precisely as possible. Any numerical
value, however, inherently contains certain errors necessarily
resulting from the standard deviation found in their respective
testing measurements.
[0259] The terms "a," "an," "the" and similar referents used in the
context of describing the invention (especially in the context of
the following claims) are to be construed to cover both the
singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. Recitation of ranges of values
herein is merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range. Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein is intended
merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed. No
language in the specification should be construed as indicating any
non-claimed element essential to the practice of the invention.
[0260] Groupings of alternative elements or embodiments of the
invention disclosed herein are not to be construed as limitations.
Each group member may be referred to and claimed individually or in
any combination with other members of the group or other elements
found herein. It is anticipated that one or more members of a group
may be included in, or deleted from, a group for reasons of
convenience and/or patentability. When any such inclusion or
deletion occurs, the specification is deemed to contain the group
as modified thus fulfilling the written description of all Markush
groups used in the appended claims.
[0261] Certain embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Of course, variations on these described embodiments
will become apparent to those of ordinary skill in the art upon
reading the foregoing description. The inventor expects skilled
artisans to employ such variations as appropriate, and the
inventors intend for the invention to be practiced otherwise than
specifically described herein. Accordingly, this invention includes
all modifications and equivalents of the subject matter recited in
the claims appended hereto as permitted by applicable law.
Moreover, any combination of the above-described elements in all
possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by
context.
[0262] Specific embodiments disclosed herein may be further limited
in the claims using consisting of or consisting essentially of
language. When used in the claims, whether as filed or added per
amendment, the transition term "consisting of" excludes any
element, step, or ingredient not specified in the claims. The
transition term "consisting essentially of" limits the scope of a
claim to the specified materials or steps and those that do not
materially affect the basic and novel characteristic(s).
Embodiments of the invention so claimed are inherently or expressly
described and enabled herein.
[0263] Furthermore, numerous references have been made to patents
and printed publications throughout this specification. Each of the
above-cited references and printed publications are individually
incorporated herein by reference in their entirety.
[0264] In closing, it is to be understood that the embodiments of
the invention disclosed herein are illustrative of the principles
of the present invention. Other modifications that may be employed
are within the scope of the invention. Thus, by way of example, but
not of limitation, alternative configurations of the present
invention may be utilized in accordance with the teachings herein.
Accordingly, the present invention is not limited to that precisely
as shown and described.
Sequence CWU 1
1
15211296PRTClostridium botulinum 1Met Pro Phe Val Asn Lys Gln Phe
Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15 Val Asp Ile Ala Tyr Ile
Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30 Val Lys Ala Phe
Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45 Asp Thr
Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60
Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65
70 75 80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu
Phe Glu 85 90 95 Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu
Thr Ser Ile Val 100 105 110 Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr
Ile Asp Thr Glu Leu Lys 115 120 125 Val Ile Asp Thr Asn Cys Ile Asn
Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140 Arg Ser Glu Glu Leu Asn
Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155 160 Ile Gln Phe
Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175 Arg
Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185
190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu
195 200 205 Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala
His Glu 210 215 220 Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala
Ile Asn Pro Asn225 230 235 240 Arg Val Phe Lys Val Asn Thr Asn Ala
Tyr Tyr Glu Met Ser Gly Leu 245 250 255 Glu Val Ser Phe Glu Glu Leu
Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270 Phe Ile Asp Ser Leu
Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285 Lys Phe Lys
Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300 Gly
Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310
315 320 Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys
Leu 325 330 335 Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr
Thr Glu Asp 340 345 350 Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg
Lys Thr Tyr Leu Asn 355 360 365 Phe Asp Lys Ala Val Phe Lys Ile Asn
Ile Val Pro Lys Val Asn Tyr 370 375 380 Thr Ile Tyr Asp Gly Phe Asn
Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400 Phe Asn Gly Gln
Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415 Lys Asn
Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430
Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys 435
440 445 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe
Phe 450 455 460 Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys
Gly Glu Glu465 470 475 480 Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala
Glu Glu Asn Ile Ser Leu 485 490 495 Asp Leu Ile Gln Gln Tyr Tyr Leu
Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510 Glu Asn Ile Ser Ile Glu
Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525 Glu Leu Met Pro
Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540 Leu Asp
Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu545 550 555
560 His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu
565 570 575 Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr
Val Lys 580 585 590 Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu
Gly Trp Val Glu 595 600 605 Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr
Ser Glu Val Ser Thr Thr 610 615 620 Asp Lys Ile Ala Asp Ile Thr Ile
Ile Ile Pro Tyr Ile Gly Pro Ala625 630 635 640 Leu Asn Ile Gly Asn
Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645 650 655 Ile Phe Ser
Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala 660 665 670 Ile
Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys 675 680
685 Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu
690 695 700 Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu
Ala Lys705 710 715 720 Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys
Met Lys Glu Ala Leu 725 730 735 Glu Asn Gln Ala Glu Ala Thr Lys Ala
Ile Ile Asn Tyr Gln Tyr Asn 740 745 750 Gln Tyr Thr Glu Glu Glu Lys
Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765 Leu Ser Ser Lys Leu
Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770 775 780 Asn Lys Phe
Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met785 790 795 800
Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys 805
810 815 Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile
Gly 820 825 830 Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu
Ser Thr Asp 835 840 845 Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn
Gln Arg Leu Leu Ser 850 855 860 Thr Phe Thr Glu Tyr Ile Lys Asn Ile
Ile Asn Thr Ser Ile Leu Asn865 870 875 880 Leu Arg Tyr Glu Ser Asn
His Leu Ile Asp Leu Ser Arg Tyr Ala Ser 885 890 895 Lys Ile Asn Ile
Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn 900 905 910 Gln Ile
Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu 915 920 925
Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser 930
935 940 Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn
Asn945 950 955 960 Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser
Gly Trp Lys Val 965 970 975 Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr
Leu Gln Asp Thr Gln Glu 980 985 990 Ile Lys Gln Arg Val Val Phe Lys
Tyr Ser Gln Met Ile Asn Ile Ser 995 1000 1005 Asp Tyr Ile Asn Arg
Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu 1010 1015 1020 Asn Asn
Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro1025 1030
1035 1040Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met
Phe Lys 1045 1050 1055 Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile
Trp Ile Lys Tyr Phe 1060 1065 1070 Asn Leu Phe Asp Lys Glu Leu Asn
Glu Lys Glu Ile Lys Asp Leu Tyr 1075 1080 1085 Asp Asn Gln Ser Asn
Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr 1090 1095 1100 Leu Gln
Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn1105 1110
1115 1120Lys Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met
Tyr Leu 1125 1130 1135 Lys Gly Pro Arg Gly Ser Val Met Thr Thr Asn
Ile Tyr Leu Asn Ser 1140 1145 1150 Ser Leu Tyr Arg Gly Thr Lys Phe
Ile Ile Lys Lys Tyr Ala Ser Gly 1155 1160 1165 Asn Lys Asp Asn Ile
Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val 1170 1175 1180 Val Val
Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala1185 1190
1195 1200Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val
Gly Asn 1205 1210 1215 Leu Ser Gln Val Val Val Met Lys Ser Lys Asn
Asp Gln Gly Ile Thr 1220 1225 1230 Asn Lys Cys Lys Met Asn Leu Gln
Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245 Phe Ile Gly Phe His
Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser 1250 1255 1260 Asn Trp
Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys1265 1270
1275 1280Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg
Pro Leu 1285 1290 1295 21296PRTClostridium botulinum 2Met Pro Phe
Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15 Val
Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25
30 Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg
35 40 45 Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro
Pro Glu 50 55 60 Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr
Tyr Leu Ser Thr65 70 75 80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly
Val Thr Lys Leu Phe Glu 85 90 95 Arg Ile Tyr Ser Thr Asp Leu Gly
Arg Met Leu Leu Thr Ser Ile Val 100 105 110 Arg Gly Ile Pro Phe Trp
Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125 Val Ile Asp Thr
Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140 Arg Ser
Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155
160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Asp Val Leu Asn Leu Thr
165 170 175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro
Asp Phe 180 185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr
Asn Pro Leu Leu 195 200 205 Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala
Val Thr Leu Ala His Glu 210 215 220 Leu Ile His Ala Glu His Arg Leu
Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240 Arg Val Phe Lys Val
Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255 Glu Val Ser
Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270 Phe
Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280
285 Lys Phe Lys Asp Val Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Ile
290 295 300 Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys
Glu Lys305 310 315 320 Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe
Ser Val Asp Lys Leu 325 330 335 Lys Phe Asp Lys Leu Tyr Lys Met Leu
Thr Glu Ile Tyr Thr Glu Asp 340 345 350 Asn Phe Val Asn Phe Phe Lys
Val Ile Asn Arg Lys Thr Tyr Leu Asn 355 360 365 Phe Asp Lys Ala Val
Phe Arg Ile Asn Ile Val Pro Asp Glu Asn Tyr 370 375 380 Thr Ile Lys
Asp Gly Phe Asn Leu Lys Gly Ala Asn Leu Ser Thr Asn385 390 395 400
Phe Asn Gly Gln Asn Thr Glu Ile Asn Ser Arg Asn Phe Thr Arg Leu 405
410 415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val
Arg 420 425 430 Gly Ile Ile Pro Phe Lys Thr Lys Ser Leu Asp Glu Gly
Tyr Asn Lys 435 440 445 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn
Trp Asp Leu Phe Phe 450 455 460 Ser Pro Ser Glu Asp Asn Phe Thr Asn
Asp Leu Asp Lys Val Glu Glu465 470 475 480 Ile Thr Ala Asp Thr Asn
Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495 Asp Leu Ile Gln
Gln Tyr Tyr Leu Thr Phe Asp Phe Asp Asn Glu Pro 500 505 510 Glu Asn
Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525
Glu Pro Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530
535 540 Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe
Glu545 550 555 560 His Gly Asp Ser Arg Ile Ile Leu Thr Asn Ser Ala
Glu Glu Ala Leu 565 570 575 Leu Lys Pro Asn Val Ala Tyr Thr Phe Phe
Ser Ser Lys Tyr Val Lys 580 585 590 Lys Ile Asn Lys Ala Val Glu Ala
Phe Met Phe Leu Asn Trp Ala Glu 595 600 605 Glu Leu Val Tyr Asp Phe
Thr Asp Glu Thr Asn Glu Val Thr Thr Met 610 615 620 Asp Lys Ile Ala
Asp Ile Thr Ile Ile Val Pro Tyr Ile Gly Pro Ala625 630 635 640 Leu
Asn Ile Gly Asn Met Leu Ser Lys Gly Glu Phe Val Glu Ala Ile 645 650
655 Ile Phe Thr Gly Val Val Ala Met Leu Glu Phe Ile Pro Glu Tyr Ala
660 665 670 Leu Pro Val Phe Gly Thr Phe Ala Ile Val Ser Tyr Ile Ala
Asn Lys 675 680 685 Val Leu Thr Val Gln Thr Ile Asn Asn Ala Leu Ser
Lys Arg Asn Glu 690 695 700 Lys Trp Asp Glu Val Tyr Lys Tyr Thr Val
Thr Asn Trp Leu Ala Lys705 710 715 720 Val Asn Thr Gln Ile Asp Leu
Ile Arg Glu Lys Met Lys Lys Ala Leu 725 730 735 Glu Asn Gln Ala Glu
Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750 Gln Tyr Thr
Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765 Leu
Ser Ser Lys Leu Asn Glu Ser Ile Asn Ser Ala Met Ile Asn Ile 770 775
780 Asn Lys Phe Leu Asp Gln Cys Ser Val Ser Tyr Leu Met Asn Ser
Met785 790 795 800 Ile Pro Tyr Ala Val Lys Arg Leu Lys Asp Phe Asp
Ala Ser Val Arg 805 810 815 Asp Val Leu Leu Lys Tyr Ile Tyr Asp Asn
Arg Gly Thr Leu Val Leu 820 825 830 Gln Val Asp Arg Leu Lys Asp Glu
Val Asn Asn Thr Leu Ser Ala Asp 835 840 845 Ile Pro Phe Gln Leu Ser
Lys Tyr Val Asp Asn Lys Lys Leu Leu Ser 850 855 860 Thr Phe Thr Glu
Tyr Ile Lys Asn Ile Val Asn Thr Ser Ile Leu Ser865 870 875 880 Ile
Val Tyr Lys Lys Asp Asp Leu Ile Asp Leu Ser Arg Tyr Gly Ala 885 890
895 Lys Ile Asn Ile Gly Asp Arg Val Tyr Tyr Asp Ser Ile Asp Lys Asn
900 905 910 Gln Ile Lys Leu Ile Asn Leu Glu Ser Ser Thr Ile Glu Val
Ile Leu 915 920 925 Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn
Phe Ser Thr Ser 930 935 940 Phe Trp Ile Lys Ile Pro Lys Tyr Phe Ser
Lys Ile Asn Leu Asn Asn945 950 955 960 Glu Tyr Thr Ile Ile Asn Cys
Ile Glu Asn Asn Ser Gly Trp Lys Val 965 970 975 Ser Leu Asn Tyr Gly
Glu Ile Ile Trp Thr Leu Gln Asp Asn Lys Gln 980 985 990 Asn Ile Gln
Arg Val Val Phe Lys Tyr Ser Gln Met Val Asn Ile Ser 995 1000 1005
Asp Tyr Ile Asn Arg Trp Ile Phe
Val Thr Ile Thr Asn Asn Arg Leu 1010 1015 1020 Thr Lys Ser Lys Ile
Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro1025 1030 1035 1040Ile
Ser Asn Leu Gly Asn Ile His Ala Ser Asn Lys Ile Met Phe Lys 1045
1050 1055 Leu Asp Gly Cys Arg Asp Pro Arg Arg Tyr Ile Met Ile Lys
Tyr Phe 1060 1065 1070 Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu
Ile Lys Asp Leu Tyr 1075 1080 1085 Asp Ser Gln Ser Asn Ser Gly Ile
Leu Lys Asp Phe Trp Gly Asn Tyr 1090 1095 1100 Leu Gln Tyr Asp Lys
Pro Tyr Tyr Met Leu Asn Leu Phe Asp Pro Asn1105 1110 1115 1120Lys
Tyr Val Asp Val Asn Asn Ile Gly Ile Arg Gly Tyr Met Tyr Leu 1125
1130 1135 Lys Gly Pro Arg Gly Ser Val Val Thr Thr Asn Ile Tyr Leu
Asn Ser 1140 1145 1150 Thr Leu Tyr Glu Gly Thr Lys Phe Ile Ile Lys
Lys Tyr Ala Ser Gly 1155 1160 1165 Asn Glu Asp Asn Ile Val Arg Asn
Asn Asp Arg Val Tyr Ile Asn Val 1170 1175 1180 Val Val Lys Asn Lys
Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala1185 1190 1195 1200Gly
Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn 1205
1210 1215 Leu Ser Gln Val Val Val Met Lys Ser Lys Asp Asp Gln Gly
Ile Arg 1220 1225 1230 Asn Lys Cys Lys Met Asn Leu Gln Asp Asn Asn
Gly Asn Asp Ile Gly 1235 1240 1245 Phe Ile Gly Phe His Leu Tyr Asp
Asn Ile Ala Lys Leu Val Ala Ser 1250 1255 1260 Asn Trp Tyr Asn Arg
Gln Val Gly Lys Ala Ser Arg Thr Phe Gly Cys1265 1270 1275 1280Ser
Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Ser Ser Leu 1285
1290 1295 31292PRTClostridium botulinum 3Met Pro Phe Val Asn Lys
Pro Phe Asn Tyr Arg Asp Pro Gly Asn Gly1 5 10 15 Val Asp Ile Ala
Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30 Val Lys
Ala Phe Lys Ile His Glu Gly Val Trp Val Ile Pro Glu Arg 35 40 45
Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50
55 60 Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser
Thr65 70 75 80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Ile Lys
Leu Phe Asp 85 90 95 Arg Ile Tyr Ser Thr Gly Leu Gly Arg Met Leu
Leu Ser Phe Ile Val 100 105 110 Lys Gly Ile Pro Phe Trp Gly Gly Ser
Thr Ile Asp Thr Glu Leu Lys 115 120 125 Val Ile Asp Thr Asn Cys Ile
Asn Val Ile Glu Pro Gly Gly Ser Tyr 130 135 140 Arg Ser Glu Glu Leu
Asn Leu Val Ile Thr Gly Pro Ser Ala Asp Ile145 150 155 160 Ile Gln
Phe Glu Cys Lys Ser Phe Gly His Asp Val Phe Asn Leu Thr 165 170 175
Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180
185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu
Leu 195 200 205 Gly Ala Gly Thr Phe Ala Thr Asp Pro Ala Val Thr Leu
Ala His Glu 210 215 220 Leu Ile His Ala Ala His Arg Leu Tyr Gly Ile
Ala Ile Asn Pro Asn225 230 235 240 Arg Val Leu Lys Val Lys Thr Asn
Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255 Glu Val Ser Phe Glu Glu
Leu Arg Thr Phe Gly Gly Asn Asp Thr Asn 260 265 270 Phe Ile Asp Ser
Leu Trp Gln Lys Lys Phe Ser Arg Asp Ala Tyr Asp 275 280 285 Asn Leu
Gln Asn Ile Ala Arg Ile Leu Asn Glu Ala Lys Thr Ile Val 290 295 300
Gly Thr Thr Thr Pro Leu Gln Tyr Met Lys Asn Ile Phe Ile Arg Lys305
310 315 320 Tyr Phe Leu Ser Glu Asp Ala Ser Gly Lys Ile Ser Val Asn
Lys Ala 325 330 335 Ala Phe Lys Glu Phe Tyr Arg Val Leu Thr Arg Gly
Phe Thr Glu Leu 340 345 350 Glu Phe Val Asn Pro Phe Lys Val Ile Asn
Arg Lys Thr Tyr Leu Asn 355 360 365 Phe Asp Lys Ala Val Phe Arg Ile
Asn Ile Val Pro Asp Glu Asn Tyr 370 375 380 Thr Ile Asn Glu Gly Phe
Asn Leu Glu Gly Ala Asn Ser Asn Gly Gln385 390 395 400 Asn Thr Glu
Ile Asn Ser Arg Asn Phe Thr Arg Leu Lys Asn Phe Thr 405 410 415 Gly
Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg Gly Ile Ile Pro 420 425
430 Phe Lys Thr Lys Ser Leu Asp Glu Gly Tyr Asn Lys Ala Leu Asn Tyr
435 440 445 Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser Pro
Ser Glu 450 455 460 Asp Asn Phe Thr Asn Asp Leu Asp Lys Val Glu Glu
Ile Thr Ala Asp465 470 475 480 Thr Asn Ile Glu Ala Ala Glu Glu Asn
Ile Ser Ser Asp Leu Ile Gln 485 490 495 Gln Tyr Tyr Leu Thr Phe Asp
Phe Asp Asn Glu Pro Glu Asn Ile Ser 500 505 510 Ile Glu Asn Leu Ser
Ser Asp Ile Ile Gly Gln Leu Glu Pro Met Pro 515 520 525 Asn Ile Glu
Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys Tyr 530 535 540 Thr
Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu His Gly Asp Ser545 550
555 560 Arg Ile Ile Leu Thr Asn Ser Ala Glu Glu Ala Leu Leu Lys Pro
Asn 565 570 575 Val Ala Tyr Thr Phe Phe Ser Ser Lys Tyr Val Lys Lys
Ile Asn Lys 580 585 590 Ala Val Glu Ala Val Ile Phe Leu Ser Trp Ala
Glu Glu Leu Val Tyr 595 600 605 Asp Phe Thr Asp Glu Thr Asn Glu Val
Thr Thr Met Asp Lys Ile Ala 610 615 620 Asp Ile Thr Ile Ile Val Pro
Tyr Ile Gly Pro Ala Leu Asn Ile Gly625 630 635 640 Asn Met Val Ser
Lys Gly Glu Phe Val Glu Ala Ile Leu Phe Thr Gly 645 650 655 Val Val
Ala Leu Leu Glu Phe Ile Pro Glu Tyr Ser Leu Pro Val Phe 660 665 670
Gly Thr Phe Ala Ile Val Ser Tyr Ile Ala Asn Lys Val Leu Thr Val 675
680 685 Gln Thr Ile Asn Asn Ala Leu Ser Lys Arg Asn Glu Lys Trp Asp
Glu 690 695 700 Val Tyr Lys Tyr Thr Val Thr Asn Trp Leu Ala Lys Val
Asn Thr Gln705 710 715 720 Ile Asp Leu Ile Arg Glu Lys Met Lys Lys
Ala Leu Glu Asn Gln Ala 725 730 735 Glu Ala Thr Arg Ala Ile Ile Asn
Tyr Gln Tyr Asn Gln Tyr Thr Glu 740 745 750 Glu Glu Lys Asn Asn Ile
Asn Phe Asn Ile Asp Asp Leu Ser Ser Lys 755 760 765 Leu Asn Arg Ser
Ile Asn Arg Ala Met Ile Asn Ile Asn Lys Phe Leu 770 775 780 Asp Gln
Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile Pro Tyr Ala785 790 795
800 Val Lys Arg Leu Lys Asp Phe Asp Ala Ser Val Arg Asp Val Leu Leu
805 810 815 Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Leu Gln Val
Asp Arg 820 825 830 Leu Lys Asp Glu Val Asn Asn Thr Leu Ser Ala Asp
Ile Pro Phe Gln 835 840 845 Leu Ser Lys Tyr Val Asn Asp Lys Lys Leu
Leu Ser Thr Phe Thr Glu 850 855 860 Tyr Ile Lys Asn Ile Val Asn Thr
Ser Ile Leu Ser Ile Val Tyr Lys865 870 875 880 Lys Asp Asp Leu Ile
Asp Leu Ser Arg Tyr Gly Ala Lys Ile Asn Ile 885 890 895 Gly Asp Arg
Val Tyr Tyr Asp Ser Ile Asp Lys Asn Gln Ile Lys Leu 900 905 910 Ile
Asn Leu Glu Ser Ser Thr Ile Glu Val Ile Leu Lys Asn Ala Ile 915 920
925 Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser Phe Trp Ile Lys
930 935 940 Ile Pro Lys Tyr Phe Ser Lys Ile Asn Leu Asn Asn Glu Tyr
Thr Ile945 950 955 960 Ile Asn Cys Ile Glu Asn Asn Ser Gly Trp Lys
Val Ser Leu Asn Tyr 965 970 975 Gly Glu Ile Ile Trp Thr Leu Gln Asp
Asn Lys Gln Asn Ile Gln Arg 980 985 990 Val Val Phe Lys Tyr Ser Gln
Met Val Asn Ile Ser Asp Tyr Ile Asn 995 1000 1005 Arg Trp Met Phe
Val Thr Ile Thr Asn Asn Arg Leu Thr Lys Ser Lys 1010 1015 1020 Ile
Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro Ile Ser Asn Leu1025
1030 1035 1040Gly Asn Ile His Ala Ser Asn Lys Ile Met Phe Lys Leu
Asp Gly Cys 1045 1050 1055 Arg Asp Pro Arg Arg Tyr Ile Met Ile Lys
Tyr Phe Asn Leu Phe Asp 1060 1065 1070 Lys Glu Leu Asn Glu Lys Glu
Ile Lys Asp Leu Tyr Asp Ser Gln Ser 1075 1080 1085 Asn Pro Gly Ile
Leu Lys Asp Phe Trp Gly Asn Tyr Leu Gln Tyr Asp 1090 1095 1100 Lys
Pro Tyr Tyr Met Leu Asn Leu Phe Asp Pro Asn Lys Tyr Val Asp1105
1110 1115 1120Val Asn Asn Ile Gly Ile Arg Gly Tyr Met Tyr Leu Lys
Gly Pro Arg 1125 1130 1135 Gly Ser Val Met Thr Thr Asn Ile Tyr Leu
Asn Ser Thr Leu Tyr Met 1140 1145 1150 Gly Thr Lys Phe Ile Ile Lys
Lys Tyr Ala Ser Gly Asn Glu Asp Asn 1155 1160 1165 Ile Val Arg Asn
Asn Asp Arg Val Tyr Ile Asn Val Val Val Lys Asn 1170 1175 1180 Lys
Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly Val Glu Lys1185
1190 1195 1200Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu
Ser Gln Val 1205 1210 1215 Val Val Met Lys Ser Lys Asp Asp Gln Gly
Ile Arg Asn Lys Cys Lys 1220 1225 1230 Met Asn Leu Gln Asp Asn Asn
Gly Asn Asp Ile Gly Phe Val Gly Phe 1235 1240 1245 His Leu Tyr Asp
Asn Ile Ala Lys Leu Val Ala Ser Asn Trp Tyr Asn 1250 1255 1260 Arg
Gln Val Gly Lys Ala Ser Arg Thr Phe Gly Cys Ser Trp Glu Phe1265
1270 1275 1280Ile Pro Val Asp Asp Gly Trp Gly Glu Ser Ser Leu 1285
1290 41296PRTClostridium botulinum 4Met Pro Leu Val Asn Gln Gln Ile
Asn Tyr Tyr Asp Pro Val Asn Gly1 5 10 15 Val Asp Ile Ala Tyr Ile
Lys Ile Pro Asn Ala Gly Lys Met Gln Pro 20 25 30 Val Lys Ala Phe
Lys Ile His Asn Lys Val Trp Val Ile Pro Glu Arg 35 40 45 Asp Ile
Phe Thr Asn Pro Glu Glu Val Asp Leu Asn Pro Pro Pro Glu 50 55 60
Ala Lys Gln Val Pro Ile Ser Tyr Tyr Asp Ser Ala Tyr Leu Ser Thr65
70 75 80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Ile Lys Leu
Phe Glu 85 90 95 Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu
Ile Ser Ile Val 100 105 110 Arg Gly Ile Pro Phe Trp Gly Gly Gly Lys
Ile Asp Thr Glu Leu Lys 115 120 125 Val Ile Asp Thr Asn Cys Ile Asn
Ile Ile Gln Leu Asp Asp Ser Tyr 130 135 140 Arg Ser Glu Glu Leu Asn
Leu Ala Ile Ile Gly Pro Ser Ala Asn Ile145 150 155 160 Ile Glu Ser
Gln Cys Ser Ser Phe Arg Asp Asp Val Leu Asn Leu Thr 165 170 175 Arg
Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185
190 Thr Val Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu
195 200 205 Gly Ala Gly Lys Phe Ala Gln Asp Pro Ala Val Ala Leu Ala
His Glu 210 215 220 Leu Ile His Ala Glu His Arg Leu Tyr Gly Ile Ala
Ile Asn Thr Asn225 230 235 240 Arg Val Phe Lys Val Asn Thr Asn Ala
Tyr Tyr Glu Met Ala Gly Leu 245 250 255 Glu Val Ser Leu Glu Glu Leu
Ile Thr Phe Gly Gly Asn Asp Ala Lys 260 265 270 Phe Ile Asp Ser Leu
Gln Lys Lys Glu Phe Ser Leu Tyr Tyr Tyr Asn 275 280 285 Lys Phe Lys
Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300 Gly
Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310
315 320 Tyr Leu Leu Ser Glu Asp Ala Thr Gly Lys Phe Leu Val Asp Arg
Leu 325 330 335 Lys Phe Asp Glu Leu Tyr Lys Leu Leu Thr Glu Ile Tyr
Thr Glu Asp 340 345 350 Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg
Lys Thr Tyr Leu Asn 355 360 365 Phe Asp Lys Ala Val Phe Lys Ile Asn
Ile Val Pro Asp Val Asn Tyr 370 375 380 Thr Ile His Asp Gly Phe Asn
Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400 Phe Asn Gly Gln
Asn Ile Glu Ile Asn Asn Lys Asn Phe Asp Lys Leu 405 410 415 Lys Asn
Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430
Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Glu Gly Tyr Asn Lys 435
440 445 Ala Leu Asn Glu Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe
Phe 450 455 460 Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asp Lys
Val Glu Glu465 470 475 480 Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala
Glu Glu Asn Ile Ser Leu 485 490 495 Asp Leu Ile Gln Gln Tyr Tyr Leu
Asn Phe Asn Phe Asp Asn Glu Pro 500 505 510 Glu Asn Thr Ser Ile Glu
Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525 Glu Pro Met Pro
Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540 Leu Asn
Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Lys545 550 555
560 His Ser Asn Ser Arg Ile Ile Leu Thr Asn Ser Ala Lys Glu Ala Leu
565 570 575 Leu Lys Pro Asn Ile Val Tyr Thr Phe Phe Ser Ser Lys Tyr
Ile Lys 580 585 590 Ala Ile Asn Lys Ala Val Glu Ala Val Thr Phe Val
Asn Trp Ile Glu 595 600 605 Asn Leu Val Tyr Asp Phe Thr Asp Glu Thr
Asn Glu Val Ser Thr Met 610 615 620 Asp Lys Ile Ala Asp Ile Thr Ile
Val Ile Pro Tyr Ile Gly Pro Ala625 630 635 640 Leu Asn Ile Gly Asn
Met Ile Tyr Lys Gly Glu Phe Val Glu Ala Ile 645 650 655 Ile Phe Ser
Gly Ala Val Ile Leu Leu Glu Ile Val Pro Glu Ile Ala 660 665 670 Leu
Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Val Ser Asn Lys 675 680
685 Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu
690 695 700 Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu
Ala Ile705 710 715 720 Val Asn Thr Gln Ile Asn Leu Ile Arg Glu Lys
Met Lys Lys Ala Leu 725
730 735 Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr
Asn 740 745 750 Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn
Ile Asp Asp 755 760 765 Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Ser
Ala Met Ile Asn Ile 770 775 780 Asn Lys Phe Leu Asp Gln Cys Ser Val
Ser Tyr Leu Met Asn Ser Met785 790 795 800 Ile Pro Tyr Ala Val Lys
Arg Leu Lys Asp Phe Asp Ala Ser Val Arg 805 810 815 Asp Val Leu Leu
Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly 820 825 830 Gln Val
Asn Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Ala Asp 835 840 845
Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Lys Lys Leu Leu Ser 850
855 860 Thr Phe Thr Glu Tyr Ile Lys Asn Ile Thr Asn Ala Ser Ile Leu
Ser865 870 875 880 Ile Val Tyr Lys Asp Asp Asp Leu Ile Asp Leu Ser
Arg Tyr Gly Ala 885 890 895 Glu Ile Tyr Asn Gly Asp Lys Val Tyr Tyr
Asn Ser Ile Asp Lys Asn 900 905 910 Gln Ile Arg Leu Ile Asn Leu Glu
Ser Ser Thr Ile Glu Val Ile Leu 915 920 925 Lys Lys Ala Ile Val Tyr
Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser 930 935 940 Phe Trp Ile Arg
Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn945 950 955 960 Glu
Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val 965 970
975 Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Phe Gln Asp Thr Gln Glu
980 985 990 Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn
Ile Ser 995 1000 1005 Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile
Thr Asn Asn Arg Ile 1010 1015 1020 Thr Lys Ser Lys Ile Tyr Ile Asn
Gly Arg Leu Ile Asp Gln Lys Pro1025 1030 1035 1040Ile Ser Asn Leu
Gly Asn Ile His Ala Ser Asn Lys Ile Met Phe Lys 1045 1050 1055 Leu
Asp Gly Cys Arg Asp Pro His Arg Tyr Ile Val Ile Lys Tyr Phe 1060
1065 1070 Asn Leu Phe Asp Lys Glu Leu Ser Glu Lys Glu Ile Lys Asp
Leu Tyr 1075 1080 1085 Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys Asp
Phe Trp Gly Asp Tyr 1090 1095 1100 Leu Gln Tyr Asp Lys Ser Tyr Tyr
Met Leu Asn Leu Tyr Asp Pro Asn1105 1110 1115 1120Lys Tyr Val Asp
Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu 1125 1130 1135 Lys
Gly Pro Arg Asp Asn Val Met Thr Thr Asn Ile Tyr Leu Asn Ser 1140
1145 1150 Ser Leu Tyr Met Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala
Ser Gly 1155 1160 1165 Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg
Val Tyr Ile Asn Val 1170 1175 1180 Val Val Lys Asn Lys Glu Tyr Arg
Leu Ala Thr Asn Ala Ser Gln Ala1185 1190 1195 1200Gly Val Glu Lys
Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn 1205 1210 1215 Leu
Ser Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr 1220
1225 1230 Asn Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp
Ile Gly 1235 1240 1245 Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala
Lys Leu Val Ala Ser 1250 1255 1260 Asn Trp Tyr Asn Arg Gln Ile Glu
Arg Ser Ser Arg Thr Leu Gly Cys1265 1270 1275 1280Ser Trp Glu Phe
Ile Pro Val Asp Asp Gly Trp Arg Glu Arg Pro Leu 1285 1290 1295
5206PRTHomo sapiens 5Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu
Glu Glu Met Gln Arg1 5 10 15 Arg Ala Asp Gln Leu Ala Asp Glu Ser
Leu Glu Ser Thr Arg Arg Met 20 25 30 Leu Gln Leu Val Glu Glu Ser
Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45 Met Leu Asp Glu Gln
Gly Glu Gln Leu Asp Arg Val Glu Glu Gly Met 50 55 60 Asn His Ile
Asn Gln Asp Met Lys Glu Ala Glu Lys Asn Leu Lys Asp65 70 75 80 Leu
Gly Lys Cys Cys Gly Leu Phe Ile Cys Pro Cys Asn Lys Leu Lys 85 90
95 Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val
100 105 110 Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln
Met Ala 115 120 125 Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp
Ala Arg Glu Asn 130 135 140 Glu Met Asp Glu Asn Leu Glu Gln Val Ser
Gly Ile Ile Gly Asn Leu145 150 155 160 Arg His Met Ala Leu Asp Met
Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175 Gln Ile Asp Arg Ile
Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile 180 185 190 Asp Glu Ala
Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200 205 6206PRTHomo
sapiens 6Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met
Gln Arg1 5 10 15 Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser
Thr Arg Arg Met 20 25 30 Leu Gln Leu Val Glu Glu Ser Lys Asp Ala
Gly Ile Arg Thr Leu Val 35 40 45 Met Leu Asp Glu Gln Gly Glu Gln
Leu Glu Arg Ile Glu Glu Gly Met 50 55 60 Asp Gln Ile Asn Lys Asp
Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65 70 75 80 Leu Gly Lys Phe
Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys 85 90 95 Ser Ser
Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val 100 105 110
Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala 115
120 125 Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu
Asn 130 135 140 Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile
Gly Asn Leu145 150 155 160 Arg His Met Ala Leu Asp Met Gly Asn Glu
Ile Asp Thr Gln Asn Arg 165 170 175 Gln Ile Asp Arg Ile Met Glu Lys
Ala Asp Ser Asn Lys Thr Arg Ile 180 185 190 Asp Glu Ala Asn Gln Arg
Ala Thr Lys Met Leu Gly Ser Gly 195 200 205 7206PRTMacaca mulatta
7Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg1 5
10 15 Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg
Met 20 25 30 Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg
Thr Leu Val 35 40 45 Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg
Ile Glu Glu Gly Met 50 55 60 Asp Gln Ile Asn Lys Asp Met Lys Glu
Ala Glu Lys Asn Leu Thr Asp65 70 75 80 Leu Gly Lys Phe Cys Gly Leu
Cys Val Cys Pro Cys Asn Lys Leu Lys 85 90 95 Ser Ser Asp Ala Tyr
Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val 100 105 110 Val Ala Ser
Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala 115 120 125 Ile
Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn 130 135
140 Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile Gly Asn
Leu145 150 155 160 Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp
Thr Gln Asn Arg 165 170 175 Gln Ile Asp Arg Ile Met Glu Lys Ala Asp
Ser Asn Lys Thr Arg Ile 180 185 190 Asp Glu Ala Asn Gln Arg Ala Thr
Lys Met Leu Gly Ser Gly 195 200 205 8206PRTRattus norvegicus 8Met
Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg1 5 10
15 Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met
20 25 30 Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr
Leu Val 35 40 45 Met Leu Asp Glu Gln Gly Glu Gln Leu Asp Arg Val
Glu Glu Gly Met 50 55 60 Asn His Ile Asn Gln Asp Met Lys Glu Ala
Glu Lys Asn Leu Lys Asp65 70 75 80 Leu Gly Lys Cys Cys Gly Leu Phe
Ile Cys Pro Cys Asn Lys Leu Lys 85 90 95 Ser Ser Asp Ala Tyr Lys
Lys Ala Trp Gly Asn Asn Gln Asp Gly Val 100 105 110 Val Ala Ser Gln
Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala 115 120 125 Ile Ser
Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn 130 135 140
Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu145
150 155 160 Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln
Asn Arg 165 170 175 Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn
Lys Thr Arg Ile 180 185 190 Asp Glu Ala Asn Gln Arg Ala Thr Lys Met
Leu Gly Ser Gly 195 200 205 9206PRTRattus norvegicus 9Met Ala Glu
Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg1 5 10 15 Arg
Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25
30 Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val
35 40 45 Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu
Gly Met 50 55 60 Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys
Asn Leu Thr Asp65 70 75 80 Leu Gly Lys Phe Cys Gly Leu Cys Val Cys
Pro Cys Asn Lys Leu Lys 85 90 95 Ser Ser Asp Ala Tyr Lys Lys Ala
Trp Gly Asn Asn Gln Asp Gly Val 100 105 110 Val Ala Ser Gln Pro Ala
Arg Val Val Asp Glu Arg Glu Gln Met Ala 115 120 125 Ile Ser Gly Gly
Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn 130 135 140 Glu Met
Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu145 150 155
160 Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg
165 170 175 Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr
Arg Ile 180 185 190 Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly
Ser Gly 195 200 205 10206PRTMus musculus 10Met Ala Glu Asp Ala Asp
Met Arg Asn Glu Leu Glu Glu Met Gln Arg1 5 10 15 Arg Ala Asp Gln
Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30 Leu Gln
Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45
Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50
55 60 Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr
Asp65 70 75 80 Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn
Lys Leu Lys 85 90 95 Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn
Asn Gln Asp Gly Val 100 105 110 Val Ala Ser Gln Pro Ala Arg Val Val
Asp Glu Arg Glu Gln Met Ala 115 120 125 Ile Ser Gly Gly Phe Ile Arg
Arg Val Thr Asn Asp Ala Arg Glu Asn 130 135 140 Glu Met Asp Glu Asn
Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu145 150 155 160 Arg His
Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175
Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile 180
185 190 Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195
200 205 11206PRTGallus gallus 11Met Ala Glu Asp Ala Asp Met Arg Asn
Glu Leu Glu Glu Met Gln Arg1 5 10 15 Arg Ala Asp Gln Leu Ala Asp
Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30 Leu Gln Leu Val Glu
Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45 Met Leu Asp
Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60 Asp
Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65 70 75
80 Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys
85 90 95 Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp
Gly Val 100 105 110 Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg
Glu Gln Met Ala 115 120 125 Ile Ser Gly Gly Phe Ile Arg Arg Val Thr
Asn Asp Ala Arg Glu Asn 130 135 140 Glu Met Asp Glu Asn Leu Glu Gln
Val Ser Gly Ile Ile Gly Asn Leu145 150 155 160 Arg His Met Ala Leu
Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175 Gln Ile Asp
Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile 180 185 190 Asp
Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200 205
12204PRTCarassius auratus 12Met Ala Glu Asp Ala Asp Met Arg Asn Glu
Leu Ser Asp Met Gln Gln1 5 10 15 Arg Ala Asp Gln Leu Ala Asp Glu
Ser Leu Glu Ser Thr Arg Arg Met 20 25 30 Leu Gln Leu Val Glu Glu
Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45 Met Leu Asp Glu
Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60 Asp Gln
Ile Asn Lys Asp Met Lys Asp Ala Glu Lys Asn Leu Asn Asp65 70 75 80
Leu Gly Lys Phe Cys Gly Leu Cys Ser Cys Pro Cys Asn Lys Met Lys 85
90 95 Ser Gly Gly Ser Lys Ala Trp Gly Asn Asn Gln Asp Gly Val Val
Ala 100 105 110 Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met
Ala Ile Ser 115 120 125 Gly Gly Phe Ile Arg Arg Val Thr Asp Asp Ala
Arg Glu Asn Glu Met 130 135 140 Asp Glu Asn Leu Glu Gln Val Gly Gly
Ile Ile Gly Asn Leu Arg His145 150 155 160 Met Ala Leu Asp Met Gly
Asn Glu Ile Asp Thr Gln Asn Arg Gln Ile 165 170 175 Asp Arg Ile Met
Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile Asp Glu 180 185 190 Ala Asn
Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200 13203PRTCarassius
auratus 13Met Ala Asp Glu Ala Asp Met Arg Asn Glu Leu Thr Asp Met
Gln Ala1 5 10 15 Arg Ala Asp Gln Leu Gly Asp Glu Ser Leu Glu Ser
Thr Arg Arg Met 20 25 30 Leu Gln Leu Val Glu Glu Ser Lys Asp Ala
Gly Ile Arg Thr Leu Val 35 40 45 Met Leu Asp Glu Gln Gly Glu Gln
Leu Glu Arg Ile Glu Glu Gly Met 50 55 60 Asp Gln Ile Asn Lys Asp
Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65 70
75 80 Leu Gly Asn Leu Cys Gly Leu Cys Pro Cys Pro Cys Asn Lys Leu
Lys 85 90 95 Gly Gly Gly Gln Ser Trp Gly Asn Asn Gln Asp Gly Val
Val Ser Ser 100 105 110 Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln
Met Ala Ile Ser Gly 115 120 125 Gly Phe Ile Arg Arg Val Thr Asn Asp
Ala Arg Glu Asn Glu Met Asp 130 135 140 Glu Asn Leu Glu Gln Val Gly
Ser Ile Ile Gly Asn Leu Arg His Met145 150 155 160 Ala Leu Asp Met
Gly Asn Glu Ile Asp Thr Gln Asn Arg Gln Ile Asp 165 170 175 Arg Ile
Met Asp Met Ala Asp Ser Asn Lys Thr Arg Ile Asp Glu Ala 180 185 190
Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200 14204PRTDanio
rerio 14Met Ala Glu Asp Ser Asp Met Arg Asn Glu Leu Ala Asp Met Gln
Gln1 5 10 15 Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr
Arg Arg Met 20 25 30 Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly
Ile Arg Thr Leu Val 35 40 45 Met Leu Asp Glu Gln Gly Glu Gln Leu
Glu Arg Ile Glu Glu Gly Met 50 55 60 Asp Gln Ile Asn Lys Asp Met
Lys Asp Ala Glu Lys Asn Leu Asn Asp65 70 75 80 Leu Gly Lys Phe Cys
Gly Leu Cys Ser Cys Pro Cys Asn Lys Met Lys 85 90 95 Ser Gly Ala
Ser Lys Ala Trp Gly Asn Asn Gln Asp Gly Val Val Ala 100 105 110 Ser
Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala Ile Ser 115 120
125 Gly Gly Phe Ile Arg Arg Val Thr Asp Asp Ala Arg Glu Asn Glu Met
130 135 140 Asp Glu Asn Leu Glu Gln Val Gly Gly Ile Ile Gly Asn Leu
Arg His145 150 155 160 Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr
Gln Asn Arg Gln Ile 165 170 175 Asp Arg Ile Met Glu Lys Ala Asp Ser
Asn Lys Thr Arg Ile Asp Glu 180 185 190 Ala Asn Gln Arg Ala Thr Lys
Met Leu Gly Ser Gly 195 200 15203PRTDanio rerio 15Met Ala Asp Glu
Ser Asp Met Arg Asn Glu Leu Asn Asp Met Gln Ala1 5 10 15 Arg Ala
Asp Gln Leu Gly Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30
Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35
40 45 Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly
Met 50 55 60 Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn
Leu Thr Asp65 70 75 80 Leu Gly Asn Leu Cys Gly Leu Cys Pro Cys Pro
Cys Asn Lys Leu Lys 85 90 95 Gly Gly Gly Gln Ser Trp Gly Asn Asn
Gln Asp Gly Val Val Ser Ser 100 105 110 Gln Pro Ala Arg Val Val Asp
Glu Arg Glu Gln Met Ala Ile Ser Gly 115 120 125 Gly Phe Ile Arg Arg
Val Thr Asn Asp Ala Arg Glu Asn Glu Met Asp 130 135 140 Glu Asn Leu
Glu Gln Val Gly Ser Ile Ile Gly Asn Leu Arg His Met145 150 155 160
Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg Gln Ile Asp 165
170 175 Arg Ile Met Asp Met Ala Asp Ser Asn Lys Thr Arg Ile Asp Glu
Ala 180 185 190 Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200
16210PRTTorpedo marmorata 16Met Glu Asn Ser Val Glu Asn Ser Met Asp
Pro Arg Ser Glu Gln Glu1 5 10 15 Glu Met Gln Arg Cys Ala Asp Gln
Ile Thr Asp Glu Ser Leu Glu Ser 20 25 30 Thr Arg Arg Met Leu Gln
Leu Val Glu Glu Ser Lys Asp Ala Gly Ile 35 40 45 Arg Thr Leu Val
Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile 50 55 60 Glu Glu
Gly Met Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys65 70 75 80
Asn Leu Ser Asp Leu Gly Lys Cys Cys Gly Leu Cys Ser Cys Pro Cys 85
90 95 Asn Lys Leu Lys Asn Phe Glu Ala Gly Gly Ala Tyr Lys Lys Val
Trp 100 105 110 Gly Asn Asn Gln Asp Gly Val Val Ala Ser Gln Pro Ala
Arg Val Met 115 120 125 Asp Asp Arg Glu Gln Met Ala Met Ser Gly Gly
Tyr Ile Arg Arg Ile 130 135 140 Thr Asp Asp Ala Arg Glu Asn Glu Met
Glu Glu Asn Leu Asp Gln Val145 150 155 160 Gly Ser Ile Ile Gly Asn
Leu Arg His Met Ala Leu Asp Met Ser Asn 165 170 175 Glu Ile Gly Ser
Gln Asn Ala Gln Ile Asp Arg Ile Val Val Lys Gly 180 185 190 Asp Met
Asn Lys Ala Arg Ile Asp Glu Ala Asn Lys His Ala Thr Lys 195 200 205
Met Leu 210 17206PRTXenopus laevis 17Met Ala Asp Asp Ala Asp Met
Arg Asn Glu Leu Glu Glu Met Gln Arg1 5 10 15 Arg Ala Asp Gln Leu
Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30 Leu Gln Tyr
Val Glu Gly Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45 Met
Leu Asp Glu Gln Gly Glu Gln Leu Asp Arg Val Glu Glu Gly Met 50 55
60 Asn His Ile Asn Gln Asp Met Lys Glu Ala Glu Lys Asn Leu Lys
Asp65 70 75 80 Leu Gly Lys Cys Cys Gly Leu Phe Ile Cys Pro Cys Asn
Lys Leu Lys 85 90 95 Ser Ser Gly Ala Tyr Asn Lys Ala Trp Gly Asn
Asn Gln Asp Gly Val 100 105 110 Val Ala Ser Gln Pro Ala Arg Val Val
Asp Glu Arg Glu Gln Met Ala 115 120 125 Ile Ser Gly Gly Phe Val Arg
Arg Val Thr Asn Asp Ala Arg Glu Thr 130 135 140 Glu Met Asp Glu Asn
Leu Glu Gln Val Gly Gly Ile Ile Gly Asn Leu145 150 155 160 Arg His
Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175
Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Ala Arg Ile 180
185 190 Asp Glu Ala Asn Lys His Ala Thr Lys Met Leu Gly Ser Gly 195
200 205 18206PRTXenopus laevis 18Met Ala Asp Asp Ala Asp Met Arg
Asn Glu Leu Glu Glu Met Gln Arg1 5 10 15 Arg Ala Asp Gln Leu Ala
Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30 Leu Gln Tyr Val
Glu Gly Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45 Met Leu
Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60
Glu Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65
70 75 80 Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys
Leu Lys 85 90 95 Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn
Gln Asp Gly Val 100 105 110 Val Ala Ser Gln Pro Ala Arg Val Val Asp
Glu Arg Glu Gln Met Ala 115 120 125 Ile Ser Gly Gly Phe Val Arg Arg
Val Thr Asn Asp Ala Arg Glu Thr 130 135 140 Glu Met Asp Glu Asn Leu
Glu Gln Val Gly Gly Ile Ile Gly Asn Leu145 150 155 160 Arg His Met
Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175 Gln
Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Ala Arg Ile 180 185
190 Asp Glu Ala Asn Lys His Ala Thr Lys Met Leu Gly Ser Gly 195 200
205 19212PRTStrongylocentrotus purpuratus 19Met Glu Asp Gln Asn Asp
Met Asn Met Arg Ser Glu Leu Glu Glu Ile1 5 10 15 Gln Met Gln Ser
Asn Met Gln Thr Asp Glu Ser Leu Glu Ser Thr Arg 20 25 30 Arg Met
Leu Gln Met Ala Glu Glu Ser Gln Asp Met Gly Ile Lys Thr 35 40 45
Leu Val Met Leu Asp Glu Gln Gly Glu Gln Leu Asp Arg Ile Glu Glu 50
55 60 Gly Met Asp Gln Ile Asn Thr Asp Met Arg Glu Ala Glu Lys Asn
Leu65 70 75 80 Thr Gly Leu Glu Lys Cys Cys Gly Ile Cys Val Cys Pro
Trp Lys Lys 85 90 95 Leu Gly Asn Phe Glu Lys Gly Asp Asp Tyr Lys
Lys Thr Trp Lys Gly 100 105 110 Asn Asp Asp Gly Lys Val Asn Ser His
Gln Pro Met Arg Met Glu Asp 115 120 125 Asp Arg Asp Gly Cys Gly Gly
Asn Ala Ser Met Ile Thr Arg Ile Thr 130 135 140 Asn Asp Ala Arg Glu
Asp Glu Met Asp Glu Asn Leu Thr Gln Val Ser145 150 155 160 Ser Ile
Val Gly Asn Leu Arg His Met Ala Ile Asp Met Gln Ser Glu 165 170 175
Ile Gly Ala Gln Asn Ser Gln Val Gly Arg Ile Thr Ser Lys Ala Glu 180
185 190 Ser Asn Glu Gly Arg Ile Asn Ser Ala Asp Lys Arg Ala Lys Asn
Ile 195 200 205 Leu Arg Asn Lys 210 20212PRTDrosophila melanogaster
20Met Pro Ala Asp Pro Ser Glu Glu Val Ala Pro Gln Val Pro Lys Thr1
5 10 15 Glu Leu Glu Glu Leu Gln Ile Asn Ala Gln Gly Val Ala Asp Glu
Ser 20 25 30 Leu Glu Ser Thr Arg Arg Met Leu Ala Leu Cys Glu Glu
Ser Lys Glu 35 40 45 Ala Gly Ile Arg Thr Leu Val Ala Leu Asp Asp
Gln Gly Glu Gln Leu 50 55 60 Asp Arg Ile Glu Glu Gly Met Asp Gln
Ile Asn Ala Asp Met Arg Glu65 70 75 80 Ala Glu Lys Asn Leu Ser Gly
Met Glu Lys Cys Cys Gly Ile Cys Val 85 90 95 Leu Pro Cys Asn Lys
Ser Gln Ser Phe Lys Glu Asp Asp Gly Thr Trp 100 105 110 Lys Gly Asn
Asp Asp Gly Lys Val Val Asn Asn Gln Pro Gln Arg Val 115 120 125 Met
Asp Asp Arg Asn Gly Met Met Ala Gln Ala Gly Tyr Ile Gly Arg 130 135
140 Ile Thr Asn Asp Ala Arg Glu Asp Glu Met Glu Glu Asn Met Gly
Gln145 150 155 160 Val Asn Thr Met Ile Gly Asn Leu Arg Asn Met Ala
Leu Asp Met Gly 165 170 175 Ser Glu Leu Glu Asn Gln Asn Arg Gln Ile
Asp Arg Ile Asn Arg Lys 180 185 190 Gly Glu Ser Asn Glu Ala Arg Ile
Ala Val Ala Asn Gln Arg Ala His 195 200 205 Gln Leu Leu Lys 210
21212PRTHirudo medicinalis 21Met Ala Lys Asp Ile Lys Pro Lys Pro
Ala Asn Gly Arg Asp Ser Pro1 5 10 15 Thr Asp Leu Gln Glu Ile Gln
Leu Gln Met Asn Ala Ile Thr Asp Asp 20 25 30 Ser Leu Glu Ser Thr
Arg Arg Met Leu Ala Met Cys Glu Glu Ser Lys 35 40 45 Asp Ala Gly
Ile Arg Thr Leu Val Met Leu Asp Glu Gln Gly Glu Gln 50 55 60 Leu
Asp Arg Ile Glu Glu Gly Met Asp Gln Ile Asn Gln Asp Met Arg65 70 75
80 Asp Ala Glu Lys Asn Leu Glu Gly Met Glu Lys Cys Cys Gly Leu Cys
85 90 95 Ile Leu Pro Trp Lys Arg Thr Lys Asn Phe Asp Lys Gly Ala
Glu Trp 100 105 110 Asn Lys Gly Asp Glu Gly Lys Val Asn Thr Asp Gly
Pro Arg Leu Val 115 120 125 Val Gly Asp Gly Asn Met Gly Pro Ser Gly
Gly Phe Ile Thr Lys Ile 130 135 140 Thr Asn Asp Ala Arg Glu Glu Glu
Met Glu Gln Asn Met Gly Glu Val145 150 155 160 Ser Asn Met Ile Ser
Asn Leu Arg Asn Met Ala Val Asp Met Gly Ser 165 170 175 Glu Ile Asp
Ser Gln Asn Arg Gln Val Asp Arg Ile Asn Asn Lys Met 180 185 190 Thr
Ser Asn Gln Leu Arg Ile Ser Asp Ala Asn Lys Arg Ala Ser Lys 195 200
205 Leu Leu Lys Glu 210 22212PRTLoligo pealei 22Met Ser Ala Asn Gly
Glu Val Glu Val Pro Lys Thr Glu Leu Glu Glu1 5 10 15 Ile Gln Gln
Gln Cys Asn Gln Val Thr Asp Asp Ser Leu Glu Ser Thr 20 25 30 Arg
Arg Met Leu Asn Met Cys Glu Glu Ser Lys Glu Ala Gly Ile Arg 35 40
45 Thr Leu Val Met Leu Asp Glu Gln Gly Glu Gln Leu Asp Arg Ile Glu
50 55 60 Glu Gly Leu Asp Gln Ile Asn Gln Asp Met Lys Asp Ala Glu
Lys Asn65 70 75 80 Leu Glu Gly Met Glu Lys Cys Cys Gly Leu Cys Val
Leu Pro Trp Lys 85 90 95 Arg Gly Lys Ser Phe Glu Lys Ser Gly Asp
Tyr Ala Asn Thr Trp Lys 100 105 110 Lys Asp Asp Asp Gly Pro Thr Asn
Thr Asn Gly Pro Arg Val Thr Val 115 120 125 Gly Asp Gln Asn Gly Met
Gly Pro Ser Ser Gly Tyr Val Thr Arg Ile 130 135 140 Thr Asn Asp Ala
Arg Glu Asp Asp Met Glu Asn Asn Met Lys Glu Val145 150 155 160 Ser
Ser Met Ile Gly Asn Leu Arg Asn Met Ala Ile Asp Met Gly Asn 165 170
175 Glu Ile Gly Ser Gln Asn Arg Gln Val Asp Arg Ile Gln Gln Lys Ala
180 185 190 Glu Ser Asn Glu Ser Arg Ile Asp Glu Ala Asn Lys Lys Ala
Thr Lys 195 200 205 Leu Leu Lys Asn 210 23220PRTLymnaea stagnalis
23Met Thr Thr Asn Gly Glu Ile Leu Pro Val Gly Glu Glu Glu Glu Glu1
5 10 15 Glu Leu Gly Glu Asp Ala Leu Leu Arg Lys Gln Ile Asp Cys Asn
Thr 20 25 30 Asn Glu Ser Leu Glu Ser Thr Arg Arg Met Leu Ser Leu
Cys Glu Glu 35 40 45 Ser Lys Glu Ala Gly Ile Lys Thr Leu Val Met
Leu Asp Glu Gln Gly 50 55 60 Glu Gln Leu Asp Arg Ile Glu Glu Gly
Met Gly Gln Ile Asn Gln Asp65 70 75 80 Met Arg Asp Ala Glu Lys Asn
Leu Glu Gly Leu Glu Lys Cys Cys Gly 85 90 95 Leu Cys Val Leu Pro
Trp Lys Arg Ser Lys Asn Phe Glu Lys Gly Ser 100 105 110 Asp Tyr Asn
Lys Thr Trp Lys Ala Ser Glu Asp Gly Lys Ile Asn Thr 115 120 125 Asn
Gly Pro Arg Leu Val Val Asp Gln Gly Asn Gly Ser Gly Pro Thr 130 135
140 Gly Gly Tyr Ile Thr Arg Ile Thr Asn Asp Ala Arg Glu Asp Glu
Met145 150 155 160 Glu Gln Asn Ile Gly Glu Val Ala Gly Met Val Ser
Asn Leu Arg Asn 165 170 175 Met Ala Val Asp Met Gly Asn Glu Ile Glu
Ser Gln Asn Lys Gln Leu 180 185 190 Asp Arg Ile Asn Gln Lys Gly Gly
Ser Leu Asn Val Arg Val Asp Glu 195 200 205 Ala Asn Lys Arg Ala Asn
Arg Ile Leu Arg Lys Gln 210 215 220 24207PRTCaenorhabditis elegans
24Met Ser Gly Asp Asp Asp Ile Pro Glu Gly Leu Glu Ala Ile Asn Leu1
5 10 15 Lys Met Asn Ala Thr Thr Asp Asp Ser Leu Glu Ser Thr Arg Arg
Met 20 25 30 Leu Ala Leu Cys Glu Glu Ser Lys Glu Ala Gly Ile Lys
Thr Leu Val 35 40 45 Met Leu Asp
Asp Gln Gly Glu Gln Leu Glu Arg Cys Glu Gly Ala Leu 50 55 60 Asp
Thr Ile Asn Gln Asp Met Lys Glu Ala Glu Asp His Leu Lys Gly65 70 75
80 Met Glu Lys Cys Cys Gly Leu Cys Val Leu Pro Trp Asn Lys Thr Asp
85 90 95 Asp Phe Glu Lys Thr Glu Phe Ala Lys Ala Trp Lys Lys Asp
Asp Asp 100 105 110 Gly Gly Val Ile Ser Asp Gln Pro Arg Ile Thr Val
Gly Asp Ser Ser 115 120 125 Met Gly Pro Gln Gly Gly Tyr Ile Thr Lys
Ile Thr Asn Asp Ala Arg 130 135 140 Glu Asp Glu Met Asp Glu Asn Val
Gln Gln Val Ser Thr Met Val Gly145 150 155 160 Asn Leu Arg Asn Met
Ala Ile Asp Met Ser Thr Glu Val Ser Asn Gln 165 170 175 Asn Arg Gln
Leu Asp Arg Ile His Asp Lys Ala Gln Ser Asn Glu Val 180 185 190 Arg
Val Glu Ser Ala Asn Lys Arg Ala Lys Asn Leu Ile Thr Lys 195 200 205
25808PRTHomo sapiens 25Met Gly Ala Pro Ala Cys Ala Leu Ala Leu Cys
Val Ala Val Ala Ile1 5 10 15 Val Ala Gly Ala Ser Ser Glu Ser Leu
Gly Thr Glu Gln Arg Val Val 20 25 30 Gly Arg Ala Ala Glu Val Pro
Gly Pro Glu Pro Gly Gln Gln Glu Gln 35 40 45 Leu Val Phe Gly Ser
Gly Asp Ala Val Glu Leu Ser Cys Pro Pro Pro 50 55 60 Gly Gly Gly
Pro Met Gly Pro Thr Val Trp Val Lys Asp Gly Thr Gly65 70 75 80 Leu
Val Pro Ser Glu Arg Val Leu Val Gly Pro Gln Arg Leu Gln Val 85 90
95 Leu Asn Ala Ser His Glu Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg
100 105 110 Leu Thr Gln Arg Val Leu Cys His Phe Ser Val Arg Val Thr
Asp Ala 115 120 125 Pro Ser Ser Gly Asp Asp Glu Asp Gly Glu Asp Glu
Ala Glu Asp Thr 130 135 140 Gly Val Asp Thr Gly Ala Pro Tyr Trp Thr
Arg Pro Glu Arg Met Asp145 150 155 160 Lys Lys Leu Leu Ala Val Pro
Ala Ala Asn Thr Val Arg Phe Arg Cys 165 170 175 Pro Ala Ala Gly Asn
Pro Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly 180 185 190 Arg Glu Phe
Arg Gly Glu His Arg Ile Gly Gly Ile Lys Leu Arg His 195 200 205 Gln
Gln Trp Ser Leu Val Met Glu Ser Val Val Pro Ser Asp Arg Gly 210 215
220 Asn Tyr Thr Cys Val Val Glu Asn Lys Phe Gly Ser Ile Arg Gln
Thr225 230 235 240 Tyr Thr Leu Asp Val Leu Glu Arg Ser Pro His Arg
Pro Ile Leu Gln 245 250 255 Ala Gly Leu Pro Ala Asn Gln Thr Ala Val
Leu Gly Ser Asp Val Glu 260 265 270 Phe His Cys Lys Val Tyr Ser Asp
Ala Gln Pro His Ile Gln Trp Leu 275 280 285 Lys His Val Glu Val Asn
Gly Ser Lys Val Gly Pro Asp Gly Thr Pro 290 295 300 Tyr Val Thr Val
Leu Lys Ser Trp Ile Ser Glu Ser Val Glu Ala Asp305 310 315 320 Val
Arg Leu Arg Leu Ala Asn Val Ser Glu Arg Asp Gly Gly Glu Tyr 325 330
335 Leu Cys Arg Ala Thr Asn Phe Ile Gly Val Ala Glu Lys Ala Phe Trp
340 345 350 Leu Ser Val His Gly Pro Arg Ala Ala Glu Glu Glu Leu Val
Glu Ala 355 360 365 Asp Glu Ala Gly Ser Val Tyr Ala Gly Ile Leu Ser
Tyr Gly Val Gly 370 375 380 Phe Phe Leu Phe Ile Leu Val Val Ala Ala
Val Thr Leu Cys Arg Leu385 390 395 400 Arg Ser Pro Pro Lys Lys Gly
Leu Gly Ser Pro Thr Val His Lys Ile 405 410 415 Ser Arg Phe Pro Leu
Lys Arg Gln Val Ser Leu Glu Ser Asn Ala Ser 420 425 430 Met Ser Ser
Asn Thr Pro Leu Val Arg Ile Ala Arg Leu Ser Ser Gly 435 440 445 Glu
Gly Pro Thr Leu Ala Asn Val Ser Glu Leu Glu Leu Pro Ala Asp 450 455
460 Pro Lys Trp Glu Leu Ser Arg Ala Arg Leu Thr Leu Gly Lys Pro
Leu465 470 475 480 Gly Glu Gly Cys Phe Gly Gln Val Val Met Ala Glu
Ala Ile Gly Ile 485 490 495 Asp Lys Asp Arg Ala Ala Lys Pro Val Thr
Val Ala Val Lys Met Leu 500 505 510 Lys Asp Asp Ala Thr Asp Lys Asp
Leu Ser Asp Leu Val Ser Glu Met 515 520 525 Glu Met Met Lys Met Ile
Gly Lys His Lys Asn Ile Ile Asn Leu Leu 530 535 540 Gly Ala Cys Thr
Gln Gly Gly Pro Leu Tyr Val Leu Val Glu Tyr Ala545 550 555 560 Ala
Lys Gly Asn Leu Arg Glu Phe Leu Arg Ala Arg Arg Pro Pro Gly 565 570
575 Leu Asp Tyr Ser Phe Asp Thr Cys Lys Pro Pro Glu Glu Gln Leu Thr
580 585 590 Phe Lys Asp Leu Val Ser Cys Ala Tyr Gln Val Ala Arg Gly
Met Glu 595 600 605 Tyr Leu Ala Ser Gln Lys Cys Ile His Arg Asp Leu
Ala Ala Arg Asn 610 615 620 Val Leu Val Thr Glu Asp Asn Val Met Lys
Ile Ala Asp Phe Gly Leu625 630 635 640 Ala Arg Asp Val His Asn Leu
Asp Tyr Tyr Lys Lys Thr Thr Asn Gly 645 650 655 Arg Leu Pro Val Lys
Trp Met Ala Pro Glu Ala Leu Phe Asp Arg Val 660 665 670 Tyr Thr His
Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu 675 680 685 Ile
Phe Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val Glu Glu 690 695
700 Leu Phe Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro Ala
Asn705 710 715 720 Cys Thr His Asp Leu Tyr Met Ile Met Arg Glu Cys
Trp His Ala Ala 725 730 735 Pro Ser Gln Arg Pro Thr Phe Lys Gln Leu
Val Glu Asp Leu Asp Arg 740 745 750 Val Leu Thr Val Thr Ser Thr Asp
Glu Tyr Leu Asp Leu Ser Ala Pro 755 760 765 Phe Glu Gln Tyr Ser Pro
Gly Gly Gln Asp Thr Pro Ser Ser Ser Ser 770 775 780 Ser Gly Asp Asp
Ser Val Phe Ala His Asp Leu Leu Pro Pro Ala Pro785 790 795 800 Pro
Ser Ser Gly Gly Ser Arg Thr 805 26806PRTHomo sapiens 26Met Gly Ala
Pro Ala Cys Ala Leu Ala Leu Cys Val Ala Val Ala Ile1 5 10 15 Val
Ala Gly Ala Ser Ser Glu Ser Leu Gly Thr Glu Gln Arg Val Val 20 25
30 Gly Arg Ala Ala Glu Val Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln
35 40 45 Leu Val Phe Gly Ser Gly Asp Ala Val Glu Leu Ser Cys Pro
Pro Pro 50 55 60 Gly Gly Gly Pro Met Gly Pro Thr Val Trp Val Lys
Asp Gly Thr Gly65 70 75 80 Leu Val Pro Ser Glu Arg Val Leu Val Gly
Pro Gln Arg Leu Gln Val 85 90 95 Leu Asn Ala Ser His Glu Asp Ser
Gly Ala Tyr Ser Cys Arg Gln Arg 100 105 110 Leu Thr Gln Arg Val Leu
Cys His Phe Ser Val Arg Val Thr Asp Ala 115 120 125 Pro Ser Ser Gly
Asp Asp Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr 130 135 140 Gly Val
Asp Thr Gly Ala Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp145 150 155
160 Lys Lys Leu Leu Ala Val Pro Ala Ala Asn Thr Val Arg Phe Arg Cys
165 170 175 Pro Ala Ala Gly Asn Pro Thr Pro Ser Ile Ser Trp Leu Lys
Asn Gly 180 185 190 Arg Glu Phe Arg Gly Glu His Arg Ile Gly Gly Ile
Lys Leu Arg His 195 200 205 Gln Gln Trp Ser Leu Val Met Glu Ser Val
Val Pro Ser Asp Arg Gly 210 215 220 Asn Tyr Thr Cys Val Val Glu Asn
Lys Phe Gly Ser Ile Arg Gln Thr225 230 235 240 Tyr Thr Leu Asp Val
Leu Glu Arg Ser Pro His Arg Pro Ile Leu Gln 245 250 255 Ala Gly Leu
Pro Ala Asn Gln Thr Ala Val Leu Gly Ser Asp Val Glu 260 265 270 Phe
His Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Leu 275 280
285 Lys His Val Glu Val Asn Gly Ser Lys Val Gly Pro Asp Gly Thr Pro
290 295 300 Tyr Val Thr Val Leu Lys Thr Ala Gly Ala Asn Thr Thr Asp
Lys Glu305 310 315 320 Leu Glu Val Leu Ser Leu His Asn Val Thr Phe
Glu Asp Ala Gly Glu 325 330 335 Tyr Thr Cys Leu Ala Gly Asn Ser Ile
Gly Phe Ser His His Ser Ala 340 345 350 Trp Leu Val Val Leu Pro Ala
Glu Glu Glu Leu Val Glu Ala Asp Glu 355 360 365 Ala Gly Ser Val Tyr
Ala Gly Ile Leu Ser Tyr Gly Val Gly Phe Phe 370 375 380 Leu Phe Ile
Leu Val Val Ala Ala Val Thr Leu Cys Arg Leu Arg Ser385 390 395 400
Pro Pro Lys Lys Gly Leu Gly Ser Pro Thr Val His Lys Ile Ser Arg 405
410 415 Phe Pro Leu Lys Arg Gln Val Ser Leu Glu Ser Asn Ala Ser Met
Ser 420 425 430 Ser Asn Thr Pro Leu Val Arg Ile Ala Arg Leu Ser Ser
Gly Glu Gly 435 440 445 Pro Thr Leu Ala Asn Val Ser Glu Leu Glu Leu
Pro Ala Asp Pro Lys 450 455 460 Trp Glu Leu Ser Arg Ala Arg Leu Thr
Leu Gly Lys Pro Leu Gly Glu465 470 475 480 Gly Cys Phe Gly Gln Val
Val Met Ala Glu Ala Ile Gly Ile Asp Lys 485 490 495 Asp Arg Ala Ala
Lys Pro Val Thr Val Ala Val Lys Met Leu Lys Asp 500 505 510 Asp Ala
Thr Asp Lys Asp Leu Ser Asp Leu Val Ser Glu Met Glu Met 515 520 525
Met Lys Met Ile Gly Lys His Lys Asn Ile Ile Asn Leu Leu Gly Ala 530
535 540 Cys Thr Gln Gly Gly Pro Leu Tyr Val Leu Val Glu Tyr Ala Ala
Lys545 550 555 560 Gly Asn Leu Arg Glu Phe Leu Arg Ala Arg Arg Pro
Pro Gly Leu Asp 565 570 575 Tyr Ser Phe Asp Thr Cys Lys Pro Pro Glu
Glu Gln Leu Thr Phe Lys 580 585 590 Asp Leu Val Ser Cys Ala Tyr Gln
Val Ala Arg Gly Met Glu Tyr Leu 595 600 605 Ala Ser Gln Lys Cys Ile
His Arg Asp Leu Ala Ala Arg Asn Val Leu 610 615 620 Val Thr Glu Asp
Asn Val Met Lys Ile Ala Asp Phe Gly Leu Ala Arg625 630 635 640 Asp
Val His Asn Leu Asp Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu 645 650
655 Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr
660 665 670 His Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu
Ile Phe 675 680 685 Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val
Glu Glu Leu Phe 690 695 700 Lys Leu Leu Lys Glu Gly His Arg Met Asp
Lys Pro Ala Asn Cys Thr705 710 715 720 His Asp Leu Tyr Met Ile Met
Arg Glu Cys Trp His Ala Ala Pro Ser 725 730 735 Gln Arg Pro Thr Phe
Lys Gln Leu Val Glu Asp Leu Asp Arg Val Leu 740 745 750 Thr Val Thr
Ser Thr Asp Glu Tyr Leu Asp Leu Ser Ala Pro Phe Glu 755 760 765 Gln
Tyr Ser Pro Gly Gly Gln Asp Thr Pro Ser Ser Ser Ser Ser Gly 770 775
780 Asp Asp Ser Val Phe Ala His Asp Leu Leu Pro Pro Ala Pro Pro
Ser785 790 795 800 Ser Gly Gly Ser Arg Thr 805 27694PRTHomo sapiens
27Met Gly Ala Pro Ala Cys Ala Leu Ala Leu Cys Val Ala Val Ala Ile1
5 10 15 Val Ala Gly Ala Ser Ser Glu Ser Leu Gly Thr Glu Gln Arg Val
Val 20 25 30 Gly Arg Ala Ala Glu Val Pro Gly Pro Glu Pro Gly Gln
Gln Glu Gln 35 40 45 Leu Val Phe Gly Ser Gly Asp Ala Val Glu Leu
Ser Cys Pro Pro Pro 50 55 60 Gly Gly Gly Pro Met Gly Pro Thr Val
Trp Val Lys Asp Gly Thr Gly65 70 75 80 Leu Val Pro Ser Glu Arg Val
Leu Val Gly Pro Gln Arg Leu Gln Val 85 90 95 Leu Asn Ala Ser His
Glu Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg 100 105 110 Leu Thr Gln
Arg Val Leu Cys His Phe Ser Val Arg Val Thr Asp Ala 115 120 125 Pro
Ser Ser Gly Asp Asp Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr 130 135
140 Gly Val Asp Thr Gly Ala Pro Tyr Trp Thr Arg Pro Glu Arg Met
Asp145 150 155 160 Lys Lys Leu Leu Ala Val Pro Ala Ala Asn Thr Val
Arg Phe Arg Cys 165 170 175 Pro Ala Ala Gly Asn Pro Thr Pro Ser Ile
Ser Trp Leu Lys Asn Gly 180 185 190 Arg Glu Phe Arg Gly Glu His Arg
Ile Gly Gly Ile Lys Leu Arg His 195 200 205 Gln Gln Trp Ser Leu Val
Met Glu Ser Val Val Pro Ser Asp Arg Gly 210 215 220 Asn Tyr Thr Cys
Val Val Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr225 230 235 240 Tyr
Thr Leu Asp Val Leu Glu Arg Ser Pro His Arg Pro Ile Leu Gln 245 250
255 Ala Gly Leu Pro Ala Asn Gln Thr Ala Val Leu Gly Ser Asp Val Glu
260 265 270 Phe His Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln
Trp Leu 275 280 285 Lys His Val Glu Val Asn Gly Ser Lys Val Gly Pro
Asp Gly Thr Pro 290 295 300 Tyr Val Thr Val Leu Lys Val Ser Leu Glu
Ser Asn Ala Ser Met Ser305 310 315 320 Ser Asn Thr Pro Leu Val Arg
Ile Ala Arg Leu Ser Ser Gly Glu Gly 325 330 335 Pro Thr Leu Ala Asn
Val Ser Glu Leu Glu Leu Pro Ala Asp Pro Lys 340 345 350 Trp Glu Leu
Ser Arg Ala Arg Leu Thr Leu Gly Lys Pro Leu Gly Glu 355 360 365 Gly
Cys Phe Gly Gln Val Val Met Ala Glu Ala Ile Gly Ile Asp Lys 370 375
380 Asp Arg Ala Ala Lys Pro Val Thr Val Ala Val Lys Met Leu Lys
Asp385 390 395 400 Asp Ala Thr Asp Lys Asp Leu Ser Asp Leu Val Ser
Glu Met Glu Met 405 410 415 Met Lys Met Ile Gly Lys His Lys Asn Ile
Ile Asn Leu Leu Gly Ala 420 425 430 Cys Thr Gln Gly Gly Pro Leu Tyr
Val Leu Val Glu Tyr Ala Ala Lys 435 440 445 Gly Asn Leu Arg Glu Phe
Leu Arg Ala Arg Arg Pro Pro Gly Leu Asp 450 455 460 Tyr Ser Phe Asp
Thr Cys Lys Pro Pro Glu Glu Gln Leu Thr Phe Lys465 470 475 480 Asp
Leu Val Ser Cys Ala Tyr Gln Val Ala Arg Gly Met Glu Tyr Leu 485 490
495 Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn Val Leu
500 505 510 Val Thr Glu Asp Asn Val Met Lys Ile Ala Asp Phe Gly Leu
Ala Arg 515 520 525 Asp Val His Asn Leu Asp Tyr Tyr
Lys Lys Thr Thr Asn Gly Arg Leu 530 535 540 Pro Val Lys Trp Met Ala
Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr545 550 555 560 His Gln Ser
Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe 565 570 575 Thr
Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe 580 585
590 Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro Ala Asn Cys Thr
595 600 605 His Asp Leu Tyr Met Ile Met Arg Glu Cys Trp His Ala Ala
Pro Ser 610 615 620 Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu
Asp Arg Val Leu625 630 635 640 Thr Val Thr Ser Thr Asp Glu Tyr Leu
Asp Leu Ser Ala Pro Phe Glu 645 650 655 Gln Tyr Ser Pro Gly Gly Gln
Asp Thr Pro Ser Ser Ser Ser Ser Gly 660 665 670 Asp Asp Ser Val Phe
Ala His Asp Leu Leu Pro Pro Ala Pro Pro Ser 675 680 685 Ser Gly Gly
Ser Arg Thr 690 28604PRTHomo sapiens 28Ala Gln Arg Arg Lys Glu Arg
Glu Glu Leu Ala Gln Gln Tyr Glu Ala1 5 10 15 Ile Leu Arg Glu Cys
Gly His Gly Arg Phe Gln Trp Thr Leu Tyr Phe 20 25 30 Val Leu Gly
Leu Ala Leu Met Ala Asp Gly Val Glu Val Phe Val Val 35 40 45 Gly
Phe Val Leu Pro Ser Ala Glu Lys Asp Met Cys Leu Ser Asp Ser 50 55
60 Asn Lys Gly Met Leu Gly Leu Ile Val Tyr Leu Gly Met Met Val
Gly65 70 75 80 Ala Phe Leu Trp Gly Gly Leu Ala Asp Arg Leu Gly Arg
Arg Gln Cys 85 90 95 Leu Leu Ile Ser Leu Ser Val Asn Ser Val Phe
Ala Phe Phe Ser Ser 100 105 110 Phe Val Gln Gly Tyr Gly Thr Phe Leu
Phe Cys Arg Leu Leu Ser Gly 115 120 125 Val Gly Ile Gly Gly Ser Ile
Pro Ile Val Phe Ser Tyr Phe Ser Glu 130 135 140 Phe Leu Ala Gln Glu
Lys Arg Gly Glu His Leu Ser Trp Leu Cys Met145 150 155 160 Phe Trp
Met Ile Gly Gly Val Tyr Ala Ala Ala Met Ala Trp Ala Ile 165 170 175
Ile Pro His Tyr Gly Trp Ser Phe Gln Met Gly Ser Ala Tyr Gln Phe 180
185 190 His Ser Trp Arg Val Phe Val Leu Val Cys Ala Phe Pro Ser Val
Phe 195 200 205 Ala Ile Gly Ala Leu Thr Thr Gln Pro Glu Ser Pro Arg
Phe Phe Leu 210 215 220 Glu Asn Gly Lys His Asp Glu Ala Trp Met Val
Leu Lys Gln Val His225 230 235 240 Asp Thr Asn Met Arg Ala Lys Gly
His Pro Glu Arg Val Phe Ser Val 245 250 255 Thr His Ile Lys Thr Ile
His Gln Glu Asp Glu Leu Ile Glu Ile Gln 260 265 270 Ser Asp Thr Gly
Thr Trp Tyr Gln Arg Trp Gly Val Arg Ala Leu Ser 275 280 285 Leu Gly
Gly Gln Val Trp Gly Asn Phe Leu Ser Cys Phe Gly Pro Glu 290 295 300
Tyr Arg Arg Ile Thr Leu Met Met Met Gly Val Trp Phe Thr Met Ser305
310 315 320 Phe Ser Tyr Tyr Gly Leu Thr Val Trp Phe Pro Asp Met Ile
Arg His 325 330 335 Leu Gln Ala Val Asp Tyr Ala Ser Arg Thr Lys Val
Phe Pro Gly Glu 340 345 350 Arg Val Glu His Val Thr Phe Asn Phe Thr
Leu Glu Asn Gln Ile His 355 360 365 Arg Gly Gly Gln Tyr Phe Asn Asp
Lys Phe Ile Gly Leu Arg Leu Lys 370 375 380 Ser Val Ser Phe Glu Asp
Ser Leu Phe Glu Glu Cys Tyr Phe Glu Asp385 390 395 400 Val Thr Ser
Ser Asn Thr Phe Phe Arg Asn Cys Thr Phe Ile Asn Thr 405 410 415 Val
Phe Tyr Asn Thr Asp Leu Phe Glu Tyr Lys Phe Val Asn Ser Arg 420 425
430 Leu Ile Asn Ser Thr Phe Leu His Asn Lys Glu Gly Cys Pro Leu Asp
435 440 445 Val Thr Gly Thr Gly Glu Gly Ala Tyr Met Val Tyr Phe Val
Ser Phe 450 455 460 Leu Gly Thr Leu Ala Val Leu Pro Gly Asn Ile Val
Ser Ala Leu Leu465 470 475 480 Met Asp Lys Ile Gly Arg Leu Arg Met
Leu Ala Gly Ser Ser Val Met 485 490 495 Ser Cys Val Ser Cys Phe Phe
Leu Ser Phe Gly Asn Ser Glu Ser Ala 500 505 510 Met Ile Ala Leu Leu
Cys Leu Phe Gly Gly Val Ser Ile Ala Ser Trp 515 520 525 Asn Ala Leu
Asp Val Leu Thr Val Glu Leu Tyr Pro Ser Asp Lys Arg 530 535 540 Thr
Thr Ala Phe Gly Phe Leu Asn Ala Leu Cys Lys Leu Ala Ala Val545 550
555 560 Leu Gly Ile Ser Ile Phe Thr Ser Phe Val Gly Ile Thr Lys Ala
Ala 565 570 575 Pro Ile Leu Phe Ala Ser Ala Ala Leu Ala Leu Gly Ser
Ser Leu Ala 580 585 590 Leu Lys Leu Pro Glu Thr Arg Gly Gln Val Leu
Gln 595 600 29683PRTHomo sapiens 29Met Asp Asp Tyr Lys Tyr Gln Asp
Asn Tyr Gly Gly Tyr Ala Pro Ser1 5 10 15 Asp Gly Tyr Tyr Arg Gly
Asn Glu Ser Asn Pro Glu Glu Asp Ala Gln 20 25 30 Ser Asp Val Thr
Glu Gly His Asp Glu Glu Asp Glu Ile Tyr Glu Gly 35 40 45 Glu Tyr
Gln Gly Ile Pro His Pro Asp Asp Val Lys Ala Lys Gln Ala 50 55 60
Lys Met Ala Pro Ser Arg Met Asp Ser Leu Arg Gly Gln Thr Asp Leu65
70 75 80 Met Ala Glu Arg Leu Glu Asp Glu Glu Gln Leu Ala His Gln
Tyr Glu 85 90 95 Thr Ile Met Asp Glu Cys Gly His Gly Arg Phe Gln
Trp Ile Leu Phe 100 105 110 Phe Val Leu Gly Leu Ala Leu Met Ala Asp
Gly Val Glu Val Phe Val 115 120 125 Val Ser Phe Ala Leu Pro Ser Ala
Glu Lys Asp Met Cys Leu Ser Ser 130 135 140 Ser Lys Lys Gly Met Leu
Gly Met Ile Val Tyr Leu Gly Met Met Ala145 150 155 160 Gly Ala Phe
Ile Leu Gly Gly Leu Ala Asp Lys Leu Gly Arg Lys Arg 165 170 175 Val
Leu Ser Met Ser Leu Ala Val Asn Ala Ser Phe Ala Ser Leu Ser 180 185
190 Ser Phe Val Gln Gly Tyr Gly Ala Phe Leu Phe Cys Arg Leu Ile Ser
195 200 205 Gly Ile Gly Ile Gly Gly Ala Leu Pro Ile Val Phe Ala Tyr
Phe Ser 210 215 220 Glu Phe Leu Ser Arg Glu Lys Arg Gly Glu His Leu
Ser Trp Leu Gly225 230 235 240 Ile Phe Trp Met Thr Gly Gly Leu Tyr
Ala Ser Ala Met Ala Trp Ser 245 250 255 Ile Ile Pro His Tyr Gly Trp
Gly Phe Ser Met Gly Thr Asn Tyr His 260 265 270 Phe His Ser Trp Arg
Val Phe Val Ile Val Cys Ala Leu Pro Cys Thr 275 280 285 Val Ser Met
Val Ala Leu Lys Phe Met Pro Glu Ser Pro Arg Phe Leu 290 295 300 Leu
Glu Met Gly Lys His Asp Glu Ala Trp Met Ile Leu Lys Gln Val305 310
315 320 His Asp Thr Asn Met Arg Ala Lys Gly Thr Pro Glu Lys Val Phe
Thr 325 330 335 Val Ser Asn Ile Lys Thr Pro Lys Gln Met Asp Glu Phe
Ile Glu Ile 340 345 350 Gln Ser Ser Thr Gly Thr Trp Tyr Gln Arg Trp
Leu Val Arg Phe Lys 355 360 365 Thr Ile Phe Lys Gln Val Trp Asp Asn
Ala Leu Tyr Cys Val Met Gly 370 375 380 Pro Tyr Arg Met Asn Thr Leu
Ile Leu Ala Val Val Trp Phe Ala Met385 390 395 400 Ala Phe Ser Tyr
Tyr Gly Leu Thr Val Trp Phe Pro Asp Met Ile Arg 405 410 415 Tyr Phe
Gln Asp Glu Glu Tyr Lys Ser Lys Met Lys Val Phe Phe Gly 420 425 430
Glu His Val Tyr Gly Ala Thr Ile Asn Phe Thr Met Glu Asn Gln Ile 435
440 445 His Gln His Gly Lys Leu Val Asn Asp Lys Phe Thr Arg Met Tyr
Phe 450 455 460 Lys His Val Leu Phe Glu Asp Thr Phe Phe Asp Glu Cys
Tyr Phe Glu465 470 475 480 Asp Val Thr Ser Thr Asp Thr Tyr Phe Lys
Asn Cys Thr Ile Glu Ser 485 490 495 Thr Ile Phe Tyr Asn Thr Asp Leu
Tyr Glu His Lys Phe Ile Asn Cys 500 505 510 Arg Phe Ile Asn Ser Thr
Phe Leu Glu Gln Lys Glu Gly Cys His Met 515 520 525 Asp Leu Glu Gln
Asp Asn Asp Phe Leu Ile Tyr Leu Val Ser Phe Leu 530 535 540 Gly Ser
Leu Ser Val Leu Pro Gly Asn Ile Ile Ser Ala Leu Leu Met545 550 555
560 Asp Arg Ile Gly Arg Leu Lys Met Ile Gly Gly Ser Met Leu Ile Ser
565 570 575 Ala Val Cys Cys Phe Phe Leu Phe Phe Gly Asn Ser Glu Ser
Ala Met 580 585 590 Ile Gly Trp Gln Cys Leu Phe Cys Gly Thr Ser Ile
Ala Ala Trp Asn 595 600 605 Ala Leu Asp Val Ile Thr Val Glu Leu Tyr
Pro Thr Asn Gln Arg Ala 610 615 620 Thr Ala Phe Gly Ile Leu Asn Gly
Leu Cys Lys Phe Gly Ala Ile Leu625 630 635 640 Gly Asn Thr Ile Phe
Ala Ser Phe Val Gly Ile Thr Lys Val Val Pro 645 650 655 Ile Leu Leu
Ala Ala Ala Ser Leu Val Gly Gly Gly Leu Ile Ala Leu 660 665 670 Arg
Leu Pro Glu Thr Arg Glu Gln Val Leu Ile 675 680 30727PRTHomo
sapiens 30Met Glu Asp Ser Tyr Lys Asp Arg Thr Ser Leu Met Lys Gly
Ala Lys1 5 10 15 Asp Ile Ala Arg Glu Val Lys Lys Gln Thr Val Lys
Lys Val Asn Gln 20 25 30 Ala Val Asp Arg Ala Gln Asp Glu Tyr Thr
Gln Arg Ser Tyr Ser Arg 35 40 45 Phe Gln Asp Glu Glu Asp Asp Asp
Asp Tyr Tyr Pro Ala Gly Glu Thr 50 55 60 Tyr Asn Gly Glu Ala Asn
Asp Asp Glu Gly Ser Ser Glu Ala Thr Glu65 70 75 80 Gly His Asp Glu
Asp Asp Glu Ile Tyr Glu Gly Glu Tyr Gln Gly Ile 85 90 95 Pro Ser
Met Asn Gln Ala Lys Asp Ser Ile Val Ser Val Gly Gln Pro 100 105 110
Lys Gly Asp Glu Tyr Lys Asp Arg Arg Glu Leu Glu Ser Glu Arg Arg 115
120 125 Ala Asp Glu Glu Glu Leu Ala Gln Gln Tyr Glu Leu Ile Ile Gln
Glu 130 135 140 Cys Gly His Gly Arg Phe Gln Trp Ala Leu Phe Phe Val
Leu Gly Met145 150 155 160 Ala Leu Met Ala Asp Gly Val Glu Val Phe
Val Val Gly Phe Val Leu 165 170 175 Pro Ser Ala Glu Thr Asp Leu Cys
Ile Pro Asn Ser Gly Ser Gly Trp 180 185 190 Leu Gly Ser Ile Val Tyr
Leu Gly Met Met Val Gly Ala Phe Phe Trp 195 200 205 Gly Gly Leu Ala
Asp Lys Val Gly Arg Lys Gln Ser Leu Leu Ile Cys 210 215 220 Met Ser
Val Asn Gly Phe Phe Ala Phe Leu Ser Ser Phe Val Gln Gly225 230 235
240 Tyr Gly Phe Phe Leu Phe Cys Arg Leu Leu Ser Gly Phe Gly Ile Gly
245 250 255 Gly Ala Ile Pro Thr Val Phe Ser Tyr Phe Ala Glu Val Leu
Ala Arg 260 265 270 Glu Lys Arg Gly Glu His Leu Ser Trp Leu Cys Met
Phe Trp Met Ile 275 280 285 Gly Gly Ile Tyr Ala Ser Ala Met Ala Trp
Ala Ile Ile Pro His Tyr 290 295 300 Gly Trp Ser Phe Ser Met Gly Ser
Ala Tyr Gln Phe His Ser Trp Arg305 310 315 320 Val Phe Val Ile Val
Cys Ala Leu Pro Cys Val Ser Ser Val Val Ala 325 330 335 Leu Thr Phe
Met Pro Glu Ser Pro Arg Phe Leu Leu Glu Val Gly Lys 340 345 350 His
Asp Glu Ala Trp Met Ile Leu Lys Leu Ile His Asp Thr Asn Met 355 360
365 Arg Ala Arg Gly Gln Pro Glu Lys Val Phe Thr Val Asn Lys Ile Lys
370 375 380 Thr Pro Lys Gln Ile Asp Glu Leu Ile Glu Ile Glu Ser Asp
Thr Gly385 390 395 400 Thr Trp Tyr Arg Arg Cys Phe Val Arg Ile Arg
Thr Glu Leu Tyr Gly 405 410 415 Ile Trp Leu Thr Phe Met Arg Cys Phe
Asn Tyr Pro Val Arg Asp Asn 420 425 430 Thr Ile Lys Leu Thr Ile Val
Trp Phe Thr Leu Ser Phe Gly Tyr Tyr 435 440 445 Gly Leu Ser Val Trp
Phe Pro Asp Val Ile Lys Pro Leu Gln Ser Asp 450 455 460 Glu Tyr Ala
Leu Leu Thr Arg Asn Val Glu Arg Asp Lys Tyr Ala Asn465 470 475 480
Phe Thr Ile Asn Phe Thr Met Glu Asn Gln Ile His Thr Gly Met Glu 485
490 495 Tyr Asp Asn Gly Arg Phe Ile Gly Val Lys Phe Lys Ser Val Thr
Phe 500 505 510 Lys Asp Ser Val Phe Lys Ser Cys Thr Phe Glu Asp Val
Thr Ser Val 515 520 525 Asn Thr Tyr Phe Lys Asn Cys Thr Phe Ile Asp
Thr Val Phe Asp Asn 530 535 540 Thr Asp Phe Glu Pro Tyr Lys Phe Ile
Asp Ser Glu Phe Lys Asn Cys545 550 555 560 Ser Phe Phe His Asn Lys
Thr Gly Cys Gln Ile Thr Phe Asp Asp Asp 565 570 575 Tyr Ser Ala Tyr
Trp Ile Tyr Phe Val Asn Phe Leu Gly Thr Leu Ala 580 585 590 Val Leu
Pro Gly Asn Ile Val Ser Ala Leu Leu Met Asp Arg Ile Gly 595 600 605
Arg Leu Thr Met Leu Gly Gly Ser Met Val Leu Ser Gly Ile Ser Cys 610
615 620 Phe Phe Leu Trp Phe Gly Thr Ser Glu Ser Met Met Ile Gly Met
Leu625 630 635 640 Cys Leu Tyr Asn Gly Leu Thr Ile Ser Ala Trp Asn
Ser Leu Asp Val 645 650 655 Val Thr Val Glu Leu Tyr Pro Thr Asp Arg
Arg Ala Thr Gly Phe Gly 660 665 670 Phe Leu Asn Ala Leu Cys Lys Ala
Ala Ala Val Leu Gly Asn Leu Ile 675 680 685 Phe Gly Ser Leu Val Ser
Ile Thr Lys Ser Ile Pro Ile Leu Leu Ala 690 695 700 Ser Thr Val Leu
Val Cys Gly Gly Leu Val Gly Leu Cys Leu Pro Asp705 710 715 720 Thr
Arg Thr Gln Val Leu Met 725 31742PRTHomo sapiens 31Met Glu Glu Gly
Phe Arg Asp Arg Ala Ala Phe Ile Arg Gly Ala Lys1 5 10 15 Asp Ile
Ala Lys Glu Val Lys Lys His Ala Ala Lys Lys Val Val Lys 20 25 30
Gly Leu Asp Arg Val Gln Asp Glu Tyr Ser Arg Arg Ser Tyr Ser Arg 35
40 45 Phe Glu Glu Glu Asp Asp Asp Asp Asp Phe Pro Ala Pro Ser Asp
Gly 50 55 60 Tyr Tyr Arg Gly Glu Gly Thr Gln Asp Glu Glu Glu Gly
Gly Ala Ser65 70 75 80 Ser Asp Ala Thr Glu Gly His Asp Glu Asp Asp
Glu Ile Tyr Glu Gly 85 90 95 Glu Tyr Gln Asp Ile Pro Arg Ala Glu
Ser Gly Gly Lys Gly Glu Arg 100 105 110 Met Ala Asp Gly Ala Pro Leu
Ala Gly Val Arg Gly Gly Leu Ser Asp 115 120
125 Gly Glu Gly Pro Pro Gly Gly Arg Gly Glu Ala Gln Arg Arg Lys Glu
130 135 140 Arg Glu Glu Leu Ala Gln Gln Tyr Glu Ala Ile Leu Arg Glu
Cys Gly145 150 155 160 His Gly Arg Phe Gln Trp Thr Leu Tyr Phe Val
Leu Gly Leu Ala Leu 165 170 175 Met Ala Asp Gly Val Glu Val Phe Val
Val Gly Phe Val Leu Pro Ser 180 185 190 Ala Glu Lys Asp Met Cys Leu
Ser Asp Ser Asn Lys Gly Met Leu Gly 195 200 205 Leu Ile Val Tyr Leu
Gly Met Met Val Gly Ala Phe Leu Trp Gly Gly 210 215 220 Leu Ala Asp
Arg Leu Gly Arg Arg Gln Cys Leu Leu Ile Ser Leu Ser225 230 235 240
Val Asn Ser Val Phe Ala Phe Phe Ser Ser Phe Val Gln Gly Tyr Gly 245
250 255 Thr Phe Leu Phe Cys Arg Leu Leu Ser Gly Val Gly Ile Gly Gly
Ser 260 265 270 Ile Pro Ile Val Phe Ser Tyr Phe Ser Glu Phe Leu Ala
Gln Glu Lys 275 280 285 Arg Gly Glu His Leu Ser Trp Leu Cys Met Phe
Trp Met Ile Gly Gly 290 295 300 Val Tyr Ala Ala Ala Met Ala Trp Ala
Ile Ile Pro His Tyr Gly Trp305 310 315 320 Ser Phe Gln Met Gly Ser
Ala Tyr Gln Phe His Ser Trp Arg Val Phe 325 330 335 Val Leu Val Cys
Ala Phe Pro Ser Val Phe Ala Ile Gly Ala Leu Thr 340 345 350 Thr Gln
Pro Glu Ser Pro Arg Phe Phe Leu Glu Asn Gly Lys His Asp 355 360 365
Glu Ala Trp Met Val Leu Lys Gln Val His Asp Thr Asn Met Arg Ala 370
375 380 Lys Gly His Pro Glu Arg Val Phe Ser Val Thr His Ile Lys Thr
Ile385 390 395 400 His Gln Glu Asp Glu Leu Ile Glu Ile Gln Ser Asp
Thr Gly Thr Trp 405 410 415 Tyr Gln Arg Trp Gly Val Arg Ala Leu Ser
Leu Gly Gly Gln Val Trp 420 425 430 Gly Asn Phe Leu Ser Cys Phe Gly
Pro Glu Tyr Arg Arg Ile Thr Leu 435 440 445 Met Met Met Gly Val Trp
Phe Thr Met Ser Phe Ser Tyr Tyr Gly Leu 450 455 460 Thr Val Trp Phe
Pro Asp Met Ile Arg His Leu Gln Ala Val Asp Tyr465 470 475 480 Ala
Ser Arg Thr Lys Val Phe Pro Gly Glu Arg Val Gly His Val Thr 485 490
495 Phe Asn Phe Thr Leu Glu Asn Gln Ile His Arg Gly Gly Gln Tyr Phe
500 505 510 Asn Asp Lys Phe Ile Gly Leu Arg Leu Lys Ser Val Ser Phe
Glu Asp 515 520 525 Ser Leu Phe Glu Glu Cys Tyr Phe Glu Asp Val Thr
Ser Ser Asn Thr 530 535 540 Phe Phe Arg Asn Cys Thr Phe Ile Asn Thr
Val Phe Tyr Asn Thr Asp545 550 555 560 Leu Phe Glu Tyr Lys Phe Val
Asn Ser Arg Leu Ile Asn Ser Thr Phe 565 570 575 Leu His Asn Lys Glu
Gly Cys Pro Leu Asp Val Thr Gly Thr Gly Glu 580 585 590 Gly Ala Tyr
Met Val Tyr Phe Val Ser Phe Leu Gly Thr Leu Ala Val 595 600 605 Leu
Pro Gly Asn Ile Val Ser Ala Leu Leu Met Asp Lys Ile Gly Arg 610 615
620 Leu Arg Met Leu Ala Gly Ser Ser Val Met Ser Cys Val Ser Cys
Phe625 630 635 640 Phe Leu Ser Phe Gly Asn Ser Glu Ser Ala Met Ile
Ala Leu Leu Cys 645 650 655 Leu Phe Gly Gly Val Ser Ile Ala Ser Trp
Asn Ala Leu Asp Val Leu 660 665 670 Thr Val Glu Leu Tyr Pro Ser Asp
Lys Arg Thr Thr Ala Phe Gly Phe 675 680 685 Leu Asn Ala Leu Cys Lys
Leu Ala Ala Val Leu Gly Ile Ser Ile Phe 690 695 700 Thr Ser Phe Val
Gly Ile Thr Lys Ala Ala Pro Ile Leu Phe Ala Ser705 710 715 720 Ala
Ala Leu Ala Leu Gly Ser Ser Leu Ala Leu Lys Leu Pro Glu Thr 725 730
735 Arg Gly Gln Val Leu Gln 740 327PRTArtificial SequenceSNAP-25
antigen having a free carboxyl-terminus at the P1 residue of the
scissile bond of the BoNT/A cleavage site 32Arg Ile Asp Glu Ala Asn
Gln1 5 338PRTArtificial SequenceSNAP-25 antigen having a free
carboxyl-terminus at the P1 residue of the scissile bond of the
BoNT/A cleavage site 33Thr Arg Ile Asp Glu Ala Asn Gln1 5
349PRTArtificial SequenceSNAP-25 antigen having a free
carboxyl-terminus at the P1 residue of the scissile bond of the
BoNT/A cleavage site 34Lys Thr Arg Ile Asp Glu Ala Asn Gln1 5
3510PRTArtificial SequenceSNAP-25 antigen having a free
carboxyl-terminus at the P1 residue of the scissile bond of the
BoNT/A cleavage site 35Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln1 5
10 3611PRTArtificial SequenceSNAP-25 antigen having a free
carboxyl-terminus at the P1 residue of the scissile bond of the
BoNT/A cleavage site 36Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln1
5 10 3712PRTArtificial SequenceSNAP-25 antigen having a free
carboxyl-terminus at the P1 residue of the scissile bond of the
BoNT/A cleavage site 37Asp Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn
Gln1 5 10 3813PRTArtificial SequenceSNAP-25 antigen having a free
carboxylated carboxyl-terminus at the P1 residue of the scissile
bond of the BoNT/A cleavage site 38Cys Asp Ser Asn Lys Thr Arg Ile
Asp Glu Ala Asn Gln1 5 10 397PRTArtificial SequenceSNAP-25 antigen
having a free carboxyl-terminus at the P1 residue of the scissile
bond of the BoNT/A cleavage site 39Arg Ile Asp Glu Ala Asn Lys1 5
408PRTArtificial SequenceSNAP-25 antigen having a free
carboxyl-terminus at the P1 residue of the scissile bond of the
BoNT/A cleavage site 40Ala Arg Ile Asp Glu Ala Asn Lys1 5
419PRTArtificial SequenceSNAP-25 antigen having a free
carboxyl-terminus at the P1 residue of the scissile bond of the
BoNT/A cleavage site 41Lys Ala Arg Ile Asp Glu Ala Asn Lys1 5
4210PRTArtificial SequenceSNAP-25 antigen having a free
carboxyl-terminus at the P1 residue of the scissile bond of the
BoNT/A cleavage site 42Asn Lys Ala Arg Ile Asp Glu Ala Asn Lys1 5
10 4311PRTArtificial SequenceSNAP-25 antigen having a free
carboxyl-terminus at the P1 residue of the scissile bond of the
BoNT/A cleavage site 43Met Asn Lys Ala Arg Ile Asp Glu Ala Asn Lys1
5 10 4412PRTArtificial SequenceSNAP-25 antigen having a free
carboxyl-terminus at the P1 residue of the scissile bond of the
BoNT/A cleavage site 44Asp Met Asn Lys Ala Arg Ile Asp Glu Ala Asn
Lys1 5 10 4513PRTArtificial SequenceSNAP-25 antigen having a free
carboxylated carboxyl-terminus at the P1 residue of the scissile
bond of the BoNT/A cleavage site 45Cys Asp Met Asn Lys Ala Arg Ile
Asp Glu Ala Asn Lys1 5 10 4611PRTArtificial SequenceSNAP-25 antigen
46Cys Gly Gly Gly Arg Ile Asp Glu Ala Asn Gln1 5 10
4711PRTArtificial SequenceSNAP-25 antigen 47Cys Gly Gly Gly Arg Ile
Asp Glu Ala Asn Lys1 5 10 4888PRTArtificial
SequenceBirA-HisTag?-SNAP-25-134-197 48Met Gly Gly Gly Leu Asn Asp
Ile Phe Glu Ala Gln Lys Ile Glu Trp1 5 10 15 His His His His His
His His His Ile Arg Arg Val Thr Asn Asp Ala 20 25 30 Arg Glu Asn
Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile 35 40 45 Gly
Asn Leu Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr 50 55
60 Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn
Lys65 70 75 80 Thr Arg Ile Asp Glu Ala Asn Gln 85 4997PRTArtificial
SequenceBirA-HisTag?-SNAP-25-134-206 49Met Gly Gly Gly Leu Asn Asp
Ile Phe Glu Ala Gln Lys Ile Glu Trp1 5 10 15 His His His His His
His His His Ile Arg Arg Val Thr Asn Asp Ala 20 25 30 Arg Glu Asn
Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile 35 40 45 Gly
Asn Leu Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr 50 55
60 Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn
Lys65 70 75 80 Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met
Leu Gly Ser 85 90 95 Gly5016PRTArtificial SequenceBirA peptide
50Gly Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His1
5 10 15 517570DNAArtificial SequencepQBI-25/GFP-BoNT/A-LC
expression construct. 51gacggatcgg gagatctccc gatcccctat ggtcgactct
cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag
gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg
ctgcttcgcc tcgaggcctg gccattgcat acgttgtatc 240catatcataa
tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt
300gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
agcccatata 360tggagttccg cgttacataa cttacggtaa atggcccgcc
tggctgaccg cccaacgacc 420cccgcccatt gacgtcaata atgacgtatg
ttcccatagt aacgccaata gggactttcc 480attgacgtca atgggtggag
tatttacggt aaactgccca cttggcagta catcaagtgt 540atcatatgcc
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
600atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
gtattagtca 660tcgctattac catggtgatg cggttttggc agtacatcaa
tgggcgtgga tagcggtttg 720actcacgggg atttccaagt ctccacccca
ttgacgtcaa tgggagtttg ttttggcacc 780aaaatcaacg ggactttcca
aaatgtcgta acaactccgc cccattgacg caaatgggcg 840gtaggcgtgt
acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg
900cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac
cgatccagcc 960tccgcgggcc accatggagg gcccggttac cggtaccgga
tccagatatc tgggcggccg 1020ctcagcaagc ttcgcgaatt cgggaggcgg
aggtggagct agcaaaggag aagaactctt 1080cactggagtt gtcccaattc
ttgttgaatt agatggtgat gttaacggcc acaagttctc 1140tgtcagtgga
gagggtgaag gtgatgcaac atacggaaaa cttaccctga agttcatctg
1200cactactggc aaactgcctg ttccatggcc aacactagtc actactctgt
gctatggtgt 1260tcaatgcttt tcaagatacc cggatcatat gaaacggcat
gactttttca agagtgccat 1320gcccgaaggt tatgtacagg aaaggaccat
cttcttcaaa gatgacggca actacaagac 1380acgtgctgaa gtcaagtttg
aaggtgatac ccttgttaat agaatcgagt taaaaggtat 1440tgacttcaag
gaagatggca acattctggg acacaaattg gaatacaact ataactcaca
1500caatgtatac atcatggcag acaaacaaaa gaatggaatc aaagtgaact
tcaagacccg 1560ccacaacatt gaagatggaa gcgttcaact agcagaccat
tatcaacaaa atactccaat 1620tggcgatggc cctgtccttt taccagacaa
ccattacctg tccacacaat ctgccctttc 1680gaaagatccc aacgaaaaga
gagaccacat ggtccttctt gagtttgtaa cagctgctgg 1740gattacacat
ggcatggatg aactgtacaa catcgatgga ggcggaggtg gaccttttgt
1800taataaacaa tttaattata aagatcctgt aaatggtgtt gatattgctt
atataaaaat 1860tccaaatgca ggacaaatgc aaccagtaaa agcttttaaa
attcataata aaatatgggt 1920tattccagaa agagatacat ttacaaatcc
tgaagaagga gatttaaatc caccaccaga 1980agcaaaacaa gttccagttt
catattatga ttcaacatat ttaagtacag ataatgaaaa 2040agataattat
ttaaagggag ttacaaaatt atttgagaga atttattcaa ctgatcttgg
2100aagaatgttg ttaacatcaa tagtaagggg aataccattt tggggtggaa
gtacaataga 2160tacagaatta aaagttattg atactaattg tattaatgtg
atacaaccag atggtagtta 2220tagatcagaa gaacttaatc tagtaataat
aggaccctca gctgatatta tacagtttga 2280atgtaaaagc tttggacatg
aagttttgaa tcttacgcga aatggttatg gctctactca 2340atacattaga
tttagcccag attttacatt tggttttgag gagtcacttg aagttgatac
2400aaatcctctt ttaggtgcag gcaaatttgc tacagatcca gcagtaacat
tagcacatga 2460acttatacat gctggacata gattatatgg aatagcaatt
aatccaaata gggtttttaa 2520agtaaatact aatgcctatt atgaaatgag
tgggttagaa gtaagctttg aggaacttag 2580aacatttggg ggacatgatg
caaagtttat agatagttta caggaaaacg aatttcgtct 2640atattattat
aataagttta aagatatagc aagtacactt aataaagcta aatcaatagt
2700aggtactact gcttcattac agtatatgaa aaatgttttt aaagagaaat
atctcctatc 2760tgaagataca tctggaaaat tttcggtaga taaattaaaa
tttgataagt tatacaaaat 2820gttaacagag atttacacag aggataattt
tgttaagttt tttaaagtac ttaacagaaa 2880aacatatttg aattttgata
aagccgtatt taagataaat atagtaccta aggtaaatta 2940cacaatatat
gatggattta atttaagaaa tacaaattta gcagcaaact ttaatggtca
3000aaatacagaa attaataata tgaattttac taaactaaaa aattttactg
gattgtttga 3060attttataag ttgctatgtg taagagggat aatcacttcg
aaatgaacgc gttggcccta 3120ttctatagtg tcacctaaat gctagagctc
gctgatcagc ctcgactgtg ccttctagtt 3180gccagccatc tgttgtttgc
ccctcccccg tgccttcctt gaccctggaa ggtgccactc 3240ccactgtcct
ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
3300ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
gacaatagca 3360ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
ggaaagaacc agctggggct 3420ctagggggta tccccacgcg ccctgtagcg
gcgcattaag cgcggcgggt gtggtggtta 3480cgcgcagcgt gaccgctaca
cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 3540cttcctttct
cgccacgttc gccggctttc cccgtcaagc tctaaatcgg ggcatccctt
3600tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
tagggtgatg 3660gttcacgtag tgggccatcg ccctgataga cggtttttcg
ccctttgacg ttggagtcca 3720cgttctttaa tagtggactc ttgttccaaa
ctggaacaac actcaaccct atctcggtct 3780attcttttga tttataaggg
attttgggga tttcggccta ttggttaaaa aatgagctga 3840tttaacaaaa
atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
3900gtccccaggc tccccaggca ggcagaagta tgcaaagcat gcatctcaat
tagtcagcaa 3960ccaggtgtgg aaagtcccca ggctccccag caggcagaag
tatgcaaagc atgcatctca 4020attagtcagc aaccatagtc ccgcccctaa
ctccgcccat cccgccccta actccgccca 4080gttccgccca ttctccgccc
catggctgac taattttttt tatttatgca gaggccgagg 4140ccgcctctgc
ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
4200tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
gagacaggat 4260gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
aggttctccg gccgcttggg 4320tggagaggct attcggctat gactgggcac
aacagacaat cggctgctct gatgccgccg 4380tgttccggct gtcagcgcag
gggcgcccgg ttctttttgt caagaccgac ctgtccggtg 4440ccctgaatga
actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
4500cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
ctattgggcg 4560aagtgccggg gcaggatctc ctgtcatctc accttgctcc
tgccgagaaa gtatccatca 4620tggctgatgc aatgcggcgg ctgcatacgc
ttgatccggc tacctgccca ttcgaccacc 4680aagcgaaaca tcgcatcgag
cgagcacgta ctcggatgga agccggtctt gtcgatcagg 4740atgatctgga
cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
4800cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
ttgccgaata 4860tcatggtgga aaatggccgc ttttctggat tcatcgactg
tggccggctg ggtgtggcgg 4920accgctatca ggacatagcg ttggctaccc
gtgatattgc tgaagagctt ggcggcgaat 4980gggctgaccg cttcctcgtg
ctttacggta tcgccgctcc cgattcgcag cgcatcgcct 5040tctatcgcct
tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
5100agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
atgaaaggtt 5160gggcttcgga atcgttttcc gggacgccgg ctggatgatc
ctccagcgcg gggatctcat 5220gctggagttc ttcgcccacc ccaacttgtt
tattgcagct tataatggtt acaaataaag 5280caatagcatc acaaatttca
caaataaagc atttttttca ctgcattcta gttgtggttt 5340gtccaaactc
atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
5400ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
caattccaca 5460caacatacga gccggaagca taaagtgtaa agcctggggt
gcctaatgag tgagctaact 5520cacattaatt gcgttgcgct cactgcccgc
tttccagtcg ggaaacctgt cgtgccagct 5580gcattaatga atcggccaac
gcgcggggag aggcggtttg cgtattgggc gctcttccgc 5640ttcctcgctc
actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
5700ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
agaacatgtg 5760agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
gcgttgctgg cgtttttcca 5820taggctccgc ccccctgacg agcatcacaa
aaatcgacgc tcaagtcaga ggtggcgaaa 5880cccgacagga ctataaagat
accaggcgtt tccccctgga agctccctcg tgcgctctcc 5940tgttccgacc
ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
6000gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc
gctccaagct 6060gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
gccttatccg gtaactatcg 6120tcttgagtcc aacccggtaa gacacgactt
atcgccactg gcagcagcca ctggtaacag 6180gattagcaga gcgaggtatg
taggcggtgc tacagagttc ttgaagtggt ggcctaacta 6240cggctacact
agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
6300aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
gtggtttttt 6360tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
caagaagatc ctttgatctt 6420ttctacgggg tctgacgctc agtggaacga
aaactcacgt taagggattt tggtcatgag 6480attatcaaaa aggatcttca
cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 6540ctaaagtata
tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
6600tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
tcgtgtagat 6660aactacgata cgggagggct taccatctgg ccccagtgct
gcaatgatac cgcgagaccc 6720acgctcaccg gctccagatt tatcagcaat
aaaccagcca gccggaaggg ccgagcgcag 6780aagtggtcct gcaactttat
ccgcctccat ccagtctatt aattgttgcc gggaagctag 6840agtaagtagt
tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
6900ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
gatcaaggcg 6960agttacatga tcccccatgt tgtgcaaaaa agcggttagc
tccttcggtc ctccgatcgt 7020tgtcagaagt aagttggccg cagtgttatc
actcatggtt atggcagcac tgcataattc 7080tcttactgtc atgccatccg
taagatgctt ttctgtgact ggtgagtact caaccaagtc 7140attctgagaa
tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
7200taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
cttcggggcg 7260aaaactctca aggatcttac cgctgttgag atccagttcg
atgtaaccca ctcgtgcacc 7320caactgatct tcagcatctt ttactttcac
cagcgtttct gggtgagcaa aaacaggaag 7380gcaaaatgcc gcaaaaaagg
gaataagggc gacacggaaa tgttgaatac tcatactctt 7440cctttttcaa
tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
7500tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
gaaaagtgcc 7560acctgacgtc 757052682PRTArtificial SequenceGFP-BoNT/A
light chain amino acid sequence. 52Ala Ser Lys Gly Glu Glu Leu Phe
Thr Gly Val Val Pro Ile Leu Val1 5 10 15 Glu Leu Asp Gly Asp Val
Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp
Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60
Cys Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg65
70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln
Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr
Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg
Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile
Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly145 150 155 160 Ile Lys Val
Asn Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln
Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185
190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser
195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu
Tyr Asn Ile Asp225 230 235 240 Gly Gly Gly Gly Gly Pro Phe Val Asn
Lys Gln Phe Asn Tyr Lys Asp 245 250 255 Pro Val Asn Gly Val Asp Ile
Ala Tyr Ile Lys Ile Pro Asn Ala Gly 260 265 270 Gln Met Gln Pro Val
Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val 275 280 285 Ile Pro Glu
Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn 290 295 300 Pro
Pro Pro Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr305 310
315 320 Tyr Leu Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val
Thr 325 330 335 Lys Leu Phe Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg
Met Leu Leu 340 345 350 Thr Ser Ile Val Arg Gly Ile Pro Phe Trp Gly
Gly Ser Thr Ile Asp 355 360 365 Thr Glu Leu Lys Val Ile Asp Thr Asn
Cys Ile Asn Val Ile Gln Pro 370 375 380 Asp Gly Ser Tyr Arg Ser Glu
Glu Leu Asn Leu Val Ile Ile Gly Pro385 390 395 400 Ser Ala Asp Ile
Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val 405 410 415 Leu Asn
Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe 420 425 430
Ser Pro Asp Phe Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr 435
440 445 Asn Pro Leu Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val
Thr 450 455 460 Leu Ala His Glu Leu Ile His Ala Gly His Arg Leu Tyr
Gly Ile Ala465 470 475 480 Ile Asn Pro Asn Arg Val Phe Lys Val Asn
Thr Asn Ala Tyr Tyr Glu 485 490 495 Met Ser Gly Leu Glu Val Ser Phe
Glu Glu Leu Arg Thr Phe Gly Gly 500 505 510 His Asp Ala Lys Phe Ile
Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu 515 520 525 Tyr Tyr Tyr Asn
Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala 530 535 540 Lys Ser
Ile Val Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val545 550 555
560 Phe Lys Glu Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser
565 570 575 Val Asp Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile 580 585 590 Tyr Thr Glu Asp Asn Phe Val Lys Phe Phe Lys Val
Leu Asn Arg Lys 595 600 605 Thr Tyr Leu Asn Phe Asp Lys Ala Val Phe
Lys Ile Asn Ile Val Pro 610 615 620 Lys Val Asn Tyr Thr Ile Tyr Asp
Gly Phe Asn Leu Arg Asn Thr Asn625 630 635 640 Leu Ala Ala Asn Phe
Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn 645 650 655 Phe Thr Lys
Leu Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu 660 665 670 Leu
Cys Val Arg Gly Ile Ile Thr Ser Lys 675 680 536259DNAArtificial
SequencepQBI-25/GFP expression construct. 53gacggatcgg gagatctccc
gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat
ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat
ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
180ttagggttag gcgttttgcg ctgcttcgcc tcgaggcctg gccattgcat
acgttgtatc 240catatcataa tatgtacatt tatattggct catgtccaac
attaccgcca tgttgacatt 300gattattgac tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata 360tggagttccg cgttacataa
cttacggtaa atggcccgcc tggctgaccg cccaacgacc 420cccgcccatt
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
480attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
catcaagtgt 540atcatatgcc aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt 600atgcccagta catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca 660tcgctattac catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg 720actcacgggg
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
780aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
caaatgggcg 840gtaggcgtgt acggtgggag gtctatataa gcagagctcg
tttagtgaac cgtcagatcg 900cctggagacg ccatccacgc tgttttgacc
tccatagaag acaccgggac cgatccagcc 960tccgcgggcc accatggagg
gcccggttac cggtaccgga tccagatatc tgggcggccg 1020ctcagcaagc
ttcgcgaatt cgggaggcgg aggtggagct agcaaaggag aagaactctt
1080cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaacggcc
acaagttctc 1140tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa
cttaccctga agttcatctg 1200cactactggc aaactgcctg ttccatggcc
aacactagtc actactctgt gctatggtgt 1260tcaatgcttt tcaagatacc
cggatcatat gaaacggcat gactttttca agagtgccat 1320gcccgaaggt
tatgtacagg aaaggaccat cttcttcaaa gatgacggca actacaagac
1380acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat agaatcgagt
taaaaggtat 1440tgacttcaag gaagatggca acattctggg acacaaattg
gaatacaact ataactcaca 1500caatgtatac atcatggcag acaaacaaaa
gaatggaatc aaagtgaact tcaagacccg 1560ccacaacatt gaagatggaa
gcgttcaact agcagaccat tatcaacaaa atactccaat 1620tggcgatggc
cctgtccttt taccagacaa ccattacctg tccacacaat ctgccctttc
1680gaaagatccc aacgaaaaga gagaccacat ggtccttctt gagtttgtaa
cagctgctgg 1740gattacacat ggcatggatg aactgtacaa catcgatgga
ggcggaggtg gatgaacgcg 1800ttggccctat tctatagtgt cacctaaatg
ctagagctcg ctgatcagcc tcgactgtgc 1860cttctagttg ccagccatct
gttgtttgcc cctcccccgt gccttccttg accctggaag 1920gtgccactcc
cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
1980ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
gattgggaag 2040acaatagcag gcatgctggg gatgcggtgg gctctatggc
ttctgaggcg gaaagaacca 2100gctggggctc tagggggtat ccccacgcgc
cctgtagcgg cgcattaagc gcggcgggtg 2160tggtggttac gcgcagcgtg
accgctacac ttgccagcgc cctagcgccc gctcctttcg 2220ctttcttccc
ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
2280gcatcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
aaacttgatt 2340agggtgatgg ttcacgtagt gggccatcgc cctgatagac
ggtttttcgc cctttgacgt 2400tggagtccac gttctttaat agtggactct
tgttccaaac tggaacaaca ctcaacccta 2460tctcggtcta ttcttttgat
ttataaggga ttttggggat ttcggcctat tggttaaaaa 2520atgagctgat
ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
2580gtgtggaaag tccccaggct ccccaggcag gcagaagtat gcaaagcatg
catctcaatt 2640agtcagcaac caggtgtgga aagtccccag gctccccagc
aggcagaagt atgcaaagca 2700tgcatctcaa ttagtcagca accatagtcc
cgcccctaac tccgcccatc ccgcccctaa 2760ctccgcccag ttccgcccat
tctccgcccc atggctgact aatttttttt atttatgcag 2820aggccgaggc
cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
2880gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
tctgatcaag 2940agacaggatg aggatcgttt cgcatgattg aacaagatgg
attgcacgca ggttctccgg 3000ccgcttgggt ggagaggcta ttcggctatg
actgggcaca acagacaatc ggctgctctg 3060atgccgccgt gttccggctg
tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc 3120tgtccggtgc
cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
3180cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
gactggctgc 3240tattgggcga agtgccgggg caggatctcc tgtcatctca
ccttgctcct gccgagaaag 3300tatccatcat ggctgatgca atgcggcggc
tgcatacgct tgatccggct acctgcccat 3360tcgaccacca agcgaaacat
cgcatcgagc gagcacgtac tcggatggaa gccggtcttg 3420tcgatcagga
tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
3480ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
gatgcctgct 3540tgccgaatat catggtggaa aatggccgct tttctggatt
catcgactgt ggccggctgg 3600gtgtggcgga ccgctatcag gacatagcgt
tggctacccg tgatattgct gaagagcttg 3660gcggcgaatg ggctgaccgc
ttcctcgtgc tttacggtat cgccgctccc gattcgcagc 3720gcatcgcctt
ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
3780gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
ccgccttcta 3840tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
tggatgatcc tccagcgcgg 3900ggatctcatg ctggagttct tcgcccaccc
caacttgttt attgcagctt ataatggtta 3960caaataaagc aatagcatca
caaatttcac aaataaagca tttttttcac tgcattctag 4020ttgtggtttg
tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
4080ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
atccgctcac 4140aattccacac aacatacgag ccggaagcat aaagtgtaaa
gcctggggtg cctaatgagt 4200gagctaactc acattaattg cgttgcgctc
actgcccgct ttccagtcgg gaaacctgtc 4260gtgccagctg cattaatgaa
tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 4320ctcttccgct
tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
4380atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
acgcaggaaa 4440gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
aaaaaggccg cgttgctggc 4500gtttttccat aggctccgcc cccctgacga
gcatcacaaa aatcgacgct caagtcagag 4560gtggcgaaac ccgacaggac
tataaagata ccaggcgttt ccccctggaa gctccctcgt 4620gcgctctcct
gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
4680aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt
aggtcgttcg 4740ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
gaccgctgcg ccttatccgg 4800taactatcgt cttgagtcca acccggtaag
acacgactta tcgccactgg cagcagccac 4860tggtaacagg attagcagag
cgaggtatgt aggcggtgct acagagttct tgaagtggtg 4920gcctaactac
ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
4980taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
ctggtagcgg 5040tggttttttt gtttgcaagc agcagattac gcgcagaaaa
aaaggatctc aagaagatcc 5100tttgatcttt tctacggggt ctgacgctca
gtggaacgaa aactcacgtt aagggatttt 5160ggtcatgaga ttatcaaaaa
ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 5220taaatcaatc
taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
5280tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
gactccccgt 5340cgtgtagata actacgatac gggagggctt accatctggc
cccagtgctg caatgatacc 5400gcgagaccca cgctcaccgg ctccagattt
atcagcaata aaccagccag ccggaagggc 5460cgagcgcaga agtggtcctg
caactttatc cgcctccatc cagtctatta attgttgccg 5520ggaagctaga
gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
5580aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
gttcccaacg 5640atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
gcggttagct ccttcggtcc 5700tccgatcgtt gtcagaagta agttggccgc
agtgttatca ctcatggtta tggcagcact 5760gcataattct cttactgtca
tgccatccgt aagatgcttt tctgtgactg gtgagtactc 5820aaccaagtca
ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
5880acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
gaaaacgttc 5940ttcggggcga aaactctcaa ggatcttacc gctgttgaga
tccagttcga tgtaacccac 6000tcgtgcaccc aactgatctt cagcatcttt
tactttcacc agcgtttctg ggtgagcaaa 6060aacaggaagg caaaatgccg
caaaaaaggg aataagggcg acacggaaat gttgaatact 6120catactcttc
ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
6180atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
catttccccg 6240aaaagtgcca cctgacgtc 625954245PRTArtificial
SequenceGFP amino acid sequence. 54Ala Ser Lys Gly Glu Glu Leu Phe
Thr Gly Val Val Pro Ile Leu Val1 5 10 15 Glu Leu Asp Gly Asp Val
Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp
Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60
Cys Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg65
70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln
Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr
Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg
Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile
Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly145 150 155 160 Ile Lys Val
Asn Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln
Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185
190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser
195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu
Tyr Asn Ile Asp225 230 235 240 Gly Gly Gly Gly Gly 245
554PRTArtificial SequenceG-spacer flexible spacer 55Gly Gly Gly
Gly1 565PRTArtificial SequenceG-spacer flexible spacer 56Gly Gly
Gly Gly Ser1 5 574PRTArtificial SequenceA-spacer flexible spacer
57Ala Ala Ala Ala1 585PRTArtificial SequenceA-spacer flexible
spacer 58Ala Ala Ala Ala Val1 5 59821PRTHomo sapiens 59Met Val Ser
Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15 Thr
Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25
30 Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu
35 40 45 Val Tyr Val Ala Ala Pro Gly Glu Ser Leu Glu Val Arg Cys
Leu Leu 50 55 60 Lys Asp Ala Ala Val Ile Ser Trp Thr Lys Asp Gly
Val His Leu Gly65 70 75 80 Pro Asn Asn Arg Thr Val Leu Ile Gly Glu
Tyr Leu Gln Ile Lys Gly 85 90 95 Ala Thr Pro Arg Asp Ser Gly Leu
Tyr Ala Cys Thr Ala Ser Arg Thr 100 105 110 Val Asp Ser Glu Thr Trp
Tyr Phe Met Val Asn Val Thr Asp Ala Ile 115 120 125 Ser Ser Gly Asp
Asp Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val 130 135 140 Ser Glu
Asn Ser Asn Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu145 150 155
160 Lys Met Glu Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys
165 170
175 Phe Arg Cys Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu
180 185 190 Lys Asn Gly Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly
Tyr Lys 195 200 205 Val Arg Asn Gln His Trp Ser Leu Ile Met Glu Ser
Val Val Pro Ser 210 215 220 Asp Lys Gly Asn Tyr Thr Cys Val Val Glu
Asn Glu Tyr Gly Ser Ile225 230 235 240 Asn His Thr Tyr His Leu Asp
Val Val Glu Arg Ser Pro His Arg Pro 245 250 255 Ile Leu Gln Ala Gly
Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly 260 265 270 Asp Val Glu
Phe Val Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile 275 280 285 Gln
Trp Ile Lys His Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp 290 295
300 Gly Leu Pro Tyr Leu Lys Val Leu Lys Ala Ala Gly Val Asn Thr
Thr305 310 315 320 Asp Lys Glu Ile Glu Val Leu Tyr Ile Arg Asn Val
Thr Phe Glu Asp 325 330 335 Ala Gly Glu Tyr Thr Cys Leu Ala Gly Asn
Ser Ile Gly Ile Ser Phe 340 345 350 His Ser Ala Trp Leu Thr Val Leu
Pro Ala Pro Gly Arg Glu Lys Glu 355 360 365 Ile Thr Ala Ser Pro Asp
Tyr Leu Glu Ile Ala Ile Tyr Cys Ile Gly 370 375 380 Val Phe Leu Ile
Ala Cys Met Val Val Thr Val Ile Leu Cys Arg Met385 390 395 400 Lys
Asn Thr Thr Lys Lys Pro Asp Phe Ser Ser Gln Pro Ala Val His 405 410
415 Lys Leu Thr Lys Arg Ile Pro Leu Arg Arg Gln Val Thr Val Ser Ala
420 425 430 Glu Ser Ser Ser Ser Met Asn Ser Asn Thr Pro Leu Val Arg
Ile Thr 435 440 445 Thr Arg Leu Ser Ser Thr Ala Asp Thr Pro Met Leu
Ala Gly Val Ser 450 455 460 Glu Tyr Glu Leu Pro Glu Asp Pro Lys Trp
Glu Phe Pro Arg Asp Lys465 470 475 480 Leu Thr Leu Gly Lys Pro Leu
Gly Glu Gly Cys Phe Gly Gln Val Val 485 490 495 Met Ala Glu Ala Val
Gly Ile Asp Lys Asp Lys Pro Lys Glu Ala Val 500 505 510 Thr Val Ala
Val Lys Met Leu Lys Asp Asp Ala Thr Glu Lys Asp Leu 515 520 525 Ser
Asp Leu Val Ser Glu Met Glu Met Met Lys Met Ile Gly Lys His 530 535
540 Lys Asn Ile Ile Asn Leu Leu Gly Ala Cys Thr Gln Asp Gly Pro
Leu545 550 555 560 Tyr Val Ile Val Glu Tyr Ala Ser Lys Gly Asn Leu
Arg Glu Tyr Leu 565 570 575 Arg Ala Arg Arg Pro Pro Gly Met Glu Tyr
Ser Tyr Asp Ile Asn Arg 580 585 590 Val Pro Glu Glu Gln Met Thr Phe
Lys Asp Leu Val Ser Cys Thr Tyr 595 600 605 Gln Leu Ala Arg Gly Met
Glu Tyr Leu Ala Ser Gln Lys Cys Ile His 610 615 620 Arg Asp Leu Ala
Ala Arg Asn Val Leu Val Thr Glu Asn Asn Val Met625 630 635 640 Lys
Ile Ala Asp Phe Gly Leu Ala Arg Asp Ile Asn Asn Ile Asp Tyr 645 650
655 Tyr Lys Lys Thr Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala Pro
660 665 670 Glu Ala Leu Phe Asp Arg Val Tyr Thr His Gln Ser Asp Val
Trp Ser 675 680 685 Phe Gly Val Leu Met Trp Glu Ile Phe Thr Leu Gly
Gly Ser Pro Tyr 690 695 700 Pro Gly Ile Pro Val Glu Glu Leu Phe Lys
Leu Leu Lys Glu Gly His705 710 715 720 Arg Met Asp Lys Pro Ala Asn
Cys Thr Asn Glu Leu Tyr Met Met Met 725 730 735 Arg Asp Cys Trp His
Ala Val Pro Ser Gln Arg Pro Thr Phe Lys Gln 740 745 750 Leu Val Glu
Asp Leu Asp Arg Ile Leu Thr Leu Thr Thr Asn Glu Glu 755 760 765 Tyr
Leu Asp Leu Ser Gln Pro Leu Glu Gln Tyr Ser Pro Ser Tyr Pro 770 775
780 Asp Thr Arg Ser Ser Cys Ser Ser Gly Asp Asp Ser Val Phe Ser
Pro785 790 795 800 Asp Pro Met Pro Tyr Glu Pro Cys Leu Pro Gln Tyr
Pro His Ile Asn 805 810 815 Gly Ser Val Lys Thr 820 60822PRTHomo
sapiens 60Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr
Met Ala1 5 10 15 Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val
Glu Asp Thr Thr 20 25 30 Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr
Gln Ile Ser Gln Pro Glu 35 40 45 Val Tyr Val Ala Ala Pro Gly Glu
Ser Leu Glu Val Arg Cys Leu Leu 50 55 60 Lys Asp Ala Ala Val Ile
Ser Trp Thr Lys Asp Gly Val His Leu Gly65 70 75 80 Pro Asn Asn Arg
Thr Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly 85 90 95 Ala Thr
Pro Arg Asp Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr 100 105 110
Val Asp Ser Glu Thr Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile 115
120 125 Ser Ser Gly Asp Asp Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe
Val 130 135 140 Ser Glu Asn Ser Asn Asn Lys Arg Ala Pro Tyr Trp Thr
Asn Thr Glu145 150 155 160 Lys Met Glu Lys Arg Leu His Ala Val Pro
Ala Ala Asn Thr Val Lys 165 170 175 Phe Arg Cys Pro Ala Gly Gly Asn
Pro Met Pro Thr Met Arg Trp Leu 180 185 190 Lys Asn Gly Lys Glu Phe
Lys Gln Glu His Arg Ile Gly Gly Tyr Lys 195 200 205 Val Arg Asn Gln
His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser 210 215 220 Asp Lys
Gly Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile225 230 235
240 Asn His Thr Tyr His Leu Asp Val Val Glu Arg Ser Pro His Arg Pro
245 250 255 Ile Leu Gln Ala Gly Leu Pro Ala Asn Ala Ser Thr Val Val
Gly Gly 260 265 270 Asp Val Glu Phe Val Cys Lys Val Tyr Ser Asp Ala
Gln Pro His Ile 275 280 285 Gln Trp Ile Lys His Val Glu Lys Asn Gly
Ser Lys Tyr Gly Pro Asp 290 295 300 Gly Leu Pro Tyr Leu Lys Val Leu
Lys His Ser Gly Ile Asn Ser Ser305 310 315 320 Asn Ala Glu Val Leu
Ala Leu Phe Asn Val Thr Glu Ala Asp Ala Gly 325 330 335 Glu Tyr Ile
Cys Lys Val Ser Asn Tyr Ile Gly Gln Ala Asn Gln Ser 340 345 350 Ala
Trp Leu Thr Val Leu Pro Lys Gln Gln Ala Pro Gly Arg Glu Lys 355 360
365 Glu Ile Thr Ala Ser Pro Asp Tyr Leu Glu Ile Ala Ile Tyr Cys Ile
370 375 380 Gly Val Phe Leu Ile Ala Cys Met Val Val Thr Val Ile Leu
Cys Arg385 390 395 400 Met Lys Asn Thr Thr Lys Lys Pro Asp Phe Ser
Ser Gln Pro Ala Val 405 410 415 His Lys Leu Thr Lys Arg Ile Pro Leu
Arg Arg Gln Val Thr Val Ser 420 425 430 Ala Glu Ser Ser Ser Ser Met
Asn Ser Asn Thr Pro Leu Val Arg Ile 435 440 445 Thr Thr Arg Leu Ser
Ser Thr Ala Asp Thr Pro Met Leu Ala Gly Val 450 455 460 Ser Glu Tyr
Glu Leu Pro Glu Asp Pro Lys Trp Glu Phe Pro Arg Asp465 470 475 480
Lys Leu Thr Leu Gly Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val 485
490 495 Val Met Ala Glu Ala Val Gly Ile Asp Lys Asp Lys Pro Lys Glu
Ala 500 505 510 Val Thr Val Ala Val Lys Met Leu Lys Asp Asp Ala Thr
Glu Lys Asp 515 520 525 Leu Ser Asp Leu Val Ser Glu Met Glu Met Met
Lys Met Ile Gly Lys 530 535 540 His Lys Asn Ile Ile Asn Leu Leu Gly
Ala Cys Thr Gln Asp Gly Pro545 550 555 560 Leu Tyr Val Ile Val Glu
Tyr Ala Ser Lys Gly Asn Leu Arg Glu Tyr 565 570 575 Leu Arg Ala Arg
Arg Pro Pro Gly Met Glu Tyr Ser Tyr Asp Ile Asn 580 585 590 Arg Val
Pro Glu Glu Gln Met Thr Phe Lys Asp Leu Val Ser Cys Thr 595 600 605
Tyr Gln Leu Ala Arg Gly Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile 610
615 620 His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Thr Glu Asn Asn
Val625 630 635 640 Met Lys Ile Ala Asp Phe Gly Leu Ala Arg Asp Ile
Asn Asn Ile Asp 645 650 655 Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu
Pro Val Lys Trp Met Ala 660 665 670 Pro Glu Ala Leu Phe Asp Arg Val
Tyr Thr His Gln Ser Asp Val Trp 675 680 685 Ser Phe Gly Val Leu Met
Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro 690 695 700 Tyr Pro Gly Ile
Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly705 710 715 720 His
Arg Met Asp Lys Pro Ala Asn Cys Thr Asn Glu Leu Tyr Met Met 725 730
735 Met Arg Asp Cys Trp His Ala Val Pro Ser Gln Arg Pro Thr Phe Lys
740 745 750 Gln Leu Val Glu Asp Leu Asp Arg Ile Leu Thr Leu Thr Thr
Asn Glu 755 760 765 Glu Tyr Leu Asp Leu Ser Gln Pro Leu Glu Gln Tyr
Ser Pro Ser Tyr 770 775 780 Pro Asp Thr Arg Ser Ser Cys Ser Ser Gly
Asp Asp Ser Val Phe Ser785 790 795 800 Pro Asp Pro Met Pro Tyr Glu
Pro Cys Leu Pro Gln Tyr Pro His Ile 805 810 815 Asn Gly Ser Val Lys
Thr 820 61769PRTHomo sapiens 61Met Val Ser Trp Gly Arg Phe Ile Cys
Leu Val Val Val Thr Met Ala1 5 10 15 Thr Leu Ser Leu Ala Arg Pro
Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30 Leu Glu Pro Glu Glu
Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu 35 40 45 Val Tyr Val
Ala Ala Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu 50 55 60 Lys
Asp Ala Ala Val Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly65 70 75
80 Pro Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly
85 90 95 Ala Thr Pro Arg Asp Ser Gly Leu Tyr Ala Cys Thr Ala Ser
Arg Thr 100 105 110 Val Asp Ser Glu Thr Trp Tyr Phe Met Val Asn Val
Thr Asp Ala Ile 115 120 125 Ser Ser Gly Asp Asp Glu Asp Asp Thr Asp
Gly Ala Glu Asp Phe Val 130 135 140 Ser Glu Asn Ser Asn Asn Lys Arg
Ala Pro Tyr Trp Thr Asn Thr Glu145 150 155 160 Lys Met Glu Lys Arg
Leu His Ala Val Pro Ala Ala Asn Thr Val Lys 165 170 175 Phe Arg Cys
Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu 180 185 190 Lys
Asn Gly Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys 195 200
205 Val Arg Asn Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser
210 215 220 Asp Lys Gly Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly
Ser Ile225 230 235 240 Asn His Thr Tyr His Leu Asp Val Val Glu Arg
Ser Pro His Arg Pro 245 250 255 Ile Leu Gln Ala Gly Leu Pro Ala Asn
Ala Ser Thr Val Val Gly Gly 260 265 270 Asp Val Glu Phe Val Cys Lys
Val Tyr Ser Asp Ala Gln Pro His Ile 275 280 285 Gln Trp Ile Lys His
Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp 290 295 300 Gly Leu Pro
Tyr Leu Lys Val Leu Lys His Ser Gly Ile Asn Ser Ser305 310 315 320
Asn Ala Glu Val Leu Ala Leu Phe Asn Val Thr Glu Ala Asp Ala Gly 325
330 335 Glu Tyr Ile Cys Lys Val Ser Asn Tyr Ile Gly Gln Ala Asn Gln
Ser 340 345 350 Ala Trp Leu Thr Val Leu Pro Lys Gln Gln Ala Pro Gly
Arg Glu Lys 355 360 365 Glu Ile Thr Ala Ser Pro Asp Tyr Leu Glu Ile
Ala Ile Tyr Cys Ile 370 375 380 Gly Val Phe Leu Ile Ala Cys Met Val
Val Thr Val Ile Leu Cys Arg385 390 395 400 Met Lys Asn Thr Thr Lys
Lys Pro Asp Phe Ser Ser Gln Pro Ala Val 405 410 415 His Lys Leu Thr
Lys Arg Ile Pro Leu Arg Arg Gln Val Thr Val Ser 420 425 430 Ala Glu
Ser Ser Ser Ser Met Asn Ser Asn Thr Pro Leu Val Arg Ile 435 440 445
Thr Thr Arg Leu Ser Ser Thr Ala Asp Thr Pro Met Leu Ala Gly Val 450
455 460 Ser Glu Tyr Glu Leu Pro Glu Asp Pro Lys Trp Glu Phe Pro Arg
Asp465 470 475 480 Lys Leu Thr Leu Gly Lys Pro Leu Gly Glu Gly Cys
Phe Gly Gln Val 485 490 495 Val Met Ala Glu Ala Val Gly Ile Asp Lys
Asp Lys Pro Lys Glu Ala 500 505 510 Val Thr Val Ala Val Lys Met Leu
Lys Asp Asp Ala Thr Glu Lys Asp 515 520 525 Leu Ser Asp Leu Val Ser
Glu Met Glu Met Met Lys Met Ile Gly Lys 530 535 540 His Lys Asn Ile
Ile Asn Leu Leu Gly Ala Cys Thr Gln Asp Gly Pro545 550 555 560 Leu
Tyr Val Ile Val Glu Tyr Ala Ser Lys Gly Asn Leu Arg Glu Tyr 565 570
575 Leu Arg Ala Arg Arg Pro Pro Gly Met Glu Tyr Ser Tyr Asp Ile Asn
580 585 590 Arg Val Pro Glu Glu Gln Met Thr Phe Lys Asp Leu Val Ser
Cys Thr 595 600 605 Tyr Gln Leu Ala Arg Gly Met Glu Tyr Leu Ala Ser
Gln Lys Cys Ile 610 615 620 His Arg Asp Leu Ala Ala Arg Asn Val Leu
Val Thr Glu Asn Asn Val625 630 635 640 Met Lys Ile Ala Asp Phe Gly
Leu Ala Arg Asp Ile Asn Asn Ile Asp 645 650 655 Tyr Tyr Lys Lys Thr
Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala 660 665 670 Pro Glu Ala
Leu Phe Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp 675 680 685 Ser
Phe Gly Val Leu Met Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro 690 695
700 Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu
Gly705 710 715 720 His Arg Met Asp Lys Pro Ala Asn Cys Thr Asn Glu
Leu Tyr Met Met 725 730 735 Met Arg Asp Cys Trp His Ala Val Pro Ser
Gln Arg Pro Thr Phe Lys 740 745 750 Gln Leu Val Glu Asp Leu Asp Arg
Ile Leu Thr Leu Thr Thr Asn Glu 755 760 765 Ile 62709PRTHomo
sapiens 62Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr
Met Ala1 5 10 15 Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val
Glu Asp Thr Thr 20 25 30 Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr
Gln Ile Ser Gln Pro Glu 35 40 45 Val Tyr Val Ala Ala Pro Gly Glu
Ser Leu Glu Val Arg Cys Leu Leu 50 55 60 Lys Asp Ala Ala Val Ile
Ser Trp Thr Lys Asp Gly Val His
Leu Gly65 70 75 80 Pro Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr Leu
Gln Ile Lys Gly 85 90 95 Ala Thr Pro Arg Asp Ser Gly Leu Tyr Ala
Cys Thr Ala Ser Arg Thr 100 105 110 Val Asp Ser Glu Thr Trp Tyr Phe
Met Val Asn Val Thr Asp Ala Ile 115 120 125 Ser Ser Gly Asp Asp Glu
Asp Asp Thr Asp Gly Ala Glu Asp Phe Val 130 135 140 Ser Glu Asn Ser
Asn Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu145 150 155 160 Lys
Met Glu Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys 165 170
175 Phe Arg Cys Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu
180 185 190 Lys Asn Gly Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly
Tyr Lys 195 200 205 Val Arg Asn Gln His Trp Ser Leu Ile Met Glu Ser
Val Val Pro Ser 210 215 220 Asp Lys Gly Asn Tyr Thr Cys Val Val Glu
Asn Glu Tyr Gly Ser Ile225 230 235 240 Asn His Thr Tyr His Leu Asp
Val Val Ala Pro Gly Arg Glu Lys Glu 245 250 255 Ile Thr Ala Ser Pro
Asp Tyr Leu Glu Ile Ala Ile Tyr Cys Ile Gly 260 265 270 Val Phe Leu
Ile Ala Cys Met Val Val Thr Val Ile Leu Cys Arg Met 275 280 285 Lys
Asn Thr Thr Lys Lys Pro Asp Phe Ser Ser Gln Pro Ala Val His 290 295
300 Lys Leu Thr Lys Arg Ile Pro Leu Arg Arg Gln Val Thr Val Ser
Ala305 310 315 320 Glu Ser Ser Ser Ser Met Asn Ser Asn Thr Pro Leu
Val Arg Ile Thr 325 330 335 Thr Arg Leu Ser Ser Thr Ala Asp Thr Pro
Met Leu Ala Gly Val Ser 340 345 350 Glu Tyr Glu Leu Pro Glu Asp Pro
Lys Trp Glu Phe Pro Arg Asp Lys 355 360 365 Leu Thr Leu Gly Lys Pro
Leu Gly Glu Gly Cys Phe Gly Gln Val Val 370 375 380 Met Ala Glu Ala
Val Gly Ile Asp Lys Asp Lys Pro Lys Glu Ala Val385 390 395 400 Thr
Val Ala Val Lys Met Leu Lys Asp Asp Ala Thr Glu Lys Asp Leu 405 410
415 Ser Asp Leu Val Ser Glu Met Glu Met Met Lys Met Ile Gly Lys His
420 425 430 Lys Asn Ile Ile Asn Leu Leu Gly Ala Cys Thr Gln Asp Gly
Pro Leu 435 440 445 Tyr Val Ile Val Glu Tyr Ala Ser Lys Gly Asn Leu
Arg Glu Tyr Leu 450 455 460 Arg Ala Arg Arg Pro Pro Gly Met Glu Tyr
Ser Tyr Asp Ile Asn Arg465 470 475 480 Val Pro Glu Glu Gln Met Thr
Phe Lys Asp Leu Val Ser Cys Thr Tyr 485 490 495 Gln Leu Ala Arg Gly
Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile His 500 505 510 Arg Asp Leu
Ala Ala Arg Asn Val Leu Val Thr Glu Asn Asn Val Met 515 520 525 Lys
Ile Ala Asp Phe Gly Leu Ala Arg Asp Ile Asn Asn Ile Asp Tyr 530 535
540 Tyr Lys Lys Thr Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala
Pro545 550 555 560 Glu Ala Leu Phe Asp Arg Val Tyr Thr His Gln Ser
Asp Val Trp Ser 565 570 575 Phe Gly Val Leu Met Trp Glu Ile Phe Thr
Leu Gly Gly Ser Pro Tyr 580 585 590 Pro Gly Ile Pro Val Glu Glu Leu
Phe Lys Leu Leu Lys Glu Gly His 595 600 605 Arg Met Asp Lys Pro Ala
Asn Cys Thr Asn Glu Leu Tyr Met Met Met 610 615 620 Arg Asp Cys Trp
His Ala Val Pro Ser Gln Arg Pro Thr Phe Lys Gln625 630 635 640 Leu
Val Glu Asp Leu Asp Arg Ile Leu Thr Leu Thr Thr Asn Glu Glu 645 650
655 Tyr Leu Asp Leu Ser Gln Pro Leu Glu Gln Tyr Ser Pro Ser Tyr Pro
660 665 670 Asp Thr Arg Ser Ser Cys Ser Ser Gly Asp Asp Ser Val Phe
Ser Pro 675 680 685 Asp Pro Met Pro Tyr Glu Pro Cys Leu Pro Gln Tyr
Pro His Ile Asn 690 695 700 Gly Ser Val Lys Thr705 63707PRTHomo
sapiens 63Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr
Met Ala1 5 10 15 Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val
Glu Asp Thr Thr 20 25 30 Leu Glu Pro Glu Asp Ala Ile Ser Ser Gly
Asp Asp Glu Asp Asp Thr 35 40 45 Asp Gly Ala Glu Asp Phe Val Ser
Glu Asn Ser Asn Asn Lys Arg Ala 50 55 60 Pro Tyr Trp Thr Asn Thr
Glu Lys Met Glu Lys Arg Leu His Ala Val65 70 75 80 Pro Ala Ala Asn
Thr Val Lys Phe Arg Cys Pro Ala Gly Gly Asn Pro 85 90 95 Met Pro
Thr Met Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Gln Glu 100 105 110
His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His Trp Ser Leu Ile 115
120 125 Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys Val
Val 130 135 140 Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His Leu
Asp Val Val145 150 155 160 Glu Arg Ser Pro His Arg Pro Ile Leu Gln
Ala Gly Leu Pro Ala Asn 165 170 175 Ala Ser Thr Val Val Gly Gly Asp
Val Glu Phe Val Cys Lys Val Tyr 180 185 190 Ser Asp Ala Gln Pro His
Ile Gln Trp Ile Lys His Val Glu Lys Asn 195 200 205 Gly Ser Lys Tyr
Gly Pro Asp Gly Leu Pro Tyr Leu Lys Val Leu Lys 210 215 220 Ala Ala
Gly Val Asn Thr Thr Asp Lys Glu Ile Glu Val Leu Tyr Ile225 230 235
240 Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly
245 250 255 Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu Thr Val
Leu Pro 260 265 270 Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser Pro
Asp Tyr Leu Glu 275 280 285 Ile Ala Ile Tyr Cys Ile Gly Val Phe Leu
Ile Ala Cys Met Val Val 290 295 300 Thr Val Ile Leu Cys Arg Met Lys
Asn Thr Thr Lys Lys Pro Asp Phe305 310 315 320 Ser Ser Gln Pro Ala
Val His Lys Leu Thr Lys Arg Ile Pro Leu Arg 325 330 335 Arg Gln Val
Thr Val Ser Ala Glu Ser Ser Ser Ser Met Asn Ser Asn 340 345 350 Thr
Pro Leu Val Arg Ile Thr Thr Arg Leu Ser Ser Thr Ala Asp Thr 355 360
365 Pro Met Leu Ala Gly Val Ser Glu Tyr Glu Leu Pro Glu Asp Pro Lys
370 375 380 Trp Glu Phe Pro Arg Asp Lys Leu Thr Leu Gly Lys Pro Leu
Gly Glu385 390 395 400 Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala
Val Gly Ile Asp Lys 405 410 415 Asp Lys Pro Lys Glu Ala Val Thr Val
Ala Val Lys Met Leu Lys Asp 420 425 430 Asp Ala Thr Glu Lys Asp Leu
Ser Asp Leu Val Ser Glu Met Glu Met 435 440 445 Met Lys Met Ile Gly
Lys His Lys Asn Ile Ile Asn Leu Leu Gly Ala 450 455 460 Cys Thr Gln
Asp Gly Pro Leu Tyr Val Ile Val Glu Tyr Ala Ser Lys465 470 475 480
Gly Asn Leu Arg Glu Tyr Leu Arg Ala Arg Arg Pro Pro Gly Met Glu 485
490 495 Tyr Ser Tyr Asp Ile Asn Arg Val Pro Glu Glu Gln Met Thr Phe
Lys 500 505 510 Asp Leu Val Ser Cys Thr Tyr Gln Leu Ala Arg Gly Met
Glu Tyr Leu 515 520 525 Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala
Ala Arg Asn Val Leu 530 535 540 Val Thr Glu Asn Asn Val Met Lys Ile
Ala Asp Phe Gly Leu Ala Arg545 550 555 560 Asp Ile Asn Asn Ile Asp
Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu 565 570 575 Pro Val Lys Trp
Met Ala Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr 580 585 590 His Gln
Ser Asp Val Trp Ser Phe Gly Val Leu Met Trp Glu Ile Phe 595 600 605
Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe 610
615 620 Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro Ala Asn Cys
Thr625 630 635 640 Asn Glu Leu Tyr Met Met Met Arg Asp Cys Trp His
Ala Val Pro Ser 645 650 655 Gln Arg Pro Thr Phe Lys Gln Leu Val Glu
Asp Leu Asp Arg Ile Leu 660 665 670 Thr Leu Thr Thr Asn Glu Glu Glu
Lys Lys Val Ser Gly Ala Val Asp 675 680 685 Cys His Lys Pro Pro Cys
Asn Pro Ser His Leu Pro Cys Val Leu Ala 690 695 700 Val Asp Gln705
64706PRTHomo sapiens 64Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val
Val Val Thr Met Ala1 5 10 15 Thr Leu Ser Leu Ala Arg Pro Ser Phe
Ser Leu Val Glu Asp Thr Thr 20 25 30 Leu Glu Pro Glu Gly Ala Pro
Tyr Trp Thr Asn Thr Glu Lys Met Glu 35 40 45 Lys Arg Leu His Ala
Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys 50 55 60 Pro Ala Gly
Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly65 70 75 80 Lys
Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn 85 90
95 Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly
100 105 110 Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn
His Thr 115 120 125 Tyr His Leu Asp Val Val Glu Arg Ser Pro His Arg
Pro Ile Leu Gln 130 135 140 Ala Gly Leu Pro Ala Asn Ala Ser Thr Val
Val Gly Gly Asp Val Glu145 150 155 160 Phe Val Cys Lys Val Tyr Ser
Asp Ala Gln Pro His Ile Gln Trp Ile 165 170 175 Lys His Val Glu Lys
Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro 180 185 190 Tyr Leu Lys
Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu 195 200 205 Ile
Glu Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu 210 215
220 Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser
Ala225 230 235 240 Trp Leu Thr Val Leu Pro Ala Pro Gly Arg Glu Lys
Glu Ile Thr Ala 245 250 255 Ser Pro Asp Tyr Leu Glu Ile Ala Ile Tyr
Cys Ile Gly Val Phe Leu 260 265 270 Ile Ala Cys Met Val Val Thr Val
Ile Leu Cys Arg Met Lys Asn Thr 275 280 285 Thr Lys Lys Pro Asp Phe
Ser Ser Gln Pro Ala Val His Lys Leu Thr 290 295 300 Lys Arg Ile Pro
Leu Arg Arg Gln Val Thr Val Ser Ala Glu Ser Ser305 310 315 320 Ser
Ser Met Asn Ser Asn Thr Pro Leu Val Arg Ile Thr Thr Arg Leu 325 330
335 Ser Ser Thr Ala Asp Thr Pro Met Leu Ala Gly Val Ser Glu Tyr Glu
340 345 350 Leu Pro Glu Asp Pro Lys Trp Glu Phe Pro Arg Asp Lys Leu
Thr Leu 355 360 365 Gly Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val
Val Met Ala Glu 370 375 380 Ala Val Gly Ile Asp Lys Asp Lys Pro Lys
Glu Ala Val Thr Val Ala385 390 395 400 Val Lys Met Leu Lys Asp Asp
Ala Thr Glu Lys Asp Leu Ser Asp Leu 405 410 415 Val Ser Glu Met Glu
Met Met Lys Met Ile Gly Lys His Lys Asn Ile 420 425 430 Ile Asn Leu
Leu Gly Ala Cys Thr Gln Asp Gly Pro Leu Tyr Val Ile 435 440 445 Val
Glu Tyr Ala Ser Lys Gly Asn Leu Arg Glu Tyr Leu Arg Ala Arg 450 455
460 Arg Pro Pro Gly Met Glu Tyr Ser Tyr Asp Ile Asn Arg Val Pro
Glu465 470 475 480 Glu Gln Met Thr Phe Lys Asp Leu Val Ser Cys Thr
Tyr Gln Leu Ala 485 490 495 Arg Gly Met Glu Tyr Leu Ala Ser Gln Lys
Cys Ile His Arg Asp Leu 500 505 510 Ala Ala Arg Asn Val Leu Val Thr
Glu Asn Asn Val Met Lys Ile Ala 515 520 525 Asp Phe Gly Leu Ala Arg
Asp Ile Asn Asn Ile Asp Tyr Tyr Lys Lys 530 535 540 Thr Thr Asn Gly
Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu545 550 555 560 Phe
Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp Ser Phe Gly Val 565 570
575 Leu Met Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile
580 585 590 Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly His Arg
Met Asp 595 600 605 Lys Pro Ala Asn Cys Thr Asn Glu Leu Tyr Met Met
Met Arg Asp Cys 610 615 620 Trp His Ala Val Pro Ser Gln Arg Pro Thr
Phe Lys Gln Leu Val Glu625 630 635 640 Asp Leu Asp Arg Ile Leu Thr
Leu Thr Thr Asn Glu Glu Tyr Leu Asp 645 650 655 Leu Ser Gln Pro Leu
Glu Gln Tyr Ser Pro Ser Tyr Pro Asp Thr Arg 660 665 670 Ser Ser Cys
Ser Ser Gly Asp Asp Ser Val Phe Ser Pro Asp Pro Met 675 680 685 Pro
Tyr Glu Pro Cys Leu Pro Gln Tyr Pro His Ile Asn Gly Ser Val 690 695
700 Lys Thr705 65705PRTHomo sapiens 65Met Val Ser Trp Gly Arg Phe
Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15 Thr Leu Ser Leu Ala
Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30 Leu Glu Pro
Glu Glu Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu 35 40 45 Val
Tyr Val Ala Ala Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu 50 55
60 Lys Asp Ala Ala Val Ile Ser Trp Thr Lys Asp Gly Val His Leu
Gly65 70 75 80 Pro Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr Leu Gln
Ile Lys Gly 85 90 95 Ala Thr Pro Arg Asp Ser Gly Leu Tyr Ala Cys
Thr Ala Ser Arg Thr 100 105 110 Val Asp Ser Glu Thr Trp Tyr Phe Met
Val Asn Val Thr Asp Ala Ile 115 120 125 Ser Ser Gly Asp Asp Glu Asp
Asp Thr Asp Gly Ala Glu Asp Phe Val 130 135 140 Ser Glu Asn Ser Asn
Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu145 150 155 160 Lys Met
Glu Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys 165 170 175
Phe Arg Cys Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu 180
185 190 Lys Asn Gly Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr
Lys 195 200 205 Val Arg Asn Gln His Trp Ser Leu Ile Met Glu Ser Val
Val Pro Ser 210 215 220 Asp Lys Gly Asn Tyr Thr Cys Val Val Glu Asn
Glu Tyr Gly Ser Ile225 230 235 240 Asn His Thr Tyr His Leu Asp Val
Val Glu Arg Ser Pro His Arg Pro 245
250 255 Ile Leu Gln Ala Gly Leu Pro Ala Asn Ala Ser Thr Val Val Gly
Gly 260 265 270 Asp Val Glu Phe Val Cys Lys Val Tyr Ser Asp Ala Gln
Pro His Ile 275 280 285 Gln Trp Ile Lys His Val Glu Lys Asn Gly Ser
Lys Tyr Gly Pro Asp 290 295 300 Gly Leu Pro Tyr Leu Lys Val Leu Lys
Val Ser Ala Glu Ser Ser Ser305 310 315 320 Ser Met Asn Ser Asn Thr
Pro Leu Val Arg Ile Thr Thr Arg Leu Ser 325 330 335 Ser Thr Ala Asp
Thr Pro Met Leu Ala Gly Val Ser Glu Tyr Glu Leu 340 345 350 Pro Glu
Asp Pro Lys Trp Glu Phe Pro Arg Asp Lys Leu Thr Leu Gly 355 360 365
Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala 370
375 380 Val Gly Ile Asp Lys Asp Lys Pro Lys Glu Ala Val Thr Val Ala
Val385 390 395 400 Lys Met Leu Lys Asp Asp Ala Thr Glu Lys Asp Leu
Ser Asp Leu Val 405 410 415 Ser Glu Met Glu Met Met Lys Met Ile Gly
Lys His Lys Asn Ile Ile 420 425 430 Asn Leu Leu Gly Ala Cys Thr Gln
Asp Gly Pro Leu Tyr Val Ile Val 435 440 445 Glu Tyr Ala Ser Lys Gly
Asn Leu Arg Glu Tyr Leu Arg Ala Arg Arg 450 455 460 Pro Pro Gly Met
Glu Tyr Ser Tyr Asp Ile Asn Arg Val Pro Glu Glu465 470 475 480 Gln
Met Thr Phe Lys Asp Leu Val Ser Cys Thr Tyr Gln Leu Ala Arg 485 490
495 Gly Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala
500 505 510 Ala Arg Asn Val Leu Val Thr Glu Asn Asn Val Met Lys Ile
Ala Asp 515 520 525 Phe Gly Leu Ala Arg Asp Ile Asn Asn Ile Asp Tyr
Tyr Lys Lys Thr 530 535 540 Thr Asn Gly Arg Leu Pro Val Lys Trp Met
Ala Pro Glu Ala Leu Phe545 550 555 560 Asp Arg Val Tyr Thr His Gln
Ser Asp Val Trp Ser Phe Gly Val Leu 565 570 575 Met Trp Glu Ile Phe
Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro 580 585 590 Val Glu Glu
Leu Phe Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys 595 600 605 Pro
Ala Asn Cys Thr Asn Glu Leu Tyr Met Met Met Arg Asp Cys Trp 610 615
620 His Ala Val Pro Ser Gln Arg Pro Thr Phe Lys Gln Leu Val Glu
Asp625 630 635 640 Leu Asp Arg Ile Leu Thr Leu Thr Thr Asn Glu Glu
Tyr Leu Asp Leu 645 650 655 Ser Gln Pro Leu Glu Gln Tyr Ser Pro Ser
Tyr Pro Asp Thr Arg Ser 660 665 670 Ser Cys Ser Ser Gly Asp Asp Ser
Val Phe Ser Pro Asp Pro Met Pro 675 680 685 Tyr Glu Pro Cys Leu Pro
Gln Tyr Pro His Ile Asn Gly Ser Val Lys 690 695 700 Thr705
66704PRTHomo sapiens 66Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val
Val Val Thr Met Ala1 5 10 15 Thr Leu Ser Leu Ala Arg Pro Ser Phe
Ser Leu Val Glu Asp Thr Thr 20 25 30 Leu Glu Pro Glu Gly Ala Pro
Tyr Trp Thr Asn Thr Glu Lys Met Glu 35 40 45 Lys Arg Leu His Ala
Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys 50 55 60 Pro Ala Gly
Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly65 70 75 80 Lys
Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn 85 90
95 Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly
100 105 110 Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn
His Thr 115 120 125 Tyr His Leu Asp Val Val Glu Arg Ser Pro His Arg
Pro Ile Leu Gln 130 135 140 Ala Gly Leu Pro Ala Asn Ala Ser Thr Val
Val Gly Gly Asp Val Glu145 150 155 160 Phe Val Cys Lys Val Tyr Ser
Asp Ala Gln Pro His Ile Gln Trp Ile 165 170 175 Lys His Val Glu Lys
Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro 180 185 190 Tyr Leu Lys
Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu 195 200 205 Ile
Glu Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu 210 215
220 Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser
Ala225 230 235 240 Trp Leu Thr Val Leu Pro Ala Pro Gly Arg Glu Lys
Glu Ile Thr Ala 245 250 255 Ser Pro Asp Tyr Leu Glu Ile Ala Ile Tyr
Cys Ile Gly Val Phe Leu 260 265 270 Ile Ala Cys Met Val Val Thr Val
Ile Leu Cys Arg Met Lys Asn Thr 275 280 285 Thr Lys Lys Pro Asp Phe
Ser Ser Gln Pro Ala Val His Lys Leu Thr 290 295 300 Lys Arg Ile Pro
Leu Arg Arg Gln Val Ser Ala Glu Ser Ser Ser Ser305 310 315 320 Met
Asn Ser Asn Thr Pro Leu Val Arg Ile Thr Thr Arg Leu Ser Ser 325 330
335 Thr Ala Asp Thr Pro Met Leu Ala Gly Val Ser Glu Tyr Glu Leu Pro
340 345 350 Glu Asp Pro Lys Trp Glu Phe Pro Arg Asp Lys Leu Thr Leu
Gly Lys 355 360 365 Pro Leu Gly Glu Gly Cys Phe Gly Gln Val Val Met
Ala Glu Ala Val 370 375 380 Gly Ile Asp Lys Asp Lys Pro Lys Glu Ala
Val Thr Val Ala Val Lys385 390 395 400 Met Leu Lys Asp Asp Ala Thr
Glu Lys Asp Leu Ser Asp Leu Val Ser 405 410 415 Glu Met Glu Met Met
Lys Met Ile Gly Lys His Lys Asn Ile Ile Asn 420 425 430 Leu Leu Gly
Ala Cys Thr Gln Asp Gly Pro Leu Tyr Val Ile Val Glu 435 440 445 Tyr
Ala Ser Lys Gly Asn Leu Arg Glu Tyr Leu Arg Ala Arg Arg Pro 450 455
460 Pro Gly Met Glu Tyr Ser Tyr Asp Ile Asn Arg Val Pro Glu Glu
Gln465 470 475 480 Met Thr Phe Lys Asp Leu Val Ser Cys Thr Tyr Gln
Leu Ala Arg Gly 485 490 495 Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile
His Arg Asp Leu Ala Ala 500 505 510 Arg Asn Val Leu Val Thr Glu Asn
Asn Val Met Lys Ile Ala Asp Phe 515 520 525 Gly Leu Ala Arg Asp Ile
Asn Asn Ile Asp Tyr Tyr Lys Lys Thr Thr 530 535 540 Asn Gly Arg Leu
Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp545 550 555 560 Arg
Val Tyr Thr His Gln Ser Asp Val Trp Ser Phe Gly Val Leu Met 565 570
575 Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val
580 585 590 Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly His Arg Met Asp
Lys Pro 595 600 605 Ala Asn Cys Thr Asn Glu Leu Tyr Met Met Met Arg
Asp Cys Trp His 610 615 620 Ala Val Pro Ser Gln Arg Pro Thr Phe Lys
Gln Leu Val Glu Asp Leu625 630 635 640 Asp Arg Ile Leu Thr Leu Thr
Thr Asn Glu Glu Tyr Leu Asp Leu Ser 645 650 655 Gln Pro Leu Glu Gln
Tyr Ser Pro Ser Tyr Pro Asp Thr Arg Ser Ser 660 665 670 Cys Ser Ser
Gly Asp Asp Ser Val Phe Ser Pro Asp Pro Met Pro Tyr 675 680 685 Glu
Pro Cys Leu Pro Gln Tyr Pro His Ile Asn Gly Ser Val Lys Thr 690 695
700 67680PRTHomo sapiens 67Met Val Ser Trp Gly Arg Phe Ile Cys Leu
Val Val Val Thr Met Ala1 5 10 15 Thr Leu Ser Leu Ala Arg Pro Ser
Phe Ser Leu Val Glu Asp Thr Thr 20 25 30 Leu Glu Pro Glu Asp Ala
Ile Ser Ser Gly Asp Asp Glu Asp Asp Thr 35 40 45 Asp Gly Ala Glu
Asp Phe Val Ser Glu Asn Ser Asn Asn Lys Arg Ala 50 55 60 Pro Tyr
Trp Thr Asn Thr Glu Lys Met Glu Lys Arg Leu His Ala Val65 70 75 80
Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala Gly Gly Asn Pro 85
90 95 Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Gln
Glu 100 105 110 His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His Trp
Ser Leu Ile 115 120 125 Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn
Tyr Thr Cys Val Val 130 135 140 Glu Asn Glu Tyr Gly Ser Ile Asn His
Thr Tyr His Leu Asp Val Val145 150 155 160 Glu Arg Ser Pro His Arg
Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn 165 170 175 Ala Ser Thr Val
Val Gly Gly Asp Val Glu Phe Val Cys Lys Val Tyr 180 185 190 Ser Asp
Ala Gln Pro His Ile Gln Trp Ile Lys His Val Glu Lys Asn 195 200 205
Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu Lys Val Leu Lys 210
215 220 His Ser Gly Ile Asn Ser Ser Asn Ala Glu Val Leu Ala Leu Phe
Asn225 230 235 240 Val Thr Glu Ala Asp Ala Gly Glu Tyr Ile Cys Lys
Val Ser Asn Tyr 245 250 255 Ile Gly Gln Ala Asn Gln Ser Ala Trp Leu
Thr Val Leu Pro Lys Gln 260 265 270 Gln Ala Pro Gly Arg Glu Lys Glu
Ile Thr Ala Ser Pro Asp Tyr Leu 275 280 285 Glu Ile Ala Ile Tyr Cys
Ile Gly Val Phe Leu Ile Ala Cys Met Val 290 295 300 Val Thr Val Ile
Leu Cys Arg Met Lys Asn Thr Thr Lys Lys Pro Asp305 310 315 320 Phe
Ser Ser Gln Pro Ala Val His Lys Leu Thr Lys Arg Ile Pro Leu 325 330
335 Arg Arg Gln Val Thr Val Ser Ala Glu Ser Ser Ser Ser Met Asn Ser
340 345 350 Asn Thr Pro Leu Val Arg Ile Thr Thr Arg Leu Ser Ser Thr
Ala Asp 355 360 365 Thr Pro Met Leu Ala Gly Val Ser Glu Tyr Glu Leu
Pro Glu Asp Pro 370 375 380 Lys Trp Glu Phe Pro Arg Asp Lys Leu Thr
Leu Gly Lys Pro Leu Gly385 390 395 400 Glu Gly Cys Phe Gly Gln Val
Val Met Ala Glu Ala Val Gly Ile Asp 405 410 415 Lys Asp Lys Pro Lys
Glu Ala Val Thr Val Ala Val Lys Met Leu Lys 420 425 430 Asp Asp Ala
Thr Glu Lys Asp Leu Ser Asp Leu Val Ser Glu Met Glu 435 440 445 Met
Met Lys Met Ile Gly Lys His Lys Asn Ile Ile Asn Leu Leu Gly 450 455
460 Ala Cys Thr Gln Asp Gly Pro Leu Tyr Val Ile Val Glu Tyr Ala
Ser465 470 475 480 Lys Gly Asn Leu Arg Glu Tyr Leu Arg Ala Arg Arg
Pro Pro Gly Met 485 490 495 Glu Tyr Ser Tyr Asp Ile Asn Arg Val Pro
Glu Glu Gln Met Thr Phe 500 505 510 Lys Asp Leu Val Ser Cys Thr Tyr
Gln Leu Ala Arg Gly Met Glu Tyr 515 520 525 Leu Ala Ser Gln Lys Cys
Ile His Arg Asp Leu Ala Ala Arg Asn Val 530 535 540 Leu Val Thr Glu
Asn Asn Val Met Lys Ile Ala Asp Phe Gly Leu Ala545 550 555 560 Arg
Asp Ile Asn Asn Ile Asp Tyr Tyr Lys Lys Thr Thr Asn Gly Arg 565 570
575 Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg Val Tyr
580 585 590 Thr His Gln Ser Asp Val Trp Ser Phe Gly Val Leu Met Trp
Glu Ile 595 600 605 Phe Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro
Val Glu Glu Leu 610 615 620 Phe Lys Leu Leu Lys Glu Gly His Arg Met
Asp Lys Pro Ala Asn Cys625 630 635 640 Thr Asn Glu Leu Tyr Met Met
Met Arg Asp Cys Trp His Ala Val Pro 645 650 655 Ser Gln Arg Pro Thr
Phe Lys Gln Leu Val Glu Asp Leu Asp Arg Ile 660 665 670 Leu Thr Leu
Thr Thr Asn Glu Ile 675 680 68396PRTHomo sapiens 68Met Val Ser Trp
Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15 Thr Leu
Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30
Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu 35
40 45 Val Tyr Val Ala Ala Pro Gly Glu Ser Leu Glu Val Arg Cys Leu
Leu 50 55 60 Lys Asp Ala Ala Val Ile Ser Trp Thr Lys Asp Gly Val
His Leu Gly65 70 75 80 Pro Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr
Leu Gln Ile Lys Gly 85 90 95 Ala Thr Pro Arg Asp Ser Gly Leu Tyr
Ala Cys Thr Ala Ser Arg Thr 100 105 110 Val Asp Ser Glu Thr Trp Tyr
Phe Met Val Asn Val Thr Asp Ala Ile 115 120 125 Ser Ser Gly Asp Asp
Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val 130 135 140 Ser Glu Asn
Ser Asn Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu145 150 155 160
Lys Thr Glu Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys 165
170 175 Phe Arg Cys Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp
Leu 180 185 190 Lys Asn Gly Lys Glu Phe Lys Gln Glu His Arg Ile Gly
Gly Tyr Lys 195 200 205 Val Arg Asn Gln His Trp Ser Leu Ile Met Glu
Ser Val Val Pro Ser 210 215 220 Asp Lys Gly Asn Tyr Thr Cys Val Val
Glu Asn Glu Tyr Gly Ser Ile225 230 235 240 Asn His Thr Tyr His Leu
Asp Val Val Glu Arg Ser Pro His Arg Pro 245 250 255 Ile Leu Gln Ala
Gly Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly 260 265 270 Asp Val
Glu Phe Val Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile 275 280 285
Gln Trp Ile Lys His Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp 290
295 300 Gly Leu Pro Tyr Leu Lys Val Leu Lys Ala Ala Gly Val Asn Thr
Thr305 310 315 320 Asp Lys Glu Ile Glu Val Leu Tyr Ile Arg Asn Val
Thr Phe Glu Asp 325 330 335 Ala Gly Glu Tyr Thr Cys Leu Ala Gly Asn
Ser Ile Gly Ile Ser Phe 340 345 350 His Ser Ala Trp Leu Thr Val Leu
Pro Gly Ile Tyr Cys Ser Phe Ser 355 360 365 Leu Gly Phe Phe Pro Phe
Ser Trp Leu Thr Ala Ile Lys Leu Thr Gln 370 375 380 Leu Leu Leu Ser
Glu Met Ala Pro Phe Ile Leu Ala385 390 395 69317PRTHomo sapiens
69Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1
5 10 15 Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr
Thr 20 25 30 Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr Gln Ile Ser
Gln Pro Glu 35 40 45 Val Tyr Val Ala Ala Pro Gly Glu Ser Leu Glu
Val Arg Cys Leu Leu 50 55 60 Lys Asp Ala Ala Val Ile Ser Trp Thr
Lys Asp Gly Val His
Leu Gly65 70 75 80 Pro Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr Leu
Gln Ile Lys Gly 85 90 95 Ala Thr Pro Arg Asp Ser Gly Leu Tyr Ala
Cys Thr Ala Ser Arg Thr 100 105 110 Val Asp Ser Glu Thr Trp Tyr Phe
Met Val Asn Val Thr Asp Ala Ile 115 120 125 Ser Ser Gly Asp Asp Glu
Asp Asp Thr Asp Gly Ala Glu Asp Phe Val 130 135 140 Ser Glu Asn Ser
Asn Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu145 150 155 160 Lys
Met Glu Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys 165 170
175 Phe Arg Cys Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu
180 185 190 Lys Asn Gly Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly
Tyr Lys 195 200 205 Val Arg Asn Gln His Trp Ser Leu Ile Met Glu Ser
Val Val Pro Ser 210 215 220 Asp Lys Gly Asn Tyr Thr Cys Val Val Glu
Asn Glu Tyr Gly Ser Ile225 230 235 240 Asn His Thr Tyr His Leu Asp
Val Val Glu Arg Ser Pro His Arg Pro 245 250 255 Ile Leu Gln Ala Gly
Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly 260 265 270 Asp Val Glu
Phe Val Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile 275 280 285 Gln
Trp Ile Lys His Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp 290 295
300 Gly Leu Pro Tyr Leu Lys Val Leu Lys Val Arg Thr Phe305 310 315
70266PRTHomo sapiens 70Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val
Val Val Thr Met Ala1 5 10 15 Thr Leu Ser Leu Ala Arg Pro Ser Phe
Ser Leu Val Glu Asp Thr Thr 20 25 30 Leu Glu Pro Glu Glu Pro Pro
Thr Lys Tyr Gln Ile Ser Gln Pro Glu 35 40 45 Val Tyr Val Ala Ala
Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu 50 55 60 Lys Asp Ala
Ala Val Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly65 70 75 80 Pro
Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly 85 90
95 Ala Thr Pro Arg Asp Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr
100 105 110 Val Asp Ser Glu Thr Trp Tyr Phe Met Val Asn Val Thr Asp
Ala Ile 115 120 125 Ser Ser Gly Asp Asp Glu Asp Asp Thr Asp Gly Ala
Glu Asp Phe Val 130 135 140 Ser Glu Asn Ser Asn Asn Lys Arg Ala Pro
Tyr Trp Thr Asn Thr Glu145 150 155 160 Lys Met Glu Lys Arg Leu His
Ala Val Pro Ala Ala Asn Thr Val Lys 165 170 175 Phe Arg Cys Pro Ala
Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu 180 185 190 Lys Asn Gly
Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys 195 200 205 Val
Arg Asn Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser 210 215
220 Asp Lys Gly Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser
Ile225 230 235 240 Asn His Thr Tyr His Leu Asp Val Val Gly Glu Ser
Ala Ser Pro Arg 245 250 255 Val Ala Ala Ala Tyr Gln Pro Ile Leu Ala
260 265 71336DNAMus musculus 71caggtgaagc tgcaggagtc tggcgctgag
ttggtgaaac ctggggcttc agtgaagata 60tcctgcaagg cttctggcta catcttcact
gaccatgctc ttcactgggt gaggcagaag 120cctgaacagg gcctggaatg
gattgggtat atttttcccg gaaatggtaa tattgagtac 180aatgagaagt
tcaagggcaa ggccacactg actgcagaca aatcctccag tactgcctac
240atgcagctca acagcctgac atctggagat tctgcaatgt atttctgtaa
aaagatggac 300tactggggcc aagggaccac ggtcaccgtc tcctca
33672112PRTMus musculus 72Gln Val Lys Leu Gln Glu Ser Gly Ala Glu
Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Ile Phe Thr Asp His 20 25 30 Ala Leu His Trp Val Arg
Gln Lys Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Phe
Pro Gly Asn Gly Asn Ile Glu Tyr Asn Glu Lys Phe 50 55 60 Lys Gly
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Gln Leu Asn Ser Leu Thr Ser Gly Asp Ser Ala Met Tyr Phe Cys 85
90 95 Lys Lys Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser 100 105 110 73336DNAMus musculus 73caggtgaagc tgcaggagtc
tggcgctgag ttggtgaaac ctggggcttc agtgaagatc 60tcctgcaagg cttctggtta
caccttcact gaccattcta ttcactgggt gaagcagaag 120cctggacagg
gcctagaatg gattggatat ctttttcccg gaaatggtaa ttttgaatat
180aatgagaaat tcaagggcaa ggccacactg actgcagaca aatcctccag
cactgcctac 240atgcacctca acagcctgac atctgaggat tctgcagtgt
atttctgtaa aaagatggac 300tactggggcc aagggaccac ggtcaccgtc tcctca
33674112PRTMus musculus 74Gln Val Lys Leu Gln Glu Ser Gly Ala Glu
Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Ser Ile His Trp Val Lys
Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Leu Phe
Pro Gly Asn Gly Asn Phe Glu Tyr Asn Glu Lys Phe 50 55 60 Lys Gly
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85
90 95 Lys Lys Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser 100 105 110 75336DNAMus musculus 75caggttcagc tgcagcagtc
cgacgctgag ttggtgaaac ctggggcttc agtgaagata 60tcctgcaggg cttctggcta
caccttcact gaccattcta ttcactgggt gaagcagcag 120cctggccagg
gcctggaatg gatcggatat atttttcccg gaaatggaaa tattgaatac
180aatgacaaat tcaagggcaa ggccacactg actgcagaca aatcctccgg
cactgcctac 240atgcagctca acagcctgac atctgaggat tctgcagtgt
atttctgtaa aaggatgggg 300tactggggtc aaggaacctc agtcaccgtc tcctca
33676112PRTMus musculus 76Gln Val Gln Leu Gln Gln Ser Asp Ala Glu
Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Arg Ala
Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Ser Ile His Trp Val Lys
Gln Gln Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Phe
Pro Gly Asn Gly Asn Ile Glu Tyr Asn Asp Lys Phe 50 55 60 Lys Gly
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Gly Thr Ala Tyr 65 70 75 80
Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85
90 95 Lys Arg Met Gly Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
Ser 100 105 110 77336DNAMus musculus 77caggtcaagc tgcaggagtc
tggcgctgag ttggtgaaac ctggggcttc agtgaagatc 60tcctgcaagg cttctggcta
caccttcact gaccattcta ttcactgggt gaagcagaag 120cctggacagg
gcctagaatg gattggatat ctttttcccg gaaatggtaa ttttgagtac
180aatgaaaaat tcaagggcaa ggccacactg actgcagaca aatcctccag
cactgtctac 240atgtacctca acagcctgac atctgaggat tctgcagtgt
atttctgtaa aaggatgggg 300tactggggcc aagggaccac ggtcaccgtc tcctca
33678112PRTMus musculus 78Gln Val Lys Leu Gln Glu Ser Gly Ala Glu
Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Ser Ile His Trp Val Lys
Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Leu Phe
Pro Gly Asn Gly Asn Phe Glu Tyr Asn Glu Lys Phe 50 55 60 Lys Gly
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr 65 70 75 80
Met Tyr Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85
90 95 Lys Arg Met Gly Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser 100 105 110 79336DNAMus musculus 79caggtcaagc tgcaggagtc
tggacctgaa ctggtaaagc ctggggcttc agtgaagatg 60tcctgcaagg cttctggata
cacattcact aactatgtta tacactgggt gaagcaaaag 120cctgggcagg
gccttgagtg gattggatat attaatcctt acaatgatgg ctctaagtac
180aatgagaagt tcaaaggcaa ggcctcactg acttcagaca aatcctccag
cacagcctac 240atggagctca gcagcctgac ctctgaggac tctgcggtct
attactgtgc aagaatggac 300tactggggcc aagggaccac ggtcaccgtc tcctca
33680112PRTMus musculus 80Gln Val Gln Leu Gln Glu Ser Gly Pro Glu
Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Ile His Trp Val Lys
Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn
Pro Tyr Asn Asp Gly Ser Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly
Lys Ala Ser Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Met Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
Ser 100 105 110 81336DNAMus musculus 81caggtcaagc tgcaggagtc
tggacctgaa ctggtaaagc ctggggcttc agtgaagatg 60tcctgcaagg cttctggata
cacattcact aactatgtta tacactgggt gaagcaaaag 120cctgggcagg
gccttgagtg gattggatat attaatcctt acaatgatgg ctctaagtac
180aatgagaagt tcaaaggcaa ggcctcactg acttcagaca aatcctccag
cacagcctac 240atggagctca gcagcctgac ctctgaggac tctgcggtct
attactgtgc aagaatgggg 300tactggggcc aagggaccac ggtcaccgtc tcctca
33682112PRTMus musculus 82Gln Val Gln Leu Gln Glu Ser Gly Pro Glu
Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Ile His Trp Val Lys
Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn
Pro Tyr Asn Asp Gly Ser Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly
Lys Ala Ser Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Met Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
Ser 100 105 110 83342DNAMus musculus 83gatgttttga tgacccaaac
tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctagtca
gagcattgta catagtaatg gaaacaccta tttagaatgg 120tacctgcaga
aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt
180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac
actcaagatc 240agcagagtgg aggctgagga tctgggagtt tattactgct
ttcaaggttc acatgttcct 300cctacgttcg gtgctgggac caagctggag
ctgaaacggg ct 34284113PRTMus musculus 84Asp Val Leu Met Thr Gln Thr
Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15 Asp Gln Ala Ser Ile
Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn
Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro
Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys
Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Ala Gly Thr
Lys Leu Glu Leu Lys 100 105 110 Arg85324DNAMus musculus
85gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc
60atcacatgtc gaacaactga aaatatttac agttattttg tatggtctca gcagagacag
120ggaaaatctc ctcagctccg ggtctataat gcaaaatcct tagcagaagg
tgtgccatca 180agtttcaatg tcagtgtatc aggcacacag ttttctctga
agatcaatag cctgcagcct 240gaagattttg ggacttatca ctgtcaacac
cattatggta ctccgtacac gttcggaggg 300gggaccaggc tggaaataag acgg
32486108PRTMus musculus 86Asp Ile Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Ala Ser Val Gly1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg
Thr Thr Glu Asn Ile Tyr Ser Tyr 20 25 30 Phe Val Trp Ser Gln Gln
Arg Gln Gly Lys Ser Pro Gln Leu Arg Val 35 40 45 Tyr Asn Ala Lys
Ser Leu Ala Glu Gly Val Pro Ser Ser Phe Asn Val 50 55 60 Ser Val
Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro65 70 75 80
Glu Asp Phe Gly Thr Tyr His Cys Gln His His Tyr Gly Thr Pro Tyr 85
90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Arg Arg 100 105
87339DNAMus musculus 87gatgttttg atgacccaaac tccactcact ttgtcggtta
ccattggaca accagcttcc 60atctcttgc aagtccagtca gagcctctta tatactaatg
gaaaaaccta tttgacttgg 120ttattccag aggccaggcca gtctccaaaa
cgcctaatct atctggtgtc tgaattggac 180tctggagtc cctgacaggtt
cagtggcagt ggttcaggga cagatttcac actggaaatc 240accagagtg
gaggctgagga tttgggagtt tattactgct tgcagagtgc acattttcca
300ttcacgttc ggctcgggcac caagctggaa atcaaacgg 33988113PRTMus
musculus 88Asp Val Leu Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr
Ile Gly1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser
Leu Leu Tyr Thr 20 25 30 Asn Gly Lys Thr Tyr Leu Thr Trp Leu Phe
Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val
Ser Glu Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Glu Ile65 70 75 80 Thr Arg Val Glu
Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ser 85 90 95 Ala His
Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110
Arg89339DNAMus musculus 89gatgttgtga tgacccaaac tccactcact
ctgtcggtga ccattggaca accagcgttc 60atctcttgca agtccagtca gagcctcttt
aacactaatg gcaaaaccta tttgacttgg 120ttaattcaga ggccaggcca
gtctccacag cgcctgatct atctggtgtc caaattggac 180tctggcgtcc
cggacaggtt cagtggcagt ggctcaggga cagatttcac actgaaaatc
240agcagagtgg aggctgagga tctgggagtt tattactgcc tgcagagtag
ccattttccg 300tttacgttcg gctcgggcac caagctggaa atcaaacgg
33990113PRTMus musculus 90Asp Val Val Met Thr Gln Thr Pro Leu Thr
Leu Ser Val Thr Ile Gly1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys
Ser Ser Gln Ser Leu Leu Asn Thr 20 25 30 Asn Gly Lys Thr Tyr Leu
Thr Trp Leu Ile Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Arg Leu
Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ser 85
90 95 Ser His Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
Lys 100 105 110 Arg 91327DNAMus musculus 91gatgttgtgc taactcagtc
tcctgccacc ctgtctgtga ctccaggaga tagagtcagt 60ctttcctgca gggccagcca
aaatattggc aactacctac actggtatca acagaaatca 120catgagtctc
caaggcttct catcaagtat gcttcccagt ccatctctgg gatcccctcc
180aggttcagtg gcagtggatc agtcacagat ttcactctca atatcaacag
tgtggagact 240gaagattttg gaatgtattt ctgtcaacag agtgacacct
ggcctctcac gttcggtgct 300gggaccaagc tggagctgaa acgggct
32792108PRTMus musculus 92Asp Val Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Val Thr Pro Gly1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg
Ala Ser Gln Asn Ile Gly Asn Tyr 20 25 30 Leu His Trp Tyr Gln Gln
Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser
Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Val Thr Asp Phe Thr Leu Asn Ile Asn Ser Val Glu Thr65 70 75 80
Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asp Thr Trp Pro Leu 85
90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105
938PRTMus musculus 93Thr Phe Thr Asp His Ser Ile His1 5 948PRTMus
musculus 94Thr Phe Thr Asn Tyr Val Ile His1 5 958PRTMus musculus
95Ile Phe Thr Asp His Ala Leu His1 5 9617PRTMus musculus 96Tyr Ile
Phe Pro Gly Asn Gly Asn Ile Glu Tyr Asn Asp Lys Phe Lys1 5 10 15
Gly9717PRTMus musculus 97Tyr Leu Phe Pro Gly Asn Gly Asn Phe Glu
Tyr Asn Glu Lys Phe Lys1 5 10 15 Gly9817PRTMus musculus 98Tyr Ile
Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Asn Glu Lys Phe Lys1 5 10 15
Gly9917PRTMus musculus 99Tyr Ile Phe Pro Gly Asn Gly Asn Ile Glu
Tyr Asn Glu Lys Phe Lys1 5 10 15 Gly1005PRTMus musculus 100Lys Arg
Met Gly Tyr1 5 1015PRTMus musculus 101Lys Lys Met Asp Tyr1 5
1025PRTMus musculus 102Ala Arg Met Asp Tyr1 5 10316PRTMus musculus
103Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu1
5 10 15 10411PRTMus musculus 104Arg Thr Thr Glu Asn Ile Tyr Ser Tyr
Phe Val1 5 10 10516PRTMus musculus 105Lys Ser Ser Gln Ser Leu Leu
Tyr Thr Asn Gly Lys Thr Tyr Leu Thr1 5 10 15 10616PRTMus musculus
106Lys Ser Ser Gln Ser Leu Leu Asn Thr Asn Gly Lys Thr Tyr Leu Thr1
5 10 15 10711PRTMus musculus 107Arg Ala Ser Gln Asn Ile Gly Asn Tyr
Leu His1 5 10 1087PRTMus musculus 108Lys Val Ser Asn Arg Phe Ser1 5
1097PRTMus musculus 109Asn Ala Lys Ser Leu Ala Glu1 5 1107PRTMus
musculus 110Leu Val Ser Glu Leu Asp Ser1 5 1117PRTMus musculus
111Leu Val Ser Lys Leu Asp Ser1 5 1127PRTMus musculus 112Tyr Ala
Ser Gln Ser Ile Ser1 5 1139PRTMus musculus 113Phe Gln Gly Ser His
Val Pro Pro Thr1 5 1149PRTMus musculus 114Gln His His Tyr Gly Thr
Pro Tyr Thr1 5 1159PRTMus musculus 115Leu Gln Ser Ala His Phe Pro
Phe Thr1 5 1169PRTMus musculus 116Leu Gln Ser Ser His Phe Pro Phe
Thr1 5 1179PRTMus musculus 117Gln Gln Ser Asp Thr Trp Pro Leu Thr1
5 1185PRTMus musculus 118Asp His Ala Leu His1 5 1195PRTMus musculus
119Asp His Ser Ile His1 5 1205PRTMus musculus 120Asn Tyr Val Ile
His1 5 1219PRTMus musculus 121Ile Phe Pro Gly Asn Gly Asn Ile Glu1
5 1229PRTMus musculus 122Leu Phe Pro Gly Asn Gly Asn Phe Glu1 5
1239PRTMus musculus 123Ile Asn Pro Tyr Asn Asp Gly Ser Lys1 5
12411PRTMus musculus 124His Leu Ala Asn Thr Tyr Tyr Tyr Phe Asp
Tyr1 5 10 1255PRTMus musculus 125Ser Asn Gly Asn Thr1 5 1265PRTMus
musculus 126Glu Asn Ile Tyr Ser1 5 1275PRTMus musculus 127Thr Ser
Gly Tyr Ser1 5 1285PRTMus musculus 128Gln Asp Ile Lys Ser1 5
1295PRTMus musculus 129Gln Asn Ile Gly Asn1 5 1304654DNAHomo
sapiens 130ggcggcggct ggaggagagc gcggtggaga gccgagcggg cgggcggcgg
gtgcggagcg 60ggcgagggag cgcgcgcggc cgccacaaag ctcgggcgcc gcggggctgc
atgcggcgta 120cctggcccgg cgcggcgact gctctccggg ctggcggggg
ccggccgcga gccccggggg 180ccccgaggcc gcagcttgcc tgcgcgctct
gagccttcgc aactcgcgag caaagtttgg 240tggaggcaac gccaagcctg
agtcctttct tcctctcgtt ccccaaatcc gagggcagcc 300cgcgggcgtc
atgcccgcgc tcctccgcag cctggggtac gcgtgaagcc cgggaggctt
360ggcgccggcg aagacccaag gaccactctt ctgcgtttgg agttgctccc
cgcaaccccg 420ggctcgtcgc tttctccatc ccgacccacg cggggcgcgg
ggacaacaca ggtcgcggag 480gagcgttgcc attcaagtga ctgcagcagc
agcggcagcg cctcggttcc tgagcccacc 540gcaggctgaa ggcattgcgc
gtagtccatg cccgtagagg aagtgtgcag atgggattaa 600cgtccacatg
gagatatgga agaggaccgg ggattggtac cgtaaccatg gtcagctggg
660gtcgtttcat ctgcctggtc gtggtcacca tggcaacctt gtccctggcc
cggccctcct 720tcagtttagt tgaggatacc acattagagc cagaagagcc
accaaccaaa taccaaatct 780ctcaaccaga agtgtacgtg gctgcgccag
gggagtcgct agaggtgcgc tgcctgttga 840aagatgccgc cgtgatcagt
tggactaagg atggggtgca cttggggccc aacaatagga 900cagtgcttat
tggggagtac ttgcagataa agggcgccac gcctagagac tccggcctct
960atgcttgtac tgccagtagg actgtagaca gtgaaacttg gtacttcatg
gtgaatgtca 1020cagatgccat ctcatccgga gatgatgagg atgacaccga
tggtgcggaa gattttgtca 1080gtgagaacag taacaacaag agagcaccat
actggaccaa cacagaaaag atggaaaagc 1140ggctccatgc tgtgcctgcg
gccaacactg tcaagtttcg ctgcccagcc ggggggaacc 1200caatgccaac
catgcggtgg ctgaaaaacg ggaaggagtt taagcaggag catcgcattg
1260gaggctacaa ggtacgaaac cagcactgga gcctcattat ggaaagtgtg
gtcccatctg 1320acaagggaaa ttatacctgt gtagtggaga atgaatacgg
gtccatcaat cacacgtacc 1380acctggatgt tgtggagcga tcgcctcacc
ggcccatcct ccaagccgga ctgccggcaa 1440atgcctccac agtggtcgga
ggagacgtag agtttgtctg caaggtttac agtgatgccc 1500agccccacat
ccagtggatc aagcacgtgg aaaagaacgg cagtaaatac gggcccgacg
1560ggctgcccta cctcaaggtt ctcaaggccg ccggtgttaa caccacggac
aaagagattg 1620aggttctcta tattcggaat gtaacttttg aggacgctgg
ggaatatacg tgcttggcgg 1680gtaattctat tgggatatcc tttcactctg
catggttgac agttctgcca gcgcctggaa 1740gagaaaagga gattacagct
tccccagact acctggagat agccatttac tgcatagggg 1800tcttcttaat
cgcctgtatg gtggtaacag tcatcctgtg ccgaatgaag aacacgacca
1860agaagccaga cttcagcagc cagccggctg tgcacaagct gaccaaacgt
atccccctgc 1920ggagacaggt aacagtttcg gctgagtcca gctcctccat
gaactccaac accccgctgg 1980tgaggataac aacacgcctc tcttcaacgg
cagacacccc catgctggca ggggtctccg 2040agtatgaact tccagaggac
ccaaaatggg agtttccaag agataagctg acactgggca 2100agcccctggg
agaaggttgc tttgggcaag tggtcatggc ggaagcagtg ggaattgaca
2160aagacaagcc caaggaggcg gtcaccgtgg ccgtgaagat gttgaaagat
gatgccacag 2220agaaagacct ttctgatctg gtgtcagaga tggagatgat
gaagatgatt gggaaacaca 2280agaatatcat aaatcttctt ggagcctgca
cacaggatgg gcctctctat gtcatagttg 2340agtatgcctc taaaggcaac
ctccgagaat acctccgagc ccggaggcca cccgggatgg 2400agtactccta
tgacattaac cgtgttcctg aggagcagat gaccttcaag gacttggtgt
2460catgcaccta ccagctggcc agaggcatgg agtacttggc ttcccaaaaa
tgtattcatc 2520gagatttagc agccagaaat gttttggtaa cagaaaacaa
tgtgatgaaa atagcagact 2580ttggactcgc cagagatatc aacaatatag
actattacaa aaagaccacc aatgggcggc 2640ttccagtcaa gtggatggct
ccagaagccc tgtttgatag agtatacact catcagagtg 2700atgtctggtc
cttcggggtg ttaatgtggg agatcttcac tttagggggc tcgccctacc
2760cagggattcc cgtggaggaa ctttttaagc tgctgaagga aggacacaga
atggataagc 2820cagccaactg caccaacgaa ctgtacatga tgatgaggga
ctgttggcat gcagtgccct 2880cccagagacc aacgttcaag cagttggtag
aagacttgga tcgaattctc actctcacaa 2940ccaatgagga atacttggac
ctcagccaac ctctcgaaca gtattcacct agttaccctg 3000acacaagaag
ttcttgttct tcaggagatg attctgtttt ttctccagac cccatgcctt
3060acgaaccatg ccttcctcag tatccacaca taaacggcag tgttaaaaca
tgaatgactg 3120tgtctgcctg tccccaaaca ggacagcact gggaacctag
ctacactgag cagggagacc 3180atgcctccca gagcttgttg tctccacttg
tatatatgga tcagaggagt aaataattgg 3240aaaagtaatc agcatatgtg
taaagattta tacagttgaa aacttgtaat cttccccagg 3300aggagaagaa
ggtttctgga gcagtggact gccacaagcc accatgtaac ccctctcacc
3360tgccgtgcgt actggctgtg gaccagtagg actcaaggtg gacgtgcgtt
ctgccttcct 3420tgttaatttt gtaataattg gagaagattt atgtcagcac
acacttacag agcacaaatg 3480cagtatatag gtgctggatg tatgtaaata
tattcaaatt atgtataaat atatattata 3540tatttacaag gagttatttt
ttgtattgat tttaaatgga tgtcccaatg cacctagaaa 3600attggtctct
ctttttttaa tagctatttg ctaaatgctg ttcttacaca taatttctta
3660attttcaccg agcagaggtg gaaaaatact tttgctttca gggaaaatgg
tataacgtta 3720atttattaat aaattggtaa tatacaaaac aattaatcat
ttatagtttt ttttgtaatt 3780taagtggcat ttctatgcag gcagcacagc
agactagtta atctattgct tggacttaac 3840tagttatcag atcctttgaa
aagagaatat ttacaatata tgactaattt ggggaaaatg 3900aagttttgat
ttatttgtgt ttaaatgctg ctgtcagacg attgttctta gacctcctaa
3960atgccccata ttaaaagaac tcattcatag gaaggtgttt cattttggtg
tgcaaccctg 4020tcattacgtc aacgcaacgt ctaactggac ttcccaagat
aaatggtacc agcgtcctct 4080taaaagatgc cttaatccat tccttgagga
cagaccttag ttgaaatgat agcagaatgt 4140gcttctctct ggcagctggc
cttctgcttc tgagttgcac attaatcaga ttagcctgta 4200ttctcttcag
tgaattttga taatggcttc cagactcttt ggcgttggag acgcctgtta
4260ggatcttcaa gtcccatcat agaaaattga aacacagagt tgttctgctg
atagttttgg 4320ggatacgtcc atctttttaa gggattgctt tcatctaatt
ctggcaggac ctcaccaaaa 4380gatccagcct catacctaca tcagacaaaa
tatcgccgtt gttccttctg tactaaagta 4440ttgtgttttg ctttggaaac
acccactcac tttgcaatag ccgtgcaaga tgaatgcaga 4500ttacactgat
cttatgtgtt acaaaattgg agaaagtatt taataaaacc tgttaatttt
4560tatactgaca ataaaaatgt ttctacagat attaatgtta acaagacaaa
ataaatgtca 4620cgcaacttat ttttttaata aaaaaaaaaa aaaa
46541314657DNAHomo sapiens 131ggcggcggct ggaggagagc gcggtggaga
gccgagcggg cgggcggcgg gtgcggagcg 60ggcgagggag cgcgcgcggc cgccacaaag
ctcgggcgcc gcggggctgc atgcggcgta 120cctggcccgg cgcggcgact
gctctccggg ctggcggggg ccggccgcga gccccggggg 180ccccgaggcc
gcagcttgcc tgcgcgctct gagccttcgc aactcgcgag caaagtttgg
240tggaggcaac gccaagcctg agtcctttct tcctctcgtt ccccaaatcc
gagggcagcc 300cgcgggcgtc atgcccgcgc tcctccgcag cctggggtac
gcgtgaagcc cgggaggctt 360ggcgccggcg aagacccaag gaccactctt
ctgcgtttgg agttgctccc cgcaaccccg 420ggctcgtcgc tttctccatc
ccgacccacg cggggcgcgg ggacaacaca ggtcgcggag 480gagcgttgcc
attcaagtga ctgcagcagc agcggcagcg cctcggttcc tgagcccacc
540gcaggctgaa ggcattgcgc gtagtccatg cccgtagagg aagtgtgcag
atgggattaa 600cgtccacatg gagatatgga agaggaccgg ggattggtac
cgtaaccatg gtcagctggg 660gtcgtttcat ctgcctggtc gtggtcacca
tggcaacctt gtccctggcc cggccctcct 720tcagtttagt tgaggatacc
acattagagc cagaagagcc accaaccaaa taccaaatct 780ctcaaccaga
agtgtacgtg gctgcgccag gggagtcgct agaggtgcgc tgcctgttga
840aagatgccgc cgtgatcagt tggactaagg atggggtgca cttggggccc
aacaatagga 900cagtgcttat tggggagtac ttgcagataa agggcgccac
gcctagagac tccggcctct 960atgcttgtac tgccagtagg actgtagaca
gtgaaacttg gtacttcatg gtgaatgtca 1020cagatgccat ctcatccgga
gatgatgagg atgacaccga tggtgcggaa gattttgtca 1080gtgagaacag
taacaacaag agagcaccat actggaccaa cacagaaaag atggaaaagc
1140ggctccatgc tgtgcctgcg gccaacactg tcaagtttcg ctgcccagcc
ggggggaacc 1200caatgccaac catgcggtgg ctgaaaaacg ggaaggagtt
taagcaggag catcgcattg 1260gaggctacaa ggtacgaaac cagcactgga
gcctcattat ggaaagtgtg gtcccatctg 1320acaagggaaa ttatacctgt
gtagtggaga atgaatacgg gtccatcaat cacacgtacc 1380acctggatgt
tgtggagcga tcgcctcacc ggcccatcct ccaagccgga ctgccggcaa
1440atgcctccac agtggtcgga ggagacgtag agtttgtctg caaggtttac
agtgatgccc 1500agccccacat ccagtggatc aagcacgtgg aaaagaacgg
cagtaaatac gggcccgacg 1560ggctgcccta cctcaaggtt ctcaagcact
cggggataaa tagttccaat gcagaagtgc 1620tggctctgtt caatgtgacc
gaggcggatg ctggggaata tatatgtaag gtctccaatt 1680atatagggca
ggccaaccag tctgcctggc tcactgtcct gccaaaacag caagcgcctg
1740gaagagaaaa ggagattaca gcttccccag actacctgga gatagccatt
tactgcatag 1800gggtcttctt aatcgcctgt atggtggtaa cagtcatcct
gtgccgaatg aagaacacga 1860ccaagaagcc agacttcagc agccagccgg
ctgtgcacaa gctgaccaaa cgtatccccc 1920tgcggagaca ggtaacagtt
tcggctgagt ccagctcctc catgaactcc aacaccccgc 1980tggtgaggat
aacaacacgc ctctcttcaa cggcagacac ccccatgctg gcaggggtct
2040ccgagtatga acttccagag gacccaaaat gggagtttcc aagagataag
ctgacactgg 2100gcaagcccct gggagaaggt tgctttgggc aagtggtcat
ggcggaagca gtgggaattg 2160acaaagacaa gcccaaggag gcggtcaccg
tggccgtgaa gatgttgaaa gatgatgcca 2220cagagaaaga cctttctgat
ctggtgtcag agatggagat gatgaagatg attgggaaac 2280acaagaatat
cataaatctt cttggagcct gcacacagga tgggcctctc tatgtcatag
2340ttgagtatgc ctctaaaggc aacctccgag aatacctccg agcccggagg
ccacccggga 2400tggagtactc ctatgacatt aaccgtgttc ctgaggagca
gatgaccttc aaggacttgg 2460tgtcatgcac ctaccagctg gccagaggca
tggagtactt ggcttcccaa aaatgtattc 2520atcgagattt agcagccaga
aatgttttgg taacagaaaa caatgtgatg aaaatagcag 2580actttggact
cgccagagat atcaacaata tagactatta caaaaagacc accaatgggc
2640ggcttccagt caagtggatg gctccagaag ccctgtttga tagagtatac
actcatcaga 2700gtgatgtctg gtccttcggg gtgttaatgt gggagatctt
cactttaggg ggctcgccct 2760acccagggat tcccgtggag gaacttttta
agctgctgaa ggaaggacac agaatggata 2820agccagccaa ctgcaccaac
gaactgtaca tgatgatgag ggactgttgg catgcagtgc 2880cctcccagag
accaacgttc aagcagttgg tagaagactt ggatcgaatt ctcactctca
2940caaccaatga ggaatacttg gacctcagcc aacctctcga acagtattca
cctagttacc 3000ctgacacaag aagttcttgt tcttcaggag atgattctgt
tttttctcca gaccccatgc 3060cttacgaacc atgccttcct cagtatccac
acataaacgg cagtgttaaa acatgaatga 3120ctgtgtctgc ctgtccccaa
acaggacagc actgggaacc tagctacact gagcagggag 3180accatgcctc
ccagagcttg ttgtctccac ttgtatatat ggatcagagg agtaaataat
3240tggaaaagta atcagcatat gtgtaaagat ttatacagtt gaaaacttgt
aatcttcccc 3300aggaggagaa gaaggtttct ggagcagtgg actgccacaa
gccaccatgt aacccctctc 3360acctgccgtg cgtactggct gtggaccagt
aggactcaag gtggacgtgc gttctgcctt 3420ccttgttaat tttgtaataa
ttggagaaga tttatgtcag cacacactta cagagcacaa 3480atgcagtata
taggtgctgg atgtatgtaa atatattcaa attatgtata aatatatatt
3540atatatttac aaggagttat tttttgtatt gattttaaat ggatgtccca
atgcacctag 3600aaaattggtc tctctttttt taatagctat ttgctaaatg
ctgttcttac acataatttc 3660ttaattttca ccgagcagag gtggaaaaat
acttttgctt tcagggaaaa tggtataacg 3720ttaatttatt aataaattgg
taatatacaa aacaattaat catttatagt tttttttgta 3780atttaagtgg
catttctatg caggcagcac agcagactag ttaatctatt gcttggactt
3840aactagttat cagatccttt gaaaagagaa tatttacaat atatgactaa
tttggggaaa 3900atgaagtttt gatttatttg tgtttaaatg ctgctgtcag
acgattgttc ttagacctcc 3960taaatgcccc atattaaaag aactcattca
taggaaggtg tttcattttg gtgtgcaacc 4020ctgtcattac gtcaacgcaa
cgtctaactg gacttcccaa gataaatggt accagcgtcc 4080tcttaaaaga
tgccttaatc cattccttga ggacagacct tagttgaaat gatagcagaa
4140tgtgcttctc tctggcagct ggccttctgc ttctgagttg cacattaatc
agattagcct 4200gtattctctt cagtgaattt tgataatggc ttccagactc
tttggcgttg gagacgcctg 4260ttaggatctt caagtcccat catagaaaat
tgaaacacag agttgttctg ctgatagttt 4320tggggatacg tccatctttt
taagggattg ctttcatcta attctggcag gacctcacca 4380aaagatccag
cctcatacct acatcagaca aaatatcgcc gttgttcctt ctgtactaaa
4440gtattgtgtt ttgctttgga aacacccact cactttgcaa tagccgtgca
agatgaatgc 4500agattacact gatcttatgt gttacaaaat tggagaaagt
atttaataaa acctgttaat 4560ttttatactg acaataaaaa tgtttctaca
gatattaatg ttaacaagac aaaataaatg 4620tcacgcaact tattttttta
ataaaaaaaa aaaaaaa 46571322781DNAHomo sapiens 132tgactgcagc
agcagcggca gcgcctcggt tcctgagccc accgcaggct gaaggcattg 60cgcgtagtcc
atgcccgtag aggaagtgtg cagatgggat taacgtccac atggagatat
120ggaagaggac cggggattgg taccgtaacc atggtcagct ggggtcgttt
catctgcctg 180gtcgtggtca ccatggcaac cttgtccctg gcccggccct
ccttcagttt agttgaggat 240accacattag agccagaaga gccaccaacc
aaataccaaa tctctcaacc agaagtgtac 300gtggctgcgc caggggagtc
gctagaggtg cgctgcctgt tgaaagatgc cgccgtgatc 360agttggacta
aggatggggt gcacttgggg cccaacaata ggacagtgct tattggggag
420tacttgcaga taaagggcgc cacgcctaga gactccggcc tctatgcttg
tactgccagt 480aggactgtag acagtgaaac ttggtacttc atggtgaatg
tcacagatgc catctcatcc 540ggagatgatg aggatgacac cgatggtgcg
gaagattttg tcagtgagaa cagtaacaac 600aagagagcac catactggac
caacacagaa aagatggaaa agcggctcca tgctgtgcct 660gcggccaaca
ctgtcaagtt tcgctgccca gccgggggga acccaatgcc aaccatgcgg
720tggctgaaaa acgggaagga gtttaagcag gagcatcgca ttggaggcta
caaggtacga 780aaccagcact ggagcctcat tatggaaagt gtggtcccat
ctgacaaggg aaattatacc 840tgtgtagtgg agaatgaata cgggtccatc
aatcacacgt accacctgga tgttgtggag 900cgatcgcctc accggcccat
cctccaagcc ggactgccgg caaatgcctc cacagtggtc 960ggaggagacg
tagagtttgt ctgcaaggtt tacagtgatg cccagcccca catccagtgg
1020atcaagcacg tggaaaagaa cggcagtaaa tacgggcccg acgggctgcc
ctacctcaag 1080gttctcaagc actcggggat aaatagttcc aatgcagaag
tgctggctct gttcaatgtg 1140accgaggcgg atgctgggga atatatatgt
aaggtctcca attatatagg gcaggccaac 1200cagtctgcct ggctcactgt
cctgccaaaa cagcaagcgc ctggaagaga aaaggagatt 1260acagcttccc
cagactacct ggagatagcc atttactgca taggggtctt cttaatcgcc
1320tgtatggtgg taacagtcat cctgtgccga atgaagaaca cgaccaagaa
gccagacttc 1380agcagccagc cggctgtgca caagctgacc aaacgtatcc
ccctgcggag acaggtaaca 1440gtttcggctg agtccagctc ctccatgaac
tccaacaccc cgctggtgag gataacaaca 1500cgcctctctt caacggcaga
cacccccatg ctggcagggg tctccgagta tgaacttcca 1560gaggacccaa
aatgggagtt tccaagagat aagctgacac tgggcaagcc cctgggagaa
1620ggttgctttg ggcaagtggt catggcggaa gcagtgggaa ttgacaaaga
caagcccaag 1680gaggcggtca ccgtggccgt gaagatgttg aaagatgatg
ccacagagaa agacctttct 1740gatctggtgt cagagatgga gatgatgaag
atgattggga aacacaagaa tatcataaat 1800cttcttggag cctgcacaca
ggatgggcct ctctatgtca tagttgagta tgcctctaaa 1860ggcaacctcc
gagaatacct ccgagcccgg aggccacccg ggatggagta ctcctatgac
1920attaaccgtg ttcctgagga gcagatgacc ttcaaggact tggtgtcatg
cacctaccag 1980ctggccagag gcatggagta cttggcttcc caaaaatgta
ttcatcgaga tttagcagcc 2040agaaatgttt tggtaacaga
aaacaatgtg atgaaaatag cagactttgg actcgccaga 2100gatatcaaca
atatagacta ttacaaaaag accaccaatg ggcggcttcc agtcaagtgg
2160atggctccag aagccctgtt tgatagagta tacactcatc agagtgatgt
ctggtccttc 2220ggggtgttaa tgtgggagat cttcacttta gggggctcgc
cctacccagg gattcccgtg 2280gaggaacttt ttaagctgct gaaggaagga
cacagaatgg ataagccagc caactgcacc 2340aacgaactgt acatgatgat
gagggactgt tggcatgcag tgccctccca gagaccaacg 2400ttcaagcagt
tggtagaaga cttggatcga attctcactc tcacaaccaa tgagatctga
2460aagtttatgg cttcattgag aaactgggaa aagttggtca ggcgcagtgg
ctcatgcctg 2520taatcccagc actttgggag gccgaggcag gcggatcatg
aggtcaggag ttccagacca 2580gcctggccaa catggtgaaa ccctgtctct
actaaagata caaaaaatta gccgggcgtg 2640ttggtgtgca cctgtaatcc
cagctactcc gggaggctga ggcaggagag tcacttgaac 2700cggggaggcg
gaggttgcag tgagccgaga tcatgccatt gcattccagc cttggcgaca
2760gagcgagact ccgtctcaaa a 27811333821DNAHomo sapiens
133tgactgcagc agcagcggca gcgcctcggt tcctgagccc accgcaggct
gaaggcattg 60cgcgtagtcc atgcccgtag aggaagtgtg cagatgggat taacgtccac
atggagatat 120ggaagaggac cggggattgg taccgtaacc atggtcagct
ggggtcgttt catctgcctg 180gtcgtggtca ccatggcaac cttgtccctg
gcccggccct ccttcagttt agttgaggat 240accacattag agccagaaga
gccaccaacc aaataccaaa tctctcaacc agaagtgtac 300gtggctgcgc
caggggagtc gctagaggtg cgctgcctgt tgaaagatgc cgccgtgatc
360agttggacta aggatggggt gcacttgggg cccaacaata ggacagtgct
tattggggag 420tacttgcaga taaagggcgc cacgcctaga gactccggcc
tctatgcttg tactgccagt 480aggactgtag acagtgaaac ttggtacttc
atggtgaatg tcacagatgc catctcatcc 540ggagatgatg aggatgacac
cgatggtgcg gaagattttg tcagtgagaa cagtaacaac 600aagagagcac
catactggac caacacagaa aagatggaaa agcggctcca tgctgtgcct
660gcggccaaca ctgtcaagtt tcgctgccca gccgggggga acccaatgcc
aaccatgcgg 720tggctgaaaa acgggaagga gtttaagcag gagcatcgca
ttggaggcta caaggtacga 780aaccagcact ggagcctcat tatggaaagt
gtggtcccat ctgacaaggg aaattatacc 840tgtgtagtgg agaatgaata
cgggtccatc aatcacacgt accacctgga tgttgtggcg 900cctggaagag
aaaaggagat tacagcttcc ccagactacc tggagatagc catttactgc
960ataggggtct tcttaatcgc ctgtatggtg gtaacagtca tcctgtgccg
aatgaagaac 1020acgaccaaga agccagactt cagcagccag ccggctgtgc
acaagctgac caaacgtatc 1080cccctgcgga gacaggtaac agtttcggct
gagtccagct cctccatgaa ctccaacacc 1140ccgctggtga ggataacaac
acgcctctct tcaacggcag acacccccat gctggcaggg 1200gtctccgagt
atgaacttcc agaggaccca aaatgggagt ttccaagaga taagctgaca
1260ctgggcaagc ccctgggaga aggttgcttt gggcaagtgg tcatggcgga
agcagtggga 1320attgacaaag acaagcccaa ggaggcggtc accgtggccg
tgaagatgtt gaaagatgat 1380gccacagaga aagacctttc tgatctggtg
tcagagatgg agatgatgaa gatgattggg 1440aaacacaaga atatcataaa
tcttcttgga gcctgcacac aggatgggcc tctctatgtc 1500atagttgagt
atgcctctaa aggcaacctc cgagaatacc tccgagcccg gaggccaccc
1560gggatggagt actcctatga cattaaccgt gttcctgagg agcagatgac
cttcaaggac 1620ttggtgtcat gcacctacca gctggccaga ggcatggagt
acttggcttc ccaaaaatgt 1680attcatcgag atttagcagc cagaaatgtt
ttggtaacag aaaacaatgt gatgaaaata 1740gcagactttg gactcgccag
agatatcaac aatatagact attacaaaaa gaccaccaat 1800gggcggcttc
cagtcaagtg gatggctcca gaagccctgt ttgatagagt atacactcat
1860cagagtgatg tctggtcctt cggggtgtta atgtgggaga tcttcacttt
agggggctcg 1920ccctacccag ggattcccgt ggaggaactt tttaagctgc
tgaaggaagg acacagaatg 1980gataagccag ccaactgcac caacgaactg
tacatgatga tgagggactg ttggcatgca 2040gtgccctccc agagaccaac
gttcaagcag ttggtagaag acttggatcg aattctcact 2100ctcacaacca
atgaggaata cttggacctc agccaacctc tcgaacagta ttcacctagt
2160taccctgaca caagaagttc ttgttcttca ggagatgatt ctgttttttc
tccagacccc 2220atgccttacg aaccatgcct tcctcagtat ccacacataa
acggcagtgt taaaacatga 2280atgactgtgt ctgcctgtcc ccaaacagga
cagcactggg aacctagcta cactgagcag 2340ggagaccatg cctcccagag
cttgttgtct ccacttgtat atatggatca gaggagtaaa 2400taattggaaa
agtaatcagc atatgtgtaa agatttatac agttgaaaac ttgtaatctt
2460ccccaggagg agaagaaggt ttctggagca gtggactgcc acaagccacc
atgtaacccc 2520tctcacctgc cgtgcgtact ggctgtggac cagtaggact
caaggtggac gtgcgttctg 2580ccttccttgt taattttgta ataattggag
aagatttatg tcagcacaca cttacagagc 2640acaaatgcag tatataggtg
ctggatgtat gtaaatatat tcaaattatg tataaatata 2700tattatatat
ttacaaggag ttattttttg tattgatttt aaatggatgt cccaatgcac
2760ctagaaaatt ggtctctctt tttttaatag ctatttgcta aatgctgttc
ttacacataa 2820tttcttaatt ttcaccgagc agaggtggaa aaatactttt
gctttcaggg aaaatggtat 2880aacgttaatt tattaataaa ttggtaatat
acaaaacaat taatcattta tagttttttt 2940tgtaatttaa gtggcatttc
tatgcaggca gcacagcaga ctagttaatc tattgcttgg 3000acttaactag
ttatcagatc ctttgaaaag agaatattta caatatatga ctaatttggg
3060gaaaatgaag ttttgattta tttgtgttta aatgctgctg tcagacgatt
gttcttagac 3120ctcctaaatg ccccatatta aaagaactca ttcataggaa
ggtgtttcat tttggtgtgc 3180aaccctgtca ttacgtcaac gcaacgtcta
actggacttc ccaagataaa tggtaccagc 3240gtcctcttaa aagatgcctt
aatccattcc ttgaggacag accttagttg aaatgatagc 3300agaatgtgct
tctctctggc agctggcctt ctgcttctga gttgcacatt aatcagatta
3360gcctgtattc tcttcagtga attttgataa tggcttccag actctttggc
gttggagacg 3420cctgttagga tcttcaagtc ccatcataga aaattgaaac
acagagttgt tctgctgata 3480gttttgggga tacgtccatc tttttaaggg
attgctttca tctaattctg gcaggacctc 3540accaaaagat ccagcctcat
acctacatca gacaaaatat cgccgttgtt ccttctgtac 3600taaagtattg
tgttttgctt tggaaacacc cactcacttt gcaatagccg tgcaagatga
3660atgcagatta cactgatctt atgtgttaca aaattggaga aagtatttaa
taaaacctgt 3720taatttttat actgacaata aaaatgtttc tacagatatt
aatgttaaca agacaaaata 3780aatgtcacgc aacttatttt tttaataaaa
aaaaaaaaaa a 38211343708DNAHomo sapiens 134aatttgttga ggaatttccc
cctagccttg accccttgac agctcccgct cctactcagt 60gctggggaga agtagggagg
ccttaagcga agagatgggt ctgcactttg gaggagccgg 120acactgttga
ctttcctgat gtgaaatcta cccaggaaca aaacaccagt gactgcagca
180gcagcggcag cgcctcggtt cctgagccca ccgcaggctg aaggcattgc
gcgtagtcca 240tgcccgtaga ggaagtgtgc agatgggatt aacgtccaca
tggagatatg gaagaggacc 300ggggattggt accgtaacca tggtcagctg
gggtcgtttc atctgcctgg tcgtggtcac 360catggcaacc ttgtccctgg
cccggccctc cttcagttta gttgaggata ccacattaga 420gccagaagat
gccatctcat ccggagatga tgaggatgac accgatggtg cggaagattt
480tgtcagtgag aacagtaaca acaagagagc accatactgg accaacacag
aaaagatgga 540aaagcggctc catgctgtgc ctgcggccaa cactgtcaag
tttcgctgcc cagccggggg 600gaacccaatg ccaaccatgc ggtggctgaa
aaacgggaag gagtttaagc aggagcatcg 660cattggaggc tacaaggtac
gaaaccagca ctggagcctc attatggaaa gtgtggtccc 720atctgacaag
ggaaattata cctgtgtagt ggagaatgaa tacgggtcca tcaatcacac
780gtaccacctg gatgttgtgg agcgatcgcc tcaccggccc atcctccaag
ccggactgcc 840ggcaaatgcc tccacagtgg tcggaggaga cgtagagttt
gtctgcaagg tttacagtga 900tgcccagccc cacatccagt ggatcaagca
cgtggaaaag aacggcagta aatacgggcc 960cgacgggctg ccctacctca
aggttctcaa ggccgccggt gttaacacca cggacaaaga 1020gattgaggtt
ctctatattc ggaatgtaac ttttgaggac gctggggaat atacgtgctt
1080ggcgggtaat tctattggga tatcctttca ctctgcatgg ttgacagttc
tgccagcgcc 1140tggaagagaa aaggagatta cagcttcccc agactacctg
gagatagcca tttactgcat 1200aggggtcttc ttaatcgcct gtatggtggt
aacagtcatc ctgtgccgaa tgaagaacac 1260gaccaagaag ccagacttca
gcagccagcc ggctgtgcac aagctgacca aacgtatccc 1320cctgcggaga
caggtaacag tttcggctga gtccagctcc tccatgaact ccaacacccc
1380gctggtgagg ataacaacac gcctctcttc aacggcagac acccccatgc
tggcaggggt 1440ctccgagtat gaacttccag aggacccaaa atgggagttt
ccaagagata agctgacact 1500gggcaagccc ctgggagaag gttgctttgg
gcaagtggtc atggcggaag cagtgggaat 1560tgacaaagac aagcccaagg
aggcggtcac cgtggccgtg aagatgttga aagatgatgc 1620cacagagaaa
gacctttctg atctggtgtc agagatggag atgatgaaga tgattgggaa
1680acacaagaat atcataaatc ttcttggagc ctgcacacag gatgggcctc
tctatgtcat 1740agttgagtat gcctctaaag gcaacctccg agaatacctc
cgagcccgga ggccacccgg 1800gatggagtac tcctatgaca ttaaccgtgt
tcctgaggag cagatgacct tcaaggactt 1860ggtgtcatgc acctaccagc
tggccagagg catggagtac ttggcttccc aaaaatgtat 1920tcatcgagat
ttagcagcca gaaatgtttt ggtaacagaa aacaatgtga tgaaaatagc
1980agactttgga ctcgccagag atatcaacaa tatagactat tacaaaaaga
ccaccaatgg 2040gcggcttcca gtcaagtgga tggctccaga agccctgttt
gatagagtat acactcatca 2100gagtgatgtc tggtccttcg gggtgttaat
gtgggagatc ttcactttag ggggctcgcc 2160ctacccaggg attcccgtgg
aggaactttt taagctgctg aaggaaggac acagaatgga 2220taagccagcc
aactgcacca acgaactgta catgatgatg agggactgtt ggcatgcagt
2280gccctcccag agaccaacgt tcaagcagtt ggtagaagac ttggatcgaa
ttctcactct 2340cacaaccaat gaggaggaga agaaggtttc tggagcagtg
gactgccaca agccaccatg 2400taacccctct cacctgccgt gcgtactggc
tgtggaccag taggactcaa ggtggacgtg 2460cgttctgcct tccttgttaa
ttttgtaata attggagaag atttatgtca gcacacactt 2520acagagcaca
aatgcagtat ataggtgctg gatgtatgta aatatattca aattatgtat
2580aaatatatat tatatattta caaggagtta ttttttgtat tgattttaaa
tggatgtccc 2640aatgcaccta gaaaattggt ctctcttttt ttaatagcta
tttgctaaat gctgttctta 2700cacataattt cttaattttc accgagcaga
ggtggaaaaa tacttttgct ttcagggaaa 2760atggtataac gttaatttat
taataaattg gtaatataca aaacaattaa tcatttatag 2820ttttttttgt
aatttaagtg gcatttctat gcaggcagca cagcagacta gttaatctat
2880tgcttggact taactagtta tcagatcctt tgaaaagaga atatttacaa
tatatgacta 2940atttggggaa aatgaagttt tgatttattt gtgtttaaat
gctgctgtca gacgattgtt 3000cttagacctc ctaaatgccc catattaaaa
gaactcattc ataggaaggt gtttcatttt 3060ggtgtgcaac cctgtcatta
cgtcaacgca acgtctaact ggacttccca agataaatgg 3120taccagcgtc
ctcttaaaag atgccttaat ccattccttg aggacagacc ttagttgaaa
3180tgatagcaga atgtgcttct ctctggcagc tggccttctg cttctgagtt
gcacattaat 3240cagattagcc tgtattctct tcagtgaatt ttgataatgg
cttccagact ctttggcgtt 3300ggagacgcct gttaggatct tcaagtccca
tcatagaaaa ttgaaacaca gagttgttct 3360gctgatagtt ttggggatac
gtccatcttt ttaagggatt gctttcatct aattctggca 3420ggacctcacc
aaaagatcca gcctcatacc tacatcagac aaaatatcgc cgttgttcct
3480tctgtactaa agtattgtgt tttgctttgg aaacacccac tcactttgca
atagccgtgc 3540aagatgaatg cagattacac tgatcttatg tgttacaaaa
ttggagaaag tatttaataa 3600aacctgttaa tttttatact gacaataaaa
atgtttctac agatattaat gttaacaaga 3660caaaataaat gtcacgcaac
ttattttttt aataaaaaaa aaaaaaaa 37081354103DNAHomo sapiens
135gagcacacat tgcctcactg aagtggctgc acgtatctga gtcctgtagc
tactgtttta 60tctctgtttc ttaaaagtat gcttttaaaa agattagcct cacacatttc
tgtggaccgg 120tctggtggta tcacctggga ctctgaggtg aggatggaag
gatttagcag ataatgaaaa 180agaactctgt ttgcgcacat ttgagaggct
gaaaaatggt tttatcccac ttgggctgga 240gtgatttggc attggggaag
attccctgac tcgccaatct ctttccttta gtgactgcag 300cagcagcggc
agcgcctcgg ttcctgagcc caccgcaggc tgaaggcatt gcgcgtagtc
360catgcccgta gaggaagtgt gcagatggga ttaacgtcca catggagata
tggaagagga 420ccggggattg gtaccgtaac catggtcagc tggggtcgtt
tcatctgcct ggtcgtggtc 480accatggcaa ccttgtccct ggcccggccc
tccttcagtt tagttgagga taccacatta 540gagccagaag gagcaccata
ctggaccaac acagaaaaga tggaaaagcg gctccatgct 600gtgcctgcgg
ccaacactgt caagtttcgc tgcccagccg gggggaaccc aatgccaacc
660atgcggtggc tgaaaaacgg gaaggagttt aagcaggagc atcgcattgg
aggctacaag 720gtacgaaacc agcactggag cctcattatg gaaagtgtgg
tcccatctga caagggaaat 780tatacctgtg tagtggagaa tgaatacggg
tccatcaatc acacgtacca cctggatgtt 840gtggagcgat cgcctcaccg
gcccatcctc caagccggac tgccggcaaa tgcctccaca 900gtggtcggag
gagacgtaga gtttgtctgc aaggtttaca gtgatgccca gccccacatc
960cagtggatca agcacgtgga aaagaacggc agtaaatacg ggcccgacgg
gctgccctac 1020ctcaaggttc tcaaggccgc cggtgttaac accacggaca
aagagattga ggttctctat 1080attcggaatg taacttttga ggacgctggg
gaatatacgt gcttggcggg taattctatt 1140gggatatcct ttcactctgc
atggttgaca gttctgccag cgcctggaag agaaaaggag 1200attacagctt
ccccagacta cctggagata gccatttact gcataggggt cttcttaatc
1260gcctgtatgg tggtaacagt catcctgtgc cgaatgaaga acacgaccaa
gaagccagac 1320ttcagcagcc agccggctgt gcacaagctg accaaacgta
tccccctgcg gagacaggta 1380acagtttcgg ctgagtccag ctcctccatg
aactccaaca ccccgctggt gaggataaca 1440acacgcctct cttcaacggc
agacaccccc atgctggcag gggtctccga gtatgaactt 1500ccagaggacc
caaaatggga gtttccaaga gataagctga cactgggcaa gcccctggga
1560gaaggttgct ttgggcaagt ggtcatggcg gaagcagtgg gaattgacaa
agacaagccc 1620aaggaggcgg tcaccgtggc cgtgaagatg ttgaaagatg
atgccacaga gaaagacctt 1680tctgatctgg tgtcagagat ggagatgatg
aagatgattg ggaaacacaa gaatatcata 1740aatcttcttg gagcctgcac
acaggatggg cctctctatg tcatagttga gtatgcctct 1800aaaggcaacc
tccgagaata cctccgagcc cggaggccac ccgggatgga gtactcctat
1860gacattaacc gtgttcctga ggagcagatg accttcaagg acttggtgtc
atgcacctac 1920cagctggcca gaggcatgga gtacttggct tcccaaaaat
gtattcatcg agatttagca 1980gccagaaatg ttttggtaac agaaaacaat
gtgatgaaaa tagcagactt tggactcgcc 2040agagatatca acaatataga
ctattacaaa aagaccacca atgggcggct tccagtcaag 2100tggatggctc
cagaagccct gtttgataga gtatacactc atcagagtga tgtctggtcc
2160ttcggggtgt taatgtggga gatcttcact ttagggggct cgccctaccc
agggattccc 2220gtggaggaac tttttaagct gctgaaggaa ggacacagaa
tggataagcc agccaactgc 2280accaacgaac tgtacatgat gatgagggac
tgttggcatg cagtgccctc ccagagacca 2340acgttcaagc agttggtaga
agacttggat cgaattctca ctctcacaac caatgaggaa 2400tacttggacc
tcagccaacc tctcgaacag tattcaccta gttaccctga cacaagaagt
2460tcttgttctt caggagatga ttctgttttt tctccagacc ccatgcctta
cgaaccatgc 2520cttcctcagt atccacacat aaacggcagt gttaaaacat
gaatgactgt gtctgcctgt 2580ccccaaacag gacagcactg ggaacctagc
tacactgagc agggagacca tgcctcccag 2640agcttgttgt ctccacttgt
atatatggat cagaggagta aataattgga aaagtaatca 2700gcatatgtgt
aaagatttat acagttgaaa acttgtaatc ttccccagga ggagaagaag
2760gtttctggag cagtggactg ccacaagcca ccatgtaacc cctctcacct
gccgtgcgta 2820ctggctgtgg accagtagga ctcaaggtgg acgtgcgttc
tgccttcctt gttaattttg 2880taataattgg agaagattta tgtcagcaca
cacttacaga gcacaaatgc agtatatagg 2940tgctggatgt atgtaaatat
attcaaatta tgtataaata tatattatat atttacaagg 3000agttattttt
tgtattgatt ttaaatggat gtcccaatgc acctagaaaa ttggtctctc
3060tttttttaat agctatttgc taaatgctgt tcttacacat aatttcttaa
ttttcaccga 3120gcagaggtgg aaaaatactt ttgctttcag ggaaaatggt
ataacgttaa tttattaata 3180aattggtaat atacaaaaca attaatcatt
tatagttttt tttgtaattt aagtggcatt 3240tctatgcagg cagcacagca
gactagttaa tctattgctt ggacttaact agttatcaga 3300tcctttgaaa
agagaatatt tacaatatat gactaatttg gggaaaatga agttttgatt
3360tatttgtgtt taaatgctgc tgtcagacga ttgttcttag acctcctaaa
tgccccatat 3420taaaagaact cattcatagg aaggtgtttc attttggtgt
gcaaccctgt cattacgtca 3480acgcaacgtc taactggact tcccaagata
aatggtacca gcgtcctctt aaaagatgcc 3540ttaatccatt ccttgaggac
agaccttagt tgaaatgata gcagaatgtg cttctctctg 3600gcagctggcc
ttctgcttct gagttgcaca ttaatcagat tagcctgtat tctcttcagt
3660gaattttgat aatggcttcc agactctttg gcgttggaga cgcctgttag
gatcttcaag 3720tcccatcata gaaaattgaa acacagagtt gttctgctga
tagttttggg gatacgtcca 3780tctttttaag ggattgcttt catctaattc
tggcaggacc tcaccaaaag atccagcctc 3840atacctacat cagacaaaat
atcgccgttg ttccttctgt actaaagtat tgtgttttgc 3900tttggaaaca
cccactcact ttgcaatagc cgtgcaagat gaatgcagat tacactgatc
3960ttatgtgtta caaaattgga gaaagtattt aataaaacct gttaattttt
atactgacaa 4020taaaaatgtt tctacagata ttaatgttaa caagacaaaa
taaatgtcac gcaacttatt 4080tttttaataa aaaaaaaaaa aaa
41031364306DNAHomo sapiens 136ggcggcggct ggaggagagc gcggtggaga
gccgagcggg cgggcggcgg gtgcggagcg 60ggcgagggag cgcgcgcggc cgccacaaag
ctcgggcgcc gcggggctgc atgcggcgta 120cctggcccgg cgcggcgact
gctctccggg ctggcggggg ccggccgcga gccccggggg 180ccccgaggcc
gcagcttgcc tgcgcgctct gagccttcgc aactcgcgag caaagtttgg
240tggaggcaac gccaagcctg agtcctttct tcctctcgtt ccccaaatcc
gagggcagcc 300cgcgggcgtc atgcccgcgc tcctccgcag cctggggtac
gcgtgaagcc cgggaggctt 360ggcgccggcg aagacccaag gaccactctt
ctgcgtttgg agttgctccc cgcaaccccg 420ggctcgtcgc tttctccatc
ccgacccacg cggggcgcgg ggacaacaca ggtcgcggag 480gagcgttgcc
attcaagtga ctgcagcagc agcggcagcg cctcggttcc tgagcccacc
540gcaggctgaa ggcattgcgc gtagtccatg cccgtagagg aagtgtgcag
atgggattaa 600cgtccacatg gagatatgga agaggaccgg ggattggtac
cgtaaccatg gtcagctggg 660gtcgtttcat ctgcctggtc gtggtcacca
tggcaacctt gtccctggcc cggccctcct 720tcagtttagt tgaggatacc
acattagagc cagaagagcc accaaccaaa taccaaatct 780ctcaaccaga
agtgtacgtg gctgcgccag gggagtcgct agaggtgcgc tgcctgttga
840aagatgccgc cgtgatcagt tggactaagg atggggtgca cttggggccc
aacaatagga 900cagtgcttat tggggagtac ttgcagataa agggcgccac
gcctagagac tccggcctct 960atgcttgtac tgccagtagg actgtagaca
gtgaaacttg gtacttcatg gtgaatgtca 1020cagatgccat ctcatccgga
gatgatgagg atgacaccga tggtgcggaa gattttgtca 1080gtgagaacag
taacaacaag agagcaccat actggaccaa cacagaaaag atggaaaagc
1140ggctccatgc tgtgcctgcg gccaacactg tcaagtttcg ctgcccagcc
ggggggaacc 1200caatgccaac catgcggtgg ctgaaaaacg ggaaggagtt
taagcaggag catcgcattg 1260gaggctacaa ggtacgaaac cagcactgga
gcctcattat ggaaagtgtg gtcccatctg 1320acaagggaaa ttatacctgt
gtagtggaga atgaatacgg gtccatcaat cacacgtacc 1380acctggatgt
tgtggagcga tcgcctcacc ggcccatcct ccaagccgga ctgccggcaa
1440atgcctccac agtggtcgga ggagacgtag agtttgtctg caaggtttac
agtgatgccc 1500agccccacat ccagtggatc aagcacgtgg aaaagaacgg
cagtaaatac gggcccgacg 1560ggctgcccta cctcaaggtt ctcaaggttt
cggctgagtc cagctcctcc atgaactcca 1620acaccccgct ggtgaggata
acaacacgcc tctcttcaac ggcagacacc cccatgctgg 1680caggggtctc
cgagtatgaa cttccagagg acccaaaatg ggagtttcca agagataagc
1740tgacactggg caagcccctg ggagaaggtt gctttgggca agtggtcatg
gcggaagcag 1800tgggaattga caaagacaag cccaaggagg cggtcaccgt
ggccgtgaag atgttgaaag 1860atgatgccac agagaaagac ctttctgatc
tggtgtcaga gatggagatg atgaagatga 1920ttgggaaaca caagaatatc
ataaatcttc ttggagcctg cacacaggat gggcctctct 1980atgtcatagt
tgagtatgcc tctaaaggca acctccgaga atacctccga gcccggaggc
2040cacccgggat ggagtactcc tatgacatta accgtgttcc tgaggagcag
atgaccttca 2100aggacttggt gtcatgcacc taccagctgg ccagaggcat
ggagtacttg gcttcccaaa 2160aatgtattca tcgagattta gcagccagaa
atgttttggt aacagaaaac aatgtgatga 2220aaatagcaga ctttggactc
gccagagata tcaacaatat agactattac aaaaagacca 2280ccaatgggcg
gcttccagtc aagtggatgg ctccagaagc cctgtttgat agagtataca
2340ctcatcagag tgatgtctgg tccttcgggg tgttaatgtg ggagatcttc
actttagggg 2400gctcgcccta cccagggatt cccgtggagg aactttttaa
gctgctgaag gaaggacaca 2460gaatggataa gccagccaac tgcaccaacg
aactgtacat
gatgatgagg gactgttggc 2520atgcagtgcc ctcccagaga ccaacgttca
agcagttggt agaagacttg gatcgaattc 2580tcactctcac aaccaatgag
gaatacttgg acctcagcca acctctcgaa cagtattcac 2640ctagttaccc
tgacacaaga agttcttgtt cttcaggaga tgattctgtt ttttctccag
2700accccatgcc ttacgaacca tgccttcctc agtatccaca cataaacggc
agtgttaaaa 2760catgaatgac tgtgtctgcc tgtccccaaa caggacagca
ctgggaacct agctacactg 2820agcagggaga ccatgcctcc cagagcttgt
tgtctccact tgtatatatg gatcagagga 2880gtaaataatt ggaaaagtaa
tcagcatatg tgtaaagatt tatacagttg aaaacttgta 2940atcttcccca
ggaggagaag aaggtttctg gagcagtgga ctgccacaag ccaccatgta
3000acccctctca cctgccgtgc gtactggctg tggaccagta ggactcaagg
tggacgtgcg 3060ttctgccttc cttgttaatt ttgtaataat tggagaagat
ttatgtcagc acacacttac 3120agagcacaaa tgcagtatat aggtgctgga
tgtatgtaaa tatattcaaa ttatgtataa 3180atatatatta tatatttaca
aggagttatt ttttgtattg attttaaatg gatgtcccaa 3240tgcacctaga
aaattggtct ctcttttttt aatagctatt tgctaaatgc tgttcttaca
3300cataatttct taattttcac cgagcagagg tggaaaaata cttttgcttt
cagggaaaat 3360ggtataacgt taatttatta ataaattggt aatatacaaa
acaattaatc atttatagtt 3420ttttttgtaa tttaagtggc atttctatgc
aggcagcaca gcagactagt taatctattg 3480cttggactta actagttatc
agatcctttg aaaagagaat atttacaata tatgactaat 3540ttggggaaaa
tgaagttttg atttatttgt gtttaaatgc tgctgtcaga cgattgttct
3600tagacctcct aaatgcccca tattaaaaga actcattcat aggaaggtgt
ttcattttgg 3660tgtgcaaccc tgtcattacg tcaacgcaac gtctaactgg
acttcccaag ataaatggta 3720ccagcgtcct cttaaaagat gccttaatcc
attccttgag gacagacctt agttgaaatg 3780atagcagaat gtgcttctct
ctggcagctg gccttctgct tctgagttgc acattaatca 3840gattagcctg
tattctcttc agtgaatttt gataatggct tccagactct ttggcgttgg
3900agacgcctgt taggatcttc aagtcccatc atagaaaatt gaaacacaga
gttgttctgc 3960tgatagtttt ggggatacgt ccatcttttt aagggattgc
tttcatctaa ttctggcagg 4020acctcaccaa aagatccagc ctcataccta
catcagacaa aatatcgccg ttgttccttc 4080tgtactaaag tattgtgttt
tgctttggaa acacccactc actttgcaat agccgtgcaa 4140gatgaatgca
gattacactg atcttatgtg ttacaaaatt ggagaaagta tttaataaaa
4200cctgttaatt tttatactga caataaaaat gtttctacag atattaatgt
taacaagaca 4260aaataaatgt cacgcaactt atttttttaa taaaaaaaaa aaaaaa
43061374303DNAHomo sapiens 137ggcggcggct ggaggagagc gcggtggaga
gccgagcggg cgggcggcgg gtgcggagcg 60ggcgagggag cgcgcgcggc cgccacaaag
ctcgggcgcc gcggggctgc atgcggcgta 120cctggcccgg cgcggcgact
gctctccggg ctggcggggg ccggccgcga gccccggggg 180ccccgaggcc
gcagcttgcc tgcgcgctct gagccttcgc aactcgcgag caaagtttgg
240tggaggcaac gccaagcctg agtcctttct tcctctcgtt ccccaaatcc
gagggcagcc 300cgcgggcgtc atgcccgcgc tcctccgcag cctggggtac
gcgtgaagcc cgggaggctt 360ggcgccggcg aagacccaag gaccactctt
ctgcgtttgg agttgctccc cgcaaccccg 420ggctcgtcgc tttctccatc
ccgacccacg cggggcgcgg ggacaacaca ggtcgcggag 480gagcgttgcc
attcaagtga ctgcagcagc agcggcagcg cctcggttcc tgagcccacc
540gcaggctgaa ggcattgcgc gtagtccatg cccgtagagg aagtgtgcag
atgggattaa 600cgtccacatg gagatatgga agaggaccgg ggattggtac
cgtaaccatg gtcagctggg 660gtcgtttcat ctgcctggtc gtggtcacca
tggcaacctt gtccctggcc cggccctcct 720tcagtttagt tgaggatacc
acattagagc cagaaggagc accatactgg accaacacag 780aaaagatgga
aaagcggctc catgctgtgc ctgcggccaa cactgtcaag tttcgctgcc
840cagccggggg gaacccaatg ccaaccatgc ggtggctgaa aaacgggaag
gagtttaagc 900aggagcatcg cattggaggc tacaaggtac gaaaccagca
ctggagcctc attatggaaa 960gtgtggtccc atctgacaag ggaaattata
cctgtgtagt ggagaatgaa tacgggtcca 1020tcaatcacac gtaccacctg
gatgttgtgg agcgatcgcc tcaccggccc atcctccaag 1080ccggactgcc
ggcaaatgcc tccacagtgg tcggaggaga cgtagagttt gtctgcaagg
1140tttacagtga tgcccagccc cacatccagt ggatcaagca cgtggaaaag
aacggcagta 1200aatacgggcc cgacgggctg ccctacctca aggttctcaa
ggccgccggt gttaacacca 1260cggacaaaga gattgaggtt ctctatattc
ggaatgtaac ttttgaggac gctggggaat 1320atacgtgctt ggcgggtaat
tctattggga tatcctttca ctctgcatgg ttgacagttc 1380tgccagcgcc
tggaagagaa aaggagatta cagcttcccc agactacctg gagatagcca
1440tttactgcat aggggtcttc ttaatcgcct gtatggtggt aacagtcatc
ctgtgccgaa 1500tgaagaacac gaccaagaag ccagacttca gcagccagcc
ggctgtgcac aagctgacca 1560aacgtatccc cctgcggaga caggtttcgg
ctgagtccag ctcctccatg aactccaaca 1620ccccgctggt gaggataaca
acacgcctct cttcaacggc agacaccccc atgctggcag 1680gggtctccga
gtatgaactt ccagaggacc caaaatggga gtttccaaga gataagctga
1740cactgggcaa gcccctggga gaaggttgct ttgggcaagt ggtcatggcg
gaagcagtgg 1800gaattgacaa agacaagccc aaggaggcgg tcaccgtggc
cgtgaagatg ttgaaagatg 1860atgccacaga gaaagacctt tctgatctgg
tgtcagagat ggagatgatg aagatgattg 1920ggaaacacaa gaatatcata
aatcttcttg gagcctgcac acaggatggg cctctctatg 1980tcatagttga
gtatgcctct aaaggcaacc tccgagaata cctccgagcc cggaggccac
2040ccgggatgga gtactcctat gacattaacc gtgttcctga ggagcagatg
accttcaagg 2100acttggtgtc atgcacctac cagctggcca gaggcatgga
gtacttggct tcccaaaaat 2160gtattcatcg agatttagca gccagaaatg
ttttggtaac agaaaacaat gtgatgaaaa 2220tagcagactt tggactcgcc
agagatatca acaatataga ctattacaaa aagaccacca 2280atgggcggct
tccagtcaag tggatggctc cagaagccct gtttgataga gtatacactc
2340atcagagtga tgtctggtcc ttcggggtgt taatgtggga gatcttcact
ttagggggct 2400cgccctaccc agggattccc gtggaggaac tttttaagct
gctgaaggaa ggacacagaa 2460tggataagcc agccaactgc accaacgaac
tgtacatgat gatgagggac tgttggcatg 2520cagtgccctc ccagagacca
acgttcaagc agttggtaga agacttggat cgaattctca 2580ctctcacaac
caatgaggaa tacttggacc tcagccaacc tctcgaacag tattcaccta
2640gttaccctga cacaagaagt tcttgttctt caggagatga ttctgttttt
tctccagacc 2700ccatgcctta cgaaccatgc cttcctcagt atccacacat
aaacggcagt gttaaaacat 2760gaatgactgt gtctgcctgt ccccaaacag
gacagcactg ggaacctagc tacactgagc 2820agggagacca tgcctcccag
agcttgttgt ctccacttgt atatatggat cagaggagta 2880aataattgga
aaagtaatca gcatatgtgt aaagatttat acagttgaaa acttgtaatc
2940ttccccagga ggagaagaag gtttctggag cagtggactg ccacaagcca
ccatgtaacc 3000cctctcacct gccgtgcgta ctggctgtgg accagtagga
ctcaaggtgg acgtgcgttc 3060tgccttcctt gttaattttg taataattgg
agaagattta tgtcagcaca cacttacaga 3120gcacaaatgc agtatatagg
tgctggatgt atgtaaatat attcaaatta tgtataaata 3180tatattatat
atttacaagg agttattttt tgtattgatt ttaaatggat gtcccaatgc
3240acctagaaaa ttggtctctc tttttttaat agctatttgc taaatgctgt
tcttacacat 3300aatttcttaa ttttcaccga gcagaggtgg aaaaatactt
ttgctttcag ggaaaatggt 3360ataacgttaa tttattaata aattggtaat
atacaaaaca attaatcatt tatagttttt 3420tttgtaattt aagtggcatt
tctatgcagg cagcacagca gactagttaa tctattgctt 3480ggacttaact
agttatcaga tcctttgaaa agagaatatt tacaatatat gactaatttg
3540gggaaaatga agttttgatt tatttgtgtt taaatgctgc tgtcagacga
ttgttcttag 3600acctcctaaa tgccccatat taaaagaact cattcatagg
aaggtgtttc attttggtgt 3660gcaaccctgt cattacgtca acgcaacgtc
taactggact tcccaagata aatggtacca 3720gcgtcctctt aaaagatgcc
ttaatccatt ccttgaggac agaccttagt tgaaatgata 3780gcagaatgtg
cttctctctg gcagctggcc ttctgcttct gagttgcaca ttaatcagat
3840tagcctgtat tctcttcagt gaattttgat aatggcttcc agactctttg
gcgttggaga 3900cgcctgttag gatcttcaag tcccatcata gaaaattgaa
acacagagtt gttctgctga 3960tagttttggg gatacgtcca tctttttaag
ggattgcttt catctaattc tggcaggacc 4020tcaccaaaag atccagcctc
atacctacat cagacaaaat atcgccgttg ttccttctgt 4080actaaagtat
tgtgttttgc tttggaaaca cccactcact ttgcaatagc cgtgcaagat
4140gaatgcagat tacactgatc ttatgtgtta caaaattgga gaaagtattt
aataaaacct 4200gttaattttt atactgacaa taaaaatgtt tctacagata
ttaatgttaa caagacaaaa 4260taaatgtcac gcaacttatt tttttaataa
aaaaaaaaaa aaa 43031383011DNAHomo sapiens 138ggcggcggct ggaggagagc
gcggtggaga gccgagcggg cgggcggcgg gtgcggagcg 60ggcgagggag cgcgcgcggc
cgccacaaag ctcgggcgcc gcggggctgc atgcggcgta 120cctggcccgg
cgcggcgact gctctccggg ctggcggggg ccggccgcga gccccggggg
180ccccgaggcc gcagcttgcc tgcgcgctct gagccttcgc aactcgcgag
caaagtttgg 240tggaggcaac gccaagcctg agtcctttct tcctctcgtt
ccccaaatcc gagggcagcc 300cgcgggcgtc atgcccgcgc tcctccgcag
cctggggtac gcgtgaagcc cgggaggctt 360ggcgccggcg aagacccaag
gaccactctt ctgcgtttgg agttgctccc cgcaaccccg 420ggctcgtcgc
tttctccatc ccgacccacg cggggcgcgg ggacaacaca ggtcgcggag
480gagcgttgcc attcaagtga ctgcagcagc agcggcagcg cctcggttcc
tgagcccacc 540gcaggctgaa ggcattgcgc gtagtccatg cccgtagagg
aagtgtgcag atgggattaa 600cgtccacatg gagatatgga agaggaccgg
ggattggtac cgtaaccatg gtcagctggg 660gtcgtttcat ctgcctggtc
gtggtcacca tggcaacctt gtccctggcc cggccctcct 720tcagtttagt
tgaggatacc acattagagc cagaagatgc catctcatcc ggagatgatg
780aggatgacac cgatggtgcg gaagattttg tcagtgagaa cagtaacaac
aagagagcac 840catactggac caacacagaa aagatggaaa agcggctcca
tgctgtgcct gcggccaaca 900ctgtcaagtt tcgctgccca gccgggggga
acccaatgcc aaccatgcgg tggctgaaaa 960acgggaagga gtttaagcag
gagcatcgca ttggaggcta caaggtacga aaccagcact 1020ggagcctcat
tatggaaagt gtggtcccat ctgacaaggg aaattatacc tgtgtagtgg
1080agaatgaata cgggtccatc aatcacacgt accacctgga tgttgtggag
cgatcgcctc 1140accggcccat cctccaagcc ggactgccgg caaatgcctc
cacagtggtc ggaggagacg 1200tagagtttgt ctgcaaggtt tacagtgatg
cccagcccca catccagtgg atcaagcacg 1260tggaaaagaa cggcagtaaa
tacgggcccg acgggctgcc ctacctcaag gttctcaagc 1320actcggggat
aaatagttcc aatgcagaag tgctggctct gttcaatgtg accgaggcgg
1380atgctgggga atatatatgt aaggtctcca attatatagg gcaggccaac
cagtctgcct 1440ggctcactgt cctgccaaaa cagcaagcgc ctggaagaga
aaaggagatt acagcttccc 1500cagactacct ggagatagcc atttactgca
taggggtctt cttaatcgcc tgtatggtgg 1560taacagtcat cctgtgccga
atgaagaaca cgaccaagaa gccagacttc agcagccagc 1620cggctgtgca
caagctgacc aaacgtatcc ccctgcggag acaggtaaca gtttcggctg
1680agtccagctc ctccatgaac tccaacaccc cgctggtgag gataacaaca
cgcctctctt 1740caacggcaga cacccccatg ctggcagggg tctccgagta
tgaacttcca gaggacccaa 1800aatgggagtt tccaagagat aagctgacac
tgggcaagcc cctgggagaa ggttgctttg 1860ggcaagtggt catggcggaa
gcagtgggaa ttgacaaaga caagcccaag gaggcggtca 1920ccgtggccgt
gaagatgttg aaagatgatg ccacagagaa agacctttct gatctggtgt
1980cagagatgga gatgatgaag atgattggga aacacaagaa tatcataaat
cttcttggag 2040cctgcacaca ggatgggcct ctctatgtca tagttgagta
tgcctctaaa ggcaacctcc 2100gagaatacct ccgagcccgg aggccacccg
ggatggagta ctcctatgac attaaccgtg 2160ttcctgagga gcagatgacc
ttcaaggact tggtgtcatg cacctaccag ctggccagag 2220gcatggagta
cttggcttcc caaaaatgta ttcatcgaga tttagcagcc agaaatgttt
2280tggtaacaga aaacaatgtg atgaaaatag cagactttgg actcgccaga
gatatcaaca 2340atatagacta ttacaaaaag accaccaatg ggcggcttcc
agtcaagtgg atggctccag 2400aagccctgtt tgatagagta tacactcatc
agagtgatgt ctggtccttc ggggtgttaa 2460tgtgggagat cttcacttta
gggggctcgc cctacccagg gattcccgtg gaggaacttt 2520ttaagctgct
gaaggaagga cacagaatgg ataagccagc caactgcacc aacgaactgt
2580acatgatgat gagggactgt tggcatgcag tgccctccca gagaccaacg
ttcaagcagt 2640tggtagaaga cttggatcga attctcactc tcacaaccaa
tgagatctga aagtttatgg 2700cttcattgag aaactgggaa aagttggtca
ggcgcagtgg ctcatgcctg taatcccagc 2760actttgggag gccgaggcag
gcggatcatg aggtcaggag ttccagacca gcctggccaa 2820catggtgaaa
ccctgtctct actaaagata caaaaaatta gccgggcgtg ttggtgtgca
2880cctgtaatcc cagctactcc gggaggctga ggcaggagag tcacttgaac
cggggaggcg 2940gaggttgcag tgagccgaga tcatgccatt gcattccagc
cttggcgaca gagcgagact 3000ccgtctcaaa a 30111394286DNAHomo sapiens
139gtcgcgggca gctggcgccg cgcggtcctg ctctgccggt cgcacggacg
caccggcggg 60ccgccggccg gagggacggg gcgggagctg ggcccgcgga cagcgagccg
gagcgggagc 120cgcgcgtagc gagccgggct ccggcgctcg ccagtctccc
gagcggcgcc cgcctcccgc 180cggtgcccgc gccgggccgt ggggggcagc
atgcccgcgc gcgctgcctg aggacgccgc 240ggcccccgcc cccgccatgg
gcgcccctgc ctgcgccctc gcgctctgcg tggccgtggc 300catcgtggcc
ggcgcctcct cggagtcctt ggggacggag cagcgcgtcg tggggcgagc
360ggcagaagtc ccgggcccag agcccggcca gcaggagcag ttggtcttcg
gcagcgggga 420tgctgtggag ctgagctgtc ccccgcccgg gggtggtccc
atggggccca ctgtctgggt 480caaggatggc acagggctgg tgccctcgga
gcgtgtcctg gtggggcccc agcggctgca 540ggtgctgaat gcctcccacg
aggactccgg ggcctacagc tgccggcagc ggctcacgca 600gcgcgtactg
tgccacttca gtgtgcgggt gacagacgct ccatcctcgg gagatgacga
660agacggggag gacgaggctg aggacacagg tgtggacaca ggggcccctt
actggacacg 720gcccgagcgg atggacaaga agctgctggc cgtgccggcc
gccaacaccg tccgcttccg 780ctgcccagcc gctggcaacc ccactccctc
catctcctgg ctgaagaacg gcagggagtt 840ccgcggcgag caccgcattg
gaggcatcaa gctgcggcat cagcagtgga gcctggtcat 900ggaaagcgtg
gtgccctcgg accgcggcaa ctacacctgc gtcgtggaga acaagtttgg
960cagcatccgg cagacgtaca cgctggacgt gctggagcgc tccccgcacc
ggcccatcct 1020gcaggcgggg ctgccggcca accagacggc ggtgctgggc
agcgacgtgg agttccactg 1080caaggtgtac agtgacgcac agccccacat
ccagtggctc aagcacgtgg aggtgaatgg 1140cagcaaggtg ggcccggacg
gcacacccta cgttaccgtg ctcaagacgg cgggcgctaa 1200caccaccgac
aaggagctag aggttctctc cttgcacaac gtcacctttg aggacgccgg
1260ggagtacacc tgcctggcgg gcaattctat tgggttttct catcactctg
cgtggctggt 1320ggtgctgcca gccgaggagg agctggtgga ggctgacgag
gcgggcagtg tgtatgcagg 1380catcctcagc tacggggtgg gcttcttcct
gttcatcctg gtggtggcgg ctgtgacgct 1440ctgccgcctg cgcagccccc
ccaagaaagg cctgggctcc cccaccgtgc acaagatctc 1500ccgcttcccg
ctcaagcgac aggtgtccct ggagtccaac gcgtccatga gctccaacac
1560accactggtg cgcatcgcaa ggctgtcctc aggggagggc cccacgctgg
ccaatgtctc 1620cgagctcgag ctgcctgccg accccaaatg ggagctgtct
cgggcccggc tgaccctggg 1680caagcccctt ggggagggct gcttcggcca
ggtggtcatg gcggaggcca tcggcattga 1740caaggaccgg gccgccaagc
ctgtcaccgt agccgtgaag atgctgaaag acgatgccac 1800tgacaaggac
ctgtcggacc tggtgtctga gatggagatg atgaagatga tcgggaaaca
1860caaaaacatc atcaacctgc tgggcgcctg cacgcagggc gggcccctgt
acgtgctggt 1920ggagtacgcg gccaagggta acctgcggga gtttctgcgg
gcgcggcggc ccccgggcct 1980ggactactcc ttcgacacct gcaagccgcc
cgaggagcag ctcaccttca aggacctggt 2040gtcctgtgcc taccaggtgg
cccggggcat ggagtacttg gcctcccaga agtgcatcca 2100cagggacctg
gctgcccgca atgtgctggt gaccgaggac aacgtgatga agatcgcaga
2160cttcgggctg gcccgggacg tgcacaacct cgactactac aagaagacga
ccaacggccg 2220gctgcccgtg aagtggatgg cgcctgaggc cttgtttgac
cgagtctaca ctcaccagag 2280tgacgtctgg tcctttgggg tcctgctctg
ggagatcttc acgctggggg gctccccgta 2340ccccggcatc cctgtggagg
agctcttcaa gctgctgaag gagggccacc gcatggacaa 2400gcccgccaac
tgcacacacg acctgtacat gatcatgcgg gagtgctggc atgccgcgcc
2460ctcccagagg cccaccttca agcagctggt ggaggacctg gaccgtgtcc
ttaccgtgac 2520gtccaccgac gagtacctgg acctgtcggc gcctttcgag
cagtactccc cgggtggcca 2580ggacaccccc agctccagct cctcagggga
cgactccgtg tttgcccacg acctgctgcc 2640cccggcccca cccagcagtg
ggggctcgcg gacgtgaagg gccactggtc cccaacaatg 2700tgaggggtcc
ctagcagccc accctgctgc tggtgcacag ccactccccg gcatgagact
2760cagtgcagat ggagagacag ctacacagag ctttggtctg tgtgtgtgtg
tgtgcgtgtg 2820tgtgtgtgtg tgtgcacatc cgcgtgtgcc tgtgtgcgtg
cgcatcttgc ctccaggtgc 2880agaggtaccc tgggtgtccc cgctgctgtg
caacggtctc ctgactggtg ctgcagcacc 2940gaggggcctt tgttctgggg
ggacccagtg cagaatgtaa gtgggcccac ccggtgggac 3000ccccgtgggg
cagggagctg ggcccgacat ggctccggcc tctgcctttg caccacggga
3060catcacaggg tgggcctcgg cccctcccac acccaaagct gagcctgcag
ggaagcccca 3120catgtccagc accttgtgcc tggggtgtta gtggcaccgc
ctccccacct ccaggctttc 3180ccacttccca ccctgcccct cagagactga
aattacgggt acctgaagat gggagccttt 3240accttttatg caaaaggttt
attccggaaa ctagtgtaca tttctataaa tagatgctgt 3300gtatatggta
tatatacata tatatatata acatatatgg aagaggaaaa ggctggtaca
3360acggaggcct gcgaccctgg gggcacagga ggcaggcatg gccctgggcg
gggcgtgggg 3420gggcgtggag ggaggcccca gggggtctca cccatgcaag
cagaggacca gggccttttc 3480tggcaccgca gttttgtttt aaaactggac
ctgtatattt gtaaagctat ttatgggccc 3540ctggcactct tgttcccaca
ccccaacact tccagcattt agctggccac atggcggaga 3600gttttaattt
ttaacttatt gacaaccgag aaggtttatc ccgccgatag agggacggcc
3660aagaatgtac gtccagcctg ccccggagct ggaggatccc ctccaagcct
aaaaggttgt 3720taatagttgg aggtgattcc agtgaagata ttttatttcc
tttgtccttt ttcaggagaa 3780ttagatttct ataggatttt tctttaggag
atttattttt tggacttcaa agcaagctgg 3840tattttcata caaattcttc
taattgctgt gtgtcccagg cagggagacg gtttccaggg 3900aggggccggc
cctgtgtgca ggttccgatg ttattagatg ttacaagttt atatatatct
3960atatatataa tttattgagt ttttacaaga tgtatttgtt gtagacttaa
cacttcttac 4020gcaatgcttc tagagtttta tagcctggac tgctaccttt
caaagcttgg agggaagccg 4080tgaattcagt tggttcgttc tgtactgtta
ctgggccctg agtctgggca gctgtccctt 4140gcttgcctgc agggccatgg
ctcagggtgg tctcttcttg gggcccagtg catggtggcc 4200agaggtgtca
cccaaaccgg caggtgcgat tttgttaacc cagcgacgaa ctttccgaaa
4260aataaagaca cctggttgct aacctg 42861403950DNAHomo sapiens
140gtcgcgggca gctggcgccg cgcggtcctg ctctgccggt cgcacggacg
caccggcggg 60ccgccggccg gagggacggg gcgggagctg ggcccgcgga cagcgagccg
gagcgggagc 120cgcgcgtagc gagccgggct ccggcgctcg ccagtctccc
gagcggcgcc cgcctcccgc 180cggtgcccgc gccgggccgt ggggggcagc
atgcccgcgc gcgctgcctg aggacgccgc 240ggcccccgcc cccgccatgg
gcgcccctgc ctgcgccctc gcgctctgcg tggccgtggc 300catcgtggcc
ggcgcctcct cggagtcctt ggggacggag cagcgcgtcg tggggcgagc
360ggcagaagtc ccgggcccag agcccggcca gcaggagcag ttggtcttcg
gcagcgggga 420tgctgtggag ctgagctgtc ccccgcccgg gggtggtccc
atggggccca ctgtctgggt 480caaggatggc acagggctgg tgccctcgga
gcgtgtcctg gtggggcccc agcggctgca 540ggtgctgaat gcctcccacg
aggactccgg ggcctacagc tgccggcagc ggctcacgca 600gcgcgtactg
tgccacttca gtgtgcgggt gacagacgct ccatcctcgg gagatgacga
660agacggggag gacgaggctg aggacacagg tgtggacaca ggggcccctt
actggacacg 720gcccgagcgg atggacaaga agctgctggc cgtgccggcc
gccaacaccg tccgcttccg 780ctgcccagcc gctggcaacc ccactccctc
catctcctgg ctgaagaacg gcagggagtt 840ccgcggcgag caccgcattg
gaggcatcaa gctgcggcat cagcagtgga gcctggtcat 900ggaaagcgtg
gtgccctcgg accgcggcaa ctacacctgc gtcgtggaga acaagtttgg
960cagcatccgg cagacgtaca cgctggacgt gctggagcgc tccccgcacc
ggcccatcct 1020gcaggcgggg ctgccggcca accagacggc ggtgctgggc
agcgacgtgg agttccactg 1080caaggtgtac agtgacgcac agccccacat
ccagtggctc aagcacgtgg aggtgaatgg 1140cagcaaggtg ggcccggacg
gcacacccta cgttaccgtg ctcaaggtgt ccctggagtc 1200caacgcgtcc
atgagctcca acacaccact ggtgcgcatc gcaaggctgt cctcagggga
1260gggccccacg ctggccaatg tctccgagct cgagctgcct gccgacccca
aatgggagct 1320gtctcgggcc cggctgaccc tgggcaagcc ccttggggag
ggctgcttcg gccaggtggt 1380catggcggag gccatcggca ttgacaagga
ccgggccgcc aagcctgtca ccgtagccgt
1440gaagatgctg aaagacgatg ccactgacaa ggacctgtcg gacctggtgt
ctgagatgga 1500gatgatgaag atgatcggga aacacaaaaa catcatcaac
ctgctgggcg cctgcacgca 1560gggcgggccc ctgtacgtgc tggtggagta
cgcggccaag ggtaacctgc gggagtttct 1620gcgggcgcgg cggcccccgg
gcctggacta ctccttcgac acctgcaagc cgcccgagga 1680gcagctcacc
ttcaaggacc tggtgtcctg tgcctaccag gtggcccggg gcatggagta
1740cttggcctcc cagaagtgca tccacaggga cctggctgcc cgcaatgtgc
tggtgaccga 1800ggacaacgtg atgaagatcg cagacttcgg gctggcccgg
gacgtgcaca acctcgacta 1860ctacaagaag acgaccaacg gccggctgcc
cgtgaagtgg atggcgcctg aggccttgtt 1920tgaccgagtc tacactcacc
agagtgacgt ctggtccttt ggggtcctgc tctgggagat 1980cttcacgctg
gggggctccc cgtaccccgg catccctgtg gaggagctct tcaagctgct
2040gaaggagggc caccgcatgg acaagcccgc caactgcaca cacgacctgt
acatgatcat 2100gcgggagtgc tggcatgccg cgccctccca gaggcccacc
ttcaagcagc tggtggagga 2160cctggaccgt gtccttaccg tgacgtccac
cgacgagtac ctggacctgt cggcgccttt 2220cgagcagtac tccccgggtg
gccaggacac ccccagctcc agctcctcag gggacgactc 2280cgtgtttgcc
cacgacctgc tgcccccggc cccacccagc agtgggggct cgcggacgtg
2340aagggccact ggtccccaac aatgtgaggg gtccctagca gcccaccctg
ctgctggtgc 2400acagccactc cccggcatga gactcagtgc agatggagag
acagctacac agagctttgg 2460tctgtgtgtg tgtgtgtgcg tgtgtgtgtg
tgtgtgtgca catccgcgtg tgcctgtgtg 2520cgtgcgcatc ttgcctccag
gtgcagaggt accctgggtg tccccgctgc tgtgcaacgg 2580tctcctgact
ggtgctgcag caccgagggg cctttgttct ggggggaccc agtgcagaat
2640gtaagtgggc ccacccggtg ggacccccgt ggggcaggga gctgggcccg
acatggctcc 2700ggcctctgcc tttgcaccac gggacatcac agggtgggcc
tcggcccctc ccacacccaa 2760agctgagcct gcagggaagc cccacatgtc
cagcaccttg tgcctggggt gttagtggca 2820ccgcctcccc acctccaggc
tttcccactt cccaccctgc ccctcagaga ctgaaattac 2880gggtacctga
agatgggagc ctttaccttt tatgcaaaag gtttattccg gaaactagtg
2940tacatttcta taaatagatg ctgtgtatat ggtatatata catatatata
tataacatat 3000atggaagagg aaaaggctgg tacaacggag gcctgcgacc
ctgggggcac aggaggcagg 3060catggccctg ggcggggcgt gggggggcgt
ggagggaggc cccagggggt ctcacccatg 3120caagcagagg accagggcct
tttctggcac cgcagttttg ttttaaaact ggacctgtat 3180atttgtaaag
ctatttatgg gcccctggca ctcttgttcc cacaccccaa cacttccagc
3240atttagctgg ccacatggcg gagagtttta atttttaact tattgacaac
cgagaaggtt 3300tatcccgccg atagagggac ggccaagaat gtacgtccag
cctgccccgg agctggagga 3360tcccctccaa gcctaaaagg ttgttaatag
ttggaggtga ttccagtgaa gatattttat 3420ttcctttgtc ctttttcagg
agaattagat ttctatagga tttttcttta ggagatttat 3480tttttggact
tcaaagcaag ctggtatttt catacaaatt cttctaattg ctgtgtgtcc
3540caggcaggga gacggtttcc agggaggggc cggccctgtg tgcaggttcc
gatgttatta 3600gatgttacaa gtttatatat atctatatat ataatttatt
gagtttttac aagatgtatt 3660tgttgtagac ttaacacttc ttacgcaatg
cttctagagt tttatagcct ggactgctac 3720ctttcaaagc ttggagggaa
gccgtgaatt cagttggttc gttctgtact gttactgggc 3780cctgagtctg
ggcagctgtc ccttgcttgc ctgcagggcc atggctcagg gtggtctctt
3840cttggggccc agtgcatggt ggccagaggt gtcacccaaa ccggcaggtg
cgattttgtt 3900aacccagcga cgaactttcc gaaaaataaa gacacctggt
tgctaacctg 39501412085DNAHomo sapiens 141atgggcgccc ctgcctgcgc
cctcgcgctc tgcgtggccg tggccatcgt ggccggcgcc 60tcctcggagt ccttggggac
ggagcagcgc gtcgtggggc gagcggcaga agtcccgggc 120ccagagcccg
gccagcagga gcagttggtc ttcggcagcg gggatgctgt ggagctgagc
180tgtcccccgc ccgggggtgg tcccatgggg cccactgtct gggtcaagga
tggcacaggg 240ctggtgccct cggagcgtgt cctggtgggg ccccagcggc
tgcaggtgct gaatgcctcc 300cacgaggact ccggggccta cagctgccgg
cagcggctca cgcagcgcgt actgtgccac 360ttcagtgtgc gggtgacaga
cgctccatcc tcgggagatg acgaagacgg ggaggacgag 420gctgaggaca
caggtgtgga cacaggggcc ccttactgga cacggcccga gcggatggac
480aagaagctgc tggccgtgcc ggccgccaac accgtccgct tccgctgccc
agccgctggc 540aaccccactc cctccatctc ctggctgaag aacggcaggg
agttccgcgg cgagcaccgc 600attggaggca tcaagctgcg gcatcagcag
tggagcctgg tcatggaaag cgtggtgccc 660tcggaccgcg gcaactacac
ctgcgtcgtg gagaacaagt ttggcagcat ccggcagacg 720tacacgctgg
acgtgctgga gcgctccccg caccggccca tcctgcaggc ggggctgccg
780gccaaccaga cggcggtgct gggcagcgac gtggagttcc actgcaaggt
gtacagtgac 840gcacagcccc acatccagtg gctcaagcac gtggaggtga
acggcagcaa ggtgggcccg 900gacggcacac cctacgttac cgtgctcaag
gtgtccctgg agtccaacgc gtccatgagc 960tccaacacac cactggtgcg
catcgcaagg ctgtcctcag gggagggccc cacgctggcc 1020aatgtctccg
agctcgagct gcctgccgac cccaaatggg agctgtctcg ggcccggctg
1080accctgggca agccccttgg ggagggctgc ttcggccagg tggtcatggc
ggaggccatc 1140ggcattgaca aggaccgggc cgccaagcct gtcaccgtag
ccgtgaagat gctgaaagac 1200gatgccactg acaaggacct gtcggacctg
gtgtctgaga tggagatgat gaagatgatc 1260gggaaacaca aaaacatcat
caacctgctg ggcgcctgca cgcagggcgg gcccctgtac 1320gtgctggtgg
agtacgcggc caagggtaac ctgcgggagt ttctgcgggc gcggcggccc
1380ccgggcctgg actactcctt cgacacctgc aagccgcccg aggagcagct
caccttcaag 1440gacctggtgt cctgtgccta ccaggtggcc cggggcatgg
agtacttggc ctcccagaag 1500tgcatccaca gggacctggc tgcccgcaat
gtgctggtga ccgaggacaa cgtgatgaag 1560atcgcagact tcgggctggc
ccgggacgtg cacaacctcg actactacaa gaagacaacc 1620aacggccggc
tgcccgtgaa gtggatggcg cctgaggcct tgtttgaccg agtctacact
1680caccagagtg acgtctggtc ctttggggtc ctgctctggg agatcttcac
gctggggggc 1740tccccgtacc ccggcatccc tgtggaggag ctcttcaagc
tgctgaagga gggccaccgc 1800atggacaagc ccgccaactg cacacacgac
ctgtacatga tcatgcggga gtgctggcat 1860gccgcgccct cccagaggcc
caccttcaag cagctggtgg aggacctgga ccgtgtcctt 1920accgtgacgt
ccaccgacga gtacctggac ctgtcggcgc ctttcgagca gtactccccg
1980ggtggccagg acacccccag ctccagctcc tcaggggacg actccgtgtt
tgcccacgac 2040ctgctgcccc cggccccacc cagcagtggg ggctcgcgga cgtga
20851424431DNAHomo sapiens 142aggcggggct ggagtggtgg aaggggggtg
gcaggtctgc attgccgctt ccctggtgcc 60gggagcagtc gccgctgccg cctccgcccg
cggccgggac ccccgtcctc gcccgggact 120ccttacccgg ggaacctaga
ccaggtctcc agaggcttgt ggaagagaag caggcgaccc 180ttcctgagtt
atcctggctt agcctcccaa tctggctccc cttccccttc ccattcccct
240gctccccctg tcccttcccc atccacccaa ctgaactggg tataggtcaa
agctcctctc 300tttccttttc cttcctaggc actcattggc taggacctgt
ttgctctttt ttttgtgccc 360agagatactg gaacacgctt catctaagta
actgtgggga ggggtctttt tgactctaca 420agtccttgag caaaaagctg
aaaaagaagc aggaggtgga gaagacccag tgaagtgccc 480caagccccat
catggaagag ggcttccgag accgggcagc tttcatccgt ggggccaaag
540acattgctaa ggaagtcaaa aagcatgcgg ccaagaaggt ggtgaagggc
ctggacagag 600tccaggacga atattcccga agatcgtact cccgctttga
ggaggaggat gatgatgatg 660acttccctgc tcccagtgat ggttattacc
gaggagaagg gacccaggat gaggaggaag 720gtggtgcatc cagtgatgct
actgagggcc atgacgagga tgatgagatc tatgaagggg 780aatatcaggg
cattccccgg gcagagtctg ggggcaaagg cgagcggatg gcagatgggg
840cgcccctggc tggagtaagg gggggcttga gtgatgggga gggtccccct
gggggccggg 900gggaggcaca acgacggaaa gaacgagaag aactggccca
acagtatgaa gccatcctac 960gggagtgtgg ccacggccgc ttccagtgga
cactgtattt tgtgcttggt ctggcgctga 1020tggctgacgg tgtggaggtc
tttgtggtgg gcttcgtgct gcccagcgct gagaaagaca 1080tgtgcctgtc
cgactccaac aaaggcatgc taggcctcat cgtctacctg ggcatgatgg
1140tgggagcctt cctctgggga ggtctggctg accggctggg tcggaggcag
tgtctgctca 1200tctcgctctc agtcaacagc gtcttcgcct tcttctcatc
ttttgtccag ggttacggca 1260ctttcctctt ctgccgccta ctttctgggg
ttgggattgg agggtccatc cccattgtct 1320tctcctattt ctccgagttt
ctggcccagg agaaacgagg ggagcatttg agctggctct 1380gcatgttttg
gatgattggt ggcgtgtacg cagctgctat ggcctgggcc atcatccccc
1440actatgggtg gagttttcag atgggttctg cctaccagtt ccacagctgg
agggtcttcg 1500tcctcgtctg cgcctttcct tctgtgtttg ccattggggc
tctgaccacg cagcctgaga 1560gcccccgttt cttcctagag aatggaaagc
atgatgaggc ctggatggtg ctgaagcagg 1620tccatgatac caacatgcga
gccaaaggac atcctgagcg agtgttctca gtaacccaca 1680ttaagacgat
tcatcaggag gatgaattga ttgagatcca gtcggacaca gggacctggt
1740accagcgctg gggggtccgg gccttgagcc taggggggca ggtttggggg
aattttctct 1800cctgttttgg tcccgaatat cggcgcatca ctctgatgat
gatgggtgtg tggttcacca 1860tgtcattcag ctactatggc ctgaccgtct
ggtttcctga catgatccgc catctccagg 1920cagtggacta cgcatcccgc
accaaagtgt tccccgggga gcgcgtagag catgtaactt 1980ttaacttcac
gttggagaat cagatccacc gaggcgggca gtacttcaat gacaagttca
2040ttgggctgcg gctcaagtca gtgtcctttg aggattccct gtttgaagag
tgttattttg 2100aggatgtcac atccagcaac acgtttttcc gcaactgcac
attcatcaac actgtgttct 2160ataacactga cctgttcgag tacaagtttg
tgaacagccg tctgataaac agtacattcc 2220tgcacaacaa ggagggctgc
ccgctagacg tgacagggac gggcgaaggt gcctacatgg 2280tatactttgt
gagcttcctg gggacactgg cagtgcttcc tgggaatatc gtgtctgccc
2340tgctcatgga caagatcggc aggctcagaa tgcttgctgg ctccagcgtg
atgtcctgtg 2400tctcctgctt cttcctgtct tttgggaaca gtgagtcggc
catgatcgct ctgctctgcc 2460tttttggcgg ggtcagcatt gcatcctgga
atgcgctgga cgtgttgact gttgaactct 2520acccctcaga caagaggacc
acagcttttg gcttcctgaa tgccctgtgt aagctggcag 2580ctgtgctggg
gatcagcatc ttcacatcct tcgtgggaat caccaaggct gcacccatcc
2640tctttgcctc agctgccctt gcccttggca gctctctggc cctgaagctg
cctgagaccc 2700gggggcaggt gctgcagtga aggggtctct agggctttgg
gattggcagg cacactgtga 2760gaccaacaac tccttccttc ccctccctgc
cctgccatcc tgacctccag agccctcact 2820ccccactccc cgtgtttggt
gtcttagctg tgtgtgcgtg tgcgtgtgca tgtgtgtaaa 2880ccccgtgggc
agggactaca gggaaggctc cttcatccca gttttgagat gaagctgtac
2940tccccatttc ccactgccct tgactttgca caagagaagg ctgagcccca
tccttctccc 3000cctgttagag aggggccctt gcttccctgt tccaggggtt
ccagaatagg cttcctgcct 3060tccccatcat tccctctgcc taggccctgg
tgaaaccaca ggtatgcaat tatgctaggg 3120gctggggctc tggtgtagac
catggaccaa aagaacttct tagagtctga agagtgggcc 3180tcgggtgccc
tctcacatct cctgttggat gctgggggag aagcaataaa cctcagccct
3240ctggcctcca ctttcctctc aatttgggct gcaaatatga agcctgaatt
ttatgaaatt 3300agctttctga ttcttattta ttaatagatt aagttctgag
gcagctccgc aggactgtgt 3360gtgaatgtgt atgtatactt acatatgtgt
gtgcatgtgc catggggcgg ggggtatcac 3420tatactgtcc tcaaatataa
gccaagggta atttcagcgg atgcacacac aaccctgcct 3480cccacagttc
ctcccctaat ctggtttctg tgttgagcct gggatggagg agccctaggc
3540cagcctggga taagagtccc acagtctagg gagatctgag ggcatccgac
aaggcccatc 3600tccttccctc ctcaagaagc agaggcctcc tctggagtga
gaggctccac ccactacagc 3660acaggcggga atagcacagc tgccctccca
tgctccctac ctgtcccctc acagggaggg 3720gagcagggga gggaaagaaa
ccaggcatct ggtcaaacca gcagatcaaa aagcacaaag 3780agctggggca
gaggcaggaa gcaggggccc tcctggcagc tcctctgagt ggggagaggt
3840tgggcagtga gtgagggacc cctaatgcag ggactagaag cctcagtttc
cccattttac 3900ccttccacac aatagcctct gtaggttagg ctgccccatc
ccaccctact ctgtgtggct 3960gctttctttg gtgccctccc ctcaccccac
tgtagctgtg acgtgttgta gtttttagat 4020gtttgtaaaa tgtttaaaaa
aatgttaaaa ggaaaaaagt gaaaataaca aaaaagaaaa 4080tcaaaattca
ccttcgtcat gctgcgtcca gtgccccaac cctgtggtca ctctccccat
4140tttgtaacac tgtaccaggt ggtgactgtt taactctttg gtgtctgtgc
tcaaaagact 4200gccttctcca gtgcccagtg tatgagtgtg tgccctgtgc
ccttgtccct cactccccac 4260atgctggacg tagccctctt cctcgcaccc
ctgggaggga cccatccatc tcccttgctc 4320tcctggggaa ccctaaaccc
aactctgttg atgtgaaaaa tgcagtgaaa aatattgacg 4380aaaaataaaa
cggaaacaaa tcctcaaaat acaaaaaaaa aaaaaaaaaa a 44311435311DNAHomo
sapiens 143agcataacct tcggtggcag gacaaatcag gccagcacgc agtctgccaa
gtcctgctcg 60ctccctgtca agaaaaacag ctggatccat ttctaatcaa cacttcccaa
cgcaacactt 120ctgagtctct gaaggagacc agagcttgaa actttccaga
cttccaacag acatcgagtg 180caaaaggata tttaggttgt ctttgcacaa
atctggttga tttgagagat aaaggggggg 240ggaaccagtg tgactttcac
ctaagaagtc acatgaacat atttcacatt tgaactacat 300aatgaatgat
ggttattgaa atagcccaaa cctctaccac agagcgaggg atatagctca
360aggggcaacc aggcagtcgc agaaccaagg aatggatgac tacaagtatc
aggacaatta 420tgggggctat gctcccagtg atggctatta ccgcggcaat
gagtccaacc cagaagaaga 480tgcacagagt gatgtcaccg aaggccatga
tgaggaagac gagatctatg agggcgagta 540ccagggtatc cctcacccag
atgatgtcaa ggccaagcag gccaagatgg cgccctccag 600aatggacagc
cttcggggcc agacagacct gatggctgag aggctggaag atgaggagca
660gttggcccac cagtacgaga ccatcatgga tgagtgtggc catggccgct
tccagtggat 720cctctttttc gtcttgggtt tggccctgat ggccgatggg
gtggaagtgt tcgtggtgag 780ttttgccctg cccagtgcag agaaggacat
gtgtctgtcc agttccaaaa aaggaatgct 840agggatgata gtctacttgg
gaatgatggc gggcgccttc atcctgggag gcctggctga 900taagctggga
aggaagcgag tcctcagcat gtctctggcc gtcaatgcct ccttcgcctc
960cctctcttcc ttcgtgcagg gatatggagc cttcctcttc tgccgactca
tctcaggcat 1020cggtattggg ggtgctctac cgattgtttt tgcctatttt
tctgaattct tgtctcggga 1080gaagcgagga gaacacctca gttggctggg
catcttctgg atgactgggg gcctgtacgc 1140atctgccatg gcctggagca
tcatcccaca ctatggctgg ggcttcagca tggggaccaa 1200ttaccacttc
catagctgga gagtgtttgt catcgtctgt gctctgccct gcaccgtgtc
1260catggtggcc ctgaagttca tgccagagag cccaaggttt ctgctagaga
tgggcaaaca 1320tgatgaagcc tggatgattc tcaagcaagt ccatgacacc
aacatgagag ctaaggggac 1380cccagagaaa gtgttcacgg tttccaacat
caaaactccc aagcaaatgg atgaattcat 1440tgagatccaa agttcaacag
gaacctggta ccagcgctgg ctggtcagat tcaagaccat 1500tttcaagcag
gtctgggata atgccctgta ctgtgtgatg gggccctaca gaatgaatac
1560actgattctg gccgtggttt ggtttgccat ggcattcagt tactatggac
tgacagtttg 1620gtttcctgat atgatccgct attttcaaga tgaagaatac
aagtctaaaa tgaaggtgtt 1680ttttggtgag catgtgtacg gcgccacaat
caacttcacg atggaaaatc agatccacca 1740acatgggaaa cttgtgaatg
ataagttcac aagaatgtac tttaaacatg tactctttga 1800ggacacattc
tttgacgagt gctattttga agacgtaaca tcaacagata cctacttcaa
1860aaattgtacc attgaatcaa ccatctttta caacacagac ctctacgagc
acaagttcat 1920caactgtcgg tttatcaact ccaccttcct ggagcagaag
gagggctgcc acatggactt 1980ggagcaagat aatgacttcc tgatttacct
cgtcagcttc ctgggcagcc tgtctgtctt 2040acccgggaac atcatttctg
ccctgctcat ggatagaatt ggaaggctca agatgattgg 2100tggctccatg
ctaatctctg cagtctgctg cttcttcctg ttttttggca acagtgagtc
2160tgcaatgatc ggctggcagt gcctgttctg tgggacaagc attgcagcct
ggaatgctct 2220ggatgtgatc acagtggagc tgtatcccac caaccagaga
gcaacagcct tcggcattct 2280caatggatta tgcaaatttg gcgccatcct
gggaaacacc atctttgctt cttttgttgg 2340gataaccaaa gtggtcccca
tccttctggc tgctgcttct ctggttgggg gtggcctgat 2400tgcccttcga
ctgccagaga ctcgagaaca ggtcctgatg tgaacaacct atgggaaaag
2460gaaaggtcga gagaatcttg tccaggacac tgaaatgcat ccacacttcc
tgcctatcac 2520ggtccggagg acaccttgga tagcacggga ggagaagttg
actttgtgac ccctagttta 2580ggacccactt cagctgtcaa tatgtttgta
actcaggtga ctgatttggg ggtgccctga 2640gccaccctta gaatcacaga
gctgcgtgtt taacttcaag tcttcccagt ccaaggcagg 2700gagaggattc
tccagtgagt gcacacacta tgcgaggagc aagcatttct ctaagtcaag
2760tgcaaggact taacttgcgt ttgaaaagga attagagggt cagaaacacc
caggttcctc 2820cagaaagctc cttggagccc aacaacttaa caaatcaact
tggctggaag ttagagtcat 2880tatatgaaga ttgggcttga agtatatatt
tttgcattta aaagtatcac ctatcatatt 2940ttccactcga aaattgacat
agtagcattg aggatactct gatctagaaa gccaagtatt 3000tgagcaacat
ctatagagat ctacttttct cctatgtctc ctaggctttc catgataatt
3060aggtaataca tttaagaagg atatttattt ctgttttgct ctattcaaag
aaacggaatg 3120ggatagttat tctgtaaact aagtttgtat ataactttat
ttgggtttaa tttccacaac 3180tggtatctgc aaatattgcc agcattttag
ccatattttg ggagaacttg gtgtttgagg 3240tcccaggaaa tgaggtctga
tcaaatgaaa tgcaagcaca atttcttaca gccatttaac 3300tttctgttgg
gaggatgaat taacaaactc acattgtgca gtctgcttaa tccaggcact
3360tttctttgtg caggtgtagt gagtagttac ttctctccct tacacagatg
acttgtgaaa 3420ctcaagctca ccatcttcag tgctggcatt ttactttgcc
actacccaaa aacaatgtga 3480gatgtgttca gtggcctctg gtactctttg
caggcaagaa tcaaacaaca tggggactga 3540gggaaggatg gggaagtgta
gccacagttc ttccaaatgt aaatactttt tgtttgttct 3600agtggtaaaa
tgcaaatgca atccatattt gttaggatgg tcaggtctca tgagaaatct
3660atgctatgtg tccagagctt ttgaaacaga gtccattgga gtgggagtta
gggagtgtag 3720tggatgccaa atatgttttt cttcagtgct taagagaact
gtttcctgaa gtccagcttt 3780gaacataaac aggggtgtgg gttgggggag
gagcttagga caaacctctc tgatgaaggt 3840cagcaataga ctgaagtctt
gactgcatgg aagaggaaaa acatcagaac tgtctgacaa 3900tggaggggac
agtgagctac gcacaactgc cagcggaggt gaacttgcac ctgcccaggc
3960cggatgaaca tcagcctgca agaactagtt gtttgagttg atttgcagtg
ctctcaatgg 4020gcaagtgcca cattttccct ggcagagatc tccaaaaatt
taaaacagaa taataatggc 4080tatatcgagt gttttctcag tattggagaa
atgcttaggt cctatgatag cttcgggaca 4140tctttctgta attttcctca
attaacgggt tggtaggggt aaatcttatg acacctttcc 4200accgtcgatt
tgagatcagt tttaatggtt aaaatgttta ctctccttct gtcaaccctc
4260acctttttat ttacacccct cccttttttt ctgtacaggg agagaagaca
tattgactct 4320gactggacac cctgattcct ccaaatatat ataccactgt
gtattaatct ttctctcagt 4380gttttatagg agtactaaca tttattgctc
tgtcaataat gaaaggctcg atgtaatata 4440gctgtaattt actttccata
tgaatacagt ggctaggttc ataaaagaga attgtgtgag 4500tctgggatta
ccacatctaa aacattattc tttaatggga taatacaatt cattgagcag
4560ctaccactta aaaaacttgc aggacagtta gagcctgcat ttctagttaa
gatggatctt 4620gtaaatttaa aattggatta acattggagt gctggggtgg
ctgcaataat ttgggggcta 4680actccatttg gtttccaaga tctcacttct
gcattatctt tatggctctt taaaccagcc 4740acctagccaa tcaagggcaa
ttcccatctc atccatcact caggtctttg taaagggtgc 4800agccaagctc
tgcagacttt tgcaggattg tctagcctga gtaccgggct acttcttaaa
4860tgccgtcact cctgctgaga taaatgcgtc tttaaaaata gtctctgtgg
caggtcactg 4920ggggacaatg tacagcattc tggccatcca cttctttttc
acttcatgtt ctaccccaag 4980agactcccga tgtcggctgt ggagggttaa
agggatgagg ctttcctttg tttagcaaat 5040ctgttcacag ttcttgatga
tgtattttat gatgcccagc ttggaaatag ttgctttcca 5100tagtctcaac
tgtattgtgt catctcctga tgctgatttt tgatcttttg ttttattaaa
5160aataattagt gaaagaggtg tgcctatctg tgaagtttgt agtacatcat
cctgaggtca 5220tgtaacaagt aaaccccaac ccagcgttcc ctcctacgtt
gtgttagttc attaaaacta 5280aataataaaa ataactgtaa gaaaacctta a
53111442362DNAHomo sapiens 144cactcagggc aagggtgtcc gacggctgga
gcgttctgtt ttgaacccaa agtggatgat 60gctgtcagag ctgaactact gaaaggaggc
tgtgaaaatt tcccatcttc tcattggcca 120tcagttgaga taagatggaa
gactcttaca aggataggac ttcactgatg aagggtgcca 180aggacattgc
cagagaggtg aagaaacaaa cagtaaagaa ggtgaatcaa gctgtggacc
240gagcccagga tgaatacacc cagaggtcct acagtcggtt ccaagatgaa
gaagatgatg 300atgactacta cccggctgga gaaacctata atggtgaggc
caacgatgac gaaggctcaa 360gtgaagccac tgaggggcat gatgaagatg
atgagatcta tgagggggag tatcagggca 420tccccagtat gaaccaagcg
aaggacagca tcgtgtcagt ggggcagccc aagggcgatg 480agtacaagga
ccgacgggag ctggaatcag aaaggagagc tgacgaggaa gagttagccc
540agcagtatga gctgataatc
caagaatgcg gtcatggtcg ttttcagtgg gcccttttct 600tcgtcctggg
catggctctt atggcagacg gtgtagaggt gtttgtcgtt ggcttcgtgt
660tacccagtgc tgagacagac ctctgcatcc caaattcagg atctggatgg
ctaggcagca 720tagtgtacct cgggatgatg gtgggggcgt tcttctgggg
aggactggca gacaaagtgg 780gaaggaaaca gtctcttctg atttgcatgt
ctgtcaacgg attctttgcc ttcctttctt 840catttgtcca aggttatggc
ttctttctct tctgtcgctt actttctgga ttcgggattg 900gaggagccat
acccactgtg ttctcgtact ttgctgaagt cctggcccgg gaaaagcggg
960gcgaacactt gagctggctc tgcatgttct ggatgatcgg tggcatctac
gcctctgcca 1020tggcctgggc catcatcccg cactacgggt ggagcttcag
catgggatcg gcctaccagt 1080ttcacagttg gcgtgtgttt gtcatcgtct
gtgcactccc ctgtgtctcc tccgtggtgg 1140ccctcacatt catgcctgaa
agcccacgat tcttgttgga ggttggaaaa catgatgaag 1200cttggatgat
tctgaagtta attcatgaca ccaacatgag agcccggggt cagcctgaga
1260aggtcttcac ggtaaacaaa ataaaaactc ctaaacaaat agatgagctg
attgaaattg 1320agagtgacac aggaacatgg tataggaggt gttttgttcg
gatccgcacc gagctgtacg 1380gaatttggtt gacttttatg agatgtttca
actacccagt cagggataat acaataaagc 1440ttacaattgt ttggttcacc
ctgtcctttg ggtactatgg attatccgtt tggttccctg 1500atgtcattaa
acctctgcag tccgatgaat atgcattgct aaccagaaat gtggagagag
1560ataaatatgc aaatttcact attaacttta caatggaaaa tcagattcat
actggaatgg 1620aatacgacaa tggcagattc ataggggtca agttcaaatc
tgtaactttc aaagactctg 1680tttttaagtc ctgcaccttt gaggatgtaa
cttcagtgaa cacctacttc aagaactgca 1740catttattga cactgttttt
gacaacacag attttgagcc atataaattc attgacagtg 1800aatttaaaaa
ctgctcgttt tttcacaaca agacgggatg tcagattacc tttgatgatg
1860actatagtgc ctactggatt tattttgtca actttctggg gacattggca
gtattgccag 1920ggaacattgt gtctgctctg ctgatggaca gaattgggcg
cttaacaatg ctaggtggct 1980ctatggtgct ttcggggatc agctgtttct
tcctttggtt cggcaccagt gaatccatga 2040tgataggcat gctgtgtctg
tacaatggat tgaccatctc agcctggaac tctcttgacg 2100tggtcactgt
ggaactgtac cccacagacc ggagggcaac aggctttggc ttcttaaatg
2160cgctatgcaa ggcagcagcc gtcctgggaa acttaatatt tggctctctg
gtcagcatca 2220ccaaatcaat ccccatcctg ctggcttcta ctgtgctcgt
gtgtggagga ctcgttgggc 2280tgtgcctgcc tgacacacga acccaggttc
tgatgtaatg ggaaaaaaag ccatccttcc 2340tgcgtttctt cctcctgccc tg
23621452053DNAHomo sapiens 145catctttgat gagggcagag ctcacgttgc
attgaagacg aaacctcggg gaggtcaggc 60gctgtctttc cttccctccc tgctcggcgg
ctccaccaca gttgcaacct gcagaggccc 120ggagaacaca accctcccga
gaagcccagg tccagagcca aacccgtcac tgacccccca 180gcccaggcgc
ccagccactc cccaccgcta ccatggccga agacgcagac atgcgcaatg
240agctggagga gatgcagcga agggctgacc agttggctga tgagtcgctg
gaaagcaccc 300gtcgtatgct gcaactggtt gaagagagta aagatgctgg
tatcaggact ttggttatgt 360tggatgaaca aggagaacaa ctcgatcgtg
tcgaagaagg catgaaccat atcaaccaag 420acatgaagga ggctgagaaa
aatttaaaag atttagggaa atgctgtggc cttttcatat 480gtccttgtaa
caagcttaaa tcaagtgatg cttacaaaaa agcctggggc aataatcagg
540acggagtggt ggccagccag cctgctcgtg tagtggacga acgggagcag
atggccatca 600gtggcggctt catccgcagg gtaacaaatg atgcccgaga
aaatgaaatg gatgaaaacc 660tagagcaggt gagcggcatc atcgggaacc
tccgtcacat ggccctggat atgggcaatg 720agatcgatac acagaatcgc
cagatcgaca ggatcatgga gaaggctgat tccaacaaaa 780ccagaattga
tgaggccaac caacgtgcaa caaagatgct gggaagtggt taagtgtgcc
840cacccgtgtt ctcctccaaa tgctgtcggg caagatagct ccttcatgct
tttctcatgg 900tattatctag taggtctgca cacataacac acatcagtcc
acccccattg tgaatgttgt 960cctgtgtcat ctgtcagctt cccaacaata
ctttgtgtct tttgttctct cttggtctct 1020ttctttccaa aggttgtaca
tagtggtcat ttggtggctc taactccttg atgtcttgag 1080tttcattttt
cattttctct cctcggtggc atttgctgaa taacaacaat ttaggaatgc
1140tcaatgtgct gttgattctt tcaatccaca gtattgttct tgtaaaactg
tgacattcca 1200cagagttact gccacggtcc tttgagtgtc aggctctgaa
tctctcaaaa tgtgccgtct 1260ttggttcctc atggctgtta tctgtcttta
tgatttcatg attagacaat gtggaattac 1320ataacaggca ttgcactaaa
agtgatgtga tttatgcatt tatgcatgag aactaaatag 1380atttttagat
tcctacttaa acaaaaactt tccatgacag tagcatactg atgagacaac
1440acacacacac acaaaacaac agcaacaaca acagaacaac aacaaagcat
gctcagtatt 1500gagacactgt caagattaag ttataccagc aaaagtgcag
tagtgtcact tttttcctgt 1560caatatatag agacttctaa atcataatca
tcctttttta aaaaaaagaa ttttaaaaaa 1620gatggatttg acacactcac
catttaatca tttccagcaa aatatatgtt tggctgaaat 1680tatgtcaaat
ggatgtaata tagggtttgt ttgctgcttt tgatggctac gttttggaga
1740gagcaatctt gctgtgaaac agtgtggatg taaattttat aaggctgact
cttactaacc 1800accatttccc ctgtggtttg ttatcagtac aattctttgt
tgcttaatct agagctatgc 1860acaccaaatt gctgagatgt ttagtagctg
ataaagaaac cttttaaaaa aataatataa 1920atgaatgaaa tataaactgt
gagataaata tcattatagc atgtaatatt aaattcctcc 1980tgtctcctct
gtcagtttgt gaagtgattg acattttgta gctagtttaa aattattaaa
2040aattatagac tcc 20531462053DNAHomo sapiens 146catctttgat
gagggcagag ctcacgttgc attgaagacg aaacctcggg gaggtcaggc 60gctgtctttc
cttccctccc tgctcggcgg ctccaccaca gttgcaacct gcagaggccc
120ggagaacaca accctcccga gaagcccagg tccagagcca aacccgtcac
tgacccccca 180gcccaggcgc ccagccactc cccaccgcta ccatggccga
agacgcagac atgcgcaatg 240agctggagga gatgcagcga agggctgacc
agttggctga tgagtcgctg gaaagcaccc 300gtcgtatgct gcaactggtt
gaagagagta aagatgctgg tatcaggact ttggttatgt 360tggatgaaca
aggagaacaa ctggaacgca ttgaggaagg gatggaccaa atcaataagg
420acatgaaaga agcagaaaag aatttgacgg acctaggaaa attctgcggg
ctttgtgtgt 480gtccctgtaa caagcttaaa tcaagtgatg cttacaaaaa
agcctggggc aataatcagg 540acggagtggt ggccagccag cctgctcgtg
tagtggacga acgggagcag atggccatca 600gtggcggctt catccgcagg
gtaacaaatg atgcccgaga aaatgaaatg gatgaaaacc 660tagagcaggt
gagcggcatc atcgggaacc tccgtcacat ggccctggat atgggcaatg
720agatcgatac acagaatcgc cagatcgaca ggatcatgga gaaggctgat
tccaacaaaa 780ccagaattga tgaggccaac caacgtgcaa caaagatgct
gggaagtggt taagtgtgcc 840cacccgtgtt ctcctccaaa tgctgtcggg
caagatagct ccttcatgct tttctcatgg 900tattatctag taggtctgca
cacataacac acatcagtcc acccccattg tgaatgttgt 960cctgtgtcat
ctgtcagctt cccaacaata ctttgtgtct tttgttctct cttggtctct
1020ttctttccaa aggttgtaca tagtggtcat ttggtggctc taactccttg
atgtcttgag 1080tttcattttt cattttctct cctcggtggc atttgctgaa
taacaacaat ttaggaatgc 1140tcaatgtgct gttgattctt tcaatccaca
gtattgttct tgtaaaactg tgacattcca 1200cagagttact gccacggtcc
tttgagtgtc aggctctgaa tctctcaaaa tgtgccgtct 1260ttggttcctc
atggctgtta tctgtcttta tgatttcatg attagacaat gtggaattac
1320ataacaggca ttgcactaaa agtgatgtga tttatgcatt tatgcatgag
aactaaatag 1380atttttagat tcctacttaa acaaaaactt tccatgacag
tagcatactg atgagacaac 1440acacacacac acaaaacaac agcaacaaca
acagaacaac aacaaagcat gctcagtatt 1500gagacactgt caagattaag
ttataccagc aaaagtgcag tagtgtcact tttttcctgt 1560caatatatag
agacttctaa atcataatca tcctttttta aaaaaaagaa ttttaaaaaa
1620gatggatttg acacactcac catttaatca tttccagcaa aatatatgtt
tggctgaaat 1680tatgtcaaat ggatgtaata tagggtttgt ttgctgcttt
tgatggctac gttttggaga 1740gagcaatctt gctgtgaaac agtgtggatg
taaattttat aaggctgact cttactaacc 1800accatttccc ctgtggtttg
ttatcagtac aattctttgt tgcttaatct agagctatgc 1860acaccaaatt
gctgagatgt ttagtagctg ataaagaaac cttttaaaaa aataatataa
1920atgaatgaaa tataaactgt gagataaata tcattatagc atgtaatatt
aaattcctcc 1980tgtctcctct gtcagtttgt gaagtgattg acattttgta
gctagtttaa aattattaaa 2040aattatagac tcc 20531475PRTArtificial
SequenceSNAP-25 antigen having a free carboxyl-terminus at the P1
residue of the scissile bond of the BoNT/A cleavage site 147Asp Glu
Ala Asn Gln1 5 1486PRTArtificial SequenceSNAP-25 antigen having a
free carboxyl-terminus at the P1 residue of the scissile bond of
the BoNT/A cleavage site 148Ile Asp Glu Ala Asn Gln1 5 149360DNAMus
musculus 149caggtgaagc tgcaggagtc tggacctgaa ctggtaaagc ctggggcttc
agtgaagatg 60tcctgcaagg cttctggata cacattcact aactatgtta tacactgggt
gaagcaaaag 120cctgggcagg gccttgagtg gattggatat attaatcctt
acaatgatgg ctctaagtac 180aatgagaagt tcaaaggcaa ggcctcactg
acttcagaca aatcctccag cacagcctac 240atggagctca gcagcctgac
ctctgaggac tctgcggtct attactgtgc aagacatctc 300gctaatacct
actactactt tgactactgg ggccaaggca ccactctcac agtctcctca
360150120PRTMus musculus 150Gln Val Gln Leu Gln Glu Ser Gly Pro Glu
Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Ile His Trp Val Lys
Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn
Pro Tyr Asn Asp Gly Ser Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly
Lys Ala Ser Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95 Ala Arg His Leu Ala Asn Thr Tyr Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 1201515PRTMus
musculus 151Ala Arg Met Gly Tyr1 515213PRTMus musculus 152Ala Arg
His Leu Ala Asn Thr Tyr Tyr Tyr Phe Asp Tyr1 5 10
* * * * *