Complexes of an3-interacting proteins and their use for plant growth

US 20120324602A1
(19) United States
(12) Patent Application Publication (10) Pub. No.: US 2012/0324602 A1
De Jaeger et al.
(54)
(43) Pub. Date:
COMPLEXES OF AN3-INTERACTING
Dec. 20, 2012
Publication Classi?cation
PROTEINS AND THEIR USE FOR PLANT
GROWTH PROMOTION
(51)
(75) Inventors: Geert De Jaeger, Evergem (BE); Dirk
Inzé, Moorsel-Aalst (BE); Aurine
Verkest, Gent (BE)
(73)
Assigneez BASF PLANT SCIENCE COMPANY
Int. Cl.
C07K 19/00
A01H 1/06
A01p 21/00
A01N37/46
(52)
(2006.01)
(200601)
(200601)
(2006.01)
U.S. Cl. ....................... .. 800/290; 530/370; 504/335
GMBH, LUDWIGSHAFEN (DE)
(21) Appl. No.:
13/579,708
(57)
ABSTRACT
(22)
PCT Filed:
Feb. 21, 2011
(86)
PCT No.:
PCT/EP11/52525
The present invention relates to protein complexes based on
AN3-interactors, more speci?cally interactors that are plant
§ 371 (6X1),
(2)’ (4) Date,
Aug 30, 2012
Variants subunits of the SWl/SNF complex, and proteins that
interact With those subunits, preferably in anAN3 free protein
complex. It relates further to the use of the complexes to
(30)
Foreign Application Priority Data
Feb. 22, 2010
(EP) ................................ .. 10154219.9
promote plant growth, and to a method for stimulating the
complex formation, by overexpressing at least one, prefer
ably at least tWo members ofa complex.
Patent Application Publication
4321
O5
0O
\I
Dec. 20, 2012
US 2012/0324602 A1
US 2012/0324602 A1
COMPLEXES OF AN3-INTERAC TING
PROTEINS AND THEIR USE FOR PLANT
GROWTH PROMOTION
[0001]
The present invention relates to protein complexes
based on AN3-interactors, more speci?cally interactors that
are plant variants subunits of the SWI/SNF complex, and
proteins that interact With those subunits, preferably in an
AN3 free protein complex. It relates further to the use of the
complexes to promote plant growth, and to a method for
stimulating the complex formation, by overexpressing at least
one, preferably at least tWo members of a complex.
[0002] The demand for more plant derived products has
spectacularly increased. In the near future the challenge for
agriculture Will be to ful?ll the groWing demands for feed and
Dec. 20, 2012
interacting proteins in Arabidopsis Zhaliana cell suspension
cultures. The tandem af?nity puri?cation (TAP) technology
combined With mass spectrometry (MS) based protein iden
ti?cation resulted in the isolation and identi?cation of 25 AN3
interacting proteins that may function in the regulation of
plant groWth (Table 1). We isolated several proteins belong
ing to multiprotein complexes. Moreover, many interactors
are completely uncharacteriZed. Reports on feW of the AN3
interactors shoW that they are implicated in several develop
mental processes (Wagner & MeyeroWitZ, 2002; Meagher et
al., 2005; SarnoWski et al., 2005; Hurtado et al., 2006; KWon
et al., 2006) but so far none of the identi?ed genes have been
associated With stimulation of plant groWth.
TABLE 1
food in a sustainable manner. Moreover plants start to play an
important role as energy sources. To cope With these major
Interactors of AN3 identi?ed by TAP analysis on
challenges, a profound increase in plant yield Will have to be
achieved. Biomass production is a multi-factorial system in
cell suspension cultures.
Table l. 35S-AN3 (8 experiment )
Which a plethora of processes are fed into the activity of
meristems that give rise to neW cells, tissues, and organs.
Although a considerable amount of research on yield perfor
mance is being performed little is knoWn about the molecular
AT number
Protein name
TAP
total
C-GS
N-GS
AT5G28640
AN3
4
3
1
netWorks underpinning yield (Van Camp, 2005). Many genes
AT4G16143
AT3G06720
importin alpha-2 (IMPA2)
importin alpha-1 subunit (IMPAl)
5
4
4
4
1
/
have been described in Arabidopsis Zhaliana that, When
mutated or ectopically expressed, result in the formation of
AT5G53480
importin beta-2
4
4
/
AT1G09270
AT2G2 8290
importin alpha-1 subunit (IMPA4)
chromatin remodeling protein
1
8
1
4
/
4
“intrinsic yield genes” are involved in many different pro
cesses Whose interrelationship is mostly unknown.
AT3G60830
AT2G46020
actin-related protein 7 (ARP7)
SNF2 protein (BRM)
7
6
4
4
3
2
[0003] One of these “intrinsic yield genes”, AN3 (also
AT1G18450
AT1G21700
actin-related protein 4 (ARP4)
SWIRM domain-containing protein
4
4
4
4
/
/
4
4
/
6
4
2
6
3
3
4
3
2
4
3
/
/
/
2
1
1
/
1
1
/
1
1
1
1
/
/
1
1
1
/
/
1
1
/
1
1
/
1
1
1
/
larger structures, such as leaves or roots. These so-called
knoWn as GIFl), Was identi?ed in search of GRF (groWth
regulating factor) interactors (Kim and Kende, 2004) and by
analysis of narroW-leaf Arabidopsis mutants (Horiguchi et
al., 2005). AN3 is a homolog of the human SYT (synovial
sarcoma translocation) protein and is encoded by a small gene
family in the Arabidopsis genome. SYT is a transcription
co-activator Whose biological function, despite the implica
tion of its chromosomal translocation in tumorigenesis, is still
unclear (Clark et al., 1994; de Bruijn et al., 1996). Using the
yeast GAL4 system, AN3 Was shoWn to possess transactiva
tion activity (Kim and Kende, 2004). This together With yeast
tWo-hybrid and in vitro binding assays demonstrating inter
(SYD)
(SWI3C)
AT5G14170
SWIB complex BAF60b domain-
containing protein (SWp73B)
AT4G17330
G2484-1, agenet domain-
containing protein
AT3G15000
expressed protein, similar to DAG
protein
AT5G55210
AT5G17510
AT2G16570
expressed protein
expressed protein
ATASE (Gln Phosphoribosyl
AT4G35550
homeobox-leucine Zipper protein
Pyrophospate Amidotransferase 1)
(HB—2)/HD—ZIP protein
AT1G20670
DNA-binding bromodomain-
containing protein
action of AN3 With several GRFs (Kim and Kende, 2004;
Horiguchi et al., 2005), suggests a role of AN3 as transcrip
tion co-activator of GRFs. GRF (groWth regulating factor)
AT3G55220
AT2G46340
splicing factor
phytochrome A supressor spal
genes occur in the genomes of all seed plants thus far exam
AT5G13030
AT5G17330
expressed protein
GAD (Glutamate decarboxylase);
AT1G80480
PTAC17 (PLASTID
ined and encode putative transcription factors that play a
regulatory role in groWth and development of leaves (Kim et
al., 2003). In support of a GRF and AN3 transcription activa
tor and co-activator complex, grf and an3 mutants display
similar phenotypes, and combinations of grf and an3 muta
tions shoWed a cooperative effect (Kim and Kende, 2004).
The an3 mutant narroW-leaf phenotype is shoWn to result of a
reduction in cell numbers. Moreover, ectopic expression of
AN3 resulted in transgenic plants With larger leaves consist
ing of more cells, indicating that AN3 controls both cell
number and organ siZe (Horiguchi et al., 2005). Although the
function of AN3 in plant groWth regulation is not knoWn,
these results shoW that AN3 ful?lls the requirements of an
(SPAl)
calmodulin binding
TRANSCRIPTIONALLY
ACTIVE17)
AT1G43800
AT5G45620
acyl-(acyl-carrier-protein)
desaturase
26S proteasome regulatory subunit
(RPN9)
TAP total gives the total number oftime that an interactor Was co-puri?ed;
C-GS and N-GS refers to Whether a C or N terminal GS-tag Was used in the experiment.
[0006] Several of the AN3p interactors Were homologues of
subunits of the SWI/SNF type chromatin remodeling com
plex (Thaete et al., 1999; Ishida et al., 2004). Recently, it Was
“intrinsic yield gene”.
shoWn in mammalian cells that the SWI/SNF ATP-dependent
[0004]
chromatin remodeling complex plays an important role in cell
differentiation and proliferation in mammalian cells (Ries
al., 2006) but so far none of the identi?ed genes have
been associated With stimulation of plant groWth.
[0005]
In our ambition to decipher the molecular netWork
underpinning yield enhancement mechanism a genome-Wide
protein centered approach Was undertaken to study AN3
man et al., 2009) Surprisingly We found that plant variants of
subunits of the SWI/SNF complex, and their interactors play
an important role in plant groWth, and can be used to increase
US 2012/0324602 A1
Dec. 20, 2012
plant yield. A ?rst aspect of the invention is an isolated protein
AN3-protein free protein complex, comprising at least
complex, preferably anAN3p-free protein complex, compris
ARP7p or a variant thereof and a protein selected from the
ing at least a plant variant of a SWI/SNF3 subunit, said
subunit capable of interacting With AN3p, and one of more
group encoded by AT3G20050, AT5G14240, AT4G22320,
AT5G26360, AT3G02530, AT3G18190, AT3G03960,
proteins interacting With said variant SWI/SNF3 subunit.
[0007] AnAN3p-free protein complex, as used here, means
that AN3p is not present in the complex as isolated; hoWever,
AT3G08580, AT4G14880 and AT1G07820, or a variant
one or more subunits of the complex may be capable of
tein complex, preferably an isolatedAN3 -protein free protein
interacting WithAN3, andAN3 may be capable of interacting
complex, comprising at least SWp73 Bp or a variant thereof
and a protein selected from the group encoded by
With the complex as a Whole. In a preferred embodiment, the
complex according to the invention is not longer capable of
interacting With AN3, Whereby the protein interacting With
the plant variant of the SWI/SNF3 subunit directly or indi
rectly inhibits binding of AN3p to said variant. Direct inhibi
tion of AN3p binding may be caused by, as a non-limiting
example, by binding to the same domain; indirect inhibition
of AN3p binding may be caused, as a non-limiting example,
by conformational changes in said variant upon binding With
its interactor. Plant variants of SWI/SNF chromatin remodel
ing complex subunits are knoWn to the person skilled in the
art, and have been described, amongst others, by JerZ
manoWski (2007), hereby incorporated by reference. Vari
thereof.
[0010]
Another preferred embodiment is an isolated pro
AT2G47620, AT2G33610, AT3G17590, AT4G34430,
AT1G32730, AT3G22990, AT1G06500, AT1G47128,
AT3G18380, AT3G06010, AT1G58025, AT5G03290,
AT5G55040, AT3G50000, AT4G28520, AT5G44120 and
AT4G22320, or a variant thereof.
[0011]
Still another preferred embodiment is an isolated
protein complex, preferably an isolatedAN3 -protein free pro
tein complex, comprising at least SWI3Cp and a protein
selected from the group encoded by AT3G01890,
AT1G76380, AT3G03460, AT4G22320, AT1G11840,
AT4G14880 and AT4G04740, or a variant thereof.
ants, as used here, are including, but not limited to homo
[0012]
logues, orthologues and paralogues of said cell cycle related
proteins. “Homologues” of a protein encompass peptides,
tein complex according to the invention to modulate plant
groWth and/or plant yield. Preferably, said modulation is an
Another aspect of the invention is the use of a pro
oligopeptides, polypeptides, proteins and enZymes having
increase of plant groWth and/or yield. Preferably, increase of
amino acid substitutions, deletions and/or insertions relative
groWth is measured as an increase of biomass production.
“Yield” refers to a situation Where only a part of the plant,
to the unmodi?ed protein in question and having similar
biological and functional activity as the unmodi?ed protein
from Which they are derived. Orthologues and paralogues
encompass evolutionary concepts used to describe the ances
tral relationships of genes. Paralogues are genes Within the
same species that have originated through duplication of an
ancestral gene; orthologues are genes from different organ
isms that have originated through speciation, and are also
derived from a common ancestral gene. Preferably, said
homologue, orthologue or paralogue has a sequence identity
at protein level of at least 30%, preferably at least 40%,
preferably 50%, 51%, 52%, 53%, 54% or 55%, 56%, 57%,
preferably an economical important part of the plant, such as
the leaves, roots or seeds, is increased in biomass. The term
“increase” as used here means least a 5%, 6%, 7%, 8%, 9% or
10%, preferably at least 15% or 20%, more preferably 25%,
30%, 35% or 40% more yield and/or groWth in comparison to
control plants as de?ned herein. Increase of plant groWth, as
used here, is preferably measured as increase of any one or
more of total plant biomass, leaf biomass, root biomass and
seed biomass. In one preferred embodiment, said increase is
an increase in total plant biomass. In a preferred embodiment,
said plant is a crop plant, preferably a monocot or a cereal,
58%, 59%, preferably 60%, 61%, 62%, 63%, 64%, 65%,
even more preferably it is a cereal selected from the group
66%, 67%, 68%, 69%, more preferably 70%, 71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, 79%, even more preferably
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%
most preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%,
consisting of rice, maiZe, Wheat, barley, millet, rye, sorghum
97%, 98%, 99% or more as measured in a BLASTp (Altschul
et al., 1997; Altschul et al., 2005). A plant as used here can be
any plant. In one preferred embodiment, said plant is Arabi
and oats.
[0013]
Still another aspect of the invention is a method to
promote the formation of a protein complex according to the
inventions, comprising the overexpression of at least one
protein, preferably at least tWo proteins of said complex.
rice, maiZe, Wheat, barley, millet, rye, sorghum and oats.
[0008] Preferably, said plant variant of the SWI/SNF chro
Overexpression of a target gene can be obtained by transfer of
a genetic construct, intended for said overexpression into a
plant. The transfer of foreign genes into the genome of a plant
is called transformation. Transformation of plant species is a
fairly routine technique knoWn to the person skilled in the art.
Advantageously, any of several transformation methods may
matin remodelling complex is selected from the group con
be used to introduce the gene of interest into a suitable ances
dopsis Zhaliana. In another preferred embodiment, said plant
is a crop plant, preferably a monocot or a cereal, even more
preferably it is a cereal selected from the group consisting of
sisting of proteins encoded by AT1G18450 (ARP4),
tor cell. The methods described for the transformation and
AT3G60830
regeneration of plants from plant tissues or plant cells may be
(ARP7),
AT5G14170
(SWp73B)
and
AT1G21700 (SWI3C), or a variant thereof.
[0009] On preferred embodiment is an isolated protein
complex, preferably an isolated AN3 -protein free protein
complex, comprising at least ARP4p or a variant thereof and
a protein selected from the group encoded by AT5G45600,
AT1G76380, AT3G01890, AT5G26360, AT5G14240,
AT1G47128, AT2G27100, AT5G55040, AT3G03460 and
AT1G54390, or a variant thereof. Another preferred embodi
ment is an isolated protein complex, preferably an isolated
utiliZed for transient or for stable transformation. Transfor
mation methods include, but are not limited to agrobaclerium
mediated transformation, the use of liposomes, electropora
tion, chemicals that increase free DNA uptake, injection of
the DNA directly into the plant, particle gun bombardment,
transformation using viruses or pollen and microprojection.
Preferably, said overexpression results in an increase of plant
groWth and/ or yield. Increase of plant groWth and/or yield is
measured by comparing the test plant, comprising a gene used
US 2012/0324602 A1
according to the invention, With the parental, non-trans
formed plant, grown under the same conditions as control.
[0014] Still another aspect of the invention is a method to
inhibit the formation of a protein complex according to the
inventions, comprising the repression of the expression of at
least one protein, preferably at least tWo proteins of said
complex. Inhibition of complex formation can be desirable in
cases Where the complex exerts a groWth limiting effect.
Repression of expression of a target gene can be obtained by
transfer of a genetic construct, intended for said repression of
Dec. 20, 2012
and Pro35S:SWI3C-GS-containing plant transformation vec
tors Were obtained by Multisite GateWay LR reaction
betWeen
pEntryL4R1iPro35S,
pEntryL1L2-ARP4(—),
pEntryL 1 L2 -ARP7 (—),
pEntryL1L2-SWp73B(—)
or
pEntryL1L2-SWI3C(—), and pEntryR2L3-GS and the desti
nation vector pKCTAP, respectively (Van Leene et al., 2007).
To obtain the Pro35S:GS-ARP4, Pro35S:GS-ARP7, Pro35S:
GS-SWp73B and Pro35S:GS-SWI3C vectors Multisite LR
recombination
betWeen
pEntryL4L3 -Pro3 5S
and
pEntryL1L2-ARP4(+), pEntryL1L2-ARP7(+), pEntryL1 L2
expression into a plant. Methods for repressing the expression
SWp73B(+) or pEntryL1L2-SWI3C(+) With pKNGSTAP
in plants are knoWn to the person skilled in the art and include,
occurred.
[0018] All entry and destination vectors Were checked by
sequence analysis. Expression vectors Were transformed to
but are not limited to the use of RNAi, anti-sense RNA and
gene silencing.
Agrobaclerium Zumefaciens strain C58C1RifR (pMP90) by
BRIEF DESCRIPTION OF THE FIGURES
[0015]
FIG. 1: leaf phenotype of 2 SWIRM overexpressing
lines. A) total rosette area. B) area of individual leaves. Plants
Were groWn in vitro for 21 days. SWIRM is an alternative
name for SWI3C.
electroporation. Transformed bacteria Were selected on yeast
extract broth plates containing 100 ug/mL rifampicin, 40
ug/mL gentamicin, and 100 ug/mL spectinomycin.
Cell Suspension Cultivation
[0019] Wild-type and transgenic Arabidopsis Zhaliana cell
EXAMPLES
suspension PSB-D cultures Were maintained in 50 mL
Materials and Methods to the Examples
MSMO medium (4.43 g/L MSMO, Sigma-Aldrich), 30 g/L
sucrose, 0.5 mg/L NAA, 0.05 mg/L kinetin, pH 5.7 adjusted
With 1M KOH) at 250 C. in the dark, by gentle agitation (130
Vector Construction for AN3 Interactors
rpm). Every 7 days the cells Were subcultured in fresh
[0016]
medium at a 1/10 dilution.
Construction of N- and C-terminal GS-tagged GFP
and AN3 under the control of the 35S (CaMV) promoter Was
Cell Culture Transformation
obtained by Multisite GateWay LR reactions. The coding
regions, Without (—) and With (+) stopcodon, Were ampli?ed
by polymerase chain reaction (PCR) and cloned into the
baclerium co-cultivation as described previously (Van Leene
GateWay pDONR221 vector (Invitrogen) resulting in
et al., 2007). The A grobaclerium culture exponentially groW
pEntryL1L2-GFP(—), pEntryL1L2-GFP(+), pEntryLlL2
ing in YEB (OD600 betWeen 1.0 and 1.5) Was Washed three
times by centrifugation (10 min at 5000 rpm) With an equal
volume MSMO medium and resuspended in cell suspension
groWing medium until an OD600 of 1.0. TWo days after sub
AN3 (—) and pEntryL1L2-AN3(+). The Pro35S:GFP-GS- and
Pro3S:AN3-GS-containing plant transformation vectors Were
obtained by Multisite GateWay LR reaction betWeen
pEntryL4R1-Pro35S, pEntryL1L2-GFP(—) or pEntryLlL2
AN3 (—), and pEntryR2L3-GS and the destination vector
pKCTAP, respectively (Van Leene et al., 2007). To obtain the
Pro35S:GS-GFP and Pro35S:GS-AN3 vectors Multisite LR
recombination
betWeen
pEntryL4L3 -Pro35S
and
pEntryL1L2-GFP(+) or pEntryL1L2-AN3(+) With pKNG
STAP occurred. All entry and destination vectors Were
[0020]
The Arabidopsis culture Was transformed by Agro
cultivation, 3 mL suspension culture Was incubated With 200
pL Washed A grobacleria and 200 uM acetoseringone, for 48
h in the dark at 250 C. With gentle agitation (130 rpm). TWo
days after co-cultivation, 7 mL MSMO containing a mix of
three antibiotics (25 ug/mL kanamycin, 500 ug/mL carbeni
cellin, and 500 ug/mL vancomycin) Was added to the cell
cultures and groWn further in suspension under standard con
checked by sequence analysis. Expression vectors Were trans
formed to A grobaclerium Zumefaciens strain C58C1RifR
(pMP90) by electroporation. Transformed bacteria Were
selected on yeast extract broth plates containing 100 ug/mL
ditions (250 C., 130 rpm and continuous darkness). The stable
transgenic cultures Were selected by sequentional dilution in
rifampicin, 40 ug/mL gentamicin, and 100 ug/mL spectino
co-cultivation. After counter selecting the bacteria, the trans
genic plant cells Were further subcultured Weekly in a 1:5
ratio in 50 mL MSMO medium containing 25 ug/mL kana
mycin.
Vector Construction for ARP4, ARP7, SWp73B and SWI3C
Interactors
[0017] Construction of N- and C-terminal GS-tagged
ARP4, ARP7, SWp73B and SWI3C under the control of the
35S (CaMV) promoter Was obtained by Multisite GateWay
a 1:5 and 1:10 ratio in 50 mL fresh MSMO medium contain
ing the antibiotics mix, respectively at 11, and 18 days post
mycin for tWo more Weeks. Thereafter the cells Were Weekly
subcultured in fresh medium at a 1/ 10 dilution.
Expression Analysis of Cell Suspension Cultures
[0021] Transgene expression Was analyZed in a total pro
tein extract derived from exponentially groWing cells, har
vested tWo days after subculturing. Equal amounts of total
LR reactions. The coding regions, Without (—) and With (+)
stopcodon, Were ampli?ed by polymerase chain reaction
(PCR) and cloned into the GateWay pDONR221 vector (Invit
protein Were separated on 12% SDS-PAGE gels and blotted
rogen) resulting in pEntryL1L2-ARP4(—), pEntryLlL2
onto Immobilon-P membranes (Millipore, Bedford, Mass.).
ARP4(+), pEntryL1L2-ARP7(—), pEntryL1L2-ARP7(+),
pEntryL1L2-SWp73B(—),
pEntryL1L2-SWp73B(+),
Protein gel blots Were blocked in 3% skim milk in 20 mM
pEntryL1L2-SWI3C(—) and pEntryL1L2-SWI3C(+). The
Pro35S:ARP4-GS-, Pro35S:ARP7-GS-, Pro35S:SWp73B-GS
Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Triton X-100. For
detection of GS-tagged proteins, blots Were incubated With
human blood plasma folloWed by incubation With anti -human
US 2012/0324602 A1
IgG coupled to horseradish peroxidase (HRP; GE-Health
care). Protein gel blots Were developed by Chemiluminiscent
detection (Perkin Elmer, NorWalk, Conn.).
Protein Extract Preparation
[0022]
Cell material (15 g) Was grinded to homogeneity in
liquid nitrogen. Crude protein extract Were prepared in an
equal volume (W/v) of extraction buffer (25 mM Tris-HCl, pH
7.6, 15 mM MgCl2, 5 mM EGTA, 150 mM NaCl, 15 mM
p-nitrophenylphosphate, 60 mM [3-glycerophosphate, 0.1%
(v/v) Nonidet P-40 (NP-40), 0.1 mM sodium vanadate, 1 mM
NaF, 1 mM DTT, 1 mM PMSF, 10 ug/mL leupeptin, 10
ug/mL aprotinin, 5 ug/mL antipain, 5 ug/mL chymostatin, 5
ug/mL pepstatin, 10 ug/mL soybean trypsin inhibitor, 0.1
mM benZamidine, 1 [1M trans-epoxysuccinyl-L-leucyla
mido-(4-guanidino)butane (E64), 5% (v/v) ethylene glycol)
using an Ultra-Turrax T25 mixer (IKA Works, Wilmington,
NC.) at 4° C. The soluble protein fraction Was obtained by a
tWo-step centrifugation at 36900 g for 20 min and at 178000
g for 45 min, at 4° C. The extract Was passed through a 0.45
pm ?lter (Alltech, Deer?eld, Ill.) and the protein content Was
determined With the Protein Assay kit (Bio-Rad, Hercules,
Calif.).
Tandem Af?nity Puri?cation
[0023] Puri?cations Were performed as described by
Biirckstiimmer et al. (2006), With some modi?cations.
Brie?y, 200 mg total protein extract Was incubated for 1 h at
4° C. under gentle rotation With 100 pL IgG Sepharose 6 Fast
FloW FloW beads (GE-Healthcare, Little Chalfont, UK), pre
equilibrated With 3 mL extraction buffer. The IgG Sepharose
beads Were transferred to a 1 mL Mobicol column (MoBiTec,
Goettingen, Germany) and Washed With 10 mL IgG Wash
buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% NP-40,
5% ethylene glycol) and 5 mL Tobacco (Nicoliana Zabacum
L.) Etch Virus (TEV) buffer (10 mM Tris-HCl, pH 8.0, 150
mM NaCl, 0.1% (v/v) NP-40, 0.5 mM EDTA, 1 mM PMSF,
1 [1M E64, 5% (v/v) ethylene glycol). Bound complexes Were
eluted via AcTEV digest (2x100 U, Invitrogen) for 1 h at 16°
C. The IgG eluted fraction Was incubated for 1 h at 4° C. under
gentle rotation With 100 pL Streptavidin resin (Stratagene, La
Jolla, Calif.), pre-equilibrated With 3 mL TEV buffer. The
Streptavidin beads Were packed in a Mobicol column, and
Washed With 10 mL TEV buffer. Bound complexes Were
eluted With 1 mL streptavidin elution buffer (10 mM Tris
HCl, pH 8.0, 150 mM NaCl, 0.1% (v/v) NP-40, 0.5 mM
EDTA, 1 mM PMSF, 1 [1M E64, 5% (v/v) ethylene glycol, 20
mM Desthiobiotin), and precipitated using TCA (25% v/v).
The protein pellet Was Washed tWice With ice-cold aceton
containing 50 mM HCl, redissolved in sample buffer and
separated on 4- 1 2% gradient NuPAGE gels (Invitro gen). Pro
teins Were visualiZed With colloidal Coomassie brilliant blue
staining.
Dec. 20, 2012
Leene et al., 2007). Per microtiterplate Well, dehydrated gel
particles Were rehydrated in 20 [LL digest buffer containing
250 ng trypsin (MS Gold; Promega, Madison, Wis.), 50 mM
NH4HCO3 and 10% CH3CN (v/v) for 30 min at 4° C. After
adding 10 pL of a buffer containing 50 mM NH4HCO3 and
10% CH3CN (v/v), proteins Were digested at 37° C. for 3
hours. The resulting peptides Were concentrated and desalted
With microcolumn solid phase tips (PerfectPureTM C18 tip,
200 mL bed volume; Eppendorf, Hamburg, Germany) and
eluted directly onto a MALDI target plate (Opti-TOFTM384
Well Insert; Applied Biosystems, Foster City, Calif.) using
1.2 pL of 50% CH3CN: 0.1% CF3COOH solution saturated
With ot-cyano-4-hydroxycinnamic acid and spiked With 20
fmole/ [1L Glu1 -Fibrinopeptide B (Sigma-Aldrich), 20 fmole/
[1L des-Pro2-Bradykinin (Sigma-Aldrich), and 20 fmole/ [1L
Adrenocorticotropic Hormone Fragment 18-39 human
(Sigma-Aldrich).
Acquisition of Mass Spectra
[0025] A MALDI-tandem MS instrument (4800 Proteom
ics AnalyZer; Applied Biosystems) Was used to acquire pep
tide mass ?ngerprints and subsequent 1 kV CID fragmenta
tion spectra of selected peptides. Peptide mass spectra and
peptide sequence spectra Were obtained using the settings
essentially as presented in Van Leene et al. (2007). Each
MALDI plate Was calibrated according to the manufacturers’
speci?cations. All peptide mass ?ngerprinting (PMF) spectra
Were internally calibrated With three internal standards at m/ Z
963.516 (des-Pro2-Bradykinin), m/Z 1570.677 (Glu1 -Fibrin
opeptide B), and m/Z 2465, 198 (Adrenocorticotropic Hor
mone Fragment 18-39) resulting in an average mass accuracy
of 5 ppm:10 ppm for each analyZed peptide spot on the
analyZed MALDI targets. Using the individual PMF spectra,
up to sixteen peptides, exceeding a signal-to-noise ratio of 20
that passed through a mass exclusion ?lter Were submitted to
fragmentation analysis.
MS-Based Protein Homology Identi?cation
[0026] PMF spectra and the peptide sequence spectra of
each sample Were processed using the accompanied softWare
suite (GPS Explorer 3.6, Applied Biosystems) With parameter
settings essentially as described in Van Leene et al. (2007).
Data search ?les Were generated and submitted for protein
homology identi?cation by using a local database search
engine (Mascot 2.1, Matrix Science). An in-house nonredun
dantArabidopsis protein database called SNAPS Arabidopsis
Zhaliana version 0.4 (SNAPS:Simple Nonredundant Assem
bly of Protein Sequences, 77488 sequence entries, 30468560
residues; available at http://WWW.ptools.ua.ac.be/ snaps) Was
compiled from nine public databases. Protein homology
identi?cations of the top hit (?rst rank) With a relative score
exceeding 95% probability Were retained. Additional positive
identi?cations (second rank and more) Were retained When
the score exceeded the 98% probability threshold.
Proteolysis and Peptide Isolation
[0024] After destaining, gel slabs Were Washed for 1 hour in
H2O, polypeptide disul?de bridges Were reduced for 40 min
in 25 mL of 6.66 mM DTT in 50 mM NH4HCO3 and sequen
tially the thiol groups Were alkylated for 30 min in 25 mL 55
mM IAM in 50 mM NH4HCO3 . After Washing the gel slabs 3
times With Water, complete lanes from the protein gels Were
cut into slices, collected in microtiter plates and treated essen
tially as described before With minor modi?cations (Van
Example 1
Identi?cation of AN3 Interactors
[0027] In order to identify the interaction partners of AN3
in vivo, We performed tandem a?inity (TAP) puri?cations on
N- and C-terminal GS-fusions of AN3 ectopically expressed
under control of the constitutive 35 SCaMV promoter in trans
genic Arabidopsis suspension cultures. TWo independent
US 2012/0324602 A1
Dec. 20, 2012
TAP puri?cations were performed on extracts from AN3-GS
TABLE 3
and GS-AN3 lines, harvested two days after sub-culturing
into fresh medium. The af?nity puri?ed proteins were sepa
Interactors of ARP7, identi?ed by TAP analysis on
cell suspension cultures.
Table 3. 35S-ARP7 (4 experiments)
rated on a 4-12% NuPAGE gel and stained with Coomassie
Brilliant Blue. Protein bands were cut, in-gel digested with
trypsin and subjected to MALDI-TOF/TOF mass spectrom
etry for protein identi?cation. After subtracting background
proteins, identi?ed by the control puri?cations (Van Leene et
al., 2007), from the obtained hit list we identi?ed 25 AN3
interacting proteins, other than AN3 itself (Table 1). 9 pro
teins were identi?ed only in one out of 8 TAP experiments.
AT number
Protein name
AT3G60830
AT3G20050
ARP7
TAP
total
TCP-l (Arabidopsis thaliana T
C-GS
N-GS
4
2
complex protein 1 alpha subunit)
AT5G14240
AT5G55210
protein coding
expressed protein, similar to
At4g22320
Example 2
Identi?cation of ARP4 Interactors
[0028] ARP4 interactors were identi?ed according to the
methods described above. The results are summarized in
AT4G223 20
AT5G2 63 60
AT3G02530
AT3G1 8190
AT3G03960
AT 1 G1 8450
AT3G0 85 80
AT4G148 80
Table 2. Apart from proteins, already identi?ed in the AN3
complex (Table 1), several novel interactors were identi?ed.
chaperonin
chaperonin
chaperonin, similar to At3 g03960
chaperonin, similar to At3g18190
ARP4
AAC1 (ADP/ATP CARRIER 1)
OASAl (O-ACETYLSERINE
(THIOL) LYASE (OAS-TL)
ISOFORM A1)
AT1G07820
TABLE 2
unknown, similar to At5g55210
HIS4
TAP total gives the total number oftime that an interactor was co-puri?ed;
C-GS and N-GS refers to whether a C or N terminal GS-tag was used in the experiment.
Interactors of ARP4, identi?ed by TAP analysis on
cell suspension cultures.
Table 2. 35S-ARP4 (4 experiments)
C-GS
N-GS
ARP4
ARP7
4
4
2
2
2
2
TAF14B, GAS41
SWp73B
SWI3C
DNA-binding bromodomain-
2
2
/
2
Protein name
AT1G18450
AT3G60830
AT5G45600
AT5G14170
AT1G21700
AT1G20670
Example 4
TAP
total
AT number
2
2
2
2
2
/
/
/
2
2
/
containing protein
ATlG7 63 80
DNA-binding bromodomain-
containing protein
Identi?cation of SWp73B Interactors
[0030] SWp73B interactors were identi?ed according to the
methods described above. The results are summariZed in
Table 4. Except for SYD, all AN3 interacting proteins of the
SWI/SNF complex are interacting with SWp73B. Apart from
those proteins, most of the other proteins show only interac
tion with SWp73B and not with the other proteins of the
SWI/SNF complex used in the tap tag experiments (ARP4,
ARP7 and SWI3C)
AT2G46020
BRM
2
2
/
AT3G01890
AT5G2 63 60
Swp73A
chaperonin
2
2
2
/
/
2
AT5G14240
AT5G17510
protein coding
expressed protein
2
2
/
2
2
/
RD21 (RES PONSIVE TO
2
2
/
TABLE 4
2
1
1
1
1
/
Interactors of SWp73B, identi?ed by TAP analysis on
cell suspension cultures.
1
1
/
1
1
1
/
/
1
AT1G47128
DEHYDRATION 21)
AT2G27100
AT5G55040
SE (SERRATE)
DNA-binding bromodomain-
AT5G55210
expressed protein, similar to
Table 4. 35S-Swp73B (5 experiment )
containing protein
At4g22320
AT3G03460
AT1G54390
unknown protein
PHD ?nger protein
TAP total gives the total number of time that an interactor was co-puri?ed;
C-GS and N-GS refers to whether a C or N terminal GS-tag was used in the experiment.
AT number
Protein name
AT5G14170
AT2G47620
AT1G20670
Swp738
DNA-binding bromodomain
containing protein, similar to
At5g55040
Example 3
Identi?cation of ARP7 Interactors
[0029] ARP7 interactors were identi?ed according to the
methods described above. The results are summarized in
AT2G33610
AT3G60830
AT3G17590
AT1G21700
AT4G34430
AT1G32730
AT5G17510
AT3G22990
AT5G55210
con?rming the reliability of the Tap-tag method. At5g55210
was also identi?ed as AN3 as well as ARP4 interactor (Table
1 & 2).
electron carrier
expressed protein
LFR (armadillo-repeat protein)
expressed protein, similar to
At4g22320
Table 3. ARP4 and At5g55210 were also identi?ed as AN3
interactors (Table 1). It is interesting to note that the ARP4
ARP7 interaction is also identi?ed using the ARP4 screening,
ARP7
BSH
SWI3C
SWI3D
AT2G46020
AT1G18450
AT1G06500
AT1G47128
BRM
ARP4
unknown protein
RD21 (RESPONSIVE TO
DEHYDRATION 21)
TAP
total
5
5
5
C-GS
N-GS
w
US 2012/0324602 A1
Dec. 20, 2012
TABLE 4-continued
Example 6
Interactors of Swp73B, identi?ed by TAP analysis on
cell suspension cultures.
Table 4. 35S-Swp73B (5 experiments)
Overexpression Studies of SWI3C
[0032] Several SWI3C overexpressing lines ofArabidopsis
Zhaliana (Ecotype Columbia) were isolated and analyzed for
growth characteristics. Amongst the 13 SWI3C overexpress
ing lines that were analyzed, 8 showed clearly development of
bigger leaves; the bigger leaves are correlated with a higher
expression of SWI3C. The detailed analysis of two SWI3C
overexpressing lines is shown in FIG. 1, demonstrating that in
TAP
total
C-GS
N-GS
transcription factor
2
/
2
AT3G06010
CHR12
1
/
1
AT1G5 8025
DNA-binding bromodomain-
1
1
/
1
1
1
1
/
1
the overexpressing lines both the individual leaves as well as
the total rosette area is larger than for the control.
1
/
1
REFERENCES
1
1
1
/
/
/
1
1
1
AT number
Protein name
AT3G183 80
containing protein
AT5G03290
AT5G55040
isocitrate dehydrogenase
DNA-binding bromodomain-
containing protein, similar to
At1g20670
AT3G50000
CKA2 (casein kinase II alpha chain
2)
AT4G28520
AT5G44120
AT4G223 20
CRU3 (CRUCIFERIN 3)
CRU1 (CRUCIFERINA)
unknown, similar to At5g55210
TAP total gives the total number of time that an interactor was co-puri?ed;
C-GS and N-GS refers to whether a C or N terminal GS-tag was used in the experiment.
Identi?cation of SWI3C Interactors
substitution matrices, FEBS J. 272, 5101-5109.
[0035] Burckstummer T, Bennett K L, Preradovic A,
do interact with ARP4 are also interacting with SWI3C. The
interaction between ARP4 and SWI3C is con?rmed in both
experiments.
interaction proteomics in mammalian cells. Nat Methods
3: 1013-1019.
[0036] Clark J, Rocques P J, Crew A J, Gill S, Shipley J,
ChanA M, Guterson B A, Cooper C S (1994) Identi?cation
ofnovel genes, SYT and SSX, in the tQ(;19)(p1 1 .2;q1 1 .2)
7: 502-508 de Bruijn D R, Baats E, Zechner U, de Leeuw B,
Balemans M, Olde Weghuis D, Hirning-Folz U, Geurts van
Kessel A G (1996) Isolation and characterization of the
mouse homolog of SYT, a gene implicated in the develop
ment of human synovial sarcomas. Oncogene 13: 643-648
Interactors of SWI3C, identi?ed by TAP analysis on
cell suspension cultures.
Table 5. 35S-SWI3C (5 experiments)
Protein name
TAP
total
C-GS
N-GS
AT1G21700
SWI3C
5
2
3
AT5G14170
Swp73B
5
2
3
AT2G46020
AT3G60830
AT 1 G1 8450
BRM
ARP7
ARP4
5
5
5
2
2
2
3
3
3
AT3G01890
AT5G17510
Swp73A
expressed protein, similar to
5
5
2
2
3
3
5
2
3
4
2
2
4
2
2
1
1
1
/
1
1
1
/
/
1
1
/
1
/
1
At3g03460
DNA-binding bromodomain-
containing protein, similar to
At1g763 80
DNA-binding bromodomain-
containing protein, similar to
expressed protein, similar to
At4g22320
AT3G03460
AT4G223 20
AT 1 G1 1840
unknown, similar to At5g17510
unknown, similar to At5 g55 210
GLX1 (GLYOXALASE I
AT4G148 80
OASA1 (O-ACETYLSERINE
AT4G04740
IS OFORM A1)
CPK23 (calcium-dependent protein
[0037] Horiguchi G, Kim G-T, Tsukaya H (2005) The tran
scription factor AtGRFS and the transcription coactivator
AN3 regulate cell proliferation in leaf primordial of Am
bidopsis Zhaliana. Plant J 43: 68-78
[0038]
Hurtado L, Farrona S, Reyes J C (2006) The putative
SWI/SNF complex subunit BRAHMA activates ?ower
homeotic genes in Arabidopsis Zhaliana. Plant Mol Biol
62: 291-304
[0039] Ishida M, Tanaka S, Ohki M, Ohta T (2004) Tran
scriptional co-activator activity of SYT is negatively regu
lated by BRM and Brgl. Genes Cells 9: 419-428
[0040] Jerzmanowski A (2007) SWI/SNF chromatin
remodeling and linker histones in plants. Biochim Biophys
Acta. 1769: 330-345.
At1g20670
AT5G55210
Schutze G, Hantschel O, Superti-Firga G, BauchA (2006)
An ef?cient tandem a?inity puri?cation procedure for
translocation found in human synovial sarcoma. Nat Genet
TABLE 5
AT 1 G7 63 80
3389-3402.
Example 5
withARP4 and with SWI3C; all AN3 interacting proteins that
AT 1 G20670
Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs, Nucleic Acids Res. 25,
[0034] Altschul, S. E, Wootton, J. C., Gertz, E. M., Agar
wala, R., Morgulis, A., Schaffer, A. A. andYu,Y. K. (2005).
Protein database searches using compositionally adjusted
[0031] SWI3C interactors were identi?ed according to the
methods described above. The results are summarized in
Table 5. There is a strong similarity in interactors identi?ed
AT number
[0033] Altschul, S. E, Madden, T. L., Schaffer, A. A.,
Zhang, J., Zhang, Z., Miller, W. and Lipman, D. L. (1997),
HOMOLOG)
(THIOL) LYASE (OAS-TL)
[0041] Kim J H, Choi D, Kende H (2003) The AtGRF
family of putative transcription factors is involved in leaf
and cotyledon growth in Arabidopsis. Plant J 36: 94-104
[0042] Kim J H, Kende H (2004) A transcriptional coacti
vator, AtGIFl, is involved in regulating leaf growth and
morphology in Arabidopsis. Proc Natl Acad Sci USA
1001: 13374-13379
kinase 23)
TAP total gives the total number oftime that an interactor was copuri?ed;
C-GS and N-GS refers to whether a C or N terminal GS-tag was used in the experiment.
[0043] Kwon C S, Hibara K-I, P?uger J, Bezhani S, Metha
H, Aida M, Tasaka M, Wagner D (2006) A role for chro
matin remodeling in regulation of CUC gene expression in
the Arabidopsis cotyledon boundary. Development 133:
3223-3230
US 2012/0324602 A1
Dec. 20, 2012
7
[0044]
MeagherRB, Deal RB, KandasamyMK, McKin-
[0047] Van Camp W (2005) Yield enhancement genes:
ney E C (2005) Nuclear actin-related proteins as epigenetic
regulators of deYelopmgm' P1amPhyS1O1.139: 1579-1585
[0045]
Samowski T J, Rios G, Jasik J, SW1eZeWsk1 S, Kac-
-
-
seeds for growth. Curr Opin Biotech 16: 147-153
[0048] Van Leene J, Stals H, Eeckhout D, Persiau G, Van
..
-
De SllJke E, Van lsterdael G, De Clercq A, Bonnet E,
ZanoWsk1 S, L1 Y, Kwiatkowska A, PaWhkoWska K,
L
KoZbial M, KoncZ C, JerZmanoWski A (2005) SW13 sub-
A211;
units of putative SWl/SNF chromatin-remodeling com-
k
K R
.
N H
d . kX K D V..1d
T
2A gl?lmene A’ V enoeni 1 h 1e ,IJD ii], ’
e
m
’
arazyn
’
an
nc een_ ’ HZ?
’ _ lt'
plexes play distinct roles duringArabidopsis development.
ters E’ De Jaeger G (2007) A tandem af?mty pun?canon'
Plant Cell 17. 2454_2472
Thaetec BrenD Monaghanp Whitehouses Ren_
nie G, Rayner E, Cooper C S, GoodWin G (1999) Functional domains of the SYT and SYT-SSX synovial sarcoma
translocation proteins and co-localiZation With the SNP
protein BRM in the nucleus. Hum Mol Genet 8: 585-591
based technology platform to study the cell cycle 1nterac
tome in Arabidopsis Zhaliana. M01
Proteomics 61
12264238
[0049] Wagner D, MeyeroWitZ E M (2002) SPLAYED, a
novel SWl/SNF ATPase homolog, controls reproductive
development in Arabidopsis. Current Biol 12: 85-94
SEQUENCE LISTING
<l60> NUMBER OF SEQ ID NOS:
59
<21o> SEQ ID No 1
<211> LENGTH: 21o
<212> TYPE: PRT
<2l3> ORGANISM: Arabidopsis thaliana
<22o> FEATURE:
<221> NAME/KEY: MISCEFEATURE
<222>
LOCATION:
(1) . . (21o)
<223> OTHER INFORMATION: AT5G2864O
<4oo> SEQUENCE:
1
Met Gln Gln His Leu Met Gln Met Gln Pro Met Met Ala Gly Tyr Tyr
1
5
1o
15
Pro Ser Asn Val Thr Ser Asp His Ile Gln Gln Tyr Leu Asp Glu Asn
2o
25
3o
Lys Ser Leu Ile Leu Lys Ile Val Glu Ser Gln Asn Ser Gly Lys Leu
35
4o
45
Ser Glu Cys Ala Glu Asn Gln Ala Arg Leu Gln Arg Asn Leu Met Tyr
50
55
6O
Leu Ala Ala Ile Ala Asp Ser Gln Pro Gln Pro Pro Ser Val His Ser
65
7o
75
so
Gln Tyr Gly Ser Ala Gly Gly Gly Met Ile Gln Gly Glu Gly Gly Ser
85
9o
95
His Tyr Leu Gln Gln Gln Gln Ala Thr Gln Gln Gln Gln Met Thr Gln
100
105
110
Gln Ser Leu Met Ala Ala Arg Ser Ser Met Leu Tyr Ala Gln Gln Gln
115
120
125
Gln Gln Gln Gln Pro Tyr Ala Thr Leu Gln His Gln Gln Leu His His
130
135
140
Ser Gln Leu Gly Met Ser Ser Ser Ser Gly Gly Gly Gly Ser Ser Gly
145
150
155
160
Leu His Ile Leu Gln Gly Glu Ala Gly Gly Phe His Asp Phe Gly Arg
165
170
175
Gly Lys Pro Glu Met Gly Ser Gly Gly Gly Gly Glu Gly Arg Gly Gly
180
185
190
Ser Ser Gly Asp Gly Gly Glu Thr Leu Tyr Leu Lys Ser Ser Asp Asp
195
Gly Asn
210
200
205
US 2012/0324602 A1
Dec. 20, 2012
—cont inued
<210> SEQ ID NO 2
<211> LENGTH: 535
<212> TYPE: PRT
<2l3> ORGANISM: Arabidopsis thaliana
<220> FEATURE:
<22l> NAME/KEY: miscifeature
<222>
LOCATION:
(1) . . (535)
<223> OTHER INFORMATION: AT4G16143
<400> SEQUENCE: 2
Met Ser Leu Arg Pro Asn Ala Lys Thr Glu Val Arg Arg Asn Arg
1
5
1O
15
Tyr
Lys Val Ala Val Asp Ala Glu Glu Gly Arg Arg Arg Arg Glu Asp
Asn
2O
25
30
Met Val Glu Ile Arg Lys Ser Lys Arg Glu Glu Ser Leu Gln Lys
35
4O
45
Lys
Arg Arg Glu Gly Leu Gln Ala Asn Gln Leu Pro Gln Phe Ala Pro
Ser
50
55
60
Pro Val Pro Ala Ser Ser Thr Val Glu Lys Lys Leu Glu Ser Leu Pro
65
80
Ala Met Val Gly Gly Val Trp Ser Asp Asp Arg Ser Leu Gln Leu Glu
85
9O
95
Ala Thr Thr Gln Phe Arg Lys Leu Leu Ser Ile Glu Arg Ser Pro Pro
100
105
110
Ile Glu Glu Val Ile Asp Ala Gly Val Val Pro Arg Phe Val Glu Phe
115
120
125
Leu Thr Arg Glu Asp Tyr Pro Gln Leu Gln Phe Glu Ala Ala Trp Ala
130
135
140
Leu Thr Asn Ile Ala Ser Gly Thr Ser Glu Asn Thr Lys Val Val Ile
145
150
155
160
Glu His Gly Ala Val Pro Ile Phe Val Gln Leu Leu Ala Ser Gln Ser
165
170
175
Asp Asp Val Arg Glu Gln Ala Val Trp Ala Leu Gly Asn Val Ala Gly
180
185
190
Asp Ser Pro Arg Cys Arg Asp Leu Val Leu Gly Gln Gly Ala Leu
195
200
Ile
205
Pro Leu Leu Ser Gln Leu Asn Glu His Ala Lys Leu Ser Met Leu
210
215
220
Arg
Asn Ala Thr Trp Thr Leu Ser Asn Phe Cys Arg Gly Lys Pro Gln Pro
225
230
235
240
Pro Phe Asp Gln Val Arg Pro Ala Leu Pro Ala Leu Glu Arg Leu Ile
245
250
255
Ser Thr Asp Glu Glu Val Leu Thr Asp Ala Cys Trp Ala Leu Ser
260
265
270
Tyr Leu Ser Asp Gly Thr Asn Asp Lys Ile Gln Ser Val Ile Glu
2'75
Gly
280
Ala
285
Val Val Pro Arg Leu Val Glu Leu Leu Gln His Gln Ser Pro Ser
290
295
300
Val Leu Ile Pro Ala Leu Arg Ser Ile Gly Asn Ile Val Thr Gly
Asp
305
320
310
315
Asp Leu Gln Thr Gln Cys Val Ile Ser His Gly Ala Leu Leu Ser
325
330
Leu
335
Leu Ser Leu Leu Thr His Asn His Lys Lys Ser Ile Lys Lys Glu Ala
340
345
350
US 2012/0324602 A1
Dec. 20, 2012
—cont inued
Cys Trp Thr Ile Ser Asn Ile Thr Ala Gly Asn Arg Asp Gln Ile Gln
355
360
365
Ala Val Cys Glu Ala Gly Leu Ile Cys Pro Leu Val Asn Leu Leu Gln
3'70
375
380
Asn Ala Glu Phe Asp Ile Lys Lys Glu Ala Ala Trp Ala Ile Ser Asn
385
390
395
400
Ala Thr Ser Gly Gly Ser Pro Asp Gln Ile Lys Tyr Met Val Glu Gln
405
410
415
Gly Val Val Lys Pro Leu Cys Asp Leu Leu Val Cys Pro Asp Pro Arg
420
425
430
Ile Ile Thr Val Cys Leu Glu Gly Leu Glu Asn Ile Leu Lys Val Gly
435
440
445
Glu Ala Glu Lys Val Thr Gly Asn Thr Gly Asp Val Asn Phe Tyr Ala
450
455
460
Gln Leu Ile Asp Asp Ala Glu Gly Leu Glu Lys Ile Glu Asn Leu Gln
465
470
475
480
Ser His Asp Asn Ser Glu Ile Tyr Glu Lys Ala Val Lys Ile Leu Glu
485
490
495
Thr Tyr Trp Leu Glu Glu Glu Asp Glu Thr Leu Pro Pro Gly Asp Pro
500
505
510
Ser Ala Gln Gly Phe Gln Phe Gly Gly Gly Asn Asp Ala Ala Val Pro
515
520
525
Pro Gly Gly Phe Asn Phe Gln
530
535
<2ll> LENGTH: 532
<2l2> TYPE: PRT
<2l3> ORGANISM: Arabidopsis thaliana
<220> FEATURE:
<22l> NAME/KEY: miscifeature
<222>
LOCATION:
(l) . . (532)
<223> OTHER INFORMATION: AT3GO672O
<400> SEQUENCE: 3
Met Ser Leu Arg Pro Asn Ala Lys Thr Glu Val Arg Arg Asn Arg Tyr
l
5
l0
l5
Lys Val Ala Val Asp Ala Glu Glu Gly Arg Arg Arg Arg Glu Asp Asn
2O
25
3O
Met Val Glu Ile Arg Lys Ser Lys Arg Glu Glu Ser Leu Met Lys Lys
35
4O
45
Arg Arg Glu Gly Met Gln Ala Leu Gln Gly Phe Pro Ser Ala Ser Ala
50
55
6O
Ala Ser Val Asp Lys Lys Leu Asp Ser Leu Lys Asp Met Val Ala Gly
65
7O
75
80
Val Trp Ser Asp Asp Pro Ala Leu Gln Leu Glu Ser Thr Thr Gln Phe
85
9O
95
Arg Lys Leu Leu Ser Ile Glu Arg Ser Pro Pro Ile Glu Glu Val Ile
100
105
110
Ser Ala Gly Val Val Pro Arg Phe Val Glu Phe Leu Lys Lys Glu Asp
115
120
125
Tyr Pro Ala Ile Gln Phe Glu Ala Ala Trp Ala Leu Thr Asn Ile Ala
130
135
140
Ser Gly Thr Ser Asp His Thr Lys Val Val Ile Asp His Asn Ala Val
145
150
155
160
US 2012/0324602 A1
Dec. 20, 2012
10
—cont inued
Pro Ile Phe Val Gln Leu Leu Ala Ser Pro Ser Asp Asp Val Arg Glu
165
170
175
Gln Ala Val Trp Ala Leu Gly Asn Val Ala Gly Asp Ser Pro Arg Cys
180
185
190
Arg Asp Leu Val Leu Gly Cys Gly Ala Leu Leu Pro Leu Leu Asn Gln
195
200
205
Leu Asn Glu His Ala Lys Leu Ser Met Leu Arg Asn Ala Thr Trp Thr
210
215
220
Leu Ser Asn Phe Cys Arg Gly Lys Pro Gln Pro His Phe Asp Gln Val
225
230
235
240
Lys Pro Ala Leu Pro Ala Leu Glu Arg Leu Ile His Ser Asp Asp Glu
245
250
255
Glu Val Leu Thr Asp Ala Cys Trp Ala Leu Ser Tyr Leu Ser Asp Gly
260
265
270
Thr Asn Asp Lys Ile Gln Thr Val Ile Gln Ala Gly Val Val Pro Lys
275
280
285
Leu Val Glu Leu Leu Leu His His Ser Pro Ser Val Leu Ile Pro Ala
290
295
300
Leu Arg Thr Val Gly Asn Ile Val Thr Gly Asp Asp Ile Gln Thr Gln
305
310
315
320
Cys Val Ile Asn Ser Gly Ala Leu Pro Cys Leu Ala Asn Leu Leu Thr
325
330
335
Gln Asn His Lys Lys Ser Ile Lys Lys Glu Ala Cys Trp Thr Ile Ser
340
345
350
Asn Ile Thr Ala Gly Asn Lys Asp Gln Ile Gln Thr Val Val Glu Ala
355
360
365
Asn Leu Ile Ser Pro Leu Val Ser Leu Leu Gln Asn Ala Glu Phe Asp
370
375
380
Ile Lys Lys Glu Ala Ala Trp Ala Ile Ser Asn Ala Thr Ser Gly Gly
385
390
395
400
Ser His Asp Gln Ile Lys Tyr Leu Val Glu Gln Gly Cys Ile Lys Pro
405
410
415
Leu Cys Asp Leu Leu Val Cys Pro Asp Pro Arg Ile Ile Thr Val Cys
420
425
430
Leu Glu Gly Leu Glu Asn Ile Leu Lys Val Gly Glu Ala Glu Lys Asn
435
440
445
Leu Gly His Thr Gly Asp Met Asn Tyr Tyr Ala Gln Leu Ile Asp Asp
450
455
460
Ala Glu Gly Leu Glu Lys Ile Glu Asn Leu Gln Ser His Asp Asn Asn
465
470
475
480
Glu Ile Tyr Glu Lys Ala Val Lys Ile Leu Glu Thr Tyr Trp Leu Glu
485
490
495
Glu Glu Asp Asp Glu Thr Gln Gln Pro Pro Gly Val Asp Gly Ser Gln
500
505
510
Ala Gly Phe Gln Phe Gly Gly Asn Gln Ala Pro Val Pro Ser Gly Gly
515
Phe Asn Phe Ser
530
<210> SEQ ID NO 4
<211> LENGTH: 8'70
<212> TYPE: PRT
520
525
US 2012/0324602 A1
Dec. 20, 2012
11
—cont inued
ORGANISM: Arabidopsis thaliana
FEATURE:
NAME/KEY: miscifeature
LOCATION: (1) ~ ~ (870)
OTHER INFORMATION: AT5G5348O
<400> SEQUENCE: 4
Met Ala Met Glu Val Thr Gln Leu Leu Ile Asn Ala Gln Ser Ile
1
5
15
Gly
Thr Val
Arg Lys His
Ala Glu Glu Ser Leu
20
Gln Asn Leu Ala
Glu
Lys
Lys
Gln Phe Gln Glu
Gly
Glu Leu Ala Asn
25
Gly
Phe Leu Leu Ser Leu Ala
35
Asp
Asp
40
45
Arg Lys
Gly
Pro Val
Asp
Ser
Asp
Ala
Lys
Glu Gln His
Arg Lys Tyr
Leu Ala Leu
85
Asp
Met Ser Thr
Lys
Leu Ala
Leu Val Leu
Lys
50
Asn Ala Leu
65
Arg Trp
Leu Leu
Lys
Ser Gln Ile
Thr Leu Ser Ala Pro Val Pro
Lys
Val Ala
Gly
Asp
Lys
Asp
Met Asn Ala Ala Glu
185
Leu Ala Ala Thr
195
Arg
Ala Leu
Ala Asn Phe Asn Asn
Asp
Tyr
Tyr
Met Gln
Met Glu
215
Arg Asp Tyr
Lys
Asp
Val
Phe Ala Gln
Arg
Val Val
Arg
Gln Ala Ala Phe
240
Tyr
Glu
Lys
Lys
Ala Val
Ile Leu Glu Glu
Leu Ala His
255
Arg
Asp
Trp
Ser Ser Ile
285
Cys
Tyr Gly Gly
Glu Phe Ala
Gly
Gln Ala Leu Pro
Gly
270
295
Asp
Val Pro
305
Cys
Phe
Tyr
Phe Thr
310
Asp
Glu
Gly
Ala
340
Leu Val Ala
355
Arg
Lys
315
Leu Val Pro Leu Leu Leu Glu Thr Leu Leu
325
330
Gly
Gly
Ile Met
280
290
Leu
Asn Thr
190
265
275
Leu
Ile
Glu Ser Val Ala Leu Gln Ala Ile Glu Phe
Ser
Ile Leu
Glu
Ile Phe Asn Ile Thr Ala
Asp
Lys
235
260
Glu Glu Ile
Glu
220
Leu Val Ser Ile Ala Ser Thr Tyr
245
250
Asp
Gly
Met Ala Leu
230
Cys
Cys
205
Glu Ala Thr Leu Ser Pro Glu Val
Glu
Leu
160
200
210
Asp
Gln
170
180
225
Gly Tyr
Val Val Glu Gln Glu His Val Asn
Gly
Thr Ala Val Val Gln
Asp
Lys
155
165
Asp
Ser Thr Ala
110
125
150
Glu Val Ser Pro
Asp
Arg
Ile Glu Leu Pro Gln
120
Gln Ala Thr Leu Glu Thr Leu
145
Cys
Val
Pro Glu Leu Ile Val Ser Leu Leu Ser Asn Ile His Gln Leu Pro
130
135
140
Ala His Val
Arg
Ala Phe
95
105
Ser Gln Val Ile Ala
115
Arg
90
100
Trp
Glu Leu Val Gln
Trp
Lys
320
Gln Glu Glu
360
Gly Asp Asp
Gln
335
Asn Ile Ala Met Ala
345
Ala Val
Asp
Gly Gly
Thr
Cys
350
Ile Val Pro His Val
365
US 2012/0324602 A1
Dec. 20, 2012
12
—cont inued
Met Pro Phe Ile Glu Glu Lys Ile Ser Lys Pro Asp Trp Arg Glu Arg
370
375
380
Glu Ala Ala Thr Tyr Ala Phe Gly Ser Ile Leu Glu Gly Pro Ser Ala
385
390
395
400
Asp Lys Leu Met Ala Ile Val Asn Ala Ala Leu Thr Phe Met Leu Asn
405
410
415
Ala Leu Thr Asn Asp Pro Ser Asn His Val Lys Asp Thr Thr Ala Trp
420
425
430
Thr Leu Gly Arg Ile Phe Glu Phe Leu His Gly Ser Thr Ile Glu Thr
435
440
445
Pro Ile Ile Asn Gln Ala Asn Cys Gln Gln Ile Ile Thr Val Leu Ile
450
455
460
Gln Ser Met Asn Asp Ala Pro Asn Val Ala Glu Lys Ala Cys Gly Ala
465
470
475
480
Leu Tyr Phe Leu Ala Gln Gly Tyr Glu Asp Ile Gly Pro Ser Ser Pro
485
490
495
Leu Thr Pro Phe Phe Gln Glu Ile Ile Lys Ser Leu Leu Ala Val Ala
500
505
510
His Arg Glu Asp Ala Thr Glu Ser Arg Leu Arg Thr Ala Ala Tyr Glu
515
520
525
Ala Leu Asn Glu Val Val Arg Cys Ser Thr Asp Glu Thr Ser Thr Met
530
535
540
Val Leu Gln Leu Val Pro Val Ile Met Met Glu Leu His Asn Thr Leu
545
550
555
560
Glu Gly Glu Lys Leu Ser Leu Asp Glu Arg Glu Lys Gln Asn Glu Leu
565
570
575
Gln Gly Leu Leu Cys Gly Cys Leu Gln Val Ile Ile Gln Lys Leu Gly
580
585
590
Ser Glu Pro Thr Lys Ser Lys Phe Met Glu Tyr Ala Asp Gln Met Met
595
600
605
Gly Leu Phe Leu Arg Val Phe Gly Cys Arg Ser Ala Thr Ala His Glu
610
615
620
Glu Ala Met Leu Ala Ile Gly Ala Leu Ala Tyr Ala Ala Gly Pro Asn
625
630
635
640
Phe Ala Lys Tyr Met Pro Glu Phe Tyr Lys Tyr Leu Glu Met Gly Leu
645
650
655
Gln Asn Phe Glu Glu Tyr Gln Val Cys Ala Val Thr Val Gly Val Val
660
665
670
Gly Asp Val Cys Arg Ala Leu Glu Asp Lys Ile Leu Pro Tyr Cys Asp
675
680
685
Gly Ile Met Thr Gln Leu Leu Lys Asp Leu Ser Ser Asn Gln Leu His
690
695
700
Arg Ser Val Lys Pro Pro Ile Phe Ser Cys Phe Gly Asp Ile Ala Leu
705
710
715
720
Ala Ile Gly Glu Asp Phe Asp Lys Tyr Trp Arg Tyr Ser Met Pro Met
725
730
735
Leu Gln Ser Ala Ala Glu Leu Ser Ala His Ser Ala Gly Ala Asp Asp
740
745
750
Glu Met Thr Glu Tyr Thr Asn Ser Leu Arg Asn Gly Ile Leu Glu Ala
755
760
765
Tyr Ser Gly Ile Phe Gln Gly Phe Lys Asn Ser Ala Lys Thr Gln Leu
770
775
780
US 2012/0324602 A1
Dec. 20, 2012
13
—cont inued
Leu Ile Pro Phe Ala Pro His Ile Leu Gln Phe Leu Asp Ser Ile Tyr
785
790
795
800
Met Glu Lys Asp Met Asp Glu Val Val Met Lys Thr Ala Ile Gly Val
805
810
815
Leu Gly Asp Leu Ala Asp Thr Leu Gly Ser His Val Gly Gly Leu Ile
820
825
830
Gln Gln Ser Val Ser Ser Lys Glu Phe Leu Asn Glu Cys Leu Ser Ser
835
840
845
Glu Asp His Thr Ile Lys Glu Ala Ala Glu Trp Ala Lys His Ala Ile
850
855
860
Thr Arg Ala Ile Ser Val
865
870
<2ll> LENGTH: 538
<2l2> TYPE: PRT
<2l3> ORGANISM: Arabidopsis thaliana
<220> FEATURE:
<22l> NAME/KEY: miscifeature
<222>
LOCATION:
(1) . . (538)
<223> OTHER INFORMATION: AT1GO927O
<400> SEQUENCE: 5
Met Ser Leu Arg Pro Ser Thr Arg Ala Glu Leu Arg Lys Lys Ile Tyr
1
5
1O
15
Lys Thr Gly Val Asp Ala Asp Glu Ala Arg Arg Arg Arg Glu Asp Asn
2O
25
3O
Leu Val Glu Ile Arg Lys Asn Lys Arg Glu Asp Ser Leu Leu Lys Lys
35
4O
45
Arg Arg Glu Gly Met Met Leu Gln Gln Gln Leu Pro Leu Gly Ala Gly
50
55
6O
Leu Asp Gly Pro Gln Thr Ala Ala Ala Val Glu Lys Arg Leu Glu Gly
65
7O
75
8O
Ile Pro Met Met Val Gln Gly Val Tyr Ser Asp Asp Pro Gln Ala Gln
85
9O
95
Leu Glu Ala Thr Thr Gln Phe Arg Lys Leu Leu Ser Ile Glu Arg Ser
100
105
110
Pro Pro Ile Asp Glu Val Ile Lys Ala Gly Val Ile Pro Arg Phe Val
115
120
125
Glu Phe Leu Gly Arg His Asp His Pro Gln Leu Gln Phe Glu Ala Ala
130
135
140
Trp Ala Leu Thr Asn Val Ala Ser Gly Thr Ser Asp His Thr Arg Val
145
150
155
160
Val Ile Glu Gln Gly Ala Val Pro Ile Phe Val Lys Leu Leu Thr Ser
165
170
175
Ala Ser Asp Asp Val Arg Glu Gln Ala Val Trp Ala Leu Gly Asn Val
180
185
190
Ala Gly Asp Ser Pro Asn Cys Arg Asn Leu Val Leu Asn Tyr Gly Ala
195
200
205
Leu Glu Pro Leu Leu Ala Gln Leu Asn Glu Asn Ser Lys Leu Ser Met
210
215
220
Leu Arg Asn Ala Thr Trp Thr Leu Ser Asn Phe Cys Arg Gly Lys Pro
225
230
235
240
Pro Thr Pro Phe Glu Gln Val Lys Pro Ala Leu Pro Ile Leu Arg Gln
US 2012/0324602 A1
Dec. 20, 2012
14
—cont inued
245
250
255
Leu Ile Tyr Leu Asn Asp Glu Glu Val Leu Thr Asp Ala Cys Trp Ala
260
265
270
Leu Ser Tyr Leu Ser Asp Gly Pro Asn Asp Lys Ile Gln Ala Val Ile
2'75
280
285
Glu Ala Gly Val Cys Pro Arg Leu Val Glu Leu Leu Gly His Gln Ser
290
295
300
Pro Thr Val Leu Ile Pro Ala Leu Arg Thr Val Gly Asn Ile Val Thr
305
310
315
320
Gly Asp Asp Ser Gln Thr Gln Phe Ile Ile Glu Ser Gly Val Leu Pro
325
330
335
His Leu Tyr Asn Leu Leu Thr Gln Asn His Lys Lys Ser Ile Lys Lys
340
345
350
Glu Ala Cys Trp Thr Ile Ser Asn Ile Thr Ala Gly Asn Lys Leu Gln
355
360
365
Ile Glu Ala Val Val Gly Ala Gly Ile Ile Leu Pro Leu Val His Leu
370
375
380
Leu Gln Asn Ala Glu Phe Asp Ile Lys Lys Glu Ala Ala Trp Ala Ile
385
390
395
400
Ser Asn Ala Thr Ser Gly Gly Ser His Glu Gln Ile Gln Tyr Leu Val
405
410
415
Thr Gln Gly Cys Ile Lys Pro Leu Cys Asp Leu Leu Ile Cys Pro Asp
420
425
430
Pro Arg Ile Val Thr Val Cys Leu Glu Gly Leu Glu Asn Ile Leu Lys
435
440
445
Val Gly Glu Ala Asp Lys Glu Met Gly Leu Asn Ser Gly Val Asn Leu
450
455
460
Tyr Ala Gln Ile Ile Glu Glu Ser Asp Gly Leu Asp Lys Val Glu Asn
465
470
475
480
Leu Gln Ser His Asp Asn Asn Glu Ile Tyr Glu Lys Ala Val Lys Ile
485
490
495
Leu Glu Arg Tyr Trp Ala Glu Glu Glu Glu Glu Gln Ile Leu Gln Asp
500
505
510
Gly Gly Asn Asp Asn Ser Gln Gln Ala Phe Asn Phe Gly Asn Asn Pro
515
520
525
Ala Ala Pro Val Gly Gly Phe Lys Phe Ala
530
535
<2ll> LENGTH:
3574
<2l3> ORGANISM: Arabidopsis thaliana
<220> FEATURE:
<22l> NAME/KEY: miscifeature
<222>
LOCATION:
(l) . . (3574)
<223> OTHER INFORMATION: AT2G2829O
<400> SEQUENCE: 6
Met Thr Ser Ser Ser His Asn Ile Glu Leu Glu Ala Ala Lys Phe Leu
1
5
l0
15
His Lys Leu Ile Gln Asp Ser Lys Asp Glu Pro Ala Lys Leu Ala Thr
20
25
30
Lys Leu Tyr Val Ile Leu Gln His Met Lys Thr Ser Gly Lys Glu Asn
35
40
45
US 2012/0324602 A1
Dec. 20, 2012
15
—cont inued
Thr Met Pro Tyr Gln Val Ile Ser Arg Ala Met Asp Thr Val Val Asn
50
55
60
Gln His Gly Leu Asp Ile Glu Ala Leu Lys Ser Ser Cys Leu Pro His
65
'70
'75
80
Pro Gly Gly Thr Gln Thr Glu Asp Ser Gly Ser Ala His Leu Ala Gly
85
90
95
Ser Ser Gln Ala Val Gly Val Ser Asn Glu Gly Lys Ala Thr Leu Val
100
105
110
Glu Asn Glu Met Thr Lys Tyr Asp Ala Phe Thr Ser Gly Arg Gln Leu
115
120
125
Gly Gly Ser Asn Ser Ala Ser Gln Thr Phe Tyr Gln Gly Ser Gly Thr
130
135
140
Gln Ser Asn Arg Ser Phe Asp Arg Glu Ser Pro Ser Asn Leu Asp Ser
145
150
155
160
Thr Ser Gly Ile Ser Gln Pro His Asn Arg Ser Glu Thr Met Asn Gln
165
170
175
Arg Asp Val Lys Ser Ser Gly Lys Arg Lys Arg Gly Glu Ser Ser Leu
180
185
190
Ser Trp Asp Gln Asn Met Asp Asn Ser Gln Ile Phe Asp Ser His Lys
195
200
205
Ile Asp Asp Gln Thr Gly Glu Val Ser Lys Ile Glu Met Pro Gly Asn
210
215
220
Ser Gly Asp Ile Arg Asn Leu His Val Gly Leu Ser Ser Asp Ala Phe
225
230
235
240
Thr Thr Pro Gln Cys Gly Trp Gln Ser Ser Glu Ala Thr Ala Ile Arg
245
250
255
Pro Ala Ile His Lys Glu Pro Gly Asn Asn Val Ala Gly Glu Gly Phe
260
265
270
Leu Pro Ser Gly Ser Pro Phe Arg Glu Gln Gln Leu Lys Gln Leu Arg
275
280
285
Ala Gln Cys Leu Val Phe Leu Ala Leu Arg Asn Gly Leu Val Pro Lys
290
295
300
Lys Leu His Val Glu Ile Ala Leu Arg Asn Thr Phe Arg Glu Glu Asp
305
310
315
320
Gly Phe Arg Gly Glu Leu Phe Asp Pro Lys Gly Arg Thr His Thr Ser
325
330
335
Ser Asp Leu Gly Gly Ile Pro Asp Val Ser Ala Leu Leu Ser Arg Thr
340
345
350
Asp Asn Pro Thr Gly Arg Leu Asp Glu Met Asp Phe Ser Ser Lys Glu
355
360
365
Thr Glu Arg Ser Arg Leu Gly Glu Lys Ser Phe Ala Asn Thr Val Phe
3'70
375
380
Ser Asp Gly Gln Lys Leu Leu Ala Ser Arg Ile Pro Ser Ser Gln Ala
385
390
395
400
Gln Thr Gln Val Ala Val Ser His Ser Gln Leu Thr Phe Ser Pro Gly
405
410
415
Leu Thr Lys Asn Thr Pro Ser Glu Met Val Gly Trp Thr Gly Val Ile
420
425
430
Lys Thr Asn Asp Leu Ser Thr Ser Ala Val Gln Leu Asp Glu Phe His
435
440
445
Ser Ser Asp Glu Glu Glu Gly Asn Leu Gln Pro Ser Pro Lys Tyr Thr
450
455
460
US 2012/0324602 A1
Dec. 20, 2012
16
—cont inued
Met Ser Gln Lys Trp Ile Met Gly Arg Gln Asn Lys Arg Leu Leu Val
465
470
475
480
Asp Arg Ser Trp Ser Leu Lys Gln Gln Lys Ala Asp Gln Ala Ile Gly
485
490
495
Ser Arg Phe Asn Glu Leu Lys Glu Ser Val Ser Leu Ser Asp Asp Ile
500
505
510
Ser Ala Lys Thr Lys Ser Val Ile Glu Leu Lys Lys Leu Gln Leu Leu
515
520
525
Asn Leu Gln Arg Arg Leu Arg Ser Glu Phe Val Tyr Asn Phe Phe Lys
530
535
540
Pro Ile Ala Thr Asp Val Glu His Leu Lys Ser Tyr Lys Lys His Lys
545
550
555
560
His Gly Arg Arg Ile Lys Gln Leu Glu Lys Tyr Glu Gln Lys Met Lys
565
570
575
Glu Glu Arg Gln Arg Arg Ile Arg Glu Arg Gln Lys Glu Phe Phe Gly
580
585
590
Gly Leu Glu Val His Lys Glu Lys Leu Glu Asp Leu Phe Lys Val Arg
595
600
605
Arg Glu Arg Leu Lys Gly Phe Asn Arg Tyr Ala Lys Glu Phe His Lys
610
615
620
Arg Lys Glu Arg Leu His Arg Glu Lys Ile Asp Lys Ile Gln Arg Glu
625
630
635
640
Lys Ile Asn Leu Leu Lys Ile Asn Asp Val Glu Gly Tyr Leu Arg Met
645
650
655
Val Gln Asp Ala Lys Ser Asp Arg Val Lys Gln Leu Leu Lys Glu Thr
660
665
670
Glu Lys Tyr Leu Gln Lys Leu Gly Ser Lys Leu Lys Glu Ala Lys Leu
675
680
685
Leu Thr Ser Arg Phe Glu Asn Glu Ala Asp Glu Thr Arg Thr Ser Asn
690
695
700
Ala Thr Asp Asp Glu Thr Leu Ile Glu Asn Glu Asp Glu Ser Asp Gln
705
710
715
720
Ala Lys His Tyr Leu Glu Ser Asn Glu Lys Tyr Tyr Leu Met Ala His
725
730
735
Ser Ile Lys Glu Asn Ile Asn Glu Gln Pro Ser Ser Leu Val Gly Gly
740
745
750
Lys Leu Arg Glu Tyr Gln Met Asn Gly Leu Arg Trp Leu Val Ser Leu
755
760
765
Tyr Asn Asn His Leu Asn Gly Ile Leu Ala Asp Glu Met Gly Leu Gly
770
775
780
Lys Thr Val Gln Val Ile Ser Leu Ile Cys Tyr Leu Met Glu Thr Lys
785
790
795
800
Asn Asp Arg Gly Pro Phe Leu Val Val Val Pro Ser Ser Val Leu Pro
805
810
815
Gly Trp Gln Ser Glu Ile Asn Phe Trp Ala Pro Ser Ile His Lys Ile
820
825
830
Val Tyr Cys Gly Thr Pro Asp Glu Arg Arg Lys Leu Phe Lys Glu Gln
835
840
845
Ile Val His Gln Lys Phe Asn Val Leu Leu Thr Thr Tyr Glu Tyr Leu
850
855
860
Met Asn Lys His Asp Arg Pro Lys Leu Ser Lys Ile His Trp His Tyr
US 2012/0324602 A1
Dec. 20, 2012
17
—cont inued
865
870
875
880
Ile Ile Ile Asp Glu Gly His Arg Ile Lys Asn Ala Ser Cys Lys Leu
885
890
895
Asn Ala Asp Leu Lys His Tyr Val Ser Ser His Arg Leu Leu Leu Thr
900
905
910
Gly Thr Pro Leu Gln Asn Asn Leu Glu Glu Leu Trp Ala Leu Leu Asn
915
920
925
Phe Leu Leu Pro Asn Ile Phe Asn Ser Ser Glu Asp Phe Ser Gln Trp
930
935
940
Phe Asn Lys Pro Phe Gln Ser Asn Gly Glu Ser Ser Ala Glu Glu Ala
945
950
955
960
Leu Leu Ser Glu Glu Glu Asn Leu Leu Ile Ile Asn Arg Leu His Gln
965
970
975
Val Leu Arg Pro Phe Val Leu Arg Arg Leu Lys His Lys Val Glu Asn
980
985
Glu Leu Pro Glu Lys Ile Glu Arg
995
Tyr Gln
1010
Ile Gly
1025
Leu Arg
1040
Glu Glu
1055
Val Arg
1070
Lys Leu
1085
Thr Arg
1100
Tyr Lys
1115
Gly Ala
1130
Ile Phe
1145
Gln Ala
1160
Gln Val
1175
Lys Lys
1190
Glu Glu
1205
Asn Gln
1220
Glu Asp
1235
Lys Glu
1250
990
Leu Ile Arg Cys Glu
1000
Lys Leu Leu Met Lys
1015
Asn Ala Lys Ser Arg
1030
Asn Ile Cys Asn His
1045
Val Asn Asn Ile Ile
1060
Leu Cys Gly Lys Leu
1075
Lys Ala Thr Asp His
1090
Leu Leu Asp Val Met
1105
Tyr Leu Arg Leu Asp
1120
Leu Ile Asp Gly Phe
1135
Leu Leu Ser Ile Arg
1150
Ala Asp Thr Val Ile
1165
Asp Leu Gln Ala Gln
1180
Asp Val Leu Val Leu
1195
Gln Val Arg Ala Ser
1210
Ser Ile Thr Ala Gly
1225
Arg Lys Glu Tyr Leu
1240
Glu Asp Ala Pro Val
1255
Ala Ser Ala
1005
Arg Val Glu Asp Asn
Leu Gly Ser
1020
Ala Val His Asn Ser
Val Met Glu
1035
Pro Tyr Leu Ser Gln
Leu His Ser
1050
Pro Lys His Phe Leu
Pro Pro Ile
1065
Glu Met Leu Asp Arg
Met Leu Pro
1080
Arg Val Leu Phe Phe
Ser Thr Met
1095
Glu Asp Tyr Leu Thr
Leu Lys Gly
1110
Gly Gln Thr Ser Gly
Gly Asp Arg
1125
Asn Lys Ser Gly Ser
Pro Phe Phe
1140
Ala Gly Gly Val Gly
Val Asn Leu
1155
Leu Phe Asp Thr Asp
Trp Asn Pro
1170
Ala Arg Ala His Arg
Ile Gly Gln
1185
Arg Phe Glu Thr Val
Asn Ser Val
1200
Ala Glu His Lys Leu
Gly Val Ala
1215
Phe Phe Asp Asn Asn
Thr Ser Ala
1230
Glu Ser Leu Leu Arg
Glu Ser Lys
1245
Leu Asp Asp Asp Ala
1260
Leu Asn Asp
US 2012/0324602 A1
Dec. 20, 2012
18
—cont inued
Leu Ile
1265
Asp Lys
1280
Val His
1295
Pro Ser
1310
Thr Met
1325
Val Gly
1340
His Gln
1355
Glu Glu
1370
Glu Ser
1385
Ala Arg Arg Glu Ser
1270
Gln Arg Lys Glu Asn
1285
Gly Pro Gly Ser Asp
1300
Arg Leu Val Thr Glu
1315
Lys Leu Asn Asp Val
1330
Met Lys Arg Lys Asp
1345
Tyr Gly Arg Gly Lys
1360
Lys Leu Thr Glu Glu
1375
Pro Asp Ser Pro Gln
1390
Glu Ile Asp Ile Phe
Glu Ser Ile
1275
Glu Met Glu Thr Trp
Asn Thr Leu
1290
Ser Phe Ala His Ile
Pro Ser Ile
1305
Asp Asp Leu Lys Leu
Leu Tyr Glu
1320
Pro Met Val Ala Lys
Glu Ser Thr
1335
Gly Ser Met Gly Gly
Leu Asp Thr
1350
Arg Ala Arg Glu Val
Arg Ser Tyr
1365
Glu Phe Glu Lys Leu
Cys Gln Thr
1380
Gly Lys Gly Glu Gly
Ser Glu Arg
1395
Ser Leu Ala Asn Asp Thr Ser Asn Ile Pro Val Glu Asn Ser Ser
1400
1405
1410
Asp Thr
1415
Pro Met
1430
Thr Gln
1445
Lys Ala
1460
Ala Thr
1475
Val Ser
1490
Ser Leu
1505
Gln Ser
1520
Gly Arg
1535
Val Glu
1550
Thr Glu
1565
Phe Ala
1580
Lys Ala
1595
Val His
1610
Lys Asp
1625
Gln Thr
1640
Leu Leu Pro Thr Ser
1420
Glu Pro Val Arg Pro
1435
Pro Ile Lys Arg Gly
1450
Leu Thr Pro Val Ser
1465
Gly Asn Ala Ile Ser
1480
Ser Asp Lys Arg Leu
1495
Ala Leu Thr Ser Pro
1510
Leu Pro Ala Ser Pro
1525
Gly Arg Ser Arg Gly
1540
Gly Val Leu His Gly
1555
Thr Ala Thr Ser Leu
1570
Leu Pro Arg Ser Ala
1585
Asn Glu Gly Ser Thr
1600
Ser Ala Thr Thr Ala
1615
Leu Asp Ala Pro Pro
1630
Leu Asn Val Leu Glu
1645
Pro Thr Gln Ala Ile
Thr Val Gln
1425
Gln Ser His Thr Leu
Lys Glu Glu
1440
Arg Gly Arg Pro Lys
Arg Thr Asp
1455
Leu Ser Ala Val Ser
Arg Thr Gln
1470
Ser Ala Ala Thr Gly
Leu Asp Phe
1485
Glu Ala Ala Ser His
Pro Thr Ser
1500
Asp Leu Ser Gly Pro
Pro Gly Phe
1515
Ala Pro Thr Pro Ile
Arg Gly Arg
1530
Arg Gly Ala Gly Arg
Gly Arg Arg
1545
Ser Asn Ser Ser Ile
Thr Gln Arg
1560
Ala Ser Asp Ala Glu
Ala Thr Lys
1575
Ser Glu Ile Val Ser
Arg Val Pro
1590
Ser Asn Pro Asp Gln
Val Ser Pro
1605
Leu Arg Ser Asp Lys
Ala Ala Asp
1620
Gly Phe Asp Ser Gly
Ser His Val
1635
Asn Ser Ser Glu Arg
1650
Lys Ala Phe