U.S. patent application number 14/187713 was filed with the patent office on 2014-11-13 for tal effector-mediated dna modification.
This patent application is currently assigned to Iowa State University Research Foundation, Inc.. The applicant listed for this patent is Iowa State University Research Foundation, Inc., Regents of the University of Minnesota. Invention is credited to Adam J. Bogdanove, Daniel F. Voytas, Feng Zhang.
Application Number | 20140335592 14/187713 |
Document ID | / |
Family ID | 43825298 |
Filed Date | 2014-11-13 |
United States Patent
Application |
20140335592 |
Kind Code |
A1 |
Voytas; Daniel F. ; et
al. |
November 13, 2014 |
TAL EFFECTOR-MEDIATED DNA MODIFICATION
Abstract
Materials and Methods related to gene targeting (e.g., gene
targeting with transcription activator-like effector nucleases;
"TALENS") are provided.
Inventors: |
Voytas; Daniel F.; (Falcon
Heights, MN) ; Bogdanove; Adam J.; (Ithaca, NY)
; Zhang; Feng; (Plymouth, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Iowa State University Research Foundation, Inc.
Regents of the University of Minnesota |
Ames
Minneapolis |
IA
MN |
US
US |
|
|
Assignee: |
Iowa State University Research
Foundation, Inc.
Ames
IA
Regents of the University of Minnesota
Minneapolis
MN
|
Family ID: |
43825298 |
Appl. No.: |
14/187713 |
Filed: |
February 24, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13738381 |
Jan 10, 2013 |
8697853 |
|
|
14187713 |
|
|
|
|
12965590 |
Dec 10, 2010 |
8586363 |
|
|
13738381 |
|
|
|
|
61285324 |
Dec 10, 2009 |
|
|
|
61352108 |
Jun 7, 2010 |
|
|
|
61366685 |
Jul 22, 2010 |
|
|
|
Current U.S.
Class: |
435/196 |
Current CPC
Class: |
C12N 15/1082 20130101;
C12N 15/63 20130101; A61P 31/12 20180101; C12N 15/01 20130101; C12N
15/62 20130101; C12N 15/8213 20130101; C12N 9/22 20130101; C12N
15/102 20130101; C12Y 301/21004 20130101 |
Class at
Publication: |
435/196 |
International
Class: |
C12N 9/22 20060101
C12N009/22 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant
nos. 0820831 and 0504304, awarded by the National Science
Foundation. The government has certain rights in the invention.
Claims
1. (canceled)
2. A first transcription activator-like (TAL) effector endonuclease
monomer comprising: (i) a FokI endonuclease domain, and (ii) a TAL
effector domain comprising a plurality of TAL effector repeat
sequences, wherein the plurality of TAL effector repeat sequences
of the first TAL effector endonuclease monomer, in combination,
bind to a first nucleotide sequence in a target DNA sequence in a
cell, wherein the first TAL effector endonuclease monomer is
capable of forming a dimer with a second TAL effector endonuclease
monomer comprising a FokI endonuclease domain and a TAL effector
domain having a plurality of TAL effector repeat sequences that, in
combination, bind to a second nucleotide sequence in the target DNA
sequence, wherein the dimer is formed between the FokI domain of
the first TAL effector endonuclease monomer and the FokI domain of
the second TAL effector endonuclease monomer when the TAL effector
domain of the first TAL effector endonuclease monomer is bound to
the first nucleotide sequence and the TAL effector domain of the
second TAL effector endonuclease monomer is bound to the second
nucleotide sequence, wherein the first nucleotide sequence and the
second nucleotide sequence are different and are separated by a
spacer sequence, and wherein the dimer cleaves the target DNA
sequence within the cell.
3. The first TAL effector endonuclease monomer of claim 2, wherein
the target DNA sequence is in a promoter region.
4. The first TAL effector endonuclease monomer of claim 2, wherein
the TAL effector domain comprises 15 or more DNA binding
repeats.
5. The first TAL effector endonuclease monomer of claim 2, wherein
each DNA binding repeat comprises a repeat variable-diresidue (RVD)
that determines recognition of a base pair in the target DNA
sequence, wherein each DNA binding repeat is responsible for
recognizing one base pair in the target DNA sequence, and wherein
the RVD comprises one or more of: HD for recognizing C; NG for
recognizing T; NI for recognizing A; NN for recognizing G; NS for
recognizing A; HG for recognizing T; IG for recognizing T; NK for
recognizing G; HA for recognizing C; ND for recognizing C; HI for
recognizing C; HN for recognizing G; and NA for recognizing G.
6. The first TAL effector endonuclease monomer of claim 5, wherein
each DNA binding repeat comprises a RVD that determines recognition
of a base pair in the target DNA sequence, wherein each DNA binding
repeat is responsible for recognizing one base pair in the target
DNA sequence, and wherein the RVD comprises one or more of: HA for
recognizing C; ND for recognizing C; HI for recognizing C; HN for
recognizing G; NA for recognizing G; and NK for recognizing G; and
one or more of: HD for recognizing C; NG for recognizing T; NI for
recognizing A; NN for recognizing G or A; NS for recognizing A or C
or G or T; HG for recognizing T; and IG for recognizing T.
7. The first TAL effector endonuclease monomer of claim 1, wherein
the spacer sequence is 12 to 30 nucleotides in length.
8. The first TAL effector endonuclease monomer of claim 1, wherein
the spacer sequence is 18 nucleotides in length.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
12/965,590, filed on Dec. 10, 2010, which claims benefit of
priority from U.S. Provisional Application Ser. No. 61/285,324,
filed on Dec. 10, 2009, U.S. Provisional Application Ser. No.
61/352,108, filed on Jun. 7, 2010, and U.S. Provisional Application
Ser. No. 61/366,685, filed on Jul. 22, 2010, all of which are
incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0003] The present document relates to methods for gene targeting,
and particularly to methods that include the use of transcription
activator-like (TAL) effector sequences.
BACKGROUND
[0004] The ability to modify chromosomes through homologous
recombination (gene targeting) has been a long sought goal of
biologists. In plants, for example, gene targeting may help to
discern the function of plant genes, opening up new possibilities
for crop improvement. For example, with gene targeting it is
possible to carry out the genetic surgery required to reorchestrate
metabolic pathways to create high value crops, including seed with
altered oil or carbohydrate profiles, food with enhanced
nutritional qualities, or plants with increased resistance to
disease and stress. In animals (e.g., mammals), gene targeting may
be used for treatment of disease. For example, gene targeting may
be used to engineer corrections in genes that are defective due to
various types of mutations. Efficient methods for such gene
targeting have been difficult to achieve.
SUMMARY
[0005] TAL effectors of plant pathogenic bacteria in the genus
Xanthomonas play important roles in disease, or trigger defense, by
binding host DNA and activating effector-specific host genes (see,
e.g., Gu et al. (2005) Nature 435:1122; Yang et al. (2006) Proc.
Natl. Acad. Sci. USA 103:10503; Kay et al. (2007) Science 318:648;
Sugio et al. (2007) Proc. Natl. Acad. Sci. USA 104:10720; and Romer
et al. (2007) Science 318:645). Specificity depends on an
effector-variable number of imperfect, typically 34 amino acid
repeats (Schornack et al. (2006) J. Plant Physiol. 163:256).
Polymorphisms are present primarily at repeat positions 12 and 13,
which are referred to herein as the repeat variable-diresidue
(RVD).
[0006] The present document is based in part on the fact that the
RVDs of TAL effectors correspond to the nucleotides in their target
sites in a direct, linear fashion, one RVD to one nucleotide, with
some degeneracy and no apparent context dependence. This surprising
finding represents a novel mechanism for protein-DNA recognition
that enables target site prediction for new target specific TAL
effector. As described herein, these proteins may be useful in
research and biotechnology as targeted chimeric nucleases that can
facilitate homologous recombination in genome engineering (e.g., to
add or enhance traits useful for biofuels or biorenewables in
plants). These proteins also may be useful as, for example,
transcription factors, and especially for therapeutic applications
requiring a very high level of specificity such as therapeutics
against pathogens (e.g., viruses) as non limiting examples.
[0007] In one aspect, this document features a method for modifying
the genetic material of a cell, comprising (a) providing a cell
containing a target DNA sequence; and (b) introducing a
transcription activator-like (TAL) effector -DNA modifying enzyme
into the cell, the TAL effector-DNA modifying enzyme comprising (i)
a DNA modifying enzyme domain that can modify double stranded DNA,
and (ii) a TAL effector domain comprising a plurality of TAL
effector repeat sequences that, in combination, bind to a specific
nucleotide sequence in the target DNA sequence, such that the TAL
effector-DNA modifying enzyme modifies the target DNA within or
adjacent to the specific nucleotide sequence in the cell or progeny
thereof. The method can further comprise providing to the cell a
nucleic acid comprising a sequence homologous to at least a portion
of the target DNA sequence, such that homologous recombination
occurs between the target DNA sequence and the nucleic acid. The
cell can be a eukaryotic cell, a mammalian cell, a plant cell, or a
prokaryotic cell. The target DNA can be chromosomal DNA. The
introducing can comprise transfecting the cell with a vector
encoding the TAL effector-DNA modifying enzyme, mechanically
injecting the TAL effector-DNA modifying enzyme into the cell as a
protein, delivering the TAL effector-DNA modifying enzyme into the
cell as a protein by means of the bacterial type III secretion
system, or introducing the TAL effector-DNA modifying enzyme into
the cell as a protein by electroporation. The DNA modifying enzyme
can be an endonuclease (e.g., a type II restriction endonuclease,
such as FokI).
[0008] The TAL effector domain that binds to a specific nucleotide
sequence within the target DNA can comprise 10 or more DNA binding
repeats, and preferably 15 or more DNA binding repeats. Each DNA
binding repeat can include a repeat variable-diresidue (RVD) that
determines recognition of a base pair in the target DNA sequence,
wherein each DNA binding repeat is responsible for recognizing one
base pair in the target DNA sequence, and wherein the RVD comprises
one or more of: HD for recognizing C; NG for recognizing T; NI for
recognizing A; NN for recognizing G or A; NS for recognizing A or C
or G or T; N* for recognizing C or T, where * represents a gap in
the second position of the RVD; HG for recognizing T; H* for
recognizing T, where * represents a gap in the second position of
the RVD; IG for recognizing T; NK for recognizing G; HA for
recognizing C; ND for recognizing C; HI for recognizing C; HN for
recognizing G; NA for recognizing G; SN for recognizing G or A; and
YG for recognizing T. Each DNA binding repeat can comprise a RVD
that determines recognition of a base pair in the target DNA
sequence, wherein each DNA binding repeat is responsible for
recognizing one base pair in the target DNA sequence, and wherein
the RVD comprises one or more of: HA for recognizing C; ND for
recognizing C; HI for recognizing C; HN for recognizing G; NA for
recognizing G; SN for recognizing G or A; YG for recognizing T; and
NK for recognizing G, and one or more of: HD for recognizing C; NG
for recognizing T; NI for recognizing A; NN for recognizing G or A;
NS for recognizing A or C or G or T; N* for recognizing C or T,
wherein * represents a gap in the second position of the RVD; HG
for recognizing T; H* for recognizing T, wherein * represents a gap
in the second position of the RVD; and IG for recognizing T.
[0009] In another aspect, this document features a method for
generating a nucleic acid encoding a TAL effector specific for a
selected nucleotide sequence, comprising: (1) linearizing a starter
plasmid with PspXI, the starter plasmid comprising a nucleotide
sequence that encodes a first TAL effector DNA binding repeat
domain having a repeat variable-diresidue (RVD) specific for the
first nucleotide of the selected nucleotide sequence, wherein the
first TAL effector DNA binding repeat domain has a unique PspXI
site at its 3' end; (2) ligating into the starter plasmid PspXI
site a DNA module encoding one or more TAL effector DNA binding
repeat domains that have RVDs specific for the next nucleotide(s)
of the selected nucleotide sequence, wherein the DNA module has
XhoI sticky ends; and (3) repeating steps (1) and (2) until the
nucleic acid encodes a TAL effector capable of binding to the
selected nucleotide sequence. The method can further comprise,
after the ligating, determining the orientation of the DNA module
in the PspXI site. The method can comprise repeating steps (1) and
(2) from one to 30 times.
[0010] In another aspect, this document features a method for
generating a nucleic acid encoding a transcription activator-like
effector endonuclease (TALEN), comprising (a) identifying a first
nucleotide sequence in the genome of a cell; and (b) synthesizing a
nucleic acid encoding a TALEN that comprises (i) a plurality of DNA
binding repeats that, in combination, bind to the first unique
nucleotide sequence, and (ii) an endonuclease that generates a
double-stranded cut at a position within or adjacent to the first
nucleotide sequence, wherein each DNA binding repeat comprises a
RVD that determines recognition of a base pair in the target DNA,
wherein each DNA binding repeat is responsible for recognizing one
base pair in the target DNA, and wherein the TALEN comprises one or
more of the following RVDs: HD for recognizing C; NG for
recognizing T; NI for recognizing A; NN for recognizing G or A; NS
for recognizing A or C or G or T; N* for recognizing C or T; HG for
recognizing T; H* for recognizing T; IG for recognizing T; NK for
recognizing G; HA for recognizing C; ND for recognizing C; HI for
recognizing C; HN for recognizing G; NA for recognizing G; SN for
recognizing G or A; and YG for recognizing T.
[0011] The TALEN can comprises one or more of the following RVDs:
HA for recognizing C; ND for recognizing C; HI for recognizing C;
HN for recognizing G; NA for recognizing G; SN for recognizing G or
A; YG for recognizing T; and NK for recognizing G, and one or more
of: HD for recognizing C; NG for recognizing T; NI for recognizing
A; NN for recognizing G or A; NS for recognizing A or C or G or T;
N* for recognizing C or T; HG for recognizing T; H* for recognizing
T; and IG for recognizing T.
[0012] The first nucleotide sequence can meet at least one of the
following criteria: i) is a minimum of 15 bases long and is
oriented from 5' to 3' with a T immediately preceding the site at
the 5' end; ii) does not have a T in the first (5') position or an
A in the second position; iii) ends in T at the last (3') position
and does not have a G at the next to last position; and iv) has a
base composition of 0-63% A, 11-63% C, 0-25% G, and 2-42% T.
[0013] The method can comprise identifying a first nucleotide
sequence and a second nucleotide sequence in the genome of the
cell, wherein the first and second nucleotide sequences meet at
least one of the criteria set forth above and are separated by
15-18 bp. The endonuclease can generate a double-stranded cut
between the first and second nucleotide sequences.
[0014] In another embodiment, this document features a TALEN
comprising an endonuclease domain and a TAL effector DNA binding
domain specific for a target DNA, wherein the DNA binding domain
comprises a plurality of DNA binding repeats, each repeat
comprising a RVD that determines recognition of a base pair in the
target DNA, wherein each DNA binding repeat is responsible for
recognizing one base pair in the target DNA, and wherein the TALEN
comprises one or more of the following RVDs: HD for recognizing C;
NG for recognizing T; NI for recognizing A; NN for recognizing G or
A; NS for recognizing A or C or G or T; N* for recognizing C or T;
HG for recognizing T; H* for recognizing T; IG for recognizing T;
NK for recognizing G; HA for recognizing C; ND for recognizing C;
HI for recognizing C; HN for recognizing G; NA for recognizing G;
SN for recognizing G or A; and YG for recognizing T. The TALEN can
comprise one or more of the following RVDs: HA for recognizing C;
ND for recognizing C; HI for recognizing C; HN for recognizing G;
NA for recognizing G; SN for recognizing G or A; YG for recognizing
T; and NK for recognizing G, and one or more of: HD for recognizing
C; NG for recognizing T; NI for recognizing A; NN for recognizing G
or A; NS for recognizing A or C or G or T; N* for recognizing C or
T; HG for recognizing T; H* for recognizing T; and IG for
recognizing T. The endonuclease domain can be from a type II
restriction endonuclease (e.g., FokI).
[0015] In still another aspect, this document features a TALEN
comprising an endonuclease domain and a TAL effector domain,
wherein the amino acid sequence of said TALEN is selected from the
group consisting of SEQ ID NO:33 to SEQ ID NO:55, SEQ ID NO:72, and
SEQ ID NO:73.
[0016] This document also features a method for generating an
animal, comprising: providing a eukaryotic cell comprising a target
DNA sequence into which it is desired to introduce a genetic
modification; generating a double-stranded cut within the target
DNA sequence with a TALEN comprising an endonuclease domain and a
TAL effector domain that binds to the target DNA sequence; and
generating an animal from the cell or progeny thereof in which a
double-stranded cut has occurred. The method can further comprise
introducing into the cell an exogenous nucleic acid comprising a
sequence homologous to at least a portion of the target DNA,
wherein the introducing is under conditions that permit homologous
recombination to occur between the exogenous nucleic acid and the
target DNA sequence in the cell or progeny thereof; and generating
an animal from the cell or progeny thereof in which homologous
recombination has occurred. The animal can be a mammal. The genetic
modification can comprise a substitution, an insertion, or a
deletion.
[0017] In yet another aspect, this document features a method for
generating a plant, comprising providing a plant cell comprising a
target DNA sequence into which it is desired to introduce a
preselected genetic modification; generating a double-stranded cut
within the target DNA sequence with a TALEN comprising an
endonuclease domain and a TAL effector domain that binds to the
target DNA sequence; and generating a plant from the cell or
progeny thereof in which a double-stranded cut has occurred. The
method can further comprise introducing into the plant cell an
exogenous nucleic acid comprising a sequence homologous to at least
a portion of the target DNA sequence, wherein the introducing is
under conditions that permit homologous recombination to occur
between the exogenous nucleic acid and the target DNA sequence in
the cell or progeny thereof; and generating a plant from the cell
or progeny thereof in which homologous recombination has
occurred.
[0018] In another aspect, this document features a method for
targeted genetic recombination in a cell, comprising introducing
into the cell a nucleic acid encoding a TAL effector endonuclease
targeted to a selected DNA target sequence; inducing expression of
the TAL effector endonuclease within the cell; and identifying a
cell in which the selected DNA target sequence exhibits a mutation.
The mutation can be selected from the group consisting of deletion
of genetic material, insertion of genetic material, and both
deletion and insertion of genetic material. The method can further
comprise introducing donor DNA into the cell. The cell can be an
insect cell, a plant cell, a fish cell, or a mammalian cell.
[0019] In another aspect, this document features a method for
generating a TAL effector having enhanced targeting capacity for a
target DNA, comprising generating a nucleic acid encoding a TAL
effector that comprises DNA binding domain having a plurality of
DNA binding repeats, wherein each repeat comprises a RVD that
determines recognition of a base pair in the target DNA, wherein
each DNA binding repeat is responsible for recognizing one base
pair in the target DNA, wherein the generating comprises
incorporating a nucleic acid encoding a variant 0th DNA binding
repeat sequence with specificity for A, C, or G, thus eliminating
the requirement for T at position -1 of the binding site.
[0020] In another aspect, this document features a method for
generating a TAL effector having enhanced targeting capacity for a
target DNA, comprising generating a nucleic acid encoding a TAL
effector that comprises DNA binding domain having a plurality of
DNA binding repeats, wherein each repeat comprises a RVD that
determines recognition of a base pair in the target DNA, wherein
each DNA binding repeat is responsible for recognizing one base
pair in the target DNA, wherein the generating comprises
incorporating one or more nucleic acids encoding TAL effector DNA
binding domains that contain RVDs having enhanced specificity for
G, and wherein said RVDs are selected from the group consisting of
RN, R*, NG, NH, KN, K*, NA, NT, DN, D*, NL, NM, EN, E*, NV, NC, QN,
Q*, NR, NP, HN, H*, NK, NY, SN, S*, ND, NR, TN, T*, NE, NF, YN, Y*,
and NQ, wherein * indicates a gap at the second position of the
RVD.
[0021] This document also features a method for producing a
polypeptide that selectively recognizes at least one base pair in a
target DNA sequence, comprising synthesizing a polypeptide
comprising a repeat domain, wherein the repeat domain comprises at
least one repeat unit derived from a transcription activator-like
(TAL) effector, wherein the repeat unit comprises a hypervariable
region which determines recognition of a base pair in the target
DNA sequence, wherein the repeat unit is responsible for the
recognition of one base pair in the DNA sequence, and wherein the
hypervariable region comprises a member selected from the group
consisting of: (a) HD for recognition of C/G; (b) NI for
recognition of A/T; (c) NG for recognition of T/A; (d) NS for
recognition of C/G or A/T or T/A or G/C; (e) NN for recognition of
G/C or A/T; (f) IG for recognition of T/A; (g) N for recognition of
C/G; (h) HG for recognition of C/G or T/A; (i) H for recognition of
T/A; and (j) NK for recognition of G/C. In addition, this document
features a polypeptide produced by the above method, and a DNA
comprising a coding sequence for the polypeptide produced by the
method. Also featured is an expression cassette comprising a
promoter operably linked to the above-mentioned DNA, and a
non-human host cell comprising the expression cassette. In another
aspect, this document features a transformed, non-human organism
comprising the expression cassette.
[0022] In still another aspect, this document features a method for
selectively recognizing a base pair in a DNA sequence by a
polypeptide, comprising constructing a polypeptide comprising a
repeat domain, wherein the repeat domain comprises at least one
repeat unit derived from a TAL effector, wherein the repeat unit
comprises a hypervariable region which determines recognition of a
base pair in the DNA sequence, wherein the repeat unit is
responsible for the recognition of one base pair in the DNA
sequence, and wherein the hypervariable region comprises a member
selected from the group consisting of (a) HD for recognition of
C/G; (b) NI for recognition of A/T; (c) NG for recognition of T/A;
(d) NS for recognition of C/G or A/T or T/A or G/C; (e) NN for
recognition of G/C or A/T; (f) IG for recognition of T/A; (g) N for
recognition of C/G; (h) HG for recognition of C/G or T/A; (i) H for
recognition of T/A; and (j) NK for recognition of G/C .
[0023] This document also features a method of modulating
expression of a target gene in a cell, wherein cells are provided
which contain a polypeptide wherein the polypeptide comprises a
repeat domain, wherein the repeat domain comprises at least one
repeat unit derived from a TAL effector, wherein the repeat unit
comprises a hypervariable region which determines recognition of a
base pair in a DNA sequence, wherein the repeat unit is responsible
for the recognition of one base pair in the DNA sequence, and
wherein the hypervariable region comprises a member selected from
the group consisting of (a) HD for recognition of C/G; (b) NI for
recognition of A/T; (c) NG for recognition of T/A; (d) NS for
recognition of C/G or A/T or T/A or G/C; (e) NN for recognition of
G/C or A/T; (f) IG for recognition of T/A; (g) N for recognition of
C/G; (h) HG for recognition of C/G or T/A; (i) H for recognition of
T/A; and (j) NK for recognition of G/C.
[0024] In another aspect, this document features a polypeptide
comprising a repeat domain, wherein the repeat domain comprises at
least one repeat unit derived from a TAL effector, wherein the
repeat unit comprises a hypervariable region which determines
recognition of a base pair in a DNA sequence, wherein the repeat
unit is responsible for the recognition of one base pair in the DNA
sequence, and wherein the hypervariable region comprises a member
selected from the group consisting of (a) HD for recognition of
C/G; (b) NI for recognition of A/T; (c) NG for recognition of T/A;
(d) NS for recognition of C/G or A/T or T/A or G/C; (e) NN for
recognition of G/C or A/T; (f) IG for recognition of T/A; (g) N for
recognition of C/G; (h) HG for recognition of C/G or T/A; (i) H for
recognition of T/A; and (j) NK for recognition of G/C. This
document also features a DNA comprising a coding sequence for the
above-mentioned polypeptide.
[0025] In another aspect, this document features a DNA which is
modified to include a base pair located in a target DNA sequence so
that the base pair can be specifically recognized by a polypeptide
comprising a repeat domain, wherein the repeat domain comprises at
least one repeat unit derived from a TAL effector, wherein the
repeat unit comprises a hypervariable region which determines
recognition of a base pair in the DNA sequence, wherein the repeat
unit is responsible for the recognition of one base pair in the DNA
sequence, and wherein, to receive a selective and determined
recognition by the hypervariable region, the base pair is selected
from the group consisting of (a) C/G for recognition by HD; (b) A/T
for recognition by NI; (c) T/A for recognition by NG; (d) CT or A/T
or T/A or G/C for recognition by NS; (e) G/C or A/T for recognition
by NN; (f) T/A for recognition by IG; (g) C/G or T/A for
recognition by N; (h) T/A for recognition by HG; (i) T/A for
recognition by H; and (j) G/C for recognition by NK. Also featured
are a vector comprising the above-mentioned DNA, a non-human host
cell comprising the DNA, and a transformed, non-human organism
comprising the DNA.
[0026] In yet another aspect, this document features a method for
producing a DNA comprising a target DNA sequence that is
selectively recognized by a polypeptide comprising a repeat domain,
wherein the repeat domain comprises at least one repeat unit
derived from a TAL effector, wherein the repeat unit comprises a
hypervariable region which determines recognition of a base pair in
the target DNA sequence, and wherein the repeat unit is responsible
for the recognition of one base pair in the target DNA sequence,
the method comprising synthesizing a DNA comprising a base pair
that is capable of being recognized by the repeat unit, wherein the
base pair is selected from the group consisting of (a) C/G for
recognition by HD; (b) A/T for recognition by NI; (c) T/A for
recognition by NG; (d) CT or A/T or T/A or G/C for recognition by
NS; (e) G/C or A/T for recognition by NN; (f) T/A for recognition
by IG; (g) C/G or T/A for recognition by N; (h) T/A for recognition
by HG; (i) T/A for recognition by H; and (j) G/C for recognition by
NK.
[0027] In one aspect, the present document features a method for
modifying the genetic material of a plant cell. The method can
include (a) introducing into the plant cell (i) a first recombinant
nucleic acid comprising a modified target nucleotide sequence,
wherein the modified target nucleotide sequence comprises one or
more modifications in nucleotide sequence with respect to a
corresponding target nucleotide sequence present in the plant cell,
and wherein the target nucleotide sequence further comprises a
recognition site for a sequence-specific TAL effector endonuclease
(TALEN); and (ii) a second recombinant nucleic acid comprising a
nucleotide sequence encoding the sequence-specific transcription
activator-like (TAL) effector endonuclease; (b) generating a plant
containing the plant cell; (c) analyzing cells, seed, or tissue
obtained from the plant, or progeny thereof, for recombination at
the target nucleotide sequence. The method can further include
introducing into the plant cell (iii) a third recombinant nucleic
acid comprising a nucleotide sequence encoding a selectable marker;
and determining if the plant or progeny thereof expresses the
selectable marker. The method can further include the step of
screening the plant or progeny thereof for the absence of the
selectable marker. The nucleotide sequence encoding the selectable
marker may or may not be flanked on one or both sides by a sequence
that is similar or identical to a sequence that is endogenous to
the plant cell (e.g., a sequence at the site of cleavage for a
second sequence-specific nuclease). The nucleotide sequence
encoding the selectable marker may be flanked on both sides by
recognition sites for a sequence-specific recombinase. The method
can further include the step of out-crossing the plant, with or
without the step of screening progeny of the out-cross for the
absence of the selectable marker. The first and second recombinant
nucleic acids can be simultaneously introduced into the plant cell.
One or both of the recombinant nucleic acids can be linearized
prior to the introducing step. The first and second recombinant
nucleic acids may be present in the same construct.
[0028] In another aspect, the present document features another
method for modifying the genetic material of a cell. The method can
include providing a primary cell containing chromosomal target DNA
sequence in which it is desired to have homologous recombination
occur; providing a TALEN comprising an endonuclease domain that can
cleave double stranded DNA, and a TAL effector domain comprising a
plurality of TAL effector repeat sequences that, in combination,
bind to a specific nucleotide sequence within the target DNA in the
cell; and contacting the target DNA sequence with the TALEN in the
cell such that the TALEN cleaves both strands of a nucleotide
sequence within or adjacent to the target DNA sequence in the cell.
The method can further include providing a nucleic acid comprising
a sequence homologous to at least a portion of the target DNA, such
that homologous recombination occurs between the target DNA
sequence and the nucleic acid. The target DNA sequence can be
endogenous to the cell. The cell can be a plant cell, a mammalian
cell, a fish cell, an insect cell or cell lines derived from these
organisms for in vitro cultures or primary cells taken directly
from living tissue and established for in vitro culture. The
contacting can include transfecting the cell with a vector
comprising a TALEN coding sequence, and expressing the TALEN
protein in the cell, mechanically injecting a TALEN protein into
the cell, delivering a TAL effector endonuclease protein into the
cell by means of the bacterial type III secretion system, or
introducing a TALEN protein into the cell by electroporation. The
endonuclease domain can be from a type II restriction endonuclease
(e.g., FokI). The TAL effector domain that binds to a specific
nucleotide sequence within the target DNA can include 10 or more
DNA binding repeats, more preferably 15 or more DNA binding
repeats. The cell can be from any prokaryotic or eukaryotic
organism.
[0029] In another aspect, the present document features a method
for designing a sequence specific TALEN capable of cleaving DNA at
a specific location. The method can include identifying a first
unique endogenous chromosomal nucleotide sequence adjacent to a
second nucleotide sequence at which it is desired to introduce a
double-stranded cut; and designing a sequence specific TALEN
comprising (a) a plurality of DNA binding repeat domains that, in
combination, bind to the first unique endogenous chromosomal
nucleotide sequence, and (b) an endonuclease that generates a
double-stranded cut at the second nucleotide sequence.
[0030] The present document also features a TALEN comprising an
endonuclease domain and a TAL effector DNA binding domain specific
for a particular DNA sequence. The TALEN can further include a
purification tag. The endonuclease domain can be from a type II
restriction endonuclease (e.g., FokI).
[0031] In another aspect, the present document features a method
for generating a genetically modified animal into which a desired
nucleic acid has been introduced. The method can include providing
a primary cell comprising an endogenous chromosomal target DNA
sequence into which it is desired to introduce the nucleic acid;
generating a double-stranded cut within the endogenous chromosomal
target DNA sequence with a TALEN comprising an endonuclease domain
and a TAL effector domain that binds to the endogenous chromosomal
target DNA sequence; introducing an exogenous nucleic acid
comprising a sequence homologous to at least a portion of the
endogenous chromosomal target DNA into the primary cell under
conditions that permit homologous recombination to occur between
the exogenous nucleic acid and the endogenous chromosomal target
DNA; and generating an animal from the primary cell in which
homologous recombination has occurred. The animal can be a mammal.
The homologous sequence can be a nucleotide sequence selected from
the group consisting of a nucleotide sequence that disrupts a gene
after homologous recombination, a nucleotide sequence that replaces
a gene after homologous recombination, a nucleotide sequence that
introduces a point mutation into a gene after homologous
recombination, and a nucleotide sequence that introduces a
regulatory site after homologous recombination.
[0032] In still another aspect, the present document features a
method for generating a genetically modified plant in which a
desired nucleic acid has been introduced. The method can include
providing a plant cell comprising an endogenous target DNA sequence
into which it is desired to introduce the nucleic acid; generating
a double-stranded cut within the endogenous target DNA sequence
with a TALEN comprising an endonuclease domain and a TAL effector
domain that binds to the endogenous target nucleotide sequence;
introducing an exogenous nucleic acid comprising a sequence
homologous to at least a portion of the endogenous target DNA into
the plant cell under conditions that permit homologous
recombination to occur between the exogenous nucleic acid and the
endogenous target DNA; and generating a plant from the plant cell
in which homologous recombination has occurred.
[0033] In another aspect, the present document features a method
for targeted genetic recombination in a cell. The method can
include introducing into the cell a nucleic acid molecule encoding
a TALEN targeted to a selected DNA target sequence; inducing
expression of the TALEN within the cell; and identifying a cell in
which the selected DNA target sequence exhibits a mutation. The
mutation can be selected from the group consisting of a deletion of
genetic material, an insertion of genetic material, and both a
deletion and an insertion of genetic material. The method can
further include introducing donor DNA into the cell. The cell can
be an insect cell, a plant cell, a fish cell, or a mammalian
cell.
[0034] In yet another aspect, the present document features a
method for generating a nucleic acid encoding a sequence specific
TALEN, comprising (1) selecting a starter plasmid comprising a
nucleotide sequence that encodes a first TAL effector DNA binding
repeat domain having a RVD specific for the first nucleotide of a
selected nucleotide sequence, wherein the first TAL effector DNA
binding repeat domain has a unique PspXI site at its 3' end; (2)
linearizing the starter plasmid with PspXI; (3) ligating into the
PspXI site a DNA module encoding one or more TAL effector DNA
binding repeat domains that have RVDs specific for the next
nucleotide(s) of the selected nucleotide sequence, wherein the DNA
module has XhoI sticky ends; and (4) repeating steps (2) and (3)
until the nucleic acid encodes a TALEN capable of binding to the
selected nucleotide sequence. In some cases, the method can further
include, after the ligating in step (3), checking the orientation
of the DNA module in the PspXI site.
[0035] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used to practice the invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0036] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0037] FIGS. 1A-1D depict the TAL effector-DNA recognition cipher.
FIG. 1A is a diagram of a generic TAL effector, showing the repeat
region (open boxes) and a representative repeat sequence (SEQ ID
NO:1) with the RVD underlined. FIG. 1B is a diagram showing best
pattern matches (low entropy alignments) for various TAL effector
RVDs and target gene promoter sequences (SEQ ID NOS:2-11). An
asterisk indicates a deletion at residue 13. FIG. 1C is a diagram
showing RVD-nucleotide associations in the alignments in B, plus
ten more alignments obtained by scanning all rice promoters with 40
additional X. oryzae TAL effectors, retaining for each effector the
best alignment for which the downstream gene was activated during
infection. FIG. 1D is a diagram showing flanking nucleotide
frequencies for the 20 TAL effector target sites. Positions are
relative to the 5' end of the target site; N, length of target
site. Logos were generated using WebLogo.
[0038] FIGS. 2A and 2B provide evidence that OsHen1 is activated by
Tal1c of Xanthomonas oryzae pv. oryzicola strain BLS256. FIG. 2A is
a picture of semi quantitative RT-PCR results, showing relative
transcript abundance of OsHen1, with an actin gene for reference,
in rice leaves 24 hours after inoculation with BLS256 marker
exchange mutant M51, M51 carrying the empty cosmid vector (ev), M51
carrying cosmid pIJF92, which contains tal1a, tal1b, and tal1c, and
the wild type (WT) strain. FIG. 2B is a schematic based on mapping
of the single marker exchange mutation in M51 by rescue and end
sequencing of a marker-containing XmaI fragment. The genome region,
the coordinates of the rescued fragment, and the coordinates of the
BLS256 genomic fragment contained in cosmid pIJF92 are shown.
[0039] FIG. 3 is a reference AvrBs3 amino acid sequence (SEQ ID
NO:12).
[0040] FIG. 4 is a reference AvrBs3 nucleic acid sequence (SEQ ID
NO:13).
[0041] FIG. 5 is a map of a TAL nuclease expression vector.
[0042] FIG. 6 is a map of a target reporter plasmid.
[0043] FIG. 7 is a diagram of the schematic architecture of TAL
nucleases. The recognition sites of TAL DNA binding domain are
represented as capital letters, while the spacer sequence is
indicated in lowercase.
[0044] FIG. 8 is the amino acid sequence (SEQ ID NO:16) of the 17
and a half tandem repeats of the AvrBs3 recognition domain.
Hypervariable amino acids at positions 12 and 13 are boxed.
[0045] FIG. 9 is a diagram showing a scheme for a yeast assay to
test TAL effectiveness.
[0046] FIG. 10 is a graph plotting yeast assay results of AvrBs3
TAL nuclease.
[0047] FIG. 11 is a diagram showing a schematic representation of
single, double, or triple AsvBs3 repeat modules and a cloning
vector.
[0048] FIGS. 12A and 12B depict a single representative TAL
effector repeat (FIG. 12A), as well as a representative truncated
repeat (FIG. 12B) that is present at the end of the repeat region
in most TAL effectors. Nucleotide and encoded amino acid sequences
as shown. Ns represent nucleotides encoding the RVDs, which are
indicated as "XX." Numbers are given for the amino acid positions.
Sequences are taken from tal1c.
[0049] FIG. 13 is a schematic depicting the tal1c gene and the
process by which the repeat region was reduced to a single,
truncated repeat, resulting in pCS487, also shown. M, MscI site; S,
SphI site.
[0050] FIG. 14 is a schematic depicting introduction of a
translationally silent mutation at the end of the original
truncated repeat in pCS487 to create a PspXI and XhoI site,
yielding pCS489. Sequences of codons 18-21 in the original repeat
(SEQ ID NO:6) and the mutated repeat (SEQ ID NO:8) are shown. The
encoded amino acid sequence (SEQ ID NO:7) was not changed by the
mutation. The mutated nucleotides are italicized.
[0051] FIG. 15 is a map of pCS488, which is a kanamycin resistant
plasmid encoding only the N- and C-terminal portions of tal1c,
without the repeat region, in the Gateway entry vector pENTR-D
(Invitrogen, Carlsbad, Calif.).
[0052] FIG. 16 is a map of the single repeat starter plasmid
designated pCS493, which encodes a repeat having the RVD NI. Three
other plasmids, designated pCS494, pCS495, and pCS496, were
identical except for the RVDs they encode (given at right).
[0053] FIG. 17A depicts nucleotide and encoded amino acid sequences
for a single repeat module with the RVD NI. The 5' XhoI compatible
cohesive end, the MscI site, and the 3' PspXI/XhoI compatible
cohesive end are underlined. The RVD and the nucleotides encoding
it are in bold type. Three other repeat modules were constructed
that are identical to that shown except for the RVD encoding
sequences, which encode HD, NI, and NG, respectively. FIG. 17B is a
map of the single repeat module plasmid designated pCS502, which
contains the repeat encoding sequence shown in FIG. 17A. Plasmids
designated pCS503, pCS504, and pCS505 also were generated, and are
identical to pCS502 except for the RVDs they encode (given at
right).
[0054] FIG. 18A depicts nucleotide and encoded amino acid sequences
for a single repeat module with RVD NI, in which nucleotide
substitutions (italicized) prevent reconstitution of the XhoI site
at the 5' end following ligation into a PspXI/XhoI site and destroy
the internal MscI site. The RVD and its encoding nucleotides are in
bold type. Three additional repeat modules were constructed that
are identical to that shown except for the RVD encoding sequences,
which encode HD, NI, and NG, respectively. FIG. 18B is a schematic
of a three repeat module assembled by sequentially ligating
additional repeat modules into a single repeat module plasmid. The
MscI site in the first repeat and the PspXI site at the 3' end
remain unique, and the entire module is flanked by two XhoI
sites.
[0055] FIG. 19 is a list of the complete set of one-, two-, and
three-repeat module plasmids.
[0056] FIG. 20 is a flow chart depicting the steps in a method that
can be used to assemble any sequence of repeats into the tal1c
"backbone" to generate a custom TAL effector gene.
[0057] FIGS. 21A and 21B are schematics depicting assembly of
repeat modules in construction of TAL endonucleases that will
target the nucleotide sequences shown. In FIG. 21A, repeat modules
from plasmids designated pCS519, pCS524, pCS537, pCS551, pCS583,
and pCS529 are sequentially added to the sequence in the starter
plasmid designated pCS493, resulting in plasmids designated pMAT55,
pMAT56, pMAT57, pMAT58, pMAT59, and pMAT60. In FIG. 21B, repeat
modules from plasmids designated pCS530, pCS533, pCS522, and pCS541
are sequentially added to the sequence in the plasmid designated
pMAT1, resulting in plasmids designated pMAT61, pMAT62, pMAT63, and
pMAT64.
[0058] FIG. 22A is a schematic of a TAL effector protein. BamHI
fragments (denoted by B's) were fused to the catalytic domain of
the FokI endonuclease to create TALENs. N, N-terminus; NLS, nuclear
localization signal; B, BamHI site, AD, acidic activation domain.
FIG. 22B is a graph plotting activity of TALENs constructed with
TAL effectors AvrBs3 and PthXo1. Avr-FokI, AvrBs3 TALEN; Pth-FokI,
PthXo1 TALEN, Avr-FokI and Pth-FokI, AvrBs3 and PthXo1 fusions to a
catalytically inactive version of FokI (Bitinaite et al. (1998)
Proc. Natl. Acad. Sci. USA 95:10570-10575); ZFN, zinc finger
nuclease containing the Zif268 DNA binding domain (Porteus and
Baltimore (2003) Science 300:763).
[0059] FIG. 23 is a reference PthXo1 amino acid sequence (SEQ ID
NO:31).
[0060] FIG. 24 is a reference PthXo1 nucleic acid sequence (SEQ ID
NO:32).
[0061] FIG. 25 is a diagram of the pFZ85 vector.
[0062] FIG. 26 shows the amino acid sequence of avrBs3_TALEN (SEQ
ID NO:33).
[0063] FIG. 27 shows the amino acid sequence of pthXo1_TALEN (SEQ
ID NO:34).
[0064] FIG. 28A is a graph plotting activity of AvrBs3 and PthXo1
TALENS on targets with different spacer lengths. ZFN,
Zif268-derived zinc finger nuclease. FIG. 28B is a graph plotting
activity of a heterodimeric TALEN. Activity in yeast containing
PthXo1-FokI and AvrBs3-FokI expression vectors and a plasmid with a
target consisting of recognition sites for each, in head to tail
orientation separated by 15 bp is shown (Avr-FokI, Pth-FokI). Also
shown for reference is activity of AvrBs3 (Avr-FokI) and PthXo1
(Pth-FokI) TALENS individually and Zif268 (ZFN) on their respective
targets. As a negative control, a yeast culture with only the
target site plasmid for Avr-FokI, Pth-FokI was assayed for LacZ
activity (denoted as (-)).
[0065] FIG. 29A is a table showing the RVD sequences of individual
custom TALENs and their respective DNA recognition sequences. FIG.
29B is a graph plotting the activity of custom TALENs. (-),
negative control with target site plasmids only; ZFN, zinc finger
nuclease positive control.
[0066] FIG. 30 is a depiction of the nucleotide and RVD frequencies
at the termini of 20 target and TAL effector pairs.
[0067] FIG. 31 is a schematic of the Golden Gate cloning system
[Engler et al. (2008) PLoS One 3:e3647; and Engler et al. (2009)
PLoS One 4:e5553].
[0068] FIGS. 32A and 32B depict a set of 58 plasmids for assembly
and cloning of custom TAL effector repeat encoding arrays using the
Golden Gate cloning approach as described herein. Tet, tetracycline
resistance gene, a marker for plasmid selection; spec,
spectinomycin resistance gene, a marker for plasmid selection; amp,
ampicillin resistance gene, a marker for plasmid selection.
[0069] FIG. 33 is a schematic of a method for assembly and cloning
of custom TAL effector repeat encoding arrays by the Golden Gate
approach using the set of plasmids shown in FIG. 32. For
illustration purposes, assembly of an arbitrary repeat array is
shown. spec, spectinomycin resistance gene, a marker for plasmid
selection; amp, ampicillin resistance gene, a marker for plasmid
selection.
[0070] FIGS. 34A-34U show the amino acid sequences of TALENs
generated as described in Example 9 herein. FIG. 34A,
telomerase-TALEN124; FIG. 34B, gridlock-TALEN105; FIG. 34C,
adh1-TALEN58; FIG. 34D, adh1-TALEN63; FIG. 34E, adh1-TALEN68; FIG.
34F, adh1-TALEN73; FIG. 34G, adh1-TALEN89; FIG. 34H,
gridlock-TALEN106; FIG. 34I, adh1-TALEN64; FIG. 34J, adh1-TALEN69;
FIG. 34K, adh1-TALEN74; FIG. 34L, tt4-TALEN90; FIG. 34M,
telomerase-TALEN121; FIG. 34N, telomerase-TALEN126; FIG. 34O,
gridlock-TALEN107; FIG. 34P, gridlock-TALEN117; FIG. 34Q,
telomerase-TALEN131; FIG. 34R, telomerase-TALEN136; FIG. 34S,
adh1-TALEN60; FIG. 34 T, tt4-TALEN85; FIG. 34U,
gridlock-TALEN102.
[0071] FIG. 35 is a graph plotting TALEN activity as measured by
the yeast assay using custom TALEN monomers of increasing length
(9-, 10-, 12-, 13-, 15-, 16-, 17-, or 18 mers). The TALENs were
targeted to Arabidopsis and zebrafish genes, as indicated.
[0072] FIG. 36A is a diagram showing two different DNA target
sequences from the Arabidopsis ADH1 gene that are targeted by two
TALEN pairs. FIG. 36B is a graph plotting yeast assay data for
functional TALEN pairs that target the Arabidopsis ADH1 gene.
[0073] FIG. 37A is a schematic of a restriction endonuclease assay
used to detect TALEN-induced mutations in Arabidopsis protoplasts.
FIG. 37B shows the sequences of nine clones from undigested DNA in
the restriction endonuclease assay. Six of the clones have
mutations introduced by non-homologous end joining (NHEJ).
[0074] FIG. 38A shows 0th repeat sequences of several
phylogenetically distinct TAL effectors, AvrHah1 from Xanthomonas
gardneri, AvrBs3 from X. campestris pv. vesicatoria, PthXo1 from X.
oryzae pv. oryzae, PthA from X. citri, and Tal1c from X. oryzae pv.
oryzicola. Polymorphic positions are boxed. FIG. 38B is a schematic
showing the 0th and 1st repeats of PthXo1. The "0th" repeat
immediately precedes the 1st repeat, shows 35% identity, and has a
similar predicted secondary structure. The RVD of the 1st repeat
and the candidate analogous residues of the 0th repeat are
underlined. *, gap; H, helix; E, extended. The structure was
predicted using JPred (Cole et al. (2008) Nucl. Acids Res.
36:W197-W201).
[0075] FIG. 39 shows a western blot of total protein isolated from
human embryonic kidney 293T cells transfected with plasmids
encoding VS-tagged TAL effector proteins AvrBs3, PthXo1, and Tal1c,
as indicated, following immunodetection using a mouse-antiV5
antibody. Immunolabeled actin is shown as a control for equivalent
loading in each lane.
[0076] FIG. 40A shows the amino acid sequence of TALEN
HPRT-3254-17, and FIG. 40B shows the amino acid sequence of TALEN
HPRT-3286-20r.
[0077] FIG. 41A is a schematic showing the TALEN-targeted site in
the human chromosomal HPRT gene. Binding sites for the HPRT-3254-17
and HPRT-3286-20r TALENs, the Bpu10I site in the spacer between
those sites, and the primer sites for amplification of the region
are indicated. Coordinates at the bottom give distance in base
pairs from the first nucleotide of the coding sequence. FIG. 41B
shows the results of Bpu10I digestion of products of PCR
amplification of the region shown in FIG. 41A using genomic DNA
isolated from TALEN-treated and untreated cells as templates.
Genomic DNA was digested with Bpu10I prior to amplification. DNA
fragments were separated by agarose gel electrophoresis and
visualized using ethidium bromide.
DETAILED DESCRIPTION
[0078] The present patent application provides materials and
methods related to sequence specific DNA recognition mediated by
TAL effectors. As described herein, the primary amino acid
sequences of TAL effectors dictate the nucleotide sequences to
which they bind. The inventors have found that relationships
between TAL effector amino acid sequences and their DNA target
sequences are direct, enabling target site prediction for TAL
effectors, and also allowing for TAL effector customization to bind
to particular nucleotide sequences. Such prediction and
customization can be harnessed for a variety of purposes. In one
example, particular TAL effector sequences can be fused to
endonuclease sequences, allowing for endonuclease targeting to
specific DNA sequences, and subsequent cutting of the DNA at or
near the targeted sequences. Cuts (i.e., double-stranded breaks) in
DNA can dramatically increase the frequency of homologous
recombination. Thus, in combination with DNA constructs that carry
sequences having a high degree of sequence similarity to a
particular target DNA sequence, TALENs can be used to facilitate
site directed mutagenesis in complex genomes, that is, to knock out
or alter gene function, or to add genes or other sequences with
great precision and high efficiency.
[0079] Thus, included in the subject matter provided herein are,
inter alia, materials and methods for making genetically modified
organisms (including, without limitation, plants, fungi,
Drosophila, nematodes, zebrafish, mice, other mammals and humans).
Such methods can include, for example, transfecting a cell with
several recombinant nucleic acids. For example, a cell (e.g., a
eukaryotic cell) can be transformed with a first recombinant
nucleic acid construct containing a donor nucleotide sequence that
includes alterations relative to a corresponding target nucleotide
sequence found within the cell, and a second recombinant nucleic
acid construct encoding a TAL-nuclease. In some embodiments, the
cell also can be transformed with a third recombinant nucleic acid
construct encoding a selectable marker. A nucleic acid sequence
from the donor nucleic acid construct can become incorporated into
the genome of the transformed cell as described herein. For
example, plant cells produced using methods as described herein can
be grown to produce plants having the altered donor nucleotide
sequence incorporated into their genomes. Seeds from such plants
can be used to produce plants having a phenotype such as, for
example, an altered growth characteristic (e.g., increased
resistance or tolerance to various biotic and abiotic stresses),
altered appearance (e.g., altered color or height), or altered
composition (e.g., increased or decreased levels of carbon,
nitrogen, oil, protein, carbohydrate (e.g., sugar or starch), amino
acid, fatty acid, or secondary metabolites) with respect to
unmodified plants.
Polynucleotides and Polypeptides
[0080] Isolated nucleic acids and polypeptides are provided herein.
The terms "nucleic acid" and "polynucleotide" are used
interchangeably, and refer to both RNA and DNA, including cDNA,
genomic DNA, synthetic (e.g., chemically synthesized) DNA, and DNA
(or RNA) containing nucleic acid analogs. Polynucleotides can have
any three-dimensional structure. A nucleic acid can be
double-stranded or single-stranded (i.e., a sense strand or an
antisense single strand). Non-limiting examples of polynucleotides
include genes, gene fragments, exons, introns, messenger RNA
(mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant
polynucleotides, branched polynucleotides, plasmids, vectors,
isolated DNA of any sequence, isolated RNA of any sequence, nucleic
acid probes, and primers, as well as nucleic acid analogs.
[0081] The polypeptides of the present document (such as TAL
effector-DNA modifying enzyme as non-limiting example) can be
introduced in a cell by using a vector encoding said polypeptides
for example or as polypeptides per se by using delivery vectors
associated or combined with any cellular permeabilization
techniques such as sonoporation or electroporation or derivatives
of these techniques.
[0082] As used herein, "isolated," when in reference to a nucleic
acid, refers to a nucleic acid that is separated from other nucleic
acids that are present in a genome, e.g., a plant genome, including
nucleic acids that normally flank one or both sides of the nucleic
acid in the genome. The term "isolated" as used herein with respect
to nucleic acids also includes any non-naturally-occurring
sequence, since such non-naturally-occurring sequences are not
found in nature and do not have immediately contiguous sequences in
a naturally-occurring genome.
[0083] An isolated nucleic acid can be, for example, a DNA
molecule, provided one of the nucleic acid sequences normally found
immediately flanking that DNA molecule in a naturally-occurring
genome is removed or absent. Thus, an isolated nucleic acid
includes, without limitation, a DNA molecule that exists as a
separate molecule (e.g., a chemically synthesized nucleic acid, or
a cDNA or genomic DNA fragment produced by PCR or restriction
endonuclease treatment) independent of other sequences, as well as
DNA that is incorporated into a vector, an autonomously replicating
plasmid, a virus (e.g., a pararetrovirus, a retrovirus, lentivirus,
adenovirus, or herpes virus), or the genomic DNA of a prokaryote or
eukaryote. In addition, an isolated nucleic acid can include a
recombinant nucleic acid such as a DNA molecule that is part of a
hybrid or fusion nucleic acid. A nucleic acid existing among
hundreds to millions of other nucleic acids within, for example,
cDNA libraries or genomic libraries, or gel slices containing a
genomic DNA restriction digest, is not to be considered an isolated
nucleic acid.
[0084] A nucleic acid can be made by, for example, chemical
synthesis or polymerase chain reaction (PCR). PCR refers to a
procedure or technique in which target nucleic acids are amplified.
PCR can be used to amplify specific sequences from DNA as well as
RNA, including sequences from total genomic DNA or total cellular
RNA. Various PCR methods are described, for example, in PCR Primer:
A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring
Harbor Laboratory Press, 1995. Generally, sequence information from
the ends of the region of interest or beyond is employed to design
oligonucleotide primers that are identical or similar in sequence
to opposite strands of the template to be amplified. Various PCR
strategies also are available by which site-specific nucleotide
sequence modifications can be introduced into a template nucleic
acid.
[0085] Isolated nucleic acids also can be obtained by mutagenesis.
For example, a donor nucleic acid sequence can be mutated using
standard techniques, including oligonucleotide-directed mutagenesis
and site-directed mutagenesis through PCR. See, Short Protocols in
Molecular Biology, Chapter 8, Green Publishing Associates and John
Wiley & Sons, edited by Ausubel et al., 1992.
[0086] The term "polypeptide" as used herein refers to a compound
of two or more subunit amino acids regardless of post-translational
modification (e.g., phosphorylation or glycosylation). The subunits
may be linked by peptide bonds or other bonds such as, for example,
ester or ether bonds. The term "amino acid" refers to either
natural and/or unnatural or synthetic amino acids, including D/L
optical isomers.
[0087] By "isolated" or "purified" with respect to a polypeptide it
is meant that the polypeptide is separated to some extent from the
cellular components with which it is normally found in nature
(e.g., other polypeptides, lipids, carbohydrates, and nucleic
acids). An purified polypeptide can yield a single major band on a
non-reducing polyacrylamide gel. A purified polypeptide can be at
least about 75% pure (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%,
99%, or 100% pure). Purified polypeptides can be obtained by, for
example, extraction from a natural source, by chemical synthesis,
or by recombinant production in a host cell or transgenic plant,
and can be purified using, for example, affinity chromatography,
immunoprecipitation, size exclusion chromatography, and ion
exchange chromatography. The extent of purification can be measured
using any appropriate method, including, without limitation, column
chromatography, polyacrylamide gel electrophoresis, or
high-performance liquid chromatography.
Recombinant Constructs
[0088] Recombinant nucleic acid constructs (e.g., vectors) also are
provided herein. A "vector" is a replicon, such as a plasmid,
phage, or cosmid, into which another DNA segment may be inserted so
as to bring about the replication of the inserted segment.
Generally, a vector is capable of replication when associated with
the proper control elements. Suitable vector backbones include, for
example, those routinely used in the art such as plasmids, viruses,
artificial chromosomes, BACs, YACs, or PACs. The term "vector"
includes cloning and expression vectors, as well as viral vectors
and integrating vectors. An "expression vector" is a vector that
includes one or more expression control sequences, and an
"expression control sequence" is a DNA sequence that controls and
regulates the transcription and/or translation of another DNA
sequence. Suitable expression vectors include, without limitation,
plasmids and viral vectors derived from, for example,
bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses,
cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses, and
adeno-associated viruses. Numerous vectors and expression systems
are commercially available from such corporations as Novagen
(Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La
Jolla, Calif.), and Invitrogen/Life Technologies (Carlsbad,
Calif.).
[0089] The terms "regulatory region," "control element," and
"expression control sequence" refer to nucleotide sequences that
influence transcription or translation initiation and rate, and
stability and/or mobility of the transcript or polypeptide product.
Regulatory regions include, without limitation, promoter sequences,
enhancer sequences, response elements, protein recognition sites,
inducible elements, promoter control elements, protein binding
sequences, 5' and 3' untranslated regions (UTRs), transcriptional
start sites, termination sequences, polyadenylation sequences,
introns, and other regulatory regions that can reside within coding
sequences, such as secretory signals, Nuclear Localization
Sequences (NLS) and protease cleavage sites.
[0090] As used herein, "operably linked" means incorporated into a
genetic construct so that expression control sequences effectively
control expression of a coding sequence of interest. A coding
sequence is "operably linked" and "under the control" of expression
control sequences in a cell when RNA polymerase is able to
transcribe the coding sequence into RNA, which if an mRNA, then can
be translated into the protein encoded by the coding sequence.
Thus, a regulatory region can modulate, e.g., regulate, facilitate
or drive, transcription in the plant cell, plant, or plant tissue
in which it is desired to express a modified target nucleic
acid.
[0091] A promoter is an expression control sequence composed of a
region of a DNA molecule, typically within 100 nucleotides upstream
of the point at which transcription starts (generally near the
initiation site for RNA polymerase II). Promoters are involved in
recognition and binding of RNA polymerase and other proteins to
initiate and modulate transcription. To bring a coding sequence
under the control of a promoter, it typically is necessary to
position the translation initiation site of the translational
reading frame of the polypeptide between one and about fifty
nucleotides downstream of the promoter. A promoter can, however, be
positioned as much as about 5,000 nucleotides upstream of the
translation start site, or about 2,000 nucleotides upstream of the
transcription start site. A promoter typically comprises at least a
core (basal) promoter. A promoter also may include at least one
control element such as an upstream element. Such elements include
upstream activation regions (UARs) and, optionally, other DNA
sequences that affect transcription of a polynucleotide such as a
synthetic upstream element.
[0092] The choice of promoters to be included depends upon several
factors, including, but not limited to, efficiency, selectability,
inducibility, desired expression level, and cell or tissue
specificity. For example, tissue-, organ- and cell-specific
promoters that confer transcription only or predominantly in a
particular tissue, organ, and cell type, respectively, can be used.
In some embodiments, promoters specific to vegetative tissues such
as the stem, parenchyma, ground meristem, vascular bundle, cambium,
phloem, cortex, shoot apical meristem, lateral shoot meristem, root
apical meristem, lateral root meristem, leaf primordium, leaf
mesophyll, or leaf epidermis can be suitable regulatory regions. In
some embodiments, promoters that are essentially specific to seeds
("seed-preferential promoters") can be useful. Seed-specific
promoters can promote transcription of an operably linked nucleic
acid in endosperm and cotyledon tissue during seed development.
Alternatively, constitutive promoters can promote transcription of
an operably linked nucleic acid in most or all tissues of a plant,
throughout plant development. Other classes of promoters include,
but are not limited to, inducible promoters, such as promoters that
confer transcription in response to external stimuli such as
chemical agents, developmental stimuli, or environmental
stimuli.
[0093] A basal promoter is the minimal sequence necessary for
assembly of a transcription complex required for transcription
initiation. Basal promoters frequently include a "TATA box" element
that may be located between about 15 and about 35 nucleotides
upstream from the site of transcription initiation. Basal promoters
also may include a "CCAAT box" element (typically the sequence
CCAAT) and/or a GGGCG sequence, which can be located between about
40 and about 200 nucleotides, typically about 60 to about 120
nucleotides, upstream from the transcription start site.
[0094] Non-limiting examples of promoters that can be included in
the nucleic acid constructs provided herein include the cauliflower
mosaic virus (CaMV) 35S transcription initiation region, the 1' or
2' promoters derived from T-DNA of Agrobacterium tumefaciens,
promoters from a maize leaf-specific gene described by Busk ((1997)
Plant J. 11:1285-1295), kn1-related genes from maize and other
species, and transcription initiation regions from various plant
genes such as the maize ubiquitin-1 promoter.
[0095] A 5' untranslated region (UTR) is transcribed, but is not
translated, and lies between the start site of the transcript and
the translation initiation codon and may include the +1 nucleotide.
A 3' UTR can be positioned between the translation termination
codon and the end of the transcript. UTRs can have particular
functions such as increasing mRNA message stability or translation
attenuation. Examples of 3' UTRs include, but are not limited to
polyadenylation signals and transcription termination sequences. A
polyadenylation region at the 3'-end of a coding region can also be
operably linked to a coding sequence. The polyadenylation region
can be derived from the natural gene, from various other plant
genes, or from an Agrobacterium T-DNA.
[0096] The vectors provided herein also can include, for example,
origins of replication, and/or scaffold attachment regions (SARs).
In addition, an expression vector can include a tag sequence
designed to facilitate manipulation or detection (e.g.,
purification or localization) of the expressed polypeptide. Tag
sequences, such as green fluorescent protein (GFP), glutathione
S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or
Flag.TM. tag (Kodak, New Haven, Conn.) sequences typically are
expressed as a fusion with the encoded polypeptide. Such tags can
be inserted anywhere within the polypeptide, including at either
the carboxyl or amino terminus.
[0097] By "delivery vector" or "delivery vectors" is intended any
delivery vector which can be used in the presently described
methods to put into cell contact or deliver inside cells or
subcellular compartments agents/chemicals and molecules (proteins
or nucleic acids). It includes, but is not limited to liposomal
delivery vectors, viral delivery vectors, drug delivery vectors,
chemical carriers, polymeric carriers, lipoplexes, polyplexes,
dendrimers, microbubbles (ultrasound contrast agents),
nanoparticles, emulsions or other appropriate transfer vectors.
These delivery vectors allow delivery of molecules, chemicals,
macromolecules (genes, proteins), or other vectors such as
plasmids, peptides developed by Diatos. In these cases, delivery
vectors are molecule carriers. By "delivery vector" or "delivery
vectors" is also intended delivery methods to perform transfection.
[0098] The terms "vector" or "vectors" refer to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. A "vector" in the present document includes, but
is not limited to, a viral vector, a plasmid, a RNA vector or a
linear or circular DNA or RNA molecule which may consists of a
chromosomal, non chromosomal, semi-synthetic or synthetic nucleic
acids. Preferred vectors are those capable of autonomous
replication (episomal vector) and/or expression of nucleic acids to
which they are linked (expression vectors). Large numbers of
suitable vectors are known to those of skill in the art and
commercially available.
[0099] Viral vectors include retrovirus, adenovirus, parvovirus
(e.g. adenoassociated viruses), coronavirus, negative strand RNA
viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus
(e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g.
measles and Sendai), positive strand RNA viruses such as
picornavirus and alphavirus, and double-stranded DNA viruses
including adenovirus, herpesvirus (e.g., Herpes Simplex virus types
1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g.,
vaccinia, fowlpox and canarypox). Other viruses include Norwalk
virus, togavirus, flavivirus, reoviruses, papovavirus,
hepadnavirus, and hepatitis virus, for example. Examples of
retroviruses include: avian leukosis-sarcoma, mammalian C-type,
B-type viruses, D type viruses, HTLV-BLV group, lentivirus,
spumavirus (Coffin, J. M., Retroviridae: The viruses and their
replication, In Fundamental Virology, Third Edition, B. N. Fields,
et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
[0100] By "lentiviral vector" is meant HIV-Based lentivirus vectors
that are very promising for gene delivery because of their
relatively large packaging capacity, reduced immunogenicity and
their ability to stably transduce with high efficiency a large
range of different cell types. Lentiviral vectors are usually
generated following transient transfection of three (packaging,
envelope and transfer) or more plasmids into producer cells. Like
HIV, lentiviral vectors enter the target cell through the
interaction of viral surface glycoproteins with receptors on the
cell surface. On entry, the viral RNA undergoes reverse
transcription, which is mediated by the viral reverse transcriptase
complex. The product of reverse transcription is a double-stranded
linear viral DNA, which is the substrate for viral integration in
the DNA of infected cells. Said lentiviral vectors can be
"non-integrative" or "integrative". [0101] By "integrative
lentiviral vectors (or LV)", is meant such vectors as non limiting
example, that are able to integrate the genome of a target cell.
[0102] At the opposite by "non integrative lentiviral vectors (or
NILV)" is meant efficient gene delivery vectors that do not
integrate the genome of a target cell through the action of the
virus integrase.
[0103] One type of preferred vector is an episome, i.e., a nucleic
acid capable of extra-chromosomal replication. Preferred vectors
are those capable of autonomous replication and/or expression of
nucleic acids to which they are linked. Vectors capable of
directing the expression of genes to which they are operatively
linked are referred to herein as "expression vectors. A vector
according to the present document comprises, but is not limited to,
a YAC (yeast artificial chromosome), a BAC (bacterial artificial),
a baculovirus vector, a phage, a phagemid, a cosmid, a viral
vector, a plasmid, a RNA vector or a linear or circular DNA or RNA
molecule which may consist of chromosomal, non chromosomal,
semi-synthetic or synthetic DNA. In general, expression vectors of
utility in recombinant DNA techniques are often in the form of
"plasmids" which refer generally to circular double stranded DNA
loops which, in their vector form are not bound to the chromosome.
Large numbers of suitable vectors are known to those of skill in
the art. Vectors can comprise selectable markers, for example:
neomycin phosphotransferase, histidinol dehydrogenase,
dihydrofolate reductase, hygromycin phosphotransferase, herpes
simplex virus thymidine kinase, adenosine deaminase, glutamine
synthetase, and hypoxanthine-guanine phosphoribosyl transferase for
eukaryotic cell culture; TRP 1 for S. cerevisiae; tetracyclin,
rifampicin or ampicillin resistance in E. coli. Preferably said
vectors are expression vectors, wherein a sequence encoding a
polypeptide of interest is placed under control of appropriate
transcriptional and translational control elements to permit
production or synthesis of said polypeptide. Therefore, said
polynucleotide is comprised in an expression cassette. More
particularly, the vector comprises a replication origin, a promoter
operatively linked to said encoding polynucleotide, a ribosome
binding site, a RNA-splicing site (when genomic DNA is used), a
polyadenylation site and a transcription termination site. It also
can comprise an enhancer or silencer elements. Selection of the
promoter will depend upon the cell in which the polypeptide is
expressed. Suitable promoters include tissue specific and/or
inducible promoters. Examples of inducible promoters are:
eukaryotic metallothionine promoter which is induced by increased
levels of heavy metals, prokaryotic lacZ promoter which is induced
in response to isopropyl-.beta.-D-thiogalacto-pyranoside (IPTG) and
eukaryotic heat shock promoter which is induced by increased
temperature. Examples of tissue specific promoters are skeletal
muscle creatine kinase, prostate-specific antigen (PSA),
.alpha.-antitrypsin protease, human surfactant (SP) A and B
proteins, .beta.-casein and acidic whey protein genes.
[0104] Inducible promoters may be induced by pathogens or stress,
more preferably by stress like cold, heat, UV light, or high ionic
concentrations (reviewed in Potenza et al. (2004) In vitro Cell Dev
Biol 40:1-22). Inducible promoter may be induced by chemicals
[reviewed in Moore et al. (2006); Padidam (2003); Wang et al.
(2003); and Zuo and Chua (2000)].
[0105] Delivery vectors and vectors can be associated or combined
with any cellular permeabilization techniques such as sonoporation
or electroporation or derivatives of these techniques.
[0106] It will be understood that more than one regulatory region
may be present in a recombinant polynucleotide, e.g., introns,
enhancers, upstream activation regions, and inducible elements.
[0107] Recombinant nucleic acid constructs can include a
polynucleotide sequence inserted into a vector suitable for
transformation of cells (e.g., plant cells or animal cells).
Recombinant vectors can be made using, for example, standard
recombinant DNA techniques (see, e.g., Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.).
[0108] A recombinant nucleic acid sequence as described herein can
integrate into the genome of a cell via illegitimate (i.e., random,
non-homologous, non site-specific) recombination, or a recombinant
nucleic acid sequence as described herein can be adapted to
integrate into the genome of a cell via homologous recombination.
Nucleic acid sequences adapted for integration via homologous
recombination are flanked on both sides with sequences that are
similar or identical to endogenous target nucleotide sequences,
which facilitates integration of the recombinant nucleic acid at
the particular site(s) in the genome containing the endogenous
target nucleotide sequences. Nucleic acid sequences adapted for
integration via homologous recombination also can include a
recognition site for a sequence-specific nuclease. Alternatively,
the recognition site for a sequence-specific nuclease can be
located in the genome of the cell to be transformed. Donor nucleic
acid sequences as described below typically are adapted for
integration via homologous recombination.
[0109] In some embodiments, a nucleic acid encoding a selectable
marker also can be adapted to integrate via homologous
recombination, and thus can be flanked on both sides with sequences
that are similar or identical to endogenous sequences within the
plant genome (e.g., endogenous sequences at the site of cleavage
for a sequence-specific nuclease). In some cases, nucleic acid
containing coding sequence for a selectable marker also can include
a recognition site for a sequence-specific nuclease. In these
embodiments, the recognition site for the sequence-specific
nuclease can be the same as or different from that contained within
the donor nucleic acid sequence (i.e., can be recognized by the
same nuclease as the donor nucleic acid sequence, or recognized by
a different nuclease than the donor nucleic acid sequence).
[0110] In some cases, a recombinant nucleic acid sequence can be
adapted to integrate into the genome of a cell via site-specific
recombination. As used herein, "site-specific" recombination refers
to recombination that occurs when a nucleic acid sequence is
targeted to a particular site(s) within a genome not by homology
between sequences in the recombinant nucleic acid and sequences in
the genome, but rather by the action of recombinase enzymes that
recognize specific nucleic acid sequences and catalyze the
reciprocal exchange of DNA strands between these sites.
Site-specific recombination thus refers to the enzyme-mediated
cleavage and ligation of two defined nucleotide sequences. Any
suitable site-specific recombination system can be used, including,
for example, the Cre-lox system or the FLP-FRT system. In such
embodiments, a nucleic acid encoding a recombinase enzyme may be
introduced into a cell in addition to a donor nucleotide sequence
and a nuclease-encoding sequence, and in some cases, a selectable
marker sequence. See, e.g., U.S. Pat. No. 4,959,317.
Sequence-Specific Endonucleases
[0111] Sequence-specific nucleases and recombinant nucleic acids
encoding the sequence-specific endonucleases are provided herein.
The sequence-specific endonucleases can include TAL effector DNA
binding domains and endonuclease domains. Thus, nucleic acids
encoding such sequence-specific endonucleases can include a
nucleotide sequence from a sequence-specific TAL effector linked to
a nucleotide sequence from a nuclease.
[0112] TAL effectors are proteins of plant pathogenic bacteria that
are injected by the pathogen into the plant cell, where they travel
to the nucleus and function as transcription factors to turn on
specific plant genes. The primary amino acid sequence of a TAL
effector dictates the nucleotide sequence to which it binds. Thus,
target sites can be predicted for TAL effectors, and TAL effectors
also can be engineered and generated for the purpose of binding to
particular nucleotide sequences, as described herein.
[0113] Fused to the TAL effector-encoding nucleic acid sequences
are sequences encoding a nuclease or a portion of a nuclease,
typically a nonspecific cleavage domain from a type II restriction
endonuclease such as FokI (Kim et al. (1996) Proc. Natl. Acad. Sci.
USA 93:1156-1160). Other useful endonucleases may include, for
example, HhaI, HindIII, Nod, BbvCI, EcoRI, Bg/I, and AlwI. The fact
that some endonucleases (e.g., FokI) only function as dimers can be
capitalized upon to enhance the target specificity of the TAL
effector. For example, in some cases each FokI monomer can be fused
to a TAL effector sequence that recognizes a different DNA target
sequence, and only when the two recognition sites are in close
proximity do the inactive monomers come together to create a
functional enzyme. By requiring DNA binding to activate the
nuclease, a highly site-specific restriction enzyme can be
created.
[0114] A sequence-specific TALEN as provided herein can recognize a
particular sequence within a preselected target nucleotide sequence
present in a cell. Thus, in some embodiments, a target nucleotide
sequence can be scanned for nuclease recognition sites, and a
particular nuclease can be selected based on the target sequence.
In other cases, a TALEN can be engineered to target a particular
cellular sequence. A nucleotide sequence encoding the desired TALEN
can be inserted into any suitable expression vector, and can be
linked to one or more expression control sequences. For example, a
nuclease coding sequence can be operably linked to a promoter
sequence that will lead to constitutive expression of the
endonuclease in the species of plant to be transformed.
Alternatively, an endonuclease coding sequence can be operably
linked to a promoter sequence that will lead to conditional
expression (e.g., expression under certain nutritional conditions).
For example, a cauliflower mosaic virus 35S promoter can be used
for constitutive expression. Other constitutive promoters include,
without limitation, the nopaline synthase promoter, the ubiquitin
promoter, and the actin promoter. In some embodiments, an
artificial estrogen-induced promoter for can be used conditional
expression, and high levels of transcription can be achieved when a
plant is exposed to estrogen. Other conditional promoters that can
be used include, for example, heat-inducible heat shock gene
promoters, and light-regulated promoters such as that from the gene
encoding the large subunit of ribulose bisphosphate
carboxylase.
[0115] For purposes of therapy, the TAL effector-DNA modifying
enzyme of the present document and a pharmaceutically acceptable
excipient are administered in a therapeutically effective amount.
Such a combination is said to be administered in a "therapeutically
effective amount" if the amount administered is physiologically
significant. An agent is physiologically significant if its
presence results in a detectable change in the physiology of the
recipient. In the present context, an agent is physiologically
significant if its presence results in a decrease in the severity
of one or more symptoms of the targeted disease and in a genome
correction of the lesion or abnormality. Vectors comprising
targeting DNA and/or nucleic acid encoding a TAL effector-DNA
modifying enzyme can be introduced into a cell by a variety of
methods (e.g., injection, direct uptake, projectile bombardment,
liposomes, electroporation). TAL effector-DNA modifying enzymes can
be stably or transiently expressed into cells using expression
vectors. Techniques of expression in eukaryotic cells are well
known to those in the art. (See Current Protocols in Human
Genetics: Chapter 12 "Vectors For Gene Therapy" and Chapter 13
"Delivery Systems for Gene Therapy").
[0116] In one further aspect of the present document, the TAL
effector-DNA modifying enzyme is substantially non-immunogenic,
i.e., engender little or no adverse immunological response. A
variety of methods for ameliorating or eliminating deleterious
immunological reactions of this sort can be used. In a preferred
embodiment, the TAL effector-DNA modifying enzyme is substantially
free of N-formyl methionine. Another way to avoid unwanted
immunological reactions is to conjugate TAL effector-DNA modifying
enzyme to polyethylene glycol ("PEG") or polypropylene glycol
("PPG") (preferably of 500 to 20,000 daltons average molecular
weight (MW)). Conjugation with PEG or PPG, as described by Davis et
al. (U.S. Pat. No. 4,179,337) for example, can provide
non-immunogenic, physiologically active, water soluble TAL
effector-DNA modifying enzyme conjugates with anti-viral activity.
Similar methods also using a polyethylene--polypropylene glycol
copolymer are described in Saifer et al. (U.S. Pat. No.
5,006,333).
Donor Vectors
[0117] Also provided herein are recombinant nucleic acids including
donor nucleotide sequences. A donor nucleotide sequence can include
a variant sequence having one or more modifications (i.e.,
substitutions, deletions, or insertions) with respect to a
preselected target nucleotide sequence found endogenously within
the genome of a cell to be transformed (also referred to herein as
a "modified target nucleotide sequence"). The variant sequence
within the donor nucleic acid typically is flanked on both sides
with sequences that are similar or identical to the endogenous
target nucleotide sequence within the cell. The flanking sequences
can have any suitable length, and typically are at least 50
nucleotides in length (e.g., at least 50 nucleotides, at least 75
nucleotides, at least 100 nucleotides, at least 200 nucleotides, at
least 250 nucleotides, at least 300 nucleotides, at least 500
nucleotides, at least 750 nucleotides, at least 1000 nucleotides,
from about 50 to about 5000 nucleotides, from about 100 to 2500
nucleotides, from about 100 to about 1000 nucleotides, from about
100 to 500 nucleotides, from about 200 to about 500 nucleotides, or
from about 250 to 400 nucleotides). Thus, homologous recombination
can occur between the recombinant donor nucleic acid construct and
the endogenous target on both sides of the variant sequence, such
that the resulting cell's genome contains the variant sequence
within the context of endogenous sequences from, for example, the
same gene. A donor nucleotide sequence can be generated to target
any suitable sequence within a genome. In a plant, for example, a
donor nucleotide sequence can be targeted to a lipid biosynthetic
gene, carbohydrate biosynthetic gene, seed storage protein gene,
disease or pest resistance gene, stress tolerance gene, drought
tolerance gene, or a gene that produces an anti-nutritional. In
addition, the donor nucleotide sequence contains a recognition site
for a sequence-specific nuclease, as described herein.
Selectable Markers
[0118] Some of the methods provided herein include the use of a
third recombinant nucleic acid encoding a selectable or screenable
marker. A nucleotide sequence encoding a polypeptide that results
in a selectable trait can be incorporated into an expression vector
containing one or more expression control sequences. For example,
an expression vector can include sequence encoding a selectable
marker operably linked to a promoter sequence that will lead to
constitutive expression in the plant cell to be transformed.
Suitable selectable markers can include, without limitation,
polypeptides conferring resistance to an antibiotic such as
kanamycin, G418, bleomycin, ampicillin, or hygromycin, or an
herbicide such as glufosinate, chlorosulfuron, or
phosphinothricin.
[0119] In embodiments for use in plants, for example, a selectable
marker can confer resistance to an herbicide that inhibits the
growing point or meristem, such as an imidazolinone or a
sulfonylurea. Exemplary polypeptides in this category code for
mutant ALS and AHAS enzymes as described, for example, in U.S. Pat.
Nos. 5,767,366 and 5,928,937. U.S. Pat. Nos. 4,761,373 and
5,013,659 are directed to plants resistant to various imidazolinone
or sulfonamide herbicides. U.S. Pat. No. 4,975,374 relates to plant
cells and plants containing a gene encoding a mutant glutamine
synthetase (GS) resistant to inhibition by herbicides that are
known to inhibit GS, e.g., phosphinothricin and methionine
sulfoximine. U.S. Pat. No. 5,162,602 discloses plants resistant to
inhibition by cyclohexanedione and aryloxyphenoxypropanoic acid
herbicides. The resistance is conferred by an altered acetyl
coenzyme A carboxylase (ACCase).
[0120] Polypeptides for resistance to glyphosate (sold under the
trade name Roundup.RTM.) also are suitable for use in plants. See,
for example, U.S. Pat. Nos. 4,940,835 and 4,769,061. U.S. Pat. No.
5,554,798 discloses transgenic glyphosate resistant maize plants,
in which resistance is conferred by an altered
5-enolpyruvyl-3-phosphoshikimate (EPSP) synthase. Such polypeptides
can confer resistance to glyphosate herbicidal compositions
including, without limitation, glyphosate salts such as the
trimethylsulphonium salt, the isopropylamine salt, the sodium salt,
the potassium salt and the ammonium salt. See, e.g., U.S. Pat. Nos.
6,451,735 and 6,451,732.
[0121] Polypeptides for resistance to phosphono compounds such as
glufosinate ammonium or phosphinothricin, and pyridinoxy or phenoxy
propionic acids and cyclohexones also are suitable. See, for
example, European Publication No. 0 242 246, as well as U.S. Pat.
Nos. 5,879,903, 5,276,268, and 5,561,236.
[0122] Other herbicides include those that inhibit photosynthesis,
such as triazine and benzonitrile (nitrilase). See, e.g., U.S. Pat.
No. 4,810,648. Other herbicides include 2,2-dichloropropionic acid,
sethoxydim, haloxyfop, imidazolinone herbicides, sulfonylurea
herbicides, triazolopyrimidine herbicides, s-triazine herbicides
and bromoxynil. Also suitable are herbicides that confer resistance
to a protox enzyme. See, e.g., U.S. Patent Publication No.
20010016956 and U.S. Pat. No. 6,084,155.
[0123] In some embodiments, a recombinant nucleic acid encoding a
selectable marker can be adapted to integrate into the genome of a
cell (e.g., a plant cell or an animal cell) by site-specific
recombination. For example, a sequence encoding a selectable marker
can be flanked by recognition sequences for a recombinase such as,
e.g., Cre or FLP. In other embodiments, a recombinant nucleic acid
encoding a selectable marker can be adapted for integration into a
plant genome by homologous recombination. In such nucleic acids,
the sequence encoding the selectable marker can be flanked by
sequences that are similar or identical to endogenous nucleotide
sequences found within the genome of the plant cell into which the
recombinant nucleic acid is to be introduced. At least one of the
endogenous sequences can be at the cleavage site for a
sequence-specific nuclease. The nucleic acid encoding the
selectable marker also can contain a recognition site for a
sequence-specific nuclease. The nuclease can be the same
sequence-specific nuclease as that which is targeted to the donor
nucleotide sequence, or a sequence-specific nuclease that is
different from that targeted to the donor nucleotide sequence. In
still other embodiments, a recombinant nucleic acid encoding a
selectable marker can be adapted for integration into the genome of
a plant cell by illegitimate recombination. Such nucleic acids
typically lack the flanking sequences and nuclease recognition
sites that are contained within nucleic acids adapted for
homologous or site-specific recombination as described herein.
Methods
[0124] One or more of the constructs provided herein can be used to
transform cells and/or a DNA modifying enzyme can be introduced
into cells, such that a genetically modified organism (e.g., a
plant or an animal) is generated. Thus, genetically modified
organisms and cells containing the nucleic acids and/or
polypeptides described herein also are provided. In some
embodiments, a transformed cell has a recombinant nucleic acid
construct integrated into its genome, i.e., can be stably
transformed. Stably transformed cells typically retain the
introduced nucleic acid sequence with each cell division. A
construct can integrate in a homologous manner, such that a
nucleotide sequence endogenous to the transformed cell is replaced
by the construct, where the construct contains a sequence that
corresponds to the endogenous sequence, but that contains one or
more modifications with respect to the endogenous sequence. It is
noted that while a plant or animal containing such a modified
endogenous sequence may be termed a "genetically modified organism"
(GMO) herein, the modified endogenous sequence is not considered a
transgene. A construct also can integrate in an illegitimate
manner, such that it integrates randomly into the genome of the
transformed cell.
[0125] Alternatively, a cell can be transiently transformed, such
that the construct is not integrated into its genome. For example,
a plasmid vector containing a TALEN coding sequence can be
introduced into a cell, such that the TALEN coding sequence is
expressed but the vector is not stably integrated in the genome.
Transiently transformed cells typically lose some or all of the
introduced nucleic acid construct with each cell division, such
that the introduced nucleic acid cannot be detected in daughter
cells after sufficient number of cell divisions. Nevertheless,
expression of the TALEN coding sequence is sufficient to achieve
homologous recombination between a donor sequence and an endogenous
target sequence. Both transiently transformed and stably
transformed cells can be useful in the methods described
herein.
[0126] With particular respect to genetically modified plant cells,
cells used in the methods described herein can constitute part or
all of a whole plant. Such plants can be grown in a manner suitable
for the species under consideration, either in a growth chamber, a
greenhouse, or in a field. Genetically modified plants can be bred
as desired for a particular purpose, e.g., to introduce a
recombinant nucleic acid into other lines, to transfer a
recombinant nucleic acid to other species or for further selection
of other desirable traits. Alternatively, genetically modified
plants can be propagated vegetatively for those species amenable to
such techniques. Progeny includes descendants of a particular plant
or plant line. Progeny of an instant plant include seeds formed on
F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5, F.sub.6 and subsequent
generation plants, or seeds formed on BC.sub.1, BC.sub.2, BC.sub.3,
and subsequent generation plants, or seeds formed on
F.sub.1BC.sub.1, F.sub.1BC.sub.2, F.sub.1BC.sub.3, and subsequent
generation plants. Seeds produced by a genetically modified plant
can be grown and then selfed (or outcrossed and selfed) to obtain
seeds homozygous for the nucleic acid construct.
[0127] Genetically modified cells (e.g., plant cells or animal
cells) can be grown in suspension culture, or tissue or organ
culture, if desired. For the purposes of the methods provided
herein, solid and/or liquid tissue culture techniques can be used.
When using solid medium, cells can be placed directly onto the
medium or can be placed onto a filter film that is then placed in
contact with the medium. When using liquid medium, cells can be
placed onto a floatation device, e.g., a porous membrane that
contacts the liquid medium. Solid medium typically is made from
liquid medium by adding agar. For example, a solid medium can be
Murashige and Skoog (MS) medium containing agar and a suitable
concentration of an auxin, e.g., 2,4-dichlorophenoxyacetic acid
(2,4-D), and a suitable concentration of a cytokinin, e.g.,
kinetin.
[0128] A cell can be transformed with one recombinant nucleic acid
construct or with a plurality (e.g., 2, 3, 4, or 5) of recombinant
nucleic acid constructs. If multiple constructs are utilized, they
can be transformed simultaneously or sequentially. Techniques for
transforming a wide variety of species are known in the art. The
polynucleotides and/or recombinant vectors described herein can be
introduced into the genome of a host using any of a number of known
methods, including electroporation, microinjection, and biolistic
methods. Alternatively, polynucleotides or vectors can be combined
with suitable T-DNA flanking regions and introduced into a
conventional Agrobacterium tumefaciens host vector. Such
Agrobacterium tumefaciens-mediated transformation techniques,
including disarming and use of binary vectors, are well known in
the art. Other gene transfer and transformation techniques include
protoplast transformation through calcium or PEG,
electroporation-mediated uptake of naked DNA, liposome-mediated
transfection, electroporation, viral vector-mediated
transformation, and microprojectile bombardment (see, e.g., U.S.
Pat. Nos. 5,538,880, 5,204,253, 5,591,616, and 6,329,571). If a
plant cell or tissue culture is used as the recipient tissue for
transformation, plants can be regenerated from transformed cultures
using techniques known to those skilled in the art.
[0129] In some embodiments, a DNA modifying enzyme (e.g., a TALEN)
can be directly introduced into a cell. For example, a polypeptide
can be introduced into a cell by mechanical injection, by delivery
via a bacterial type III secretion system, by electroporation, or
by Agrobacterium mediated transfer. See, e.g., Vergunst et al.
(2000) Science 290:979-982 for a discussion of the Agrobacterium
VirB/D4 transport system, and its use to mediate transfer of a
nucleoprotein T complex into plant cells.
[0130] With further respect to plants, the polynucleotides, vectors
and polypeptides described herein can be introduced into a number
of monocotyledonous and dicotyledonous plants and plant cell
systems, including dicots such as safflower, alfalfa, soybean,
coffee, amaranth, rapeseed (high erucic acid and canola), peanut or
sunflower, as well as monocots such as oil palm, sugarcane, banana,
sudangrass, corn, wheat, rye, barley, oat, rice, millet, or
sorghum. Also suitable are gymnosperms such as fir and pine.
[0131] Thus, the methods described herein can be utilized with
dicotyledonous plants belonging, for example, to the orders
Magniolales, Illiciales, Laurales, Piperales, Aristochiales,
Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae,
Trochodendrales, Hamamelidales, Eucomiales, Leitneriales,
Myricales, Fagales, Casuarinales, Caryophyllales, Batales,
Polygonales, Plumbaginales, Dilleniales, Theales, Malvales,
Urticales, Lecythidales, Violates, Salicales, Capparales, Ericales,
Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales,
Haloragales, Myrtales, Cornales, Proteales, Santales, Rafflesiales,
Celastrales, Euphorbiales, Rhamnales, Sapindales, Juglandales,
Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales,
Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales,
Dipsacales, and Asterales. The methods described herein also can be
utilized with monocotyledonous plants such as those belonging to
the orders Alismatales, Hydrocharitales, Najadales, Triuridales,
Commelinales, Eriocaulales, Restionales, Poales, Juncales,
Cyperales, Typhales, Bromeliales, Zingiberales, Arecales,
Cyclanthales, Pandanales, Arales, Lilliales, and Orchidales, or
with plants belonging to Gymnospermae, e.g., Pinales, Ginkgoales,
Cycadales and Gnetales.
[0132] The methods can be used over a broad range of plant species,
including species from the dicot genera Atropa, Alseodaphne,
Anacardium, Arachis, Beilschmiedia, Brassica, Carthamus, Cocculus,
Croton, Cucumis, Citrus, Citrullus, Capsicum, Catharanthus, Cocos,
Coffea, Cucurbita, Daucus, Duguetia, Eschscholzia, Ficus, Fragaria,
Glaucium, Glycine, Gossypium, Helianthus, Hevea, Hyoscyamus,
Lactuca, Landolphia, Linum, Litsea, Lycopersicon, Lupinus, Manihot,
Majorana, Malus, Medicago, Nicotiana, Olea, Parthenium, Papaver,
Persea, Phaseolus, Pistacia, Pisum, Pyrus, Prunus, Raphanus,
Ricinus, Senecio, Sinomenium, Stephania, Sinapis, Solanum,
Theobroma, Trifolium, Trigonella, Vicia, Vinca, Vitis, and Vigna;
the monocot genera Allium, Andropogon, Aragrostis, Asparagus,
Avena, Cynodon, Elaeis, Festuca, Festulolium, Heterocallis,
Hordeum, Lemna, Lolium, Musa, Oryza, Panicum, Pannesetum, Phleum,
Poa, Secale, Sorghum, Triticum, and Zea; or the gymnosperm genera
Abies, Cunninghamia, Picea, Pinus, and Pseudotsuga.
[0133] A transformed cell, callus, tissue, or plant can be
identified and isolated by selecting or screening the engineered
cells for particular traits or activities, e.g., those encoded by
marker genes or antibiotic resistance genes. Such screening and
selection methodologies are well known to those having ordinary
skill in the art. In addition, physical and biochemical methods can
be used to identify transformants. These include Southern analysis
or PCR amplification for detection of a polynucleotide; Northern
blots, S1 RNase protection, primer-extension, or RT-PCR
amplification for detecting RNA transcripts; enzymatic assays for
detecting enzyme or ribozyme activity of polypeptides and
polynucleotides; and protein gel electrophoresis, Western blots,
immunoprecipitation, and enzyme-linked immunoassays to detect
polypeptides. Other techniques such as in situ hybridization,
enzyme staining, and immunostaining also can be used to detect the
presence or expression of polypeptides and/or polynucleotides.
Methods for performing all of the referenced techniques are well
known. Polynucleotides that are stably incorporated into plant
cells can be introduced into other plants using, for example,
standard breeding techniques.
[0134] In the context of the present document, "eukaryotic cells"
refer to a fungal, yeast, plant or animal cell or a cell line
derived from the organisms listed below and established for in
vitro culture. More preferably, the fungus can be of the genus
Aspergillus, Penicillium, Acremonium, Trichoderma, Chrysoporium,
Mortierella, Kluyveromyces or Pichia. More preferably, the fungus
can be of the species Aspergillus niger, Aspergillus nidulans,
Aspergillus oryzae, Aspergillus terreus, Penicillium chrysogenum,
Penicillium citrinum, Acremonium Chrysogenum, Trichoderma reesei,
Mortierella alpine, Chrysosporium lucknowense, Kluyveromyces
lactis, Pichia pastoris or Pichia ciferrii.
[0135] The plant can be of the genus Arabidospis, Nicotiana,
Solanum, Lactuca, Brassica, Oryza, Asparagus, Pisum, Medicago, Zea,
Hordeum, Secale, Triticum, Capsicum, Cucumis, Cucurbita, Citrullis,
Citrus, or Sorghum. More preferably, the plant can be of the
species Arabidospis thaliana, Nicotiana tabaccum, Solanum
lycopersicum, Solanum tuberosum, Solanum melongena, Solanum
esculentum, Lactuca saliva, Brassica napus, Brassica oleracea,
Brassica rapa, Oryza glaberrima, Oryza sativa, Asparagus
officinalis, Pisum sativum, Medicago sativa, Zea mays, Hordeum
vulgare, Secale cereal, Triticum aestivum, Triticum durum, Capsicum
sativus, Cucurbita pepo, Citrullus lanatus, Cucumis melo, Citrus
aurantifolia, Citrus maxima, Citrus medica, or Citrus
reticulata.
[0136] The animal cell can be of the genus Homo, Rattus, Mus, Sus,
Bos, Danio, Canis, Felis, Equus, Salmo, Oncorhynchus, Gallus,
Meleagris, Drosophila, or Caenorhabditis; more preferably, the
animal cell can be of the species Homo sapiens, Rattus norvegicus,
Mus musculus, Sus scrofa, Bos taurus, Danio rerio, Canis lupus,
Felis catus, Equus caballus, Oncorhynchus mykiss, Gallus gallus, or
Meleagris gallopavo; the animal cell can be a fish cell from Salmo
salar, Teleost fish or zebrafish species as non-limiting examples.
The animal cell also can be an insect cell from Drosophila
melanogaster as a non-limiting example; the animal cell can also be
a worm cell from Caenorhabditis elegans as a non-limiting
example.
[0137] In the present document, the cell can be a plant cell, a
mammalian cell, a fish cell, an insect cell or cell lines derived
from these organisms for in vitro cultures or primary cells taken
directly from living tissue and established for in vitro culture.
As non limiting examples cell lines can be selected from the group
consisting of CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells;
NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562
cells, U-937 cells; MRCS cells; IMR90 cells; Jurkat cells; HepG2
cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec
cells; Molt 4 cells.
[0138] All these cell lines can be modified by the method of the
present document to provide cell line models to produce, express,
quantify, detect, study a gene or a protein of interest; these
models can also be used to screen biologically active molecules of
interest in research and production in various fields such as
chemical, biofuels, therapeutics and agronomy as non-limiting
examples.
[0139] The present document also provides methods for harnessing
the sequence-specific DNA binding domains within TAL effectors to,
for example, alter the genetic material within cells, to modulate
gene expression, and to target pathogenic sequences in, e.g.,
anti-viral therapies. For example, in some embodiments, the present
document provides methods for modifying cellular genetic material.
In some embodiments, the methods include introducing a polypeptide
containing a TAL effector DNA binding domain, or a nucleic acid
encoding such a polypeptide, into a cell. The TAL effector DNA
binding domain can be fused to all or a portion of a DNA modifying
enzyme (e.g., an endonuclease). In some embodiments, the methods
include introducing two or more recombinant nucleic acids into a
cell. A first recombinant nucleic acid contains a donor nucleotide
sequence that includes one or more modifications (i.e.,
substitutions, deletions, or insertions) with respect to a
corresponding, preselected target nucleotide sequence found in the
cell. The donor nucleotide sequence can undergo homologous
recombination with the endogenous target nucleotide sequence, such
that the endogenous sequence or a portion thereof is replaced with
the donor sequence or a portion thereof. The target nucleotide
sequence typically includes a recognition site for a
sequence-specific TALEN. In some cases, a target nucleotide
sequence can include recognition sites for two or more distinct
TALENs (e.g., two opposed target sequences that are distinct, such
that TALENs having distinct DNA sequence binding specificity can be
used). In such cases, the specificity of DNA cleavage can be
increased as compared to cases in which only one target sequence
(or multiple copies of the same target sequence) is used.
[0140] A second recombinant nucleic acid contains a nucleotide
sequence encoding a sequence specific TALEN that binds to the
recognition site in the target nucleotide sequence. In some cases,
the donor nucleotide sequence and the nucleotide sequence encoding
the sequence-specific nuclease can be contained in the same nucleic
acid construct. Alternatively, the donor nucleotide sequence and
the TALEN coding sequence can be contained in separate constructs,
or the TALEN polypeptide can be produced and introduced directly
into a cell.
[0141] In some embodiments, a third recombinant nucleic acid
containing a nucleotide sequence encoding a selectable marker also
may be used. The second and third recombinant nucleic acids may
undergo recombination with endogenous sequences and thus integrate
into the genome of the cell. These recombination events can be
illegitimate (i.e., random), or they can occur through homologous
recombination or through site-specific recombination. The
recombinant nucleic acids can be simultaneously or sequentially
transformed into the cell, and can be linearized prior to
transformation.
[0142] When the cell is a plant cell, the methods provided herein
can further include steps such as generating a plant containing the
transformed cell, generating progeny of the plant, selecting or
screening for plants expressing the selectable marker (if
included), generating progeny of the selected plants, and testing
the plants (e.g., tissue, seed, precursor cells, or whole plants)
or progeny of the plants for recombination at the target nucleotide
sequence. In some cases, the methods can include out-crossing the
selected plants to remove the selectable marker, and/or screening
the selected or out-crossed plants for the absence of the
sequence-specific nuclease.
[0143] In some embodiments, the present document provides methods
for modifying the genetic material of a cell, e.g., a prokaryotic
cell, an animal cell, or a plant cell. The methods can include
introducing into the cell a first recombinant nucleic acid
containing a modified target nucleotide sequence that includes one
or more modifications in nucleotide sequence with respect to a
corresponding target nucleotide sequence present in the cell, as
well as a recognition site for a sequence-specific TALEN, and a
second recombinant nucleic acid containing a nucleotide sequence
encoding the sequence-specific TALEN. When the cell is a plant
cell, a plant containing the cell can be generated, and cells,
seed, or tissue obtained from the plant (or progeny thereof) can be
analyzed for recombination at the target nucleotide sequence. The
first and second recombinant nucleic acids can be simultaneously or
serially transformed into the cell, and one or both may be
linearized prior to transformation. In some cases, the first and
second recombinant nucleic acids can be present in the same
construct.
[0144] In some cases, the method also can include introducing into
the cell a third recombinant nucleic acid containing a nucleotide
sequence encoding a selectable marker, and determining whether the
cell, an organism generated from the cell, or progeny thereof
expresses the selectable marker. The method further can include
screening the cell, the organism or progeny thereof for the absence
of the selectable marker. The nucleotide sequence encoding the
selectable marker may or may not be flanked on both sides by
nucleotide sequences that are similar or identical to nucleotide
sequences endogenous to the cell at the site of cleavage for a
second sequence-specific nuclease, or by recognition sites for a
sequence-specific recombinase. In some cases, the method also can
include the step of out-crossing the organism. Progeny of the
out-cross can be screened for the absence of the selectable
marker.
[0145] The present document also provides methods for modifying the
genetic material of a cell (e.g., a plant cell or an animal cell),
comprising providing a cell containing a target DNA sequence, e.g.,
a chromosomal, mitochondrial, or chloroplast sequence, in which it
is desired to have homologous recombination occur, providing a
TALEN that contains a DNA modifying enzyme domain (e.g., an
endonuclease domain) and a TAL effector domain having a plurality
of TAL effector repeats that, in combination, bind to a specific
nucleotide sequence within the target DNA sequence, providing a
nucleic acid containing a sequence homologous to at least a portion
of the target DNA, and contacting the target DNA sequence in the
cell with the TAL endonuclease such that both strands of a
nucleotide sequence within or adjacent to the target DNA sequence
in the cell are cleaved. Such cleavage can enhance the frequency of
homologous recombination at the target DNA sequence. The target DNA
sequence can be endogenous to the cell. The methods can include
introducing into the cell a vector containing a cDNA encoding the
TAL endonuclease, and expressing a TAL endonuclease protein in the
cell. In some cases, the TAL endonuclease protein itself can be
introduced into the cell, for example, by mechanical injection, by
delivery via a bacterial type III secretion system, by
electroporation, or by Agrobacterium mediated transfer.
[0146] The methods described herein can be used in a variety of
situations. In agriculture, for example, methods described herein
are useful to facilitate homologous recombination at a target site
can be used to remove a previously integrated transgene (e.g., a
herbicide resistance transgene) from a plant line, variety, or
hybrid. The methods described herein also can be used to modify an
endogenous gene such that the enzyme encoded by the gene confers
herbicide resistance, e.g., modification of an endogenous
5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase gene such that
the modified enzyme confers resistance to glyphosate herbicides. As
another example, the methods described herein are useful to
facilitate homologous recombination at regulatory regions for one
or more endogenous genes in a plant or mammal metabolic pathway
(e.g., fatty acid biosynthesis), such that expression of such genes
is modified in a desired manner. The methods described herein are
useful to facilitate homologous recombination in an animal (e.g., a
rat or a mouse) in one or more endogenous genes of interest
involved in, as non-limiting examples, metabolic and internal
signaling pathways such as those encoding cell-surface markers,
genes identified as being linked to a particular disease, and any
genes known to be responsible for a particular phenotype of an
animal cell.
[0147] The present document also provides methods for designing
sequence-specific TAL effectors capable of interacting with
particular DNA sequences (e.g., TALENs capable of cleaving DNA at
specific locations). The methods can include identifying a target
nucleotide sequence (e.g., an endogenous chromosomal sequence, a
mitochondrial DNA sequence, or a chloroplast DNA sequence) at which
it is desired to have TAL effector binding (e.g., a sequence
adjacent to a second nucleotide sequence at which it is desired to
introduce a double-stranded cut), and designing a sequence specific
TAL effector that contains a plurality of DNA binding repeats that,
in combination, bind to the target sequence. As described herein,
TAL effectors include a number of imperfect repeats that determine
the specificity with which they interact with DNA. Each repeat
binds to a single base, depending on the particular di-amino acid
sequence at residues 12 and 13 of the repeat. Thus, by engineering
the repeats within a TAL effector (e.g., using standard techniques
or the techniques described herein), particular DNA sites can be
targeted. Such engineered TAL effectors can be used, for example,
as transcription factors targeted to particular DNA sequences. A
diagram of a generic TAL effector is shown in FIG. 1A, with the
repeat region indicated by open boxes, and the RVD in the
representative repeat sequence (SEQ ID NO:1) underlined.
[0148] Examples of RVDs and their corresponding target nucleotides
are shown in Table 1A (See, also, PCT Publication No.
WO2010/079430).
TABLE-US-00001 TABLE 1A RVD Nucleotide HD C NG T NI A NN G or A NS
A or C or G N* C or T HG T H* T IG T *Denotes a gap in the repeat
sequence corresponding to a lack of an amino acid residue at the
second position of the RVD.
Other RVDs and their corresponding target nucleotides are shown in
Table 1B.
TABLE-US-00002 TABLE 1B RVD Nucleotide HA C ND C NK G HI C HN G NA
G SN G or A YG T
[0149] When it is desired to have sequence-specific DNA cleavage,
for example, a sequence-specific TALEN can be designed to contain
(a) a plurality of DNA binding repeat domains that, in combination,
bind to the endogenous chromosomal nucleotide sequence, and (b) an
endonuclease that generates a double-stranded cut at the second
nucleotide sequence. Such sequence-specific DNA cleavage can be
useful to enhance homologous recombination, as described herein.
Other uses for TALENs include, for example, as therapeutics against
viruses. TALENs can be engineered to target particular viral
sequences, cleaving the viral DNA and reducing or abolishing
virulence.
[0150] The materials and methods provided herein can be used to
modify the sequence of a particular gene in a targeted manner. A
gene may contain a plurality of sequences to which an engineered
TAL effector could be targeted. As described herein, however,
certain target sequences may be more effectively targeted. For
example, as set forth in Example 9, sequences having particular
characteristics may be more effectively targeted by TAL effectors.
Thus, the methods provided herein can include identifying target
sequences that meet particular criteria. These include sequences
that: i) have a minimum length of 15 bases and an orientation from
5' to 3' with a T immediately preceding the site at the 5' end; ii)
do not have a T in the first (5') position or an A in the second
position; iii) end in T at the last (3') position and do not have a
G at the next to last position; and iv) have a base composition of
0-63% A, 11-63% C, 0-25% G, and 2-42% T.
[0151] Since TALENs as described herein generally work as dimers,
some embodiments of the methods provided herein can include
identifying a first genomic nucleotide sequence and a second
genomic nucleotide sequence in a cell, wherein the first and second
nucleotide sequences meet at least one of the criteria set forth
above and are separated by 15-18 bp. In some cases, one TALEN
polypeptide can bind to each nucleotide sequences, and the
endonuclease contained in the TALEN can cleave within the 15-18 bp
spacer.
[0152] The present document also provides methods for generating
genetically modified animals into which a desired nucleic acid has
been introduced. Such methods can include obtaining a cell
containing an endogenous chromosomal target DNA sequence into which
it is desired to introduce the nucleic acid, introducing into the
cell a TALEN to generate a double-stranded cut within the
endogenous chromosomal target DNA sequence, introducing into the
cell an exogenous nucleic acid containing a sequence homologous to
at least a portion of the endogenous chromosomal target DNA, where
the introduction is done under conditions that permit homologous
recombination to occur between the exogenous nucleic acid and the
endogenous chromosomal target DNA, and generating an animal from
the primary cell in which homologous recombination has occurred.
The homologous nucleic acid can include, e.g., a nucleotide
sequence that disrupts a gene after homologous recombination, a
nucleotide sequence that replaces a gene after homologous
recombination, a nucleotide sequence that introduces a point
mutation into a gene after homologous recombination, or a
nucleotide sequence that introduces a regulatory site after
homologous recombination.
[0153] The methods provided herein also can be used to generate
genetically modified plants in which a desired nucleic acid has
been introduced. Such methods can include obtaining a plant cell
containing an endogenous target DNA sequence into which it is
desired to introduce the nucleic acid, introducing a TALEN to
generate a double-stranded cut within the endogenous target DNA
sequence, introducing into the plant cell an exogenous nucleic acid
containing a sequence homologous to at least a portion of the
endogenous target DNA, where the introducing is under conditions
that permit homologous recombination to occur between the exogenous
nucleic acid and the endogenous target DNA, and generating a plant
from the plant cell in which homologous recombination has
occurred.
[0154] The DNA in cells generated by the TALEN-facilitated
homologous recombination methods provided herein is modified, as
compared to cells that have not undergone such methods, and cells
containing the modified DNA are referred to as "genetically
modified." It is noted, however, that organisms containing such
cells may not be considered GMO for regulatory purposes, since such
a modification involves a homologous recombination and not random
integration of a transgene. Thus, using the TALEN-facilitated
methods described herein to generate genetic modifications may be
advantageous in that, for example, standard regulatory procedures
along with their associated time and cost may be avoided.
[0155] Other methods of targeted genetic recombination, as provided
herein, can include introducing into a cell (e.g., a plant cell,
insect cell, teleost fish cell, or animal cell) a nucleic acid
molecule encoding a TALEN targeted to a selected DNA target
sequence, inducing expression of the TALEN within the cell, and
identifying a recombinant cell in which the selected DNA target
sequence exhibits a mutation (e.g., a deletion of genetic material,
an insertion of genetic material, or both a deletion and an
insertion of genetic material). A donor DNA also can be introduced
into the cell.
[0156] In some embodiments, a monomeric TALEN can be used. TALENs
as described herein typically function as dimers across a bipartite
recognition site with a spacer, such that two TAL effector domains
are each fused to a catalytic domain of the FokI restriction
enzyme, the DNA recognition sites for each resulting TALEN are
separated by a spacer sequence, and binding of each TALEN monomer
to the recognition site allows FokI to dimerize and create a
double-strand break within the spacer (see, e.g., Moscou and
Bogdanove (2009) Science 326:1501). Monomeric TALENs also can be
constructed, however, such that single TAL effectors are fused to a
nuclease that does not require dimerization to function. One such
nuclease, for example, is a single-chain variant of FokI in which
the two monomers are expressed as a single polypeptide (Minczuk et
al. (2008) Nucleic Acids Res. 36:3926-3938). Other naturally
occurring or engineered monomeric nucleases also can serve this
role. The DNA recognition domain used for a monomeric TALEN can be
derived from a naturally occurring TAL effector. Alternatively, the
DNA recognition domain can be engineered to recognize a specific
DNA target. Engineered single-chain TALENs may be easier to
construct and deploy, as they require only one engineered DNA
recognition domain.
[0157] In some embodiments, a dimeric DNA sequence-specific
nuclease can be generated using two different DNA binding domains
(e.g., one TAL effector binding domain and one binding domain from
another type of molecule). As set forth above, the TALENs described
herein typically function as dimers across a bipartite recognition
site with a spacer. This nuclease architecture also can be used for
target-specific nucleases generated from, for example, one TALEN
monomer and one zinc finger nuclease monomer. In such cases, the
DNA recognition sites for the TALEN and zinc finger nuclease
monomers can be separated by a spacer of appropriate length.
Binding of the two monomers can allow FokI to dimerize and create a
double-strand break within the spacer sequence. DNA binding domains
other than zinc fingers, such as homeodomains, myb repeats or
leucine zippers, also can be fused to FokI and serve as a partner
with a TALEN monomer to create a functional nuclease.
[0158] In some embodiments, a TAL effector can be used to target
other protein domains (e.g., non-nuclease protein domains) to
specific nucleotide sequences. For example, a TAL effector can be
linked to a protein domain from, without limitation, a DNA
interacting enzyme (e.g., a methylase, a topoisomerase, an
integrase, a transposase, or a ligase), a transcription activators
or repressor, or a protein that interacts with or modifies other
proteins such as histones. Applications of such TAL effector
fusions include, for example, creating or modifying epigenetic
regulatory elements, making site-specific insertions, deletions, or
repairs in DNA, controlling gene expression, and modifying
chromatin structure.
[0159] In some embodiments, the spacer of the target sequence can
be selected or varied to modulate TALEN specificity and activity.
The results presented herein for TALENs that function as dimers
across a bipartite recognition site with a spacer demonstrate that
TALENs can function over a range of spacer lengths, and that the
activity of TALENs varies with spacer length. See, e.g., Example 6
below. The flexibility in spacer length indicates that spacer
length can be chosen to target particular sequences (e.g., in a
genome) with high specificity. Further, the variation in activity
observed for different spacer lengths indicates that spacer length
can be chosen to achieve a desired level of TALEN activity.
[0160] In some embodiments, TALEN activity can be modulated by
varying the number and composition of repeats within the DNA
binding domain(s). As described in Example 7 herein, for example, a
PthXoI-based TALEN showed greater activity than an AvrBs3-based
TALEN. PthXoI differs from AvrBs3 both in the number and RVD
composition of its repeats. In addition, the naturally occurring
DNA recognition sites for these proteins differ in their divergence
from the respective recognition sequences predicted based on the
TAL effector DNA cipher described by Moscou and Bogdanove (supra).
Further, several custom TALENs of the same length (12 RVDs) but
with differing RVD composition differed in their activity, and a 13
RVD custom TALEN had higher activity than a 12 RVD custom TALEN.
Thus, not only can TALENs be engineered to recognize a DNA sequence
of interest, but (1) the number of repeats can be varied to
modulate activity, (2) different binding sites can be selected to
achieve different levels of activity, and (3) the composition of
RVDs and their fit to the target site (according to the cipher) can
be varied to modulate TALEN activity.
[0161] When the TALEN is in a heterodimeric form, for instance with
two different monomers including each a TAL effector domain and a
FokI nuclease catalytic domain, the RVDs can be found in equivalent
number in each of the two TAL effector domains, or each domain can
display different numbers of RVDs. For instance, if a total of 22
RVDs is used to bind DNA in a particular heterodimeric TALEN, 11
repeats can be found in each of the two TAL effector domains;
alternatively, 10 repeats can be found in one of the two TAL
effector domains and 12 in the other. The present document also
encompasses TALEN with DNA modifying enzyme domain which functions
as a monomer. In this case, all the RVDs can be found in a single
TAL effector domain, which is fused to the monomeric enzyme. In
this case, in order to have efficient binding, the number of RVDs
must be equivalent to the total number of RVDs that would be found
in an equivalent dimeric TALEN. For example, instead of having 10
repeats on two different TAL effector domains (as in the case for a
dimeric TALEN), one would have 20 repeats in a single TAL effector
domain (as in the case for a monomeric TALEN).
[0162] In a further aspect, the total number of repeats within the
dimeric or monomeric TALEN is at least 14. In another further
aspect, the total number of repeats within the dimeric or monomeric
TALEN is at least 20. In another further aspect, the total number
of repeats within the dimeric or monomeric TALEN is at least 24. In
another further aspect, the total number of repeats within the
dimeric or monomeric TALEN is at least 30.
[0163] This patent application also provides methods for generating
TAL effector proteins having enhanced targeting capacity for a
target DNA. The methods can include, for example, generating a
nucleic acid encoding a TAL effector that has a DNA binding domain
with a plurality of DNA binding repeats, each repeat containing a
RVD that determines recognition of a base pair in the target DNA,
where each DNA binding repeat is responsible for recognizing one
base pair in the target DNA. As described in Example 12 below,
relaxing the requirement for T at position -1 of the binding site
may enhance the targeting capacity for engineered TAL effector
proteins. Thus, generating a TAL effector encoding nucleic acid can
include incorporating a nucleic acid encoding a variant 0th DNA
binding repeat sequence with specificity for A, C, or G, thus
eliminating the requirement for T at position -1 of the binding
site.
[0164] In addition, methods are provided herein for generating TAL
effectors having enhanced targeting capacity for a target DNA. Such
methods can include generating a nucleic acid encoding a TAL
effector that comprises DNA binding domain having a plurality of
DNA binding repeats, each repeat containing a RVD that determines
recognition of a base pair in the target DNA. As described in
Example 12 below, the specificity of NN (the most common RVD that
recognizes G) appears to be generally weak and can vary with
context, but certain RVDs may have enhanced specificity for G.
Thus, methods provided herein can include using alternate RVDs that
may have more robust specificity for G. For example, one or more
RVDs selected from the group consisting of RN, R*, NG, NH, KN, K*,
NA, NT, DN, D*, NL, NM, EN, E*, NV, NC, QN, Q*, NR, NP, HN, H*, NK,
NY, SN, S*, ND, NR, TN, T*, NE, NF, YN, Y*, and NQ can be used,
where the asterisk indicates a gap at the second position of the
RVD.
Articles of Manufacture
[0165] The present document also provides articles of manufacture
containing, for example, nucleic acid molecules encoding TALENs,
TALEN polypeptides, compositions containing such nucleic acid
molecules or polypeptides, or TAL endonuclease engineered cell
lines. Such items can be used, for example, as research tools, or
therapeutically.
[0166] In some embodiments, an article of manufacture can include
seeds from plants generated using methods provided herein. The
seeds can be conditioned using means known in the art and packaged
using packaging material well known in the art to prepare an
article of manufacture. A package of seed can have a label e.g., a
tag or label secured to the packaging material, a label printed on
the packaging material or a label inserted within the package. The
label can indicate that the seeds contained within the package can
produce a crop of genetically modified plants, and can described
the traits that are altered by the genetic modification, relative
to unmodified plants.
OTHER DEFINITIONS
[0167] Amino acid residues or subunits in a polypeptide sequence
are designated herein according to the one-letter code, in which,
for example, Q means Gln or Glutamine residue, R means Arg or
Arginine residue and D means Asp or Aspartic acid residue. [0168]
Amino acid substitution means the replacement of one amino acid
residue with another, for instance the replacement of an Arginine
residue with a Glutamine residue in a peptide sequence is an amino
acid substitution. [0169] Nucleotides are designated as follows:
one-letter code is used for designating the base of a nucleoside: a
is adenine, t is thymine, c is cytosine, and g is guanine. For the
degenerated nucleotides, r represents g or a (purine nucleotides),
k represents g or t, s represents g or c, w represents a or t, m
represents a or c, y represents t or c (pyrimidine nucleotides), d
represents g, a or t, v represents g, a or c, b represents g, t or
c, h represents a, t or c, and n represents g, a, t or c. [0170]
The term "DNA modifying enzyme" refers to any protein which is
capable of modifying the genetic material of a cell, whatever the
level of DNA modification (cleavage, covalent interaction,
water-mediated interaction . . . ). DNA-interacting proteins (e.g.,
a methylase, a topoisomerase, an integrase, a transposase, or a
ligase), transcription activators or repressor, other proteins such
as histones, and nucleases are intended to be included in the
meaning of "DNA modifying enzyme". When comprised in a TAL
effector-DNA modifying enzyme the DNA modifying enzyme is referred
as the DNA modifying enzyme domain. [0171] The term "nuclease" is
intended to include exonucleases and endonucleases. [0172] The term
"endonuclease" refers to any wild-type or variant enzyme capable of
catalyzing the hydrolysis (cleavage) of bonds between nucleic acids
within a DNA or RNA molecule, preferably a DNA molecule.
Non-limiting examples of endonucleases include type II restriction
endonucleases such as FokI, HhaI, HindIII, Nod, BbvCI, EcoRI, Bg/I,
and AlwI. Endonucleases comprise also rare-cutting endonucleases
when having typically a polynucleotide recognition site of about
12-45 base pairs (bp) in length, more preferably of 14-45 bp.
Rare-cutting endonucleases significantly increase HR by inducing
DNA double-strand breaks (DSBs) at a defined locus (Rouet, Smih et
al. 1994; Rouet, Smih et al. 1994; Choulika, Perrin et al. 1995;
Pingoud and Silva 2007). Rare-cutting endonucleases can for example
be a homing endonuclease (Paques and Duchateau 2007), a chimeric
Zinc-Finger nuclease (ZFN) resulting from the fusion of engineered
zinc-finger domains with the catalytic domain of a restriction
enzyme such as FokI (Porteus and Carroll 2005) or a chemical
endonuclease (Eisenschmidt, Lanio et al. 2005; Arimondo, Thomas et
al. 2006; Simon, Cannata et al. 2008). In chemical endonucleases, a
chemical or peptidic cleaver is conjugated either to a polymer of
nucleic acids or to another DNA recognizing a specific target
sequence, thereby targeting the cleavage activity to a specific
sequence. Chemical endonucleases also encompass synthetic nucleases
like conjugates of orthophenanthroline, a DNA cleaving molecule,
and triplex-forming oligonucleotides (TFOs), known to bind specific
DNA sequences (Kalish and Glazer 2005). Such chemical endonucleases
are comprised in the term "endonuclease" according to the present
document. Examples of such endonuclease include I-Sce I, I-Chu I,
I-Cre I I-Csm I PI-Sce I, PI-Tli I, PI-Mtu I I-Ceu I I-Sce II,
I-Sce III, HO, PI-Civ I PI-Ctr I, PI-Aae I PI-Bsu I PI-Dha I PI-Dra
I, PI-Mav I, PI-Mch I PI-Mfu I, PI-Mfl I PI-Mga I, PI-Mgo I PI-Min
I, PI-Mka I, PI-Mle I PI-Mma I, PI-Msh I, PI-Msm I PI-Mth I PI-Mtu
I PI-Mxe I PI-Npu I PI-Pfu I, PI-Rma I PI-Spb I PI-Ssp I, PI-Fac I,
PI-Mja I PI-Pho I, PI-Tag I, PI-Thy I, PI-Tko I PI-Tsp I
I-MsoI.
[0173] The endonucleases according to the present document can be
part of a Transcription Activator-Like (TAL) effector endonuclease
(TALEN). [0174] By "TALEN" is intended a protein comprising a
Transcription Activator-like (TAL) effector binding domain and an
endonuclease domain, the fusion of both domains resulting in a
"monomeric TALEN". Some monomeric TALEN can be functional per se
and others require dimerization with another monomeric TALEN. The
dimerization can result in a homodimeric TALEN when both monomeric
TALEN are identical or can result in a heterodimeric TALEN when
monomeric TALEN are different. Two monomeric TALEN are different
when, for example, their RVDs numbers are different, and/or when
the content (i.e amino acid sequence) of at least one RVD is
different.By "TAL effector-DNA modifying enzyme" is intended a
protein comprising a Transcription Activator-Like effector binding
domain and a DNA-modifying enzyme domain.
[0175] By "variant" is intended a "variant" protein, i.e. an
protein that does not naturally exist in nature and that is
obtained by genetic engineering or by random mutagenesis, i.e. an
engineered protein. This variant protein can for example be
obtained by substitution of at least one residue in the amino acid
sequence of a wild-type, naturally-occurring, protein with a
different amino acid. Said substitution(s) can for example be
introduced by site-directed mutagenesis and/or by random
mutagenesis.
[0176] By "cell" or "cells" is intended any prokaryotic or
eukaryotic living cells, cell lines derived from these organisms
for in vitro cultures, primary cells from animal or plant
origin.
[0177] By "primary cell" or "primary cells" are intended cells
taken directly from living tissue (i.e. biopsy material) and
established for growth in vitro, that have undergone very few
population doublings and are therefore more representative of the
main functional components and characteristics of tissues from
which they are derived from, in comparison to continuous
tumorigenic or artificially immortalized cell lines. These cells
thus represent a more valuable model to the in vivo state to which
they refer. [0178] By "homologous" is intended a sequence with
enough identity to another one to lead to homologous recombination
between sequences, more particularly having at least 95% identity,
preferably 97% identity and more preferably 99%. [0179] "Identity"
refers to sequence identity between two nucleic acid molecules or
polypeptides. Identity can be determined by comparing a position in
each sequence which may be aligned for purposes of comparison. When
a position in the compared sequence is occupied by the same base,
then the molecules are identical at that position. A degree of
similarity or identity between nucleic acid or amino acid sequences
is a function of the number of identical or matching nucleotides at
positions shared by the nucleic acid sequences. Various alignment
algorithms and/or programs may be used to calculate the identity
between two sequences, including FASTA, or BLAST which are
available as a part of the GCG sequence analysis package
(University of Wisconsin, Madison, Wis.), and can be used with,
e.g., default setting. [0180] by "mutation" is intended the
substitution, deletion, insertion of one or more nucleotides/amino
acids in a polynucleotide (cDNA, gene) or a polypeptide sequence.
Said mutation can affect the coding sequence of a gene or its
regulatory sequence. It may also affect the structure of the
genomic sequence or the structure/stability of the encoded mRNA.
[0181] By "gene" is meant the basic unit of heredity, consisting of
a segment of DNA arranged in a linear manner along a chromosome,
which codes for a specific protein or segment of protein. A gene
typically includes a promoter, a 5' untranslated region, one or
more coding sequences (exons), optionally introns, a 3'
untranslated region. The gene may further comprise a terminator,
enhancers and/or silencers. [0182] The term "gene of interest"
refers to any nucleotide sequence encoding a known or putative gene
product. [0183] As used herein, the term "locus" is the specific
physical location of a DNA sequence (e.g. of a gene) on a
chromosome. The term "locus" usually refers to the specific
physical location of a target sequence on a chromosome. [0184] By
"fusion protein" is intended the result of a well-known process in
the art consisting in the joining of two or more genes which
originally encode for separate proteins, the translation of said
"fusion gene" resulting in a single polypeptide with functional
properties derived from each of the original proteins. [0185] By
"catalytic domain" is intended the protein domain or module of an
enzyme containing the active site of said enzyme; by active site is
intended the part of said enzyme at which catalysis of the
substrate occurs. Enzymes, but also their catalytic domains, are
classified and named according to the reaction they catalyze. The
Enzyme Commission number (EC number) is a numerical classification
scheme for enzymes, based on the chemical reactions they catalyze
(World Wide Web at chem.qmul.ac.uk/iubmb/enzyme/). In the scope of
the present document, any catalytic domain can be used as a partner
and be fused to a TAL effector domain to generate a chimeric fusion
protein resulting in a TAL effector-DNA modifying enzyme.
Non-limiting examples of such catalytic domains can be those of
MmeI, EsaSSII, CstMI, NucA, EndA Escherichia coli, NucM, EndA
Streptococcus pneumonia, SNase Staphylococcus aureus, SNase
Staphylococcus hyicus, SNase shigella flexneri, Bacillus subtilis
yncB, Endodeoxyribonucleasel Enterobacteria phage T7, EndoG bovine,
ttSmr DNA mismatch repair protein mutS, cleavage domain of
Metnase.
[0186] The practice of the subject matter disclosed herein will
employ, unless otherwise indicated, conventional techniques of cell
biology, cell culture, molecular biology, transgenic biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, for example, Current Protocols in Molecular
Biology (Ausubel, 2000, Wiley and son Inc, Library of Congress,
USA); Molecular Cloning: A Laboratory Manual, Third Edition,
(Sambrook et al, 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor
Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed.,
1984); U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (Harries
and Higgins eds. 1984); Transcription and Translation (Hames and
Higgins eds. 1984); Culture of Animal Cells (Freshney, Alan R.
Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986);
Perbal, A Practical Guide to Molecular Cloning (1984); the series,
Methods in Enzymology (Abelson and Simon, eds.-in-chief, Academic
Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al.
eds.) and Vol. 185, "Gene Expression Technology" (Goeddel, ed.);
Gene Transfer Vectors For Mammalian Cells (Miller and Calos eds.,
1987, Cold Spring Harbor Laboratory); Immunochemical Methods in
Cell and Molecular Biology (Mayer and Walker, eds., Academic Press,
London, 1987); Handbook of Experimental Immunology, Vols. I-IV
(Weir and Blackwell, eds., 1986); and Manipulating the Mouse
Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1986).
[0187] The above written description of the invention provides a
manner and process of making and using it such that any person
skilled in this art is enabled to make and use the same, this
enablement being provided in particular for the subject matter of
the appended claims, which make up a part of the original
description.
[0188] As used above, the phrases "selected from the group
consisting of," "chosen from," and the like include mixtures of the
specified materials.
[0189] Where a numerical limit or range is stated herein, the
endpoints are included. Also, all values and subranges within a
numerical limit or range are specifically included as if explicitly
written out.
[0190] The above description is presented to enable a person
skilled in the art to make and use the invention, and is provided
in the context of a particular application and its requirements.
Various modifications to the preferred embodiments will be readily
apparent to those skilled in the art, and the generic principles
defined herein may be applied to other embodiments and applications
without departing from the spirit and scope of the invention. Thus,
this invention is not intended to be limited to the embodiments
shown, but is to be accorded the widest scope consistent with the
principles and features disclosed herein.
[0191] Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples, which are provided herein for purposes of illustration
only, the invention being further described in the following
examples, which do not limit the scope of the invention described
in the claims unless otherwise specified.
EXAMPLES
Example 1
A Cipher Governs TAL Effector-DNA Recognition
[0192] To determine whether there is a one-to-one, linear
correspondence between RVDs and contiguous nucleotides in the TAL
target site, the predicted promoter region (i.e., the 1,000 bp
immediately preceding the annotated translational start site) of
the known target gene for each of ten TAL effectors was scanned
with the TAL effector RVD sequence for alignments that minimized
entropy (randomness) in RVD-nucleotide associations. The following
formula was used to quantify entropy, where R is the set of RVDs
for the effector, D is the set of four nucleotides (A, C, G, T),
and fi,j represents the observed frequency with which the i.sub.th
RVD associates with the j.sub.th nucleotide:
i .di-elect cons. R j .di-elect cons. D max j ( f i ) - f i , j
##EQU00001##
[0193] Multiple low entropy sites were present in each promoter.
For effector AvrBs3, however, only one mapped to the 54 bp upa20
promoter fragment identified previously as sufficient and necessary
for activation, and it coincided with the UPA box common to genes
directly activated by AvrBs3 (Kay et al., supra). Also, for
effectors PthXo1 and AvrXa27, only one site each overlapped a
polymorphism between the activated and non-activated alleles of
their respective targets, Os8N3 and Xa27. Across the alignments at
these three sites, RVD-nucleotide associations were consistent, so
the remaining alignments were selected based on those associations,
resulting in exactly one site per TAL effector-target pair (FIG. 1B
and Table 2). Each site is preceded by a T (FIG. 1D).
[0194] To assess the specificity conferred by the RVD-nucleotide
associations, a weight matrix was first generated based on the
frequencies of all RVD-nucleotide associations observed across the
ten minimal entropy TAL effector-target site alignments (FIG. 1B).
The weight matrix was then used to scan the promoter region, the
1,000 bp preceding the translational start, of each nonredundant
gene model in rice, Oryza sativa spp. japonica cv. Nipponbare
(Osal, Release 6.0, rice.plantbiology.msu.edu) for best matches to
the five TAL effectors of the rice pathogen Xanthomonas oryzae
(AvrXa27, PthXo1, PthXo6, PthXo7, and Tal1c). For AvrXa27, the
sequence upstream of Xa27 (GenBank accession AY986492) was
included. This upstream sequence is not present in Nipponbare.
Observed association frequencies were weighted at 90% and the
remaining 10% was distributed equally to frequencies of all
possible associations. Alignments were ranked using a weight matrix
score (y axis), taken as a negative log of the frequency score
derived from the RVD-nucleotide association frequencies in FIG. 1B.
Thus, the lower the score, the better the match. For PthXo1,
PthXo6, PthXo7, and Tal1c, the experimentally identified target
gene was the best or nearly best match. Better matches were not
preceded by a T, were not represented on the microarray used to
identify the target, or lacked introns and EST evidence. Scanning
the reverse complement promoter sequences yielded no better scoring
alignments than the forward sites for the known targets. This
result does not imply that TAL effectors bind to the positive
strand, but indicates that they function in a forward orientation
relative to the positive strand. The known target of the fifth
effector, AvrXa27, is the disease resistance gene Xa27 (Gu et al.,
supra). The poorer rank for this match (5,368) may reflect a
calibrated, or recent and sub-optimal host adaptation. Better
scoring sites likely comprise genes targeted by AvrXa27 for
pathogenesis.
[0195] Using the weight matrix again, ten additional alignments
were obtained by scanning all rice promoters with 40 additional X.
oryzae TAL effectors and retaining the best alignments for which
the downstream gene was activated during infection based on public
microarray data (PLEXdb.org, accession OS3) (Table 3). As with the
initial set, a T precedes each site, and no reverse-strand sites
scored better. The RVD-nucleotide association frequencies of the
total 20 alignments are shown in FIG. 1C. They constitute a
strikingly simple cipher.
[0196] The RVD-nucleotide frequencies in the expanded set of 20 TAL
effector nucleotide alignments were used to generate a new weight
matrix, and a computational script was written in Python v2.5
(www.python.org). The script can be used to scan any collection of
DNA sequences for matches to a particular TAL effector, with a
user-definable weight factor for observed vs. unobserved
RVD-nucleotide associations. See Moscou and Bogdanove (supra).
[0197] There is some degeneracy in the cipher. Strong associations
may represent anchors that account for most of the binding
affinity, with weak associations providing a measure of
flexibility. Alternatively, neighbor effects may be involved. The
latter possibility was investigated by determining the nucleotide
association frequencies of every RVD conditioned on the RVD to
either side and comparing them to the total observed
frequencies--in other words, by sorting the RVD-nucleotide pairings
according to the neighbor RVD to the left or right, and comparing
the relative frequencies of each pair thus sorted with the overall
frequency for that pair. The frequencies of the RVD-nucleotide
associations sorted by neighbor did not deviate significantly from
the total observed frequencies, suggesting that the associations
are context independent.
[0198] Sequences flanking the 20 target sites revealed no conserved
nucleotides except the T at -1, but they tend to be C-rich
following the site and G-poor throughout (FIG. 1D). With few
exceptions, sites begin within 60 bp upstream of the annotated
transcriptional start, and none are closer than 87 bp to the
translational start (Table 2 and Table 3). Additional rules
governing RVD/nucleotide associations are described in Examples 4
and 5.
[0199] Given these results, prediction of TAL effector targets in a
genome and construction of targets de novo are now possible. The
ability to predict sites will expedite identification of host genes
important in disease. The ability to construct targets holds
promise for designing durable resistance genes that are responsive
to conserved or multiple TAL effectors. Customizing TAL effectors
for arbitrary gene activation or targeting of fused proteins for
DNA modification also is possible, as described herein.
TABLE-US-00003 TABLE 2 Predicted target site features for
experimentally identified TAL effector-target pairs TATA TAL
effector Source RVDs Target gene TcS box TlS AvrXa27.sup.1
Xanthomonas oryzae 17 Xa27 (rice) 27 -7 87 pv. oryzae PXO99.sup.A
AvrBs3.sup.2 X. campestris pv. 18 Bs3 (pepper) 59 1 123 vesicatoria
AvrBs3.sup.3 X. campestris pv. 18 UPA20 72 1 150 vesicatoria
(pepper) AvrBs3.DELTA.rep16.sup.4,5 Modified AvrBs3 14 Bs3-E 85 1
136 (pepper) AvrBs3.DELTA.rep109.sup.4 Modified AvrBs3 15 Bs3
(pepper) 59 1 123 AvrHah1.sup.6 X. gardneri 14 Bs3 (pepper) 59 1
121 PthXo1.sup.7 X. oryzae pv. oryzae 24 Os8N3 (rice) 79 46 251
PXO99.sup.A PthXo6.sup.8 X. oryzae pv. oryzae 23 OsTFX1 31 -780 136
PXO99.sup.A (rice) PthXo7.sup.8 X. oryzae pv. oryzae 22
OsTFIIA.gamma.1 333 44 469 PXO99.sup.A (rice) Tal1c X. oryzae 16
OsHEN1 10 -265 217 pv. - oryzicola BLS256 (rice) RVDs,
repeat-variable diresidues; TcS, annotated transcriptional start
site; TlS, translational start site. Locations are relative to the
5' end of the target site. .sup.1Gu et al., supra .sup.2Kay et al.
(2007) Science 318: 648 .sup.3Romer et al. (2007) Science 318: 645
.sup.4Herbers et al. (1992) Nature 356: 172 .sup.5Romer et al.
(2009) Plant Physiol. .sup.6Schornack et al. (2008) New Phytologist
179: 546 .sup.7Yang et al. (2006) Proc. Natl. Acad. Sci. USA 103:
10503 .sup.8Sugio et al. (2007) Proc. Natl. Acad. Sci. USA
TABLE-US-00004 TABLE 3 Xanthomonas oryzae TAL effector candidate
targets in rice activated during infection. TATA Fold Effector
Strain RVDs Rice locus r TcS box TlS q change Tal1c BLS256 16
OsHen1 1 10 -265 217 0.01 3.3 Tal2c BLS256 27 Os03g03034 15 -16
-145 143 0.01 5.2 Tal2d BLS256 16 Os04g49194 9 27 n.p. 102 3.9E-07
29.7 Tal3b BLS256 18 Os05g27590 42 34 -1 104 3.4E-08 8.5 Tal4a
BLS256 26 Os03g37840 1 152 221 363 2.2E-04 2.6 Tal4b BLS256 14
Os09g32100 72 68 n.p. 271 8.0E-03 3.6 Tal4c BLS256 23 Os06g37080 18
31 n.p. 151 2.7E-10 17.1 Tal6 BLS256 20 Os07g47790 16 -15 -70 93
3.6E-02 21.6 PthXo1 PXO99.sup.A 24 Os8N3 1 79 46 251 1.0E-08 84.2
PthXo6 PXO99.sup.A 23 OsTFX1 2 31 -780 136 3.5E-03 2.8 PthXo7
PXO99.sup.A 22 OsTFIIA.gamma.1 7 333 44 469 1.6E-06 4.5 Tal9a
PXO99.sup.A 20 OsHen1 1 44 -3 93 0.13 8.2 Tal7a/8a PXO99.sup.A 18
Os01g68740 2 32 -197 102 1.8E-01 1.7 Tal7b/8b PXO99.sup.A 20
Os01g40290 57 -2 -276 206 1.8E-01 1.7 RVDs, repeat-variable
diresidues; r, rank out of 58,918 gene models scanned, based on the
RVD weight matrix score; TcS, annotated transcriptional start site;
n.p., not present; TlS, translational start site. Locations are
relative to the 5' end of the target site. q values are for a
comparison to mock across five time points up to 96 hours after
inoculation, replicated four times; fold change given is at 96
hours (PLEXdb, accession OS3).
Example 2
TALENs can Function in Yeast
[0200] Plasmid Construction:
[0201] The protein coding sequence of the TAL effector, AvrBs3, was
obtained by digestion from a plasmid with BamHI. A DNA fragment
encoding principally the repeat domain was excised with SphI. The
amino acid sequence of AvrBs3 can be found under GENBANK Accession
No. P14727 and SEQ ID NO:12 (FIG. 3), and the nucleic acid sequence
under Accession No. X16130 and SEQ ID NO:13 (FIG. 4). In FIG. 4,
the BamHI and SphI sites are in bold and underlined. The AvrBs3
BamHI and SphI fragments were cloned into the nuclease expression
vector pDW1789 TAL (FIG. 5) adjacent to sequences encoding the FokI
nuclease domain. To clone the AvrBs3 target site into the target
reporter plasmid, two complementary DNA oligos, containing two
AvrBs3 recognition sites arranged in an inverted orientation with
an 18 bp spacer sequence in between, were synthesized with Bg/II
and SpeI overhangs at the 5' and 3' ends, respectively. Other
reporter plasmids were made that had recognition sites with spacer
lengths of 6, 9, 12 and 15 bp. The annealed DNA oligos were cloned
into the reporter plasmid, pCP5 (FIG. 6), which was digested with
Bg/II and SpeI.
[0202] Yeast Assay:
[0203] The target reporter plasmids were transformed into the yeast
strain YPH499 (a MAT a strain), and transformants were selected on
synthetic complete medium lacking tryptophan (SC-W). The TALEN
expression plasmids were transformed into YPH500 (a MAT .alpha.
strain); and transformants were plated on SC medium lacking
histidine (SC-H). Yeast colonies carrying the target reporter
plasmid and colonies carrying the TALEN expression plasmid were
cultured overnight at 30.degree. C. in liquid SC-W and SC-H media,
respectively. The cultures were adjusted to the same OD.sub.600,
and 200 .mu.l of each were mixed into 200 .mu.l YPD medium. The
mixture was incubated at 30.degree. C. for 4 hours to allow the two
types of yeast strain to mate. The mixed culture was spun down and
resuspended in 5 ml SC-W-H media at 30 C overnight or until the
OD.sub.600 reaches a range of 0.5-1. The cells were harvested and
quantitative .beta.-galactosidase assays were performed as
described (Townsend et al. (2009) Nature 459:442-445).
[0204] Results:
[0205] The TAL-FokI fusion is a site-specific nuclease consisting
of the TAL DNA recognition domain and the non-specific FokI DNA
cleavage domain. The TAL DNA recognition domain can be engineered
to bind different DNA sequences. As described in Example 1 herein,
the DNA recognition specificity for TAL effectors, a novel class of
DNA binding domain, has been deciphered. In particular, the DNA
binding domain of TAL, effectors contain a various number of
tandem, 34-amino acid repeats, which can recognize and bind to
specific DNA sequences. Amino acid sequences of the repeats are
conserved except for two adjacent highly variable residues at
positions 12 and 13 of the repeats. These positions together
specify individual nucleotides in the DNA binding site, one repeat
to one nucleotide. The architecture of the TALENs is illustrated in
FIG. 7. The TALENs function as dimers, with each monomer composed
of engineered TAL DNA recognition repeats fused to a non-specific
cleavage domain from the FokI endonuclease. The DNA recognition
repeats can be engineered to bind target DNA sequences within a
genome of interest. TAL nuclease monomers bind to one of two DNA
half-sites that are separated by a spacer sequence. This spacing
allows the FokI monomers to dimerize and create a double-strand DNA
break (DSB) in the spacer sequence between the half-sites.
[0206] To explore the potential of the TAL effector DNA recognition
domain, experiments were conducted to determine whether native TAL
effectors can function as nucleases when fused with the FokI
nuclease domain. The yeast-based assay was carried out by using a
TAL nuclease expression construct and a target reporter construct.
As illustrated in FIG. 5, the backbone of the nuclease expression
construct contains a FokI nuclease domain and an N-terminal nuclear
localization signal (NLS) under control of the yeast TEF1 promoter.
Several restriction sites are located between the FokI nuclease
domain and the NLS motif to facilitate cloning of various TAL
effectors. The target reporter construct has a disrupted lacZ
reporter gene with a 125 bp duplication of coding sequence as shown
in FIG. 6. The duplication flanks a URA3 gene and a target sequence
(composed of two half sites and a spacer sequence) recognized by
TAL DNA binding domains. If the TALEN binds and generates DNA
double-strand breaks (DSBs) at the target site, such breaks, in
yeast, are repaired predominantly by homologous recombination
between the duplicated lacZ sequences through single strand
annealing (Haber (1995) Bioessays17:609). Recombination results in
reconstitution of a functional lacZ gene and loss of URA3
(conferring 5-fluoroorotic acid resistance). Relative cleavage
activity of TALENs was measured by determining lacZ enzyme
activity.
[0207] In these studies, a native TAL effector, AvrBs3, which had a
central nuclease repeat region as set forth in SEQ ID NO:3 (FIG. 8)
was cloned into the nuclease expression vector, and the AvrBs3
target sites (two binding sites arranged in an inverted
orientation) with an 18 bp spacer sequence were cloned into the
target reporter vector. The yeast assay was performed using the
scheme shown in FIG. 9 and described above. The results showed that
the lacZ activity from yeast cells transformed with both the AvrBs3
nuclease plasmid and the target reporter plasmid was significantly
higher (15.8-fold higher) than the control yeast cells that
contained only the target reporter plasmid (FIG. 10). No activity
was observed with nuclease fusions made with only the SphI fragment
that encodes predominantly the repeat domain. This indicated that
sequences other than the DNA binding domain are required for TALEN
activity. Reporter plasmids with spacer lengths of 6 and 9 bp also
failed to show activity, indicating that the space between the two
binding sites is critical to allow FokI to dimerize. These data
indicate that the AvrBs3 TAL nuclease can function as a
site-specific nuclease that cleaves its cognate target sequence in
yeast.
Example 3
Modular Assembly of TAL Effector Repeats for Customized TALENs
[0208] Complementary oligonucleotides corresponding to the 102
basepairs of each of four individual TAL effector repeats, each
specifying a different nucleotide, are synthesized, annealed and
cloned into a high copy bacterial cloning vector, individually and
in combinations of 2 and 3 repeats in all permutations to yield 4
single, 16 double, and 64 triple repeat modules using standard
restriction digestion and ligation techniques (e.g., as illustrated
in FIG. 11). The desired TAL effector coding sequence is assembled
by introducing the appropriate modules sequentially into a
Gateway-ready high copy bacterial cloning vector containing a
truncated form of the tal1c gene that lacks the central repeat
region except for the characteristic final half repeat. For
example, an 18 repeat TAL effector coding sequence can be assembled
by sequentially introducing 5 triple modules and 1 double module
into the truncated tal1c vector.
Example 4
A System for Modular Assembly of TAL Effector Repeats
[0209] Plasmids and methods were developed for generating custom
TAL effector-encoding genes. The functional specificity of TAL
effectors is determined by the RVDs in the repeats, as described
herein; other polymorphisms in the repeats and elsewhere in the
proteins are rare and inconsequential with regard to functional
specificity. Thus, custom TAL effector genes were generated by
replacing the repeat region of an arbitrary TAL effector gene with
repeats containing the desired RVDs. The repeat sequences outside
the RVDs matched a consensus sequence (see below). DNA fragments
encoding TAL effector repeats were sequentially assembled into
modules encoding one, two, or three repeats, and the modules were
cloned into a TAL effector gene from which the original repeats
were removed. Each encoded repeat, with the exception of the last
(half) repeat, had the sequence LTPDQVVAIASXXGGKQALETVQRLLPVLCQDHG
(SEQ ID NO:18; FIG. 12A). The last (half) repeat had the sequence
LTPDQVVAIASXXGGKQALES (SEQ ID NO:20; FIG. 12B). In both sequences,
"XX" indicates the location of the RVD. The RVDs used in the
modular repeats were NI, HD, NN, and NG, which specify binding to
A, C, G, and T, respectively. In the experiments described below,
the tal1c gene of Xanthomonas oryzae pv. oryzicola strain BLS256,
with its repeats removed, was used as the "backbone" for building
custom TAL effector genes.
[0210] The method described herein included five components: (1)
generation of single repeat starter plasmids; (2) generation of
single repeat module plasmids; (3) generation of multiple repeat
modules; (4) generation of a complete set of one-, two-, and
three-repeat module plasmids; and (5) assembly of custom TAL
effector coding sequences.
[0211] To generate single repeat starter plasmids, the talk gene
was digested with MscI and religated to remove the entire repeat
region except for the first part of the first repeat and the last
part of the last, truncated repeat, resulting in the plasmid
designated pCS487 (FIG. 13). The resulting gene encoded the RVD NI
and, like most TAL effector genes, contained two SphI sites that
flanked the repeat region. The gene contained no XhoI site.
[0212] Next, a translationally silent mutation was introduced into
pCS487 to create a unique PspXI site, which encompasses a unique
XhoI site centered on codons 19 and 20. The mutation is depicted in
FIG. 14, which shows the original and altered nucleotide sequences
for codons 18-21 (SEQ ID NO:21 and SEQ ID NO:23, respectively),
both of which encode the amino acid sequence ALES (SEQ ID NO:22).
The resulting plasmid was designated pCS489.
[0213] By further mutagenesis, three additional constructs were
generated with the RVDs HD, NN, and NG, to create the plasmids
designated pCS490, pCS491, and pCS492, respectively. The SphI
fragment encompassing the modified repeat region was transferred
from pCS489, pCS490, pCS491, and pCS492 to the kanamycin resistant
plasmid designated pCS488 (FIG. 15), which encoded only the N- and
C-terminal portions of tal1c, without the repeat region, in the
Gateway entry vector pENTR-D (Invitrogen, Carlsbad, Calif.). This
transfer resulted in the single repeat starter plasmids designated
pCS493 (FIG. 16), pCS494, pCS495, and pCS496, respectively. The
PspXI/XhoI site in the truncated repeat remained unique in these
plasmids. The TAL effector gene in pCS488 and each of its
derivatives was preceded by Shine-Dalgarno and Kozak sequences for
efficient translation in prokaryotes and eukaryotes,
respectively.
[0214] Single repeat module plasmids were then constructed. One
plasmid was generated for each of the four chosen RVDs (NI, HD, NN,
and NG). Each plasmid had a 5' compatible cohesive end that
reconstituted a XhoI but not a PspXI site when ligated into a PspXI
site, and a 3' compatible cohesive end that reconstituted both a
XhoI and a PspXI site. The plasmids were generated by cloning
annealed synthetic, complementary oligonucleotides with overhangs
(FIG. 17A) into the PspXI/XhoI site of pBluescript SK-, resulting
in plasmids designated pCS502 (FIG. 17B), pCS503, pCS504, and
pCS505, respectively. Each plasmid allowed for introduction of
additional repeats at the 3' end of the single repeat module at the
unique reconstituted PspXI site, or for excision of the repeat
module using the reconstituted XhoI sites.
[0215] Additional single repeat modules, one each for NI, HD, NN,
and NG, were generated. Each had a 5' compatible cohesive end that
did not reconstitute a PspXI or XhoI site when ligated into a PspXI
site, a 3' compatible cohesive end that reconstituted both the XhoI
and a PspXI site, and a translationally silent nucleotide
substitution that destroyed the internal MscI site (FIG. 18A).
These modules were generated by annealing synthetic, complementary
oligonucleotides with overhangs. Ligating any of these additional
single repeat modules into the unique PspXI/XhoI site of a single
repeat module plasmid (pCS502, pCS503, pCS504, or pCS505) resulted
in no new XhoI site at the 5' junction, but restoration of the
unique 3' PspXI/XhoI site, so the resulting plasmids could be
linearized for introduction of more additional repeats by cutting
with PspXI. Reiteration of this process resulted in modules
containing multiple repeats (FIG. 18B). Further, each entire
multiple repeat module could be excised using XhoI. Because the
MscI site was destroyed in the additional single repeat modules,
the MscI site in the initial repeat remained unique, and was useful
to check orientation upon subsequent subcloning of the multiple
repeat module.
[0216] Additional single repeat modules were cloned iteratively
into the single repeat module plasmids to generate, along with the
single repeat module plasmids, a complete set of all possible one-,
two-, and three-repeat modules, for a total of 84 plasmids
designated pCS502 through pCS585 (FIG. 19). Modules containing more
than three repeats (e.g., four, five, six, seven, eight, nine, ten,
or more than ten repeats) are generated in the same manner.
[0217] A method was then devised to assemble any sequence of
repeats into the tal1c "backbone" to generate a custom TAL effector
gene. The method included the following steps, which also are
depicted in FIG. 20:
[0218] (1) Choose a single repeat starter plasmid with the first
desired repeat (pCS493, pCS494, pCS495, or pCS495, encoding RVD NI,
HD, NN, or NG, respectively);
[0219] (2) linearize the plasmid with PspXI;
[0220] (3) isolate the module for the next repeat(s) from the
appropriate module plasmid (pCS502 through pCS585) using XhoI;
[0221] (4) ligate;
[0222] (5) check orientation by digestion with MscI and confirm
sequence from the 3' end using a vector based primer; and
[0223] (6) repeat steps 2-5 until all repeats are assembled.
Example 5
Library of Plasmids for Modular Assembly of TALENs
[0224] Assembly of TALEN repeats as described herein (e.g., using
the steps depicted in FIG. 20) results in numerous intermediate
plasmids containing increasing numbers of repeats. Each of these
plasmids is stored such that a library of plasmids for modular
assembly of TALENs (pMATs) is generated. For example, FIGS. 21A and
21B depict the assembly of repeat modules in construction of TAL
endonucleases that will target the nucleotide sequences shown. In
FIG. 21A, repeat modules from plasmids designated pCS519, pCS524,
pCS537, pCS551, pCS583, and pCS529 are sequentially added to the
sequence in the starter plasmid designated pCS493, resulting in
plasmids designated pMAT55, pMAT56, pMAT57, pMAT58, pMAT59, and
pMAT60. In FIG. 21B, repeat modules from plasmids designated
pCS530, pCS533, pCS522, and pCS541 are sequentially added to the
sequence in the plasmid designated pMAT1, resulting in plasmids
designated pMAT61, pMAT62, pMAT63, and pMAT64.
Example 6
Generation and Testing of Customized TALENs
[0225] The TAL DNA recognition domain was used to create TALENs
that recognize and cleave particular DNA targets (FIG. 22A), using
the system described in Examples 4 and 5. To assess TALEN function,
a yeast assay was adapted in which LacZ activity serves as an
indicator of DNA cleavage (Townsend et al., supra). In this assay,
a target plasmid and a TALEN expression plasmid are brought
together in the same cell by mating. The target plasmid has a lacZ
reporter gene with a 125-bp duplication of coding sequence. The
duplication flanks a target site recognized by a given TALEN. When
a double-strand DNA break occurs at the target site, it is repaired
through single-strand annealing between the duplicated sequences,
which creates a functional lacZ gene whose expression can be
measured using standard .beta.-galactosidase assays that provide a
quantifiable readout (FIG. 22A). This assay has been demonstrated
to be a good predictor of the ability of a ZFN to create
chromosomal mutations by NHEJ or to stimulate homologous
recombination for gene editing in higher eukaryotes (Townsend et
al., supra; and Zhang et al. (2010) Proc. Natl. Acad. Sci. USA
107:12028-12033).
[0226] Two well characterized TAL effectors were used--AvrBs3 from
the pepper pathogen Xanthomonas campestris pv. vesicatoria and
PthXo1 from the rice pathogen X. oryzae pv. oryzae (Bonas et al.
(1989) Mol. Gen. Genet. 218:127-136; and Yang et al. (2006) Proc.
Natl. Acad. Sci. USA 103:10503-10508). The amino acid sequence of
AvrBs3 can be found under GENBANK Accession No. P14727 and SEQ ID
NO:12 (FIG. 3), and the nucleic acid sequence under Accession No.
X16130 and SEQ ID NO:13 (FIG. 4). The amino acid sequence of PthXo1
can be found under GENBANK Accession No. ACD58243 and SEQ ID NO:31
(FIG. 23), and the nucleic acid sequence under Accession No.
CP000967, gene ID 6305128, and SEQ ID NO:32 (FIG. 24). The amino
acid sequence of PthXo1 under GENBANK Accession No. ACD58243 is
truncated at the N-terminus due to a misannotation of the start
codon. The complete sequence is presented in FIG. 23.
[0227] The repeat domains of both AvrBs3 and PthXo1 are encoded
entirely within a conserved SphI fragment (FIGS. 4 and 24). Both
TAL effector-encoding genes also have a BamHI restriction fragment
that encompasses the coding sequence for the repeat domain and 287
amino acids prior and 231 amino acids after (FIGS. 4 and 24; see,
also, FIG. 22A). Absent from the BamHI fragment is the TAL effector
transcriptional activation domain. Both the SphI fragments and the
BamHI fragments were fused to a DNA fragment encoding FokI that is
present in the nuclease expression vector pFZ85 (FIG. 25). The
fusion proteins between FokI nuclease and the BamHI fragments
encoded by AvrBs3 and PthXo1 are given in FIGS. 26 and 27; SEQ ID
NOS:33 and 34.
[0228] The FokI monomers must dimerize in order to cleave, but the
appropriate spacer length between the two DNA recognition sites was
unclear. For ZFNs, in which the zinc finger array is separated from
FokI by a 4-7 amino acid linker, the typical spacer between the two
recognition sites is 5-7 bp (Handel et al. (2009) Mol. Ther.
17:104-111). Since, for example, 235 amino acids separate the
repeat domain from FokI in the BamHI TALEN constructs used herein,
a variety of spacer lengths for both the BamHI and SphI constructs
(6, 9, 12, 15, and 18 bp) were used. As a positive control, a
well-characterized zinc finger nuclease with a DNA binding domain
derived from the mouse transcription factor Zif268 (Porteus and
Baltimore (2003) Science 300:763) was used. As negative controls,
the TAL effector domains were fused to a catalytically inactive
FokI variant or tested against non-cognate DNA targets.
[0229] Haploid cell types containing either TALEN expression or
target plasmid in 200 .mu.l of overnight culture were mated in YPD
medium at 30.degree. C. After 4 hours, the YPD medium was replaced
with 5 ml of selective medium and incubated overnight at 30.degree.
C. Mated cultures were lysed, ONPG substrate added, and absorbance
read at 415 nm using a 96-well plate reader (Townsend et al.,
supra). .beta.-galactosidase levels were calculated as a function
of substrate cleavage velocity. The results obtained with target
reporter constructs that had a 15 bp spacer separating the two
recognition sites are shown in FIG. 22B. All nuclease expression
constructs derived from the SphI fragment, which encoded
principally the repeat array, failed to show activity, indicating
that amino acid sequences in addition to those in the repeat array
are required for function (FIG. 22B). Robust activity, however, was
observed for both the AvrBs3 and the PthXo1 TALENs derived from the
BamHI fragment (FIG. 22B). The activity of the PthXo1 TALEN
approximated that of the ZFN positive control. The activity
required the functional FokI domain and was specific for the DNA
target recognized by a given TALEN.
[0230] Experiments also were conducted to test various distances
between the TAL effector binding sites (11 length variants between
12 and 30 bp), in order to identify spacer lengths that enable FokI
to dimerize most efficiently (FIG. 28A). Both enzymes showed two
spacer length optima--one at 15 bp and the other at either 21 bp
(AvrBs3) or 24 bp (PthXo1). For PthXo1, activity was observed for
all tested spacer lengths 13 bp and longer. Some spacer lengths for
AvrBs3 showed no activity, however, suggesting that spacer length
is critical for certain TALENs.
[0231] The above experiments tested activity of homodimeric TALENs,
which bind two identical recognition sequences placed in opposition
on either side of the spacer. Since such palindromic sites are
unlikely to occur naturally in genomic targets, experiments were
conducted to test whether TALENs could function as heterodimers.
AvrBs3 and PthXo1 recognition sites were placed in head to tail
orientation on either side of a 15 bp spacer. Activity of AvrBs3
and PthXo1 TALENS individually and Zif268 on their respective
targets was measured as controls. As a negative control, a yeast
culture with only the target site plasmid for the heterodimeric
site was assayed for LacZ activity. The resulting activity of the
heterodimeric TALEN approximated an average of the activities
observed with the two homodimeric enzymes (FIG. 28B). To test
whether repeat domains can be assembled to target TALENs to
arbitrary chromosomal sequences, two genes were chosen that were
previously targeted for mutagenesis with ZFNs--ADH1 from
Arabidopsis and gridlock from zebrafish (Foley et al. (2009) PLoS
One 4:e4348; and Zhang et al., supra). A search was conducted for
12-13 bp sequences in the coding regions that were preceded by a 5'
T and with a nucleotide composition similar to that of TAL effector
binding sites identified by Moscou and Bogdanove (supra). In ADH1
and gridlock, such sites occurred on average every 7-9 bp. Four 12
bp sites were selected in ADH1 (at positions 360, 408, 928, and 975
of the chromosomal gene sequence) and one 13 bp site in gridlock
(at position 2356 of the chromosomal gene sequence; FIG. 29A). TAL
effector repeat domains were constructed to recognize these
targets, using the most abundant RVDs from native TAL effectors (NI
for A, HD for C, NN for G, and NG for T). To construct custom
TALENs, repeats with these RVDs were synthesized individually and
assembled into modules of one, two, or three repeats as described
in Examples 4 and 5. These modules were ligated sequentially into a
derivative of the talk gene (Moscou and Bogdanove, supra) from
which the original repeats had been removed, and BamHI fragments
from these engineered TAL effectors were fused to sequences
encoding the catalytic domain of FokI in pFZ85 (FIG. 25). Five
custom TALENs targeted to ADH1 from Arabidopsis and the zebrafish
gridlock gene were created.
[0232] The resulting custom TALENs were tested in the yeast assay
as homodimeric TALENs (that is, the identical DNA binding site was
duplicated in inverse orientation on either side of a 16-18 bp
spacer), although it is noted that heterodimeric TALENs would need
to be constructed to direct cleavage at naturally occurring DNA
targets. Spacer lengths were chosen based on the distance closest
to 15 bp from the 3' end of the next neighboring (and opposing)
candidate site. Sixteen by spacers were used for ADH1-360-12,
ADH1-408-12r, and 18 bp spacers for ADH1-928-12, ADH1-975-12r, and
gridlock-2356-13r. The yeast assay was performed as described
above.
[0233] Robust nuclease activity was observed for the ADH1-360-12
and gridlock-2356-13r TALEN (FIG. 29B). The ADH1-928-12 TALEN had
modest activity that was nonetheless significantly above the
negative controls. For each TALEN that gave positive results,
nuclease activity was specific to the cognate target. These results
indicate that novel, functional TALENs can be created by assembly
of customized repeat domains.
Example 7
Naturally Occurring Target and TAL Effector Pairs Show Overall and
Positional Bias in Nucleotide and RVD Composition
[0234] The 20 paired targets and TAL effectors analyzed by Moscou
and Bogdanove (supra) were evaluated for overall composition bias
and for positional effects on nucleotide or RVD frequencies. It was
observed that sites (on the positive strand) were generally A- and
C-rich, and G-poor. The average percent A was 31.+-.16% (1 standard
deviation). The average percent C was 37.+-.13%. The average
percent G was 9.+-.8%, and the average percent T was 22.+-.10%.
Since the alignments vary in length, the analysis of positional
effects was restricted to the five positions on each end.
Strikingly, bias in the target sequences was apparent for A and
against T at positions 1 and 3, and for T at position N and
possibly 2. G was particularly rare at position N-1. This bias was
reflected by matching RVDs in the effectors, with NI being most
common at positions 1 and 3, no NG at position 1, nearly always NG
at position N, and rarely NN at position N-1 (FIG. 30).
Example 8
Method and Reagents for Rapid Assembly and Cloning of Custom TAL
Effector Repeat Arrays
[0235] The Golden Gate cloning method [Engler et al. (2008), supra;
and Engler et al. (2009), supra] employs the ability of Type IIS
restriction endonucleases (e.g. BsaI) to cut outside their
recognition sites to create custom overhangs for ordered ligation
of multiple DNA fragments simultaneously. Using this method,
several DNA fragments can be fused into an array in a specific
order and cloned into a desired destination vector in a single
reaction (FIG. 31).
[0236] A method and reagents for assembling custom TAL effector
repeat encoding arrays were developed based on the Golden Gate
system. When BsaI sites are positioned on either side of a TAL
effector repeat coding sequence, cleavage releases a repeat
fragment flanked by 4-bp overhangs. Because the cleavage site is
not sequence-specific, by staggering, repeat clones can be released
with ordered, complementary overhangs (sticky ends), enabling the
ordered assembly of multi-repeat arrays.
[0237] A library of 58 plasmids (FIGS. 32A and 32B) was generated
to allow the simultaneous assembly of up to 10 repeat units into
"subarrays," followed by simultaneous assembly of one, two, or
three of these subarrays together with a final truncated repeat
into a complete, custom array. Ten staggered sets of four
fragments, each fragment in a set encoding a repeat module with a
different one of the four most common RVDs, HD, NG, NI, and NN,
were synthesized and cloned into a vector carrying the tetracycline
resistance gene, for a total of 40 plasmids. Four more fragments
that encoded the terminal truncated TAL effector repeat of 20 amino
acids, each fragment encoding a different one of the four most
common RVDs, were synthesized and cloned into a different vector
carrying the spectinomycin resistance gene to yield four more
plasmids, designated as "last repeat plasmids," FIG. 32A). All
fragments in the staggered sets are flanked by BsaI sites in the
vector so that cleavage with BsaI releases the fragments with
different sticky ends that allow for assembly in the appropriate
order; that is, i.e. the overhang at the 3' end of a fragment for
repeat module 1 is complementary only to the overhang at the 5' end
of the fragment for repeat module 2, the overhang at the 3' end of
repeat module 2 is complementary only to the overhang at the 5' end
of repeat module 3, and so on. The fragments in the last repeat
plasmids are flanked by sites for a different Type IIS restriction
endonuclease, Esp3I. Fourteen additional plasmids, described
following, were constructed as destination vectors to receive
assembled subarrays.
[0238] The first destination vector, plasmid pFUS_A was constructed
to receive the first subarray of 10 repeats to be assembled into a
final array of 21 or fewer repeats (counting the final, truncated
repeat). pFUS_A was constructed such that cleavage by BsaI creates
an overhang on one side complementary to the overhang at the 5' end
of the first repeat module and an overhang at the other side
complementary to the overhang at the 3' end of the 10th repeat
module. To receive a second subarray of 10 or fewer repeats to be
assembled into such a final array, destination vector plasmids
pFUS_B1, pFUS_B2, pFUS_B3, pFUS_B4, pFUS_B5, pFUS_B6, pFUS_B7,
pFUS_B8, pFUS_B9, and pFUS_B10 were constructed that when cleaved
by BsaI have overhangs respectively complementary to the overhang
at the 5' end of the first repeat module and the 3' end of the
repeat module for the corresponding numbered position (e.g., the
pFUS_B6 overhang for the 3' end of the subarray matches the
overhang of the four repeat module fragments for position 6).
Arrays cloned in pFUS_A and the pFUS_B series of plasmids are
flanked by Esp3I sites in the vector and when released by digestion
with Esp3I the arrays have unique complementary overhangs that
allow for them to be ligated in order along with a final truncated
repeat fragment into destination vector pTAL, which encodes a TALEN
missing the repeat region. pTAL was constructed so that cleavage
with Esp3I allows insertion of the repeat array at the correct
location and in the correct orientation by virtue of an overhang at
one end that is complementary to the overhang at the 5' end of the
first ten repeat subarray and an overhang at the other end
complementary to the overhang at the 3' end of the final truncated
repeat fragment (FIG. 33).
[0239] The final two destination vector plasmids, pFUS_A30A and
pFUS_A30B were constructed to receive the first and second ten
repeat subarrays to be assembled into a final array of 22-31
repeats. pFUS_A30A and pFUS_A30B were constructed such that
digestion with Esp3I releases the arrays with the appropriate
complementary overhangs such that the arrays can be ligated in
order along with a third array from a pFUS_B vector and a final
truncated repeat fragment from a last repeat plasmid, released
similarly by digestion with Esp3I, into pTAL (FIG. 32).
[0240] All destination vectors have the LacZ gene cloned in between
the Type IIS restriction endonuclease sites, allowing for
blue-white screening for recombinants. Except for pTAL, which
carries a gene for ampicillin resistance, all the destination
vectors carry a gene for spectinomycin resistance.
[0241] To rapidly construct a custom TAL effector repeat array
using these reagents, the following method was established. In the
first step, the appropriate individual RVD module plasmids for the
necessary subarrays of ten or fewer repeats are mixed together with
the appropriate destination vector in one tube. T4 DNA ligase and
BsaI endonuclease are added and the reaction is incubated in a PCR
machine for 10 cycles of 5 minutes at 37.degree. C. and 10 minutes
at 16.degree. C., the respective optimal temperatures for the two
enzymes. The reaction mixture is then treated with the
PLASMID-SAFET.TM. nuclease to hydrolyze all linear dsDNA fragments
in order to prevent cloning of shorter, incomplete arrays by in
vivo recombination, and then the mixture is used to transform
chemically competent E. coli cells. The resulting recombinant
plasmids are isolated and the correct constructs confirmed. Then,
in the second step, the confirmed plasmids from the first step are
mixed together with the appropriate last repeat plasmid and pTAL,
and the digestion and ligation reaction cycle carried out as in the
first step. Finally, the reaction products are introduced into E.
coli, and the full length, final array construct is isolated and
confirmed. The protocol can be completed by one person within a
week's time.
[0242] Expression constructs for TALENS 85, 102 and 117 in Table
4A, as well as TALENS HPRT-3254-17 and HPRT-3286-20r, described in
Example 14 below, were made using the method and reagents described
in this example.
[0243] Repeat arrays cloned in pTAL are subcloned readily into
other TAL effector gene contexts using the conserved SphI
restriction endonuclease sites that flank the repeat region.
Example 9
Custom TALEN Data Show Initial Support for "Rules" and a
Correlation Between RVD Number and Activity
[0244] Example 6 describes experiments conducted to engineer the
TALEN DNA binding domain so that it can recognize unique DNA
sequences. As described, these custom TALENs recognized sites in
the Arabidopsis ADH1 and zebrafish gridlock genes. Additional
custom TAL effector DNA binding domains were engineered to
recognize not only sites in these genes, but also in the TT4 gene
from Arabidopsis, and telomerase from zebrafish (Foley et al.,
supra; and Zhang et al., supra). These custom TALENs were made
using the methods described in Examples 3, 4 and 8. In engineering
the custom TALENs, the observed compositional and positional biases
were adopted as design principles or "rules." First, a search was
conducted for sequences in the coding regions that were preceded by
a 5' T and at least 15 bp in length, and that had a nucleotide
composition consistent with the averages noted above. Specifically,
only those sites with 0-63% A, 11-63% C, 0-25% G, and 2-42% T were
selected. Such sites occurred on average every 7-9 bp. Sites were
then selected that conformed to the observed positional biases
described above. From this set, two pairs of binding sites in each
gene were identified that were 15-19 bp in length and separated by
15-18 bp, so that binding of the engineered TALENs would allow FokI
to dimerize. The modular assembly methods (Examples 3 and 4)
generated partial length constructs.
[0245] In total, 21 intermediate and full length TALENs designed to
target 16 nucleotide sequences, each with an array of nine repeats
or longer. The amino acid sequences of these TALENs are provided in
FIGS. 34A-34U (SEQ ID NOS:35-55). These 21 TALENs were tested for
their ability to cleave DNA using the yeast assay described in
Examples 2 and 6. Activity data are shown in FIG. 35 and summarized
in Table 4A.
[0246] Some of the intermediate, partial length TALENs correspond
to targets that break the rules for nucleotide composition and
terminal T. Table 4A shows length, conformity to these two rules,
and activity relative to that of ZFN268 for each TALEN. The results
reveal a general trend that increasing the length of the RVD array
increases activity of the resulting TALEN. This suggests that there
is a minimal number of RVDs that are needed before a DNA target can
be recognized in vivo. Further, conformity to the rules appears to
be important. Of the six TALENs showing no detectable activity, two
violated the target composition rule, two did not end in NG, and
another broke both rules (one obeyed both rules). Three of the
eight TALENs with activity less than 25% of ZFN268 violated one of
the rules, and one of four TALENs with activity 25-50% of ZFN268
did not have an RVD sequence ending in NG. It is noted that TALENs
with activity 50% or greater than that of ZFN268 obeyed all the
rules, and for TALENs of the same length, rule breakers generally
had less activity than obedient arrays. Consistent with the overall
trend regarding length, even for intermediates that broke no rules,
the corresponding full length TALENs had higher activity (Table 4A
and FIG. 35). Variation in spacer length due to TALEN length
differences on the same target may have contributed to this
observation, but some range of spacer lengths is tolerated
(Christian et al., supra).
[0247] Some complexities in the data were apparent. For example,
activity varied among obedient TALENs of the same length, some
short arrays had moderately high activity, and some long arrays
that were obedient had little or no activity (Table 4B).
Nonetheless, the results provided support for the conclusions that
1) generally a greater number of repeats results in greater
activity, and 2) conformity to composition and positional bias
rules is important for activity. Therefore, the following design
principles were derived. [0248] TAL effector binding sites are
designed to be a minimum of 15 bases long and oriented from 5' to
3' with a T immediately preceding the site at the 5' end. [0249] A
site may not have a T in the first (5') position or an A in the
second position. [0250] A site must end in T (3'), and may not have
a G at the next to last position. [0251] The base composition of
the site must fall within specified ranges (average.+-.two standard
deviations): A 0-63%, C 11-63%, G 0-25%, and T 2-42%.
TABLE-US-00005 [0251] TABLE 4A Activity, conformity to rules, and
length of TALENs tested in the yeast assay. Names from Christian et
al. % Ends Gene TALEN (supra) RVDs Activity GATC in NG RVD
sequence.sup.1 telomerase 124 9 - N Y HD NN NN NG NG NG NN HD NG
gridlock 105 10 + N N NI HD HD HD HD NG HD NG HD HD ADH1 58
ADH1-360-12 12 ++ Y N NI NG HD NI NI NN NI NG NG HD NG HD ADH1 63
ADH1-408-12r 12 - Y N HD HD HD NI NN NI NI NN NG NI NI NI ADH1 68
ADH1-928-12 12 + Y Y HD HD NN NN NI NG NN HD NG HD HD NG ADH1 73
ADH1-975-12r 12 - N N NI NN NI HD NI NI NI HD HD NI HD NI TT4 89 12
- Y N NN NN HD NI HD NG NN HD NG NI NI HD gridlock 106
gridlock-2356-13r 13 ++ Y Y NI HD HD HD HD NG HD NG HD HD NN HD NG
ADH1 64 15 + Y Y HD HD HD NI NN NI NI NN NG NI NI NI HD NI NG ADH1
69 15 +++ Y Y ND ND NN NN NI NG NN HD NG HD HD NG HD NG NG ADH1 74
15 ++ Y Y NI NN NI HD NI NI NI HD HD NI HD NI NI HD NG TT4 90 15 -
Y Y NN NN HD NI HD NG NN HD NG NI NI HD HD HD NG telomerase 121 15
+ Y Y HD NG NG NN NG HD HD NN HD NI NG NN NI NG NG telomerase 126
15 - N Y HD NN NN NG NG NG NN HD NG NI NG HD NN NG NG gridlock 107
16 ++++ Y Y NI HD HD HD HD NG HD NG HD HD NN HD NG NG HD NG
gridlock 117 16 ++ Y Y HD HD HD NN NN NI NI NN HD HD NN NI HD NN HD
NG telomerase 131 16 + Y Y NI NG NG HD HD HD HD NI HD NN NI NN HD
NG HD NG telomerase 136 17 + N Y NI NN NI HD NI NN NN NI NI NN NG
NN NN NI NN HD NG ADH1 60 18 +++++ Y Y NI NG HD NI NI NN NI HG NG
HD NG HD NG NG HD NI HD NG TT4 85 18 + Y Y NI HD NG HD HD NN HD HD
NG NN NI NI NN HD NI HD NI NG gridlock 102 18 + Y N NN NN HD NG HD
NI HD HD NG NI HD NI NI HD NN NI HD NI .sup.1Target sequences
tested consist of inverted repeats of the corresponding nucleotide
sequence, where HD, NG, NI, and NN correspond to C, T, A, and G,
respectively, separated by a spacer sequence of 16-18 bp.
TABLE-US-00006 TABLE 4B Excerpt of Table 4A, sorted by activity
level % Ends RVDs Activity GATC in NG 9 - n y 12 - y n 12 - n n 12
- y n 15 - y y 15 - n y 10 + n n 12 + y y 15 + y y 15 + y y 16 + y
Y 17 + n y 18 + y y 18 + y n 12 ++ y n 13 ++ y y 15 ++ y y 16 ++ y
y 15 +++ y y 16 ++++ y y 18 +++++ y y
Example 10
Heterodimeric TALEN Pairs Cleave their Intended Naturally Occurring
Target Sequences in the Yeast Assay
[0252] The data in Examples 2, 6 and 9 demonstrate that custom
TALENs can be engineered to recognize novel target DNA sequences.
The yeast activity data for the custom TALENs was gathered using
individual TALEN monomers that recognized a homodimeric target
site. That is, the target sequence of the TALEN was duplicated in
inverse orientation on either side of a 15-18 bp spacer. Cleavage
of endogenous chromosomal sequences, however, generally would
require that two different custom TALENs recognize two different
sequences on either side of a spacer. As described in Example 6,
this ability was demonstrated for the AvrBs3 and PthXo1 TALENS
together using a corresponding chimeric target site in the yeast
assay. We tested whether two different custom TALENs could
recognize and cleave a naturally occurring DNA sequence. Using the
yeast assay described in Example 2, custom TALENs designed to
cleave two different target sequences in the Arabidopsis ADH1 gene
were assayed for activity on these targets. The DNA sequences of
the target sites and the corresponding TALENs are shown in FIG.
36A. The amino acid sequences of the TALENs are provided in FIG.
34. The beta-galactosidase activity obtained in the yeast assay is
plotted in the graph shown in FIG. 36B. The activity of the TALENs
on their naturally occurring target sequence was significantly
above the negative controls, indicating that TALENs can be
engineered to recognize and cleave endogenous target DNA
sequences.
Example 11
TALENs Cleave Native Genes in Arabidopsis and Introduce Mutations
by Imprecise Non-Homologous End-Joining
[0253] One of the active TALEN pairs designed to recognize a target
sequence in the Arabidopsis ADH1 gene was tested to determine
whether it can bind, cleave and mutate chromosomal DNA. Each of the
individual ADH1 TALENs comprising this pair (pTALENs 69 and 74) was
cloned into the plant expression vector pFZ14, which places the
TALENs under the control of the constitutive 35S promoter (Zhang et
al., supra). The resulting constructs were then introduced into
Arabidopsis protoplasts by electroporation. After 48 hours, genomic
DNA was isolated and digested with Tth1111. A Tth1111 cleavage site
is located in the spacer sequence between the two TALEN recognition
sites (FIG. 37A). Cleavage of the chromosomal DNA by the TALEN
would be expected to introduce mutations by imprecise
non-homologous end joining (NHEJ), which would result in failure to
cleave by Tthl 111. A 375 bp fragment encompassing the TALEN
recognition site was then PCR amplified. The PCR product was
digested again with Tth1111 to remove most of the remaining genomic
DNA that was not modified by TALEN-mediated NHEJ. The digestion
products were then run on an agarose gel. An uncleaved PCR product
was observed, and such uncleaved PCR products are diagnostic of
nuclease activity (in this case TALEN activity) at the endogenous
target sequence (Zhang et al., supra). The uncut DNA was cloned and
analyzed by DNA sequencing. The sequencing of nine independent
clones revealed that six carried mutations introduced by NHEJ (FIG.
37B). Thus, TALENS cleave endogenous chromosomal loci and introduce
DNA double strand breaks and mutations.
Example 12
Enhancing Targeting Capacity
[0254] At the core of the TAL effector DNA cipher, the four most
common RVDs each have apparent one-to-one specificity for the four
nucleotides, based on association frequencies. This is markedly so
for HD, NG, and NI, but less so for NN (FIG. 1C). NN associates
most frequently with G, but almost as commonly with A, and
sometimes with C or T. For a randomly assembled TAL effector with
NN at four locations in a 13 RVD sequence, having G at all
corresponding positions in an artificial target gave the best
activity (Boch et al. (2009) Science 326:1509-1512). A reduced but
did not abolish activity, and C and T eliminated detectable
activity. A drastic loss of activity was observed when C, T, or A
was substituted for G at just the first position in the binding
site for the 24 RVD effector PthXo1, which is an NN (Romer et al.
(2010) New Phytol. 187:1048-1057). This was in contrast, however,
to the observation that the much shorter AvrHah1 (14 RVDs) begins
with an NN that aligns with A, and the 23 RVD effector PthXo6 has
three NNs in a row at positions 4-6 that each align with A, yet
both of these proteins are highly active (see, Schornack et al.
(2008) New Phytol. 179:546-556; and Romer et al., supra). Thus the
specificity of NN for G appears to be generally weak and can vary
with context.
[0255] The observed invariance of the thymine immediately preceding
TAL effector target sites is a requirement for several effectors
[Boch et al., supra; Romer et al., supra; and Romer et al. (2009)
Plant Physiol. 150:1697-1712]. The amino acid sequence immediately
preceding the repeat region in TAL effectors, which is highly
conserved (FIG. 38A), shares significant similarity with the
repeat, both in amino acid sequence and in predicted secondary
structure (FIG. 38B and Bodganove et al. (2010) Curr. Opin. Plant
Biol. 13:394-401). It was hypothesized that this sequence, termed
the "0.sup.th" repeat, is the basis for the requirement for T at
position -1 of the binding site, and that residues in the
RVD-analogous position (FIG. 38B) specify the nucleotide.
[0256] Based on these findings, it was hypothesized that by
incorporating repeats with high specificity for G, and by relaxing
the requirement for T at -1, targeting capacity for engineered TAL
effector proteins can be enhanced. Experiments were initiated to
test novel and rare RVDs for more robust specificity for G than NN
displays, and to replace the RVD-analogous residues of the 0.sup.th
repeat with common RVDs.
[0257] Novel and Rare RVDs for Robust Specificity for G:
[0258] The modules disclosed above (see, e.g., Example 4) used four
particular RVDs (NI, HD, NN, and NG) to specify binding to the four
nucleotide bases (A, C, G, and T, respectively). Repeats containing
other RVDs also may be useful, and may have increased specificity
and/or affinity for the four bases as compared to NI, HD, NN, and
NG. Toward improving specificity for G, several repeats encoding
novel and rare RVDs were constructed. The rare RVDs NK, HN, and NA
associated with G, suggesting that N may be important as one or the
other of the residues (FIG. 1C). Thus, a broad set of derivatives
encoding repeats having the RVDs shown in Table 5 were constructed.
The left column lists RVDs having a polar amino acid (R, K, D, E,
Q, H, S, T, or Y) at position 12 and N at position 13. The right
columns list combinations of N in the first position with any of 17
other amino acids (G, L, V, R, K, D, E, Q, H, T, M, C, P, Y, W, or
F) in the second position of the RVD. To account for the
possibility of greater specificity without N, repeats also were
made with a polar amino acid (R, K, D, E, Q, H, S, T, or Y) at
position 12 and a gap (*) at position 13 (middle column).
[0259] Novel artificial RVDs are tested for function in a
quantitative reporter gene based assay for transcriptional
activation activity of TAL effectors, such as a GUS or dual
luciferase reporter based, Agrobacterium-mediated transient
expression assay in Nicotiana benthamiana, or in the lacZ reporter
based TALEN assay in Saccharomyces cerevisiae, described above
(see, e.g., Example 2) Repeat modules containing RVDs to be tested
are incorporated into a TAL effector or TALEN with measurable and
sub-saturation levels of activity, and the resulting proteins are
tested for differences in activity on a set of DNA targets with
integrated permutations of all four nucleotides at corresponding
positions. In particular, beginning with the PthXo1 variant(s)
minimally active in the in planta and yeast assays and responsive
to mismatches at three added repeats, TALENs containing each of the
novel and rare repeats (in homomeric threes) are tested in vivo
against targets with G at each of the corresponding positions. For
any that show increased activity, the assays are repeated with
targets permutated to the other nucleotides at those positions, to
ascertain specificity.
TABLE-US-00007 TABLE 5 RVDs to be tested.sup.a Polar + N Polar* N +
all RN R* NG NH KN K* NA NT DN D* NL NM EN E* NV NC QN Q* NR NP HN
H* NK NY SN S* ND NW TN T* NE NF YN Y* NQ .sup.aN*, NG, and NS nt
association frequencies are known. An asterisk represents a gap
corresponding to the 2.sup.nd position in the RVD (i.e., the
13.sup.th position of the consensus repeat sequence).
[0260] Common RVD Substitutions for the RVD-Analogous Position of
the 0.sup.th Repeat to Relax Specificity of Tat Position -1:
[0261] Secondary structure predictions and alignment of the
0.sup.th repeat and repeat consensus sequences suggested that
positions occupied by KR* (asterisk denotes a gap) in the 0.sup.th
repeat were analogous to the RVD and were therefore the residues
that specify the T at -1. Variants of PthXo1 with substitutions of
HD, NG, NI, and NN for KR and separately for R* were constructed in
the Tal1c "backbone" construct described above. Activities of these
variants are compared to the wild type effector in the in planta
and yeast assays using targets with corresponding nucleotides at
position -1, namely, C, T, A, and G, respectively. Additional
variants of PthXo1 are constructed that have S, the residue at
position 11 of the consensus repeat sequence, substituted for the K
at position 11 of the 0.sup.th repeat. And other variants are
constructed that have this substitution combined with a
substitution of K, the residue at position 16 of the consensus
repeat sequence, for the V at position 15 of the 0.sup.th repeat
(Table 6). A proximal TATA box for TAL effector activity may be
included. In addition, PthXo1 is useful for this experiment because
unlike AvrBs3, for which the T at -1 appears to be part of a TATA
box, the TATA box closest to the PthXo1 binding site is 46 bp
downstream and would not be perturbed by modifications at -1.
[0262] If the above modifications do not result in enhanced
targeting for G or increased ability to target sequences preceded
by nucleotides other than T, then a more comprehensive set of
artificial RVDs are tested for G specificity, and substitutions
other than the common RVDs are tested for the 0th repeat.
TABLE-US-00008 TABLE 6 0.sup.th repeat constructs to be made and
tested for specificity for targets with A, C, G, or T at the -1
position Native 0.sup.th repeat sequence Substitution Substitution
Substitution Substitution (specifies T at-1) specifying T
specifying A specifying C specifying G . . . KIA* . . . KIA*NGGGV .
. . . . . KIA*NIGGV . . . . . . KIA*HDGGV . . . . . . KIA*NNGGV . .
. KRGGV . . .(74).sup..dagger. (75) (76) (77) (78) . . . KIA* . . .
KIASNGGGV . . . . . . KIASNIGGV . . . . . . KIASHDGGV . . . . . .
KIASNNGGV . . . KRGGV . . .(79) (80) (81) (82) (83) . . . KIAKR* .
. . KIAKNGGGV . . . . . . KIAKNIGGV . . . . . . KIAKHDGGV . . . . .
. KIAKNNGGV . . . GGV . . .(84) (85) (86) (87) (88) . . . KIA* . .
. KIASNGGGK . . . . . . KIASNIGGK . . . . . . KIASHDGGK . . . . . .
KIASNNGGK . . . KRGGV . . .(89) (90) (91) (92) (93) Candidate and
substituted RVDs are bold. Other substitutions or modifications are
underlined. Asterisks denote a gap relative to the consensus repeat
sequence. .sup..dagger.SEQ ID NO:
Example 13
Novel Predicted Nucleotide Specific RVDs
[0263] It was observed that when the RVDs listed in Tables 1A and
1B were grouped by the second amino acid residue in the RVD (i.e.,
the 13.sup.th in the overall repeat), there was a near perfect
correlation of that amino acid with the nucleotide(s) specified by
the RVD, irrespective of the amino acid at the first position of
the RVD (Table 7). Thus, RVDs ending in a gap (denoted by an
asterisk) specify C or T, or T; RVDs ending in D specify C; RVDs
ending in G specify T; and RVDs ending in N specify G or A, or G.
It also was observed that amino acids at position 1 of the RVD were
either H, I, N, S, or Y. These observations suggested that RVD
specificity is determined by the residue in the second position,
independent of whether the residue at the first position is H, I,
N, S, or Y. Therefore, specificities were predicted for several
novel (i.e., yet unobserved) RVDs that combine residues observed at
the second position with residues H, I, S, N, or Y at the first
position. Thus, I*, S*, and Y* were predicted to specify C or T, or
T; ID, SD, and YD were predicted to specify C; SG was predicted to
specify T: and IN and YN were predicted to specify G or A, or G.
Also, although there was only one instance of K at the second
position, based on the observed specificity of NK, it was predicted
that HK, IK, SK, and YK specify G.
[0264] These novel RVDs are tested and compared to existing RVDs
for function and specificity in quantitative TAL effector and TALEN
activity assays as described in Examples 2 and 11.
TABLE-US-00009 TABLE 71 RVDs grouped and ordered by their second
residue ##STR00001## .sup.1An asterisk denotes a gap. RVD groups
with like specificities are boxed in thick lines.
Example 14
Custom TALENs Cleave Endogenous Targets in Animal Cells and
Introduce Mutations by Imprecise Non-Homologous End Joining
[0265] To test whether TALENs could be used for targeted
mutagenesis in animal cells, first, expression of TAL effectors
AvrBs3, PthXo1, and Tal1c was tested in human embryonic kidney
(HEK) 293T cells. The stop codon was removed from the AvrBs3,
PthXo1, and Tal1c encoding genes and the genes were subcloned into
mammalian expression vector pcDNA3.2/V5-DEST (Invitrogen, Carlsbad,
Calif.) in frame with the downstream sequence in that vector that
encodes the V5 epitope for protein immunodetection.
pcDNA3.2/V5-DEST places the TAL effector gene under the control of
the constitutive human cytomegalovirus (CMV) promoter. HEK 293T
cells were transfected using Lipofectamine 2000 (Invitrogen) with
the resulting plasmids individually, and after 24 hours, total
proteins were isolated from each transfected batch of cells and
subjected to polyacrylamide gel electrophoresis, western blotting
and immunolabeling using a mouse anti-V5 antibody. The labeled
proteins were detected with a goat anti-mouse antibody-horse radish
peroxidase conjugate using the SuperSignal Weat Pico
Chemiluminescent kit (ThermoScientific, Inc.). Equivalent loading
was confirmed by immunolabeling and detection of actin. Each TAL
effector protein was detectably expressed with no apparent
degradation (FIG. 39).
[0266] Next, a pair of TALENs were designed as described in Example
9 to target a sequence in the endogenous human HPRT gene, and named
HPRT-3254-17 and HPRT-3286-20r (FIG. 40A and FIG. 40B). Plasmids
pTALEN141 encoding HPRT-3254-17 and plasmid pTALEN142 encoding
HPRT-3286-20r were constructed using the Golden Gate cloning-based
method and reagents described in Example 8. The TALEN genes were
then subcloned into the mammalian expression vector pcDNA3.1(-)
(Invitrogen, Inc.), which places them under control of the
constitutive CMV promoter, yielding plasmids pTALEN141M and pTALEN
142M. HEK 293T cells were then transfected with both pTALEN141M and
pTALEN142M together and separately with pcDNA3.1(-) as a negative
control. After 72 hours, genomic DNA was isolated and digested with
restriction endonuclease Bpu10I. A Bpu10I site exists within the
spacer that separates the HPRT-3254-17 and HPRT-3286-20r binding
sites in HPRT (FIG. 41A). Following Bpu10I digestion, PCR was used
to amplify a 244 bp fragment spanning the TALEN-targeted site from
both the TALEN-treated and the control samples. The expected
fragment was amplified from both samples, indicating that Bpu10I
digestion of the genomic DNA had been incomplete. Subsequent
digestion of the PCR products with Bpu101, however, resulted in
complete cleavage of the product amplified from the control sample,
but incomplete cleavage of the product from the TALEN treated
sample (FIG. 41B). The presence of cleavage-resistant PCR product
in the TALEN-treated sample provides evidence that the endogenous
Bpu10I site was mutated in vivo as a result of imperfect repair by
non-homologous end joining of a TALEN-mediated double strand break
at the intended target in HPRT. Thus, TALENs can be used for
targeted mutagenesis in mammalian cells.
Other Embodiments
[0267] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
107134PRTArtificial Sequencesynthetic peptide 1Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser His Asp Gly Gly Lys1 5 10 15 Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30 His
Gly217DNAArtificial Sequencetarget sequence 2agaagaagag acccata
17318DNAArtificial Sequencetarget sequence 3atataaacct aaccatcc
18418DNAArtificial Sequencetarget sequence 4atataaacct gacccttt
18514DNAArtificial Sequencetarget sequence 5atataaacct ctct
14615DNAArtificial Sequencetarget sequence 6atataaacct aacca
15714DNAArtificial Sequencetarget sequence 7ataaacctaa ccat
14824DNAArtificial Sequencetarget sequence 8gcatctcccc ctactgtaca
ccac 24923DNAArtificial Sequencetarget sequence 9ataaaaggcc
ctcaccaacc cat 231022DNAArtificial Sequencetarget sequence
10ataatcccca aatcccctcc tc 221116DNAArtificial Sequencetarget
sequence 11ccccctcgct tccctt 16121164PRTXanthomonas euvesicatoria
12Met Asp Pro Ile Arg Ser Arg Thr Pro Ser Pro Ala Arg Glu Leu Leu1
5 10 15 Pro Gly Pro Gln Pro Asp Gly Val Gln Pro Thr Ala Asp Arg Gly
Val 20 25 30 Ser Pro Pro Ala Gly Gly Pro Leu Asp Gly Leu Pro Ala
Arg Arg Thr 35 40 45 Met Ser Arg Thr Arg Leu Pro Ser Pro Pro Ala
Pro Ser Pro Ala Phe 50 55 60 Ser Ala Gly Ser Phe Ser Asp Leu Leu
Arg Gln Phe Asp Pro Ser Leu65 70 75 80 Phe Asn Thr Ser Leu Phe Asp
Ser Leu Pro Pro Phe Gly Ala His His 85 90 95 Thr Glu Ala Ala Thr
Gly Glu Trp Asp Glu Val Gln Ser Gly Leu Arg 100 105 110 Ala Ala Asp
Ala Pro Pro Pro Thr Met Arg Val Ala Val Thr Ala Ala 115 120 125 Arg
Pro Pro Arg Ala Lys Pro Ala Pro Arg Arg Arg Ala Ala Gln Pro 130 135
140 Ser Asp Ala Ser Pro Ala Ala Gln Val Asp Leu Arg Thr Leu Gly
Tyr145 150 155 160 Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val
Arg Ser Thr Val 165 170 175 Ala Gln His His Glu Ala Leu Val Gly His
Gly Phe Thr His Ala His 180 185 190 Ile Val Ala Leu Ser Gln His Pro
Ala Ala Leu Gly Thr Val Ala Val 195 200 205 Lys Tyr Gln Asp Met Ile
Ala Ala Leu Pro Glu Ala Thr His Glu Ala 210 215 220 Ile Val Gly Val
Gly Lys Gln Trp Ser Gly Ala Arg Ala Leu Glu Ala225 230 235 240 Leu
Leu Thr Val Ala Gly Glu Leu Arg Gly Pro Pro Leu Gln Leu Asp 245 250
255 Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly Gly Val Thr Ala Val
260 265 270 Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro
Leu Asn 275 280 285 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
Asp Gly Gly Lys 290 295 300 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala305 310 315 320 His Gly Leu Thr Pro Gln Gln
Val Val Ala Ile Ala Ser Asn Gly Gly 325 330 335 Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 340 345 350 Gln Ala His
Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn 355 360 365 Ser
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 370 375
380 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile
Ala385 390 395 400 Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu 405 410 415 Pro Val Leu Cys Gln Ala His Gly Leu Thr
Pro Glu Gln Val Val Ala 420 425 430 Ile Ala Ser Asn Ile Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Ala 435 440 445 Leu Leu Pro Val Leu Cys
Gln Ala His Gly Leu Thr Pro Glu Gln Val 450 455 460 Val Ala Ile Ala
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val465 470 475 480 Gln
Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 485 490
495 Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
500 505 510 Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
Leu Thr 515 520 525 Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala 530 535 540 Leu Glu Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Ala His Gly545 550 555 560 Leu Thr Pro Glu Gln Val Val
Ala Ile Ala Ser His Asp Gly Gly Lys 565 570 575 Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 580 585 590 His Gly Leu
Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly 595 600 605 Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 610 615
620 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser
Asn625 630 635 640 Ser Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala
Leu Leu Pro Val 645 650 655 Leu Cys Gln Ala His Gly Leu Thr Pro Glu
Gln Val Val Ala Ile Ala 660 665 670 Ser Asn Ser Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu 675 680 685 Pro Val Leu Cys Gln Ala
His Gly Leu Thr Pro Glu Gln Val Val Ala 690 695 700 Ile Ala Ser His
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg705 710 715 720 Leu
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 725 730
735 Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
740 745 750 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
Pro Glu 755 760 765 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
Gln Ala Leu Glu 770 775 780 Thr Val Gln Arg Leu Leu Pro Val Leu Cys
Gln Ala His Gly Leu Thr785 790 795 800 Pro Gln Gln Val Val Ala Ile
Ala Ser Asn Gly Gly Gly Arg Pro Ala 805 810 815 Leu Glu Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 820 825 830 Leu Thr Pro
Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys 835 840 845 Gln
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 850 855
860 His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly
Gly865 870 875 880 Gly Arg Pro Ala Leu Glu Ser Ile Val Ala Gln Leu
Ser Arg Pro Asp 885 890 895 Pro Ala Leu Ala Ala Leu Thr Asn Asp His
Leu Val Ala Leu Ala Cys 900 905 910 Leu Gly Gly Arg Pro Ala Leu Asp
Ala Val Lys Lys Gly Leu Pro His 915 920 925 Ala Pro Ala Leu Ile Lys
Arg Thr Asn Arg Arg Ile Pro Glu Arg Thr 930 935 940 Ser His Arg Val
Ala Asp His Ala Gln Val Val Arg Val Leu Gly Phe945 950 955 960 Phe
Gln Cys His Ser His Pro Ala Gln Ala Phe Asp Asp Ala Met Thr 965 970
975 Gln Phe Gly Met Ser Arg His Gly Leu Leu Gln Leu Phe Arg Arg Val
980 985 990 Gly Val Thr Glu Leu Glu Ala Arg Ser Gly Thr Leu Pro Pro
Ala Ser 995 1000 1005 Gln Arg Trp Asp Arg Ile Leu Gln Ala Ser Gly
Met Lys Arg Ala Lys 1010 1015 1020 Pro Ser Pro Thr Ser Thr Gln Thr
Pro Asp Gln Ala Ser Leu His Ala1025 1030 1035 1040 Phe Ala Asp Ser
Leu Glu Arg Asp Leu Asp Ala Pro Ser Pro Met His 1045 1050 1055 Glu
Gly Asp Gln Thr Arg Ala Ser Ser Arg Lys Arg Ser Arg Ser Asp 1060
1065 1070 Arg Ala Val Thr Gly Pro Ser Ala Gln Gln Ser Phe Glu Val
Arg Val 1075 1080 1085 Pro Glu Gln Arg Asp Ala Leu His Leu Pro Leu
Ser Trp Arg Val Lys 1090 1095 1100 Arg Pro Arg Thr Ser Ile Gly Gly
Gly Leu Pro Asp Pro Gly Thr Pro1105 1110 1115 1120 Thr Ala Ala Asp
Leu Ala Ala Ser Ser Thr Val Met Arg Glu Gln Asp 1125 1130 1135 Glu
Asp Pro Phe Ala Gly Ala Ala Asp Asp Phe Pro Ala Phe Asn Glu 1140
1145 1150 Glu Glu Leu Ala Trp Leu Met Glu Leu Leu Pro Gln 1155 1160
134366DNAXanthomonas euvesicatoria 13gaattcaagg tgtcaaaaag
cgataggcgg aattatagat gtacttgtat gaacttatca 60acgccagttt agtgaacggg
ttcgacaaag cgaaaccaac acccaggcgc gaaagccttg 120cgccgcaatg
ctttccggca atgtgaccca gggcattgac cgaaacggcg taggaatttc
180ggaacacgac ggtaggggaa tgctctaccg cccggctacg caaaagcact
ctcgcctgcc 240agacgcgcca ctgcgtggaa ttggccgtta tgtccgctgg
cggcctcgcc gccgtagtgc 300ttgcagcgca gccttgaatg atcgaacatc
aaacatcact gtttgatagg tcgatcatga 360catcgcccat ttcgagggtc
ggcagggatt cgtgtaaaaa acagccaaaa gtgagctaac 420tcgctgtcag
cacagaaatt tttcacaacc ttctgccgat cctccatgcg ggtccgtgat
480cgccttcatg tctgcgcctc accctggtcg tcgagggttg ccaggatcac
ccgaagttgt 540gtactgccat gcggcctcgg aagctatgta ggaaccacag
accgctagtc tggaggcgac 600catgtaaaga ggtatgcctg atggatccca
ttcgttcgcg cacaccaagt cctgcccgcg 660agcttctgcc cggaccccaa
cccgatgggg ttcagccgac tgcagatcgt ggggtgtctc 720cgcctgccgg
cggccccctg gatggcttgc ccgctcggcg gacgatgtcc cggacccggc
780tgccatctcc ccctgccccc tcacctgcgt tctcggcggg cagcttcagt
gacctgttac 840gtcagttcga tccgtcactt tttaatacat cgctttttga
ttcattgcct cccttcggcg 900ctcaccatac agaggctgcc acaggcgagt
gggatgaggt gcaatcgggt ctgcgggcag 960ccgacgcccc cccacccacc
atgcgcgtgg ctgtcactgc cgcgcggccg ccgcgcgcca 1020agccggcgcc
gcgacgacgt gctgcgcaac cctccgacgc ttcgccggcc gcgcaggtgg
1080atctacgcac gctcggctac agccagcagc aacaggagaa gatcaaaccg
aaggttcgtt 1140cgacagtggc gcagcaccac gaggcactgg tcggccatgg
gtttacacac gcgcacatcg 1200ttgcgctcag ccaacacccg gcagcgttag
ggaccgtcgc tgtcaagtat caggacatga 1260tcgcagcgtt gccagaggcg
acacacgaag cgatcgttgg cgtcggcaaa cagtggtccg 1320gcgcacgcgc
tctggaggcc ttgctcacgg tggcgggaga gttgagaggt ccaccgttac
1380agttggacac aggccaactt ctcaagattg caaaacgtgg cggcgtgacc
gcagtggagg 1440cagtgcatgc atggcgcaat gcactgacgg gtgcccccct
gaacctgacc ccggagcagg 1500tggtggccat cgccagccac gatggcggca
agcaggcgct ggagacggtg cagcggctgt 1560tgccggtgct gtgccaggcc
catggcctga ccccgcagca ggtggtggcc atcgccagca 1620atggcggtgg
caagcaggcg ctggagacgg tgcagcggct gttgccggtg ctgtgccagg
1680cccatggcct gaccccgcag caggtggtgg ccatcgccag caatagcggt
ggcaagcagg 1740cgctggagac ggtgcagcgg ctgttgccgg tgctgtgcca
ggcccatggc ctgaccccgg 1800agcaggtggt ggccatcgcc agcaatggcg
gtggcaagca ggcgctggag acggtgcagc 1860ggctgttgcc ggtgctgtgc
caggcccatg gcctgacccc ggagcaggtg gtggccatcg 1920ccagcaatat
tggtggcaag caggcgctgg agacggtgca ggcgctgttg ccggtgctgt
1980gccaggccca tggcctgacc ccggagcagg tggtggccat cgccagcaat
attggtggca 2040agcaggcgct ggagacggtg caggcgctgt tgccggtgct
gtgccaggcc catggcctga 2100ccccggagca ggtggtggcc atcgccagca
atattggtgg caagcaggcg ctggagacgg 2160tgcaggcgct gttgccggtg
ctgtgccagg cccatggcct gaccccggag caggtggtgg 2220ccatcgccag
ccacgatggc ggcaagcagg cgctggagac ggtgcagcgg ctgttgccgg
2280tgctgtgcca ggcccatggc ctgaccccgg agcaggtggt ggccatcgcc
agccacgatg 2340gcggcaagca ggcgctggag acggtgcagc ggctgttgcc
ggtgctgtgc caggcccatg 2400gcctgacccc gcagcaggtg gtggccatcg
ccagcaatgg cggtggcaag caggcgctgg 2460agacggtgca gcggctgttg
ccggtgctgt gccaggccca tggcctgacc ccggagcagg 2520tggtggccat
cgccagcaat agcggtggca agcaggcgct ggagacggtg caggcgctgt
2580tgccggtgct gtgccaggcc catggcctga ccccggagca ggtggtggcc
atcgccagca 2640atagcggtgg caagcaggcg ctggagacgg tgcagcggct
gttgccggtg ctgtgccagg 2700cccatggcct gaccccggag caggtggtgg
ccatcgccag ccacgatggc ggcaagcagg 2760cgctggagac ggtgcagcgg
ctgttgccgg tgctgtgcca ggcccatggc ctgaccccgg 2820agcaggtggt
ggccatcgcc agccacgatg gcggcaagca ggcgctggag acggtgcagc
2880ggctgttgcc ggtgctgtgc caggcccatg gcctgacccc ggagcaggtg
gtggccatcg 2940ccagccacga tggcggcaag caggcgctgg agacggtgca
gcggctgttg ccggtgctgt 3000gccaggccca tggcctgacc ccgcagcagg
tggtggccat cgccagcaat ggcggcggca 3060ggccggcgct ggagacggtg
cagcggctgt tgccggtgct gtgccaggcc catggcctga 3120ccccggagca
ggtggtggcc atcgccagcc acgatggcgg caagcaggcg ctggagacgg
3180tgcagcggct gttgccggtg ctgtgccagg cccatggcct gaccccgcag
caggtggtgg 3240ccatcgccag caatggcggc ggcaggccgg cgctggagag
cattgttgcc cagttatctc 3300gccctgatcc ggcgttggcc gcgttgacca
acgaccacct cgtcgccttg gcctgcctcg 3360gcggacgtcc tgcgctggat
gcagtgaaaa agggattgcc gcacgcgccg gccttgatca 3420aaagaaccaa
tcgccgtatt cccgaacgca catcccatcg cgttgccgac cacgcgcaag
3480tggttcgcgt gctgggtttt ttccagtgcc actcccaccc agcgcaagca
tttgatgacg 3540ccatgacgca gttcgggatg agcaggcacg ggttgttaca
gctctttcgc agagtgggcg 3600tcaccgaact cgaagcccgc agtggaacgc
tccccccagc ctcgcagcgt tgggaccgta 3660tcctccaggc atcagggatg
aaaagggcca aaccgtcccc tacttcaact caaacgccgg 3720atcaggcgtc
tttgcatgca ttcgccgatt cgctggagcg tgaccttgat gcgcctagcc
3780caatgcacga gggagatcag acgcgggcaa gcagccgtaa acggtcccga
tcggatcgtg 3840ctgtcaccgg tccctccgca cagcaatcgt tcgaggtgcg
cgttcccgaa cagcgcgatg 3900cgctgcattt gcccctcagt tggagggtaa
aacgcccgcg taccagtatc gggggcggcc 3960tcccggatcc tggtacgccc
acggctgccg acctggcagc gtccagcacc gtgatgcggg 4020aacaagatga
ggaccccttc gcaggggcag cggatgattt cccggcattc aacgaagagg
4080agctcgcatg gttgatggag ctattgcctc agtgaggctc agtcggtgac
tacctgagcg 4140tcggcaggga ttggtgtaag taacctttac tgacagcgag
ttagcccact tttggctgtt 4200ttttacacaa atccctgcct cccctctggt
tgcaccacac ccgtacacca agcgcggcgg 4260cgaagcaggc accgagtggt
tccgctgcgg tgttgcgttc cctaaccagg gcggtggcta 4320tacgctcaag
ctgcgcaccg tcccggtggc gatcgacgac gaaatg 43661456DNAArtificial
Sequencetarget sequence 14tatataaacc taaccatcct cacaacttca
agttatcgga tggttaggtt tatata 561556DNAArtificial Sequencetarget
sequence 15tatataaacc taaccatccg ataacttgaa gttgtgagga tggttaggtt
tatata 56161373PRTXanthomonas euvesicatoria 16Met Asp Pro Ile Arg
Ser Arg Thr Pro Ser Pro Ala Arg Glu Leu Leu1 5 10 15 Pro Gly Pro
Gln Pro Asp Arg Val Gln Pro Thr Ala Asp Arg Gly Gly 20 25 30 Ala
Pro Pro Ala Gly Gly Pro Leu Asp Gly Leu Pro Ala Arg Arg Thr 35 40
45 Met Ser Arg Thr Arg Leu Pro Ser Pro Pro Ala Pro Ser Pro Ala Phe
50 55 60 Ser Ala Gly Ser Phe Ser Asp Leu Leu Arg Gln Phe Asp Pro
Ser Leu65 70 75 80 Leu Asp Thr Ser Leu Leu Asp Ser Met Pro Ala Val
Gly Thr Pro His 85 90 95 Thr Ala Ala Ala Pro Ala Glu Cys Asp Glu
Val Gln Ser Gly Leu Arg 100 105 110 Ala Ala Asp Asp Pro Pro Pro Thr
Val Arg Val Ala Val Thr Ala Ala 115 120 125 Arg Pro Pro Arg Ala Lys
Pro Ala Pro Arg Arg Arg Ala Ala Gln Pro 130 135 140 Ser Asp Ala Ser
Pro Ala Ala Gln Val Asp Leu Arg Thr Leu Gly Tyr145 150 155 160 Ser
Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val Gly Ser Thr Val 165 170
175 Ala Gln His His Glu Ala Leu Val Gly His Gly Phe Thr His Ala His
180 185 190 Ile Val Ala Leu Ser Arg His Pro Ala Ala Leu Gly Thr Val
Ala Val 195 200 205 Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu Ala
Thr His Glu Asp 210 215 220 Ile Val Gly Val Gly Lys Gln Trp Ser Gly
Ala Arg Ala Leu Glu Ala225 230 235 240 Leu Leu Thr Val Ala
Gly Glu Leu Arg Gly Pro Pro Leu Gln Leu Asp 245 250 255 Thr Gly Gln
Leu Val Lys Ile Ala Lys Arg Gly Gly Val Thr Ala Val 260 265 270 Glu
Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly Ala Pro Leu Asn 275 280
285 Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
290 295 300 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
Gln Ala305 310 315 320 His Gly Leu Thr Pro Ala Gln Val Val Ala Ile
Ala Ser His Asp Gly 325 330 335 Gly Lys Gln Ala Leu Glu Thr Met Gln
Arg Leu Leu Pro Val Leu Cys 340 345 350 Gln Ala His Gly Leu Pro Pro
Asp Gln Val Val Ala Ile Ala Ser Asn 355 360 365 Ile Gly Gly Lys Gln
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 370 375 380 Leu Cys Gln
Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala385 390 395 400
Ser His Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 405
410 415 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val
Ala 420 425 430 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr
Val Gln Arg 435 440 445 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
Thr Pro Asp Gln Val 450 455 460 Val Ala Ile Ala Ser Asn Gly Gly Gly
Lys Gln Ala Leu Glu Thr Val465 470 475 480 Gln Arg Leu Leu Pro Val
Leu Cys Gln Ala His Gly Leu Thr Pro Asp 485 490 495 Gln Val Val Ala
Ile Ala Ser Asn Gly Gly Lys Gln Ala Leu Glu Thr 500 505 510 Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro 515 520 525
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu 530
535 540 Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Thr His Gly
Leu545 550 555 560 Thr Pro Ala Gln Val Val Ala Ile Ala Ser His Asp
Gly Gly Lys Gln 565 570 575 Ala Leu Glu Thr Val Gln Gln Leu Leu Pro
Val Leu Cys Gln Ala His 580 585 590 Gly Leu Thr Pro Asp Gln Val Val
Ala Ile Ala Ser Asn Ile Gly Gly 595 600 605 Lys Gln Ala Leu Ala Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln 610 615 620 Ala His Gly Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly625 630 635 640 Gly
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 645 650
655 Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
660 665 670 Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
Leu Pro 675 680 685 Val Leu Cys Gln Ala His Gly Leu Thr Gln Val Gln
Val Val Ala Ile 690 695 700 Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu705 710 715 720 Leu Pro Val Leu Cys Gln Ala
His Gly Leu Thr Pro Ala Gln Val Val 725 730 735 Ala Ile Ala Ser His
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln 740 745 750 Arg Leu Leu
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln 755 760 765 Val
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr 770 775
780 Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
Gln785 790 795 800 Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
Lys Gln Ala Leu 805 810 815 Glu Thr Val Gln Arg Leu Leu Pro Val Leu
Cys Gln Ala His Gly Leu 820 825 830 Thr Pro Asp Gln Val Val Ala Ile
Ala Ser Asn Gly Gly Gly Lys Gln 835 840 845 Ala Leu Glu Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Ala His 850 855 860 Gly Leu Thr Pro
Ala Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly865 870 875 880 Lys
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln 885 890
895 Asp His Gly Leu Thr Leu Ala Gln Val Val Ala Ile Ala Ser Asn Ile
900 905 910 Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
Val Leu 915 920 925 Cys Gln Ala His Gly Leu Thr Gln Asp Gln Val Val
Ala Ile Ala Ser 930 935 940 Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro945 950 955 960 Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp Gln Val Val Ala Ile 965 970 975 Ala Ser Asn Ile Gly
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu 980 985 990 Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr Leu Asp Gln Val Val 995 1000 1005
Ala Ile Ala Ser Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
1010 1015 1020 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
Asp Gln Val1025 1030 1035 1040 Val Ala Ile Ala Ser Asn Ser Gly Gly
Lys Gln Ala Leu Glu Thr Val 1045 1050 1055 Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr Pro Asn 1060 1065 1070 Gln Val Val
Ala Ile Ala Ser Asn Gly Gly Lys Gln Ala Leu Glu Ser 1075 1080 1085
Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr
1090 1095 1100 Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg
Pro Ala Met1105 1110 1115 1120 Asp Ala Val Lys Lys Gly Leu Pro His
Ala Pro Glu Leu Ile Arg Arg 1125 1130 1135 Val Asn Arg Arg Ile Gly
Glu Arg Thr Ser His Arg Val Ala Asp Tyr 1140 1145 1150 Ala Gln Val
Val Arg Val Leu Glu Phe Phe Gln Cys His Ser His Pro 1155 1160 1165
Ala Tyr Ala Phe Asp Glu Ala Met Thr Gln Phe Gly Met Ser Arg Asn
1170 1175 1180 Gly Leu Val Gln Leu Phe Arg Arg Val Gly Val Thr Glu
Leu Glu Ala1185 1190 1195 1200 Arg Gly Gly Thr Leu Pro Pro Ala Ser
Gln Arg Trp Asp Arg Ile Leu 1205 1210 1215 Gln Ala Ser Gly Met Lys
Arg Ala Lys Pro Ser Pro Thr Ser Ala Gln 1220 1225 1230 Thr Pro Asp
Gln Ala Ser Leu His Ala Phe Ala Asp Ser Leu Glu Arg 1235 1240 1245
Asp Leu Asp Ala Pro Ser Pro Met His Glu Gly Asp Gln Thr Gly Ala
1250 1255 1260 Ser Ser Arg Lys Arg Ser Arg Ser Asp Arg Ala Val Thr
Gly Pro Ser1265 1270 1275 1280 Ala Gln His Ser Phe Glu Val Arg Val
Pro Glu Gln Arg Asp Ala Leu 1285 1290 1295 His Leu Pro Leu Ser Trp
Arg Val Lys Arg Pro Arg Thr Arg Ile Gly 1300 1305 1310 Gly Gly Leu
Pro Asp Pro Gly Thr Pro Ile Ala Ala Asp Leu Ala Ala 1315 1320 1325
Ser Ser Thr Val Met Trp Glu Gln Asp Ala Ala Pro Phe Ala Gly Ala
1330 1335 1340 Ala Asp Asp Phe Pro Ala Phe Asn Glu Glu Glu Leu Ala
Trp Leu Met1345 1350 1355 1360 Glu Leu Leu Pro Gln Ser Gly Ser Val
Gly Gly Thr Ile 1365 1370 17102DNAArtificial Sequencesynthetic
oligonucleotide 17ctgaccccgg cacaggtggt ggccatcgcc agcmaygayg
gcggcaagca ggcgctggag 60acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg
gc 1021834PRTArtificial Sequencesynthetic peptide 18Leu Thr Pro Ala
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys1 5 10 15 Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 20 25 30
His Gly1963DNAArtificial Sequencesynthetic oligonucleotide
19ctgaccccgg cacaggtggt ggccatcgcc agcmaygayg gcggcaagca ggcgctcgag
60agc 632021PRTArtificial Sequencesynthetic peptide 20Leu Thr Pro
Ala Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys1 5 10 15 Gln
Ala Leu Glu Ser 20 2112DNAXanthomonas oryzae 21gcgctggaga gc
12224PRTXanthomonas oryzae 22Ala Leu Glu Ser1 2312DNAArtificial
Sequencesynthetic oligonucleotide 23gcgctcgagt cc
1224102DNAArtificial Sequencesynthetic oligonucleotide 24tcgagacggt
gcagcggctg ttgccggtgc tgtgccagga ccatggcctg accccggacc 60aagtggtggc
catcgccagc aacattggcg gcaagcaagc gc 10225102DNAArtificial
Sequencesynthetic oligonucleotide 25tcgagcgctt gcttgccgcc
aatgttgctg gcgatggcca ccacttggtc cggggtcagg 60ccatggtcct ggcacagcac
cggcaacagc cgctgcaccg tc 1022635PRTArtificial Sequencesynthetic
peptide 26Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly Leu1 5 10 15 Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile
Gly Gly Lys Gln 20 25 30 Ala Leu Glu 35 27102DNAArtificial
Sequencesynthetic oligonucleotide 27tcgaaacggt gcagcggctg
ttgccggtgc tgtgccagga ccatggcctg accccggacc 60aagtggtggc tatcgccagc
aacattggcg gcaagcaagc gc 10228102DNAArtificial Sequencesynthetic
oligonucleotide 28tcgagcgctt gcttgccgcc aatgttgctg gcgatagcca
ccacttggtc cggggtcagg 60ccatggtcct ggcacagcac cggcaacagc cgctgcaccg
tt 1022918DNAArtificial Sequencetarget sequence 29atcaagattc
tcttcact 183015DNAArtificial Sequencetarget sequence 30cccagaagta
aacat 1531598PRTXanthomonas oryzae 31Leu Thr Pro Glu Gln Val Val
Ala Ile Ala Ser His Asp Gly Gly Lys1 5 10 15 Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30 His Gly Leu
Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly 35 40 45 Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 50 55
60 Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser
Asn65 70 75 80 Ser Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
Leu Pro Val 85 90 95 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln
Val Val Ala Ile Ala 100 105 110 Ser Asn Gly Gly Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu 115 120 125 Pro Val Leu Cys Gln Ala His
Gly Leu Thr Pro Glu Gln Val Val Ala 130 135 140 Ile Ala Ser Asn Ile
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala145 150 155 160 Leu Leu
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 165 170 175
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val 180
185 190 Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
Glu 195 200 205 Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln
Ala Leu Glu 210 215 220 Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln
Ala His Gly Leu Thr225 230 235 240 Pro Glu Gln Val Val Ala Ile Ala
Ser His Asp Gly Gly Lys Gln Ala 245 250 255 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Ala His Gly 260 265 270 Leu Thr Pro Glu
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys 275 280 285 Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 290 295 300
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly305
310 315 320 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
Leu Cys 325 330 335 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
Ile Ala Ser Asn 340 345 350 Ser Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Ala Leu Leu Pro Val 355 360 365 Leu Cys Gln Ala His Gly Leu Thr
Pro Glu Gln Val Val Ala Ile Ala 370 375 380 Ser Asn Ser Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu385 390 395 400 Pro Val Leu
Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 405 410 415 Ile
Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 420 425
430 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
435 440 445 Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
Thr Val 450 455 460 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
Leu Thr Pro Glu465 470 475 480 Gln Val Val Ala Ile Ala Ser His Asp
Gly Gly Lys Gln Ala Leu Glu 485 490 495 Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln Ala His Gly Leu Thr 500 505 510 Pro Gln Gln Val Val
Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala 515 520 525 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 530 535 540 Leu
Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys545 550
555 560 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Ala 565 570 575 His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser
Asn Gly Gly 580 585 590 Gly Arg Pro Ala Leu Glu 595
324122DNAXanthomonas oryzae 32atggatccca ttcgttcgcg cacgccaagt
cctgcccgcg agcttctgcc cggaccccaa 60ccggataggg ttcagccgac tgcagatcgg
gggggggctc cgcctgctgg cggccccctg 120gatggcttgc ccgctcggcg
gacgatgtcc cggacccggc tgccatctcc ccctgcgccc 180tcgcctgcgt
tctcggcggg cagcttcagc gatctgctcc gtcagttcga tccgtcgctt
240cttgatacat cgcttcttga ttcgatgcct gccgtcggca cgccgcatac
agcggctgcc 300ccagcagagt gcgatgaggt gcaatcgggt ctgcgtgcag
ccgatgaccc gccacccacc 360gtgcgtgtcg ctgtcactgc cgcgcggccg
ccgcgcgcca agccggcccc gcgacggcgt 420gcggcgcaac cctccgacgc
ttcgccggcc gcgcaggtgg atctacgcac gctcggctac 480agtcagcagc
agcaagagaa gatcaaaccg aaggtgggtt cgacagtggc gcagcaccac
540gaggcactgg tgggccatgg gtttacacac gcgcacatcg ttgcgctcag
ccgacacccg 600gcagcgttag ggaccgtcgc tgtcaagtat caggacatga
tcgcggcgtt accagaggcg 660acacacgaag acatcgttgg tgtcggcaaa
cagtggtccg gcgcacgcgc cctggaggcc 720ttgctcacgg tggcgggaga
gttgagaggt ccaccgttac agttggacac aggccaactt 780gtcaagattg
caaaacgtgg cggcgtgacc gcagtggagg cagtgcatgc atcgcgcaat
840gcactgacgg gtgcccccct gaacctgacc ccggcacagg tggtggccat
cgccagcaat 900aacggtggca agcaggcgct ggagacggtg cagcggctgt
tgccggtgct gtgccaggcc 960catggcctga ccccggcgca ggtggtggcc
atcgccagcc acgatggcgg caagcaggca 1020ctggagacga tgcagcggct
gttgccggtg ctgtgccagg cccatggcct gcccccggac 1080caggtggtgg
ccatcgccag caatattggc ggcaagcagg cgctggagac ggtgcagcgg
1140ctgttgccgg tgctctgcca ggcccatggc ctgaccccgg accaggtggt
ggccatcgcc 1200agccatggcg gcggcaagca ggcgctggag acggtgcagc
ggctgttgcc ggtgctctgc 1260caggcccatg gcctgacccc ggaccaggtg
gtggccatcg ccagccacga tggcggcaag 1320caggcgctgg agacggtgca
gcggctgttg ccggtgctgt gccaggccca tggcctgacc 1380ccggaccagg
tggtggccat cgccagcaat ggcggcggca agcaggcgct ggagacggtg
1440cagcggctgt tgccggtgct gtgccaggcc catggtctga ccccggacca
ggtggtggcc 1500atcgccagca atggcggcaa gcaggcgctg gagacggtgc
agcggctgtt gccggtgctg 1560tgccaggccc atggcctgac cccggaccag
gtggtggcca tcgccagcca cgatggcggc
1620aagcaggcgc tggagacggt gcagcggctg ttgccggtgc tgtgccagac
ccatggtctg 1680accccggcgc aggtggtggc catcgccagc cacgatggcg
gcaagcaggc gctggagacg 1740gtgcagcagc tgttgccggt gctgtgccag
gcccatggcc tgaccccgga ccaggtggtg 1800gccatcgcca gcaatattgg
cggcaagcag gcgctagcga cggtgcagcg gctgttgccg 1860gtgctgtgcc
aagcccatgg cctgaccccg gaccaggtgg tggccatcgc cagcaatggc
1920ggcggcaagc aggcgctgga gacggtgcag cggctgttgc cggtgctgtg
ccaggcccat 1980ggcctgaccc cggaccaggt ggtggccatc gccagcaatg
gcggcggcaa gcaggcgctg 2040gagacggtgc agcggctgtt gccggtgctg
tgccaggccc atggtctgac ccaggtgcag 2100gtggtggcca tcgccagcaa
tattggcggc aagcaggcgc tggagacggt gcagcggctg 2160ttgccggtgc
tgtgccaggc ccatggcctg accccggcgc aggtggtggc catcgccagc
2220cacgatggcg gcaagcaggc gctggagacg gtgcagcggc tgttgccggt
gctgtgccag 2280gcccatggcc tgaccccgga ccaagtggtg gccatcgcca
gcaatggcgg cggcaagcag 2340gcgctggaga cggtgcagcg gctgttgccg
gtgctgtgcc aggcccatgg cctgacccag 2400gagcaggtgg tggccatcgc
cagcaataac ggcggcaagc aggcgctgga gacggtgcag 2460cggctgttgc
cggtgctgtg ccaggcccat ggcctgaccc cggaccaggt ggtggccatc
2520gccagcaatg gcggcggcaa gcaggcgctg gagacggtgc agcggctgtt
gccggtgctg 2580tgccaggccc atggtctgac cccggcgcag gtggtggcca
tcgccagcaa tattggcggc 2640aagcaggcgc tggagacggt gcagcggctg
ttgccggtgc tgtgccagga ccatggcctg 2700accctggcgc aggtggtggc
catcgccagc aatattggcg gcaagcaggc gctggagacg 2760gtgcagcggc
tgttgccggt gctgtgccag gcacatggcc tgacccagga ccaggtggtg
2820gccatcgcca gcaatattgg cggcaagcag gcgctggaga cggtgcagcg
gctgttgccg 2880gtgctgtgcc aggaccatgg cctgaccccg gaccaggtcg
tggccatcgc cagcaatatt 2940ggcggcaagc aggcgctgga gacggtgcag
cggctgttgc cggtgctgtg ccaggaccat 3000ggcctgaccc tggaccaggt
ggtggccatc gccagcaatg gcggcaagca ggcgctggag 3060acggtgcagc
ggctgttgcc ggtgctgtgc caggaccatg gactgacccc ggaccaggtc
3120gtggccatcg ccagcaatag tggcggcaag caggcgctgg agacggtgca
gcggctgttg 3180ccggtgctgt gccaggacca tggcctgacc ccgaaccagg
tggtggccat cgccagcaat 3240ggcggcaagc aggcgctgga gagcattgtt
gcccagttat ctcgccctga tccggcgttg 3300gccgcgttga ccaacgacca
cctcgtcgcc ttggcctgcc tcggcggacg tcctgccatg 3360gatgcagtga
aaaagggatt gccgcacgcg ccggaattga tcagaagagt caatcgccgt
3420attggcgaac gcacgtccca tcgcgttgcc gactacgcgc aagtggttcg
cgtgctggag 3480tttttccagt gccactccca cccagcgtac gcatttgatg
aggccatgac gcagttcggg 3540atgagcagga acgggttggt acagctcttt
cgcagagtgg gcgtcaccga actcgaagcc 3600cgcggtggaa cgctcccccc
agcctcgcag cgttgggacc gtatcctcca ggcatcaggg 3660atgaaaaggg
ccaaaccgtc ccctacttca gctcaaacac cggatcaggc gtctttgcat
3720gcattcgccg attcgctgga gcgtgacctt gatgcgccta gcccaatgca
cgagggagat 3780cagacagggg caagcagccg taaacggtcc cgatcggatc
gtgctgtcac cggcccctcc 3840gcacagcact ctttcgaggt gcgcgttccc
gaacagcgcg atgcgctgca tttgcccctc 3900agctggaggg taaaacgccc
gcgtaccagg atcgggggcg gcctcccgga tcctggtacg 3960cccatcgctg
ccgacctggc agcgtccagc accgtgatgt gggaacaaga tgcggccccc
4020ttcgcagggg cagcggatga tttcccggca ttcaacgaag aggagctcgc
atggttgatg 4080gagctattgc ctcagtcagg ctcagtcgga gggacgatct ga
4122331341PRTArtificial Sequencesynthetic peptide 33Met Ala Ser Ser
Pro Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser
Gly Trp Ser Arg Met His Ala Asp Pro Ile Arg Ser Arg Thr 20 25 30
Pro Ser Pro Ala Arg Glu Leu Leu Pro Gly Pro Gln Pro Asp Gly Val 35
40 45 Gln Pro Thr Ala Asp Arg Gly Val Ser Pro Pro Ala Gly Gly Pro
Leu 50 55 60 Asp Gly Leu Pro Ala Arg Arg Thr Met Ser Arg Thr Arg
Leu Pro Ser65 70 75 80 Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly
Ser Phe Ser Asp Leu 85 90 95 Leu Arg Gln Phe Asp Pro Ser Leu Phe
Asn Thr Ser Leu Phe Asp Ser 100 105 110 Leu Pro Pro Phe Gly Ala His
His Thr Glu Ala Ala Thr Gly Glu Trp 115 120 125 Asp Glu Val Gln Ser
Gly Leu Arg Ala Ala Asp Ala Pro Pro Pro Thr 130 135 140 Met Arg Val
Ala Val Thr Ala Ala Arg Pro Pro Arg Ala Lys Pro Ala145 150 155 160
Pro Arg Arg Arg Ala Ala Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165
170 175 Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys
Ile 180 185 190 Lys Pro Lys Val Arg Ser Thr Val Ala Gln His His Glu
Ala Leu Val 195 200 205 Gly His Gly Phe Thr His Ala His Ile Val Ala
Leu Ser Gln His Pro 210 215 220 Ala Ala Leu Gly Thr Val Ala Val Lys
Tyr Gln Asp Met Ile Ala Ala225 230 235 240 Leu Pro Glu Ala Thr His
Glu Ala Ile Val Gly Val Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg
Ala Leu Glu Ala Leu Leu Thr Val Ala Gly Glu Leu 260 265 270 Arg Gly
Pro Pro Leu Gln Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala 275 280 285
Lys Arg Gly Gly Val Thr Ala Val Glu Ala Val His Ala Trp Arg Asn 290
295 300 Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr Pro Glu Gln Val Val
Ala305 310 315 320 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg 325 330 335 Leu Leu Pro Val Leu Cys Gln Ala His Gly
Leu Thr Pro Gln Gln Val 340 345 350 Val Ala Ile Ala Ser Asn Gly Gly
Gly Lys Gln Ala Leu Glu Thr Val 355 360 365 Gln Arg Leu Leu Pro Val
Leu Cys Gln Ala His Gly Leu Thr Pro Gln 370 375 380 Gln Val Val Ala
Ile Ala Ser Asn Ser Gly Gly Lys Gln Ala Leu Glu385 390 395 400 Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 405 410
415 Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala
420 425 430 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 435 440 445 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
Ile Gly Gly Lys 450 455 460 Gln Ala Leu Glu Thr Val Gln Ala Leu Leu
Pro Val Leu Cys Gln Ala465 470 475 480 His Gly Leu Thr Pro Glu Gln
Val Val Ala Ile Ala Ser Asn Ile Gly 485 490 495 Gly Lys Gln Ala Leu
Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys 500 505 510 Gln Ala His
Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn 515 520 525 Ile
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val 530 535
540 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile
Ala545 550 555 560 Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln Ala His Gly Leu Thr
Pro Glu Gln Val Val Ala 580 585 590 Ile Ala Ser His Asp Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys
Gln Ala His Gly Leu Thr Pro Gln Gln Val 610 615 620 Val Ala Ile Ala
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val625 630 635 640 Gln
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 645 650
655 Gln Val Val Ala Ile Ala Ser Asn Ser Gly Gly Lys Gln Ala Leu Glu
660 665 670 Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
Leu Thr 675 680 685 Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ser Gly
Gly Lys Gln Ala 690 695 700 Leu Glu Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Ala His Gly705 710 715 720 Leu Thr Pro Glu Gln Val Val
Ala Ile Ala Ser His Asp Gly Gly Lys 725 730 735 Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 740 745 750 His Gly Leu
Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly 755 760 765 Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 770 775
780 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser
His785 790 795 800 Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val 805 810 815 Leu Cys Gln Ala His Gly Leu Thr Pro Gln
Gln Val Val Ala Ile Ala 820 825 830 Ser Asn Gly Gly Gly Arg Pro Ala
Leu Glu Thr Val Gln Arg Leu Leu 835 840 845 Pro Val Leu Cys Gln Ala
His Gly Leu Thr Pro Glu Gln Val Val Ala 850 855 860 Ile Ala Ser His
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg865 870 875 880 Leu
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val 885 890
895 Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala Leu Glu Ser Ile
900 905 910 Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu
Thr Asn 915 920 925 Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg
Pro Ala Leu Asp 930 935 940 Ala Val Lys Lys Gly Leu Pro His Ala Pro
Ala Leu Ile Lys Arg Thr945 950 955 960 Asn Arg Arg Ile Pro Glu Arg
Thr Ser His Arg Val Ala Asp His Ala 965 970 975 Gln Val Val Arg Val
Leu Gly Phe Phe Gln Cys His Ser His Pro Ala 980 985 990 Gln Ala Phe
Asp Asp Ala Met Thr Gln Phe Gly Met Ser Arg His Gly 995 1000 1005
Leu Leu Gln Leu Phe Arg Arg Val Gly Val Thr Glu Leu Glu Ala Arg
1010 1015 1020 Ser Gly Thr Leu Pro Pro Ala Ser Gln Arg Trp Asp Arg
Ile Leu Gln1025 1030 1035 1040 Ala Ser Gly Met Lys Arg Ala Lys Pro
Ser Pro Thr Ser Thr Gln Thr 1045 1050 1055 Pro Asp Gln Ala Ser Leu
His Ala Phe Ala Asp Ser Leu Glu Arg Asp 1060 1065 1070 Leu Asp Ala
Pro Ser Pro Met His Glu Gly Asp Gln Thr Arg Ala Ser 1075 1080 1085
Ser Arg Lys Arg Ser Arg Ser Asp Arg Ala Val Thr Gly Pro Ser Ala
1090 1095 1100 Gln Gln Ser Phe Glu Val Arg Val Pro Glu Gln Arg Asp
Ala Leu His1105 1110 1115 1120 Leu Pro Leu Ser Trp Arg Val Lys Arg
Pro Arg Thr Ser Ile Gly Gly 1125 1130 1135 Gly Leu Pro Asp Pro Ile
Ser Arg Ser Gln Leu Val Lys Ser Glu Leu 1140 1145 1150 Glu Glu Lys
Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His 1155 1160 1165
Glu Tyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg
1170 1175 1180 Ile Leu Glu Met Lys Val Met Glu Phe Phe Met Lys Val
Tyr Gly Tyr1185 1190 1195 1200 Arg Gly Lys His Leu Gly Gly Ser Arg
Lys Pro Asp Gly Ala Ile Tyr 1205 1210 1215 Thr Val Gly Ser Pro Ile
Asp Tyr Gly Val Ile Val Asp Thr Lys Ala 1220 1225 1230 Tyr Ser Gly
Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln 1235 1240 1245
Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn
1250 1255 1260 Glu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe
Lys Phe Leu1265 1270 1275 1280 Phe Val Ser Gly His Phe Lys Gly Asn
Tyr Lys Ala Gln Leu Thr Arg 1285 1290 1295 Leu Asn His Ile Thr Asn
Cys Asn Gly Ala Val Leu Ser Val Glu Glu 1300 1305 1310 Leu Leu Ile
Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu 1315 1320 1325
Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe 1330 1335 1340
341542PRTArtificial Sequencesynthetic peptide 34Met Ala Ser Ser Pro
Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser Gly
Trp Ser Arg Met His Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30 Pro
Ser Pro Ala Arg Glu Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35 40
45 Gln Pro Thr Ala Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro Leu
50 55 60 Asp Gly Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg Leu
Pro Ser65 70 75 80 Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly Ser
Phe Ser Asp Leu 85 90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu Asp
Thr Ser Leu Leu Asp Ser 100 105 110 Met Pro Ala Val Gly Thr Pro His
Thr Ala Ala Ala Pro Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser Ala
Leu Arg Ala Ala Asp Asp Pro Pro Pro Thr 130 135 140 Val Arg Val Ala
Val Thr Ala Ala Arg Pro Pro Arg Ala Lys Pro Ala145 150 155 160 Pro
Arg Arg Arg Ala Ala Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165 170
175 Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile
180 185 190 Lys Pro Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala
Leu Val 195 200 205 Gly His Gly Phe Thr His Ala His Ile Val Ala Leu
Ser Gln His Pro 210 215 220 Ala Ala Leu Gly Thr Val Ala Val Thr Tyr
Gln His Ile Ile Thr Ala225 230 235 240 Leu Pro Glu Ala Thr His Glu
Asp Ile Val Gly Val Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg Ala
Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly Pro
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala 275 280 285 Lys
Arg Gly Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn 290 295
300 Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val
Ala305 310 315 320 Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg 325 330 335 Leu Leu Pro Val Leu Cys Gln Ala His Gly
Leu Thr Pro Ala Gln Val 340 345 350 Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala Leu Glu Thr Met 355 360 365 Gln Arg Leu Leu Pro Val
Leu Cys Gln Ala His Gly Leu Pro Pro Asp 370 375 380 Gln Val Val Ala
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu385 390 395 400 Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 405 410
415 Pro Asp Gln Val Val Ala Ile Ala Ser His Gly Gly Gly Lys Gln Ala
420 425 430 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 435 440 445 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His
Asp Gly Gly Lys 450 455 460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala465 470 475 480 His Gly Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Asn Gly Gly 485 490 495 Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 500 505 510 Gln Ala His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 515 520 525 Gly
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 530 535
540 Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser545 550 555 560 His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
Arg Leu Leu Pro 565 570
575 Val Leu Cys Gln Thr His Gly Leu Thr Pro Ala Gln Val Val Ala Ile
580 585 590 Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
Gln Leu 595 600 605 Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
Asp Gln Val Val 610 615 620 Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln
Ala Leu Ala Thr Val Gln625 630 635 640 Arg Leu Leu Pro Val Leu Cys
Gln Ala His Gly Leu Thr Pro Asp Gln 645 650 655 Val Val Ala Ile Ala
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr 660 665 670 Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro 675 680 685 Asp
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu 690 695
700 Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
Leu705 710 715 720 Thr Gln Val Gln Val Val Ala Ile Ala Ser Asn Ile
Gly Gly Lys Gln 725 730 735 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln Ala His 740 745 750 Gly Leu Thr Pro Ala Gln Val Val
Ala Ile Ala Ser His Asp Gly Gly 755 760 765 Lys Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln 770 775 780 Ala His Gly Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly785 790 795 800 Gly
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 805 810
815 Cys Gln Ala His Gly Leu Thr Gln Glu Gln Val Val Ala Ile Ala Ser
820 825 830 Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
Leu Pro 835 840 845 Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln
Val Val Ala Ile 850 855 860 Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu865 870 875 880 Leu Pro Val Leu Cys Gln Ala
His Gly Leu Thr Pro Ala Gln Val Val 885 890 895 Ala Ile Ala Ser Asn
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln 900 905 910 Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly Leu Thr Leu Ala Gln 915 920 925 Val
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr 930 935
940 Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
Gln945 950 955 960 Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
Lys Gln Ala Leu 965 970 975 Glu Thr Val Gln Arg Leu Leu Pro Val Leu
Cys Gln Asp His Gly Leu 980 985 990 Thr Pro Asp Gln Val Val Ala Ile
Ala Ser Asn Ile Gly Gly Lys Gln 995 1000 1005 Ala Leu Glu Thr Val
Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His 1010 1015 1020 Gly Leu
Thr Leu Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Lys1025 1030
1035 1040 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
Gln Asp 1045 1050 1055 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
Ala Ser Asn Ser Gly 1060 1065 1070 Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu Pro Val Leu Cys 1075 1080 1085 Gln Asp His Gly Leu
Thr Pro Asn Gln Val Val Ala Ile Ala Ser Asn 1090 1095 1100 Gly Gly
Lys Gln Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro1105 1110
1115 1120 Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val Ala
Leu Ala 1125 1130 1135 Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val
Lys Lys Gly Leu Pro 1140 1145 1150 His Ala Pro Glu Leu Ile Arg Arg
Val Asn Arg Arg Ile Gly Glu Arg 1155 1160 1165 Thr Ser His Arg Val
Ala Asp Tyr Ala Gln Val Val Arg Val Leu Glu 1170 1175 1180 Phe Phe
Gln Cys His Ser His Pro Ala Tyr Ala Phe Asp Glu Ala Met1185 1190
1195 1200 Thr Gln Phe Gly Met Ser Arg Asn Gly Leu Val Gln Leu Phe
Arg Arg 1205 1210 1215 Val Gly Val Thr Glu Leu Glu Ala Arg Gly Gly
Thr Leu Pro Pro Ala 1220 1225 1230 Ser Gln Arg Trp Asp Arg Ile Leu
Gln Ala Ser Gly Met Lys Arg Ala 1235 1240 1245 Lys Pro Ser Pro Thr
Ser Ala Gln Thr Pro Asp Gln Ala Ser Leu His 1250 1255 1260 Ala Phe
Ala Asp Ser Leu Glu Arg Asp Leu Asp Ala Pro Ser Pro Met1265 1270
1275 1280 His Glu Gly Asp Gln Thr Arg Ala Ser Ser Arg Lys Arg Ser
Arg Ser 1285 1290 1295 Asp Arg Ala Val Thr Gly Pro Ser Ala Gln Gln
Ala Val Glu Val Arg 1300 1305 1310 Val Pro Glu Gln Arg Asp Ala Leu
His Leu Pro Leu Ser Trp Arg Val 1315 1320 1325 Lys Arg Pro Arg Thr
Arg Ile Trp Gly Gly Leu Pro Asp Pro Ile Ser 1330 1335 1340 Arg Ser
Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu1345 1350
1355 1360 Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu
Ile Glu 1365 1370 1375 Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu
Glu Met Lys Val Met 1380 1385 1390 Glu Phe Phe Met Lys Val Tyr Gly
Tyr Arg Gly Lys His Leu Gly Gly 1395 1400 1405 Ser Arg Lys Pro Asp
Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp 1410 1415 1420 Tyr Gly
Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu1425 1430
1435 1440 Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu
Asn Gln 1445 1450 1455 Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp
Trp Lys Val Tyr Pro 1460 1465 1470 Ser Ser Val Thr Glu Phe Lys Phe
Leu Phe Val Ser Gly His Phe Lys 1475 1480 1485 Gly Asn Tyr Lys Ala
Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys 1490 1495 1500 Asn Gly
Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met1505 1510
1515 1520 Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys
Phe Asn 1525 1530 1535 Asn Gly Glu Ile Asn Phe 1540
351035PRTArtificial Sequencesynthetic peptide 35Met Ala Ser Ser Pro
Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser Gly
Trp Ser Arg Met His Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30 Pro
Ser Pro Ala Arg Glu Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35 40
45 Gln Pro Thr Ala Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro Leu
50 55 60 Asp Gly Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg Leu
Pro Ser65 70 75 80 Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly Ser
Phe Ser Asp Leu 85 90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu Asp
Thr Ser Leu Leu Asp Ser 100 105 110 Met Pro Ala Val Gly Thr Pro His
Thr Ala Ala Ala Pro Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser Ala
Leu Arg Ala Ala Asp Asp Pro Pro Pro Thr 130 135 140 Val Arg Val Ala
Val Thr Ala Ala Arg Pro Pro Arg Ala Lys Pro Ala145 150 155 160 Pro
Arg Arg Arg Ala Ala Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165 170
175 Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile
180 185 190 Lys Pro Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala
Leu Val 195 200 205 Gly His Gly Phe Thr His Ala His Ile Val Ala Leu
Ser Gln His Pro 210 215 220 Ala Ala Leu Gly Thr Val Ala Val Thr Tyr
Gln His Ile Ile Thr Ala225 230 235 240 Leu Pro Glu Ala Thr His Glu
Asp Ile Val Gly Val Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg Ala
Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly Pro
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala 275 280 285 Lys
Arg Gly Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn 290 295
300 Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val
Ala305 310 315 320 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg 325 330 335 Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp Gln Val 340 345 350 Val Ala Ile Ala Asn Asn Asn Gly
Gly Lys Gln Ala Leu Glu Thr Val 355 360 365 Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr Pro Asp 370 375 380 Gln Val Val Ala
Ile Ala Asn Asn Asn Gly Gly Lys Gln Ala Leu Glu385 390 395 400 Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 405 410
415 Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala
420 425 430 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 435 440 445 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn
Gly Gly Gly Lys 450 455 460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Asn Gly Gly 485 490 495 Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Asn Asn 515 520 525 Asn
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535
540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
Ala545 550 555 560 Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser Asn Gly Gly Gly Lys
Gln Ala Leu Glu Ser Ile Val Ala 595 600 605 Gln Leu Ser Arg Arg Asp
Pro Ala Leu Ala Ala Leu Thr Asn Asp His 610 615 620 Leu Val Ala Leu
Ala Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val625 630 635 640 Lys
Lys Gly Leu Pro His Ala Pro Glu Phe Ile Arg Arg Val Asn Arg 645 650
655 Arg Ile Ala Glu Arg Thr Ser His Arg Val Ala Asp Tyr Ala His Val
660 665 670 Val Arg Val Leu Glu Phe Phe Gln Cys His Ser His Pro Ala
His Ala 675 680 685 Phe Asp Glu Ala Met Thr Gln Phe Gly Met Ser Arg
His Gly Leu Val 690 695 700 Gln Leu Phe Arg Arg Val Gly Val Thr Glu
Phe Glu Ala Arg Tyr Gly705 710 715 720 Thr Leu Pro Pro Ala Ser Gln
Arg Trp Asp Arg Ile Leu Gln Ala Ser 725 730 735 Gly Met Lys Arg Ala
Lys Pro Ser Pro Thr Ser Ala Gln Thr Pro Asp 740 745 750 Gln Thr Ser
Leu His Ala Phe Ala Asp Ser Leu Glu Arg Asp Leu Asp 755 760 765 Ala
Pro Ser Pro Met His Glu Gly Asp Gln Thr Arg Ala Ser Ser Arg 770 775
780 Lys Arg Ser Arg Ser Asp Arg Ala Val Thr Gly Pro Ser Ala Gln
Gln785 790 795 800 Ala Val Glu Val Arg Val Pro Glu Gln Arg Asp Ala
Leu His Leu Pro 805 810 815 Leu Ser Trp Arg Val Lys Arg Pro Arg Thr
Arg Ile Trp Gly Gly Leu 820 825 830 Pro Asp Pro Ile Ser Arg Ser Gln
Leu Val Lys Ser Glu Leu Glu Glu 835 840 845 Lys Lys Ser Glu Leu Arg
His Lys Leu Lys Tyr Val Pro His Glu Tyr 850 855 860 Ile Glu Leu Ile
Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu865 870 875 880 Glu
Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly 885 890
895 Lys His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val
900 905 910 Gly Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala
Tyr Ser 915 920 925 Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu
Met Gln Arg Tyr 930 935 940 Val Glu Glu Asn Gln Thr Arg Asn Lys His
Ile Asn Pro Asn Glu Trp945 950 955 960 Trp Lys Val Tyr Pro Ser Ser
Val Thr Glu Phe Lys Phe Leu Phe Val 965 970 975 Ser Gly His Phe Lys
Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn 980 985 990 His Ile Thr
Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu 995 1000 1005
Ile Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val
1010 1015 1020 Arg Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe1025 1030
1035361069PRTArtificial Sequencesynthetic peptide 36Met Ala Ser Ser
Pro Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser
Gly Trp Ser Arg Met His Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30
Pro Ser Pro Ala Arg Glu Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35
40 45 Gln Pro Thr Ala Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro
Leu 50 55 60 Asp Gly Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg
Leu Pro Ser65 70 75 80 Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly
Ser Phe Ser Asp Leu 85 90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu
Asp Thr Ser Leu Leu Asp Ser 100 105 110 Met Pro Ala Val Gly Thr Pro
His Thr Ala Ala Ala Pro Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser
Ala Leu Arg Ala Ala Asp Asp Pro Pro Pro Thr 130 135 140 Val Arg Val
Ala Val Thr Ala Ala Arg Pro Pro Arg Ala Lys Pro Ala145 150 155 160
Pro Arg Arg Arg Ala Ala Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165
170 175 Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys
Ile 180 185 190 Lys Pro Lys Val Arg Ser Thr Val Ala Gln His His Glu
Ala Leu Val 195 200 205 Gly His Gly Phe Thr His Ala His Ile Val Ala
Leu Ser Gln His Pro 210 215 220 Ala Ala Leu Gly Thr Val Ala Val Thr
Tyr Gln His Ile Ile Thr Ala225 230 235 240 Leu Pro Glu Ala Thr His
Glu Asp Ile Val Gly Val Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg
Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly
Pro Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala 275 280 285
Lys Arg Gly Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn
290
295 300 Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val
Ala305 310 315 320 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg 325 330 335 Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp Gln Val 340 345 350 Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala Leu Glu Thr Val 355 360 365 Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr Pro Asp 370 375 380 Gln Val Val Ala
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu385 390 395 400 Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 405 410
415 Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala
420 425 430 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 435 440 445 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His
Asp Gly Gly Lys 450 455 460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Asn Gly Gly 485 490 495 Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His 515 520 525 Asp
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535
540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
Ala545 550 555 560 Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser His Asp Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys
Gln Asp His Gly Leu Thr Pro Asp Gln Val 610 615 620 Val Ala Ile Ala
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Ser Ile625 630 635 640 Val
Ala Gln Leu Ser Arg Arg Asp Pro Ala Leu Ala Ala Leu Thr Asn 645 650
655 Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Leu Asp
660 665 670 Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Phe Ile Arg
Arg Val 675 680 685 Asn Arg Arg Ile Ala Glu Arg Thr Ser His Arg Val
Ala Asp Tyr Ala 690 695 700 His Val Val Arg Val Leu Glu Phe Phe Gln
Cys His Ser His Pro Ala705 710 715 720 His Ala Phe Asp Glu Ala Met
Thr Gln Phe Gly Met Ser Arg His Gly 725 730 735 Leu Val Gln Leu Phe
Arg Arg Val Gly Val Thr Glu Phe Glu Ala Arg 740 745 750 Tyr Gly Thr
Leu Pro Pro Ala Ser Gln Arg Trp Asp Arg Ile Leu Gln 755 760 765 Ala
Ser Gly Met Lys Arg Ala Lys Pro Ser Pro Thr Ser Ala Gln Thr 770 775
780 Pro Asp Gln Thr Ser Leu His Ala Phe Ala Asp Ser Leu Glu Arg
Asp785 790 795 800 Leu Asp Ala Pro Ser Pro Met His Glu Gly Asp Gln
Thr Arg Ala Ser 805 810 815 Ser Arg Lys Arg Ser Arg Ser Asp Arg Ala
Val Thr Gly Pro Ser Ala 820 825 830 Gln Gln Ala Val Glu Val Arg Val
Pro Glu Gln Arg Asp Ala Leu His 835 840 845 Leu Pro Leu Ser Trp Arg
Val Lys Arg Pro Arg Thr Arg Ile Trp Gly 850 855 860 Gly Leu Pro Asp
Pro Ile Ser Arg Ser Gln Leu Val Lys Ser Glu Leu865 870 875 880 Glu
Glu Lys Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His 885 890
895 Glu Tyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg
900 905 910 Ile Leu Glu Met Lys Val Met Glu Phe Phe Met Lys Val Tyr
Gly Tyr 915 920 925 Arg Gly Lys His Leu Gly Gly Ser Arg Lys Pro Asp
Gly Ala Ile Tyr 930 935 940 Thr Val Gly Ser Pro Ile Asp Tyr Gly Val
Ile Val Asp Thr Lys Ala945 950 955 960 Tyr Ser Gly Gly Tyr Asn Leu
Pro Ile Gly Gln Ala Asp Glu Met Gln 965 970 975 Arg Tyr Val Glu Glu
Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn 980 985 990 Glu Trp Trp
Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu 995 1000 1005
Phe Val Ser Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg
1010 1015 1020 Leu Asn His Ile Thr Asn Cys Asn Gly Ala Val Leu Ser
Val Glu Glu1025 1030 1035 1040 Leu Leu Ile Gly Gly Glu Met Ile Lys
Ala Gly Thr Leu Thr Leu Glu 1045 1050 1055 Glu Val Arg Arg Lys Phe
Asn Asn Gly Glu Ile Asn Phe 1060 1065 371137PRTArtificial
Sequencesynthetic peptide 37Met Ala Ser Ser Pro Pro Lys Lys Lys Arg
Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser Gly Trp Ser Arg Met His
Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30 Pro Ser Pro Ala Arg Glu
Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35 40 45 Gln Pro Thr Ala
Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro Leu 50 55 60 Asp Gly
Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg Leu Pro Ser65 70 75 80
Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly Ser Phe Ser Asp Leu 85
90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu Asp Thr Ser Leu Leu Asp
Ser 100 105 110 Met Pro Ala Val Gly Thr Pro His Thr Ala Ala Ala Pro
Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser Ala Leu Arg Ala Ala Asp
Asp Pro Pro Pro Thr 130 135 140 Val Arg Val Ala Val Thr Ala Ala Arg
Pro Pro Arg Ala Lys Pro Ala145 150 155 160 Pro Arg Arg Arg Ala Ala
Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165 170 175 Val Asp Leu Arg
Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile 180 185 190 Lys Pro
Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val 195 200 205
Gly His Gly Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro 210
215 220 Ala Ala Leu Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr
Ala225 230 235 240 Leu Pro Glu Ala Thr His Glu Asp Ile Val Gly Val
Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu
Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly Pro Pro Leu Gln Leu Asp
Thr Gly Gln Leu Val Lys Ile Ala 275 280 285 Lys Arg Gly Gly Val Thr
Ala Met Glu Ala Val His Ala Ser Arg Asn 290 295 300 Ala Leu Thr Gly
Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val Ala305 310 315 320 Ile
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 325 330
335 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val
340 345 350 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
Thr Val 355 360 365 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp 370 375 380 Gln Val Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala Leu Glu385 390 395 400 Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln Asp His Gly Leu Thr 405 410 415 Pro Asp Gln Val Val
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala 420 425 430 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 435 440 445 Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 450 455
460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Asn Asn Asn Gly 485 490 495 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala Ser Asn 515 520 525 Ile Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535 540 Leu Cys Gln Asp
His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala545 550 555 560 Ser
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 565 570
575 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala
580 585 590 Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val 610 615 620 Val Ala Ile Ala Ser His Asp Gly Gly Lys
Gln Ala Leu Glu Thr Val625 630 635 640 Gln Arg Leu Leu Pro Val Leu
Cys Gln Asp His Gly Leu Thr Pro Asp 645 650 655 Gln Val Val Ala Ile
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu 660 665 670 Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 675 680 685 Pro
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 690 695
700 Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Arg Asp Pro Ala Leu
Ala705 710 715 720 Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys
Leu Gly Gly Arg 725 730 735 Pro Ala Leu Asp Ala Val Lys Lys Gly Leu
Pro His Ala Pro Glu Phe 740 745 750 Ile Arg Arg Val Asn Arg Arg Ile
Ala Glu Arg Thr Ser His Arg Val 755 760 765 Ala Asp Tyr Ala His Val
Val Arg Val Leu Glu Phe Phe Gln Cys His 770 775 780 Ser His Pro Ala
His Ala Phe Asp Glu Ala Met Thr Gln Phe Gly Met785 790 795 800 Ser
Arg His Gly Leu Val Gln Leu Phe Arg Arg Val Gly Val Thr Glu 805 810
815 Phe Glu Ala Arg Tyr Gly Thr Leu Pro Pro Ala Ser Gln Arg Trp Asp
820 825 830 Arg Ile Leu Gln Ala Ser Gly Met Lys Arg Ala Lys Pro Ser
Pro Thr 835 840 845 Ser Ala Gln Thr Pro Asp Gln Thr Ser Leu His Ala
Phe Ala Asp Ser 850 855 860 Leu Glu Arg Asp Leu Asp Ala Pro Ser Pro
Met His Glu Gly Asp Gln865 870 875 880 Thr Arg Ala Ser Ser Arg Lys
Arg Ser Arg Ser Asp Arg Ala Val Thr 885 890 895 Gly Pro Ser Ala Gln
Gln Ala Val Glu Val Arg Val Pro Glu Gln Arg 900 905 910 Asp Ala Leu
His Leu Pro Leu Ser Trp Arg Val Lys Arg Pro Arg Thr 915 920 925 Arg
Ile Trp Gly Gly Leu Pro Asp Pro Ile Ser Arg Ser Gln Leu Val 930 935
940 Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu Arg His Lys Leu
Lys945 950 955 960 Tyr Val Pro His Glu Tyr Ile Glu Leu Ile Glu Ile
Ala Arg Asn Ser 965 970 975 Thr Gln Asp Arg Ile Leu Glu Met Lys Val
Met Glu Phe Phe Met Lys 980 985 990 Val Tyr Gly Tyr Arg Gly Lys His
Leu Gly Gly Ser Arg Lys Pro Asp 995 1000 1005 Gly Ala Ile Tyr Thr
Val Gly Ser Pro Ile Asp Tyr Gly Val Ile Val 1010 1015 1020 Asp Thr
Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala1025 1030
1035 1040 Asp Glu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn
Lys His 1045 1050 1055 Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro
Ser Ser Val Thr Glu 1060 1065 1070 Phe Lys Phe Leu Phe Val Ser Gly
His Phe Lys Gly Asn Tyr Lys Ala 1075 1080 1085 Gln Leu Thr Arg Leu
Asn His Ile Thr Asn Cys Asn Gly Ala Val Leu 1090 1095 1100 Ser Val
Glu Glu Leu Leu Ile Gly Gly Glu Met Ile Lys Ala Gly Thr1105 1110
1115 1120 Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu
Ile Asn 1125 1130 1135 Phe381137PRTArtificial Sequencesynthetic
peptide 38Met Ala Ser Ser Pro Pro Lys Lys Lys Arg Lys Val Ser Trp
Lys Asp1 5 10 15 Ala Ser Gly Trp Ser Arg Met His Ala Asp Pro Ile
Arg Pro Arg Arg 20 25 30 Pro Ser Pro Ala Arg Glu Leu Leu Pro Gly
Pro Gln Pro Asp Arg Val 35 40 45 Gln Pro Thr Ala Asp Arg Gly Val
Ser Ala Pro Ala Gly Ser Pro Leu 50 55 60 Asp Gly Leu Pro Ala Arg
Arg Thr Val Ser Arg Thr Arg Leu Pro Ser65 70 75 80 Pro Pro Ala Pro
Ser Pro Ala Phe Ser Ala Gly Ser Phe Ser Asp Leu 85 90 95 Leu Arg
Pro Phe Asp Pro Ser Leu Leu Asp Thr Ser Leu Leu Asp Ser 100 105 110
Met Pro Ala Val Gly Thr Pro His Thr Ala Ala Ala Pro Ala Glu Trp 115
120 125 Asp Glu Ala Gln Ser Ala Leu Arg Ala Ala Asp Asp Pro Pro Pro
Thr 130 135 140 Val Arg Val Ala Val Thr Ala Ala Arg Pro Pro Arg Ala
Lys Pro Ala145 150 155 160 Pro Arg Arg Arg Ala Ala Gln Pro Ser Asp
Ala Ser Pro Ala Ala Gln 165 170 175 Val Asp Leu Arg Thr Leu Gly Tyr
Ser Gln Gln Gln Gln Glu Lys Ile 180 185 190 Lys Pro Lys Val Arg Ser
Thr Val Ala Gln His His Glu Ala Leu Val 195 200 205 Gly His Gly Phe
Thr His Ala His Ile Val Ala Leu Ser Gln His Pro 210 215 220 Ala Ala
Leu Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala225 230 235
240 Leu Pro Glu Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp
245 250 255 Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly
Glu Leu 260 265 270 Arg Gly Pro Pro Leu Gln Leu Asp Thr Gly Gln Leu
Val Lys Ile Ala 275 280 285 Lys Arg Gly Gly Val Thr Ala Met Glu Ala
Val His Ala Ser Arg Asn 290 295 300 Ala Leu Thr Gly Ala Pro Leu Asn
Leu Thr Pro Ala Gln Val Val Ala305 310 315 320 Ile Ala Ser His Asp
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 325 330 335 Leu Leu Pro
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 340 345 350 Val
Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val 355 360
365 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp
370 375 380 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala
Leu Glu385
390 395 400 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr 405 410 415 Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly
Gly Lys Gln Ala 420 425 430 Leu Glu Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly 435 440 445 Leu Thr Pro Asp Gln Val Val Ala
Ile Ala Asn Asn Asn Gly Gly Lys 450 455 460 Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu Pro Val Leu Cys Gln Asp465 470 475 480 His Gly Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly 485 490 495 Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 500 505
510 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn
515 520 525 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val 530 535 540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val
Val Ala Ile Ala545 550 555 560 Asn Asn Asn Gly Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln Asp His
Gly Leu Thr Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser Asn Gly
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu Leu Pro
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 610 615 620 Val
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val625 630
635 640 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
Asp 645 650 655 Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln
Ala Leu Glu 660 665 670 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Asp His Gly Leu Thr 675 680 685 Pro Asp Gln Val Val Ala Ile Ala Ser
Asn Ile Gly Gly Lys Gln Ala 690 695 700 Leu Glu Ser Ile Val Ala Gln
Leu Ser Arg Arg Asp Pro Ala Leu Ala705 710 715 720 Ala Leu Thr Asn
Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg 725 730 735 Pro Ala
Leu Asp Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Phe 740 745 750
Ile Arg Arg Val Asn Arg Arg Ile Ala Glu Arg Thr Ser His Arg Val 755
760 765 Ala Asp Tyr Ala His Val Val Arg Val Leu Glu Phe Phe Gln Cys
His 770 775 780 Ser His Pro Ala His Ala Phe Asp Glu Ala Met Thr Gln
Phe Gly Met785 790 795 800 Ser Arg His Gly Leu Val Gln Leu Phe Arg
Arg Val Gly Val Thr Glu 805 810 815 Phe Glu Ala Arg Tyr Gly Thr Leu
Pro Pro Ala Ser Gln Arg Trp Asp 820 825 830 Arg Ile Leu Gln Ala Ser
Gly Met Lys Arg Ala Lys Pro Ser Pro Thr 835 840 845 Ser Ala Gln Thr
Pro Asp Gln Thr Ser Leu His Ala Phe Ala Asp Ser 850 855 860 Leu Glu
Arg Asp Leu Asp Ala Pro Ser Pro Met His Glu Gly Asp Gln865 870 875
880 Thr Arg Ala Ser Ser Arg Lys Arg Ser Arg Ser Asp Arg Ala Val Thr
885 890 895 Gly Pro Ser Ala Gln Gln Ala Val Glu Val Arg Val Pro Glu
Gln Arg 900 905 910 Asp Ala Leu His Leu Pro Leu Ser Trp Arg Val Lys
Arg Pro Arg Thr 915 920 925 Arg Ile Trp Gly Gly Leu Pro Asp Pro Ile
Ser Arg Ser Gln Leu Val 930 935 940 Lys Ser Glu Leu Glu Glu Lys Lys
Ser Glu Leu Arg His Lys Leu Lys945 950 955 960 Tyr Val Pro His Glu
Tyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser 965 970 975 Thr Gln Asp
Arg Ile Leu Glu Met Lys Val Met Glu Phe Phe Met Lys 980 985 990 Val
Tyr Gly Tyr Arg Gly Lys His Leu Gly Gly Ser Arg Lys Pro Asp 995
1000 1005 Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly Val
Ile Val 1010 1015 1020 Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu
Pro Ile Gly Gln Ala1025 1030 1035 1040 Asp Glu Met Gln Arg Tyr Val
Glu Glu Asn Gln Thr Arg Asn Lys His 1045 1050 1055 Ile Asn Pro Asn
Glu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu 1060 1065 1070 Phe
Lys Phe Leu Phe Val Ser Gly His Phe Lys Gly Asn Tyr Lys Ala 1075
1080 1085 Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys Asn Gly Ala
Val Leu 1090 1095 1100 Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met
Ile Lys Ala Gly Thr1105 1110 1115 1120 Leu Thr Leu Glu Glu Val Arg
Arg Lys Phe Asn Asn Gly Glu Ile Asn 1125 1130 1135
Phe391137PRTArtificial Sequencesynthetic peptide 39Met Ala Ser Ser
Pro Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser
Gly Trp Ser Arg Met His Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30
Pro Ser Pro Ala Arg Glu Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35
40 45 Gln Pro Thr Ala Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro
Leu 50 55 60 Asp Gly Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg
Leu Pro Ser65 70 75 80 Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly
Ser Phe Ser Asp Leu 85 90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu
Asp Thr Ser Leu Leu Asp Ser 100 105 110 Met Pro Ala Val Gly Thr Pro
His Thr Ala Ala Ala Pro Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser
Ala Leu Arg Ala Ala Asp Asp Pro Pro Pro Thr 130 135 140 Val Arg Val
Ala Val Thr Ala Ala Arg Pro Pro Arg Ala Lys Pro Ala145 150 155 160
Pro Arg Arg Arg Ala Ala Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165
170 175 Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys
Ile 180 185 190 Lys Pro Lys Val Arg Ser Thr Val Ala Gln His His Glu
Ala Leu Val 195 200 205 Gly His Gly Phe Thr His Ala His Ile Val Ala
Leu Ser Gln His Pro 210 215 220 Ala Ala Leu Gly Thr Val Ala Val Thr
Tyr Gln His Ile Ile Thr Ala225 230 235 240 Leu Pro Glu Ala Thr His
Glu Asp Ile Val Gly Val Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg
Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly
Pro Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala 275 280 285
Lys Arg Gly Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn 290
295 300 Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val
Ala305 310 315 320 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg 325 330 335 Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp Gln Val 340 345 350 Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala Leu Glu Thr Val 355 360 365 Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr Pro Asp 370 375 380 Gln Val Val Ala
Ile Ala Asn Asn Asn Gly Gly Lys Gln Ala Leu Glu385 390 395 400 Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 405 410
415 Pro Asp Gln Val Val Ala Ile Ala Asn Asn Asn Gly Gly Lys Gln Ala
420 425 430 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 435 440 445 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn
Ile Gly Gly Lys 450 455 460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Asn Gly Gly 485 490 495 Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Asn Asn 515 520 525 Asn
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535
540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
Ala545 550 555 560 Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser Asn Gly Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys
Gln Asp His Gly Leu Thr Pro Asp Gln Val 610 615 620 Val Ala Ile Ala
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val625 630 635 640 Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 645 650
655 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
660 665 670 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr 675 680 685 Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly
Gly Lys Gln Ala 690 695 700 Leu Glu Ser Ile Val Ala Gln Leu Ser Arg
Arg Asp Pro Ala Leu Ala705 710 715 720 Ala Leu Thr Asn Asp His Leu
Val Ala Leu Ala Cys Leu Gly Gly Arg 725 730 735 Pro Ala Leu Asp Ala
Val Lys Lys Gly Leu Pro His Ala Pro Glu Phe 740 745 750 Ile Arg Arg
Val Asn Arg Arg Ile Ala Glu Arg Thr Ser His Arg Val 755 760 765 Ala
Asp Tyr Ala His Val Val Arg Val Leu Glu Phe Phe Gln Cys His 770 775
780 Ser His Pro Ala His Ala Phe Asp Glu Ala Met Thr Gln Phe Gly
Met785 790 795 800 Ser Arg His Gly Leu Val Gln Leu Phe Arg Arg Val
Gly Val Thr Glu 805 810 815 Phe Glu Ala Arg Tyr Gly Thr Leu Pro Pro
Ala Ser Gln Arg Trp Asp 820 825 830 Arg Ile Leu Gln Ala Ser Gly Met
Lys Arg Ala Lys Pro Ser Pro Thr 835 840 845 Ser Ala Gln Thr Pro Asp
Gln Thr Ser Leu His Ala Phe Ala Asp Ser 850 855 860 Leu Glu Arg Asp
Leu Asp Ala Pro Ser Pro Met His Glu Gly Asp Gln865 870 875 880 Thr
Arg Ala Ser Ser Arg Lys Arg Ser Arg Ser Asp Arg Ala Val Thr 885 890
895 Gly Pro Ser Ala Gln Gln Ala Val Glu Val Arg Val Pro Glu Gln Arg
900 905 910 Asp Ala Leu His Leu Pro Leu Ser Trp Arg Val Lys Arg Pro
Arg Thr 915 920 925 Arg Ile Trp Gly Gly Leu Pro Asp Pro Ile Ser Arg
Ser Gln Leu Val 930 935 940 Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu
Leu Arg His Lys Leu Lys945 950 955 960 Tyr Val Pro His Glu Tyr Ile
Glu Leu Ile Glu Ile Ala Arg Asn Ser 965 970 975 Thr Gln Asp Arg Ile
Leu Glu Met Lys Val Met Glu Phe Phe Met Lys 980 985 990 Val Tyr Gly
Tyr Arg Gly Lys His Leu Gly Gly Ser Arg Lys Pro Asp 995 1000 1005
Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly Val Ile Val
1010 1015 1020 Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile
Gly Gln Ala1025 1030 1035 1040 Asp Glu Met Gln Arg Tyr Val Glu Glu
Asn Gln Thr Arg Asn Lys His 1045 1050 1055 Ile Asn Pro Asn Glu Trp
Trp Lys Val Tyr Pro Ser Ser Val Thr Glu 1060 1065 1070 Phe Lys Phe
Leu Phe Val Ser Gly His Phe Lys Gly Asn Tyr Lys Ala 1075 1080 1085
Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys Asn Gly Ala Val Leu
1090 1095 1100 Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile Lys
Ala Gly Thr1105 1110 1115 1120 Leu Thr Leu Glu Glu Val Arg Arg Lys
Phe Asn Asn Gly Glu Ile Asn 1125 1130 1135 Phe401137PRTArtificial
Sequencesynthetic peptide 40Met Ala Ser Ser Pro Pro Lys Lys Lys Arg
Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser Gly Trp Ser Arg Met His
Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30 Pro Ser Pro Ala Arg Glu
Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35 40 45 Gln Pro Thr Ala
Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro Leu 50 55 60 Asp Gly
Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg Leu Pro Ser65 70 75 80
Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly Ser Phe Ser Asp Leu 85
90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu Asp Thr Ser Leu Leu Asp
Ser 100 105 110 Met Pro Ala Val Gly Thr Pro His Thr Ala Ala Ala Pro
Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser Ala Leu Arg Ala Ala Asp
Asp Pro Pro Pro Thr 130 135 140 Val Arg Val Ala Val Thr Ala Ala Arg
Pro Pro Arg Ala Lys Pro Ala145 150 155 160 Pro Arg Arg Arg Ala Ala
Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165 170 175 Val Asp Leu Arg
Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile 180 185 190 Lys Pro
Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val 195 200 205
Gly His Gly Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro 210
215 220 Ala Ala Leu Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr
Ala225 230 235 240 Leu Pro Glu Ala Thr His Glu Asp Ile Val Gly Val
Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu
Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly Pro Pro Leu Gln Leu Asp
Thr Gly Gln Leu Val Lys Ile Ala 275 280 285 Lys Arg Gly Gly Val Thr
Ala Met Glu Ala Val His Ala Ser Arg Asn 290 295 300 Ala Leu Thr Gly
Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val Ala305 310 315 320 Ile
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 325 330
335 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val
340 345 350 Val Ala Ile Ala Asn Asn Asn Gly Gly Lys Gln Ala Leu Glu
Thr Val 355 360 365 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp 370 375 380 Gln Val Val Ala Ile Ala Ser Asn Ile Gly
Gly Lys Gln Ala Leu Glu385 390 395 400 Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln Asp His Gly Leu Thr 405 410 415 Pro Asp Gln Val Val
Ala Ile Ala Ser His
Asp Gly Gly Lys Gln Ala 420 425 430 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 435 440 445 Leu Thr Pro Asp Gln Val
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 450 455 460 Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp465 470 475 480 His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly 485 490
495 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
500 505 510 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Asn 515 520 525 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val 530 535 540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala545 550 555 560 Ser His Asp Gly Gly Lys Gln
Ala Leu Glu Thr Val Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser
His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 610 615
620 Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr
Val625 630 635 640 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp 645 650 655 Gln Val Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala Leu Glu 660 665 670 Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr 675 680 685 Pro Asp Gln Val Val Ala
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala 690 695 700 Leu Glu Ser Ile
Val Ala Gln Leu Ser Arg Arg Asp Pro Ala Leu Ala705 710 715 720 Ala
Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg 725 730
735 Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Phe
740 745 750 Ile Arg Arg Val Asn Arg Arg Ile Ala Glu Arg Thr Ser His
Arg Val 755 760 765 Ala Asp Tyr Ala His Val Val Arg Val Leu Glu Phe
Phe Gln Cys His 770 775 780 Ser His Pro Ala His Ala Phe Asp Glu Ala
Met Thr Gln Phe Gly Met785 790 795 800 Ser Arg His Gly Leu Val Gln
Leu Phe Arg Arg Val Gly Val Thr Glu 805 810 815 Phe Glu Ala Arg Tyr
Gly Thr Leu Pro Pro Ala Ser Gln Arg Trp Asp 820 825 830 Arg Ile Leu
Gln Ala Ser Gly Met Lys Arg Ala Lys Pro Ser Pro Thr 835 840 845 Ser
Ala Gln Thr Pro Asp Gln Thr Ser Leu His Ala Phe Ala Asp Ser 850 855
860 Leu Glu Arg Asp Leu Asp Ala Pro Ser Pro Met His Glu Gly Asp
Gln865 870 875 880 Thr Arg Ala Ser Ser Arg Lys Arg Ser Arg Ser Asp
Arg Ala Val Thr 885 890 895 Gly Pro Ser Ala Gln Gln Ala Val Glu Val
Arg Val Pro Glu Gln Arg 900 905 910 Asp Ala Leu His Leu Pro Leu Ser
Trp Arg Val Lys Arg Pro Arg Thr 915 920 925 Arg Ile Trp Gly Gly Leu
Pro Asp Pro Ile Ser Arg Ser Gln Leu Val 930 935 940 Lys Ser Glu Leu
Glu Glu Lys Lys Ser Glu Leu Arg His Lys Leu Lys945 950 955 960 Tyr
Val Pro His Glu Tyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser 965 970
975 Thr Gln Asp Arg Ile Leu Glu Met Lys Val Met Glu Phe Phe Met Lys
980 985 990 Val Tyr Gly Tyr Arg Gly Lys His Leu Gly Gly Ser Arg Lys
Pro Asp 995 1000 1005 Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp
Tyr Gly Val Ile Val 1010 1015 1020 Asp Thr Lys Ala Tyr Ser Gly Gly
Tyr Asn Leu Pro Ile Gly Gln Ala1025 1030 1035 1040 Asp Glu Met Gln
Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His 1045 1050 1055 Ile
Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu 1060
1065 1070 Phe Lys Phe Leu Phe Val Ser Gly His Phe Lys Gly Asn Tyr
Lys Ala 1075 1080 1085 Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys
Asn Gly Ala Val Leu 1090 1095 1100 Ser Val Glu Glu Leu Leu Ile Gly
Gly Glu Met Ile Lys Ala Gly Thr1105 1110 1115 1120 Leu Thr Leu Glu
Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile Asn 1125 1130 1135
Phe411137PRTArtificial Sequencesynthetic peptide 41Met Ala Ser Ser
Pro Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser
Gly Trp Ser Arg Met His Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30
Pro Ser Pro Ala Arg Glu Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35
40 45 Gln Pro Thr Ala Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro
Leu 50 55 60 Asp Gly Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg
Leu Pro Ser65 70 75 80 Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly
Ser Phe Ser Asp Leu 85 90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu
Asp Thr Ser Leu Leu Asp Ser 100 105 110 Met Pro Ala Val Gly Thr Pro
His Thr Ala Ala Ala Pro Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser
Ala Leu Arg Ala Ala Asp Asp Pro Pro Pro Thr 130 135 140 Val Arg Val
Ala Val Thr Ala Ala Arg Pro Pro Arg Ala Lys Pro Ala145 150 155 160
Pro Arg Arg Arg Ala Ala Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165
170 175 Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys
Ile 180 185 190 Lys Pro Lys Val Arg Ser Thr Val Ala Gln His His Glu
Ala Leu Val 195 200 205 Gly His Gly Phe Thr His Ala His Ile Val Ala
Leu Ser Gln His Pro 210 215 220 Ala Ala Leu Gly Thr Val Ala Val Thr
Tyr Gln His Ile Ile Thr Ala225 230 235 240 Leu Pro Glu Ala Thr His
Glu Asp Ile Val Gly Val Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg
Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly
Pro Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala 275 280 285
Lys Arg Gly Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn 290
295 300 Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val
Ala305 310 315 320 Ile Ala Asn Asn Asn Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg 325 330 335 Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp Gln Val 340 345 350 Val Ala Ile Ala Asn Asn Asn Gly
Gly Lys Gln Ala Leu Glu Thr Val 355 360 365 Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr Pro Asp 370 375 380 Gln Val Val Ala
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu385 390 395 400 Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 405 410
415 Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
420 425 430 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 435 440 445 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His
Asp Gly Gly Lys 450 455 460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Asn Gly Gly 485 490 495 Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Asn Asn 515 520 525 Asn
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535
540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
Ala545 550 555 560 Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser Asn Gly Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys
Gln Asp His Gly Leu Thr Pro Asp Gln Val 610 615 620 Val Ala Ile Ala
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val625 630 635 640 Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 645 650
655 Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
660 665 670 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr 675 680 685 Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala 690 695 700 Leu Glu Ser Ile Val Ala Gln Leu Ser Arg
Arg Asp Pro Ala Leu Ala705 710 715 720 Ala Leu Thr Asn Asp His Leu
Val Ala Leu Ala Cys Leu Gly Gly Arg 725 730 735 Pro Ala Leu Asp Ala
Val Lys Lys Gly Leu Pro His Ala Pro Glu Phe 740 745 750 Ile Arg Arg
Val Asn Arg Arg Ile Ala Glu Arg Thr Ser His Arg Val 755 760 765 Ala
Asp Tyr Ala His Val Val Arg Val Leu Glu Phe Phe Gln Cys His 770 775
780 Ser His Pro Ala His Ala Phe Asp Glu Ala Met Thr Gln Phe Gly
Met785 790 795 800 Ser Arg His Gly Leu Val Gln Leu Phe Arg Arg Val
Gly Val Thr Glu 805 810 815 Phe Glu Ala Arg Tyr Gly Thr Leu Pro Pro
Ala Ser Gln Arg Trp Asp 820 825 830 Arg Ile Leu Gln Ala Ser Gly Met
Lys Arg Ala Lys Pro Ser Pro Thr 835 840 845 Ser Ala Gln Thr Pro Asp
Gln Thr Ser Leu His Ala Phe Ala Asp Ser 850 855 860 Leu Glu Arg Asp
Leu Asp Ala Pro Ser Pro Met His Glu Gly Asp Gln865 870 875 880 Thr
Arg Ala Ser Ser Arg Lys Arg Ser Arg Ser Asp Arg Ala Val Thr 885 890
895 Gly Pro Ser Ala Gln Gln Ala Val Glu Val Arg Val Pro Glu Gln Arg
900 905 910 Asp Ala Leu His Leu Pro Leu Ser Trp Arg Val Lys Arg Pro
Arg Thr 915 920 925 Arg Ile Trp Gly Gly Leu Pro Asp Pro Ile Ser Arg
Ser Gln Leu Val 930 935 940 Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu
Leu Arg His Lys Leu Lys945 950 955 960 Tyr Val Pro His Glu Tyr Ile
Glu Leu Ile Glu Ile Ala Arg Asn Ser 965 970 975 Thr Gln Asp Arg Ile
Leu Glu Met Lys Val Met Glu Phe Phe Met Lys 980 985 990 Val Tyr Gly
Tyr Arg Gly Lys His Leu Gly Gly Ser Arg Lys Pro Asp 995 1000 1005
Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly Val Ile Val
1010 1015 1020 Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile
Gly Gln Ala1025 1030 1035 1040 Asp Glu Met Gln Arg Tyr Val Glu Glu
Asn Gln Thr Arg Asn Lys His 1045 1050 1055 Ile Asn Pro Asn Glu Trp
Trp Lys Val Tyr Pro Ser Ser Val Thr Glu 1060 1065 1070 Phe Lys Phe
Leu Phe Val Ser Gly His Phe Lys Gly Asn Tyr Lys Ala 1075 1080 1085
Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys Asn Gly Ala Val Leu
1090 1095 1100 Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile Lys
Ala Gly Thr1105 1110 1115 1120 Leu Thr Leu Glu Glu Val Arg Arg Lys
Phe Asn Asn Gly Glu Ile Asn 1125 1130 1135 Phe421171PRTArtificial
Sequencesynthetic peptide 42Met Ala Ser Ser Pro Pro Lys Lys Lys Arg
Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser Gly Trp Ser Arg Met His
Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30 Pro Ser Pro Ala Arg Glu
Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35 40 45 Gln Pro Thr Ala
Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro Leu 50 55 60 Asp Gly
Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg Leu Pro Ser65 70 75 80
Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly Ser Phe Ser Asp Leu 85
90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu Asp Thr Ser Leu Leu Asp
Ser 100 105 110 Met Pro Ala Val Gly Thr Pro His Thr Ala Ala Ala Pro
Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser Ala Leu Arg Ala Ala Asp
Asp Pro Pro Pro Thr 130 135 140 Val Arg Val Ala Val Thr Ala Ala Arg
Pro Pro Arg Ala Lys Pro Ala145 150 155 160 Pro Arg Arg Arg Ala Ala
Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165 170 175 Val Asp Leu Arg
Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile 180 185 190 Lys Pro
Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val 195 200 205
Gly His Gly Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro 210
215 220 Ala Ala Leu Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr
Ala225 230 235 240 Leu Pro Glu Ala Thr His Glu Asp Ile Val Gly Val
Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu
Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly Pro Pro Leu Gln Leu Asp
Thr Gly Gln Leu Val Lys Ile Ala 275 280 285 Lys Arg Gly Gly Val Thr
Ala Met Glu Ala Val His Ala Ser Arg Asn 290 295 300 Ala Leu Thr Gly
Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val Ala305 310 315 320 Ile
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 325 330
335 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val
340 345 350 Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
Thr Val 355 360 365 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp 370 375 380 Gln Val Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala Leu Glu385 390 395 400 Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln Asp His Gly Leu Thr 405 410 415 Pro Asp Gln Val Val
Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 420 425 430 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 435 440 445
Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys 450 455
460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Asn Gly Gly 485 490 495 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala Ser His 515 520 525 Asp Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535 540 Leu Cys Gln Asp
His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala545 550 555 560 Ser
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 565 570
575 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala
580 585 590 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val 610 615 620 Val Ala Ile Ala Ser His Asp Gly Gly Lys
Gln Ala Leu Glu Thr Val625 630 635 640 Gln Arg Leu Leu Pro Val Leu
Cys Gln Asp His Gly Leu Thr Pro Asp 645 650 655 Gln Val Val Ala Ile
Ala Asn Asn Asn Gly Gly Lys Gln Ala Leu Glu 660 665 670 Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 675 680 685 Pro
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 690 695
700 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly705 710 715 720 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn
Gly Gly Gly Lys 725 730 735 Gln Ala Leu Glu Ser Ile Val Ala Gln Leu
Ser Arg Arg Asp Pro Ala 740 745 750 Leu Ala Ala Leu Thr Asn Asp His
Leu Val Ala Leu Ala Cys Leu Gly 755 760 765 Gly Arg Pro Ala Leu Asp
Ala Val Lys Lys Gly Leu Pro His Ala Pro 770 775 780 Glu Phe Ile Arg
Arg Val Asn Arg Arg Ile Ala Glu Arg Thr Ser His785 790 795 800 Arg
Val Ala Asp Tyr Ala His Val Val Arg Val Leu Glu Phe Phe Gln 805 810
815 Cys His Ser His Pro Ala His Ala Phe Asp Glu Ala Met Thr Gln Phe
820 825 830 Gly Met Ser Arg His Gly Leu Val Gln Leu Phe Arg Arg Val
Gly Val 835 840 845 Thr Glu Phe Glu Ala Arg Tyr Gly Thr Leu Pro Pro
Ala Ser Gln Arg 850 855 860 Trp Asp Arg Ile Leu Gln Ala Ser Gly Met
Lys Arg Ala Lys Pro Ser865 870 875 880 Pro Thr Ser Ala Gln Thr Pro
Asp Gln Thr Ser Leu His Ala Phe Ala 885 890 895 Asp Ser Leu Glu Arg
Asp Leu Asp Ala Pro Ser Pro Met His Glu Gly 900 905 910 Asp Gln Thr
Arg Ala Ser Ser Arg Lys Arg Ser Arg Ser Asp Arg Ala 915 920 925 Val
Thr Gly Pro Ser Ala Gln Gln Ala Val Glu Val Arg Val Pro Glu 930 935
940 Gln Arg Asp Ala Leu His Leu Pro Leu Ser Trp Arg Val Lys Arg
Pro945 950 955 960 Arg Thr Arg Ile Trp Gly Gly Leu Pro Asp Pro Ile
Ser Arg Ser Gln 965 970 975 Leu Val Lys Ser Glu Leu Glu Glu Lys Lys
Ser Glu Leu Arg His Lys 980 985 990 Leu Lys Tyr Val Pro His Glu Tyr
Ile Glu Leu Ile Glu Ile Ala Arg 995 1000 1005 Asn Ser Thr Gln Asp
Arg Ile Leu Glu Met Lys Val Met Glu Phe Phe 1010 1015 1020 Met Lys
Val Tyr Gly Tyr Arg Gly Lys His Leu Gly Gly Ser Arg Lys1025 1030
1035 1040 Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr
Gly Val 1045 1050 1055 Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr
Asn Leu Pro Ile Gly 1060 1065 1070 Gln Ala Asp Glu Met Gln Arg Tyr
Val Glu Glu Asn Gln Thr Arg Asn 1075 1080 1085 Lys His Ile Asn Pro
Asn Glu Trp Trp Lys Val Tyr Pro Ser Ser Val 1090 1095 1100 Thr Glu
Phe Lys Phe Leu Phe Val Ser Gly His Phe Lys Gly Asn Tyr1105 1110
1115 1120 Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys Asn
Gly Ala 1125 1130 1135 Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly
Glu Met Ile Lys Ala 1140 1145 1150 Gly Thr Leu Thr Leu Glu Glu Val
Arg Arg Lys Phe Asn Asn Gly Glu 1155 1160 1165 Ile Asn Phe 1170
431239PRTArtificial Sequencesynthetic peptide 43Met Ala Ser Ser Pro
Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser Gly
Trp Ser Arg Met His Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30 Pro
Ser Pro Ala Arg Glu Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35 40
45 Gln Pro Thr Ala Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro Leu
50 55 60 Asp Gly Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg Leu
Pro Ser65 70 75 80 Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly Ser
Phe Ser Asp Leu 85 90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu Asp
Thr Ser Leu Leu Asp Ser 100 105 110 Met Pro Ala Val Gly Thr Pro His
Thr Ala Ala Ala Pro Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser Ala
Leu Arg Ala Ala Asp Asp Pro Pro Pro Thr 130 135 140 Val Arg Val Ala
Val Thr Ala Ala Arg Pro Pro Arg Ala Lys Pro Ala145 150 155 160 Pro
Arg Arg Arg Ala Ala Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165 170
175 Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile
180 185 190 Lys Pro Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala
Leu Val 195 200 205 Gly His Gly Phe Thr His Ala His Ile Val Ala Leu
Ser Gln His Pro 210 215 220 Ala Ala Leu Gly Thr Val Ala Val Thr Tyr
Gln His Ile Ile Thr Ala225 230 235 240 Leu Pro Glu Ala Thr His Glu
Asp Ile Val Gly Val Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg Ala
Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly Pro
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala 275 280 285 Lys
Arg Gly Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn 290 295
300 Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val
Ala305 310 315 320 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg 325 330 335 Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp Gln Val 340 345 350 Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala Leu Glu Thr Val 355 360 365 Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr Pro Asp 370 375 380 Gln Val Val Ala
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu385 390 395 400 Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 405 410
415 Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
420 425 430 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 435 440 445 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Asn Asn
Asn Gly Gly Lys 450 455 460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Asn Ile Gly 485 490 495 Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 515 520 525 Ile
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535
540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
Ala545 550 555 560 Asn Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser Asn Gly Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys
Gln Asp His Gly Leu Thr Pro Asp Gln Val 610 615 620 Val Ala Ile Ala
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val625 630 635 640 Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 645 650
655 Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
660 665 670 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr 675 680 685 Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly
Gly Lys Gln Ala 690 695 700 Leu Glu Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly705 710 715 720 Leu Thr Pro Asp Gln Val Val
Ala Ile Ala Ser His Asp Gly Gly Lys 725 730 735 Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 740 745 750 His Gly Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly 755 760 765 Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 770 775
780 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
Asn785 790 795 800 Gly Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala
Gln Leu Ser Arg 805 810 815 Arg Asp Pro Ala Leu Ala Ala Leu Thr Asn
Asp His Leu Val Ala Leu 820 825 830 Ala Cys Leu Gly Gly Arg Pro Ala
Leu Asp Ala Val Lys Lys Gly Leu 835 840 845 Pro His Ala Pro Glu Phe
Ile Arg Arg Val Asn Arg Arg Ile Ala Glu 850 855 860 Arg Thr Ser His
Arg Val Ala Asp Tyr Ala His Val Val Arg Val Leu865 870 875 880 Glu
Phe Phe Gln Cys His Ser His Pro Ala His Ala Phe Asp Glu Ala 885 890
895 Met Thr Gln Phe Gly Met Ser Arg His Gly Leu Val Gln Leu Phe Arg
900 905 910 Arg Val Gly Val Thr Glu Phe Glu Ala Arg Tyr Gly Thr Leu
Pro Pro 915 920 925 Ala Ser Gln Arg Trp Asp Arg Ile Leu Gln Ala Ser
Gly Met Lys Arg 930 935 940 Ala Lys Pro Ser Pro Thr Ser Ala Gln Thr
Pro Asp Gln Thr Ser Leu945 950 955 960 His Ala Phe Ala Asp Ser Leu
Glu Arg Asp Leu Asp Ala Pro Ser Pro 965 970 975 Met His Glu Gly Asp
Gln Thr Arg Ala Ser Ser Arg Lys Arg Ser Arg 980 985 990 Ser Asp Arg
Ala Val Thr Gly Pro Ser Ala Gln Gln Ala Val Glu Val 995 1000 1005
Arg Val Pro Glu Gln Arg Asp Ala Leu His Leu Pro Leu Ser Trp Arg
1010 1015 1020 Val Lys Arg Pro Arg Thr Arg Ile Trp Gly Gly Leu Pro
Asp Pro Ile1025 1030 1035 1040 Ser Arg Ser Gln Leu Val Lys Ser Glu
Leu Glu Glu Lys Lys Ser Glu 1045 1050 1055 Leu Arg His Lys Leu Lys
Tyr Val Pro His Glu Tyr Ile Glu Leu Ile 1060 1065 1070 Glu Ile Ala
Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys Val 1075 1080 1085
Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly
1090 1095 1100 Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly
Ser Pro Ile1105 1110 1115 1120 Asp Tyr Gly Val Ile Val Asp Thr Lys
Ala Tyr Ser Gly Gly Tyr Asn 1125 1130 1135 Leu Pro Ile Gly Gln Ala
Asp Glu Met Gln Arg Tyr Val Glu Glu Asn 1140 1145 1150 Gln Thr Arg
Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr 1155 1160 1165
Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe
1170 1175 1180 Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His
Ile Thr Asn1185 1190 1195 1200 Cys Asn Gly Ala Val Leu Ser Val Glu
Glu Leu Leu Ile Gly Gly Glu 1205 1210 1215 Met Ile Lys Ala Gly Thr
Leu Thr Leu Glu Glu Val Arg Arg Lys Phe 1220 1225 1230 Asn Asn Gly
Glu Ile Asn Phe 1235 441239PRTArtificial Sequencesynthetic peptide
44Met Ala Ser Ser Pro Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp1
5 10 15 Ala Ser Gly Trp Ser Arg Met His Ala Asp Pro Ile Arg Pro Arg
Arg 20 25 30 Pro Ser Pro Ala Arg Glu Leu Leu Pro Gly Pro Gln Pro
Asp Arg Val 35 40 45 Gln Pro Thr Ala Asp Arg Gly Val Ser Ala Pro
Ala Gly Ser Pro Leu 50 55 60 Asp Gly Leu Pro Ala Arg Arg Thr Val
Ser Arg Thr Arg Leu Pro Ser65 70 75 80 Pro Pro Ala Pro Ser Pro Ala
Phe Ser Ala Gly Ser Phe Ser Asp Leu 85 90 95 Leu Arg Pro Phe Asp
Pro Ser Leu Leu Asp Thr Ser Leu Leu Asp Ser 100 105 110 Met Pro Ala
Val Gly Thr Pro His Thr Ala Ala Ala Pro Ala Glu Trp 115 120 125 Asp
Glu Ala Gln Ser Ala Leu Arg Ala Ala Asp Asp Pro Pro Pro Thr 130 135
140 Val Arg Val Ala Val Thr Ala Ala Arg Pro Pro Arg Ala Lys Pro
Ala145 150 155 160 Pro Arg Arg Arg Ala Ala Gln Pro Ser Asp Ala Ser
Pro Ala Ala Gln 165 170 175 Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln
Gln Gln Gln Glu Lys Ile 180 185 190 Lys Pro Lys Val Arg Ser Thr Val
Ala Gln His His Glu Ala Leu Val 195 200 205 Gly His Gly Phe Thr His
Ala His Ile Val Ala Leu Ser Gln His Pro 210 215 220 Ala Ala Leu Gly
Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala225 230 235 240 Leu
Pro Glu Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp 245 250
255 Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu
260 265 270 Arg Gly Pro Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys
Ile Ala 275 280 285 Lys Arg Gly Gly Val Thr Ala Met Glu Ala Val His
Ala Ser Arg Asn 290 295 300 Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr
Pro Ala Gln Val Val Ala305 310 315 320 Ile Ala Ser His Asp Gly Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg 325 330 335 Leu Leu Pro Val Leu
Cys Gln Asp His Gly
Leu Thr Pro Asp Gln Val 340 345 350 Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala Leu Glu Thr Val 355 360 365 Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr Pro Asp 370 375 380 Gln Val Val Ala
Ile Ala Asn Asn Asn Gly Gly Lys Gln Ala Leu Glu385 390 395 400 Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 405 410
415 Pro Asp Gln Val Val Ala Ile Ala Asn Asn Asn Gly Gly Lys Gln Ala
420 425 430 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 435 440 445 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn
Ile Gly Gly Lys 450 455 460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Asn Gly Gly 485 490 495 Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Asn Asn 515 520 525 Asn
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535
540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
Ala545 550 555 560 Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser Asn Gly Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys
Gln Asp His Gly Leu Thr Pro Asp Gln Val 610 615 620 Val Ala Ile Ala
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val625 630 635 640 Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 645 650
655 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
660 665 670 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr 675 680 685 Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly
Gly Lys Gln Ala 690 695 700 Leu Glu Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly705 710 715 720 Leu Thr Pro Asp Gln Val Val
Ala Ile Ala Ser His Asp Gly Gly Lys 725 730 735 Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 740 745 750 His Gly Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly 755 760 765 Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 770 775
780 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
Asn785 790 795 800 Gly Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala
Gln Leu Ser Arg 805 810 815 Arg Asp Pro Ala Leu Ala Ala Leu Thr Asn
Asp His Leu Val Ala Leu 820 825 830 Ala Cys Leu Gly Gly Arg Pro Ala
Leu Asp Ala Val Lys Lys Gly Leu 835 840 845 Pro His Ala Pro Glu Phe
Ile Arg Arg Val Asn Arg Arg Ile Ala Glu 850 855 860 Arg Thr Ser His
Arg Val Ala Asp Tyr Ala His Val Val Arg Val Leu865 870 875 880 Glu
Phe Phe Gln Cys His Ser His Pro Ala His Ala Phe Asp Glu Ala 885 890
895 Met Thr Gln Phe Gly Met Ser Arg His Gly Leu Val Gln Leu Phe Arg
900 905 910 Arg Val Gly Val Thr Glu Phe Glu Ala Arg Tyr Gly Thr Leu
Pro Pro 915 920 925 Ala Ser Gln Arg Trp Asp Arg Ile Leu Gln Ala Ser
Gly Met Lys Arg 930 935 940 Ala Lys Pro Ser Pro Thr Ser Ala Gln Thr
Pro Asp Gln Thr Ser Leu945 950 955 960 His Ala Phe Ala Asp Ser Leu
Glu Arg Asp Leu Asp Ala Pro Ser Pro 965 970 975 Met His Glu Gly Asp
Gln Thr Arg Ala Ser Ser Arg Lys Arg Ser Arg 980 985 990 Ser Asp Arg
Ala Val Thr Gly Pro Ser Ala Gln Gln Ala Val Glu Val 995 1000 1005
Arg Val Pro Glu Gln Arg Asp Ala Leu His Leu Pro Leu Ser Trp Arg
1010 1015 1020 Val Lys Arg Pro Arg Thr Arg Ile Trp Gly Gly Leu Pro
Asp Pro Ile1025 1030 1035 1040 Ser Arg Ser Gln Leu Val Lys Ser Glu
Leu Glu Glu Lys Lys Ser Glu 1045 1050 1055 Leu Arg His Lys Leu Lys
Tyr Val Pro His Glu Tyr Ile Glu Leu Ile 1060 1065 1070 Glu Ile Ala
Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys Val 1075 1080 1085
Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly
1090 1095 1100 Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly
Ser Pro Ile1105 1110 1115 1120 Asp Tyr Gly Val Ile Val Asp Thr Lys
Ala Tyr Ser Gly Gly Tyr Asn 1125 1130 1135 Leu Pro Ile Gly Gln Ala
Asp Glu Met Gln Arg Tyr Val Glu Glu Asn 1140 1145 1150 Gln Thr Arg
Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr 1155 1160 1165
Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe
1170 1175 1180 Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His
Ile Thr Asn1185 1190 1195 1200 Cys Asn Gly Ala Val Leu Ser Val Glu
Glu Leu Leu Ile Gly Gly Glu 1205 1210 1215 Met Ile Lys Ala Gly Thr
Leu Thr Leu Glu Glu Val Arg Arg Lys Phe 1220 1225 1230 Asn Asn Gly
Glu Ile Asn Phe 1235 451239PRTArtificial Sequencesynthetic peptide
45Met Ala Ser Ser Pro Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp1
5 10 15 Ala Ser Gly Trp Ser Arg Met His Ala Asp Pro Ile Arg Pro Arg
Arg 20 25 30 Pro Ser Pro Ala Arg Glu Leu Leu Pro Gly Pro Gln Pro
Asp Arg Val 35 40 45 Gln Pro Thr Ala Asp Arg Gly Val Ser Ala Pro
Ala Gly Ser Pro Leu 50 55 60 Asp Gly Leu Pro Ala Arg Arg Thr Val
Ser Arg Thr Arg Leu Pro Ser65 70 75 80 Pro Pro Ala Pro Ser Pro Ala
Phe Ser Ala Gly Ser Phe Ser Asp Leu 85 90 95 Leu Arg Pro Phe Asp
Pro Ser Leu Leu Asp Thr Ser Leu Leu Asp Ser 100 105 110 Met Pro Ala
Val Gly Thr Pro His Thr Ala Ala Ala Pro Ala Glu Trp 115 120 125 Asp
Glu Ala Gln Ser Ala Leu Arg Ala Ala Asp Asp Pro Pro Pro Thr 130 135
140 Val Arg Val Ala Val Thr Ala Ala Arg Pro Pro Arg Ala Lys Pro
Ala145 150 155 160 Pro Arg Arg Arg Ala Ala Gln Pro Ser Asp Ala Ser
Pro Ala Ala Gln 165 170 175 Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln
Gln Gln Gln Glu Lys Ile 180 185 190 Lys Pro Lys Val Arg Ser Thr Val
Ala Gln His His Glu Ala Leu Val 195 200 205 Gly His Gly Phe Thr His
Ala His Ile Val Ala Leu Ser Gln His Pro 210 215 220 Ala Ala Leu Gly
Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala225 230 235 240 Leu
Pro Glu Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp 245 250
255 Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu
260 265 270 Arg Gly Pro Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys
Ile Ala 275 280 285 Lys Arg Gly Gly Val Thr Ala Met Glu Ala Val His
Ala Ser Arg Asn 290 295 300 Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr
Pro Ala Gln Val Val Ala305 310 315 320 Ile Ala Ser Asn Ile Gly Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg 325 330 335 Leu Leu Pro Val Leu
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 340 345 350 Val Ala Ile
Ala Asn Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val 355 360 365 Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 370 375
380 Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu
Glu385 390 395 400 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly Leu Thr 405 410 415 Pro Asp Gln Val Val Ala Ile Ala Ser His
Asp Gly Gly Lys Gln Ala 420 425 430 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 435 440 445 Leu Thr Pro Asp Gln Val
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 450 455 460 Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp465 470 475 480 His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly 485 490
495 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
500 505 510 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Asn 515 520 525 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val 530 535 540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala545 550 555 560 Ser His Asp Gly Gly Lys Gln
Ala Leu Glu Thr Val Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser
His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 610 615
620 Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr
Val625 630 635 640 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp 645 650 655 Gln Val Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala Leu Glu 660 665 670 Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr 675 680 685 Pro Asp Gln Val Val Ala
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala 690 695 700 Leu Glu Thr Val
Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly705 710 715 720 Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 725 730
735 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
740 745 750 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His
Asp Gly 755 760 765 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys 770 775 780 Gln Asp His Gly Leu Thr Pro Asp Gln Val
Val Ala Ile Ala Ser Asn785 790 795 800 Gly Gly Gly Lys Gln Ala Leu
Glu Ser Ile Val Ala Gln Leu Ser Arg 805 810 815 Arg Asp Pro Ala Leu
Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu 820 825 830 Ala Cys Leu
Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu 835 840 845 Pro
His Ala Pro Glu Phe Ile Arg Arg Val Asn Arg Arg Ile Ala Glu 850 855
860 Arg Thr Ser His Arg Val Ala Asp Tyr Ala His Val Val Arg Val
Leu865 870 875 880 Glu Phe Phe Gln Cys His Ser His Pro Ala His Ala
Phe Asp Glu Ala 885 890 895 Met Thr Gln Phe Gly Met Ser Arg His Gly
Leu Val Gln Leu Phe Arg 900 905 910 Arg Val Gly Val Thr Glu Phe Glu
Ala Arg Tyr Gly Thr Leu Pro Pro 915 920 925 Ala Ser Gln Arg Trp Asp
Arg Ile Leu Gln Ala Ser Gly Met Lys Arg 930 935 940 Ala Lys Pro Ser
Pro Thr Ser Ala Gln Thr Pro Asp Gln Thr Ser Leu945 950 955 960 His
Ala Phe Ala Asp Ser Leu Glu Arg Asp Leu Asp Ala Pro Ser Pro 965 970
975 Met His Glu Gly Asp Gln Thr Arg Ala Ser Ser Arg Lys Arg Ser Arg
980 985 990 Ser Asp Arg Ala Val Thr Gly Pro Ser Ala Gln Gln Ala Val
Glu Val 995 1000 1005 Arg Val Pro Glu Gln Arg Asp Ala Leu His Leu
Pro Leu Ser Trp Arg 1010 1015 1020 Val Lys Arg Pro Arg Thr Arg Ile
Trp Gly Gly Leu Pro Asp Pro Ile1025 1030 1035 1040 Ser Arg Ser Gln
Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu 1045 1050 1055 Leu
Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile 1060
1065 1070 Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met
Lys Val 1075 1080 1085 Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg
Gly Lys His Leu Gly 1090 1095 1100 Gly Ser Arg Lys Pro Asp Gly Ala
Ile Tyr Thr Val Gly Ser Pro Ile1105 1110 1115 1120 Asp Tyr Gly Val
Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn 1125 1130 1135 Leu
Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu Asn 1140
1145 1150 Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys
Val Tyr 1155 1160 1165 Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe
Val Ser Gly His Phe 1170 1175 1180 Lys Gly Asn Tyr Lys Ala Gln Leu
Thr Arg Leu Asn His Ile Thr Asn1185 1190 1195 1200 Cys Asn Gly Ala
Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu 1205 1210 1215 Met
Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe 1220
1225 1230 Asn Asn Gly Glu Ile Asn Phe 1235 461239PRTArtificial
Sequencesynthetic peptide 46Met Ala Ser Ser Pro Pro Lys Lys Lys Arg
Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser Gly Trp Ser Arg Met His
Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30 Pro Ser Pro Ala Arg Glu
Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35 40 45 Gln Pro Thr Ala
Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro Leu 50 55 60 Asp Gly
Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg Leu Pro Ser65 70 75 80
Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly Ser Phe Ser Asp Leu 85
90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu Asp Thr Ser Leu Leu Asp
Ser 100 105 110 Met Pro Ala Val Gly Thr Pro His Thr Ala Ala Ala Pro
Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser Ala Leu Arg Ala Ala Asp
Asp Pro Pro Pro Thr 130 135 140 Val Arg Val Ala Val Thr Ala Ala Arg
Pro Pro Arg Ala Lys Pro Ala145 150 155 160 Pro Arg Arg Arg Ala Ala
Gln Pro Ser Asp Ala Ser
Pro Ala Ala Gln 165 170 175 Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln
Gln Gln Gln Glu Lys Ile 180 185 190 Lys Pro Lys Val Arg Ser Thr Val
Ala Gln His His Glu Ala Leu Val 195 200 205 Gly His Gly Phe Thr His
Ala His Ile Val Ala Leu Ser Gln His Pro 210 215 220 Ala Ala Leu Gly
Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala225 230 235 240 Leu
Pro Glu Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp 245 250
255 Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu
260 265 270 Arg Gly Pro Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys
Ile Ala 275 280 285 Lys Arg Gly Gly Val Thr Ala Met Glu Ala Val His
Ala Ser Arg Asn 290 295 300 Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr
Pro Ala Gln Val Val Ala305 310 315 320 Ile Ala Asn Asn Asn Gly Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg 325 330 335 Leu Leu Pro Val Leu
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 340 345 350 Val Ala Ile
Ala Asn Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val 355 360 365 Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 370 375
380 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
Glu385 390 395 400 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly Leu Thr 405 410 415 Pro Asp Gln Val Val Ala Ile Ala Ser Asn
Ile Gly Gly Lys Gln Ala 420 425 430 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 435 440 445 Leu Thr Pro Asp Gln Val
Val Ala Ile Ala Ser His Asp Gly Gly Lys 450 455 460 Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp465 470 475 480 His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly 485 490
495 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
500 505 510 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Asn Asn 515 520 525 Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val 530 535 540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala545 550 555 560 Ser His Asp Gly Gly Lys Gln
Ala Leu Glu Thr Val Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 610 615
620 Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr
Val625 630 635 640 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp 645 650 655 Gln Val Val Ala Ile Ala Ser Asn Ile Gly
Gly Lys Gln Ala Leu Glu 660 665 670 Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr 675 680 685 Pro Asp Gln Val Val Ala
Ile Ala Ser His Asp Gly Gly Lys Gln Ala 690 695 700 Leu Glu Thr Val
Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly705 710 715 720 Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys 725 730
735 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
740 745 750 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His
Asp Gly 755 760 765 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys 770 775 780 Gln Asp His Gly Leu Thr Pro Asp Gln Val
Val Ala Ile Ala Ser Asn785 790 795 800 Gly Gly Gly Lys Gln Ala Leu
Glu Ser Ile Val Ala Gln Leu Ser Arg 805 810 815 Arg Asp Pro Ala Leu
Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu 820 825 830 Ala Cys Leu
Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu 835 840 845 Pro
His Ala Pro Glu Phe Ile Arg Arg Val Asn Arg Arg Ile Ala Glu 850 855
860 Arg Thr Ser His Arg Val Ala Asp Tyr Ala His Val Val Arg Val
Leu865 870 875 880 Glu Phe Phe Gln Cys His Ser His Pro Ala His Ala
Phe Asp Glu Ala 885 890 895 Met Thr Gln Phe Gly Met Ser Arg His Gly
Leu Val Gln Leu Phe Arg 900 905 910 Arg Val Gly Val Thr Glu Phe Glu
Ala Arg Tyr Gly Thr Leu Pro Pro 915 920 925 Ala Ser Gln Arg Trp Asp
Arg Ile Leu Gln Ala Ser Gly Met Lys Arg 930 935 940 Ala Lys Pro Ser
Pro Thr Ser Ala Gln Thr Pro Asp Gln Thr Ser Leu945 950 955 960 His
Ala Phe Ala Asp Ser Leu Glu Arg Asp Leu Asp Ala Pro Ser Pro 965 970
975 Met His Glu Gly Asp Gln Thr Arg Ala Ser Ser Arg Lys Arg Ser Arg
980 985 990 Ser Asp Arg Ala Val Thr Gly Pro Ser Ala Gln Gln Ala Val
Glu Val 995 1000 1005 Arg Val Pro Glu Gln Arg Asp Ala Leu His Leu
Pro Leu Ser Trp Arg 1010 1015 1020 Val Lys Arg Pro Arg Thr Arg Ile
Trp Gly Gly Leu Pro Asp Pro Ile1025 1030 1035 1040 Ser Arg Ser Gln
Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu 1045 1050 1055 Leu
Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile 1060
1065 1070 Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met
Lys Val 1075 1080 1085 Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg
Gly Lys His Leu Gly 1090 1095 1100 Gly Ser Arg Lys Pro Asp Gly Ala
Ile Tyr Thr Val Gly Ser Pro Ile1105 1110 1115 1120 Asp Tyr Gly Val
Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn 1125 1130 1135 Leu
Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu Asn 1140
1145 1150 Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys
Val Tyr 1155 1160 1165 Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe
Val Ser Gly His Phe 1170 1175 1180 Lys Gly Asn Tyr Lys Ala Gln Leu
Thr Arg Leu Asn His Ile Thr Asn1185 1190 1195 1200 Cys Asn Gly Ala
Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu 1205 1210 1215 Met
Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe 1220
1225 1230 Asn Asn Gly Glu Ile Asn Phe 1235 471239PRTArtificial
Sequencesynthetic peptide 47Met Ala Ser Ser Pro Pro Lys Lys Lys Arg
Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser Gly Trp Ser Arg Met His
Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30 Pro Ser Pro Ala Arg Glu
Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35 40 45 Gln Pro Thr Ala
Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro Leu 50 55 60 Asp Gly
Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg Leu Pro Ser65 70 75 80
Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly Ser Phe Ser Asp Leu 85
90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu Asp Thr Ser Leu Leu Asp
Ser 100 105 110 Met Pro Ala Val Gly Thr Pro His Thr Ala Ala Ala Pro
Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser Ala Leu Arg Ala Ala Asp
Asp Pro Pro Pro Thr 130 135 140 Val Arg Val Ala Val Thr Ala Ala Arg
Pro Pro Arg Ala Lys Pro Ala145 150 155 160 Pro Arg Arg Arg Ala Ala
Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165 170 175 Val Asp Leu Arg
Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile 180 185 190 Lys Pro
Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val 195 200 205
Gly His Gly Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro 210
215 220 Ala Ala Leu Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr
Ala225 230 235 240 Leu Pro Glu Ala Thr His Glu Asp Ile Val Gly Val
Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu
Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly Pro Pro Leu Gln Leu Asp
Thr Gly Gln Leu Val Lys Ile Ala 275 280 285 Lys Arg Gly Gly Val Thr
Ala Met Glu Ala Val His Ala Ser Arg Asn 290 295 300 Ala Leu Thr Gly
Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val Ala305 310 315 320 Ile
Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 325 330
335 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val
340 345 350 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
Thr Val 355 360 365 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp 370 375 380 Gln Val Val Ala Ile Ala Ser Asn Gly Gly
Gly Lys Gln Ala Leu Glu385 390 395 400 Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln Asp His Gly Leu Thr 405 410 415 Pro Asp Gln Val Val
Ala Ile Ala Asn Asn Asn Gly Gly Lys Gln Ala 420 425 430 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 435 440 445 Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys 450 455
460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser His Asp Gly 485 490 495 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala Ser His 515 520 525 Asp Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535 540 Leu Cys Gln Asp
His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala545 550 555 560 Asn
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 565 570
575 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala
580 585 590 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val 610 615 620 Val Ala Ile Ala Ser Asn Ile Gly Gly Lys
Gln Ala Leu Glu Thr Val625 630 635 640 Gln Arg Leu Leu Pro Val Leu
Cys Gln Asp His Gly Leu Thr Pro Asp 645 650 655 Gln Val Val Ala Ile
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu 660 665 670 Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 675 680 685 Pro
Asp Gln Val Val Ala Ile Ala Asn Asn Asn Gly Gly Lys Gln Ala 690 695
700 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly705 710 715 720 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn
Ile Gly Gly Lys 725 730 735 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp 740 745 750 His Gly Leu Thr Pro Asp Gln Val
Val Ala Ile Ala Ser Asn Gly Gly 755 760 765 Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys 770 775 780 Gln Asp His Gly
Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn785 790 795 800 Gly
Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg 805 810
815 Arg Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu
820 825 830 Ala Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys
Gly Leu 835 840 845 Pro His Ala Pro Glu Phe Ile Arg Arg Val Asn Arg
Arg Ile Ala Glu 850 855 860 Arg Thr Ser His Arg Val Ala Asp Tyr Ala
His Val Val Arg Val Leu865 870 875 880 Glu Phe Phe Gln Cys His Ser
His Pro Ala His Ala Phe Asp Glu Ala 885 890 895 Met Thr Gln Phe Gly
Met Ser Arg His Gly Leu Val Gln Leu Phe Arg 900 905 910 Arg Val Gly
Val Thr Glu Phe Glu Ala Arg Tyr Gly Thr Leu Pro Pro 915 920 925 Ala
Ser Gln Arg Trp Asp Arg Ile Leu Gln Ala Ser Gly Met Lys Arg 930 935
940 Ala Lys Pro Ser Pro Thr Ser Ala Gln Thr Pro Asp Gln Thr Ser
Leu945 950 955 960 His Ala Phe Ala Asp Ser Leu Glu Arg Asp Leu Asp
Ala Pro Ser Pro 965 970 975 Met His Glu Gly Asp Gln Thr Arg Ala Ser
Ser Arg Lys Arg Ser Arg 980 985 990 Ser Asp Arg Ala Val Thr Gly Pro
Ser Ala Gln Gln Ala Val Glu Val 995 1000 1005 Arg Val Pro Glu Gln
Arg Asp Ala Leu His Leu Pro Leu Ser Trp Arg 1010 1015 1020 Val Lys
Arg Pro Arg Thr Arg Ile Trp Gly Gly Leu Pro Asp Pro Ile1025 1030
1035 1040 Ser Arg Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys
Ser Glu 1045 1050 1055 Leu Arg His Lys Leu Lys Tyr Val Pro His Glu
Tyr Ile Glu Leu Ile 1060 1065 1070 Glu Ile Ala Arg Asn Ser Thr Gln
Asp Arg Ile Leu Glu Met Lys Val 1075 1080 1085 Met Glu Phe Phe Met
Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly 1090 1095 1100 Gly Ser
Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile1105 1110
1115 1120 Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly
Tyr Asn 1125 1130 1135 Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg
Tyr Val Glu Glu Asn 1140 1145 1150 Gln Thr Arg Asn Lys His Ile Asn
Pro Asn Glu Trp Trp Lys Val Tyr 1155 1160 1165 Pro Ser Ser Val Thr
Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe 1170 1175 1180 Lys Gly
Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn1185 1190
1195 1200 Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly
Gly Glu 1205 1210 1215 Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu
Val Arg Arg Lys Phe 1220 1225 1230 Asn Asn Gly Glu Ile Asn Phe
1235 481239PRTArtificial Sequencesynthetic peptide 48Met Ala Ser
Ser Pro Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp1 5 10 15 Ala
Ser Gly Trp Ser Arg Met His Ala Asp Pro Ile Arg Pro Arg Arg 20 25
30 Pro Ser Pro Ala Arg Glu Leu Leu Pro Gly Pro Gln Pro Asp Arg Val
35 40 45 Gln Pro Thr Ala Asp Arg Gly Val Ser Ala Pro Ala Gly Ser
Pro Leu 50 55 60 Asp Gly Leu Pro Ala Arg Arg Thr Val Ser Arg Thr
Arg Leu Pro Ser65 70 75 80 Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala
Gly Ser Phe Ser Asp Leu 85 90 95 Leu Arg Pro Phe Asp Pro Ser Leu
Leu Asp Thr Ser Leu Leu Asp Ser 100 105 110 Met Pro Ala Val Gly Thr
Pro His Thr Ala Ala Ala Pro Ala Glu Trp 115 120 125 Asp Glu Ala Gln
Ser Ala Leu Arg Ala Ala Asp Asp Pro Pro Pro Thr 130 135 140 Val Arg
Val Ala Val Thr Ala Ala Arg Pro Pro Arg Ala Lys Pro Ala145 150 155
160 Pro Arg Arg Arg Ala Ala Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln
165 170 175 Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu
Lys Ile 180 185 190 Lys Pro Lys Val Arg Ser Thr Val Ala Gln His His
Glu Ala Leu Val 195 200 205 Gly His Gly Phe Thr His Ala His Ile Val
Ala Leu Ser Gln His Pro 210 215 220 Ala Ala Leu Gly Thr Val Ala Val
Thr Tyr Gln His Ile Ile Thr Ala225 230 235 240 Leu Pro Glu Ala Thr
His Glu Asp Ile Val Gly Val Gly Lys Gln Trp 245 250 255 Ser Gly Ala
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu 260 265 270 Arg
Gly Pro Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala 275 280
285 Lys Arg Gly Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn
290 295 300 Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr Pro Ala Gln Val
Val Ala305 310 315 320 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg 325 330 335 Leu Leu Pro Val Leu Cys Gln Asp His
Gly Leu Thr Pro Asp Gln Val 340 345 350 Val Ala Ile Ala Asn Asn Asn
Gly Gly Lys Gln Ala Leu Glu Thr Val 355 360 365 Gln Arg Leu Leu Pro
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 370 375 380 Gln Val Val
Ala Ile Ala Asn Asn Asn Gly Gly Lys Gln Ala Leu Glu385 390 395 400
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 405
410 415 Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
Ala 420 425 430 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Asp His Gly 435 440 445 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
Asn Gly Gly Gly Lys 450 455 460 Gln Ala Leu Glu Thr Val Gln Arg Leu
Leu Pro Val Leu Cys Gln Asp465 470 475 480 His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala Ser Asn Gly Gly 485 490 495 Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp
His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Asn Asn 515 520 525
Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530
535 540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
Ala545 550 555 560 Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser Asn Gly Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys
Gln Asp His Gly Leu Thr Pro Asp Gln Val 610 615 620 Val Ala Ile Ala
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val625 630 635 640 Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 645 650
655 Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
660 665 670 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr 675 680 685 Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala 690 695 700 Leu Glu Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly705 710 715 720 Leu Thr Pro Asp Gln Val Val
Ala Ile Ala Asn Asn Asn Gly Gly Lys 725 730 735 Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 740 745 750 His Gly Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly 755 760 765 Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 770 775
780 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
Asn785 790 795 800 Gly Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala
Gln Leu Ser Arg 805 810 815 Arg Asp Pro Ala Leu Ala Ala Leu Thr Asn
Asp His Leu Val Ala Leu 820 825 830 Ala Cys Leu Gly Gly Arg Pro Ala
Leu Asp Ala Val Lys Lys Gly Leu 835 840 845 Pro His Ala Pro Glu Phe
Ile Arg Arg Val Asn Arg Arg Ile Ala Glu 850 855 860 Arg Thr Ser His
Arg Val Ala Asp Tyr Ala His Val Val Arg Val Leu865 870 875 880 Glu
Phe Phe Gln Cys His Ser His Pro Ala His Ala Phe Asp Glu Ala 885 890
895 Met Thr Gln Phe Gly Met Ser Arg His Gly Leu Val Gln Leu Phe Arg
900 905 910 Arg Val Gly Val Thr Glu Phe Glu Ala Arg Tyr Gly Thr Leu
Pro Pro 915 920 925 Ala Ser Gln Arg Trp Asp Arg Ile Leu Gln Ala Ser
Gly Met Lys Arg 930 935 940 Ala Lys Pro Ser Pro Thr Ser Ala Gln Thr
Pro Asp Gln Thr Ser Leu945 950 955 960 His Ala Phe Ala Asp Ser Leu
Glu Arg Asp Leu Asp Ala Pro Ser Pro 965 970 975 Met His Glu Gly Asp
Gln Thr Arg Ala Ser Ser Arg Lys Arg Ser Arg 980 985 990 Ser Asp Arg
Ala Val Thr Gly Pro Ser Ala Gln Gln Ala Val Glu Val 995 1000 1005
Arg Val Pro Glu Gln Arg Asp Ala Leu His Leu Pro Leu Ser Trp Arg
1010 1015 1020 Val Lys Arg Pro Arg Thr Arg Ile Trp Gly Gly Leu Pro
Asp Pro Ile1025 1030 1035 1040 Ser Arg Ser Gln Leu Val Lys Ser Glu
Leu Glu Glu Lys Lys Ser Glu 1045 1050 1055 Leu Arg His Lys Leu Lys
Tyr Val Pro His Glu Tyr Ile Glu Leu Ile 1060 1065 1070 Glu Ile Ala
Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys Val 1075 1080 1085
Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly
1090 1095 1100 Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly
Ser Pro Ile1105 1110 1115 1120 Asp Tyr Gly Val Ile Val Asp Thr Lys
Ala Tyr Ser Gly Gly Tyr Asn 1125 1130 1135 Leu Pro Ile Gly Gln Ala
Asp Glu Met Gln Arg Tyr Val Glu Glu Asn 1140 1145 1150 Gln Thr Arg
Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr 1155 1160 1165
Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe
1170 1175 1180 Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His
Ile Thr Asn1185 1190 1195 1200 Cys Asn Gly Ala Val Leu Ser Val Glu
Glu Leu Leu Ile Gly Gly Glu 1205 1210 1215 Met Ile Lys Ala Gly Thr
Leu Thr Leu Glu Glu Val Arg Arg Lys Phe 1220 1225 1230 Asn Asn Gly
Glu Ile Asn Phe 1235 491273PRTArtificial Sequencesynthetic peptide
49Met Ala Ser Ser Pro Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp1
5 10 15 Ala Ser Gly Trp Ser Arg Met His Ala Asp Pro Ile Arg Pro Arg
Arg 20 25 30 Pro Ser Pro Ala Arg Glu Leu Leu Pro Gly Pro Gln Pro
Asp Arg Val 35 40 45 Gln Pro Thr Ala Asp Arg Gly Val Ser Ala Pro
Ala Gly Ser Pro Leu 50 55 60 Asp Gly Leu Pro Ala Arg Arg Thr Val
Ser Arg Thr Arg Leu Pro Ser65 70 75 80 Pro Pro Ala Pro Ser Pro Ala
Phe Ser Ala Gly Ser Phe Ser Asp Leu 85 90 95 Leu Arg Pro Phe Asp
Pro Ser Leu Leu Asp Thr Ser Leu Leu Asp Ser 100 105 110 Met Pro Ala
Val Gly Thr Pro His Thr Ala Ala Ala Pro Ala Glu Trp 115 120 125 Asp
Glu Ala Gln Ser Ala Leu Arg Ala Ala Asp Asp Pro Pro Pro Thr 130 135
140 Val Arg Val Ala Val Thr Ala Ala Arg Pro Pro Arg Ala Lys Pro
Ala145 150 155 160 Pro Arg Arg Arg Ala Ala Gln Pro Ser Asp Ala Ser
Pro Ala Ala Gln 165 170 175 Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln
Gln Gln Gln Glu Lys Ile 180 185 190 Lys Pro Lys Val Arg Ser Thr Val
Ala Gln His His Glu Ala Leu Val 195 200 205 Gly His Gly Phe Thr His
Ala His Ile Val Ala Leu Ser Gln His Pro 210 215 220 Ala Ala Leu Gly
Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala225 230 235 240 Leu
Pro Glu Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp 245 250
255 Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu
260 265 270 Arg Gly Pro Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys
Ile Ala 275 280 285 Lys Arg Gly Gly Val Thr Ala Met Glu Ala Val His
Ala Ser Arg Asn 290 295 300 Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr
Pro Ala Gln Val Val Ala305 310 315 320 Ile Ala Ser Asn Ile Gly Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg 325 330 335 Leu Leu Pro Val Leu
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 340 345 350 Val Ala Ile
Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val 355 360 365 Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 370 375
380 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
Glu385 390 395 400 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly Leu Thr 405 410 415 Pro Asp Gln Val Val Ala Ile Ala Ser His
Asp Gly Gly Lys Gln Ala 420 425 430 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 435 440 445 Leu Thr Pro Asp Gln Val
Val Ala Ile Ala Ser His Asp Gly Gly Lys 450 455 460 Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp465 470 475 480 His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly 485 490
495 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
500 505 510 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser His 515 520 525 Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val 530 535 540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala545 550 555 560 Ser Asn Gly Gly Gly Lys Gln
Ala Leu Glu Thr Val Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser
His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 610 615
620 Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr
Val625 630 635 640 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp 645 650 655 Gln Val Val Ala Ile Ala Asn Asn Asn Gly
Gly Lys Gln Ala Leu Glu 660 665 670 Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr 675 680 685 Pro Asp Gln Val Val Ala
Ile Ala Ser His Asp Gly Gly Lys Gln Ala 690 695 700 Leu Glu Thr Val
Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly705 710 715 720 Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys 725 730
735 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
740 745 750 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn
Gly Gly 755 760 765 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys 770 775 780 Gln Asp His Gly Leu Thr Pro Asp Gln Val
Val Ala Ile Ala Ser His785 790 795 800 Asp Gly Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val 805 810 815 Leu Cys Gln Asp His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 820 825 830 Ser Asn Gly
Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala Gln Leu 835 840 845 Ser
Arg Arg Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val 850 855
860 Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys
Lys865 870 875 880 Gly Leu Pro His Ala Pro Glu Phe Ile Arg Arg Val
Asn Arg Arg Ile 885 890 895 Ala Glu Arg Thr Ser His Arg Val Ala Asp
Tyr Ala His Val Val Arg 900 905 910 Val Leu Glu Phe Phe Gln Cys His
Ser His Pro Ala His Ala Phe Asp 915 920 925 Glu Ala Met Thr Gln Phe
Gly Met Ser Arg His Gly Leu Val Gln Leu 930 935 940 Phe Arg Arg Val
Gly Val Thr Glu Phe Glu Ala Arg Tyr Gly Thr Leu945 950 955 960 Pro
Pro Ala Ser Gln Arg Trp Asp Arg Ile Leu Gln Ala Ser Gly Met 965 970
975 Lys Arg Ala Lys Pro Ser Pro Thr Ser Ala Gln Thr Pro Asp Gln Thr
980 985 990 Ser Leu His Ala Phe Ala Asp Ser Leu Glu Arg Asp Leu Asp
Ala Pro 995 1000 1005 Ser Pro Met His Glu Gly Asp Gln Thr Arg Ala
Ser Ser Arg Lys Arg 1010 1015 1020 Ser Arg Ser Asp Arg Ala Val Thr
Gly Pro Ser Ala Gln Gln Ala Val1025 1030 1035 1040 Glu Val Arg Val
Pro Glu Gln Arg Asp Ala Leu His Leu Pro Leu Ser 1045 1050 1055 Trp
Arg Val Lys Arg Pro Arg Thr Arg Ile
Trp Gly Gly Leu Pro Asp 1060 1065 1070 Pro Ile Ser Arg Ser Gln Leu
Val Lys Ser Glu Leu Glu Glu Lys Lys 1075 1080 1085 Ser Glu Leu Arg
His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu 1090 1095 1100 Leu
Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met1105
1110 1115 1120 Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg
Gly Lys His 1125 1130 1135 Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala
Ile Tyr Thr Val Gly Ser 1140 1145 1150 Pro Ile Asp Tyr Gly Val Ile
Val Asp Thr Lys Ala Tyr Ser Gly Gly 1155 1160 1165 Tyr Asn Leu Pro
Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu 1170 1175 1180 Glu
Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys1185
1190 1195 1200 Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe
Val Ser Gly 1205 1210 1215 His Phe Lys Gly Asn Tyr Lys Ala Gln Leu
Thr Arg Leu Asn His Ile 1220 1225 1230 Thr Asn Cys Asn Gly Ala Val
Leu Ser Val Glu Glu Leu Leu Ile Gly 1235 1240 1245 Gly Glu Met Ile
Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg 1250 1255 1260 Lys
Phe Asn Asn Gly Glu Ile Asn Phe1265 1270 501273PRTArtificial
Sequencesynthetic peptide 50Met Ala Ser Ser Pro Pro Lys Lys Lys Arg
Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser Gly Trp Ser Arg Met His
Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30 Pro Ser Pro Ala Arg Glu
Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35 40 45 Gln Pro Thr Ala
Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro Leu 50 55 60 Asp Gly
Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg Leu Pro Ser65 70 75 80
Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly Ser Phe Ser Asp Leu 85
90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu Asp Thr Ser Leu Leu Asp
Ser 100 105 110 Met Pro Ala Val Gly Thr Pro His Thr Ala Ala Ala Pro
Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser Ala Leu Arg Ala Ala Asp
Asp Pro Pro Pro Thr 130 135 140 Val Arg Val Ala Val Thr Ala Ala Arg
Pro Pro Arg Ala Lys Pro Ala145 150 155 160 Pro Arg Arg Arg Ala Ala
Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165 170 175 Val Asp Leu Arg
Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile 180 185 190 Lys Pro
Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val 195 200 205
Gly His Gly Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro 210
215 220 Ala Ala Leu Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr
Ala225 230 235 240 Leu Pro Glu Ala Thr His Glu Asp Ile Val Gly Val
Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu
Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly Pro Pro Leu Gln Leu Asp
Thr Gly Gln Leu Val Lys Ile Ala 275 280 285 Lys Arg Gly Gly Val Thr
Ala Met Glu Ala Val His Ala Ser Arg Asn 290 295 300 Ala Leu Thr Gly
Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val Ala305 310 315 320 Ile
Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 325 330
335 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val
340 345 350 Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
Thr Val 355 360 365 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp 370 375 380 Gln Val Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala Leu Glu385 390 395 400 Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln Asp His Gly Leu Thr 405 410 415 Pro Asp Gln Val Val
Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala 420 425 430 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 435 440 445 Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys 450 455
460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Asn Ile Gly 485 490 495 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala Ser Asn 515 520 525 Ile Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535 540 Leu Cys Gln Asp
His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala545 550 555 560 Ser
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 565 570
575 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala
580 585 590 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val 610 615 620 Val Ala Ile Ala Ser His Asp Gly Gly Lys
Gln Ala Leu Glu Thr Val625 630 635 640 Gln Arg Leu Leu Pro Val Leu
Cys Gln Asp His Gly Leu Thr Pro Asp 645 650 655 Gln Val Val Ala Ile
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu 660 665 670 Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 675 680 685 Pro
Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala 690 695
700 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly705 710 715 720 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn
Gly Gly Gly Lys 725 730 735 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp 740 745 750 His Gly Leu Thr Pro Asp Gln Val
Val Ala Ile Ala Ser Asn Asn Gly 755 760 765 Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys 770 775 780 Gln Asp His Gly
Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His785 790 795 800 Asp
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 805 810
815 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
820 825 830 Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala
Gln Leu 835 840 845 Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn
Asp His Leu Val 850 855 860 Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala
Met Asp Ala Val Lys Lys865 870 875 880 Gly Leu Pro His Ala Pro Glu
Leu Ile Arg Arg Val Asn Arg Arg Ile 885 890 895 Gly Glu Arg Thr Ser
His Arg Val Ala Asp Tyr Ala Gln Val Val Arg 900 905 910 Val Leu Glu
Phe Phe Gln Cys His Ser His Pro Ala Tyr Ala Phe Asp 915 920 925 Glu
Ala Met Thr Gln Phe Gly Met Ser Arg Asn Gly Leu Val Gln Leu 930 935
940 Phe Arg Arg Val Gly Val Thr Glu Leu Glu Ala Arg Gly Gly Thr
Leu945 950 955 960 Pro Pro Ala Ser Gln Arg Trp Asp Arg Ile Leu Gln
Ala Ser Gly Met 965 970 975 Lys Arg Ala Lys Pro Ser Pro Thr Ser Ala
Gln Thr Pro Asp Gln Ala 980 985 990 Ser Leu His Ala Phe Ala Asp Ser
Leu Glu Arg Asp Leu Asp Ala Pro 995 1000 1005 Ser Pro Met His Glu
Gly Asp Gln Thr Arg Ala Ser Ser Arg Lys Arg 1010 1015 1020 Ser Arg
Ser Asp Arg Ala Val Thr Gly Pro Ser Ala Gln Gln Ala Val1025 1030
1035 1040 Glu Val Arg Val Pro Glu Gln Arg Asp Ala Leu His Leu Pro
Leu Ser 1045 1050 1055 Trp Arg Val Lys Arg Pro Arg Thr Arg Ile Trp
Gly Gly Leu Pro Asp 1060 1065 1070 Pro Ile Ser Arg Ser Gln Leu Val
Lys Ser Glu Leu Glu Glu Lys Lys 1075 1080 1085 Ser Glu Leu Arg His
Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu 1090 1095 1100 Leu Ile
Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met1105 1110
1115 1120 Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly
Lys His 1125 1130 1135 Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile
Tyr Thr Val Gly Ser 1140 1145 1150 Pro Ile Asp Tyr Gly Val Ile Val
Asp Thr Lys Ala Tyr Ser Gly Gly 1155 1160 1165 Tyr Asn Leu Pro Ile
Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu 1170 1175 1180 Glu Asn
Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys1185 1190
1195 1200 Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val
Ser Gly 1205 1210 1215 His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr
Arg Leu Asn His Ile 1220 1225 1230 Thr Asn Cys Asn Gly Ala Val Leu
Ser Val Glu Glu Leu Leu Ile Gly 1235 1240 1245 Gly Glu Met Ile Lys
Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg 1250 1255 1260 Lys Phe
Asn Asn Gly Glu Ile Asn Phe1265 1270 511273PRTArtificial
Sequencesynthetic peptide 51Met Ala Ser Ser Pro Pro Lys Lys Lys Arg
Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser Gly Trp Ser Arg Met His
Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30 Pro Ser Pro Ala Arg Glu
Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35 40 45 Gln Pro Thr Ala
Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro Leu 50 55 60 Asp Gly
Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg Leu Pro Ser65 70 75 80
Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly Ser Phe Ser Asp Leu 85
90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu Asp Thr Ser Leu Leu Asp
Ser 100 105 110 Met Pro Ala Val Gly Thr Pro His Thr Ala Ala Ala Pro
Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser Ala Leu Arg Ala Ala Asp
Asp Pro Pro Pro Thr 130 135 140 Val Arg Val Ala Val Thr Ala Ala Arg
Pro Pro Arg Ala Lys Pro Ala145 150 155 160 Pro Arg Arg Arg Ala Ala
Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165 170 175 Val Asp Leu Arg
Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile 180 185 190 Lys Pro
Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val 195 200 205
Gly His Gly Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro 210
215 220 Ala Ala Leu Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr
Ala225 230 235 240 Leu Pro Glu Ala Thr His Glu Asp Ile Val Gly Val
Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu
Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly Pro Pro Leu Gln Leu Asp
Thr Gly Gln Leu Val Lys Ile Ala 275 280 285 Lys Arg Gly Gly Val Thr
Ala Met Glu Ala Val His Ala Ser Arg Asn 290 295 300 Ala Leu Thr Gly
Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val Ala305 310 315 320 Ile
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 325 330
335 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val
340 345 350 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
Thr Val 355 360 365 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp 370 375 380 Gln Val Val Ala Ile Ala Ser Asn Gly Gly
Gly Lys Gln Ala Leu Glu385 390 395 400 Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln Asp His Gly Leu Thr 405 410 415 Pro Asp Gln Val Val
Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 420 425 430 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 435 440 445 Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys 450 455
460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser His Asp Gly 485 490 495 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala Ser His 515 520 525 Asp Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535 540 Leu Cys Gln Asp
His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala545 550 555 560 Ser
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 565 570
575 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala
580 585 590 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val 610 615 620 Val Ala Ile Ala Asn Asn Asn Gly Gly Lys
Gln Ala Leu Glu Thr Val625 630 635 640 Gln Arg Leu Leu Pro Val Leu
Cys Gln Asp His Gly Leu Thr Pro Asp 645 650 655 Gln Val Val Ala Ile
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu 660 665 670 Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 675 680 685 Pro
Asp Gln Val Val Ala Ile Ala Asn Asn Asn Gly Gly Lys Gln Ala 690 695
700 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly705 710 715 720 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His
Asp Gly Gly Lys 725 730 735 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp 740 745 750 His Gly Leu Thr Pro Asp Gln Val
Val Ala Ile Ala Ser Asn Gly Gly 755 760 765 Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys 770 775 780 Gln Asp His Gly
Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His785 790 795 800 Asp
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 805 810
815 Leu Cys Gln Asp
His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 820 825 830 Ser Asn
Gly Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala Gln Leu 835 840 845
Ser Arg Arg Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val 850
855 860 Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys
Lys865 870 875 880 Gly Leu Pro His Ala Pro Glu Phe Ile Arg Arg Val
Asn Arg Arg Ile 885 890 895 Ala Glu Arg Thr Ser His Arg Val Ala Asp
Tyr Ala His Val Val Arg 900 905 910 Val Leu Glu Phe Phe Gln Cys His
Ser His Pro Ala His Ala Phe Asp 915 920 925 Glu Ala Met Thr Gln Phe
Gly Met Ser Arg His Gly Leu Val Gln Leu 930 935 940 Phe Arg Arg Val
Gly Val Thr Glu Phe Glu Ala Arg Tyr Gly Thr Leu945 950 955 960 Pro
Pro Ala Ser Gln Arg Trp Asp Arg Ile Leu Gln Ala Ser Gly Met 965 970
975 Lys Arg Ala Lys Pro Ser Pro Thr Ser Ala Gln Thr Pro Asp Gln Thr
980 985 990 Ser Leu His Ala Phe Ala Asp Ser Leu Glu Arg Asp Leu Asp
Ala Pro 995 1000 1005 Ser Pro Met His Glu Gly Asp Gln Thr Arg Ala
Ser Ser Arg Lys Arg 1010 1015 1020 Ser Arg Ser Asp Arg Ala Val Thr
Gly Pro Ser Ala Gln Gln Ala Val1025 1030 1035 1040 Glu Val Arg Val
Pro Glu Gln Arg Asp Ala Leu His Leu Pro Leu Ser 1045 1050 1055 Trp
Arg Val Lys Arg Pro Arg Thr Arg Ile Trp Gly Gly Leu Pro Asp 1060
1065 1070 Pro Ile Ser Arg Ser Gln Leu Val Lys Ser Glu Leu Glu Glu
Lys Lys 1075 1080 1085 Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro
His Glu Tyr Ile Glu 1090 1095 1100 Leu Ile Glu Ile Ala Arg Asn Ser
Thr Gln Asp Arg Ile Leu Glu Met1105 1110 1115 1120 Lys Val Met Glu
Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His 1125 1130 1135 Leu
Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser 1140
1145 1150 Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser
Gly Gly 1155 1160 1165 Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met
Gln Arg Tyr Val Glu 1170 1175 1180 Glu Asn Gln Thr Arg Asn Lys His
Ile Asn Pro Asn Glu Trp Trp Lys1185 1190 1195 1200 Val Tyr Pro Ser
Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly 1205 1210 1215 His
Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile 1220
1225 1230 Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu
Ile Gly 1235 1240 1245 Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu
Glu Glu Val Arg Arg 1250 1255 1260 Lys Phe Asn Asn Gly Glu Ile Asn
Phe1265 1270 521307PRTArtificial Sequencesynthetic peptide 52Met
Ala Ser Ser Pro Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp1 5 10
15 Ala Ser Gly Trp Ser Arg Met His Ala Asp Pro Ile Arg Pro Arg Arg
20 25 30 Pro Ser Pro Ala Arg Glu Leu Leu Pro Gly Pro Gln Pro Asp
Arg Val 35 40 45 Gln Pro Thr Ala Asp Arg Gly Val Ser Ala Pro Ala
Gly Ser Pro Leu 50 55 60 Asp Gly Leu Pro Ala Arg Arg Thr Val Ser
Arg Thr Arg Leu Pro Ser65 70 75 80 Pro Pro Ala Pro Ser Pro Ala Phe
Ser Ala Gly Ser Phe Ser Asp Leu 85 90 95 Leu Arg Pro Phe Asp Pro
Ser Leu Leu Asp Thr Ser Leu Leu Asp Ser 100 105 110 Met Pro Ala Val
Gly Thr Pro His Thr Ala Ala Ala Pro Ala Glu Trp 115 120 125 Asp Glu
Ala Gln Ser Ala Leu Arg Ala Ala Asp Asp Pro Pro Pro Thr 130 135 140
Val Arg Val Ala Val Thr Ala Ala Arg Pro Pro Arg Ala Lys Pro Ala145
150 155 160 Pro Arg Arg Arg Ala Ala Gln Pro Ser Asp Ala Ser Pro Ala
Ala Gln 165 170 175 Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln Gln Gln
Gln Glu Lys Ile 180 185 190 Lys Pro Lys Val Arg Ser Thr Val Ala Gln
His His Glu Ala Leu Val 195 200 205 Gly His Gly Phe Thr His Ala His
Ile Val Ala Leu Ser Gln His Pro 210 215 220 Ala Ala Leu Gly Thr Val
Ala Val Thr Tyr Gln His Ile Ile Thr Ala225 230 235 240 Leu Pro Glu
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp 245 250 255 Ser
Gly Ala Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu 260 265
270 Arg Gly Pro Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala
275 280 285 Lys Arg Gly Gly Val Thr Ala Met Glu Ala Val His Ala Ser
Arg Asn 290 295 300 Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr Pro Ala
Gln Val Val Ala305 310 315 320 Ile Ala Ser Asn Ile Gly Gly Lys Gln
Ala Leu Glu Thr Val Gln Arg 325 330 335 Leu Leu Pro Val Leu Cys Gln
Asp His Gly Leu Thr Pro Asp Gln Val 340 345 350 Val Ala Ile Ala Asn
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val 355 360 365 Gln Arg Leu
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 370 375 380 Gln
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu385 390
395 400 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
Thr 405 410 415 Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly
Lys Gln Ala 420 425 430 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
Cys Gln Asp His Gly 435 440 445 Leu Thr Pro Asp Gln Val Val Ala Ile
Ala Ser Asn Ile Gly Gly Lys 450 455 460 Gln Ala Leu Glu Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp465 470 475 480 His Gly Leu Thr
Pro Asp Gln Val Val Ala Ile Ala Asn Asn Asn Gly 485 490 495 Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 500 505 510
Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Asn Asn 515
520 525 Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
Val 530 535 540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
Ala Ile Ala545 550 555 560 Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser Asn Ile Gly
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 610 615 620 Val Ala
Ile Ala Asn Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val625 630 635
640 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp
645 650 655 Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala
Leu Glu 660 665 670 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly Leu Thr 675 680 685 Pro Asp Gln Val Val Ala Ile Ala Asn Asn
Asn Gly Gly Lys Gln Ala 690 695 700 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly705 710 715 720 Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Asn Asn Asn Gly Gly Lys 725 730 735 Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 740 745 750 His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly 755 760
765 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
770 775 780 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Asn Asn785 790 795 800 Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
Arg Leu Leu Pro Val 805 810 815 Leu Cys Gln Asp His Gly Leu Thr Pro
Asp Gln Val Val Ala Ile Ala 820 825 830 Ser His Asp Gly Gly Lys Gln
Ala Leu Glu Thr Val Gln Arg Leu Leu 835 840 845 Pro Val Leu Cys Gln
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 850 855 860 Ile Ala Ser
Asn Gly Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala865 870 875 880
Gln Leu Ser Arg Arg Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His 885
890 895 Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala
Val 900 905 910 Lys Lys Gly Leu Pro His Ala Pro Glu Phe Ile Arg Arg
Val Asn Arg 915 920 925 Arg Ile Ala Glu Arg Thr Ser His Arg Val Ala
Asp Tyr Ala His Val 930 935 940 Val Arg Val Leu Glu Phe Phe Gln Cys
His Ser His Pro Ala His Ala945 950 955 960 Phe Asp Glu Ala Met Thr
Gln Phe Gly Met Ser Arg His Gly Leu Val 965 970 975 Gln Leu Phe Arg
Arg Val Gly Val Thr Glu Phe Glu Ala Arg Tyr Gly 980 985 990 Thr Leu
Pro Pro Ala Ser Gln Arg Trp Asp Arg Ile Leu Gln Ala Ser 995 1000
1005 Gly Met Lys Arg Ala Lys Pro Ser Pro Thr Ser Ala Gln Thr Pro
Asp 1010 1015 1020 Gln Thr Ser Leu His Ala Phe Ala Asp Ser Leu Glu
Arg Asp Leu Asp1025 1030 1035 1040 Ala Pro Ser Pro Met His Glu Gly
Asp Gln Thr Arg Ala Ser Ser Arg 1045 1050 1055 Lys Arg Ser Arg Ser
Asp Arg Ala Val Thr Gly Pro Ser Ala Gln Gln 1060 1065 1070 Ala Val
Glu Val Arg Val Pro Glu Gln Arg Asp Ala Leu His Leu Pro 1075 1080
1085 Leu Ser Trp Arg Val Lys Arg Pro Arg Thr Arg Ile Trp Gly Gly
Leu 1090 1095 1100 Pro Asp Pro Ile Ser Arg Ser Gln Leu Val Lys Ser
Glu Leu Glu Glu1105 1110 1115 1120 Lys Lys Ser Glu Leu Arg His Lys
Leu Lys Tyr Val Pro His Glu Tyr 1125 1130 1135 Ile Glu Leu Ile Glu
Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu 1140 1145 1150 Glu Met
Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly 1155 1160
1165 Lys His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr
Val 1170 1175 1180 Gly Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr
Lys Ala Tyr Ser1185 1190 1195 1200 Gly Gly Tyr Asn Leu Pro Ile Gly
Gln Ala Asp Glu Met Gln Arg Tyr 1205 1210 1215 Val Glu Glu Asn Gln
Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp 1220 1225 1230 Trp Lys
Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val 1235 1240
1245 Ser Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu
Asn 1250 1255 1260 His Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val
Glu Glu Leu Leu1265 1270 1275 1280 Ile Gly Gly Glu Met Ile Lys Ala
Gly Thr Leu Thr Leu Glu Glu Val 1285 1290 1295 Arg Arg Lys Phe Asn
Asn Gly Glu Ile Asn Phe 1300 1305 531341PRTArtificial
Sequencesynthetic peptide 53Met Ala Ser Ser Pro Pro Lys Lys Lys Arg
Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser Gly Trp Ser Arg Met His
Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30 Pro Ser Pro Ala Arg Glu
Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35 40 45 Gln Pro Thr Ala
Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro Leu 50 55 60 Asp Gly
Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg Leu Pro Ser65 70 75 80
Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly Ser Phe Ser Asp Leu 85
90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu Asp Thr Ser Leu Leu Asp
Ser 100 105 110 Met Pro Ala Val Gly Thr Pro His Thr Ala Ala Ala Pro
Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser Ala Leu Arg Ala Ala Asp
Asp Pro Pro Pro Thr 130 135 140 Val Arg Val Ala Val Thr Ala Ala Arg
Pro Pro Arg Ala Lys Pro Ala145 150 155 160 Pro Arg Arg Arg Ala Ala
Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165 170 175 Val Asp Leu Arg
Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile 180 185 190 Lys Pro
Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val 195 200 205
Gly His Gly Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro 210
215 220 Ala Ala Leu Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr
Ala225 230 235 240 Leu Pro Glu Ala Thr His Glu Asp Ile Val Gly Val
Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu
Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly Pro Pro Leu Gln Leu Asp
Thr Gly Gln Leu Val Lys Ile Ala 275 280 285 Lys Arg Gly Gly Val Thr
Ala Met Glu Ala Val His Ala Ser Arg Asn 290 295 300 Ala Leu Thr Gly
Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val Ala305 310 315 320 Ile
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 325 330
335 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val
340 345 350 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
Thr Val 355 360 365 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp 370 375 380 Gln Val Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala Leu Glu385 390 395 400 Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln Asp His Gly Leu Thr 405 410 415 Pro Asp Gln Val Val
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala 420 425 430 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 435 440 445 Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 450 455
460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Asn Asn Asn Gly 485 490 495 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala Ser Asn 515 520 525 Ile Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535
540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
Ala545 550 555 560 Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser Asn Gly Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys
Gln Asp His Gly Leu Thr Pro Asp Gln Val 610 615 620 Val Ala Ile Ala
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val625 630 635 640 Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 645 650
655 Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
660 665 670 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr 675 680 685 Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala 690 695 700 Leu Glu Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly705 710 715 720 Leu Thr Pro Asp Gln Val Val
Ala Ile Ala Ser Asn Gly Gly Gly Lys 725 730 735 Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 740 745 750 His Gly Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly 755 760 765 Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 770 775
780 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
His785 790 795 800 Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val 805 810 815 Leu Cys Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala 820 825 830 Ser Asn Ile Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu 835 840 845 Pro Val Leu Cys Gln Asp
His Gly Leu Thr Pro Asp Gln Val Val Ala 850 855 860 Ile Ala Ser His
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg865 870 875 880 Leu
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 885 890
895 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Ser Ile
900 905 910 Val Ala Gln Leu Ser Arg Arg Asp Pro Ala Leu Ala Ala Leu
Thr Asn 915 920 925 Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg
Pro Ala Leu Asp 930 935 940 Ala Val Lys Lys Gly Leu Pro His Ala Pro
Glu Phe Ile Arg Arg Val945 950 955 960 Asn Arg Arg Ile Ala Glu Arg
Thr Ser His Arg Val Ala Asp Tyr Ala 965 970 975 His Val Val Arg Val
Leu Glu Phe Phe Gln Cys His Ser His Pro Ala 980 985 990 His Ala Phe
Asp Glu Ala Met Thr Gln Phe Gly Met Ser Arg His Gly 995 1000 1005
Leu Val Gln Leu Phe Arg Arg Val Gly Val Thr Glu Phe Glu Ala Arg
1010 1015 1020 Tyr Gly Thr Leu Pro Pro Ala Ser Gln Arg Trp Asp Arg
Ile Leu Gln1025 1030 1035 1040 Ala Ser Gly Met Lys Arg Ala Lys Pro
Ser Pro Thr Ser Ala Gln Thr 1045 1050 1055 Pro Asp Gln Thr Ser Leu
His Ala Phe Ala Asp Ser Leu Glu Arg Asp 1060 1065 1070 Leu Asp Ala
Pro Ser Pro Met His Glu Gly Asp Gln Thr Arg Ala Ser 1075 1080 1085
Ser Arg Lys Arg Ser Arg Ser Asp Arg Ala Val Thr Gly Pro Ser Ala
1090 1095 1100 Gln Gln Ala Val Glu Val Arg Val Pro Glu Gln Arg Asp
Ala Leu His1105 1110 1115 1120 Leu Pro Leu Ser Trp Arg Val Lys Arg
Pro Arg Thr Arg Ile Trp Gly 1125 1130 1135 Gly Leu Pro Asp Pro Ile
Ser Arg Ser Gln Leu Val Lys Ser Glu Leu 1140 1145 1150 Glu Glu Lys
Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His 1155 1160 1165
Glu Tyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg
1170 1175 1180 Ile Leu Glu Met Lys Val Met Glu Phe Phe Met Lys Val
Tyr Gly Tyr1185 1190 1195 1200 Arg Gly Lys His Leu Gly Gly Ser Arg
Lys Pro Asp Gly Ala Ile Tyr 1205 1210 1215 Thr Val Gly Ser Pro Ile
Asp Tyr Gly Val Ile Val Asp Thr Lys Ala 1220 1225 1230 Tyr Ser Gly
Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln 1235 1240 1245
Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn
1250 1255 1260 Glu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe
Lys Phe Leu1265 1270 1275 1280 Phe Val Ser Gly His Phe Lys Gly Asn
Tyr Lys Ala Gln Leu Thr Arg 1285 1290 1295 Leu Asn His Ile Thr Asn
Cys Asn Gly Ala Val Leu Ser Val Glu Glu 1300 1305 1310 Leu Leu Ile
Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu 1315 1320 1325
Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe 1330 1335 1340
541341PRTArtificial Sequencesynthetic peptide 54Met Ala Ser Ser Pro
Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser Gly
Trp Ser Arg Met His Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30 Pro
Ser Pro Ala Arg Glu Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35 40
45 Gln Pro Thr Ala Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro Leu
50 55 60 Asp Gly Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg Leu
Pro Ser65 70 75 80 Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly Ser
Phe Ser Asp Leu 85 90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu Asp
Thr Ser Leu Leu Asp Ser 100 105 110 Met Pro Ala Val Gly Thr Pro His
Thr Ala Ala Ala Pro Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser Ala
Leu Arg Ala Ala Asp Asp Pro Pro Pro Thr 130 135 140 Val Arg Val Ala
Val Thr Ala Ala Arg Pro Pro Arg Ala Lys Pro Ala145 150 155 160 Pro
Arg Arg Arg Ala Ala Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165 170
175 Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile
180 185 190 Lys Pro Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala
Leu Val 195 200 205 Gly His Gly Phe Thr His Ala His Ile Val Ala Leu
Ser Gln His Pro 210 215 220 Ala Ala Leu Gly Thr Val Ala Val Thr Tyr
Gln His Ile Ile Thr Ala225 230 235 240 Leu Pro Glu Ala Thr His Glu
Asp Ile Val Gly Val Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg Ala
Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly Pro
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala 275 280 285 Lys
Arg Gly Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn 290 295
300 Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val
Ala305 310 315 320 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg 325 330 335 Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp Gln Val 340 345 350 Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala Leu Glu Thr Val 355 360 365 Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr Pro Asp 370 375 380 Gln Val Val Ala
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu385 390 395 400 Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 405 410
415 Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala
420 425 430 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 435 440 445 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His
Asp Gly Gly Lys 450 455 460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Asn Asn Gly 485 490 495 Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His 515 520 525 Asp
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535
540 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
Ala545 550 555 560 Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val Val Ala 580 585 590 Ile Ala Ser Asn Gly Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys
Gln Asp His Gly Leu Thr Pro Asp Gln Val 610 615 620 Val Ala Ile Ala
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val625 630 635 640 Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 645 650
655 Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
660 665 670 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr 675 680 685 Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly
Gly Lys Gln Ala 690 695 700 Leu Glu Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly705 710 715 720 Leu Thr Pro Asp Gln Val Val
Ala Ile Ala Ser Asn Asn Gly Gly Lys 725 730 735 Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 740 745 750 His Gly Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly 755 760 765 Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 770 775
780 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
Asn785 790 795 800 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val 805 810 815 Leu Cys Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala 820 825 830 Ser His Asp Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu 835 840 845 Pro Val Leu Cys Gln Asp
His Gly Leu Thr Pro Asp Gln Val Val Ala 850 855 860 Ile Ala Ser Asn
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg865 870 875 880 Leu
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 885 890
895 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Ser Ile
900 905 910 Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu
Thr Asn 915 920 925 Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg
Pro Ala Met Asp 930 935 940 Ala Val Lys Lys Gly Leu Pro His Ala Pro
Glu Leu Ile Arg Arg Val945 950 955 960 Asn Arg Arg Ile Gly Glu Arg
Thr Ser His Arg Val Ala Asp Tyr Ala 965 970 975 Gln Val Val Arg Val
Leu Glu Phe Phe Gln Cys His Ser His Pro Ala 980 985 990 Tyr Ala Phe
Asp Glu Ala Met Thr Gln Phe Gly Met Ser Arg Asn Gly 995 1000 1005
Leu Val Gln Leu Phe Arg Arg Val Gly Val Thr Glu Leu Glu Ala Arg
1010 1015 1020 Gly Gly Thr Leu Pro Pro Ala Ser Gln Arg Trp Asp Arg
Ile Leu Gln1025 1030 1035 1040 Ala Ser Gly Met Lys Arg Ala Lys Pro
Ser Pro Thr Ser Ala Gln Thr 1045 1050 1055 Pro Asp Gln Ala Ser Leu
His Ala Phe Ala Asp Ser Leu Glu Arg Asp 1060 1065 1070 Leu Asp Ala
Pro Ser Pro Met His Glu Gly Asp Gln Thr Arg Ala Ser 1075 1080 1085
Ser Arg Lys Arg Ser Arg Ser Asp Arg Ala Val Thr Gly Pro Ser Ala
1090 1095 1100 Gln Gln Ala Val Glu Val Arg Val Pro Glu Gln Arg Asp
Ala Leu His1105 1110 1115 1120 Leu Pro Leu Ser Trp Arg Val Lys Arg
Pro Arg Thr Arg Ile Trp Gly 1125 1130 1135 Gly Leu Pro Asp Pro Ile
Ser Arg Ser Gln Leu Val Lys Ser Glu Leu 1140 1145 1150 Glu Glu Lys
Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His 1155 1160 1165
Glu Tyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg
1170 1175 1180 Ile Leu Glu Met Lys Val Met Glu Phe Phe Met Lys Val
Tyr Gly Tyr1185 1190 1195 1200 Arg Gly Lys His Leu Gly Gly Ser Arg
Lys Pro Asp Gly Ala Ile Tyr 1205 1210 1215 Thr Val Gly Ser Pro Ile
Asp Tyr Gly Val Ile Val Asp Thr Lys Ala 1220 1225 1230 Tyr Ser Gly
Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln 1235 1240 1245
Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn
1250 1255 1260 Glu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe
Lys Phe Leu1265 1270 1275 1280 Phe Val Ser Gly His Phe Lys Gly Asn
Tyr Lys Ala Gln Leu Thr Arg 1285 1290 1295 Leu Asn His Ile Thr Asn
Cys Asn Gly Ala Val Leu Ser Val Glu Glu 1300 1305 1310 Leu Leu Ile
Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu 1315 1320 1325
Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe 1330 1335 1340
551341PRTArtificial Sequencesynthetic peptide 55Met Ala Ser Ser Pro
Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser Gly
Trp Ser Arg Met His Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30 Pro
Ser Pro Ala Arg Glu Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35 40
45 Gln Pro Thr Ala Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro Leu
50 55 60 Asp Gly Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg Leu
Pro Ser65 70 75 80 Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly Ser
Phe Ser Asp Leu 85 90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu Asp
Thr Ser Leu Leu Asp Ser 100 105 110 Met Pro Ala Val Gly Thr Pro His
Thr Ala Ala Ala Pro Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser Ala
Leu Arg Ala Ala Asp Asp Pro Pro Pro Thr 130 135 140 Val Arg Val Ala
Val Thr Ala Ala Arg Pro Pro Arg Ala Lys Pro Ala145 150 155 160 Pro
Arg Arg Arg Ala
Ala Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165 170 175 Val Asp Leu
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile 180 185 190 Lys
Pro Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val 195 200
205 Gly His Gly Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro
210 215 220 Ala Ala Leu Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile
Thr Ala225 230 235 240 Leu Pro Glu Ala Thr His Glu Asp Ile Val Gly
Val Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg Ala Leu Glu Ala Leu
Leu Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly Pro Pro Leu Gln Leu
Asp Thr Gly Gln Leu Val Lys Ile Ala 275 280 285 Lys Arg Gly Gly Val
Thr Ala Met Glu Ala Val His Ala Ser Arg Asn 290 295 300 Ala Leu Thr
Gly Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val Ala305 310 315 320
Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 325
330 335 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
Val 340 345 350 Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu
Glu Thr Val 355 360 365 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly Leu Thr Pro Asp 370 375 380 Gln Val Val Ala Ile Ala Ser His Asp
Gly Gly Lys Gln Ala Leu Glu385 390 395 400 Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly Leu Thr 405 410 415 Pro Asp Gln Val
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala 420 425 430 Leu Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 435 440 445
Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys 450
455 460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Asn Ile Gly 485 490 495 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala Ser His 515 520 525 Asp Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535 540 Leu Cys Gln Asp
His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala545 550 555 560 Ser
His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 565 570
575 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala
580 585 590 Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val 610 615 620 Val Ala Ile Ala Ser Asn Ile Gly Gly Lys
Gln Ala Leu Glu Thr Val625 630 635 640 Gln Arg Leu Leu Pro Val Leu
Cys Gln Asp His Gly Leu Thr Pro Asp 645 650 655 Gln Val Val Ala Ile
Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu 660 665 670 Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 675 680 685 Pro
Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala 690 695
700 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly705 710 715 720 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn
Ile Gly Gly Lys 725 730 735 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp 740 745 750 His Gly Leu Thr Pro Asp Gln Val
Val Ala Ile Ala Ser His Asp Gly 755 760 765 Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys 770 775 780 Gln Asp His Gly
Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn785 790 795 800 Asn
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 805 810
815 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
820 825 830 Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu 835 840 845 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala 850 855 860 Ile Ala Ser His Asp Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg865 870 875 880 Leu Leu Pro Val Leu Cys Gln
Asp His Gly Leu Thr Pro Asp Gln Val 885 890 895 Val Ala Ile Ala Ser
Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile 900 905 910 Val Ala Gln
Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn 915 920 925 Asp
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp 930 935
940 Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg
Val945 950 955 960 Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val
Ala Asp Tyr Ala 965 970 975 Gln Val Val Arg Val Leu Glu Phe Phe Gln
Cys His Ser His Pro Ala 980 985 990 Tyr Ala Phe Asp Glu Ala Met Thr
Gln Phe Gly Met Ser Arg Asn Gly 995 1000 1005 Leu Val Gln Leu Phe
Arg Arg Val Gly Val Thr Glu Leu Glu Ala Arg 1010 1015 1020 Gly Gly
Thr Leu Pro Pro Ala Ser Gln Arg Trp Asp Arg Ile Leu Gln1025 1030
1035 1040 Ala Ser Gly Met Lys Arg Ala Lys Pro Ser Pro Thr Ser Ala
Gln Thr 1045 1050 1055 Pro Asp Gln Ala Ser Leu His Ala Phe Ala Asp
Ser Leu Glu Arg Asp 1060 1065 1070 Leu Asp Ala Pro Ser Pro Met His
Glu Gly Asp Gln Thr Arg Ala Ser 1075 1080 1085 Ser Arg Lys Arg Ser
Arg Ser Asp Arg Ala Val Thr Gly Pro Ser Ala 1090 1095 1100 Gln Gln
Ala Val Glu Val Arg Val Pro Glu Gln Arg Asp Ala Leu His1105 1110
1115 1120 Leu Pro Leu Ser Trp Arg Val Lys Arg Pro Arg Thr Arg Ile
Trp Gly 1125 1130 1135 Gly Leu Pro Asp Pro Ile Ser Arg Ser Gln Leu
Val Lys Ser Glu Leu 1140 1145 1150 Glu Glu Lys Lys Ser Glu Leu Arg
His Lys Leu Lys Tyr Val Pro His 1155 1160 1165 Glu Tyr Ile Glu Leu
Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg 1170 1175 1180 Ile Leu
Glu Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr1185 1190
1195 1200 Arg Gly Lys His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala
Ile Tyr 1205 1210 1215 Thr Val Gly Ser Pro Ile Asp Tyr Gly Val Ile
Val Asp Thr Lys Ala 1220 1225 1230 Tyr Ser Gly Gly Tyr Asn Leu Pro
Ile Gly Gln Ala Asp Glu Met Gln 1235 1240 1245 Arg Tyr Val Glu Glu
Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn 1250 1255 1260 Glu Trp
Trp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu1265 1270
1275 1280 Phe Val Ser Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu
Thr Arg 1285 1290 1295 Leu Asn His Ile Thr Asn Cys Asn Gly Ala Val
Leu Ser Val Glu Glu 1300 1305 1310 Leu Leu Ile Gly Gly Glu Met Ile
Lys Ala Gly Thr Leu Thr Leu Glu 1315 1320 1325 Glu Val Arg Arg Lys
Phe Asn Asn Gly Glu Ile Asn Phe 1330 1335 1340 5651DNAArabidopsis
sp. 56tatcaagatt ctcttcactt ctctctgtca caccgatgtt tacttctggg a
515750DNAArabidopsis sp. 57tccggatgct cctcttgaca aggtctgtat
tgtcagttgt ggtttgtcta 505848DNAArabidopsis sp. 58ccggatgctc
ctcttgacaa ggtctgtatt gtcagttgtg gtttgtct 485940DNAArtificial
Sequencemodified Arabidopsis 59ccggatgctc ctcttgacaa ttgtcagttg
tggtttgtct 406043DNAArtificial Sequencemodified Arabidopsis
60ccggatgctc ctcttgacaa gtattgtcag ttgtggtttg tct
436133DNAArtificial Sequencemodified Arabidopsis 61ccggatgctc
ctcttgacaa ttgtggtttg tct 336243DNAArtificial Sequencemodified
Arabidopsis 62ccggatgctc ctcttgacaa ggattgtcag ttgtggtttg tct
436341DNAArtificial Sequencemodified Arabidopsis 63ccggatgctc
ctcttgacaa attgtcagtt gtggtttgtc t 416444DNAArtificial
Sequencemodified Arabidopsis 64ccggatgctc ctcttgacaa ggtattgtca
gttgtggttt gtct 446534PRTXanthomonas gardneri 65Leu Asp Thr Gly Gln
Leu Phe Lys Ile Ala Lys Arg Gly Gly Val Thr1 5 10 15 Ala Val Glu
Ala Val His Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro 20 25 30 Leu
Asn6634PRTXanthomonas campestris 66Leu Asp Thr Gly Gln Leu Leu Lys
Ile Ala Lys Arg Gly Gly Val Thr1 5 10 15 Ala Val Glu Ala Val His
Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro 20 25 30 Leu
Asn6734PRTXanthomonas oryzae 67Leu Asp Thr Gly Gln Leu Val Lys Ile
Ala Lys Arg Gly Gly Val Thr1 5 10 15 Ala Val Glu Ala Val His Ala
Ser Arg Asn Ala Leu Thr Gly Ala Pro 20 25 30 Leu
Asn6834PRTXanthomonas citri 68Leu Asp Thr Gly Gln Leu Leu Lys Ile
Ala Lys Arg Gly Gly Val Thr1 5 10 15 Ala Val Glu Ala Val His Ala
Trp Arg Asn Ala Leu Thr Gly Ala Pro 20 25 30 Leu
Asn6934PRTXanthomonas oryzae 69Leu Asp Thr Gly Gln Leu Val Lys Ile
Ala Lys Arg Gly Gly Val Thr1 5 10 15 Ala Met Glu Ala Val His Ala
Ser Arg Asn Ala Leu Thr Gly Ala Pro 20 25 30 Leu Asn
7034PRTXanthomonas oryzae 70Leu Asp Thr Gly Gln Leu Val Lys Ile Ala
Lys Arg Gly Gly Val Thr1 5 10 15 Ala Met Glu Ala Val His Ala Ser
Arg Asn Ala Leu Thr Gly Ala Pro 20 25 30 Leu Asn7134PRTXanthomonas
oryzae 71Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Asn Asn Gly
Gly Lys1 5 10 15 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Ala 20 25 30 His Gly721307PRTArtificial
Sequencesynthetic peptide 72Met Ala Ser Ser Pro Pro Lys Lys Lys Arg
Lys Val Ser Trp Lys Asp1 5 10 15 Ala Ser Gly Trp Ser Arg Met His
Ala Asp Pro Ile Arg Pro Arg Arg 20 25 30 Pro Ser Pro Ala Arg Glu
Leu Leu Pro Gly Pro Gln Pro Asp Arg Val 35 40 45 Gln Pro Thr Ala
Asp Arg Gly Val Ser Ala Pro Ala Gly Ser Pro Leu 50 55 60 Asp Gly
Leu Pro Ala Arg Arg Thr Val Ser Arg Thr Arg Leu Pro Ser65 70 75 80
Pro Pro Ala Pro Ser Pro Ala Phe Ser Ala Gly Ser Phe Ser Asp Leu 85
90 95 Leu Arg Pro Phe Asp Pro Ser Leu Leu Asp Thr Ser Leu Leu Asp
Ser 100 105 110 Met Pro Ala Val Gly Thr Pro His Thr Ala Ala Ala Pro
Ala Glu Trp 115 120 125 Asp Glu Ala Gln Ser Ala Leu Arg Ala Ala Asp
Asp Pro Pro Pro Thr 130 135 140 Val Arg Val Ala Val Thr Ala Ala Arg
Pro Pro Arg Ala Lys Pro Ala145 150 155 160 Pro Arg Arg Arg Ala Ala
Gln Pro Ser Asp Ala Ser Pro Ala Ala Gln 165 170 175 Val Asp Leu Arg
Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile 180 185 190 Lys Pro
Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val 195 200 205
Gly His Gly Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro 210
215 220 Ala Ala Leu Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr
Ala225 230 235 240 Leu Pro Glu Ala Thr His Glu Asp Ile Val Gly Val
Gly Lys Gln Trp 245 250 255 Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu
Thr Asp Ala Gly Glu Leu 260 265 270 Arg Gly Pro Pro Leu Gln Leu Asp
Thr Gly Gln Leu Val Lys Ile Ala 275 280 285 Lys Arg Gly Gly Val Thr
Ala Met Glu Ala Val His Ala Ser Arg Asn 290 295 300 Ala Leu Thr Gly
Ala Pro Leu Asn Leu Thr Pro Ala Gln Val Val Ala305 310 315 320 Ile
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 325 330
335 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val
340 345 350 Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
Thr Val 355 360 365 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp 370 375 380 Gln Val Val Ala Ile Ala Ser Asn Ile Gly
Gly Lys Gln Ala Leu Glu385 390 395 400 Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln Asp His Gly Leu Thr 405 410 415 Pro Asp Gln Val Val
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala 420 425 430 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 435 440 445 Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 450 455
460 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Asp465 470 475 480 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser His Asp Gly 485 490 495 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys 500 505 510 Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala Ser His 515 520 525 Asp Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val 530 535 540 Leu Cys Gln Asp
His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala545 550 555 560 Ser
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 565 570
575 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala
580 585 590 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg 595 600 605 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val 610 615 620 Val Ala Ile Ala Ser Asn Ile Gly Gly Lys
Gln Ala Leu Glu Thr Val625 630 635 640 Gln Arg Leu Leu Pro Val Leu
Cys Gln Asp His Gly Leu Thr Pro Asp 645 650 655 Gln Val Val Ala Ile
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu 660 665 670 Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 675 680 685 Pro
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 690 695
700 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly705 710 715 720 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn
Ile Gly Gly Lys 725 730 735 Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 740 745 750 His Gly
Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly 755 760 765
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 770
775 780 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
Asn785 790 795 800 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val 805 810 815 Leu Cys Gln Asp His Gly Leu Thr Pro Asp
Gln Val Val Ala Ile Ala 820 825 830 Ser Asn Ile Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu 835 840 845 Pro Val Leu Cys Gln Asp
His Gly Leu Thr Pro Asp Gln Val Val Ala 850 855 860 Ile Ala Ser Asn
Gly Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala865 870 875 880 Gln
Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His 885 890
895 Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val
900 905 910 Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val
Asn Arg 915 920 925 Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Asp
Tyr Ala Gln Val 930 935 940 Val Arg Val Leu Glu Phe Phe Gln Cys His
Ser His Pro Ala Tyr Ala945 950 955 960 Phe Asp Glu Ala Met Thr Gln
Phe Gly Met Ser Arg Asn Gly Leu Val 965 970 975 Gln Leu Phe Arg Arg
Val Gly Val Thr Glu Leu Glu Ala Arg Gly Gly 980 985 990 Thr Leu Pro
Pro Ala Ser Gln Arg Trp Asp Arg Ile Leu Gln Ala Ser 995 1000 1005
Gly Met Lys Arg Ala Lys Pro Ser Pro Thr Ser Ala Gln Thr Pro Asp
1010 1015 1020 Gln Ala Ser Leu His Ala Phe Ala Asp Ser Leu Glu Arg
Asp Leu Asp1025 1030 1035 1040 Ala Pro Ser Pro Met His Glu Gly Asp
Gln Thr Arg Ala Ser Ser Arg 1045 1050 1055 Lys Arg Ser Arg Ser Asp
Arg Ala Val Thr Gly Pro Ser Ala Gln Gln 1060 1065 1070 Ala Val Glu
Val Arg Val Pro Glu Gln Arg Asp Ala Leu His Leu Pro 1075 1080 1085
Leu Ser Trp Arg Val Lys Arg Pro Arg Thr Arg Ile Trp Gly Gly Leu
1090 1095 1100 Pro Asp Pro Ile Ser Arg Ser Gln Leu Val Lys Ser Glu
Leu Glu Glu1105 1110 1115 1120 Lys Lys Ser Glu Leu Arg His Lys Leu
Lys Tyr Val Pro His Glu Tyr 1125 1130 1135 Ile Glu Leu Ile Glu Ile
Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu 1140 1145 1150 Glu Met Lys
Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly 1155 1160 1165
Lys His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val
1170 1175 1180 Gly Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys
Ala Tyr Ser1185 1190 1195 1200 Gly Gly Tyr Asn Leu Pro Ile Gly Gln
Ala Asp Glu Met Gln Arg Tyr 1205 1210 1215 Val Glu Glu Asn Gln Thr
Arg Asn Lys His Ile Asn Pro Asn Glu Trp 1220 1225 1230 Trp Lys Val
Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val 1235 1240 1245
Ser Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn
1250 1255 1260 His Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu
Glu Leu Leu1265 1270 1275 1280 Ile Gly Gly Glu Met Ile Lys Ala Gly
Thr Leu Thr Leu Glu Glu Val 1285 1290 1295 Arg Arg Lys Phe Asn Asn
Gly Glu Ile Asn Phe 1300 1305 731409PRTArtificial Sequencesynthetic
peptide 73Met Ala Ser Ser Pro Pro Lys Lys Lys Arg Lys Val Ser Trp
Lys Asp1 5 10 15 Ala Ser Gly Trp Ser Arg Met His Ala Asp Pro Ile
Arg Pro Arg Arg 20 25 30 Pro Ser Pro Ala Arg Glu Leu Leu Pro Gly
Pro Gln Pro Asp Arg Val 35 40 45 Gln Pro Thr Ala Asp Arg Gly Val
Ser Ala Pro Ala Gly Ser Pro Leu 50 55 60 Asp Gly Leu Pro Ala Arg
Arg Thr Val Ser Arg Thr Arg Leu Pro Ser65 70 75 80 Pro Pro Ala Pro
Ser Pro Ala Phe Ser Ala Gly Ser Phe Ser Asp Leu 85 90 95 Leu Arg
Pro Phe Asp Pro Ser Leu Leu Asp Thr Ser Leu Leu Asp Ser 100 105 110
Met Pro Ala Val Gly Thr Pro His Thr Ala Ala Ala Pro Ala Glu Trp 115
120 125 Asp Glu Ala Gln Ser Ala Leu Arg Ala Ala Asp Asp Pro Pro Pro
Thr 130 135 140 Val Arg Val Ala Val Thr Ala Ala Arg Pro Pro Arg Ala
Lys Pro Ala145 150 155 160 Pro Arg Arg Arg Ala Ala Gln Pro Ser Asp
Ala Ser Pro Ala Ala Gln 165 170 175 Val Asp Leu Arg Thr Leu Gly Tyr
Ser Gln Gln Gln Gln Glu Lys Ile 180 185 190 Lys Pro Lys Val Arg Ser
Thr Val Ala Gln His His Glu Ala Leu Val 195 200 205 Gly His Gly Phe
Thr His Ala His Ile Val Ala Leu Ser Gln His Pro 210 215 220 Ala Ala
Leu Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala225 230 235
240 Leu Pro Glu Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp
245 250 255 Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly
Glu Leu 260 265 270 Arg Gly Pro Pro Leu Gln Leu Asp Thr Gly Gln Leu
Val Lys Ile Ala 275 280 285 Lys Arg Gly Gly Val Thr Ala Met Glu Ala
Val His Ala Ser Arg Asn 290 295 300 Ala Leu Thr Gly Ala Pro Leu Asn
Leu Thr Pro Ala Gln Val Val Ala305 310 315 320 Ile Ala Ser His Asp
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 325 330 335 Leu Leu Pro
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 340 345 350 Val
Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val 355 360
365 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp
370 375 380 Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
Leu Glu385 390 395 400 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Asp His Gly Leu Thr 405 410 415 Pro Asp Gln Val Val Ala Ile Ala Ser
Asn Gly Gly Gly Lys Gln Ala 420 425 430 Leu Glu Thr Val Gln Arg Leu
Leu Pro Val Leu Cys Gln Asp His Gly 435 440 445 Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys 450 455 460 Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp465 470 475 480
His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly 485
490 495 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
Cys 500 505 510 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
Ala Ser Asn 515 520 525 Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
Arg Leu Leu Pro Val 530 535 540 Leu Cys Gln Asp His Gly Leu Thr Pro
Asp Gln Val Val Ala Ile Ala545 550 555 560 Ser Asn Asn Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 565 570 575 Pro Val Leu Cys
Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 580 585 590 Ile Ala
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 595 600 605
Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 610
615 620 Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr
Val625 630 635 640 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
Leu Thr Pro Asp 645 650 655 Gln Val Val Ala Ile Ala Ser Asn Gly Gly
Gly Lys Gln Ala Leu Glu 660 665 670 Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Asp His Gly Leu Thr 675 680 685 Pro Asp Gln Val Val Ala
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala 690 695 700 Leu Glu Thr Val
Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly705 710 715 720 Leu
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 725 730
735 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
740 745 750 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn
Ile Gly 755 760 765 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys 770 775 780 Gln Asp His Gly Leu Thr Pro Asp Gln Val
Val Ala Ile Ala Ser His785 790 795 800 Asp Gly Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val 805 810 815 Leu Cys Gln Asp His
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 820 825 830 Ser Asn Ile
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 835 840 845 Pro
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 850 855
860 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
Arg865 870 875 880 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr
Pro Asp Gln Val 885 890 895 Val Ala Ile Ala Ser His Asp Gly Gly Lys
Gln Ala Leu Glu Thr Val 900 905 910 Gln Arg Leu Leu Pro Val Leu Cys
Gln Asp His Gly Leu Thr Pro Asp 915 920 925 Gln Val Val Ala Ile Ala
Ser His Asp Gly Gly Lys Gln Ala Leu Glu 930 935 940 Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr945 950 955 960 Pro
Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala 965 970
975 Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala
980 985 990 Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly
Gly Arg 995 1000 1005 Pro Ala Met Asp Ala Val Lys Lys Gly Leu Pro
His Ala Pro Glu Leu 1010 1015 1020 Ile Arg Arg Val Asn Arg Arg Ile
Gly Glu Arg Thr Ser His Arg Val1025 1030 1035 1040 Ala Asp Tyr Ala
Gln Val Val Arg Val Leu Glu Phe Phe Gln Cys His 1045 1050 1055 Ser
His Pro Ala Tyr Ala Phe Asp Glu Ala Met Thr Gln Phe Gly Met 1060
1065 1070 Ser Arg Asn Gly Leu Val Gln Leu Phe Arg Arg Val Gly Val
Thr Glu 1075 1080 1085 Leu Glu Ala Arg Gly Gly Thr Leu Pro Pro Ala
Ser Gln Arg Trp Asp 1090 1095 1100 Arg Ile Leu Gln Ala Ser Gly Met
Lys Arg Ala Lys Pro Ser Pro Thr1105 1110 1115 1120 Ser Ala Gln Thr
Pro Asp Gln Ala Ser Leu His Ala Phe Ala Asp Ser 1125 1130 1135 Leu
Glu Arg Asp Leu Asp Ala Pro Ser Pro Met His Glu Gly Asp Gln 1140
1145 1150 Thr Arg Ala Ser Ser Arg Lys Arg Ser Arg Ser Asp Arg Ala
Val Thr 1155 1160 1165 Gly Pro Ser Ala Gln Gln Ala Val Glu Val Arg
Val Pro Glu Gln Arg 1170 1175 1180 Asp Ala Leu His Leu Pro Leu Ser
Trp Arg Val Lys Arg Pro Arg Thr1185 1190 1195 1200 Arg Ile Trp Gly
Gly Leu Pro Asp Pro Ile Ser Arg Ser Gln Leu Val 1205 1210 1215 Lys
Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu Arg His Lys Leu Lys 1220
1225 1230 Tyr Val Pro His Glu Tyr Ile Glu Leu Ile Glu Ile Ala Arg
Asn Ser 1235 1240 1245 Thr Gln Asp Arg Ile Leu Glu Met Lys Val Met
Glu Phe Phe Met Lys 1250 1255 1260 Val Tyr Gly Tyr Arg Gly Lys His
Leu Gly Gly Ser Arg Lys Pro Asp1265 1270 1275 1280 Gly Ala Ile Tyr
Thr Val Gly Ser Pro Ile Asp Tyr Gly Val Ile Val 1285 1290 1295 Asp
Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala 1300
1305 1310 Asp Glu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn
Lys His 1315 1320 1325 Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro
Ser Ser Val Thr Glu 1330 1335 1340 Phe Lys Phe Leu Phe Val Ser Gly
His Phe Lys Gly Asn Tyr Lys Ala1345 1350 1355 1360 Gln Leu Thr Arg
Leu Asn His Ile Thr Asn Cys Asn Gly Ala Val Leu 1365 1370 1375 Ser
Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile Lys Ala Gly Thr 1380
1385 1390 Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu
Ile Asn 1395 1400 1405 Phe 748PRTArtificial Sequencesynthetic
peptide 74Lys Ile Ala Lys Arg Gly Gly Val1 5 758PRTArtificial
Sequencesynthetic peptide 75Lys Ile Ala Asn Gly Gly Gly Val1 5
768PRTArtificial Sequencesynthetic peptide 76Lys Ile Ala Asn Ile
Gly Gly Val1 5 778PRTArtificial Sequencesynthetic peptide 77Lys Ile
Ala His Asp Gly Gly Val1 5 788PRTArtificial Sequencesynthetic
peptide 78Lys Ile Ala Asn Asn Gly Gly Val1 5 798PRTArtificial
Sequencesynthetic peptide 79Lys Ile Ala Lys Arg Gly Gly Val1 5
809PRTArtificial Sequencesynthetic peptide 80Lys Ile Ala Ser Asn
Gly Gly Gly Val1 5 819PRTArtificial Sequencesynthetic peptide 81Lys
Ile Ala Ser Asn Ile Gly Gly Val1 5 829PRTArtificial
Sequencesynthetic peptide 82Lys Ile Ala Ser His Asp Gly Gly Val1 5
839PRTArtificial Sequencesynthetic peptide 83Lys Ile Ala Ser Asn
Asn Gly Gly Val1 5 848PRTArtificial Sequencesynthetic peptide 84Lys
Ile Ala Lys Arg Gly Gly Val1 5 859PRTArtificial Sequencesynthetic
peptide 85Lys Ile Ala Lys Asn Gly Gly Gly Val1 5 869PRTArtificial
Sequencesynthetic peptide 86Lys Ile Ala Lys Asn Ile Gly Gly Val1 5
879PRTArtificial Sequencesynthetic peptide 87Lys Ile Ala Lys His
Asp Gly Gly Val1 5 889PRTArtificial Sequencesynthetic peptide 88Lys
Ile Ala Lys Asn Asn Gly Gly Val1 5 898PRTArtificial
Sequencesynthetic peptide 89Lys Ile Ala Lys Arg Gly Gly Val1 5
909PRTArtificial Sequencesynthetic peptide 90Lys Ile Ala Ser Asn
Gly Gly Gly Lys1 5 919PRTArtificial Sequencesynthetic peptide 91Lys
Ile Ala Ser Asn Ile Gly Gly Lys1 5 929PRTArtificial
Sequencesynthetic peptide 92Lys Ile Ala Ser His Asp Gly Gly Lys1 5
939PRTArtificial Sequencesynthetic peptide 93Lys Ile Ala Ser Asn
Asn Gly Gly Lys1 5 94102DNAArtificial Sequencesynthetic
oligonucleotide 94ctgaccccgg cacaggtggt ggccatcgcc agcmayggng
gcggcaagca ggcgctggag 60acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg
gc 10295102DNAArtificial Sequencesynthetic oligonucleotide
95ctgaccccgg
cacaggtggt ggccatcgcc agcmaytcng gcggcaagca ggcgctggag 60acggtgcagc
ggctgttgcc ggtgctgtgc caggaccatg gc 10296102DNAArtificial
Sequencesynthetic oligonucleotide 96ctgaccccgg cacaggtggt
ggccatcgcc agcmayagyg gcggcaagca ggcgctggag 60acggtgcagc ggctgttgcc
ggtgctgtgc caggaccatg gc 10297102DNAArtificial Sequencesynthetic
oligonucleotide 97ctgaccccgg cacaggtggt ggccatcgcc agcmayathg
gcggcaagca ggcgctggag 60acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg
gc 1029834PRTArtificial Sequencesynthetic peptide 98Leu Thr Pro Ala
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys1 5 10 15 Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 20 25 30
His Gly9934PRTArtificial Sequencesynthetic peptide 99Leu Thr Pro
Ala Gln Val Val Ala Ile Ala Ser Asn Ser Gly Gly Lys1 5 10 15 Gln
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 20 25
30 His Gly10034PRTArtificial Sequencesynthetic peptide 100Leu Thr
Pro Ala Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 20
25 30 His Gly10163DNAArtificial Sequencesynthetic oligonucleotide
101ctgaccccgg cacaggtggt ggccatcgcc agcmayggng gcggcaagca
ggcgctcgag 60agc 6310263DNAArtificial Sequencesynthetic
oligonucleotide 102ctgaccccgg cacaggtggt ggccatcgcc agcmaytcng
gcggcaagca ggcgctcgag 60agc 6310363DNAArtificial Sequencesynthetic
oligonucleotide 103ctgaccccgg cacaggtggt ggccatcgcc agcmayagyg
gcggcaagca ggcgctcgag 60agc 6310463DNAArtificial Sequencesynthetic
oligonucleotide 104ctgaccccgg cacaggtggt ggccatcgcc agcmayathg
gcggcaagca ggcgctcgag 60agc 6310521PRTArtificial Sequencesynthetic
peptide 105Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Asn Gly Gly
Gly Lys1 5 10 15 Gln Ala Leu Glu Ser 20 10621PRTArtificial
Sequencesynthetic peptide 106Leu Thr Pro Ala Gln Val Val Ala Ile
Ala Ser Asn Ser Gly Gly Lys1 5 10 15 Gln Ala Leu Glu Ser 20
10721PRTArtificial Sequencesynthetic peptide 107Leu Thr Pro Ala Gln
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys1 5 10 15 Gln Ala Leu
Glu Ser 20
* * * * *