Note: Descriptions are shown in the official language in which they were submitted.
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Gene involved in epigenetic gene silencing
The present invention relates to DNA which encodes proteins that control gene
silencing,
and particularly the silencing of plant genes.
The loss of expression of previously active genes in plants, also referred to
as gene
silencing, is observed in response to developmental, environmental or unknown
signals. It
occurs at a frequency higher than that of mutations, yet it is markedly stable
during somatic
transmission. Gene silencing, initially perceived as an unwanted source of
instability of
transgene expression, is now regarded as a molecular tool to intentionally
regulate gene
expression.
It appears that chromosomal position or structure of the affected loci are
factors determining
the frequency and strength of silencing. Inactivation seems. to preferentially
affect genes
present in multiple copies and is thought to be a consequence of sequence
redundancy.
Many examples of homology-dependent gene silencing have been reported. Closer
analysis has allowed the classification of silencing events according to the
relative position
of the affected loci (cis, traps, allelic, ectopic), the origin of the
affected genes (endogenous
or transgenic), and the level of interaction (transcriptional or post-
transcriptional). While
post-transcriptional silencing seems to mainly involve the formation of
aberrant RNA
molecules and is occasionally, but not necessarily, accompanied by DNA
methylation,
silencing interfering with transcription initiation is more strictly
correlated with
hypermethylation of the DNA and possibly with alteration of chromatin
structure at the silent
loci. It is, however, not clear whether these molecular events are a
prerequisite for gene
silencing or a consequence of the silent state.
In the case of transcriptional silencing, the inactive state of silenced genes
is stably
transmitted through mitotic and meiotic divisions. As in other organisms,
traps-acting
modifier loci are assumed to be responsible for the stability of the inactive
state of the
silenced genes. Mutations in such loci resulting in mutated proteins are
expected to result in
reduced gene silencing and reactivation of previously silent loci by
interfering with the
maintenance of the silent state, or by a failure to recognize sequence
redundancy. It has
been reported that mutations in the DDM1 gene of Arabidopsis thaliana release
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transcriptional gene silencing and that this genes encodes a SW12/SNF2-like
protein
involved in chromatin remodeling. However, mutation of the DDM1 gene causes
severe
pleiotropic effects. Therefore, to be able to modify such effects making use
of gene
technology, it is necessary to identify further specific modifier loci and
characterize the
corresponding wild-type and mutant proteins. It is the main objective of the
present
invention to provide DNA comprising an open reading frame encoding such a
protein.
Trans-acting modifier loci according to the present invention can be
identified by T-DNA
insertion mutagenesis as described in Example 1 for an Arabidopsis line
carrying a heritably
inactivated, methylated hygromycin resistance gene. A mutation of a silencing
modifier
locus results in release of silencing of the hygromycin resistance gene and
restores
hygromycin resistance. Plants homozygous for the silent resistance gene are
subjected to
transformation with a selectable marker gene different from the hygromycin
resistance
gene, which is under the control of the T-DNA 1'-2' dual promoter.
Transformants are
selected and their progeny screened for hygromycin resistance. The mutant
phenotype
(hygromycin resistance) is screened for genetic co-segregation with a specific
T-DNA insert.
Cloning of the tagged gene using routine methods of recombinant DNA technology
allows
to characterize the mutant and wild-type DNA sequence of the silencing
modifier locus as
well as the encoded protein.
Within the context of the present invention reference to a gene is to be
understood as
reference to a DNA coding sequence associated with regulatory sequences, which
allow
transcription of the coding sequence into RNA such as mRNA, rRNA, tRNA, snRNA,
sense
RNA or antisense RNA. Examples of regulatory sequences are promoter sequences,
5' and
3' untranslated sequences, introns, and termination sequences.
A promoter is understood to be a DNA sequence initiating transcription of an
associated
DNA sequence, and may also include elements that act as regulators of gene
expression
such as activators, enhancers, or repressors.
Expression of a gene refers to its transcription into RNA or its transcription
and subsequent
translation into protein within a living cell. In the case of antisense
constructs expression
refers to the transcription of the antisense DNA only.
The term transformation of cells designates the introduction of nucleic acid
into a host cell,
particularly the stable integration of a DNA molecule into the genome of said
cell.
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Any part or piece of a specific nucleotide or amino acid sequence is referred
to as a
component seduence.
DNA according to the present invention comprises an open reading frame
encoding a
protein characterized by an amino acid sequence comprising a component
sequence of at
least 150 amino acid residues having 40% or more identity with SEGO ID NO: 3.
In particular
the protein encoded by the open reading frame can be described by the formula
R~-R2-R3,
wherein
-- R,, R2 and R3 constitute component sequences consisting of amino acid
residues
independently selected from the group of the amino acid residues Gly, Ala,
Val, Leu, Ile,
Phe, Pro, Ser, Thr, Cys, Met, Trp, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, and His,
-- R~ and R3 consist independently of 0 to 3000 amino acid residues;
-- R2 consists of at least 150 amino acid residues; and
-- R2 is at least 40% identical to an aligned component sequence of SEQ ID NO:
3.
In most cases the total length of the protein will be in the range of 1000 to
3000 amino acid
residues. In preferred embodiments of the invention the component sequence R2
consists
of at least 200 amino acid residues. Specific examples of the component
sequence R2 are
component sequences of SEQ ID NO: 3 represented by the following range of
amino acids:
1 - 416 (corresponding to exon 2);
418 - 583 (corresponding to exons 3 to 5);
584 - 890 (corresponding to exon 6);
892 - 1472 (corresponding to exons 7 to 9);
1007 - 1472 (corresponding to exon 9);
1473 - 1631 (corresponding to exons 10 to 12);
1632 - 1827 (corresponding to exons 13 to 15); and
1829 - 2001 (corresponding to exon 16).
In a preferred embodiment of the present invention at least one of the
component
sequences R, or R3 comprises one or more additional component sequences with a
length
of at least 50 amino acids and at least 60% identical to an aligned component
sequence of
SEQ ID NO: 3. Specific examples of such additional component sequences are
component
sequences of SEQ ID NO: 3 represented by the following range of amino acids:
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420 - (corresponding to exons
525 3 and 4);
444 - (corresponding to exon
525 4);
526 - (corresponding to exon
583 5);
892 - (corresponding to exon
971 7);
892 - (corresponding to exons
1006 7 and 8);
1473 - (corresponding to exon
1524 10);
1525 - (corresponding to exon
1576 11 );
1577 - (corresponding to exon
1631 12);
1632 - (corresponding to exons
1690 13);
1692 - (corresponding to exons
1757 14); and
1758 - (corresponding to exons
1827 15).
Particularly preferred embodiments of the DNA according to the present
invention encode a
protein having a component sequence defined by amino acids 478-490, 584-600,
617-630,
654-668, 676-690, 718-734, 776-788, 1222-1233, 1738-1749 or 1761-1770 of SEQ
ID NO:
3. Preferably, the encoded protein comprises at least two, three or more
different
representatives of said component sequences. Specific examples of said
embodiments
encode a protein characterized by the amino acid sequence of SEQ ID NO: 3, an
allelic
amino acid sequence having amino acid residue K instead of M at position 705
of SEQ ID
NO: 3, or an amino acid residue D instead of E at position 1219 of SEQ ID NO:
3.
Dynamic programming algorithms yield different kinds of alignments. In general
there exist
two approaches towards sequence alignment. Algorithms as proposed by Needleman
&
Wunsch and by Sellers align the entire length of two sequences providing a
global
alignment of the sequences. The Smith-Waterman algorithm on the other hand
yields local
alignments. A local alignment aligns the pair of regions within the sequences
that are most
similar given the choice of scoring matrix and gap penalties. This allows a
database search
to focus on the most highly conserved regions of the sequences. It also allows
similar
domains within sequences to be identified. To speed up alignments using the
Smith-
Waterman algorithm both BLAST (Basic Local Alignment Search Tool) and FASTA
place
additional restrictions on the alignments.
Within the context of the present invention alignments are conveniently
performed using
BLAST, a set of similarity search programs designed to explore all of the
available
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sequence databases regardless of whether the query is protein or DNA. Version
BLAST 2.0
(Gapped BLAST) of this search tool has been made publicly available on the
intemet
(currently http://www.ncbi.nlm.nih.gov/BLASTn. It uses a heuristic algorithm
which seeks
local as opposed to global alignments and is therefore able to detect
relationships among
sequences which share only isolated regions. The scores assigned in a BLAST
search have
a well-defined statistical interpretation. Particularly useful within the
scope of the present
invention are the blastp program allowing for the introduction of gaps in the
local sequence
alignments and the PSI-BLAST program, both programs comparing an amino acid
query
sequence against a protein sequence database, as well as a blastp variant
program
allowing local alignment of two sequences only. Said programs are preferably
run with
optional parameters set to the default values.
Sequence alignments using BLAST can also take into account whether the
substitution of
one amino acid for another is likely to conserve the physical and chemical
properties
necessary to maintain the structure and function of the protein or is more
likely to disrupt
essential structural and functional features of a protein. Such sequence
similarity is
quantified in terms of a percentage of "positive" amino acids, as compared to
the
percentage of identical amino acids and can help assigning a protein to the
correct protein
family in border-line cases.
Sequence alignments using such computer programs reveal the presence of an
ATP/GTP-
binding motif A (amino acids 460 to 467 in SEO ID N0:3), the consensus
sequence of
which is (Ala/Gly)XaaXaaXaaXaaGIyLys(Ser/Thr), wherein (Ala/Gly) indicates Ala
or Gly,
Xaa indicates any naturally occurring amino acid and (SerlThr) indicates Ser
or Thr.
Alignment additionally reveals a region (amino acid position 479 to 719 in SEQ
ID: 3),
similar to part of the ATPase/helicase domain of proteins in the SW12/SNF2
family which
are involved in chromatin remodeling but no significant overall sequence
identity with known
proteins.
Specific examples of DNA according to the present invention are described in
SEQ ID
NO: 1 and SECT ID NO: 2 encoding an Arabidopsis protein described in SEQ ID
N0:3.
Stretches of SEQ ID NO: 3 having 50 to 500 amino acids length can show between
20 and
50% sequence identity to stretches of known protein sequences after alignment.
Overall
alignments of SEQ ID NO: 3, however, result in sequence identities lower than
30%. Thus,
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the present invention defines a new protein family the members of which are
characterized
by an amino acid sequence comprising a component sequence of at least 150
amino acid
residues having 40% or more identity with an aligned component sequence of SEQ
ID
NO: 3. Preferably the amino acid sequence identity is higher than 50% or even
higher than
55%.
DNA encoding proteins belonging to the new protein family according to the
present
invention can be isolated from monocotyledonous and dicotyledonous plants.
Preferred
sources are corn, sugarbeet, sunflower, winter oilseed rape, soybean, cotton,
wheat, rice,
potato, broccoli, cauliflower, cabbage, cucumber, sweet corn, daikon, garden
beans,
lettuce, melon, pepper, squash, tomato, or watermelon. However, they can also
be isolated
from mammalian sources such as mouse or human tissues. The following general
method,
can be used, which the person skilled in the art knows to adapt to the
specific task. A single
stranded fragment of SEQ ID NO: 1 or SEQ ID NO: 2 consisting of at least 15,
preferably 20
to 30 or even more than 100 consecutive nucleotides is used as a probe to
screen a DNA
library for clones hybridizing to said fragment. The factors to be observed
for hybridization
are described in Sambrook et al, Molecular cloning: A laboratory manual, Cold
Spring
Harbor Laboratory Press, chapters 9.47-9.57 and 11.45-11.49, 1989. Hybridizing
clones are
sequenced and DNA of clones comprising a complete coding region encoding a
protein
characterized by an amino acid sequence comprising a component sequence of at
least
150 amino acid residues having 40% or more sequence identity to SEQ ID NO: 3
is purified.
Said DNA can then be further processed by a number of routine recombinant DNA
techniques such as restriction enzyme digestion, ligation, or polymerase chain
reaction
analysis.
The disclosure of SEQ ID NO: 1 and SEQ ID NO: 2 enables a person skilled in
the art to
design oligonucleotides for polymerase chain reactions which attempt to
amplify DNA
fragments from templates comprising a sequence of nucleotides characterized by
any
continuous sequence of 15 and preferably 20 to 30 or more basepairs in SEQ ID
NO: 1 or
SEQ ID NO: 2. Said nucleotides comprise a sequence of nucleotides which
represents 15
and preferably 20 to 30 or more basepairs of SEQ ID NO: 1 or SEO ID NO: 2.
Polymerase
chain reactions performed using at least one such oligonucleotide and their
amplification
products constitute another embodiment of the present invention.
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EXAMPLES:
Example 1: T DNA Insertion
Transgenic line A of Arabidopsis thaliana ecotype Zurich with a
transcriptionally silenced
locus containing multiple copies of a chimeric hygromycin phosphotransferase
gene (hpt)
has been described in Mittelsten Scheid et al, Mol Gen Genet 228: 104-112,
1991 and
Mittelsten Scheid et al, Proc Natl Acad Sci USA 93: 7114-7119, 1996. A
homozygous,
diploid genotype of said line is subjected to Agrobacterium mediated gene
transfer by in
planta vacuum infiltration (Bechtold et al., C R Acad Sci Paris Life Science
316: 1194-1199,
1993) generating more than 4000 independent T-DNA transformants. The binary
vector with
T-DNA consisting of the coding region of the bar gene transcriptionally fused
to the 1'
promoter (pi'barbi), the Agrobacterium strain (C58CIRifR) and the
transformation protocol
are described by Mengiste et al, Plant J 12: 945-948, 1997. Transformants (T1
plants) are
selected by repeated spraying of germinated seedlings with Basta solution (150
mg/I) and
grown to maturity.
Example 2: Mutant Selection
Selfed seeds (T2 families) are collected from individual transformants. Prior
to screening for
revertants of the silenced phenotype, seeds are dried for one week at room
temperature
and cold-treated at 4°C for a minimum of one week. Pooled aliquots of
approximately 1000
seeds (consisting of 50 seeds from 20 T2 families) are surface-sterilized
twice (with 5%
sodium hypochlorite containing 0.1 % Tween 80) for 7 min and washed with
sterile double-
distilled water. For selection, each aliquot is plated on 14-cm Petri dishes
containing 75 ml
germination medium (according to Masson et al, Plant J 2: 829-933, 1992)
solidified with
0.8% agar and containing 10 mg/I hygromycin B (Calbiochem). To ensure equal
distribution
during sowing, seeds are mixed with 30 ml of the same medium containing 0.4%
agar. As
positive control two seeds from a hygromycin-resistant line are sown at marked
locations on
each plate. Plates are cold-treated at 4°C for 2 days and subsequently
subjected to
alternating periods of 16 hours light at 21 °C and 8 hours darkness at
16°C. Hygromycin
resistance is evaluated each day for 8-15 days after sowing.
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Example 3: Molecular and Genetic Analysis of the Mutant
Following identification of 11 hygromycin-resistant seedlings in one of the
pools, the families
forming this pool are re-screened individually. One family contains
approximately 25%
hygromycin-resistant seedlings. Six resistant plantlets of this family are
transferred to larger
vessels containing germination medium without hygromycin. After rosette
formation and
development of the root system, plants are transferred to soil for further
growth and seed
setting. Prior to potting, tissue explants are taken from each plant to
generate callus
cultures on RCA medium (Table 1 ) with or without 10 mg/I hygromycin B. Callus
cultures are
used as a source of material for DNA and RNA analyses and for a further
confirmation of
hygromycin resistance in this tissue.
Genomic DNA is isolated using a CTAB based method as described by Mittelsten
Scheid et
al, Mol Gen Genet 244: 325-330, 1994, and incubated with restriction enzymes
BamHl,
Hpall, Mspll, Dral, EcoRV, Rcal or Hindlll. Total RNA is obtained using a
RNAeasy kit
(Qiagen) according to the supplier's recommendation. Southern and northern
blot analysis
are performed under conditions described by Church and Gilbert, Proc Natl Acad
Sci USA
81: 1991-1995, 1984, using DNA fragments labeled with 32P by random prime
labeling. The
coding region of the hpt gene, or DNA consisting of the P35S promoter, hpt
coding and
terminator region, or the coding region of the bar gene together with the 1'
promoter are
used as probes.
Northern blot analysis of 4 hygromycin-resistant siblings shows restoration of
transcription
of the hpt gene. Southern blot analysis of said siblings indicates that there
is no detectable
rearrangement within the complex hpt insert. The hpt transgene complex in the
mutant is
still hypermethylated like in the original line A, as judged by Southern blot
analysis with the
methylation-sensitive restriction enzymes Hpall and Mspl, and by genomic
sequencing of
the promoter region after treatment with bisulfate. There is also no influence
of the mutation
on the methylation of repetitive genomic DNA in contrast to that observed for
the som
mutations.
The hygromycin-resistant plants, as well as non-selected siblings from the
same family are
grown to set seeds, checked for Basta resistance in the next generation, and
scored for the
number and size of the T-DNA inserts by Southern analysis. The results
demonstrate that
the original T-DNA transformant must have contained 2 T-DNA insertions
segregating
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independently in the siblings. One insert co-segregates with the hygromycin
resistant
mutant phenotype. A plant homozygous for this insert and lacking the other T-
DNA insert, is
used for cloning the corresponding T-DNA insertion site.
Histochemical GUS staining of crosses between plants with mutant phenotype and
the
transgenic plant line GUS-TS (obtainable from Dr. H. Vaucheret, INRA,
Versailles Cedex,
France) of Arabidopsis thaliana ecotype Colombia containing a
transcriptionally silenced
locus with multiple copies of a chimeric beta-glucuronidase (gus) gene reveals
reactivation
of the silent GUS gene in the F2 progeny which are homozygous for the mom
allele.
Inbreeding of plants with the moml mutant phenotype does not result in any
morphological
abnormalities even in the 9th generation of inbreeding. This is in contrast to
the som
mutants.
Backcrossing of the mutant phenotype of moml with line A (see example 1 )
results in
immediate resilencing of the reacivated hpt gene upon introduction of a wild-
type MOM
allele in F1 hybrids. This also is in contrast to the som mutants.
Table 1: Composition of RCA medium
.~-, ~ ~ .
~ k~ ~. ~4
~:
~f~~~ ri~eti~yn
~4
=
~
a ~
- ~..~.'~'. ..w
'..,.~.srtn,y~assp;. ".,ws.th 7 r.,w ,
E~ . ~ . , auee 1_ . _. ,..w-m.Y~..~
~
MS macro 10 100 ml
x
B5 micro 10001 ml
x
ferric citrate5 ml
NT vitamins 10 ml
100 x
sucrose 10 g
MES 5 ml
agar 10 g
NAA 0.1 mg
BAP 1 mg
pH 5.8 (KOH)
ad 1 I
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, ~yv, 3'','.~~ y.;. °2~;5'z~
.a b~~dy'3' \~a:'t..e.rr;~ .,..r~~rr~,a~4re~
potassium nitrate 19 g
ammonium nitrate 16.5 g
calcium chloride (x 2 H20) 4.4 g
magnesium sulfate (x 7 H20) 3.7 g
potassium dihydrogen phosphate 1.7 g
ad 1 I
magnesium sulfate (x 1000 mg
H20)
boric acid 300 mg
zinc sulfate (x 7 H20) 200 mg
potassium iodide 75 mg
sodium molybdate (x 25 mg
2 HZO)
copper sulfate (x 5 2.5 mg
HZO)
cobalt chloride (x 6 2.5 mg
H20)
ad 100 ml
ammonium iron citrate 10 g
ad 1 I
myo-inositol 1000 mg
thiamine HCI 10 mg
ad 1 I
:. '3 F'~ , f"'~s S t 'd-..: '~ ~~
'k "~. s t ~ f 4~ ~ n ~: a,
-...,p~.'s~,..,~~,w. .,..x..,.~~~t"~.-".3, ~....H,. . .....~. , ,. ..~~,'".~"<
.~;.a~t.:~.~..:..,~.-'.~~, r~~,.~N~:
MES 14 g
pH 6 (NaOH)
ad 100 ml
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Example 4: Cloning of the "Silencing Gene"
Genomic DNA from the plant containing only the T-DNA co-segregating with the
hygromycin
resistant mutant phenotype is isolated. The DNA is subjected to TAIL (thermal
asymmetric
interlaced) PCR according to Liu et al, Plant J 8: 457-463, 1995, using 3
specific, nested
primers close to the right border of the T-DNA (5 ' -CAT CTA CGG CAA TGT ACC
AGC-3 '
(SEQ ID N0:4), 5'-GAT GGG AAT TGG CTG AGT GGC-3' (SEO ID N0:5), 5'-CAG
TTC CAA ACG TAA AAC GGC-3 ' (SEQ ID NO: 6)) which are directed outwards, and
one
of several degenerate primers which might bind in flanking plant DNA. Two out
of the
following seven degenerate primers
AD1 5' -NTC GAS TWT SGWGTT-3' (Liu et al supra; SEGO
ID NO: 7)
AD2 5' -NGT CGA SWG ANAWGA A-3 (LIU et al SUpra; SEQ
' ID NO: 8)
AD3 5' -WGT GNA GWA NCANAG A-3'(LIU et al supra; SEQ
ID NO: 9)
AD4 5 ' -WGG WAN CWG AWANGC A-3 (SEQ ID NO: 10)
'
AD5 5 ' -WCG WWG AWC ANGNCG A-3 (SEQ ID NO: 11 )
'
AD6 5'-WGC NAG TNA GWANAA G-3'(SEQ ID NO: 12)
AD7 5'-AWG CAN GNC WGANAT A-3'(SEQ ID NO: 13)
actually result in amplification of specific fragments. The larger one
obtained using AD7 is
cloned and sequenced. It contains 50 by of the T-DNA and 275 by of flanking
plant DNA. In
Southern blot analysis it is shown that this PCR fragment contains the plant
DNA flanking
the T-DNA. The PCR fragment is used to screen a genomic library (Stratagene)
of wild type
Arabidopsis thaliana ecotype Columbia. Three genomic clones hybridizing to the
PCR
fragment are identified. The genomic clones are further mapped with
restriction enzymes,
hybridized to the PCR fragment and aligned to each other. In one of the
genomic clones
obtained (p4A-11 ), the sequence found to flank the T-DNA of the insertion
mutation is
located approximately in the middle of the genomic sequence. An approximately
800 by
EcoRl-Sall fragment of p4A-11 is used to obtain the overlapping genomic clone
p5-6, and
an approximately 700 by EcoRl fragment of p5-6 is used to obtain genomic clone
p30-1
overlapping with p5-6. An approximately 700 by Hindlll fragment of p30-1 is
used to obtain
the genomic clone p33-19 overlapping with p30-1. Said clones are sequenced to
design
primers for RT-PCR. The approximately 700 by EcoRl fragment of p5-6 is further
used for
screening of a cDNA library of wild type Arabidopsis thaliana ecotype Zurich
according to
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Elledge et al, Proc Natl Acad Sci USA 88: 1731-1735, 1991). Nine cDNA clones
are
obtained and the longest clone p17-8 having a length of 2.6 kb is sequenced.
Example 5: Sequence Analysis and Alignments
Taking into account the large size of the Arabidopsis silencing gene cloned
above it cannot
be entirely excluded that the authentic nucleotide and amino acid sequences of
the gene
and protein, respectively, might deviate from the sequences given in SEQ ID
NO: 1, SEQ ID
NO: 2, and SEQ ID NO: 3 at a few positions due to mutations arising from the
cloning
procedure or due to ambiguities in the sequencing reactions. Additionally,
sequencing of
DNA derived from a different ecotype can reveal allelic differences. Thus, the
sequences of
SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 represent the corresponding genes
and
proteins of Arabidopsis thaliana ecotype Zurich, whereas genomic sequences
obtained
from Arabidopsis thaliana ecotype Columbia reveal two mismatches at nucleotide
positions
4338 (A instead of T) and 6721 (T instead of G) of SEO ID NO: 1, which result
in an amino
acid residue K instead of M at position 705 of SEQ ID NO: 3 and an amino acid
residue D
instead of E at position 1219 of SEQ ID NO: 3.
The 2.6 kb cDNA clone is analyzed sequentially from both ends and is shown to
contain
one large ORF as well as a 3' untranslated sequence.
Analysis of the genomic clones reveals that clones p4A-11 and p5-6 contain
sequences
homologous to the cDNA sequence as well as 7 intron sequences. Comparing the
genomic
sequences with the DNA sequences flanking the T-DNA insert, it turns out that
the T-DNA
insertion causes a deletion of about 2 kb of genomic DNA. The 5' end of the
deletion is
located in an intron (intron 12) and the 3' end of the deletion is located
downstream of the 3'
end of the cDNA. The sequence of 5' end of the cDNA clone terminates in the
middle of the
sequence of the genomic clone p5-6. Three independent nested RT-PCR reactions
are
performed to obtain additional cDNA sequences further upstream. The sequences
of the
primers used for these RT-PCRs are as follows:
RTl-1 5' -CTGTACATACTGAGTACAATCGGA-3' (SEQ ID NO: 14)
RT1-2 5'-GCTTCAATTCCTGCCTCAGTTGAAC-3' (SEQ ID NO: 15)
RT1-3 5' -CTCTACGTGCTTAACATCATGCGA-3' (SEQ ID NO: 16)
RT1-4 5' -CCAGCTTCTGCTACTAGAAAGTCAG-3' (SEQ ID NO: 17)
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RT2/3-15' -CTGGAGTTGCATGAAATCCTGGATG-3(SEGO NO:
' ID 18)
RT2/3-25' -GCTCTTTGTAAGCTGTTCACGAGAC-3(SEQ NO:
' ID 19)
RT2-3 5' -TCGCATGATGTTAAGCACGTAGAG-3 (SEQ NO:
' (D 20)
RT2-4 5' -GAGTACTGGTCCGTGAACAGGTAAT-3(SEQ NO:
' ID 21
)
RT3-3 5' -ATGCTTGCACAAGCATGGTCGGAAA-3(SEQ NO:
' ID 22)
RT3-4 5'-TGCAACATCGTGCATTTGCTCCAGA-3'(SEQ NO:
ID 23)
RT4-1 5' -CACAAGCATGAGTTTTTCCTTCCGG-3(SEQ NO:
' ID 24)
RT4-2 5' -CTGACTTTCTAGTAGCAGAAGCTGG-3(SEQ NO:
' ID 25)
Sequences of several parts of the genomic clones are found to be deposited in
the
Arabidopsis database (accession numbers 867281, 862563, 820434, 820425,
821274,
808967, 811993, 820116, 812496 and 810852 as end sequences of BAC, and 218494
and AA597930 as partial cDNA sequences, on 13 Apr 1999). A comparison of the
encoded
protein sequence with the Swiss Protein Database reveals partial similarity
with
ATPase/helicase proteins of the SW12/SNF2 family (amino acid position 479 to
719 in SEQ
ID NO: 3). The encoded protein consists of 2001 amino acids and is calculated
to have a
molecular weight of 219 kD and a pl of 5.1. An ATP/GTP-binding motif (amino
acid position
460 to 467 in SEQ ID NO: 3) and three nuclear localization motifs (amino acid
positions 362
to 367, 832 to 838 and 858 to 862 in SEQ ID NO: 3) are found in the encoded
protein.
Subcellular immunodetection of HA-tagged MOM protein confirms its nuclear
localization.
Similarity to the actin binding domain of chicken tensin (amino acid position
1899 to 1941 in
SEQ ID NO: 3) and a predicted membrane spanning domain (amino acid position
995 to
1015 in SEQ ID NO: 3) are also detected. Additionally, the encoded protein
contains three
types of repetitive regions or internal repeats essentially defined by amino
acid positions
177 to 350, 1462 to 1672 and 1848 to 1894 OF SEQ ID NO: 3.
Example 6: Homologous genes in other species
A putative proline/hydroxyproline-rich glycoprotein of Arabidopsis thaliana
showing partial
similarity to the MOM protein is disclosed as GenBank accession nubmer
AAD29829). The
similarity is 34-47% depending on the region and is only seen in the second
half of the
MOM protein (i.e. amino acids 1368 to 1944).
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The MOM cDNA clone is used to probe genomic DNA from turnip, tomato, tobacco,
maize,
mouse, fruit fly and man for the presence of homologous genes by Southern blot
analysis.
Hybridization under conditions of low stringency is found in all cases. Cross-
hybridizing
clones from libraries can be identified and sequenced.
A genomic library of the Brassica oleracea var. acephala (obtained from Dr.
Mark Cock,
INRA, CNRS, Lyon, France) are screened with the MOM cDNA under stringent
conditions.
Two positive clones are obtained, subcloned, and partially sequenced. Partial
sequences of
clone 1 show similarity to different regions in the MOM gene (80-86% at DNA
level and 62-
80% at amino acid level) which encode the N-terminal, ATPase, and C-terminal
parts of the
MOM protein. All three putative nuclear localization sequences of the MOM
protein are fully
conserved in clone 1. Partial sequences of clone 2 also show similarity
regions in the MOM
gene (64-76% at DNA level and 55-64% at amino acid level) which encode the
ATPase,
putative transmembrane, and C-terminal parts of the MOM protein. The sequences
of
clones 1 and 2 are not identical, suggesting the presence of, at least, two
homologous
genes in Brassica oleracea. Examples of partial sequences obtained from clone
1 and 2 are
given in SEQ ID Nos: 26-33.
Additionally a genomic library of Brassica raga (obtained from Dr. Kinya
Toriyama, Tohoku
University, Sendai, Japan) is screened with the MOM cDNA under stringent
conditions.
Positive signals hybridizing to both a 5' and a 3' part of the MOM cDNA are
obtained.
Furthermore, a genomic library of Petunia hybrida (obtained from Dr. Jan
Kooter, Vrije
Universiteit, Amsterdam, The Netherlands) is screened with MOM cDNA under less
stringent conditions. Positive signals hybridizing to both the 5' and 3' part
of the MOM cDNA
are obtained.
Example 7: Manipulating marker gene expression by antisense constructs
The 2.6 kb cDNA fragment and a 1.8 kb RT-PCR fragment amplified by a nested RT-
PCR
using primers RT1-1 and RT1-2 for the first PCR and primers RT1-3 and RT1-4
for the
second PCR, are each inversely cloned into the multiple cloning site of the
binary vector
pbarbi53 to generate antisense RNA. pbarbi53 is a modified vector of p1'barbi
and carries
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an expression cassette consisting of the 35S promoter of cauliflower mosaic
virus, a
multiple cloning site containing Xho I, SnaBl, Hpa 1 and Cla I restriction
sites and the 35S
terminator of cauliflower mosaic virus at the Hindlll site of p1'barbi. The
resulting
recombinant plasmids are introduced into Agrobacterium as described in Example
1. The
transgenic plant line GUS-TS (obtainable from Dr. H. Vaucheret, INRA,
Versailles Cedex,
France) of Arabidopsis thaliana ecotype Colombia containing a
transcriptionally silenced
locus with multiple copies of a chimeric beta-glucuronidase (gus) gene, is
transformed with
the recombinant plasmids as described in Example 1 and transformants are
selected as
described by Mengiste et al, Plant J 12: 945-948, 1997. pbarbi53 vector DNA is
used in
control transformations. The transformants are examined for reactivation of
the gus gene by
histochemical staining. A cotyledon leaf is soaked in gus staining solution
(100 mM sodium
phosphate buffer (pH 7.0), 0.05% 5-bromo-4-chloro-3-indolyl-beta-D-
glucuronidase, 0.1
sodium azide) under vacuum for 10 min and then incubated at 37°C
overnight. While strong
gus activity is observed in the plants transformed with the recombinant
plasmid carrying the
2.6 kb cDNA, plants transformed with the recombinant plasmid carrying the 1.8
kb RT-PCR
fragment or pbarbi53 do not show any gus activity above background. Therefore,
expression of the antisense RNA of the 2.6 kb cDNA mimicks the mutant
phenotype and
confirms that sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3
represent the genetic information for a component of the transcriptional gene
silencing
system.
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SEQIJINCE LISTING
<110> Novartis AG
Novartis Research Foundation
<120> Gene involved in epigenetic gene silencing
<130> S-31005A
<140>
<141>
<150> GB 9914623.5
<151> 1999-06-23
<160> 33
<170> Patentln Ver. 2.1
<210> 1
<211> 10329
<212> I~
<213> Arabidopsis thaliana
<220>
<221> intron
<222> (1009)..(1295)
<220>
<221> intron
<222> (2551)..(2673)
<220>
<221> intron
<222> (2753)..(2867)
<220>
<221> intron
<222> (3114)..(3506)
<220>
<221> intron
<222> (3681)..(3973)
<220>
<221> intron
<222> (4896)..(4975)
<220>
<221> intron
<222> (5218)..(5777)
<220>
<221> intron
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_2_
<222> (5883)..(6082)
<220>
<221> intron
<222> (7481)..(7615)
<220>
<221> intron
<222> (7772)..(7914)
<220>
<221> intron
<222> (8071)..(8153)
<220>
<221> intron
<222> (8319)..(8451)
<220>
<221> intron
<222> (8630)..(8718)
<220>
<221> intron
<222> (8919)..(9000)
<220>
<221> intron
<222> (9212)..(9284)
<400> 1
aatatttaag tttggtttat attctttcta gtaatctttg aaatattgta agagataatg 60
cttctaataa ataacattgg atttattgga attaatgtat tgaaaaaact atgcaaatac 120
tacagtgtat tttggaacga ccaaaatgat atatgtaaac tttcgttcta gtcttctaca 180
tagtgtaata ggatagcgga caaggttgat cgactctaaa cattatgggt acgtaattcc 240
gcagtggtta cagtctactg tcgaggccaa actggtaatt aaacgtttga agtttagaga 300
aatattttga tgatgagtac cacaatcaaa gatgataggt gttaatcact gtaaaaatgt 360
tgattgaata ctacgaatgc agaacatata catattttta atctctttgg aatttttgtt 420
tttgttttta tcatttttga atacacgaag agctcagtta tatttcatat tgtatatgaa 480
tttgttctat ttaatcttca attctagcaa catactctta tgctaattcg tttcatattt 540
tagtatagta taaaaattac aaatttcaaa acaaactata agtaatatac taacatagtc 600
ggtgtaacat ttcgttaatt tcacataaca tatgttaatt acatatgtac actatttttg 660
aagtatttta taacttaaaa tatataaatt taaatctaag aaatcacaag catgagtttt 720
tccttccggt aatcgtaaaa tcaaaaatcg ctcgctcgag aaacgccggt gctagaagag 780
gaaagtaccg tacataatcc tgcgaaccca attctcgtct tcttcaaact cagttttccg 840
aaaccccaaa caccgcgagg attgcatggc ctgaagaacc acttaatcga gaattgtgct 900
ggaattctca aattttccct cgcgtttttc tttcacactc tcggaatcgg aaatttccac 960
caagctccgt caagcgatag attctgacaa ttacacactt tcgcgcaggt atgcttcctt 1020
ccctgtttta ggttggtgtt aatctatcgg tgaatcgaag gttttgggcc tcgggctttg 1080
cgttttaggt ttttcagaga atcttatcta cttggggatg gatcttaggc gtttgttaga 1140
tgtaactcat tagttttgca tataggaatt ttgatttgaa agttaggtcg ccggatttgt 1200
agacattttg tttgatggtc ttcttcggtg ctcacattct ttgtttttaa gtgcttgatt 1260
tggttgctaa ggtcctttcc gttgcgtgct ctcagtgaat atgaagaaag atgaaaagat 1320
tggtttgacg gggagaacca tttacaccag atccctagca gcttcaattc ctgcctcagt 1380
tgaacaagaa acccctggtt tgaggaggtc aagccggggg acaccatcta cgaaggtaat 1440
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aactccagct tctgctacta gaaagtcaga gagactggct ccctcacctg cttcagtttc 1500
aaaaaagtcc ggtggaatcg tcaagaattc cacaccaagt tctttgcgaa ggtccaatag 1560
ggggaagact gaagtatcct tgcagagttc caaaggatca gataattcta tcaggaaagg 1620
agatacttca ccggatattg agcagagaaa ggatagtgtt gaagagtcga cagataagat 1680
caagcctata atgtcagccc gaagttacag ggcattgttt agagggaagc tcaaggaatc 1740
tgaggcatta gttgatgctt ccccaaatga agaggaacta gtagttgttg gttgttctcg 1800
ccgcatacct gcaggcaatg atgatgttca aggtaaaaca gattgtccac cacctgcaga 1860
tgcaggatca aaaaggctgc cagttgacga aactagtttg gacaagggca ctgattttcc 1920
tttgaaatca gttacggaga ccgagaagat agtgcttgat gcatccccca tagttgaaac 1980
tggggatgac agtgttatag gttcaccatc tgagaattta gagacacaaa agcttcaaga 2040
tggtaagaca gattgttcac cacctgcaaa tgcagaatcg aaaacgctgc cagttggtga 2100
aactagttta gaaaaagaat atccacaaaa gtttcaagat gataacacag attgtctacc 2160
acctgcaaat gcagaatcaa aaaggctgcc agttggcgaa actagtttag aaaaggacac 2220
tgattttcct ttgaaatcaa ctacggagac tggaaagatg gttctttatg catcccccat 2280
agttgaaact agggatgaca gcgttatatg ttcaccatct acaaatttag aaacccaaaa 2340
gcttcttgtc agtaaaactg gcttagaaac cgacatagtt ttgcctttga aaagaaaaag 2400
agacactgca gaaattgagc tggatgcatg tgctacagtt gcaaatggag atgatcacgt 2460
tatgagttct gatggggtca ttccatctcc atctgggtgc aaaaatgata atcgacctga 2520
aatgtgcaac acgtgtaaaa aacggcaaaa gtaagagttt ttttagtgtt gtctgtctat 2580
tgaaacgatc tgccaatgtt gaatgttggg cagatgggtt tgattcttag gatatatgtt 2640
ctgtattgta atgagttgtt caaaattttg aagggtcaac ggtgattgtc aaaataggag 2700
tgtttgctcc tgcattgtcc agccagttga agaatctgat aacgtgacac aggttggttt 2760
ctaattactt tcggagaccc gttaatcagt ggactcttaa atagttagat actagattta 2820
cttatccttt tacttgtaat ctgcaattct attttgcatt tgattaggat atgaaagaaa 2880
ctggaccagt tacgagcaga gaatatgagg agaacgggca aatacaacat ggtaaatcaa 2940
gtgatcccaa attctattct tcggtgtacc cagagtattg ggttcctgtg cagctatcag 3000
atgtacagct ggagcaatac tgtcagactc tcttctccaa atccttatct ctttcttcac 3060
tttcgaagat tgatcttgga gctctagaag aaactctcaa ttctgtaaga aaagtaagtt 3120
acttgatttt aaaaacactt attcttcaat gcacttgtga gttaagtacc cagttattac 3180
tggtgataag ataaagaaag caatagaaaa attgataagg tgttcaccgc attgcagcca 3240
aaaaaacaat tctgtgttcc atgctttcaa gaggttgtca cataggtgtt atgcctttct 3300
gtttgatgtt tggtagagca aaggttttgg gtctatttgt tttatgcttt tttgaaacac 3360
atagaacctg gcaaacttga cagttttggg gttgcttaga tatacgacta ttgtcggtca 3420
gcatcacatt ttctcaaggc ctctttctgc atgttaatgt gtgaatatat taaaatcttc 3480
tttatgtgtt tgcaacttgt tgacagacct gtgaccatcc atacgttatg gatgcatctt 3540
tgaaacaact gctcaccaag aatctggagt tgcatgaaat cctggatgta gaaattaaag 3600
cgagcgggaa acttcacctc cttgataaaa tgcttactca tataaaaaag aatggtttaa 3660
aagcagttgt cttctaccag gtgcattttc tattacttgc gaatgtgaat agctctatgt 3720
ttgtcatgaa tacgtcactt tgtgcattct caatatatgt gcattttctt tttgacaatg 3780
gaattctgtc ttgtattgaa atttgagtgg gatgaaagta tgctttttat cgtgcaatta 3840
tgaagtgtaa gttagccttc agcagtcagc tagcattatg agatatgctg aactaaaatg 3900
tttcttttct cttctttctt tttcgttata tgtgcctcat gtatgtttga attacagttt 3960
ttattttcag caggcaacac aaacccctga agggcttctg cttggtaata ttctcgaaga 4020
ttttgtgggt caaagatttg gtccaaaatc ttatgagcat gggatatatt cctcaaagaa 4080
gaactccgct ataaacaatt tcaacaagga gagtcaatgc tgtgttctgc tgttggaaac 4140
acgtgcctgc agtcaaacca ttaaactctt gcgagctgat gcgtttattc tttttggaag 4200
cagcttgaat ccatcgcatg atgttaagca cgtagagaag ataaaaatcg agtcatgttc 4260
tgaaagaact aagatattcc gattgtactc agtatgtaca gttgaagaaa aagccctgat 4320
tctggctagg caaaatatgc ggcaaaataa agctgtagag aacctaaacc gctctctcac 4380
gcacgcactg ctcatgtggg gggcgtcata cttatttgat aaactggatc attttcacag 4440
cagtgaaact ccagattcag gagtttcatt tgaacaatct attatggacg gcgtgattca 4500
tgaattctcg tccatacttt cttccaaagg tggagaagaa aatgaagtca agctgtgtct 4560
acttttggag gccaagcatg ctcagggaac ttacagcagt gattctactc tatttggtga 4620
agaccatatt aagttgtcag atgaagagag tccaaatata ttttggtcaa agctgttggg 4680
gggaaaaaat cctatgtgga aatacccttc agatactccc caaaggaatc gaaaacgagt 4740
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tcagtatttt gagggttctg aagcgagtcc caaaactggc gatggtggaa atgcaaagaa 4800
gcgaaagaag gcttctgatg atgtcactga tccccgggtc actgatccgc cagtagatga 4860
tgatgaaaga aaggcctctg ggaaggatca catgggtaaa atagtttaat ttctgctccg 4920
atacctctag tgttcattga ttatgcaact actttgctga ctatctttcc tacaggggct 4980
ttggagtcac caaaagtcat aacactccag tcatcatgta aatcttctgg tacagatggt 5040
acattggatg gaaatgatgc ttttggcttg tattctatgg gcagccatat ctctggaatc 5100
ccagaggata tgttagctag tcaagattgg gggaaaatac cggatgaatc acagaggagg 5160
ctccacactg ttttaaagcc gaagatggca aaactttgcc aagttttgca tctttcagta 5220
agtggccttt ttcacctcca caacttattt tagccttgca tatgcttata tatagctgat 5280
tgcaactgta gttgttacct gatttcctgt tacagccaaa tgtgagagtt ttattcttca 5340
actatatcca tccgtttaag catattttat ttcttatatc tggcttcgtt accaatgcac 5400
tgttaaaatg agcaactgct gcacaaaaca gtaggtagtt atgtgcctca tgtcattcat 5460
tgtttattga agcaaagaaa tttctgtcta ctttacatga tccatctgtg ggagtatata 5520
actatatata accttaggcc tttgtacctg gctgatcaaa gacatgtcaa aagtttatct 5580
gttcgctgtt ggtatagaaa ctaatacagt gtctgatgct attttaaggt agtcttatgt 5640
cttcacatat tggctaatag atgtttccgc tgtcgtgtcc atatacttct gtgattatca 5700
cggtgctccg tctatcaaaa ttgtactaaa aggtattttg caatgtgtga ttggttaaca 5760
gattattttg ttttcaggat gcttgcacaa gcatggtcgg aaattttctc gaatatgtta 5820
ttgaaaatca ccgaatctac gaagagccag ccactacttt tcaggcattc cagatagccc 5880
tggtatgaca gcatttactt tgataattta tgcattgttt ccttcatcat ctgcctttgt 5940
ttagaatgtc ctcagaaggc agcactcctt tagttttaac tttccaatca taggattcaa 6000
atatccatta actggccttt gatcgctgca taatatatga atagttgaca tactgaatac 6060
gttgttaata atgcattttc agagttggat tgcagccttg ttggtaaagc aaattcttag 6120
ccacaaagaa tctctggtcc gtgcaaattc tgaattagct ttcaaatgct ctagagtaga 6180
ggtggattat atttattcga tattgtcctg catgaagagt ctgttcctgg agcatacaca 6240
aggtttgcag ttcgattgct ttggtactaa ttctaaacag tcagtggtta gcacaaaact 6300
agtaaatgaa agtctctcag gggctacagt gcgtgacgaa aagattaata cgaagtcgat 6360
gcgaaatagc tcagaggatg aagagtgcat gactgagaag agatgtagcc attatagcac 6420
agcaacaaga gatatcgaaa agactattag tggcataaaa aagaaataca agaagcaagt 6480
gcaaaagctt gtacaagagc atgaggaaaa gaaaatggag ctgttaaata tgtatgcaga 6540
caagaagcag aaacttgaaa ctagtaaaag tgtggaagca gcagtaattc gtattacctg 6600
ttcacggacc agtactcaag tgggtgatct caaactgctg gatcataatt atgaaagaaa 6660
gtttgatgaa atcaaaagtg agaaaaatga atgcctcaaa agtctggagc aaatgcacga 6720
ggttgcaaag aagaagttgg ctgaggatga agcctgttgg attaatcgga taaagagctg 6780
ggcagctaaa ttaaaagttt gtgttcccat tcaaagtggc aataacaagc attttagtgg 6840
ttcatcaaac atttcccaaa atgctcctga tgtacaaatt tgcaataatg ctaacgttga 6900
agctacttac gctgatacga attgcatggc ttccaaggtt aatcaagtgc cagaagcaga 6960
aaacacatta ggaaccatgt cgggtggcag cactcaacaa gttcatgaaa tggtggatgt 7020
aagaaatgac gagacaatgg atgtctcagc tttgtctcgt gaacagctta caaagagcca 7080
gtccaatgag cacgcttcta tcactgtgcc tgagattttg attcctgctg actgtcaaga 7140
ggaatttgcg gccttgaacg tgcatttgtc agaagaccag aattgtgaca gaataacatc 7200
tgcggcatca gatgaagatg tttcatcaag ggtgccagag gtatcccagt cactcgaaaa 7260
tctttctgcc tcccccgagt tttctctaaa tagagaggag gctttggtta caacagaaaa 7320
tagaagaaca agtcatgtgg gttttgatac tgataacatt ttggaccagc agaatagaga 7380
agattgttct cttgaccaag agattcctga cgagttagcg atgcctgtgc aacatcttgc 7440
gtctgtggta gagactaggg gtgctgctga atctgatcag gtacttactg gccctgtaga 7500
atagttgatg ccttgttcat ttaatctttt ctaatgttca ttcttgcttt cttgaaaata 7560
acgggtagtg atcagatgtc tttttttctc ttattaaatt cacttttctg gacagtatgg 7620
tcaagatata tgtcctatgc cttcttcact ggctggaaag caacctgacc cagcagcaaa 7680
cactgagagc gaaaatcttg aagaagcaat tgagcctcag tctgctggtt cagaaacagt 7740
agagactact gattttgctg catcacatca ggtccctatt gaagactttc cttttttact 7800
agtttaaagt tatcaatctg tgttatgttc attctaagtt tccgtgagaa aaaggtgggg 7860
aaatgtggtt actgatcaag tctcgttgtt gttttaaatc gactcttttg acagggtgat 7920
caagttacat gtcctttgct atcttcaccg actggaaatc agcctgcgcc agaagcaaat 7980
attgaaggcc aaaatatcaa cacatcagct gagccccatg tagcgggtcc agatgcagta 8040
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gagagtggtg attatgcagt aatagatcag gttattgcct taactaaaga caaatgtctt 8100
ttgttgttta aaagtcttac atctttgtaa tgctcgttct ggatatcctg caggaaacaa 8160
tgggtgctca ggatgcatgc tctctgccat ctggatcggt tggaactcag tctgacctag 8220
gagcaaacat tgagggtcaa aatgtcacaa cagtggctca acttcccaca gatggatcag 8280
atgcagttgt aaccggtgga tctcctgtat cagatcaggt acctgcctct gctcaaggac 8340
tttcttatgt gttggtttaa aggtctagtc cttagtaatg ttgaaactaa gcaaacagtg 8400
gatagtgatc atatggttat ttttgcttgt gaatttaata tttctggaca gtgtgcccag 8460
gatgcatctc ctatgccatt atcttcgcct ggaaatcacc ctgatacagc agttaatatc 8520
gagggtttag ataacacatc agtagctgag cctcatataa gtggatcaga tgcatgtgaa 8580
atggaaattt cagaacctgg tccccaagta gagcggtcaa cctttgcaag tcagtaactg 8640
ccttgggcat ttttaagtat cacctaggtc gacatatgtg attgccaaac agctaacaag 8700
gagatgcctt ttgtgcagat cttttccatg aaggtggcgt ggagcattca gcaggtgtaa 8760
cagctcttgt tccatcactt cttaacaatg gtacggaaca gattgccgtt caacctgttc 8820
ctcaaatacc tttccctgtg ttcaacgacc cgtttctgca tgaactggag aagttgcgga 8880
gagaatcaga gaactcaaag aagacttttg aagaaaaagt cagtttccct cattacccag 8940
ttacctcttg ttttggttta ttttctagct gcccattgac tctcagttgc ttgtgagcag 9000
aaatcaatct tgaaagctga actcgagagg aagatggctg aagtacaagc agagtttcga 9060
agaaaatttc atgaggtaga agccgagcat aacaccagaa cgacaaagat agagaaggat 9120
aagaatcttg ttataatgaa caaactgttg gcgaatgcgt tcttgtccaa atgtactgac 9180
aagaaggtat ctccctcagg agctccaagg ggtaagtgtc gaataatata gcaaattggt 9240
tttaaaaata aggcgacgaa gtcataatag cactttttct ccaggtaaaa ttcagcagct 9300
agcacagaga gcagcacaag tgagtgcact gagaaattac attgctcctc agcagcttca 9360
ggcatcttct tttcctgctc ctgctctggt ttcggctcct ctgcaacttc agcaatcatc 9420
atttcctgct cctggtccgg ctcctctgca gcctcaggca tcttcgtttc cttcttcagt 9480
ctctcgtcca tcagcccttc ttctgaattt tgcggtctgt ccaatgcctc agcccagaca 9540
gcctctcata tccaacatag ctccaactcc atcagttact cctgcaacaa atccaggtct 9600
gcgttctcct gcaccacacc taaactcata tagaccatcc tcttcaactc ccgtcgccac 9660
agctactcca acctcgtcag tgcctcctca agctttgaca tattcagctg tgtcaattca 9720
gcagcagcaa gaacaacaac cgcaacagag cttgagcagt ggattgcaga gcaacaatga 9780
agtggtttgt ctttctgacg acgagtgacc taagaggaga gatggttagg gtcttagtta 9840
ttgattttta gagagttaat aatagtatat atatatatgt ataagtaggt tacctaatct 9900
ctgtcgttaa tctaatttag tgagtcagga accgactcgt tggctaaggt ctctcctttt 9960
gaaacgcaac gttctacttt catgtatata aatacagtct gatcacacaa cacaaattga 10020
tgattgaaaa tactactgat ttaactttat agaaaaccca aattatagag cgacaacttt 10080
ataaacatgt caaacttcga agttaaaatt taagacccca taattttaca attatagatt 10140
ttaatactcc aactattttg tgatgttaaa agaagtatcc gagtcttttc tttccagttt 10200
ccccaccgtc ccatgactcc cccagccagt agaaaaagcc aaaaaagtaa acaaaaagtc 10260
gttaaaaaag ttaaattaaa aaaaaaatag atagttgacg tttactaaag tgatttgaat 10320
tgaacaatt 10329
<210> 2
<211> 6571
<212> 1~
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (310)..(6312)
<400> 2
cacaagcatg agtttttcct tccggtaatc gtaaaatcaa aaatcgctcg ctcgagaaac 60
gccggtgcta gaagaggaaa gtaccgtaca taatcctgcg aacccaattc tcgtcttctt 120
CA 02369749 2001-12-05
WO 01/00801 PCT/EP00/05761
-6-
caaactcagt tttccgaaac cccaaacacc gcgaggattg catggcctga agaaccactt 180
aatcgagaat tgtgctggaa ttctcaaatt ttccctcgcg tttttctttc acactctcgg 240
aatcggaaat ttccaccaag ctccgtcaag cgatagattc tgacaattac acactttcgc 300
gcagtgaat atg aag aaa gat gaa aag att ggt ttg acg ggg aga acc att 351
Met Lys Lys Asp Glu Lys Ile Gly Leu Thr Gly Arg Thr Ile
1 5 10
tac acc aga tcc cta gca get tca att cct gcc tca gtt gaa caa gaa 399
Tyr Thr Arg Ser Leu Ala Ala Ser Ile Pro Ala Ser Val Glu Gln Glu
15 20 25 30
acc cct ggt ttg agg agg tca agc cgg ggg aca cca tct acg aag gta 447
Thr Pro Gly Leu Arg Arg Ser Ser Arg Gly Thr Pro Ser Thr Lys Val
35 40 45
ata act cca get tct get act aga aag tca gag aga ctg get ccc tca 495
Ile Thr Pro Ala Ser Ala Thr Arg Lys Ser Glu Arg Leu Ala Pro Ser
50 55 60
cct get tca gtt tca aaa aag tcc ggt gga atc gtc aag aat tcc aca 543
Pro Ala Ser Val Ser Lys Lys Ser Gly Gly Ile Val Lys Asn Ser Thr
65 70 75
cca agt tct ttg cga agg tcc aat agg ggg aag act gaa gta tcc ttg 591
Pro Ser Ser Leu Arg Arg Ser Asn Arg Gly Lys Thr Glu Val Ser Leu
80 85 90
cag agt tcc aaa gga tca gat aat tct atc agg aaa gga gat act tca 639
Gln Ser Ser Lys Gly Ser Asp Asn Ser Ile Arg Lys Gly Asp Thr Ser
95 100 105 110
ccg gat att gag cag aga aag gat agt gtt gaa gag tcg aca gat aag 687
Pro Asp Ile Glu Gln Arg Lys Asp Ser Val Glu Glu Ser Thr Asp Lys
115 120 125
atc aag cct ata atg tca gcc cga agt tac agg gca ttg ttt aga ggg 735
Ile Lys Pro Ile Met Ser Ala Arg Ser Tyr Arg Ala Leu Phe Arg Gly
130 135 140
aag ctc aag gaa tct gag gca tta gtt gat get tcc cca aat gaa gag 783
Lys Leu Lys Glu Ser Glu Ala Leu Val Asp Ala Ser Pro Asn Glu Glu
145 150 155
gaa cta gta gtt gtt ggt tgt tct cgc cgc ata cct gca ggc aat gat 831
Glu Leu Val Val Val Gly Cars Ser A~ Arg Ile Pro Ala Gly Asn Asp
160 165 170
gat gtt caa ggt aaa aca gat tgt cca cca cct gca gat gca gga tca 879
Asp Val Gln Gly Lys Thr Asp Cars Pro Pro Pro Ala Asp Ala Gly Ser
175 180 185 190
aaa agg ctg cca gtt gac gaa act agt ttg gac aag ggc act gat ttt 927
CA 02369749 2001-12-05
WO 01/00801 PCT/EP00/05761
_7_
Lys Arg Leu Pro Val Asp Glu Thr Ser Leu Asp Lys
Gly Thr Asp Phe
195 200 205
cct ttg aaa tca gtt acg gag acc gag aag ata gtg 975
ctt gat gca tcc
Pro Leu Lys Ser Val Thr Glu Thr Glu Lys Ile Val
Leu Asp Ala Ser
210 215 220
ccc ata gtt gaa act ggg gat gac agt gtt ata ggt 1023
tca cca tct gag
Pro Ile Val Glu Thr Gly Asp Asp Ser Val Ile Gly
Ser Pro Ser Glu
225 230 235
aat tta gag aca caa aag ctt caa gat ggt aag aca 1071
gat tgt tca cca
Asn Leu Glu Thr Gln Lys Leu Gln Asp Gly Lys Thr
Asp Cps Ser Pro
240 245 250
cct gca aat gca gaa tcg aaa acg ctg cca gtt ggt 1119
gaa act agt tta
Pro Ala Asn Ala Glu Ser Lys '~hr Leu Pro Val Gly
Glu Thr Ser Leu
255 260 265 270
gaa aaa gaa tat cca caa aag ttt caa gat gat aac 1167
aca gat tgt cta
Glu Lys Glu Tyr Pro Gln Lys Phe Gln Asp Asp Asn
Thr Asp Cps Leu
275 280 285
cca cct gca aat gca gaa tca aaa agg ctg cca gtt 1215
ggc gaa act agt
Pro Pro Ala Asn Ala Glu Ser Lys Arg Leu Pro Val
Gly Glu Thr Ser
290 295 300
tta gaa aag gac act gat ttt cct ttg aaa tca act 1263
acg gag act gga
Leu Glu Lys Asp Thr Asp Phe Pro Leu Lys Ser Thr
Z'hr Glu Thr Gly
305 310 315
aag atg gtt ctt tat gca tcc ccc ata gtt gaa act 1311
agg gat gac agc
Lys Met Val Leu Tyr Ala Ser Pro Ile Val Glu Thr
Arg Asp Asp Ser
320 325 330
gtt ata tgt tca cca tct aca aat tta gaa acc caa 1359
aag ctt ctt gtc
Val Ile Cps Ser Pro Ser Thr Asn Leu Glu Thr Gln
Lys Leu Leu Val
335 340 345 350
agt aaa act ggc tta gaa acc gac ata gtt ttg cct 1407
ttg aaa aga aaa
Ser Lys Thr Gly Leu Glu Thr Asp Ile Val Leu Pro
Leu Lys Arg Lys
355 360 365
aga gac act gca gaa att gag ctg gat gca tgt get 1455
aca gtt gca aat
Arg Asp Thr Ala Glu Ile Glu Leu Asp Ala Cps Ala
Thr Val Ala Asn
370 375 380
gga gat gat cac gtt atg agt tct gat ggg gtc att 1503
cca tct cca tct
Gly Asp Asp His Val Met Ser Ser Asp Gly Val Ile
Pro Ser Pro Ser
385 390 395
ggg tgc aaa aat gat aat cga cct gaa atg tgc aac 1551
acg tgt aaa aaa
Gly Cps Lys Asn Asp Asn An3 Pro Glu Met Cps Asn
Thr Cars Lys Lys
400 405 410
CA 02369749 2001-12-05
WO 01/00801 PCT/EP00/05761
_g_
cgg caa aag gtc aac ggt gat tgt caa aat agg agt gtt tgc tcc tgc 1599
Arg Gln Lys Val Asn Gly Asp Cps Gln Asn Arg Ser Val Cps Ser Cars
415 420 425 430
att gtc cag cca gtt gaa gaa tct gat aac gtg aca cag gat atg aaa 1647
Ile Val Gln Pro Val Glu Glu Ser Asp Asn Val Thr G1n Asp Met Lys
435 440 445
gaa act gga cca gtt acg agc aga gaa tat gag gag aac ggg caa ata 1695
Glu Thr Gly Pro Val Thr Ser Arg Glu Tyr Glu Glu Asn Gly Gln Ile
450 455 460
caa cat ggt aaa tca agt gat ccc aaa ttc tat tct tcg gtg tac cca 1743
Gln His Gly Lys Ser Ser Asp Pro Lys Phe Tyr Ser Ser Val Tyr Pro
465 470 475
gag tat tgg gtt cct gtg cag cta tca gat gta cag ctg gag caa tac 1791
Glu Tyr Trp Val Pro Val G1n Leu Ser Asp Val Gln Leu Glu Gln Tyr
480 485 490
tgt cag act ctc ttc tcc aaa tcc tta tct ctt tct tca ctt tcg aag 1839
Cys Gln Thr Leu Phe Ser Lys Ser Leu Ser Leu Ser Ser Leu Ser Lys
495 500 505 510
att gat ctt gga get cta gaa gaa act ctc aat tct gta aga aaa acc 1887
Ile Asp Leu Gly Ala Leu Glu Glu Thr Leu Asn Ser Val Arg Lys Thr
515 520 525
tgt gac cat cca tac gtt atg gat gca tct ttg aaa caa ctg ctc acc 1935
Cys Asp His Pro Tyr Val Met Asp Ala Ser Leu Lys Gln Leu Leu Zhr
530 535 540
aag aat ctg gag ttg cat gaa atc ctg gat gta gaa att aaa gcg agc 1983
Lys Asn Leu Glu Leu His Glu Ile Leu Asp Val Glu Ile Lys Ala Ser
545 550 555
ggg aaa ctt cac ctc ctt gat aaa atg ctt act cat ata aaa aag aat 2031
Gly Lys Leu His Leu Leu Asp Lys Met Leu Thr His Ile Lys Lys Asn
560 565 570
ggt tta aaa gca gtt gtc ttc tac cag gca aca caa acc cct gaa ggg 2079
Gly Leu Lys Ala Val Val Phe Tyr Gln Ala Thr G1n Thr Pro Glu Gly
575 580 585 590
ctt ctg ctt ggt aat att ctc gaa gat ttt gtg ggt caa aga ttt ggt 2127
Leu Leu Leu Gly Asn Ile Leu Glu Asp Phe Val Gly Gln Arg Phe Gly
595 600 605
cca aaa tct tat gag cat ggg ata tat tcc tca aag aag aac tcc get 2175
Pro Lys Ser Tyr Glu His Gly Ile 'i~r Ser Ser Lys Lys Asn Ser Ala
610 615 620
ata aac aat ttc aac aag gag agt caa tgc tgt gtt ctg ctg ttg gaa 2223
Ile Asn Asn Phe Asn Lys Glu Ser Gln Cars Cars Val Leu Leu Leu Glu
625 630 635
CA 02369749 2001-12-05
WO 01/00801 PCT/EP00/05761
_g_
aca cgt tgc agt caa acc ctc ttg cga get gat 2271
gcc att aaa gcg ttt
Thr Arg Cps Ser Gln Thr Leu Leu Arg Ala Asp
Ala Ile Lys Ala Phe
640 645 650
att ctt gga agc agc ttg tcg cat gat gtt aag 2319
ttt aat cca cac gta
Ile Leu Gly Ser Ser Leu Ser His Asp Val Lys
Phe Asn Pro His Val
655 660 665 670
gag aag aaa atc gag tca gaa aga act aag ata 2367
ata tgt tct ttc cga
Glu Lys Lys Ile Glu Ser Glu Arg Thr Lys Ile
Ile Cps Ser Phe Arg
675 680 685
ttg tac gta tgt aca gtt aaa gcc ctg att ctg 2415
tca gaa gaa get agg
Leu Tyr Val Cars Thr Val Lys Ala Leu Ile Leu
Ser Glu Glu Ala Arg
690 695 700
caa aat atg cgg caa aat aaa get gta gag aac cta 2463
aac cgc tct ctc
Gln Asn Met Arg Gln Asn Lys Ala Val Glu Asn Leu
Asn Arg Ser Leu
705 710 715
acg cac gca ctg ctc atg tgg ggg gcg tca tac tta 2511
ttt gat aaa ctg
Thr His Ala Leu Leu Met Trp Gly Ala Ser 'I'yr Leu
Phe Asp Lys Leu
720 725 730
gat cat ttt cac agc agt gaa act cca gat tca gga 2559
gtt tca ttt gaa
Asp His Phe His Ser Ser Glu 'I'hr Pro Asp Ser Gly
Val Ser Phe Glu
735 740 745 750
caa tct att atg gac ggc gtg att cat gaa ttc tcg 2607
tcc ata ctt tct
Gln Ser Ile Met Asp Gly Val Ile His Glu Phe Ser
Ser Ile Leu Ser
755 760 765
tcc aaa ggt gga gaa gaa aat gaa gtc aag ctg tgt 2655
cta ctt ttg gag
Ser Lys Gly Gly Glu Glu Asn Glu Val Lys Leu Cps
Leu Leu Leu Glu
770 775 780
gcc aag cat get cag gga act tac agc agt gat tct 2703
act cta ttt ggt
Ala Lys His Ala Gln Gly Thr Tyr Ser Ser Asp Ser
Thr Leu Phe Gly
785 790 795
gaa gac cat att aag ttg tca gat gaa gag agt cca 2751
aat ata ttt tgg
Glu Asp His Ile Lys Leu Ser Asp Glu Glu Ser Pro
Asn Ile Phe Trp
800 805 810
tca aag ctg ttg ggg gga aaa aat cct atg tgg aaa 2799
tac cct tca gat
Ser Lys Leu Leu Gly Gly Lys Asn Pro Met Trp Lys
~r Pro Ser Asp
815 820 825 830
act ccc caa agg aat cga aaa cga gtt cag tat ttt 2847
gag ggt tct gaa
Thr Pro Gln Arg Asn Arg Lys Arg Val Gln Tyr Phe
Glu Gly Ser Glu
835 840 845
gcg agt ccc aaa act ggc gat ggt gga aat gca aag 2895
aag cga aag aag
Ala Ser Pro Lys Thr Gly Asp Gly Gly Asn Ala Lys
Lys Arg Lys Lys
CA 02369749 2001-12-05
WO 01/00801 PCT/EP00/05761
-10-
850 855 860
get tct gat gat gtc act gat ccc cgg gtc act gat ccg cca gta gat 2943
Ala Ser Asp Asp Val Thr Asp Pro Arg Val Thr Asp Pro Pro Val Asp
865 870 875
gat gat gaa aga aag gcc tct ggg aag gat cac atg ggg get ttg gag 2991
Asp Asp Glu Arg Lys Ala Ser Gly Lys Asp His Met Gly Ala Leu Glu
880 885 890
tca cca aaa gtc ata aca.ctc cag tca tca tgt aaa tct tct ggt aca 3039
Ser Pro Lys Val Ile Thr Leu Gln Ser Ser Cys Lys Ser Ser Gly Thr
895 900 905 910
gat ggt aca ttg gat gga aat gat get ttt ggc ttg tat tct atg ggc 3087
Asp Gly Thr Leu Asp Gly Asn Asp Ala Phe Gly Leu Tyr Ser Met Gly
915 920 925
agc cat atc tct gga atc cca gag gat atg tta get agt caa gat tgg 3135
Ser His Ile Ser Gly Ile Pro Glu Asp Met Leu Ala Ser Gln Asp Trp
930 935 940
ggg aaa ata ccg gat gaa tca cag agg agg ctc cac act gtt tta aag 3183
Gly Lys Ile Pro Asp Glu Ser Gln Arg Arg Leu His Thr Val Leu Lys
945 950 955
ccg aag atg gca aaa ctt tgc caa gtt ttg cat ctt tca gat get tgc 3231
Pro Lys Met Ala Lys Leu Cps Gln Val Leu His Leu Ser Asp Ala Cps
960 965 970
aca agc atg gtc gga aat ttt ctc gaa tat gtt att gaa aat cac cga 3279
Thr Ser Met Val Gly Asn Phe Leu Glu Tyr Val Ile Glu Asn His Arg
975 980 985 990
atc tac gaa gag cca gcc act act ttt cag gca ttc cag ata gcc ctg 3327
Ile Tyr Glu Glu Pro Ala Thr Thr Phe Gln Ala Phe Gln Ile Ala Leu
995 1000 1005
agt tgg att gca gcc ttg ttg gta aag caa att ctt agc cac aaa gaa 3375
Ser Trp Ile Ala Ala Leu Leu Val Lys Gln Ile Leu Ser His Lys Glu
1010 1015 1020
tct ctg gtc cgt gca aat tct gaa tta get ttc aaa tgc tct aga gta 3423
Ser Leu Val Arg Ala Asn Ser Glu Leu Ala Phe Lys Cps Ser Arg Val
1025 1030 1035
gag gtg gat tat att tat tcg ata ttg tcc tgc atg aag agt ctg ttc 3471
Glu Val Asp 'I~rr Ile Tyr Ser Ile Leu Ser Cps Met Lys Ser Leu Phe
1040 1045 1050
ctg gag cat aca caa ggt ttg cag ttc gat tgc ttt ggt act aat tct 3519
Leu Glu His Thr Gln Gly Leu Gln Phe Asp Cps Phe Gly Thr Asn Ser
1055 1060 1065 1070
aaa cag tca gtg gtt agc aca aaa cta gta aat gaa agt ctc tca ggg 3567
CA 02369749 2001-12-05
WO 01/00801 PCT/EP00/05761
-11 -
Lys Gln Ser Val Val Ser Thr Lys Leu Val Asn Glu Ser Leu Ser Gly
1075 1080 1085
get aca gtg cgt gac gaa aag att aat acg aag tcg atg cga aat agc 3615
Ala Thr Val Arg Asp Glu Lys Ile Asn Thr Lys Ser Met Arg Asn Ser
1090 1095 1100
tca gag gat gaa gag tgc atg act gag aag aga tgt agc cat tat agc 3663
Ser Glu Asp Glu Glu Cps Met Thr Glu Lys Arg Cps Ser His Tyr Ser
1105 1110 1115
aca gca aca aga gat atc gaa aag act att agt ggc ata aaa aag aaa 3711
Thr Ala Thr Arg Asp Ile Glu Lys Thr Ile Ser Gly Ile Lys Lys Lys
1120 1125 1130
tac aag aag caa gtg caa aag ctt gta caa gag cat gag gaa aag aaa 3759
Tyr Lys Lys Gln Val Gln Lys Leu Val Gln Glu His Glu Glu Lys Lys
1135 1140 1145 1150
atg gag ctg tta aat atg tat gca gac aag aag cag aaa ctt gaa act 3807
Met Glu Leu Leu Asn Met Tyr Ala Asp Lys Lys Gln Lys Leu Glu Thr
1155 1160 1165
agt aaa agt gtg gaa gca gca gta att cgt att acc tgt tca cgg acc 3855
Ser Lys Ser Val Glu Ala Ala Val Ile Arg Ile Thr Cps Ser Arg Thr
1170 1175 1180
agt act caa gtg ggt gat ctc aaa ctg ctg gat cat aat tat gaa aga 3903
Ser Thr Gln Val Gly Asp Leu Lys Leu Leu Asp His Asn Z~rr Glu Arg
1185 1190 1195
aag ttt gat gaa atc aaa agt gag aaa aat gaa tgc ctc aaa agt ctg 3951
Lys Phe Asp Glu Ile Lys Ser Glu Lys Asn Glu Cys Leu Lys Ser Leu
1200 1205 1210
gag caa atg cac gag gtt gca aag aag aag ttg get gag gat gaa gcc 3999
Glu Gln Met His Glu Val Ala Lys Lys Lys Leu Ala Glu Asp Glu Ala
1215 1220 1225 1230
tgt tgg att aat cgg ata aag agc tgg gca get aaa tta aaa gtt tgt 4047
Cps Trp Ile Asn Arg Ile Lys Ser Trp Ala Ala Lys Leu Lys Val Cars
1235 1240 1245
gtt ccc att caa agt ggc aat aac aag cat ttt agt ggt tca tca aac 4095
Val Pro Ile G1n Ser Gly Asn Asn Lys His Phe Ser Gly Ser Ser Asn
1250 1255 1260
att tcc caa aat get cct gat gta caa att tgc aat aat get aac gtt 4143
Ile Ser Gln Asn Ala Pro Asp Val Gln Ile Cars Asn Asn Ala Asn Val
1265 1270 1275
gaa get act tac get gat acg aat tgc atg get tcc aag gtt aat caa 4191
Glu Ala Thr Tyr Ala Asp Thr Asn Cps Met Ala Ser Lys Val Asn Gln
1280 1285 1290
CA 02369749 2001-12-05
WO 01/00801 PCT/EP00/05761
-12-
gtg cca gaa gca gaa aac aca tta gga acc atg tcg ggt ggc agc act 4239
Val Pro Glu Ala Glu Asn Thr Leu Gly Thr Met Ser Gly Gly Ser Thr
1295 1300 1305 1310
caa caa gtt cat gaa atg gtg gat gta aga aat gac gag aca atg gat 4287
Gln Gln Val His Glu Met Val Asp Val Arg Asn Asp Glu Thr Met Asp
1315 1320 1325
gtc tca get ttg tct cgt gaa cag ctt aca aag agc cag tcc aat gag 4335
Val Ser Ala Leu Ser Arg Glu Gln Leu Thr Lys Ser Gln Ser Asn Glu
1330 1335 1340
cac get tct atc act gtg cct gag att ttg att cct get gac tgt caa 4383
His Ala Ser Ile Thr Val Pro Glu Ile Leu Ile Pro Ala Asp Cps Gln
1345 1350 1355
gag gaa ttt gcg gcc ttg aac gtg cat ttg tca gaa gac cag aat tgt 4431
Glu Glu Phe Ala Ala Leu Asn Val His Leu Ser Glu Asp Gln Asn Cps
1360 1365 1370
gac aga ata aca tct gcg gca tca gat gaa gat gtt tca tca agg gtg 4479
Asp Arg Ile Thr Ser Ala Ala Ser Asp Glu Asp Val Ser Ser Arg Val
1375 1380 1385 1390
cca gag gta tcc cag tca ctc gaa aat ctt tct gcc tcc ccc gag ttt 4527
Pro Glu Val Ser Gln Ser Leu Glu Asn Leu Ser Ala Ser Pro Glu Phe
1395 1400 1405
tct cta aat aga gag gag get ttg gtt aca aca gaa aat aga aga aca 4575
Ser Leu Asn Arg Glu Glu Ala Leu Val Thr Thr Glu Asn Arg Arg Thr
1410 1415 1420
agt cat gtg ggt ttt gat act gat aac att ttg gac cag cag aat aga 4623
Ser His Val Gly Phe Asp Thr Asp Asn Ile Leu Asp Gln Gln Asn Arg
1425 1430 1435
gaa gat tgt tct ctt gac caa gag att cct gac gag tta gcg atg cct 4671
Glu Asp Cars Ser Leu Asp Gln Glu Ile Pro Asp Glu Leu Ala Met Pro
1440 1445 1450
gtg caa cat ctt gcg tct gtg gta gag act agg ggt get get gaa tct 4719
Val Gln His Leu Ala Ser Val Val Glu Thr Arg Gly Ala Ala Glu Ser
1455 1460 1465 1470
gat cag tat ggt caa gat ata tgt cct atg cct tct tca ctg get gga 4767
Asp Gln Tyr Gly Gln Asp Ile Cars Pro Met Pro Ser Ser Leu Ala Gly
1475 1480 1485
aag caa cct gac cca gca gca aac act gag agc gaa aat ctt gaa gaa 4815
Lys Gln Pro Asp Pro Ala Ala Asn Thr Glu Ser Glu Asn Leu Glu Glu
1490 1495 1500
gca att gag cct cag tct get ggt tca gaa aca gta gag act act gat 4863
Ala Ile Glu Pro Gln Ser Ala Gly Ser Glu Thr Val Glu Thr Thr Asp
1505 1510 1515
CA 02369749 2001-12-05
WO 01/00801 PCT/EP00/05761
-13-
ttt get gca cag ggt gat caa gtt aca ttg cta 4911
tca cat tgt cct tct
Phe Ala Ala Gln Gly Asp Gln Val Thr Leu Leu
Ser His ~s Pro Ser
1520 1525 1530
tca ccg act cag cct gcg cca gaa gca gaa ggc 4959
gga aat aat att caa
Ser Pro 'hhr Gln Pro Ala Pro Glu Ala Glu Gly.Gln
Gly Asn Asn Ile
1535 1540 1545 1550
aat atc aac get gag ccc cat gta gcg gat gca 5007
aca tca ggt cca gta
Asn Ile Asn Ala Glu Pro His Val Ala Asp Ala
Thr Ser Gly Pro Val
1555 1560 1565
gag agt ggt gca gta ata gat cag gaa ggt get 5055
gat tat aca atg cag
Glu Ser Gly Ala Val Ile Asp Gln Glu Gly Ala
Asp 'I~r Thr Met Gln
1570 1575 1580
gat gca tgc cca tct gga tcg gtt gga tct gac 5103
tct ctg act cag cta
Asp Ala Cars Pro Ser Gly Ser Val Gly Ser Asp
Ser Leu Thr Gln Leu
1585 1590 1595
gga gca aac ggt caa aat gtc aca aca caa ctt 5151
att gag gtg get ccc
Gly Ala Asn Gly Gln Asn Val Thr Thr Gln Leu
Ile Glu Val Ala Pro
1600 1605 1610
aca gat gga tca gat gca gtt gta acc ggt gga tct cct gta tca gat 5199
Thr Asp Gly Ser Asp Ala Val Val Thr Gly Gly Ser Pro Val Ser Asp
1615 1620 1625 1630
cag tgt gcc cag gat gca tct cct atg cca tta tct tcg cct gga aat 5247
Gln Cys Ala Gln Asp Ala Ser Pro Met Pro Leu Ser Ser Pro Gly Asn
1635 1640 1645
cac cct gat aca gca gtt aat atc gag ggt tta gat aac aca tca gta 5295
His Pro Asp Thr Ala Val Asn Ile Glu Gly Leu Asp Asn Thr Ser Val
1650 1655 1660
get gag cct cat ata agt gga tca gat gca tgt gaa atg gaa att tca 5343
Ala Glu Pro His Ile Ser Gly Ser Asp Ala Cars Glu Met Glu Ile Ser
1665 1670 1675
gaa cct ggt ccc caa gta gag cgg tca acc ttt gca aat ctt ttc cat 5391
Glu Pro Gly Pro Gln Val Glu Arg Ser Thr Phe Ala Asn Leu Phe His
1680 1685 1690
gaa ggt ggc gtg gag cat tca gca ggt gta aca get ctt gtt cca tca 5439
Glu Gly Gly Val Glu His Ser Ala Gly Val Thr Ala Leu Val Pro Ser
1695 1700 1705 1710
ctt ctt aac aat ggt acg gaa cag att gcc gtt caa cct gtt cct caa 5487
Leu Leu Asn Asn Gly Thr Glu G1n Ile Ala Val Gln Pro Val Pro Gln
1715 1720 1725
ata cct ttc cct gtg ttc aac gac ccg ttt ctg cat gaa ctg gag aag 5535
Ile Pro Phe Pro Val Phe Asn Asp Pro Phe Leu His Glu Leu Glu Lys
CA 02369749 2001-12-05
WO 01/00801 PCT/EP00/05761
-14-
1730 1735 1740
ttg cgg aga gaa tca gag aac tca aag aag act ttt gaa gaa aaa aaa 5583
Leu Arg Arg Glu Ser Glu Asn Ser Lys Lys Thr Phe Glu Glu Lys Lys
1745 1750 1755
tca atc ttg aaa get gaa ctc gag agg aag atg get gaa gta caa gca 5631
Ser Ile Leu Lys Ala Glu Leu Glu Arg Lys Met Ala Glu Val Gln Ala
1760 1765 1770
gag ttt cga aga aaa ttt cat gag gta gaa gcc gag cat aac acc aga 5679
Glu Phe Arg Arg Lys Phe His Glu Val Glu Ala Glu His Asn Thr Arg
1775 1780 1785 1790
acg aca aag ata gag aag gat aag aat ctt gtt ata atg aac aaa ctg 5727
Thr Thr Lys Ile Glu Lys Asp Lys Asn Leu Val Ile Met Asn Lys Leu
1795 1800 1805
ttg gcg aat gcg ttc ttg tcc aaa tgt act gac aag aag gta tct ccc 5775
Leu Ala Asn Ala Phe Leu Ser Lys Cars Thr Asp Lys Lys Val Ser Pro
1810 1815 1820
tca gga get cca agg ggt aaa att cag cag cta gca cag aga gca gca 5823
Ser Gly Ala Pro Arg Gly Lys Ile Gln Gln Leu Ala Gln Arg Ala Ala
1825 1830 1835
caa gtg agt gca ctg aga aat tac att get cct cag cag ctt cag gca 5871
Gln Val Ser Ala Leu Arg Asn Tyr Ile Ala Pro Gln Gln Leu Gln Ala
1840 1845 1850
tct tct ttt cct get cct get ctg gtt tcg get cct ctg caa ctt cag 5919
Ser Ser Phe Pro Ala Pro Ala Leu Val Ser Ala Pro Leu Gln Leu Gln
1855 1860 1865 1870
caa tca tca ttt cct get cct ggt ccg get cct ctg cag cct cag gca 5967
Gln Ser Ser Phe Pro Ala Pro Gly Pro Ala Pro Leu Gln Pro Gln Ala
1875 1880 1885
tct tcg ttt cct tct tca gtc tct cgt cca tca gcc ctt ctt ctg aat 6015
Ser Ser Phe Pro Ser Ser Val Ser Arg Pro Ser Ala Leu Leu Leu Asn
1890 1895 1900
ttt gcg gtc tgt cca atg cct cag ccc aga cag cct ctc ata tcc aac 6063
Phe Ala Val Cars Pro Met Pro G1n Pro Arg Gln Pro Leu Ile Ser Asn
1905 1910 1915
ata get cca act cca tca gtt act cct gca aca aat cca ggt ctg cgt 6111
Ile Ala Pro Thr Pro Ser Val Thr Pro Ala Thr Asn Pro Gly Leu Arg
1920 1925 1930
tct cct gca cca cac cta aac tca tat aga cca tcc tct tca act ccc 6159
Ser Pro Ala Pro His Leu Asn Ser Tyr Arg Pro Ser Ser Ser Thr Pro
1935 1940 1945 1950
gtc gcc aca get act cca acc tcg tca gtg cct cct caa get ttg aca 6207
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Val Ala Thr Ala Thr Pro Thr Ser Ser Val Pro Pro Gln Ala Leu Thr
1955 1960 1965
tat tca get gtg tca att cag cag cag caa gaa caa caa ccg caa cag 6255
Tyr Ser Ala Val Ser Ile Gln Gln Gln Gln Glu Gln Gln Pro Gln Gln
1970 1975 1980
agc ttg agc agt gga ttg cag agc aac aat gaa gtg gtt tgt ctt tct 6303
Ser Leu Ser Ser Gly Leu Gln Ser Asn Asn Glu Val Val Cps Leu Ser
1985 1990 1995
gac gac gag tgacctaaga ggagagatgg ttagggtctt agttattgat 6352
Asp Asp Glu
2000
ttttagagag ttaataatag tatatatata tatgtataag taggttacct aatctctgtc 6412
gttaatctaa tttagtgagt caggaaccga ctcgttggct aaggtctctc cttttgaaac 6472
gcaacgttct actttcatgt atataaatac agtctgatca cacaacacaa attgatgatt 6532
gaaaatacta ctgatttaac ttaaaaaaaa aaaaaaaaa 6571
<210> 3
<211> 2001
<212> PRT
<213> Arabidopsis thaliana
<400> 3
Met Lys Lys Asp Glu Lys Ile Gly Leu Thr Gly Arg Thr Ile Tyr Thr
1 5 10 15
Arg Ser Leu Ala Ala Ser Ile Pro Ala Ser Val Glu Gln Glu Thr Pro
20 25 30
Gly Leu Arg Arg Ser Ser Arg Gly Thr Pro Ser Thr Lys Val Ile Thr
35 40 45
Pro Ala Ser Ala Thr Arg Lys Ser Glu Arg Leu Ala Pro Ser Pro Ala
50 55 60
Ser Val Ser Lys Lys Ser Gly Gly Ile Val Lys Asn Ser Thr Pro Ser
65 70 75 80
Ser Leu Arg Arg Ser Asn Arg Gly Lys Thr Glu Val Ser Leu Gln Ser
85 90 95
Ser Lys Gly Ser Asp Asn Ser Ile Arg Lys Gly Asp Thr Ser Pro Asp
100 105 110
Ile Glu Gln Arg Lys Asp Ser Val Glu Glu Ser Thr Asp Lys Ile Lys
115 120 125
Pro Ile Met Ser Ala Arg Ser Tyr Arg Ala heu Phe Arg Gly Lys Leu
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130 135 140
Lys Glu Ser Glu Ala Leu Val Asp Ala Ser Pro Asn Glu Glu Glu L~eu
145 150 155 160
Val Val Val Gly Cps Ser Arg Arg Ile Pro Ala Gly Asn Asp Asp Val
165 170 175
Gln Gly Lys Thr Asp Cars Pro Pro Pro Ala Asp Ala Gly Ser Lys Arg
180 185 190
Leu Pro Val Asp Glu Thr Ser Leu Asp Lys Gly Thr Asp Phe Pro Leu
195 200 205
Lys Ser Val Thr Glu Thr Glu Lys Ile Val Leu Asp Ala Ser Pro Ile
210 215 220
Val Glu Thr Gly Asp Asp Ser Val Ile Gly Ser Pro Ser Glu Asn Leu
225 230 235 240
Glu Thr Gln Lys Leu Gln Asp Gly Lys Thr Asp Cars Ser Pro Pro Ala
245 250 255
Asn Ala Glu Ser Lys Thr Leu Pro Val Gly Glu Thr Ser Leu Glu Lys
260 265 270
Glu Tyr Pro Gln Lys Phe Gln Asp Asp Asn Thr Asp Cars Leu Pro Pro
275 280 285
Ala Asn Ala Glu Ser Lys Arg Leu Pro Val Gly Glu Thr Ser Leu Glu
290 295 300
Lys Asp Thr Asp Phe Pro Leu Lys Ser Thr Thr Glu Thr Gly Lys Met
305 310 315 320
Val Leu 'I~r Ala Ser Pro Ile Val Glu Thr Arg Asp Asp Ser Val Ile
325 330 335
Cars Ser Pro Ser Thr Asn Leu Glu 2'hr Gln Lys Leu Leu Val Ser Lys
340 345 350
Thr Gly Leu Glu Thr Asp Ile Val Leu Pro Leu Lys Arg Lys Arg Asp
355 360 365
Thr Ala Glu Ile Glu Leu Asp Ala Cps Ala 'Ihr Val Ala Asn Gly Asp
370 375 380
Asp His Val Met Ser Ser Asp Gly Val Ile Pro Ser Pro Ser Gly Cps
385 390 395 400
Lys Asn Asp Asn Arg Pro Glu Met Cps Asn Thr Cps Lys Lys Arg Gln
405 410 415
Lys Val Asn Gly Asp Cys Gln Asn Arg Ser Val Cys Ser Cars Ile Val
420 425 430
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Gln Pro Val Glu Glu Ser Asp Asn Val Thr Gln Asp Met Lys Glu Thr
435 440 445
Gly Pro Val Thr Ser Arg Glu 'I~r Glu Glu Asn Gly G1n Ile Gln His
450 455 460
Gly Lys Ser Ser Asp Pro Lys Phe Tyr Ser Ser Val Tyr Pro Glu Tyr
465 470 475 480
Trp Val Pro Val Gln Leu Ser Asp Val G1n Leu Glu Gln Tyr Cps Gln
485 490 495
Thr Leu Phe Ser Lys Ser Leu Ser Leu Ser Ser Leu Ser Lys Ile Asp
500 505 510
Leu Gly Ala Leu Glu Glu Thr Leu Asn Ser Val Arg Lys 'I'hr Cps Asp
515 520 525
His Pro Tyr Val Met Asp Ala Ser Leu Lys Gln Leu Leu Thr Lys Asn
530 535 540
Lzu Glu Leu His Glu Ile Leu Asp Val Glu Ile Lys Ala Ser Gly Lys
545 550 555 560
Leu His Leu Leu Asp Lys Met Leu Thr His Ile Lys Lys Asn Gly Leu
565 570 575
Lys Ala Val Val Phe Tyr Gln Ala Thr Gln Thr Pro Glu Gly Leu Leu
580 585 590
Leu Gly Asn Ile Leu Glu Asp Phe Val Gly Gln Arg Phe Gly Pro Lys
595 600 605
Ser Tyr Glu His Gly Ile ~r Ser Ser Lys Lys Asn Ser Ala Ile Asn
610 615 620
Asn Phe Asn Lys Glu Ser Gln Cps Cys Val Leu Leu Leu Glu Thr Arg
625 630 635 640
Ala Cps Ser Gln Thr Ile Lys Leu Leu Arg Ala Asp Ala Phe Ile Leu
645 650 655
Phe Gly Ser Ser Leu Asn Pro Ser His Asp Val Lys His Val Glu Lys
660 665 670
Ile Lys Ile Glu Ser Cars Ser Glu Arg Thr Lys Ile Phe Arg Leu Tyr
675 680 685
Ser Val Cars Thr Val Glu Glu Lys Ala Leu Ile Leu Ala An3 Gln Asn
690 695 700
Met Arg Gln Asn Lys Ala Val Glu Asn Leu Asn Arg Ser Leu Thr His
705 710 715 720
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Ala Leu Leu Met Trp Gly Ala Ser Tyr Leu Phe Asp Lys Leu Asp His
725 730 735
Phe His Ser Ser Glu Thr Pro Asp Ser Gly Val Ser Phe Glu Gln Ser
740 745 750
Ile Met Asp Gly Val Ile His Glu Phe Ser Ser Ile Leu Ser Ser Lys
755 760 765
Gly Gly Glu Glu Asn Glu Val Lys Leu Cps Leu Leu Leu Glu Ala Lys
770 775 780
His Ala G1n Gly Thr 'I~rr Ser Ser Asp Ser Thr Leu Phe Gly Glu Asp
785 790 795 800
His Ile Lys Leu Ser Asp Glu Glu Ser Pro Asn Ile Phe 'IYp Ser Lys
805 810 815
Leu Leu Gly Gly Lys Asn Pro Met Trp Lys 'I~r Pro Ser Asp Thr Pro
820 825 830
Gln Arg Asn Arg Lys Arg Val Gln Tyr Phe Glu Gly Ser Glu Ala Ser
835 840 845
Pro Lys Zhr Gly Asp Gly Gly Asn Ala Lys Lys Arg Lys Lys Ala Ser
850 855 860
Asp Asp Val Thr Asp Pro Arg Val Thr Asp Pro Pro Val Asp Asp Asp
865 870 875 880
Glu Arg Lys Ala Ser Gly Lys Asp His Met Gly Ala Leu Glu Ser Pro
885 890 895
Lys Val Ile Thr Leu Gln Ser Ser Gars Lys Ser Ser Gly I'hr Asp Gly
900 905 910
Thr Leu Asp Gly Asn Asp Ala Phe Gly Leu Tyr Ser Met Gly Ser His
915 920 925
Ile Ser Gly Ile Pro Glu Asp Met Leu Ala Ser Gln Asp Trp Gly Lys
930 935 940
Ile Pro Asp Glu Ser Gln Arg Arg Leu His Thr Val Leu Lys Pro Lys
945 950 955 960
Met Ala Lys Leu Cars Gln Val Leu His Leu Ser Asp Ala Cps Thr Ser
965 970 975
Met Val Gly Asn Phe Leu Glu Tyr Val Ile Glu Asn His Arg Ile Tyr
980 985 990
Glu Glu Pro Ala Thr Thr Phe Gln Ala Phe Gln Ile Ala Leu Ser Trp
995 1000 1005
Ile Ala Ala Leu Leu Val Lys Gln Ile L~eu Ser His Lys Glu Ser Leu
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1010 1015 1020
Val Arg Ala Asn Ser Glu Leu Ala Phe Lys Cys Ser Arg Val Glu Val
025 1030 1035 1040
Asp Tyr Ile 'I~r Ser Ile Leu Ser Cps Met Lys Ser Leu Phe Leu Glu
1045 1050 1055
His Thr Gln Gly Leu Gln Phe Asp Cars Phe Gly Thr Asn Ser Lys G1n
1060 1065 1070
Ser Val Val Ser Thr Lys Leu Val Asn Glu Ser Leu Ser Gly Ala Thr
1075 1080 1085
Val Arg Asp Glu Lys Ile Asn Thr Lys Ser Met Arg Asn Ser Ser Glu
1090 1095 1100
Asp Glu Glu Cps Met Thr Glu Lys Arg Cars Ser His Tyr Ser Thr Ala
105 1110 1115 1120
'~hr Arg Asp Ile Glu Lys Thr Ile Ser Gly Ile Lys Lys Lys 'I~r Lys
1125 1130 1135
Lys Gln Val Gln Lys Leu Val Gln Glu His Glu Glu Lys Lys Met Glu
1140 1145 1150
Leu Leu Asn Met Tyr Ala Asp Lys Lys Gln Lys Leu Glu Thr Ser Lys
1155 1160 1165
Ser Val Glu Ala Ala Val Ile Arg Ile Thr Cps Ser Arg Thr Ser Thr
1170 1175 1180
Gln Val Gly Asp Leu Lys Leu Leu Asp His Asn Tyr Glu Arg Lys Phe
185 1190 1195 1200
Asp Glu Ile Lys Ser Glu Lys Asn Glu Cps Leu Lys Ser Leu Glu Gln
1205 1210 1215
Met His Glu Val Ala Lys Lys Lys Leu Ala Glu Asp Glu Ala Cps Trp
1220 1225 1230
Ile Asn Arg Ile Lys Ser Trp Ala Ala Lys Leu Lys Val Cars Val Pro
1235 1240 1245
Ile Gln Ser Gly Asn Asn Lys His Phe Ser Gly Ser Ser Asn Ile Ser
1250 1255 1260
Gln Asn Ala Pro Asp Val G1n Ile Cps Asn Asn Ala Asn Val Glu Ala
265 1270 1275 1280
Thr Tyr Ala Asp Thr Asn Cars Met Ala Ser Lys Val Asn G1n Val Pro
1285 1290 1295
Glu Ala Glu Asn Thr Leu Gly Thr Met Ser Gly Gly Ser Thr Gln G1n
1300 1305 1310
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Val His Glu Met Val Asp Val Arg Asn Asp Glu Thr Met Asp Val Ser
1315 1320 1325
Ala Leu Ser Arg Glu Gln Leu Thr Lys Ser Gln Ser Asn Glu His Ala
1330 1335 1340
Ser Ile Thr Val Pro Glu Ile Leu Ile Pro Ala Asp Cars Gln Glu Glu
345 1350 1355 1360
Phe Ala Ala Leu Asn Val His Leu Ser Glu Asp Gln Asn Cps Asp Arg
1365 1370 1375
Ile Thr Ser Ala Ala Ser Asp Glu Asp Val Ser Ser Arg Val Pro Glu
1380 1385 1390
Val Ser G1n Ser Leu Glu Asn Leu Ser Ala Ser Pro Glu Phe Ser Leu
1395 1400 1405
Asn Arg Glu Glu Ala Leu Val Thr Thr Glu Asn Arg Arg Thr Ser His
1410 1415 1420
Val Gly Phe Asp Thr Asp Asn Ile Leu Asp Gln Gln Asn Arg Glu Asp
425 1430 1435 1440
Cps Ser Leu Asp Gln Glu Ile Pro Asp Glu Leu Ala Met Pro Val Gln
1445 1450 1455
His Leu Ala Ser Val Val Glu Thr Arg Gly Ala Ala Glu Ser Asp Gln
1460 1465 1470
Tyr Gly Gln Asp Ile Cps Pro Met Pro Ser Ser Leu Ala Gly Lys Gln
1475 1480 1485
Pro Asp Pro Ala Ala Asn Thr Glu Ser Glu Asn Leu Glu Glu Ala Ile
1490 1495 1500
Glu Pro Gln Ser Ala Gly Ser Glu Thr Val Glu Thr Thr Asp Phe Ala
505 1510 1515 1520
Ala Ser His Gln Gly Asp G1n Val Thr Cars Pro Leu Leu Ser Ser Pro
1525 1530 1535
Thr Gly Asn Gln Pro Ala Pro Glu Ala Asn Ile Glu Gly Gln Asn Ile
1540 1545 1550
Asn Thr Ser Ala Glu Pro His Val Ala Gly Pro Asp Ala Val Glu Ser
1555 1560 1565
Gly Asp 'I~r Ala Val Ile Asp Gln Glu Thr Met Gly Ala Gln Asp Ala
1570 1575 1580
Cps Ser Leu Pro Ser Gly Ser Val Gly Thr Gln Ser Asp Leu Gly Ala
585 1590 1595 1600
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Asn Ile Glu Gly Gln Asn Val Thr Thr Val Ala Gln Leu Pro Thr Asp
1605 1610 1615
Gly Ser Asp Ala Val Val Thr Gly Gly Ser Pro Val Ser Asp G1n Cps
1620 1625 1630
Ala Gln Asp Ala Ser Pro Met Pro Leu Ser Ser Pro Gly Asn His Pro
1635 1640 1645
Asp Thr Ala Val Asn Ile Glu Gly Leu Asp Asn Thr Ser Val Ala Glu
1650 1655 1660
Pro His Ile Ser Gly Ser Asp Ala Cps Glu Met Glu Ile Ser Glu Pro
665 1670 1675 1680
Gly Pro Gln Val Glu Arg Ser Thr Phe Ala Asn Leu Phe His Glu Gly
1685 1690 1695
Gly Val Glu His Ser Ala Gly Val Thr Ala Leu Val Pro Ser Leu Leu
1700 1705 1710
Asn Asn Gly Thr Glu Gln Ile Ala Val Gln Pro Val Pro Gln Ile Pro
1715 1720 1725
Phe Pro Val Phe Asn Asp Pro Phe Leu His Glu Leu Glu Lys Leu Arg
1730 1735 1740
Arg Glu Ser Glu Asn Ser Lys Lys Thr Phe Glu Glu Lys Lys Ser Ile
745 1750 1755 1760
Leu Lys Ala Glu Leu Glu Arg Lys Met Ala Glu Val Gln Ala Glu Phe
1765 1770 1775
Arg Arg Lys Phe His Glu Val Glu Ala Glu His Asn Thr Arg Thr Thr
1780 1785 1790
Lys Ile Glu Lys Asp Lys Asn Leu Val Ile Met Asn Lys Leu Leu Ala
1795 1800 1805
Asn Ala Phe Leu Ser Lys Cps Thr Asp Lys Lys Val Ser Pro Ser Gly
1810 1815 1820
Ala Pro Arg Gly Lys Ile Gln Gln Leu Ala Gln Arg Ala Ala Gln Val
825 1830 1835 1840
Ser Ala Leu Arg Asn 'I~r Ile Ala Pro Gln Gln Leu Gln Ala Ser Ser
1845 1850 1855
Phe Pro Ala Pro Ala Leu Val Ser Ala Pro Lzu Gln Leu Gln Gln Ser
1860 1865 1870
Ser Phe Pro Ala Pro Gly Pro Ala Pro Leu G1n Pro Gln Ala Ser Ser
1875 1880 1885
Phe Pro Ser Ser Val Ser Arg Pro Ser Ala Leu Leu Leu Asn Phe Ala
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1890 1895 1900
Val Cps Pro Met Pro Gln Pro Arg Gln Pro Leu Ile Ser Asn Ile Ala
905 1910 1915 1920
Pro Thr Pro Ser Val Thr Pro Ala Thr Asn Pro Gly Leu Arg Ser Pro
1925 1930 1935
Ala Pro His Leu Asn Ser Tyr Arg Pro Ser Ser Ser Thr Pro Val Ala
1940 1945 1950
Thr Ala Thr Pro Thr Ser Ser Val Pro Pro Gln Ala Leu 'fhr Tyr Ser
1955 1960 1965
Ala Val Ser Ile Gln Gln Gln G1n Glu Gln Gln Pro Gln Gln Ser Leu
1970 1975 1980
Ser Ser Gly Leu Gln Ser Asn Asn Glu Val Val Cps Leu Ser Asp Asp
985 1990 1995 2000
Glu
<210> 4
<211> 21
<212> L7~1A
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 4
catctacggc aatgtaccag c 21
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 5
gatgggaatt ggctgagtgg c 21
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence
CA 02369749 2001-12-05
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-23-
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 6
cagttccaaa cgtaaaacgg c 21
<210> 7
<211> 15
<212> I1'1A
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:5ynthetic
Oligonucleotide
<400> 7
ntcgastwts gwgtt 15
<210> 8
<211> 16
<212> L~1A
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 8
ngtcgaswga nawgaa 16
<210> 9
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 9
wgtgnagwan canaga 16
<210> 10
<211> 16
<212> LIB.
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
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Oligonucleotide
<400> 10
wggwancwga wangca 16
<210> 11
<211> 16
<212> I~IA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 11
wcgwwgawca ngncga 16
<210> 12
<211> 16
<212> L~.
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:S~nthetic
Oligonucleotide
<400> 12
wgcnagtnag wanaag 16
<210> 13
<211> 16
<212> LAP.
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 13
awgcangncw ganata 16
<210> 14
<211> 24
<212> I%~1
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 14
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ctgtacatac tgagtacaat cgga 24
<210> 15
<211> 25
<212> INA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 15
gcttcaattc ctgcctcagt tgaac 25
<210> 16
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 16
ctctacgtgc ttaacatcat gcga 24
<210> 17
<211> 25
<212> I~VA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 17
ccagcttctg ctactagaaa gtcag 25
<210> 18
<211> 25
<212> I~1A
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 18
ctggagttgc atgaaatcct ggatg 25
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<210> 19
<211> 25
<212> L~lA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 19
gctctttgta agctgttcac gagac 25
<210> 20
<211> 24
<212> I~
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 20
tcgcatgatg ttaagcacgt agag 24
<210> 21
<211> 25
<212> I~
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 21
gagtactggt ccgtgaacag gtaat 25
<210> 22
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 22
atgcttgcac aagcatggtc ggaaa 25
<210> 23
<211> 25
<212> L~1A
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<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 23
tgcaacatcg tgcatttgct ccaga 25
<210> 24
<211> 25
<212> I%~
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 24
cacaagcatg agtttttcct tccgg 25
<210> 25
<211> 25
<212> L~1P.
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Oligonucleotide
<400> 25
ctgactttct agtagcagaa gctgg 25
<210> 26
<211> 519
<212> DNA
<213> Brassica oleracea
<220>
<223> seal-23
<400> 26
gaattcctgn nttacggcat ccttaataga ctgttcaaat ggaactcctg aacctggggt 60
tccactccca tggaagtgtt ccagcttatc aaataaatat gatgcccccc acatgagcaa 120
tgcatgtgtg agaggacggt ttaggttctc tagaggctta ttttgcctag caagaatcag 180
ggttttttct tcaactgtaa acactgagta caaccggaaa atcttagttc tttcagaaca 240
cgactcaacc tttatcttct ctaagagctt aacgtcatgc gatggattca ggctgcttcc 300
aaaaagtata aaagactcag cgcgtaagag tttaatgctt tgactacagg cacgtatttc 360
cagcagcaga ataaaacact cactctcctt gttgaaattg tttatagcgt tcttcttcga 420
gaggcagacc ccatgctcat aggaattttg accaaatctt tgcatcagaa aatcttcgag 480
aatattacca agcagaagcc cctcagggct atgtattgc 519
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<210> 27
<211> 419
<212> L1~.
<213> Brassica oleracea
<220>
<223> seql-27
<400> 27
gaattcagga tcaaaagggt tgccggttgg agaaactggt ttagagaaag gctctgattt 60
tcctgtggaa gtaactaagg atatagagaa gacagtggtt gattcatccc ccatggttga 120
aactgaggat ggcagtgtta taggttcacc atccgagaat ccagaaccac aaaagcttcg 180
tgacagtgaa actagcttgg aaaccgatat agacttggct ctgaaaagaa aaagagacac 240
tgcagaaatt gtgatggatg catgtacaaa tgcagatgac cgcattatga gtactgatgg 300
ggttattcct tttccacccg tgtgcacaaa tattaatcaa cccgaaaggt gtggcacatg 360
tcaaaaacgg caaaagtaag aatttccgac tgttgtctgt cgttttgaaa ccatttgcc 419
<210> 28
<211> 467
<212> INA
<213> Brassica oleracea
<220>
<223> seal-43
<400> 28
gaattctcgt ccatactttc ttccgatgtt ggagaagaaa atgaaggcaa gctgtgtcta 60
cttttggaag ccaagcatgc tcagggaagt tacagcactg atgctactct atttggtgaa 120
gaacatgtca agttatcaga tgaaagtcca aatatgtttt ggtcaaagct gttgagtgga 180
aagaacccta tgtggaaata ctgttcggat actcctcaaa ggagtcgaaa aagagtacgg 240
catcttcagg gctatgagga gactaccaaa gttggcaatg gcggaaactt aaagaagaaa 300
aagaaggctt cagatgatgt cacagtagat aacgctgaga gaaaagcctc tggaaaggat 360
cacatgggta aaacagttca cttcctgctc ctttacctct agtgttcatt gaatgttcca 420
tttactttgc ttactatctt tccttcaggg catttggagt caccaaa 467
<210> 29
<211> 490
<212> I~
<213> Brassica oleracea
<220>
<223> seal-47
<400> 29
gaattcagct tttaaaactg atctctgctc acagataatt taagagtcag tgaaaattga 60
gataaaacga accaaaactg gaggtaacag atactctgag aacaactaac cttttcttca 120
taagtcttct ttgtgttctc tgattctctc cgcagcttct ccagttcatg ctgaaatggg 180
tcactgaaca cagggaaagg tacttgagga acaggtggag tggcattctg tcccgtagca 240
ttgttaagct gtgaagaaac aggagctgtt acacctgctg gaggctccac aacaccttca 300
tcgacaacgt ctgcgtaaaa ggtattacca gattgtcagt ttctctggca aacacatacg 360
ttatacttaa atgcaaaaga gcagttactg acttgcaaag gttggttgtt ctacttgagc 420
atcaggttct gctacttcca tttcacatgc ttctgatcca gttgtgcgag gcgcagccat 480
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tgttgtgttg 490
<210> 30
<211> 515
<212> I~IP.
<213> Brassica oleracea
<220>
<223> 2-33
<400> 30
tctagagaag aggtggatta tgtatattct tttctgtact gcatgaagag tctattcgtg 60
gggcgcacac aaggtttcca agaaaagggt gaagaatgca tggctgagaa aagaggtagc 120
cattatagct cagtaaccaa ggatgttgaa aagactatta gcgacatcaa aaagaaatgc 180
agtaagagcc tgcataagct tgtacaaacc ctcgaggaag aaaagatgga cctgatgaat 240
aggaatgctg tcaagaagca ggaacttcag aattgtaaaa aggtggaagc atcatttatt 300
cgtgtcacct attcaggtat aaatactcag agcttacatg atgctctcca acggctggaa 360
tgtacttttg aaagaaagtt tgatgatctc aaaggagagt tggatgaatg ccttgaaagt 420
ttagagcaaa taaacgaggc tggaaagaag aagttggctg aagatgaagc ctgttggatt 480
agtcggatag agaaatgggc acgagctgaa ttaag 515
<210> 31
<211> 574
<212> L~.
<213> Brassica oleracea
<220>
<223> seq2-37
<400> 31
tctagaccaa actattaaac gctaaacata agaagattag atcactcgtc atcagagaga 60
cagaccacat cattgctcct ctgcaatcca ctccccaagt tctgtggttg ttcttgctgc 120
tgaataaacg catttgaata tggtaaaggg ttggagatga gaggttgtct tggttgaggc 180
attgtgcagt acggagccga agcagtatga ttcctcagtg cgcttacttg tgttgctctc 240
tgtgctagct gctggattct aactggagaa agaaaaaaag aaaaaaaagg tgttattatg 300
acttcataac cttatatctt taaaaaacaa ttatgcttct attattcgaa cacttgccca 360
ttggagttgc tgctgaggaa tgagaggaga ttctgctcgt acatttagac aagaacgcac 420
tcgacaacag cttgttcttt ataacaagat tcttcctcgt ctgtaacttc gtctttctgg 480
ctgcatgtac agcttgtacc tcatgaaact ttctctgata ctcttcttgt aattcagcta 540
tcttcttctc gaatttagct ttcaagactg cttt 574
<210> 32
<211> 466
<212> L1~
<213> Brassica oleracea
<220>
<223> seq2-53
<400> 32
tctagattgt aattttaaat ttacaacaaa ttttgaaagg gtcagcgatg agtttgcaaa 60
tctccgtgtt tcctccagca ttgctcagcc agttcaagaa cctgatcact tggcacaggt 120
CA 02369749 2001-12-05
WO 01/00801 PCT/EP00/05761
-30-
tggtttcttc ttgctttact ttggacacct gtttaatatt ggcctgtcaa atttacttat 180
ccttttactt ctaaactgca aattctggtc tgcattgcat tgtgatatga aggtatctgg 240
acccgcttca agcagagact atggggagga caggcagaat atgcaacaag ataaatcaca 300
tgaccgaaag ttgtcatcga tgtatccaga gtattgggtt ccagtgcagc tatcagatgt 360
acagatagag caatactgtc ggactctctt ctccaaatct tcatctcttt cttcgctgtc 420
gaggactgat cctgttcgag ctcttgaaca aactctcagt tctgta 466
<210> 33
<211> 417
<212> L~
<213> Brassica oleracea
<220>
<223> seq2-57
<400> 33
tctagagcaa ttgaaaccta attccgattt tgcgcgggcc agagattctt cacggttgaa 60
cttttgctta acgaaagaga ctgcaatcca aatctggaag tgcattatta agaacgtatt 120
cagcaatatt cataaattat gcaacaatca aaggccttac gttgtggcct acaaagcatg 180
gattttgtta gatattagta gctagtctaa ttcaagcaat taatggaagt ttctatccta 240
tgactggaaa gttaaacatt cccacaaaag cagtgatgcc acagatgatg aagaagaaaa 300
atgcatatac tatggaagtg aatgctatca taccacagct atctggaagg cctgcaatgt 360
tgtagctggc tctttgcaga cacggtggtt gtcaataata tattcaagaa ctttttc 417
