Note: Descriptions are shown in the official language in which they were submitted.
CA 02388851 2002-04-23
1
FORMYLGLYCINAMIDINRIBOTIDE SYNTHASE FROM PLANTS
The present invention relates to the identification of plant
formylglycinamidine ribotide synthase (E. C. 6.3.5.3.) as novel
target for herbicidal active ingredients. The present invention
furthermore relates to DNA sequences SEQ-ID No. 1, SEQ-ID No. 3
or SEQ-ID No. 5 encoding a polypeptide with formylglycinamidine
ribotide synthase activity. Moreover, the invention relates to
the use of a nucleic acid encoding a protein with
formylglycinamidine ribotide synthase activity which originates
from plants for generating an assay system for identifying
herbicidally active formylglycinamidine ribotide synthase
inhibitors. The invention furthermore relates to the use of the
nucleic acid SEQ-ID No. 1, SEQ-ID No. 3 or SEQ-ID No. 5 encoding
plant formylglycinamidine ribotide synthase for the generation of
plants with an increased resistance to formylglycinamidine
ribotide synthase inhibitors. Moreover, the invention relates to
a method of eliminating undesired vegetation by treating the
plants to be eliminated with a compound which specifically binds
to formylglycinamidine ribotide synthase encoded by a DNA
sequence SEQ-ID No. 1, SEQ-ID No. 3 or SEQ-ID No. 5 or a DNA
sequence hybridizing with this DNA sequence, and which inhibits
its function.
Plants are capable of synthesizing their cell components from
carbon dioxide, water and inorganic salts.
This process is only possible by exploiting biochemical reactions
for synthesizing organic substances. Nucleotides are synthesized
de novo in plants. Being components of the nucleic acids, they
are particularly important. Covalently bound, nucleotides
activate carbohydrates for polysaccharide biosynthesis. They
furthermore activate head groups for lipid biosynthesis.
Nucleotides are involved in virtually all metabolic pathways.
Nucleoside triphosphates, especially ATP, drive most of the
energy-requiring reactions of the cell. Adenine nucleotides are
additionally also found as components in essential factors such
as coenzyme A and in nicotinamide and flavin coenzymes, which are
involved in a large number of cellular reactions. The coupled
hydrolysis of guanosine-5'-triphosphate (GTP) defines a direction
of reaction for various cellular processes such as protein
translation, assembly of microtubuli, vesicular transport, signal
transduction and cell division. Furthermore, nucleotides
constitute the starting metabolites for the biosynthesis of
0050/50827
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- methylxanthines such as coffein and theobromin in the plant
family of the Rubiaceae and Theaceae.
Since plants depend on an effective nucleotide metabolism, it can
be assumed that enzymes which are involved in nucleotide
biosynthesis are suitable as target protein for herbicides. Thus,
there have already been described active ingredients which
inhibit de novo purine biosynthesis in plants. An example which
may be mentioned is the natural substance hydanthocidin, which,
after phosphorylation in planta inhibits adenylosuccinate
synthetase (ASS); (Siehl et al., Plant Physiol. 110(.1996),
753-758).
Other inhibitors for enzymes of purine biosynthesis are also
known for their pharmacological action in animals and
microorganisms: folate analogs inhibit, inter alia, the enzyme
GAR transformylase and have an antiproliferative,
antiinflammatory and immunosuppressant action. Mycophenolic acid
(MPA), an IMP dehydrogenase inhibitor in the GMP synthetic
pathway, has an antimicrobial, antiviral and immunosuppressant
action (Kitchin et al, Journal of the American Academy of
Dermatology 37(1997), 445-449).
Bacterial PRPP amidotransferase can be inhibited by glutamine
antagonists such as, for example, acivicin (L-(alpha
S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid).
Glutarnine antagonists are not specific to PRPP amidotransferase,
but also inhibit the formylglycinamidine ribotide synthase (E. C.
6.3.5.3.) of mammals and have an antiproliferative action on
tumor tissue (Elliot and Weber, Biochemical Pharmacology
34(1985), 243-248). In the fourth step of purine biosynthesis,
formylglycinamidine ribotide synthase converts the carboxamide
oxygen of the formylglycinamide ribonucleotide (FGAR) into an
imino group. During the hydrolysis of ATP to ADP, the amide
nitrogen is transferred from the glutamine to FGAR, :resulting in
formylglycinamidine ribonucleotide (FGAM) and glutamate, see
Figure 1. The efficacy of glutamine antagonists on plant
formylglycinamidine ribotide synthase is still to be proven.
Genes which encode formylglycinamidine ribotide synthase have
been isolated from a variety of organisms.
cDNAs which encode formylglycinamidine ribotide synthase have
been isolated and characterized from various bacterial and animal
organisms.
005x/50827 CA 02388851 2002-04-23
3
- The genes which encode most of the enzymes of the purine
biosynthesis pathway in plants have already been isolated. In the
case of formylglycinamidine ribotide synthase, only two partial
EST (expressed sequence tag) sequences from Arabidopsis thaliana
(GenBank Number AA042492, 1295 bp) and barley (GenBank
Number HV,7000235, 384 bp) exist as yet.
The suitability of an enzyme as herbicide target can be proved by
reducing the enzyme activity in transgenic plants, for example by
means of antisense technology. If this leads to reduced growth,
it can be concluded therefore that the enzyme, whose activity is
reduced, is suitable as site of action for herbicidal active
ingredients. For example, antisense inhibition of acetolactate
synthase (ALS) in transgenic potato plants and the treatment of
control plants with ALS-inhibiting herbicides leads to comparable
phenotypes (Hofgen et al., Plant Physiology 107(1995), 469-477).
It is an object of the present invention to provide proof that
formylglycinamidine ribotide synthase in plants is a suitable
herbicidal target, to isolate a complete plant cDNA encoding the
enzyme formylglycinamidine ribotide synthase and functionally
express it in bacterial or eukaryotic cells, and to produce an
efficient and simple formylglycinamidine ribotide synthase assay
system for carrying out inhibitor-enzyme binding studies.
we have found that this object is achieved by first isolating
cDNAs encoding plant formylglycinamidine ribotide synthase. The
present invention therefore relates to the isolation of cDNAs
encoding Arabidopsis thaliana, Nicotiana tabacum and Chilopsis
linearis formylglycinamidine ribotide synthase.
The present invention furthermore relates to processes for
identifying formylglycinamidine ribotide synthase inhibitors in
plants by high-throughput methods. The invention therefore
relates to the functional expression of plant formyl.glycinamidine
ribotide synthase, in particular of Arabidopsis tha3iana and
tobacco formylglycinamidine ribotide synthase in suitable
expression systems and to the use of the resulting enzymes in an
in vitro assay system for measuring formylglycinamidine ribotide
synthase activity.
We have found that this object is achieved by the isolation of
genes which encode the plant enzyme formylglycinamidine ribotide
synthase, the generation of formylglycinamidine ribotide synthase
antisense constructs, and the functional expression of
~~50/50827 CA 02388851 2002-04-23
4
formylglycinamidine ribotide synthase in bacterial or eukaryotic
cells.
It is an object of the present invention to isolate full-length
cDNAs encoding functional formylglycinamidine ribotide synthase
(E.C.6.3.5.3.) from Arabidopsis thaliana and Nicotiana tabacum
and a partial cDNA sequence from Chilopsis linearis.
A first subject-matter of the present invention is a DNA sequence
SEQ-ID No. 1 or SEQ-ID No. 3 containing the encoding region of a
plant formylglycinamidine ribotide synthase from Nicotiana
tabacum and Arabidopsis thaliana, see Examples 1 and 2.
Another subject-matter of the invention is DNA sequences which
are derived from SEQ-ID No. 1 or SEQ-ID No. 3 or which hybridize
with one of these sequences and which encode a protein which has
the biological activity of a formylglycinamidine ribotide
synthase.
Arabidopsis thaliana plants and tobacco plants of the line
Nicotiana tabacum co. Samsun NN which carry a formylglycinamidine
ribotide synthase sense or antisense construct have been
characterized in greater detail. The plants show different
degrees of retarded growth. The transgenic lines and the progeny
of the lgt and 2nd generation showed a reduced growth in soil.
Using Northern hybridization, it was detected that the RNA
quantity of the formylglycinamidine ribotide synthase was reduced
in plants with reduced growth compared with the wild type.
Furthermore, measurement of the enzyme activity detected that the
amount of formylglycinamidine ribotide synthase activity was
reduced in the transgenic lines compared with wild-type plants,
see Examples 5 and 6. The expression level and the reduction in
formylglycinamidine ribotide synthase activity correlate with the
growth retardation. This clear connection identifies
formylglycinamidine ribotide synthase for the first time
unambiguously as suitable target protein for herbicidal active
ingredients.
To be able to find efficient inhibitors of plant
formylglycinamidine ribotide synthase, it is necessary to provide
suitable assay systems with which inhibitor/enzyme binding
studies can be carried out. To this end, for example, the cDNA
sequence of formylglycinamidine ribotide synthase or suitable
fragments of the cDNA sequence of formylglycinamidine ribotide
synthase from Arabidopsis thaliana is, or are, cloned into an
expression vector (pQE, Qiagen) and overexpressed in E. coli.
0~'rJ~/50827 CA 02388851 2002-04-23
Alternatively, however, it is possible to express the expression
cassette containing a DNA subsequence of SEQ-ID No. 1 or
SEQ-ID No. 3 for example in other bacteria, in yeasts, fungi,
algae, plant cells, insect cells or mammalian cells.
5
The formylglycinamidine ribotide synthase protein which is
expressed with the aid of an expression cassette is particularly
suitable for finding inhibitors which are specific to
formylglycinamidine ribotide synthase.
To this end, for example, the plant formylglycinamidine ribotide
synthase can be employed in an enzyme assay in which the
formylglycinamidine ribotide synthase activity is determined in
the presence and absence of the active ingredient to be tested. A
comparison of the two activity determinations allows a
qualitative and quantitative statement to be made on the
inhibitory behavior of the active ingredient to be tested, see
Example 8.
The assay system according to the invention allows a multiplicity
of chemicals to be tested rapidly and simply for herbicidal
properties. Using this method, substances with a potent action
can be selected specifically and reproducibly from amongst a
large number of substances, in order that further in-depth tests
with which the skilled worker is familiar be carried out
subsequently with these substances.
Another subject-matter of the invention is a method of
identifying herbicidally active substances which inhibit the
formylglycinamidine ribotide synthase activity in plants,
consisting of the following steps:
a) the generation of transgenic plants, plant tissues or plant
cells comprising an additional DNA sequence encoding an
enzyme with formylglycinamidine ribotide synthase activity
and capable of overexpressing enzymatically active
formylglycinamidine ribotide synthase;
b) applying a substance to transgenic plants, plant cells, plant
tissues or plant parts and to untransformed plants, plant
cells, plant tissues or plant parts;
c) determining the growth or the viability of the transgenic and
the untransformed plants, plant cells, plant tissues or plant
parts after application of the chemical substance; and
.,
0050/50827 CA 02388851 2002-04-23
6
d) comparing the growth or the viability of the transgenic and
the untransformed plants, plant cells, plant tissues or plant
parts after application of the chemical substance;
where suppression of the growth or viability of the untransformed
plants, plant cells, plant tissues or plant parts without
simultaneous severe suppression of the growth or the viability of
the transgenic plants, plant cells, plant tissues or plant parts
confirms that the substance of b) is herbicidally active and
inhibits the formylglycinamidine ribotide synthase enzyme
activity in plants.
Another subject-matter of the invention is a method of
identifying plant formylglycinamidine ribotide synthase
inhibitors with a potentially herbicidal action by cloning the
gene of a plant formylglycinamidine ribotide synthase,
overexpressing it in a suitable expression cassette - for example
in insect cells - disrupting the cells and employing the cell
extract in an assay system for measuring the enzyme activity in
the presence of low-molecular-weight chemicals, either directly
or after concentration or isolation of the enzyme
formylglycinamidine ribotide synthase.
Another subject-matter of the invention is compounds with a
herbicidal action which can be identified with the
above-described assay system.
Another subject-matter of the invention is a method of
eliminating undesired vegetation, which comprises treating the
plants to be eliminated with a compound which binds specifically
to formylglycinamidine ribotide synthase encoding a DNA sequence
SEQ-ID No. 1, SEQ-ID No. 3 or SEQ-ID No. 5 or a DNA sequence
hybridizing with this DNA sequence, and which inhibits its
function.
Herbicidally active formylglycinamidine ribotide synthase
inhibitors can be employed as defoliants, desiccants, haulm
killers and, in particular, as herbicides. Weeds in the widest
sense are to be understood as meaning all plants which grow in
locations where they are undesired. Whether the active
ingredients found with the aid of the assay system according to
the invention act as total or selective herbicides depends, inter
alia, on the quantity applied.
Herbicidally active formylglycinamidine ribotide synthase
inhibitors can be used, for example, against the following weeds:
~~SU/50827 CA 02388851 2002-04-23
7
Dicotyledonous weeds of the genera:
Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis,
Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca,
Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia,
Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia,
Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis,
Papaver, Centaurea, Trifolium, Ranunculus, Taraxacum.
Monocotyledonous weeds of the genera:
Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca,
Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum,
Agropyron, Cynodon, Monochoria, Fimbristylis, Sagittaria,
Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea,
Dactyloctenium, Agrostis, Alopecurus, Apera.
A subject-matter of the invention is also expression cassettes
whose sequence encodes a formylglycinamidine ribotide synthase
from Arabidopsis thaliana, Nicotiana tabacum or Chilopsis
linearis or their functional equivalent. The nucleic acid
sequence can be, for example, a DNA or a cDNA sequence.
In addition, the expression cassettes according to the invention
comprise regulatory nucleic acid sequences which govern the
expression of the encoding sequence in the host cell. In
accordance with, a preferred embodiment, an expression cassette
according to the invention encompasses upstream, i.e. at the 5'
end of the encoding sequence, a promoter, and downstream, i.e. at
the 3' end, a polyadenylation signal and, if appropriate, other
regulatory elements which are operatively linked to the encoding
sequence for the formylglycinamidine ribotide synthase gene,
which sequence lies between the promoter and the polyadenylation
signal. Operative linkage is to be understood as meaning the
sequential arrangement of promoter, encoding sequence, terminator
and, if appropriate, other regulatory elements in such a manner
that each of the regulatory elements can function as intended
when the encoding sequence is expressed.
An expression cassette according to the invention is generated by
fusing a suitable promoter with a suitable formylglycinamidine
ribotide synthase DNA sequence and a polyadenylation signal using
customary recombination and cloning techniques as they are
described, for example, by T. Maniatis, E.F. Fritsch and
J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY (1989) and by
T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene
Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
(1984) and by Ausubel, F.M. et al., Current Protocols in
~05~~~~$27 CA 02388851 2002-04-23
Molecular Biology, Gree"ne Publishing Assoc. and
Wiley-Interscience (1987).
A subject-matter of the invention is also functionally equivalent
DNA sequences which encode a formylglycinamidine ribotide
synthase gene and which show a sequence homology with the DNA
sequence SEQ-ID No. 1, SEQ-ID No. 3 or SEQ-ID No. 5 of 40 to
100, based on the total length of the DNA sequence.
A preferred subject-matter of the invention is functionally
equivalent DNA sequences which encode a formylglycinamidine
ribotide synthase gene and which show a sequence homology with
the DNA sequence SEQ-ID No. 1, SEQ-ID No. 3 or SEQ-ID No. 5 of 60
to 100, based on the total length of the DNA sequence.
A particularly preferred subject-matter of the invention is
functionally equivalent DNA sequences which encode a
formylglycinamidine ribotide synthase gene and which show a
sequence homology with the DNA sequence SEQ-ID No. 1, SEQ-ID No.
3 or SEQ-ID No. 5 of 80 to 100, based on the total length of the
DNA sequence.
Functionally equivalent sequences which encode a
formylglycinamidine ribotide synthase gene are in accordance with
the invention those sequences which retain the desired functions,
despite a deviating nucleotide sequence. Functional equivalents
thus encompass naturally occurring variants of the sequences
described herein, but also artificial nucleotide sequences, for
example those which have been obtained by chemical synthesis and
which are adapted to suit the codon usage of a plant.
A functional equivalent is also to be understood as meaning in
particular natural or artificial mutations of an originally
isolated sequence which encodes a formylglycinamidine ribotide
synthase and which continues to show the desired function.
Mutations encompass substitutions, additions, deletions,
exchanges or insertions of one or more nucleotide residues. Thus,
the present invention for example also extends to those
nucleotide sequences which are obtained by modifying this
nucleotide sequence. The target of such a modification can be,
for example, the further delimitation of the encoding sequence
contained therein or else, for example, the introduction of
further restriction enzyme cleavage sites.
0050/50827 CA 02388851 2002-04-23
. 9
Functional equivalents are also those variants whose function is
reduced or increased compared with the starting gene, or gene
fragment.
In addition, the expression cassette according to the invention
can also be employed for the transformation of bacteria,
cyanobacteria, yeasts, filamentous fungi and algae and eukaryotic
cells (for example insect cells), with the purpose of producing
sufficient amounts of the enzyme formylglycinamidine ribotide
synthase.
Another subject-matter of the invention is a protein from
Arabidopsis thaliana, Nicotiana tabacum or Chilopsis linearis
with the amino acid sequence SEQ-ID No. 2, SEQ-ID No. 4 or
SEQ-ID No. 6, or derivatives or parts of this protein with
formylglycinamidine ribotide synthase activity.
A subject-matter of the invention is also plant proteins with
formylglycinamidine ribotide synthase activity with an amino acid
sequence homology to the Arabidopsis thaliana, Nicotiana tabacum
or Chilopsis linearis formylglycinamidine ribotide synthase with
the SEQ-ID No. 2, SEQ-ID No. 4 or SEQ-ID No. 6 of 20 - 100
identity.
Preferred are plant proteins with formylglycinamidine ribotide
synthase activity with an amino acid sequence homology to the
Arabidopsis thaliana, Nicotiana tabacum or Chilopsis linearis
formylglycinamidine ribotide synthase with the sequence SEQ-ID
No. 2, SEQ-ID No. 4 or sequence SEQ-ID No. 6 of 50 - 100
identity.
Particularly preferred are plant proteins with
formylglycinamidine ribotide synthase activity with an amino acid
sequence homology to the Arabidopsis thaliana, Nicotiana tabacum
or Chilopsis linearis formylglycinamidine ribotide synthase with
the sequence SEQ-ID No. 2, SEQ-ID No. 4 or sequence SEQ-ID No. 6
of 80 - 100 identity.
It was another object of the invention to overexpress the
formylglycinamidine ribotide synthase gene in plants in order to
generate plants which tolerate formylglycinamidine ribotide
synthase inhibitors.
Overexpression, in a plant, of the gene sequence SEQ-ID No. 1 or
SEQ-ID No. 3, which encodes a formylglycinamidine ribotide
synthase, results in an increased resistance to
, 0050/50827
CA 02388851 2002-04-23
formylglycinamidine ribotide synthase inhibitors. The transgenic
plants generated thus are also subject-matter of the invention.
Expressional efficacy of the recombinantly expressed
formylglycinamidine ribvtide synthase gene can be determined, for
example, in vitro by shoot-meristem propagation or by a
germination test. Moreover, the expression of the
formylglycinamidine ribotide synthase gene which has been altered
in terms of type and level, and its effects an the resistance to
formylglycinamidine ribotide synthase inhibitors can be tested in
greenhouse experiments using test plants.
Subject-matter of the invention are also transgenic plants,
transformed with an expression cassette containing the DNA
sequence SEQ-ID No. 1 or SEQ-ID No. 3, which have been made
tolerant to formylglycinamidine ribotide synthase inhibitors by
additionally expressing the DNA sequence SEQ-ID No. 1 or SEQ-ID
No. 3, and transgenic cells, tissues, parts and propagation
material of such plants. Especially preferred in this context are
transgenic crop plants such as, for example, barley, wheat, rye,
maize, soya, rice, cotton, sugar beet, canola, sunflowers, flax,
hemp, potatoes, tobacco, tomatoes, oilseed rape, alfalfa, lettuce
and the various tree, nut and grapevine species, and also
legumes.
Particularly preferred are sequences which ensure targeting into
the apoplast, into plastids, into the vacuole, into the
mitochondrium, into the endoplasmatic reticulum (ER), or which,
owing to the absence of suitable operative sequences, ensure that
the product remains in the compartment where it is formed, in the
cytosol, (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423).
For example, the plant expression cassette can be introduced into
the plant transformation vector pBinAR, see Example 5.
A suitable promoter of the expression cassette according to the
invention is, in principle, any promoter which is capable of
governing the expression of foreign genes in plants. It is
preferred to use, in particular, a plant gromoter or a promoter
derived from a plant virus. Particularly preferred is the
cauliflower mosaic virus CaMV 35S promoter (Franck et al., Cell
21(1980), 285-294). This promoter contains different recognition
sequences for transcriptional effectors which, in their totality,
lead to permanent and constitutive expression of the gene which
has been introduced (Benfey et al., EMBO J., 8 (1989),
2195-2202).
0050/50827
CA 02388851 2002-04-23
11
The expression cassette according to the invention may also
comprise a chemically inducible promoter which allows expression
- of the exogenous formylglycinamidine ribotide synthase gene in
the plant to be governed at a particular point in time. Such
promoters which are described in the literature and which can be
used are, inter alia, for example the PRP1 promoter (Ward et al.,
Plant.Mol. Biol. (1993) 22, 361-366), a salicylic acid-inducible
promoter (WO 95/19443), a benzenesulfonamide-inducible promoter
(EP 388186), a tetracyclin-inducible promoter (Gatz et al., Plant
J. (1992) 2, 397-404), an abscisic acid-inducible promoter
(EP0335528) or an ethanol- or cyclohexanone-inducible promoter
(WO 93/21334).
Particularly preferred promoters are furthermore those which
ensure expression in tissues or parts of the plant in which the
biosynthesis of purines or their precursors takes place.
Promoters which ensure leaf-specific expression must be mentioned
in particular. Promoters which must be mentioned are the potato
cytosolic FBPase or the potato ST-LSI promoter (Stockhaus et al.,
EMBO J., 8 (1989) 2445-245).
A foreign protein can be expressed stably in the seeds of
transgenic tobacco plants to an extent of 0.67% of the total
soluble seed protein with the aid of a seed-specific promoter
(Fiedler and Conrad, Bio/Technology 10 (1995), 1090-1094). The
expression cassette according to the invention can therefore
contain, for example, a seed-specific promoter (preferably the
phaseolin promoter, the USP promoter or the LEB4 promoter), the
LEB4 signal peptide, the gene to be expressed and an ER retention
signal.
The inserted nucleotide sequence encoding a formylglycinamidine
ribotide synthase can be produced synthetically or obtained
naturally or contain a mixture of synthetic and natural DNA
components. In general, synthetic nucleotide sequences are
generated with codons which are preferred by plants. These codons
which are preferred by plants can be determined from codons with
the highest protein frequency expressed in the plant species of
the highest interest. when preparing an expression cassette, a
variety of DNA fragments may be manipulated in order to obtain a
nucleotide sequence which expediently reads in the correct
direction and which is equipped with a correct reading frame.
Adaptors or linkers can be added to the fragments in order to
link the DNA fragments to each other.
0050/50827 CA 02388851 2002-04-23
12
Other suitable DNA sequences are artificial DNA sequences as long
as they mediate the desired property by increasing the purine
nucleotide content in the plant by overexpressing the
formylglycinamidine ribotide synthase gene. Such artificial DNA
sequences can be determined for example by backtranslating of
proteins which have formylglycinamidine ribotide synthase
activity and which have been constructed by means of molecular
modeling, or they can be determined by in vitro selection.
Especially suitable are encoding DNA sequences which have been
obtained by backtranslating a polypeptide sequence in accordance
with the host-plant-specific codon usage. The specific codon
usage can be determined readily by the skilled worker familiar
with methods of plant genetics by means of computer evaluations
of other, known genes of the plant to be transformed.
Other suitable equivalent nucleic acid sequences according to the
invention which must be mentioned are sequences which encode
fusion proteins, component of the fusion protein being a plant
formylglycinamidine ribotide synthase polypeptide or a
functionally equivalent part thereof. The second part of the
fusion protein can be, for example, another polypeptide with
enzymatic activity or an antigenic polypeptide sequence, with the
aid of which detection of formylglycinamidine ribotide synthase
expression is possible (for example myc-tag or his-tag). However,
it is preferably a regulatory protein sequence such as, for
example, a signal or transit peptide, which leads the
formylglycinamidine ribotide synthase protein to the desired site
of action.
The promoter and terminator regions according to the invention
should expediently be provided, in the direction of
transcription, with a linker or polylinker containing one or more
restriction sites for insertion of this sequence. As a rule, the
linker has 1 to 10, in most cases 1 to 8, preferably 2 to 6,
restriction sites. In general, the linker within the regulatory
regions has a size less than 100 bp, frequently less than 60 bp,
but at least 5 bp. The promoter according to the invention may be
native, or homologous, or else foreign, or heterologous, to the
host plant. The expression cassette according to the invention
comprises, in the 5'-3' direction of transcription, the promoter
according to the invention, any sequence and a region of
transcriptional termination. Various termination regions can be
exchanged for each other as desired.
Manipulations which provide suitable restriction cleavage sites
or which eliminate the excess DNA or restriction cleavage sites
may also be employed. In vitro mutagenesis, primer repair,
', 0050/50827 CA 02388851 2002-04-23
13
restriction or ligation may be used in cases where insertions,
deletions or substitutions such as, for example, transitions and
transversions, are suitable. Complementary ends of the fragments
may be provided for ligation in the case of suitable
manipulations such as, for example, restriction, chewing-back or
filling overhangs far blunt ends.
Preferred polyadenylation signals are plant polyadenylation
signals, preferably those which correspond essentially to
Agrobacterium tumefaciens T-DNA polyadenylation signals, in
particular those of the gene 3 of the T-DNA (octopine synthase)
of the Ti plasmid pTiACHS (Gielen et al., EMBO J., 3 (1984),
835), or functional equivalents.
To transform a host plant with a DNA encoding formylglycinamidine
ribotide synthase, an expression cassette according to the
invention is incorporated, as insertion, into a recombinant
vector whose vector DNA contains additional functional regulatory
signals, for example sequences for replication or integration.
Suitable vectors are described, inter alia, in "Methods in Plant
Molecular Biology and Biotechnology" (CRC Press, Chapters 6/7,
71-119).
The transfer of foreign genes into the genome of a plant is
termed transformation. It exploits the above-described methods
for transforming and regenerating plants from plant tissues or
plant cells for transient or stable transformation. Suitable
methods are the protoplast transformation by
polyethylene-glycol-induced DNA uptake, the biolistic method
using the gene gun, electroporation, incubation of dry embryos in
DNA-containing solution, microinjection and
agrobacterium-mediated gene transfer. The abovementioned methods
are described by, for example, B. Jenes et al., Techniques for
Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and
Utilization, edited by S.D. Kung and R. Wu, Academic Press
(1993), 128-143, and Potrykus Annu. Rev. Plant Physiol. Plant
Molec. Biol. 42 (1991), 205-225. The construct to be expressed is
preferably cloned into a vector which is suitable for the
transformation of Agrobacterium tumefaciens, for example pBinl9
(Bevan et al., Nucl. Acids Res. 12 (1984), 8711).
Agrobacteria transformed with an expression cassette according to
the invention can equally be used in a known manner for
transforming crop plants, in particular crop plants such as
cereals, maize, soya, rice, cotton, sugar beet, canola,
sunflowers, flax, hemp, potatoes, tobacco, tomatoes, oilseed
rape, alfalfa, lettuce and the various tree, nut and grapevine
~ i
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14
species, or else legumes, for example by bathing wounded leaves
or leaf sections in an agrobacterial suspension and subsequently
growing them in suitable media.
The purine biosynthesis site is generally the leaf tissue, so
that leaf-specific expression of the formylglycinamidine ribotide
synthase gene is meaningful. However, it is obvious that the
purine biosynthesis need not be limited to the leaf tissue, but
may also take place in all other remaining parts of the plant in
a tissue-specific fashion, for example in fatty seeds.
In addition, constitutive expression of the exogenous
formylglycinamidine ribotide synthase gene is advantageous. On
the other hand, inducible expression may also be desirable.
Using the recombination and cloning techniques cited above, the
expression cassettes according to the invention can be cloned
into suitable vectors which allow them to be multiplied, for
example in E. coli. Suitable cloning vectors are, inter alia,
pBR332, pUC series, Ml3mp series and pACYC184. Especially
suitable are binary vectors which are capable of replication both
in E. coli and in agrobacteria.
Another subject-matter of the invention relates to the use of an
expression cassette according to the invention for transforming
plants, plant cells, plant tissues or parts of plants. The
preferred purpose of the use is to increase the
formylglycinamidine ribotide synthase content in the plant.
Depending on the choice of the promoter, expression may take
place specifically in the leaves, in the seeds or in other parts
of the plant. Such transgenic plants and their propagation
material and their plant cells, tissue or parts are another
subject of the present invention.
The invention will now be illustrated by the examples which
follow, without being limited thereto.
Examples
Recombinant methods on which the use examples are based:
General cloning methods
Cloning methods such as restriction cleavages, agarose gel
electrophoresis, purification of DNA fragments, transfer of
nucleic acids to nitrocellulose and nylon membranes, linking DNA
050/50827 CA 02388851 2002-04-23
fragments, transformation of Escherichia coli cells, growing
bacteria and the sequence analysis of recombinant DNA were
carried out as described by Sambrook et al. (1989) (Cold Spring
Harbor Laboratory Press: ISBN 0-87969-309-6).
5
Screening of cDNA libraries
h-Phages of the relevant cDNA libraries were plated onto agar
plates with E. coli XL1-Blue as bacterial strain. The phage DNA
10 was transferred to nitrocellulose filters (Gelman Sciences) by
means of standard methods (Sambrook et al. (1989), Cold Spring
Harbor Laboratory Press: ISBN 0-87969-309-6) and fixed to the
filters. The hybridization probes used were PCR fragments, or DNA
fragments obtained by restriction cleavage, which were
15 radiolabeled with the aid of a "Multiprime DNA labeling system"
(Amersham Buchler) in the presence of 32P-dCTP (specific activity
3000 Ci/mmol), following the manufacturer's instructions. The
membranes were hybridized after prehybridization at 60°C in 3 x
SSPE, 0.1~ sodium dodecyl sulfate (w/v), 0.02
polyvinylpyrrolidone (w/v), 0.02 Ficoll 400 (w/v) and 50 mg/ml
calf thymus DNA for 12-16 hours. The filters were then washed for
60 minutes in 2 x SSPE, 0.1~ sodium dodecyl sulfate (w/v) at 60°C.
Positively hybridizing phages were visualized by autoradiography,
singled out by means of standard techniques (Sambrook et al.
(1989); Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6)
and transferred into plasmids (Stratagene).
Sequence analysis of recombinant DNA
Recombinant DNA molecules were sequenced using an ABI laser
fluorescence DNA sequences, following the method of Sanger
(Sanger et al., Proc. Natl. Acad. Sci. USA, 74(1977), 5463-5467).
Fragments resulting from a polymerase chain reaction were
sequenced and checked to avoid polymerase errors in constructs to
be expressed.
Analysis of total RNA from plant tissues
Total RNA from plant tissues was isolated as described by
Logemann et al.(Anal. Biochem. 163(1987), 21). For the analysis,
in each case 20 ~,g of RNA were separated in a
formaldehyde-containing 1.5$ agarose gel and transferred to nylon
membranes (Hybond, Amersham). Specific transcripts were detected
as described by Amasino (Anal. Biochem. 152(1986), 304). The DNA
fragments employed as probe were radiolabeled with a Random
Primed DNA Labeling Kit (Roche, Mannheim) and hybridized by
standard methods, see Hybond instructions, Amersham.
0050/50827
CA 02388851 2002-04-23
16
Hybridization signals were visualized by autoradiography with the
aid of Kodak X-GMAT AR films.
Unless otherwise specified, the chemicals used were analytical
grade and obtained from Fluka (Neu-Ulm), Merck (Darmstadt), Roth
(Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen).
Solutions were made with a refined, pyrogen-free water, termed H20
hereinbelow, from a Milli-Q water refining system (Millipore,
Eschborn). Restriction endonucleases, DNA-modifying enzymes and
molecular biologic kits were obtained from AGS (Heidelberg),
Amersham (Braunschweig), Biometra (Gottingen), Roche (Mannheim),
Genomed (Bad Oeynnhausen), New England Biolabs
(Schwalbach/Taunus), Novagen (Madison, Wisconsin, USA),
Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg), Qiagen (Hilden)
and Stratagene (Heidelberg). Unless otherwise specified, they
were used in accordance with the manufacturer's instructions.
The bacterial strains used hereinbelow (E. coli, XL-1 Blue) were
obtained from Stratagene. E. coli AT 2465 was obtained from the
coli genetic stock center (Yale University, New Haven). The
agrobactexial strain used for transforming plants (Agrobacterium
tumefaciens, C58C1 with plasmid pGV2260 or pGV3850kan) was
described by Deblaere et al. (Nucl. Acids Res. 13 (1985), 4777).
Alternatively, it is also possible to use the agrobacterial
strain LBA4404 (Glontech) or other suitable strains. Vectors
which can be used for cloning are pUCl9 (Vanish-Perron, Gene
33(1985), 103-119), pBluescript SK- (Stratagene), pGEM-T
(Promega), pZerO (Invitrogen), pBinl9 (Bevan et al., Nucl. Acids
Res. 12(1984), 8711-8720) and pBinAR (Hofgen and Willmitzer,
Plant Science 66 (1990), 221-230).
Example 1
Isolation of a full-length formylglycinamidine ribotide synthase
cDNA from Arabidopsis thaliana
To isolate formylglycinamidine-ribotide-synthase-encoding cDNAs,
an Arabidopsis thaliana expressed sequence tag (est) which
partially encodes formylglycinamidine ribotide synthase (GenBank
Accession number AA042492) was obtained, sequenced and used as
template for generating a hybridization probe by means of
polymerase chain reaction (PCR) in order to isolate
formylglycinamidine-ribotide-synthase-encoding cDNAs.
The reaction mixtures contained approx. 1 ng/~,1 template DNA,
0.5 ~,M of the oligonucleotides 5'-GCTGCTTCAATAAGGTTCAAC-3' and
5'-CGCTATTCCCAACGGCACACC-3', 200 ~M deoxy-nucleotides
0050/50827 CA 02388851 2002-04-23
10
17
(Pharmacia), 50 mM KC1, 10 mM Tris-HC1 (pH 8.3 at 25°C, 1.5 mM
MgCl2) and 0.02 U/~1 Taq polymerase (Perkin Elmer).
The amplification conditions were set as follows:
Annealing temperature: 50°C, 1 min
Denaturation temperature: 94°C, 1 min
Elongation temperature: 72°C, 2 min
Number of cycles: 30
The resulting 465 by fragment was used for a heterologous
screening of 6.5*105 pfu of a UniZAP XR cDNA library (Stratagene)
from Arabidopsis thaliana (whole plant) mRNA. After restriction
and sequence analysis, it was possible to identify two
distinguishable clones (purl-19 and purl-23) which encoded
reading frames with homology to formylglycinamidine ribotide
synthase from Escherichia coli, Bacillus subtilis, Saccharomyces
cerevisiae and Drosophila melanogaster. The sequence comparisons
show that both purl-19 (length: 1267 bp) and purl-23 (length:
917 bp) are partial clones encoding 3' regions of
formylglycinamidine ribotide synthase. To isolate a full-length
cDNA, it is necessary to employ a size-fractionated cDNA library
for the screening since only further partial clones were isolated
from other cDNA libraries. Therefore, a 964 by fragment obtained
by cleavage of purl-19 with EcoRI and KpnI was used for screening
5*105 pfu of an Arabidopsis thaliana cDNA library in the vector
lambda-ZAP-Express (Stratagene). The library was prepared with
RNA from entire plants. A cDNA fraction > 2 kbp long was prepared
in order to generate the cDNA library.
A positively hybridizing clone with the longest insert
{purl-48.1) was identified by means of restriction analysis and
subsequently sequenced, see SEQ-ID No. 1. The purl-48.1 cDNA has
a length of 4570 base pairs and is identical in the overlapping
region (nucleotide 3328- 4570) with the shorter purl-19 cDNA. A
continuous reading frame encodes, in purl-48.1, a polypeptide of
1407 amino acids and a calculated mass of 153 952 kDa, see SEQ-ID
No. 2. The amino acid sequence shows a similarity to
formylglycinamidine ribotide synthase from other organisms, see
Table 1. A relationship exists with human formylglycinamidine
ribotide synthase (GenBank Number AB002359) with 51.4 identity,
or with 59.7 similarity at the amino acid level. purl-48.1 shows
an N-terminal extension of 53 to 88 amino acids compared with
formylglycinamidine ribotide synthase from other organisms, which
suggests an important transit peptide. The computer -aided
evaluation of the sequence predicts a possible function for amino
acids 1-53 as signal peptide for an import into the plastids.
0050/50827
CA 02388851 2002-04-23
1$
Localization of the enzyme in plastids tallies with its function
in purine biosynthesis. The reading frame upstream of the start
methionine contains a stop codon. purl-48.1 is thus the first
full-length plant cDNA for a formylglycinamidine ribotide
synthase.
Table 1
Sequence formylglycinamidine synthase
comparison ribotide
of
10sequences organisms Saccharomyces
from various (E.coli,
cerevisiae,Drasophilamelanogaster, Sapiens,
Homo Arabidopsis
thaliana). amino
Only those acids
which
do not
correspond
to the
consensus shown.
sequence
are
1 50
15PurL_e.coli-__________-________________________-__-_-________
purL_yeast -_-________---____-_--____-_-__-____-_-__-_-______
purl Drome ----_-__-________-_____,_________-__--___--_______
purl human ----_____________________-___________-_-__--_-__--
purL-ara-48-mntsqatraalflngsnrqamllqrssmsqlwgsvrmrtsrlslnrtkav
Consensus --___-_-_-_-_____-_____-____-----_-__--__---_--__-
51 100
20
purl e.coli---------------------------_ me.-lr---a-saf-ink-1
-
purl-yeast ---------_--------_--------- tdy-lp-pka-sqf--dn-i
-
purl Drome -__-______-_____________________--_-__m-ilr-ydvqa-
purL human ---aasasstnlihl-skghispakdt--qqrtpa-m----h--vr-sg-
purL-ara-48-slrcsaqpnkpkaav-tgsfvtadelp--vekpaa-... ih-----liq
25Consensus ---------------S-----------SLM---I-EGSPVL-FYRVP-LH
101 150
purl e.colia...rfqaa-lpvhniya---hfad.-nap--dd-haq--r-1--gpa-.
purL~reast kdi--ytnstsvinelrsc-ihyvngiaq--seqdt-1--v-lt-dsa-d
purl Drome sa-ee-sv--rlreedg.av-s-rm-r--h-e.ys-qaeh--aldel-v-
purL human -g-a-ghtr-kl-gklp.-1qg-e------v-.wt-ea-p-aeetkk-m-
30
purl-ara-48--s--a-.l-kav-tkisnqi-slt--qsf-ig.l-s-lkd..e-lsv-k-
Consensus E-ANSE--LR--Q-----EYV-V-TELCYNLN--EAK-LESL-KY--LLW
151 200
purl e.coli.............. -ha-q....-k...-1-t--pgtis---sk-td-
purL_yeast iand--arql-d-v-nn--ssa-.-edty-ir-v--sgtis---sk-tn-
35purl Drome lvk---skgqs-s-qpa-q...st--sq----i---fn-s--y---c-n-
purL human lfgc--ll.ddv--e-w-.....-p--nd------n-s --t---i-sv
purl-ara-48-il.-etyepe--gtd-f-erkkqe-lhaviv------s-t-a------s-
Consensus ---QPL----NLARAS-LP---L-GS--LLLEVGPRL-F-TPWSTNAV-I
201 250
40purl e.coliahn--- n----gva-yiea..-tltn-qwqgvtae.-----m-tvf
.q-
purL_yeast ahv-----k-q-i--glal-iktvp-f-----lnd-slkcvy----qql-
purL_Drome fqnl-y.s--r-m-tst---v..t---gskapes-r-vpl-g----qcl-
purL human crat--.gp-d-v-tt---rl..s-ah-psaeve--al-t-------qhf
purl-ara-48-cra-- . t----s----l..fsk------qike-a-mv------cv-
--
Consensus ---CGLQDEV-RLER-RRYL----FGEPLLEN--AIF-A-LHDRMTE--Y
251 300
45
purl e.colifa.lddaeql-ahhq-t--ts-d-lgqg.....-q--id--lr-----ae
purL_yeast lt-ppntm-i--hee-k-lvh---t-kdtkqspkdi-s---t-------s
purl Drome -e-nt-ka--d.eql--rqa-whf--.. ----a---ri-------fnd
-
0050/50827 CA 02388851 2002-04-23
19
purl human ph...-iq-- spesm--.pl -g-.in..i- g---1----- ---------s
purl-ara-48- -qk...lv-- e-nvv--e-. ..ky--..-m -k--k---ei ---m---f-a
Consensus T-E--P--SF FT---PEPV- NVPLVP--VL EEGR-ALEKA NQELGLALD-
301 350
purl e.coli dei--lq-a- tk.-g---nd i--ym---a- ---c--ki-- a-w----eq-
purL-yeast genre-liha- vetmk-d--d ----m---v- ---c--ki-- a-wt---i--
purL Drome y-----h-1- ak--g----t -----c---- ---------r -rm----ve-
purL human w---f--kr- .q--q---st --a--1---- ---------k -qlhv--q-1
purl-ara-48- q--q---rl- redik-d--n -----i---- ---------a -nm----kpm
Consensus -DLDYYTD-F --EL-RNPT- VELFDFAQSN SEHSRHWFFN GD-VIDG-KQ
351 400
purl e.coli-----k--kn-f-ttpdhvlsayk--aavme-s--gryfadhe-g.r.y-
purL_yeast qft-----rn-hklnpeytisays--aavldsenda-ffapns-t.kewt
purl Drome ----ir--md--ahtn---tik-s-----mv--dhqtiv-ssvva-gavr
purl human vh---es-ms---ssn----lk-c-----iq-k--r--r-ed--r-srfq
purl-ara-48-d---m-ivks-w-anrn-s-ig-k-----ir--1-nq-r-11-gsvcll-
Consensus PKSLFQMI--TQE---PNNV--F-DNSSA--GFEV-FL-P--PT-P---D
401 450
purl e.colifh--pahi-mkv----h---is-w---a--s--e---eg-----a-pk--
purL_yeast stk-ripl-ikv----h----s-----a--s--e---eg-----s-tkc-
purL Drome 1-svqsdli-------m----a--s--t--t---1--vqgv---gvpi--
Purl human q--glrhvv-------f--g-c--s--t--t------vqc----ahvv--
purL-ara-48-vsardldi--------f-c--a-y---e--a------th-----sfvv-s
Consensus -QQE----LFTAETHN-PTAV-PFPGA-TG-GGRIRD--ATGRG-K--AG
451 500
purl e.colilv-fs-s--r---f-----.ed-gk-erivta-d-mt-gp1-gaafn-e-
purL_yeast ls-fs-sd-1---n---- -nigk-yhi--a-d-m---p1-saafn-e-
.
purl Drome -a-----a------k--y-p-d-k--atf-p--qvl--------------
purL human -a---f--------nl-----s-q--g-f-r--eva--------------
purL-ara-48--s-------nme-sya----ss-q--s-1----q-1-d------------
Consensus T-GYCVGNLHIPGYEQPWEDL-F-YP-N-ASPL-I-IEASNGASDYGNKF
501 550
Purl,_e.coli-r-al---f--yee--nshn-.e-1-gy-----la----ni--d-vq--.
purL_yeast -r-c----f--ltt--lnhq-ke-i-gf-----ia--f--v-pqfal-nt
purl Drome -----s-falsy--nsaadas--..d-yv--------1--mp-tmre-lp
purl human ----la-fa-sl--q..-pd-- ..--wi----------sme-d--s-ea
purl-ara-48----m-q--t--f-mr..-ps-d-..--wl------a---qidht--t--a
Consensus GEPVINGY-RT-GLKV-L--GQRE-RE-HKPIMFSGGIGT-RA-HI-KG-
551 600
purL,e.colie-nv-ak--v1---amn--1-------m.a---s------as---dn---
purL_yeast --tp-sc-iv1--qsml--1--------.a--egs-----as----n---
purL Drome -.ar-q--a-i---v----v--------eiq-sg--e---n------a--
purL_human -.-p--ev--v---v----v--------qvq-dnts----g---------
purL-ara-48-.-v---v--i---a----m-------m.v---n--e---n------a--
Consensus PIE-GMLLVK-GGP-YRIG-GGGAASSV--SGQ-DADLDF-AVQRGDPEM
601 650
purl e.coli-rrcqe--dr-wq--da----f---v----ls-amp---sd-gr-g-fel
purl-yeast -rrcqq--d---a--nn---q----v----ls-a-p---hdndl---f--
purL Drome -n-ln--v---ld---q----a---------g-------e--f---v-fs
purl -q-mn--------apkg---c-1--------g------sd-..---i-yt
human
- sq-ly--v---i-m--k---i----------c--v--iiy-..q--e---
purl-ara-48-
Consensus E-K--RVIRACVELGE-NPILSIHDQGAGGN-NVLKELV-PG-AGAKIDI
651 700
0050/50827 CA 02388851 2002-04-23
purl e.coli--ilsde-g--p----cn-s---yv-a-a---lplfd-1-----a-y--i
purL_yeast -kvlsle-g--pm---cn-s---yv-g-spqdlsif--------a-f---
purL Drome k-fql----yta--1-------nn-i-cn-----1--k--r---c-isf-
purL-human srfql----1na----------sn---lrspn-df-thusa---c-acf-
purL-ara-48--avvv--h---v----------qd-i--k-es--i-qs------lsm--i
5 Consensus RE---GDPTMS-LEIWGAEYQER-ALLV-ADARE-LEEICKRER-P-AVV
701 750
purl e.coli-ea-eelh-s-h-...............rhfdnq-i--p-dvl---t--m
purL_yeast -ha-aeqk-ive-...............-1lkt--i---mpilf--p--m
purl Drome -vv--d--v--l.ekp---dleqa-n-fnrsevs-f----ky---d---r
human --i--dr-iv-v-drec-yrrngqgd-p-t-----v-----w------rk
purL
10_ --in-g--c--i-sta-a-cskeg-....p---pav-----k---d---k
purl-ara-48-
Consensus GT-TG-GRLTL-D---APK-----L-A-P-PPPTP-DLELE-VLGKMPK-
751 800
purl e.coli-r-v-tlkak.gdalare-itia--vk---h--t-ae-t--v-ig-----
purL_yeast sretit--In-pean-seips-q--iq---n--s-g--s--i-ig-----
15purL_Drome -y--k--qtp-ke-s--k-11-de--e---s-va-g------n----c-g
purl-human eff---kppm-qp-a--p-1svhq--e------a-a---y--n------g
purl-ara-48--fkfn-i-yarep-diap-it-m---k------s-s-----------c--
Consensus T-DLQREA--L--L-LP-G--L-DAL-RVLRLP-V-SKRFLTTKVDRSVT
801 850
20purl e.coli-m--rd-m---w---v-nc---ta-ldsyy... --m----rapva---f
purL_yeast --idrd-f---w---v---g--gt-lgetiis----m-m--k--na-isa
purl Drome --i----------a----y-1-tv-hfsh...s-i-ts--t--1-g----
purL human -------------t-------val-hee-...i-a-t-1------s----
purL-ara-48--------t-----it------iaqtftd-...--g-c------i-g----
Consensus GLVAQQQCVGPLQVPLADVAVT--S---L---TGEA-AIGEQPVK-LLDP
851 900
purl e.colia-s-----g-----iaatqig-ikri-1-a---a--gh---d-g--e-v-
purL_yeast s-s-k-s---s-1-ifa-d-ks-nhi-1-a---sp-shq---sk--e-vq
purl Drome a-m--mc-----s---fv-ise-a---c---------------rmf--c-
purL human kva--___________f_1__-_r___c______-_____-____a__ce
purl-ara-48-k-m--___g_______~,,~____a_s___a_____y____e___s_m__-ai
30Consensus -A-ARLAVAEALTNLV-AKVTDL-DVK-SGNWMWAAKLPGEGAALYDA-K
901 950
purl e.coli-v-e--c----lt-pv----m--ktr-qegneeremts-1------f-rv
purL_yeast ---ld-c----v--pv----m--kmk-....ddke-t--1--n-t-f--v
purl Drome e-.cqilee-hi-------------k....-g--tiks--t----t----
purL human -m.vavma---v-v-----------r....-gt-t-r-----------v-
35purl-ara-48-- .s-amie--i-------------h....ad--v-k---n------vt-
Consensus ALG-EL-PALG-AIDGGKDSLSMAA-W---V-GE-V-APGSLVISAYAPC
951 1000
purl e.colie-vrh-i--q-..s-...e-na---i--g..--nna--at------r---
purL-yeast fnts--w--1-nrn-... sv-v-----akqetks--a---1--yn-v-
-
40Purl Drome --vrlk------g-.-a-skts--win-e...nsa---------aya-q-
purL human --ita-------h-e-r-...h--y-a--..p-qh----t----c-s---
purL-ara-48---it------- ..1-. gi--h---a.. -kr-----------g-i-
--
Consensus PD--KTVTPDLK-PTG-GDD--LLLVDLS--KG--RLGGSALAQVF-QLG
1001 1050
purl e.colidk-a-vr---q---fy-ai---v-q-k..l--y--r---------a----
45purl-yeast -ks-tvy-n-i---flesliq-hqqkediv--y--r------i-------
purL Drome kdt-n-trsdv-gka-av--s--gdg..-iq----v-------cv----i
purl human eh-----lpen-vra-si--g--kd-..-1cs---v-----vtc------
purL-ara-48--dc------py--nv--gv---i-en..-vs----i-----v--a-----
0050/50827 CA 02388851 2002-04-23
21
Consensus N-PPDLDDVA -LKG-FD-TQ ALLA-R--L- LAGHD-SDGG LLVTLLEMAF
1051 1100
purl e.coli --h--ida-i............atl--..dr--a--n----a-iq-r-a-
purL_yeast -sr---eini............dg.--lesq-tn--n----a-fqi--kn
purl Drome g-ls--r--1se -lak-knfdksveklnrpe-----a--c-w-v--ld--
purL human ------q--vpv-rvdv..............-s---a--p-1----qep-
purL-ara-48----k-inl-1asngis-..............fet--s----1---i-k-n
Consensus AGNCGL-VD---P---L--------GD----LAVLF-EELG-VLEVSATD
1101 1150
purl e.coli r----sv-aqh-1-.dcvhy--q-vsgd-f-ita--q-...-f--s-tt-
10purL_yeast -skf-ki-nen---k--isi--kpsfq-qeikii-st-ndviyans----
purL Drome --r-rstyek---pny-1-vtegf-ld----1-ngkse...l-dqplrv-
purL human -aq-lkry-d--lhclel-ht-e--pham-r-s---av... -e-pvg--
purL-ara-48--d--mek--afd-ta-ii-n-td...splie---d-i-...h---kt-f-
Consensus LEAVE--LR-AGVA-EY-G-VG-AG--SRVVVKVNG-T---VLSE-RSEL
1151 1200
purl e.coli -vw-a--tw-mqr-rd-----dq-hqaksnda----nv--s-dine-vaa
purL_yeast eqt-sk--yemq--rd--kt----fasitddr----q-a-ty--ad-mki
purl Drome ykk--r--ye-e---a------a-yns-ey-qa-..q-rgpq-v.q.-el
purl human -al-----f--dr--ae-r-va--erg-re-mg-..s-c-pptfpk.-sv
purl-ara-48--dm--d--f--e---rlas-v-m-keg-kf-he-..nw--s-i-ss.tnn
20Consensus R--WEETS-QL-KLQ-NPECAEEE---L--R-DPGL-YKL-FNP--DA--
1201 1250
purL_e.coli -yiatg----------q---shv-----------dai--h-----t--t-
purL_yeast gl-1-sq-----i---q----qm---wc-qq---nsv----t---e--fh
purl Drome tlkr-s-pvr----------se---m-cll--n--_h--------q-tas
purl human -r-pggps-r--i-----s-------d---1----------q--cs-a--
25purl-ara-48-nymsqdvk-----i----s------s---ya----p----v----a-d-t
Consensus P-E-S-ARPKVAVLREEGVNGDREMP.AAFHRAGFEVWDVTMSDLL-GRIG
1251 1300
purl e.coli -ed-ha-vac-----g----age---k-----d--rde-at--h.--q-1
purl yeast --d-i--aac-----g----aga---k-v-yheg-r---sk--ne-q---
30Purl Drome vsqy---i-p-------t---------n--h-p-llp--aa.-kr-q-v-
purL human --t---va-v-----------------avt-hp-agaelrr.-r------
purL-ara-48--_q___iv_v____-____d_________r__ep_1___qe,_y______
Consensus LD-FRGL-F-GGFSYADVLGSAKGWAASILFN-RV-SQF--FF-KRPDTF
1301 1350
purl e.coli a--v-----m-sn-...........r-li-g--.1-----..r-t-d---
35purL_yeast af-a-----flsr-...........kdii-gc-.n--s-a..r-v-eqy-
purL Drome ---i-------t-i-f--....saks---ad.....-dva-1--k-q---
purL human ---v------la-------dpnedaa-m--d-qpar-gll-r--1---y-
purL-ara-48--__i_______a_____p_p,.....q--gsldtsq---_" _e-____
Consensus SLG-CNGCQLM-LLGWVGG-------EVGP-SE--WPRFVL-HN-SGRFE
1351 1400
40
purl e.coli a-fsl-evt-...... 1-q--v--qm-ia-s-----vev.--aah-
--1
purL_yeast a-vcm-q-s-ekdns-ee-vf-n--a--k--ia------kat-sksa-q-
purL Drome c-w-t-k-......p-nr-i--gs-kdl--gc--------f--. -ek-i
purl human s-w---rv......gpg-al--r----a---v-s-----yv--.ssp--q
purl-ara-48-c-ft--t-......kd---i--k-----t-gv-a------ay-.p-egv-
Consensus -R-ASV-3-Q-----SSPS-ML-GMEGSVLP-WVAHGEGR-AF-RD-ELL
45 1401 1450
purl e.coli -a---k--va -----nf-k- --t--a---- -----tavtt es--vti---
purLTyeast ekf-kd--ec i----ny--- --rf-f---- -t------k- -n--v-----
X050/5082? CA 02388851 2002-04-23
22
purl Drome s--q-eq-vt -q----v-kp --1--1---- --q----1-- s------1--
purL human -qi-ar--a- -hwa-----p --q--1---- --g-v----- c------v--
purL-ara-48- d-mlhsd-a- ---c------ --a--f-1-- --1---a--- ---------
Consensus AHLES-GL-P LRYVDDDGNV TE-YP-NPNG SPNGIAGICS PDGRHLAMMP
1451 1495
purl e.coli ----v--tvs ns-h-..... -nw-edg--m -i-----kql g----
purL-yeast ----vc-lea ns---eg.ky -ew--yg--i -1--s--r-v g----
purL Drome ----css-y- wpyv-s--e- sptqse---q im-n--y--c vk-nq
purl human ----av-p-- wa-r-pp--. ..tltt---- qlsi-----t 1-gsc
purl-ara-48- ----c-1--- fp---t-w-- -ka.-p---- km-q---d-. 1--c
Consensus HPER-FRMWQ --WYP-SFDV E--GG-SPWL R-FRNARNW- -ES-
Example 2
Isolation of a cDNA encoding a tobacco formylglycinamidine
ribotide synthase
To isolate further plant formylglycinamidine ribotide synthase
cDNAs, an EcoRI/XhoI fragment of clone purl-48.1 was used for
screening a tobacco leaf tissue cDNA library (Nicotiana tabacum
var. Samsun NN). 19 positive clones were identified and isolated.
Following restriction analysis and sequencing, clone purl-Ntl.l
was identified. purl-Ntl.l encodes, on a partial reading frame of
3434 by - see SEQ-ID No. 3 -, a polypeptide 1017 amino acids in
length, see SEQ-ID No. 4. In the overlapping region, the
polypeptide showed similarity to Arabidopsis thaliana
formylglycinamidine ribotide synthase (purl-48.1) (80.1%
identity).
Example 3
Isolation of a Chilopsis linearis cDNA encoding a
formylglycinamidine ribotide synthase
Double-stranded cDNA was generated from Chilopsis linearis mRNA
and used to generate a cDNA library in the vector pBluescript
SKII (lambda ZAP II RI library construction kit, Stratagene).
Individual clones of this library were partially sequenced. The
sequence on the 3'-side of clone 74 chi 005 e10 showed similarity
to tobacco purl-Ntl.l and Arabidopsis thaliana purl-48.1. Clone
74 chi_005 e10 was sequenced completely. On a partial cDNA 478 by
in length, it encodes a polypeptide of 97 amino acids. At the
amino acid level, 74 chi_005 e10 has 82.4% identity with
purl-48.1 and 75.2% identity with purl-Ntl.l.
0050/50827 CA 02388851 2002-04-23
23
Example 4
Proof of the function of purl-48.I by complementation of E. coli
To prove the function of the cDNA encoded by purl-48.1, it was
cloned into suitable expression vectors (for example the pQE,
Qiagen or pET series, Novagen). To this end, fragments of various
lengths of the purl-48.1 cDNA can be generated, for example by
PCR, and ligated into the vectors which have been prepared by
treatment with restriction endonucleases. The resulting
expression constructs can be used to complement the E. coli
strain CGSC#4537 (genotype: fhuA2 , lacYl, g1nV44(AS), gal-6, ~,-,
nadB4, purL66, rpsL9, malT1(~.R), xylA7, mtlA2, DargHl; E.coli
genetic stock center, Yale University, New Haven). To this end,
the strain is transformed with the expression construct in
question and plated onto M9 minimal media without adenine. By way
of example, the minimal media should contain 0.4~ glucose, 0.2%
casamino acids, 100 ~,g/ml thiamine, 100 ~g/ml inosine, 100 ~,g/ml
biotin, 100 ~,g/ml nicotinate, 100 ~M IPTG and 50-100 ~,g/ml of the
antibiotic in question to which the expression vector mediates
resistance. In the parallel experiment, the cloning vector can be
transformed into CGSC#4537 without purl-48.1 insert as negative
control. It emerged that only the bacteria which have been
transformed with purl-48.1 expression constructs are capable of
growth on minimal media without adenine. Accordingly, the enzyme
encoded by purl-48.1 is functional and the first functional
formylglycinamidine ribotide synthase which has been isolated
from plants.
Furthermore, yeast (Saccharomyces cerevisiae) may also be used
for complementation. To this end, a yeast mutant is generated
whose Ade6 gene, which encodes the yeast formylglycinamidine
ribotide synthase, is rendered unfunctional. A method which can
be relied on for generating such a yeast mutant which is suitable
for complementation is, for example, that of Guldener et al.,
Nucleic Acids Research 24(I996), 2519-2524, applied to a suitable
starting strain such as, for example, SEY6210 (Robinson et al.,
Protoplasma 150(1989), 79-82). The yeast mutant generated thus
should not show growth on minimal media without adenine. To prove
the function of the purl-48.1-encoded cDNA, the latter is cloned
into suitable yeast expression vectors (for example pYEB112,
Riesmeier et al., EMBO J. 11(1992), 4705-4713 or pYES2,
Invitrogen). To this end, purl-48.1 cDNAs of various lengths can
be generated for example by PCR and ligated into vectors which
have been prepared by treatment with restriction endonucleases.
The resulting expression constructs are used to complement the
yeast mutant generated. To this end, the latter is transformed
0050/50827 CA 02388851 2002-04-23
24
with the expression construct and plated onto minimal media (for
example SDG media, Clontech, Matchmaker System) without adenine.
In the parallel experiment, the cloning vector is transformed
into yeast mutants without purl-48.1-insert as negative control.
It emerges that only those yeasts which have been transformed
with purl-48.1 expression constructs are capable of growth on
minimal media without adenine. Thus, the enzyme encoded by
purl-48.1 is the first functional formylglycinamidine ribotide
synthase which has been isolated from plants.
Example 5
Generation of transgenic Arabidopsis thaliana plants
To prove the suitability of formylglycinamidine ribotide synthase
as target for herbicidal active ingredients; the expression of
formylglycinamidine ribotide synthase was lowered in transgenic
Arabidopsis plants by cosuppression or antisense inhibition.
To this end, binary vectors for plant transformation were first
generated. To achieve inhibition by means of cosuppression in
Arabidopsis thaliana, clone purl-19 was cleaved with BamHI and
SalI, and the resulting 1127 kb fragment was ligated into vector
pBinAR (Hofgen and Willmitzer, Plant Science 66(1990), 221-230)
which had been cleaved with the same restriction enzymes. To
achieve antisense inhibition in Arabidopsis thaliana, clone
purl-19 was cleaved with BamHI and KpnI, and the resulting 964 by
fragment was ligated into vector pBinAR which had been cleaved
with the same restriction enzymes. The resulting binary
constructs AtSpurL and AtASpurL (Figure 2) were transformed into
Arabidopsis thaliana.
To this end, a slightly modified form of the vacuum infiltration
method of Bechtold et al. (C. R. Acad.Sci. Paris, Life Sciences
316(1993), 1194-1199) was applied.
To this end, the binary vectors AtSpurL and AtASpurL were
transformed into Agrobacterium tumefaciens C58C1:pGV2260
(Deblaere et al., Nucl. Acids. Res. 13(1984), 4777-4788). 20 ml
of an overnight culture in YEB medium with 50 ~,g/ml kanamycin
(Sambrook et al. (1989), Cold Spring Harbor Laboratory Press:
ISBN 0-87969-309-6) were diluted in 500 ml of YEB/kanamycin and
shaken overnight at 28°C. The bacteria were harvested by
centrifugation and resuspended in 500 ml of infiltration medium
(2.15 g/1 Murashige and Skoog Basal salt mixture (Sigma), 50 g/1
sucrose, 1 mg/1 nicotinic acid, 1 mg/1 pyridoxin, 10 mg/1
thiamine, 100 mg/1 myo-inositol, 1 mg/1 glycine, 44 ~M
0050/50827
CA 02388851 2002-04-23
N6-benzylaminopurine, pH 5.7). The aerial parts of flowering
Arabidopsis plants were dipped into the bacterial suspension and
left to infiltrate for 10-15 minutes at 10-15 mbar. The plants
were then grown on in the greenhouse until the seeds had matured.
5 The seeds were sterilized and then placed on solid medium
(2.15 g/1 Murashige and Skoog Basal salt mixture (Sigma), 0.1 g/1
myo-inositol, 0.5 g/1 MES, 10 g/1, 8 g/1 agar-agar, 1 mg/1
nicotinic acid, 1 mg/1 pyridoxin, 10 mg/1 thiamine, 100 mg/1
myo-inositol, 1 mg/1 glycine, pH 5.7) supplemented with 15 ~g/ml
10 hygromycin, grown under sterile conditions for 2-4 weeks and then
transferred into soil.
Culture conditions:
15 Sterile culture: 21°C, 150 ~E*m-Z*s-1, 75% atmospheric humidity
Greenhouse: daytime temperature 22°C / night temperature
18°C,
16 h photoperiod, 450 ~E*m-2*s-1, 68% atmospheric humidity.
20 Example 6
Generation of transgenic tobacco plants
To inhibit formylglycinamidine ribotide synthase in tobacco by
25 cosuppression, clone purl-Ntl.l was cleaved with Xbal and SalI,
and the resulting 3.6 kbp fragment was ligated into vector pBinAR
which had been cleaved with the same restriction enzymes. To
subject formylglycinamidine ribotide synthase in tobacco to
antisense inhibition, clone purl-Ntl.l was cleaved with Asp718
and XbaI, and the resulting 3.6 kbp fragment was ligated into
vector pBinAR which had been cleaved with the same restriction
enzymes. The resulting binary constructs NtSpurL and NtASpurL -
see Figure 2 - were transformed into tobacco.
To this end, the plasmids NtSpurL and NtASpurL were transformed
into Agrobacterium tumefaciens C58C1:pGV2260 (Deblaere et al.,
Nucl. Acids. Res. 13(1984), 4777-4788). To transform tobacco
plants (Nicotiana tabacum cv. Samsun NN), a 1:50 dilution of an
overnight culture of a positively transformed agrobacterial
colony in Murashige-Skoog medium (Murashige and Skovg, Physiol.
Plant. 15(1962), 473) supplemented with 2% sucrose (2MS medium)
was used. Leaf disks of sterile plants (in each case approx.
1 cm2) were incubated in a Petri dish for 5-10 minutes with a 1:50
agrobacterial dilution. This was followed by 2 days' incubation
in the dark at 25°C on 2MS medium with 0.8% Bacto agar.
Cultivation was continued after 2 days and at 16 hours
light/8 hours dark and continued in a weekly rhythm on MS medium
i
0050/50827 CA 02388851 2002-04-23
26
with 500 mg/1 claforan (cefotaxime-sodium), 50 mg/1 kanamycin,
1 mg/1 benzylaminopurine (BAP), 0.2 mg/1 naphthylacetic acid and
1.6 g/1 glucose. Growing shoots were transferred to MS medium
supplemented with 2~ sucrose, 250 mg/1 claforan and 0.8$ Bacto
agar. Regenerated shoots were obtained on 2MS medium with
kanamycin and claforan, transferred into soil after rooting, and,
after cultivation in a controlled-environment cabinet in a
16-hour light/8-hour dark rhythm at 60$ atmospheric humidity,
analyzed.
Example 7
Analysis of transgenic plants
Transgenic plants were examined for formylglycinamidine ribotide
synthase expression and activity, and for altered metabolite
contents and phenotypical growth characteristics. Altered
nucleotide contents can be determined, for example, by the method
of Stitt et al., FEBS Letters 145(1982), 217-222.
Lines of transgenic plants which had been transformed with the
constructs AtSpurL, AtASpurL, NtSpurL or NtASpurL are
characterized by a growth which is reduced by different degrees
in comparison with untransformed control plants. In transgenic
lines, with the above-described phenotype, a reduced amount of
purl-48.1 or purl-Ntl.l was found by RNA analysis using the
Northern blot technique. The formylglycinamidine ribotide
synthase activity can be determined by a method as described in
Example 8.
These data constitute a direct connection between reduced
formylglycinamidine ribotide synthase expression and reduced
growth of Arabidopsis thaliana and tobacco plants, respectively,
and therefore identify formylglycinamidine ribotide synthase as
suitable target protein for herbicidal active ingredients.
Example 8
Measurement of formylglycinamidine ribotide synthase activity
Formylglycinamidine ribotide synthase activity is measured by
modifying methods which have been described (Methods in
Enzymology. 51(1978), 193-201). After adapting the method to
high-throughput methods, one of the above-described in vitro
assays can be used to search for formylglycinamidine ribotide
synthase activity inhibitors. To this end, the
formylglycinamidine ribotide synthase activity can be prepared
0050/50827 CA 02388851 2002-04-23
27
from plant tissues. Alternatively, a complete or truncated plant
formylglycinamidine ribotide synthase (preferably Arabidopsis
thaliana formylglycinamidine ribotide synthase) can be produced
in a suitable prokaryotic (for example E.coli) or eukaryotic (for
example yeasts, insect cells) expression system. After disrupting
the plant tissues or cells in suitable buffers and, if
appropriate, further purification steps, for example by
chromatographic methods, the formylglycinamidine ribotide
synthase activity can be quantified. This makes it possible to
correlate the growth phenotype in transgenic lines with the
formylglycinamidine ribotide synthase activity.
This method allows known formylglycinamidine ribotide synthase
inhibitors such as glutamine antagonists, and in particular novel
plant formylglycinamidine ribotide synthase inhibitors, to be
identified.
25
35
45
CA 02388851 2002-04-23
SEQUENCE LISTING
<110> BASF Aktiengesellschaft
<120> Formylglycinamidine ribotide synthase
<130> NAE 1237-99
<140>
<141>
<160> 6
<170> PatentIn Vers. 2.0
<210> 1
<211> 4570
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (146)..(4366)
<400> 1
gaattcggca cgaggaccag acaagaactc tgtgtcgact gccgtactct cggagtcgga 60
gcttcggtcc cgttaagaca gtaacagaga aaaaccctaa accctctttt cactttgttt 120
gctgttccgg tcatatctcg cgacg atg aat acc tcc caa gca act cgt gcg 172
Met Asn Thr Ser Gln Ala Thr Arg Ala
1 5
get ctg ttt cta aac ggt tcc aat aga caa gcg atg ctt ttg cag cgg 220
Ala Leu Phe Leu Asn Gly Ser Asn Arg Gln Ala Met Leu Leu Gln Arg
15 20 25
agc tca atg agt caa ttg tgg ggt tct gtt aga atg aga act tca cgg 268
Ser Ser Met Ser Gln Leu Trp Gly Ser Val Arg Met Arg Thr Ser Arg
30 35 40
ctg tct ctg aat cga aca aaa get gtt agc ttg aga tgt tct get caa 316
Leu Ser Leu Asn Arg Thr Lys Ala Val Ser Leu Arg Cys Ser Ala Gln
45 50 55
cct aat aaa cct aaa gca get gtt tct act ggt tct ttt gtt act get 364
Pro Asn Lys Pro Lys Ala Ala Val Ser Thr Gly Ser Phe Val Thr Ala
60 65 70
1
i1
~ CA 02388851 2002-04-23
gat gag cta cca agc ttg gtt gag aaa cct get get gag gtt atc cat 412
Asp Glu Leu Pro Ser Leu Val Glu Lys Pro Ala Ala Glu Val Ile His
75 80 85
ttt tat cgc gtg cct ttg att caa gaa agc gcg aac gcg gaa ctc ctc 460
Phe Tyr Arg Val Pro Leu Ile Gln Glu Ser Ala Asn Ala Glu Leu Leu
90 95 100 105
aag get gtt caa acc aaa atc agc aac cag att gtc agt ttg act aca 508
Lys Ala Val Gln Thr Lys Ile Ser Asn Gln Ile Val Ser Leu Thr Thr
110 115 120
gaa cag tct ttc aat att ggg ctt gaa tcc aag tta aaa gat gaa aaa 556
Glu G1n Ser Phe Asn Ile Gly Leu Glu Ser Lys Leu Lys Asp Glu Lys
125 130 I35
ctt tct gtt cta aag tgg att ctt caa gaa aca tat gag cca gag aat 604
Leu Ser Val Leu Lys Trp Ile Leu Gln Glu Thr Tyr Glu Pro Glu Asn
140 145 150
tta ggt acg gat agt ttt ctt gag agg aag aag caa gaa gga ctc cat 652
Leu Gly Thr Asp Ser Phe Leu Glu Arg Lys Lys Gln Glu Gly Leu His
155 160 165
get gtt att gtt gaa gtt ggc ccc aga ttg tct ttc aca aca gca tgg 700
Ala Val Ile Val Glu Val Gly Pro Arg Leu Ser Phe Thr Thr Ala Trp
170 175 180 185
tcc acc aat gcg gtt tcg ata tgt aga gca tgt ggg tta gac gag gtg 748
Ser Thr Asn Ala Val Ser Ile Cys Arg Ala Cys Gly Leu Asp Glu Val
190 195 200
act cgc ttg gaa agg tca agg agg tac ctt ttg ttc agc aag gag cca 796
Thr Arg Leu Glu Arg Ser.Arg Arg Tyr Leu Leu Phe Ser Lys Glu Pro
205 210 215
ctt ttg gaa aat cag ata aaa gaa ttt get gca atg gtt cat gat aga 844
Leu Leu Glu Asn Gln Ile Lys Glu Phe Ala Ala Met Val His Asp Arg
220 225 230
atg acc gag tgc gtg tac act caa aag ctg gtt tca ttc gag acg aat 892
Met Thr Glu Cys Val Tyr Thr Gln Lys Leu Val Ser Phe Glu Thr Asn
235 240 245
gtg gtt cca gaa gaa gtg aag tat gtg cct gtg atg gag aag ggt cgg 940
Val Val Pro Glu Glu Val Lys Tyr Val Pro Val Met Glu Lys Gly Arg
250 255 260 265
2
CA 02388851 2002-04-23
aag gcg ctg gaa gaa atc aac cag gag atg ggt tta gcc ttt gat gag 988
Lys Ala Leu Glu Glu Ile Asn Gln Glu Met Gly Leu Ala Phe Asp Glu
270 275 280
caa gat ttg cag tat tat act aga ctt ttc aga gag gac att aag cgt 1036
Gln Asp Leu Gln Tyr Tyr Thr Arg Leu Phe Arg Glu Asp Ile Lys Arg
285 290 295
gat cct acc aat gtg gag cta ttt gat atc get caa tcc aac agc gag 1084
Asp Pro Thr Asn Val Glu Leu Phe Asp Ile Ala Gln Ser Asn Ser Glu
300 305 310
cac agt cga cac tgg ttc ttt get ggg aac atg gtt att gat gga aag 1132
His Ser Arg His Trp Phe Phe Ala Gly Asn Met Val Ile Asp Gly Lys
315 320 325
cca atg gat aag tct ctt atg cag att gta aag agc act tgg gag gca 1180
Pro Met Asp Lys Ser Leu Met Gln Ile Val Lys Ser Thr Trp Glu Ala
330 335 340 345
aat cga aat aat tct gtc att ggg ttt aag gat aac tct agt get att 1228
Asn Arg Asn Asn Ser Val Ile Gly Phe Lys Asp Asn Ser Ser Ala Ile
350 355 360
aga ggc ttt ctg gtg aac cag cta cgg cct cta ctt cct ggt tct gtg 1276
Arg Gly Phe Leu Val Asn Gln Leu Arg Pro Leu Leu Pro Gly Ser Val
365 370 375
tgc tta ctt gat gtc agt gca cgt gat ctc gac ata ttg ttc act get 1324
Cys Leu Leu Asp Val Ser Ala Arg Asp Leu Asp Ile Leu Phe Thr Ala
380 385 390
gaa acc cat aat ttc cct tgt get gtg get cct tat cct ggt get gag 1372
Glu Thr His Asn Phe Pro Cys Ala Val Ala Pro Tyr Pro Gly Ala Glu
395 400 405
aca ggt get gga ggt cga atc aga gat aca cat gca aca gga aga ggt 1420
Thr Gly Ala Gly Gly Arg Ile Arg Asp Thr His Ala Thr Gly Arg Gly
410 415 420 425
tct ttt gtg gtt gca tcc act tct ggt tac tgt gtt ggt aac ctt aac 1468
Ser Phe Val Val Ala Ser Thr Ser Gly Tyr Cys Val Gly Asn Leu Asn
430 435 440
atg gag gga tct tat get cca tgg gaa gac tcg tct ttc caa tac cca 1516
Met G1u Gly Ser Tyr Ala Pro Trp Glu Asp Ser Ser Phe Gln Tyr Pro
445 450 455
3
CA 02388851 2002-04-23
tcg aac cta gca tca cct cta cag ata ctc atc gac get agt aat ggt 1564
Ser Asn Leu Ala Ser Pro Leu Gln Ile Leu Ile Asp Ala Ser Asn G1y
460 465 470
gca tct gac tat ggt aac aaa ttt gga gag ccc atg att cag gga tat 1612
Ala Ser Asp Tyr Gly Asn Lys Phe Gly Glu Pro Met Ile Gln Gly Tyr
475 480 485
act agg act ttt gga atg aga ctg cca agt ggg gat aga cga gaa tgg 1660
Thr Arg Thr Phe Gly Met Arg Leu Pro Ser Gly Asp Arg Arg Glu Trp
490 495 500 505
ttg aag ccg att atg ttc agt gca ggg att gga cag att gat cat act 1708
Leu Lys Pro Ile Met Phe Ser Ala Gly Ile Gly Gln Ile Asp His Thr
510 515 520
cat ata act aaa ggg gag cca gag gtt ggg atg ctt gtt gtt aag att 1756
His Ile Thr Lys Gly Glu Pro Glu Val Gly Met Leu Val Val Lys Ile
525 530 535
ggt gga cct get tac cgc att ggc atg gga gga gga gca get tcg agt 1804
Gly Gly Pro Ala Tyr Arg Ile Gly Met Gly Gly Gly Ala Ala Ser Ser
540 545 550
atg gtt agt ggc cag aat gat gca gag ctt gat ttc aat get gtg caa 1852
Met Val Ser Gly Gln Asn Asp Ala Glu Leu Asp Phe Asn Ala Val Gln
555 560 565
cgt gga gat get gaa atg tct cag aag ttg tac cgt gtt gtt cgt get 1900
Arg Gly Asp Ala Glu Met Ser Gln Lys Leu Tyr Arg Val Val Arg Ala
570 575 580 585
tgt att gag atg ggt gag aag aat cct att atc agc att cat gat caa 1948
Cys Ile Glu Met Gly Glu Lys Asn Pro Ile Ile Ser Ile His Asp Gln
590 595 600
ggt get ggt gga aac tgc aat gtt gtc aag gaa att att tat cca cag 1996
Gly Ala Gly Gly Asn Cys Asn Val Val Lys Glu Ile Ile Tyr Pro Gln
605 610 615
ggt gca gag att gac ata aga gcg gtt gtt gtg ggt gat cat act atg 2044
Gly Ala Glu Ile Asp Ile Arg Ala Val Vai Val Gly Asp His Thr Met
620 625 630
tcg gtg ttg gag ata tgg gga gca gaa tat caa gag caa gat gcg att 2092
Ser Val Leu Glu Ile Trp Gly Ala Glu Tyr Gln GIu Gln Asp Ala Ile
635 640 645
4
CA 02388851 2002-04-23
ttg gtg aaa get gag agc cgg gag att ttg caa tca atc tgt aag agg 2140
Leu Val Lys Ala Glu Ser Arg Glu Ile Leu Gln Ser Ile Cys Lys Arg
650 655 660 665
gaa agg ctt tcg atg get gtg att gga aca att aat ggg ggt ggt cgc 2188
Glu Arg Leu Ser Met Ala Val Ile Gly Thr Ile Asn Gly Gly Gly Arg
670 675 680
tgt act tta att gac agc aca get gca gcg aag tgc agt aaa gaa ggg 2236
Cys Thr Leu Ile Asp Ser Thr Ala Ala Ala Lys Cys Ser Lys Glu Gly
685 690 695
cta cct cca cct cca cct get gtg gat ctt gaa ctc gag aag gtt ctt 2284
Leu Pro Pro Pro Pro Pro Ala Val Asp Leu Glu Leu Glu Lys Val Leu
700 705 710
ggt gat atg cct aag aag acg ttt aag ttc aac cgc att get tat gca 2332
Gly Asp Met Pro Lys Lys Thr Phe Lys Phe Asn Arg Ile Ala Tyr Ala
715 720 725
cgg gag cca ctt gat att get cct gga att aca ttg atg gat get ttg 2380
Arg Glu Pro Leu Asp Ile Ala Pro Gly Ile Thr Leu Met Asp Ala Leu
730 735 740 745
aaa aga gtt ctc cga tta cca tca gtt tct tca aag cgg ttc ttg aca 2428
Lys Arg Val Leu Arg Leu Pro Ser Val Ser Ser Lys Arg Phe Leu Thr
750 755 760
acc aaa gtg gat aga tgt gtg aca ggt ctt gtt get cag caa caa aca 2476
Thr Lys Val Asp Arg Cys Val Thr Gly Leu Val Ala Gln Gln Gln Thr
765 770 775
gtt ggg cca ttg cag atc acg ctt get gat gtt gca gtt att gca cag 2524
Val Gly Pro Leu Gln Ile Thr Leu Ala Asp Val Ala Val Ile Ala Gln
780 785 790
aca ttc aca gat cta aca ggt ggt gca tgt gcc att ggt gag caa ccg 2572
Thr Phe Thr Asp Leu Thr Gly Gly Ala Cys Ala Ile Gly Glu Gln Pro
795 800 805
atc aaa ggc ttg ctt gat cca aaa gcc atg get agg cta get gtt gga 2620
Ile Lys Gly Leu Leu Asp Pro Lys Ala Met Ala Arg Leu Ala Val Gly
810 815 820 825
gag get ttg aca aat ctg gtt tgg gca aag gtc act gca ctt tct gat 2668
Glu Ala Leu Thr Asn Leu Val Trp Ala Lys Val Thr Ala Leu Ser Asp
830 835 840
CA 02388851 2002-04-23
gtt aaa get agt ggt aac tgg atg tat get gcc aag ctt gaa gga gaa 2716
Val Lys Ala Ser Gly Asn Trp Met Tyr Ala Ala Lys Leu Glu Gly Glu
845 850 855
gga tca gca atg tat gat get gcg att get cta tct gaa gcg atg att 2764
Gly Ser Ala Met Tyr Asp Ala Ala Ile Ala Leu Ser Glu Ala Met Ile
860 865 870
gaa ctt ggc att gca att gat ggt gga aaa gac agt ctt tca atg gca 2812
Glu Leu Gly Ile Ala Ile Asp Gly Gly Lys Asp Ser Leu Ser Met Ala
875 880 885
get cat gcg gac ggt gag gtt gtt aaa get cca gga aat ctt gtg att 2860
Ala His Ala Asp Gly Glu Val Val Lys Ala Pro Gly Asn Leu Val Ile
890 895 900 905
agt gcc tat gtt acc tgt cca gac ata aca aag aca gtg act ccg gat 2908
Ser Ala Tyr Val Thr Cys Pro Asp Ile Thr Lys Thr Val Thr Pro Asp
910 915 920
cta aag ctc gga ggt gat gat ggt att ctc ttg cat gtt gat ttg gca 2956
Leu Lys Leu GIy Gly Asp Asp Gly Ile Leu Leu His Val Asp Leu Ala
925 930 935
aag gga aag agg aga ttg ggt gga tct gca ctg get cag gtt ttt ggt 3004
Lys Gly Lys Arg Arg Leu Gly Gly Ser Ala Leu Ala Gln Val Phe Gly
940 945 950
cag ata gga aat gac tgt cct gat ctt gat gat gtt cca tat ctg aaa 3052
Gln Ile Gly Asn Asp Cys Pro Asp Leu Asp Asp Val Pro Tyr Leu Lys
955 960 965
aac gtt ttc gat ggc gtt caa get ctc att gca gaa aac ttg gta tct 3100
Asn Val Phe Asp Gly Val GIn Ala Leu Ile Ala Glu Asn Leu Val Ser
970 975 980 985
get gga cac gac atc agt gat ggt gga ctg gta gta aca get ttg gaa 3148
Ala Gly His Asp Ile Ser Asp Gly Gly Leu Val Val Thr Ala Leu Glu
990 995 1000
atg get ttt get gga aac aaa ggt ata aat ctc gac ttg get tca aat 3196
Met Ala Phe Ala Gly Asn Lys Gly Ile Asn Leu Asp Leu Ala Ser Asn
1005 1010 1015
gga att agc ctt ttt gag act ttg ttt tct gaa gaa ctt ggt ctc gtg 3244
Gly Ile Ser Leu Phe Glu Thr Leu Phe Ser Glu Glu Leu Gly Leu Val
1020 1025 1030
6
~ CA 02388851 2002-04-23
ctt gag att agt aag aca aac ctg gac get gtg atg gaa aaa ctc cgt 3292
Leu Glu Ile Ser Lys Thr Asn Leu Asp Ala Val Met Glu Lys Leu Arg
1035 1040 1045
get ttc gat gtt act get gag atc att gga aat gtc act gat tcg cct 3340
Ala Phe Asp Val Thr Ala Glu Ile Ile Gly Asn Val Thr Asp Ser Pro
1050 1055 1060 1065
ctg ata gag gta aaa gtg gat ggg att act cat ttg agt gag aaa act 3388
Leu Ile Glu Val Lys Val Asp Gly Ile Thr His Leu Ser Glu Lys Thr
1070 1075 1080
tca ttc ctc aga gac atg tgg gaa gac acc agt ttc cag ttg gaa aag 3436
Ser Phe Leu Arg Asp Met Trp Glu Asp Thr Ser Phe Gln Leu Glu Lys
1085 1090 1095
ctg caa cga ttg gca tct tgt gta gag atg gag aaa gaa ggt ttg aag 3484
Leu Gln Arg Leu Ala Ser Cys Val Glu Met Glu Lys Glu Gly Leu Lys
1100 1105 1110
ttt agg cat gag cct aat tgg aaa ctc tca ttt att cca tcc tcg acc 3532
Phe Arg His Glu Pro Asn Trp Lys Leu Ser Phe Ile Pro Ser Ser Thr
1115 1120 1125
aac aat aat tat atg tct cag gat gtt aag cca aaa gta gca gtg atc 3580
Asn Asn Asn Tyr Met Ser Gln Asp Val Lys Pro Lys Val Ala Val Ile
1130 1135 1140 1145
cga gaa gaa ggc agc aat gga gac aga gaa atg tca get gca ttt tac 3628
Arg Glu Glu Gly Ser Asn Gly Asp Arg Glu Met Ser Ala Ala Phe Tyr
1150 1155 1160
get gcc ggt ttt gaa cct tgg gac gtg aca gtg tct gat ctt cta get 3676
Ala Ala Gly Phe Glu Pro Trp Asp Val Thr Val Ser Asp Leu Leu Ala
1165 1170 1175
gga gac atc acc ctt gat cag ttc cgc ggt att gtg ttt gtg gga ggg 3724
Gly Asp Ile Thr Leu Asp Gln Phe Arg Gly Ile Val Phe Val Gly Gly
1180 1185 1190
ttc agt tat gcg gat gtt ctc gac tca gcc aaa gga tgg get get tca 3772
Phe Ser Tyr Ala Asp Val Leu Asp Ser Ala Lys Gly Trp Ala Ala Ser
1195 1200 1205
ata agg ttc aac gag cct gtg ttg agt caa ttt cag gag ttc tac aaa 3820
Ile Arg Phe Asn Glu Pro Val Leu Ser Gln Phe Gln Glu Phe Tyr Lys
1210 1215 1220 1225
7
CA 02388851 2002-04-23
aga cca gac acg ttc agt ctt gga atc tgc aat gga tgt cag tta atg 3868
Arg Pro Asp Thr Phe Ser Leu Gly Ile Cys Asn Gly Cys Gln Leu Met
1230 1235 1240
get ctg tta gga tgg gtt cca ggt cct caa gtt ggc ggg tca ctt gac 3916
Ala Leu Leu Gly Trp Val Pro Gly Pro Gln Val Gly Gly Ser Leu Asp
1245 1250 1255
acc tcg cag ccg agg ttt gtt cac aac gaa tca gga agg ttt gag tgc 3964
Thr Ser Gln Pro Arg Phe Val His Asn Glu Ser Gly Arg Phe Glu Cys
1260 1265 ' 1270
agg ttc aca agc gtg acc ata aag gac tcg cca tcg ata atg ctg aaa 4012
Arg Phe Thr Ser Val Thr Ile Lys Asp Ser Pro Ser Ile Met Leu Lys
1275 1280 1285
gga atg gag gga agt act tta ggg gtt tgg gcg gcg cac gga gaa gga 4060
Gly Met Glu Gly Ser Thr Leu Gly Val Trp Ala Ala His Gly Glu Gly
1290 1295 1300 1305
cgg get tat ttc ccg gac gaa gga gtc ttg gac cat atg ctt cac tca 4108
Arg Ala Tyr Phe Pro Asp Glu Gly Val Leu Asp His Met Leu His Ser
1310 1315 1320
gat ttg gca cca ttg aga tac tgt gat gat gat ggg aac gtg act gaa 4156
Asp Leu Ala Pro Leu Arg Tyr Cys Asp Asp Asp Gly Asn Val Thr Glu
1325 1330 1335
gcg tac cct ttt aac ctc aat ggt tca ccg ttg gga ata gcg get ata 4204
Ala Tyr Pro Phe Asn Leu Asn Gly Ser Pro Leu Gly Ile Ala Ala Ile
1340 1345 1350
tgt tca cct gat ggg aga cat ttg gcg atg atg cct cat cct gaa cgt 4252
Cys Ser Pro Asp Gly Arg His Leu Ala Met Met Pro His Pro Glu Arg
1355 1360 1365
tgt ttc ttg atg tgg cag ttt cca tgg tac cca aca agc tgg gac gtt 4300
Cys Phe Leu Met Trp Gln Phe Pro Trp Tyr Pro Thr Ser Trp Asp Val
1370 1375 1380 1385
gag aaa get ggc ccg agc ccg tgg ttg aag atg ttc cag aat gca agg 4348
Glu Lys Ala Gly Pro Ser Pro Trp Leu Lys Met Phe Gln Asn Ala Arg
1390 1395 1400
gac tgg tta gag tcg tgt taagcgtgtt tttttagtat aatttttatc 4396
Asp Trp Leu Glu Ser Cys
1405
8
~ CA 02388851 2002-04-23
tgcatcattt tgttgtgcat ttggtgtttt gcagagtctt tgaaagttgt cgacaaatca 4456
tgtgcttttt agtgagactt gatgtgtgct ttttgtcctt cacgttgtat tcaaactctg 4516
gaaattaagc atgagttact ctcttcgatc aaaaaaaaaa aaaaaaaact cgag 4570
<210> 2
<211> 1407
<212> PRT
<213> Arabidopsis thaliana
<400> 2
Met Asn Thr Ser Gln Ala Thr Arg Ala Ala Leu Phe Leu Asn Gly Ser
1 5 10 15
Asn Arg Gln Ala Met Leu Leu Gln Arg Ser Ser Met Ser Gln Leu Trp
20 25 30
Gly Ser Val Arg Met Arg Thr Ser Arg Leu Ser Leu Asn Arg Thr Lys
35 40 45
Ala Val Ser Leu Arg Cys Ser Ala Gln Pro Asn Lys Pro Lys Ala Ala
50 55 60
Val Ser Thr Gly Ser Phe Val Thr Ala Asp Glu Leu Pro Ser Leu Val
65 70 75 80
Glu Lys Pro Ala Ala Glu Val Ile His Phe Tyr Arg Val Pro Leu Ile
85 90 95
Gln Glu Ser Ala Asn Ala Glu Leu Leu Lys Ala Val Gln Thr Lys Ile
100 105 110
Ser Asn Gln Ile Val Ser Leu Thr Thr Glu Gln Ser Phe Asn Ile Gly
115 120 125
Leu Glu Ser Lys Leu Lys Asp Glu Lys Leu Ser Val Leu Lys Trp Ile
130 135 140
Leu Gln Glu Thr Tyr Glu Pro Glu Asn Leu Gly Thr Asp Ser Phe Leu
145 150 155 160
Glu Arg Lys Lys Gln Glu Gly Leu His Ala Val Ile Val Glu Val Gly
165 170 175
Pro Arg Leu Ser Phe Thr Thr Ala Trp Ser Thr Asn Ala Val Ser Ile
9
~ CA 02388851 2002-04-23
180 185 190
Cys Arg AIa Cys Gly Leu Asp Glu Val Thr Arg Leu Glu Arg Ser Arg
195 200 205
Arg Tyr Leu Leu Phe Ser Lys Glu Pro Leu Leu Glu Asn Gln Ile Lys
210 215 220
Glu Phe Ala Ala Met Val His Asp Arg Met Thr Glu Cys Val Tyr Thr
225 230 235 240
Gln Lys Leu Val Ser Phe Glu Thr Asn Val Val Pro Glu Glu Val Lys
245 250 255
Tyr Val Pro Val Met Glu Lys Gly Arg Lys Ala Leu Glu Glu Ile Asn
26D 265 270
Gln Glu Met Gly Leu Ala Phe Asp Glu Gln Asp Leu Gln Tyr Tyr Thr
275 280 285
Arg Leu Phe Arg Glu Asp Ile Lys Arg Asp Pro Thr Asn Val Glu Leu
290 295 300
Phe Asp Ile Ala Gln Ser Asn Ser Glu His Ser Arg His Trp Phe Phe
305 310 315 320
Ala Gly Asn Met Val Ile Asp Gly Lys Pro Met Asp Lys Ser Leu Met
325 330 335
Gln Ile Val Lys Ser Thr Trp Glu~.Ala Asn Arg Asn Asn Ser Val Ile
340 345 350
Gly Phe Lys Asp Asn Ser Ser Ala Ile Arg Gly Phe Leu Val Asn Gln
355 360 365
Leu Arg Pro Leu Leu Pro Gly Ser Val Cys Leu Leu Asp Val Ser Ala
370 375 380
Arg Asp Leu Asp Ile Leu Phe Thr Ala Glu Thr His Asn Phe Pro Cys
385 390 395 400
Ala Val Ala Pro Tyr Pro Gly Ala Glu Thr Gly Ala Gly Gly Arg Ile
405 410 415
Arg Asp Thr His Ala Thr Gly Arg Gly Ser Phe Val Val Ala Ser Thr
420 425 430
Ser Gly Tyr Cys Val Gly Asn Leu Asn Met Glu Gly Ser Tyr Ala Pro
CA 02388851 2002-04-23
435 440 445
Trp Glu Asp Ser Ser Phe Gln Tyr Pro Ser Asn Leu Ala Ser Pro Leu
450 455 460
G1n Ile Leu Ile Asp Ala Ser Asn Gly Ala Ser Asp Tyr Gly Asn Lys
465 470 475 480
Phe Gly Glu Pro Met Ile Gln Gly Tyr Thr Arg Thr Phe Gly Met Arg
485 490 495
Leu Pro Ser Gly Asp Arg Arg Glu Trp Leu Lys Pro Ile Met Phe Ser
500 505 510
Ala G1y Ile Gly Gln Ile Asp His Thr His Ile Thr Lys Gly Glu Pro
515 520 525
Glu Val GIy Met Leu Val Val Lys Ile Gly Gly Pro Ala Tyr Arg Ile
530 535 540
Gly Met Gly G1y Gly Ala Ala Ser Ser Met Val Ser Gly Gln Asn Asp
545 550 555 560
Ala Glu Leu Asp Phe Asn Ala Val Gln Arg Gly Asp Ala Glu Met Ser
565 570 575
Gln Lys Leu Tyr Arg Val Val Arg Ala Cys Ile Glu Met Gly Glu Lys
580 585 590
Asn Pro I1e Ile Ser Ile His Asp Gln Gly Ala Gly Gly Asn Cys Asn
595 600 605
Val Val Lys Glu Ile Ile Tyr Pro Gln Gly Ala Glu Ile Asp Ile Arg
610 615 620
Ala Val Val Val Gly Asp His Thr Met Ser Val Leu Glu Ile Trp Gly
625 630 635 640
Ala Glu Tyr Gln Glu Gln Asp Ala Ile Leu Val Lys Ala Glu Ser Arg
645 650 655
Glu Ile Leu Gln Ser Ile Cys Lys Arg Glu Arg Leu Ser Met Ala Val
660 665 670
Ile Gly Thr Ile Asn Gly Gly Gly Arg Cys Thr Leu Ile Asp Ser Thr
675 680 685
Ala Ala Ala Lys Cys Ser Lys Glu Gly Leu Pro Pro Pro Pro Pro Ala
11
CA 02388851 2002-04-23
690 695 700
Val Asp Leu Glu Leu Glu Lys Val Leu Gly Asp Met Pro Lys Lys Thr
705 710 715 720
Phe Lys Phe Asn Arg Ile Ala Tyr Ala Arg Glu Pro Leu Asp Ile Ala
725 730 735
Pro Gly Ile Thr Leu Met Asp Ala Leu Lys Arg Val Leu Arg Leu Pro
740 745 750
Ser Val Ser Ser Lys Arg Phe Leu Thr Thr Lys Val Asp Arg Cys Val
755 760 765
Thr Gly Leu Val Ala Gln Gln Gln Thr Val Gly Pro Leu Gln Ile Thr
770 775 780
Leu Ala Asp Val Ala Val I1e Ala Gln Thr Phe Thr Asp Leu Tlir Gly
785 790 795 800
Gly Ala Cys Ala Ile Gly Glu Gln Pro Ile Lys Gly Leu Leu Asp Pro
805 810 815
Lys Ala Met Ala Arg Leu Ala Va1 Gly Glu Ala Leu Thr Asn Leu Val
820 825 830
Trp Ala Lys Val Thr Ala Leu Ser Asp Val Lys Ala Ser Gly Asn Trp
835 840 845
Met Tyr Ala A1a Lys Leu Glu Gly Glu Gly Ser Ala Met Tyr Asg Ala
850 855 860
Ala Ile Ala Leu Ser Glu Ala Met Ile Glu Leu Gly Ile Ala Ile Asp
865 870 875 880
Gly Gly Lys Asp Ser Leu Ser Met Ala Ala His Ala Asp Gly Glu Val
885 890 895
Val Lys Ala Pro Gly Asn Leu Val Ile Ser Ala Tyr Val Thr Cys Pro
900 905 910
Asp Ile Thr Lys Thr Val Thr Pro Asp Leu Lys Leu Gly Gly Asp Asp
915 920 925
G1y Ile Leu Leu His Val Asp Leu Ala Lys Gly Lys Arg Arg Leu Gly
930 935 940
Gly Ser Ala Leu Ala Gln Val Phe Gly Gln Ile Gly Asn Asp Cys Pro
12
fl
~ CA 02388851 2002-04-23
945 950 955 960
Asp Leu Asp Asp Val Pro Tyr Leu Lys Asn Val Phe Asp Gly Val Gln
965 970 975
Ala Leu Ile Ala Glu Asn Leu Val Ser Ala Gly His Asp Ile Ser Asp
980 985 990
Gly Gly Leu Val Val Thr Ala Leu Glu Met Ala Phe Ala Gly Asn Lys
995 1000 1005
Gly Ile Asn Leu Asp Leu Ala Ser Asn Gly Ile Ser Leu Phe Glu Thr
1010 1015 1020
Leu Phe Ser Glu Glu Leu GIy Leu Val Leu Glu Ile Ser Lys Thr Asn
025 1030 1035 1040
Leu Asp Ala Val Met Glu Lys Leu Arg Ala Phe Asp Val Thr Ala Glu
1045 1050 1055
Ile Ile Gly Asn Val Thr Asp Ser Pro Leu Ile Glu Val Lys Val Asp
1060 1065 1070
Gly Ile Thr His Leu Ser Glu Lys Thr Ser Phe Leu Arg Asp Met Trp
1075 1080 1085
Glu Asp Thr Ser Phe Gln Leu Glu Lys Leu Gln Arg Leu Ala Ser Cys
1090 1095 1100
Val Glu Met Glu Lys Glu Gly Leu Lys Phe Arg His Glu Pro Asn Trp
105 1110 1115 1120
Lys Leu Ser Phe Ile Pro Ser Ser Thr Asn Asn Asn Tyr Met Ser Gln
1125 1130 1135
Asp Val Lys Pro Lys Val Ala Val Ile Arg Glu Glu Gly Ser Asn Gly
1140 1145 1150
Asp Arg Glu Met Ser Ala Ala Phe Tyr Ala Ala Gly Phe Glu Pro Trp
1155 1160 1165
Asp Val Thr Val Ser Asp Leu Leu Ala Gly Asp Ile Thr Leu Asp Gln
1170 1175 1180
Phe Arg Gly Ile Val Phe Val Gly Gly Phe Ser Tyr Ala Asp Val Leu
185 1190 1195 1200
Asp Ser Ala Lys Gly Trp Ala Ala Ser Ile Arg Phe Asn Glu Pro Val
13
CA 02388851 2002-04-23
1205 1210 1215
Leu Ser Gln Phe Gln Glu Phe Tyr Lys Arg Pro Asp Thr Phe Ser Leu
1220 1225 1230
Gly Ile Cys Asn Gly Cys Gln Leu Met Ala Leu Leu Gly Trp Val Pro
1235 1240 1245
Gly Pro Gln Val Gly Gly Ser Leu Asp Thr Ser Gln Pro Arg Phe Val
1250 1255 1260
His Asn Glu Ser Gly Arg Phe Glu Cys Arg Phe Thr Ser Val Thr Ile
265 1270 1275 1280
Lys Asp Ser Pro Ser Ile Met Leu Lys Gly Met Glu Gly Ser Thr Leu
1285 1290 1295
Gly Val Trp Ala Ala His Gly Glu Gly Arg Ala Tyr Phe Pro Asp Glu
1300 1305 1310
Gly Val Leu Asp His Met Leu His Ser Asp Leu Ala Pro Leu Arg Tyr
1315 1320 1325
Cys Asp Asp Asp Gly Asn Val Thr Glu Ala Tyr Pro Phe Asn Leu Asn
1330 1335 1340
Gly Ser Pro Leu Gly Ile Ala Ala Ile Cys Ser Pro Asp Gly Arg His
345 1350 1355 1360
Leu Ala Met Met Pro His Pro Glu Arg.Cys Phe Leu Met Trp Gln Phe
1365 1370 1375
Pro Trp Tyr Pro Thr Ser Trp Asp Val Glu Lys Ala Gly Pro Ser Pro
1380 1385 1390
Trp Leu Lys Met Phe Gln Asn Ala Arg Asp Trp Leu Glu Ser Cys
1395 1400 1405
<210> 3
<211> 3434
<212> DNA
<213> Nicotiana tabacum
<220>
<221> CDS
<222> (2)..(3052)
14
m
CA 02388851 2002-04-23
<400> 3
g aat tcg cgg ccg cgc ggc cgc gaa ttc gcg gcc gca cct tat cct ggt 49
Asn Ser Arg Pro Arg Gly Arg Glu Phe Ala Ala Ala Pro Tyr Pro Gly
1 ~ 5 10 15
gcc gag aca ggt gca ggc ggc cgt atc cgg gat acc cat get act gga 97
Ala Glu Thr Gly AIa Gly Gly Arg Ile Arg Asp Thr His Ala Thr Gly
20 25 30
agg ggt tct ttt gtt gtt gca tct aca gcc gga tat tgt gtt gga aat 145
Arg Gly Ser Phe Val Val Ala Ser Thr Ala Gly Tyr Cys Val Gly Asn
35 40 45
ctt cat att gaa ggt tca tat get cct tgg gaa gat cct tct ttc aca 193
Leu His Ile Glu Gly Ser Tyr Ala Pro Trp Glu Asp Pro Ser Phe Thr
50 55 60
tac-cca gca-aat ttg get tca ccg ctg cag atc ctt att gat get agt 241
Tyr Pro Ala Asn Leu Ala Ser Pro Leu Gln Ile Leu Ile Asp Ala Ser
65 70 75 80
aat gga gca tcg gac tat ggg aac aaa ttc ggg gag cct ttg att cag 289
Asn Gly AIa Ser Asp Tyr Gly Asn Lys Phe Gly Glu Pro Leu Ile Gln
85 90 95
ggt tat tgt cga acg ttt gga atg aga ctg cca agt ggc gag agg agg 337
Gly Tyr Cys Arg Thr Phe Gly Met Arg Leu Pro Ser Gly Glu Arg Arg
100 105 110
gaa tgg ttg aag ccg atc atg ttt agt get ggc att ggg caa ata gat 385
Glu Trp Leu Lys Pro Ile Met Phe Ser Ala Gly Ile Gly Gln Ile Asp
115 120 125
cac ctt cac tta tca aag gga gaa ccc gag att ggt atg ttg gta gtt 433
His Leu His Leu Ser Lys Gly Glu Pro Glu Ile Gly Met Leu VaI Val
130 135 140
aag att gga gga cca gca tat cgt att gga atg gga ggt ggc get gca 481
Lys Ile Gly Gly Pro Ala Tyr Arg Ile Gly Met Gly Gly Gly Ala Ala
145 150 155 160
tcc agc atg gtc agt gga cag aat gat gcc gag ctt gac ttc aac gcc 529
Ser Ser Met Val Ser Gly Gln Asn Asp Ala Glu Leu Asp Phe Asn Ala
165 170 175
gtg cag cgt gga gat get gag atg gca cag aag ttg tat cgg gtt gtt 577
Val Gln Arg Gly Asp Ala Glu Met Ala Gln Lys Leu Tyr Arg Va1 Val
180 185 190
. CA 02388851 2002-04-23
cgt get tgc att gag atg ggg gac aac aac ccg atc ata agc att cat 625
Arg Ala Cys Ile Glu Met Gly Asp Asn Asn Pro Ile Ile Ser Ile His
195 200 205
gat cag ggt get ggt gga aac tgt aat gtc gtg aag gaa ata ata cat 673
Asp Gln Gly Ala Gly Gly Asn Cys Asn Val Val Lys Glu Ile Ile His
210 215 220
cca cag ggc gcc aaa att gat ata agg gca att gta gtt ggc gat cac 72I
Pro Gln Gly Ala Lys Ile Asp Ile Arg Ala Ile Val Val Gly Asp His
225 230 235 240
acg atg tct gtt ctg gaa att tgg ggt gca gaa tat cag gag caa gat 769
Thr Met Ser Val Leu Glu Ile Trp Gly Ala Glu Tyr Gln Glu Gln Asp
245 250 255
gcc ata cta gtg aag cct gaa agt cgt gat ctt ttg caa gca atc tgt 817
Ala Ile Leu Val Lys Pro Glu Ser Arg Asp Leu Leu Gln Ala Ile Cys
260 265 270
gcg agg gaa aga gtt tcc atg get gtt att gga aca att aat ggt gaa 865
Ala Arg Glu Arg Val Ser Met Ala Val Ile Gly Thr Ile Asn Gly Glu
275 280 285
ggg cgt att gtc ctg gag gat agc gtg gca att gaa aaa acc agg tct 913
Gly Arg Ile Val Leu Glu Asp Ser Val Ala Ile Glu Lys Thr Arg Ser
290 295 300
agt gga ttg cct cct cct cca cct gca gtg gat ctt gag ctt gag sag 961
Ser Gly Leu Pro Pro Pro Pro Pro Ala Val Asp Leu Glu Leu Glu Lys
305 310 315 320
gtg ctt ggc gat atg cct aaa aag aca ttt gaa ttt cgt cgc atg aac 1009
Val Leu Gly Asp Met Pro Lys Lys Thr Phe Glu Phe Arg Arg Met Asn
325 330 335
tat ctg cgt gaa cca ctt gat att get cct gca aca aca gtc tta gat 1057
Tyr Leu Arg Glu Pro Leu Asp Ile Ala Pro Ala Thr Thr Val Leu Asp
340 345 350
tca ttg aag agg gtc ctg agg ctc cct tct gtt tgt tcg aaa agg ttc 1105
Ser Leu Lys Arg Val Leu Arg Leu Pro Ser Val Cys Ser Lys Arg Phe
355 360 365
ttg acc act aaa gtt gac agg tgt gtc aca ggc ctt gtg gca cag cag 1153
Leu Thr Thr Lys Val Asp Arg Cys Val Thr Gly Leu Val Ala Gln Gln
370 375 380
16
CA 02388851 2002-04-23
caa act gtg ggc ccc ctg cag att cct ctt get gat gtt get gtt ata 1201
Gln Thr val Gly Pro Leu Gln Ile Pro Leu Ala Asp Val Ala Val Ile
385 390 395 400
get caa act tat aca gac tta act gga ggt gca tgc tca atc ggg gag 1249
Ala Gln Thr Tyr Thr Asp Leu Thr Gly Gly Ala Cys Ser Ile Gly Glu
405 410 415
cag cca ata aaa ggt ctt ttg gat cca aaa gca atg gca cgg ctg get 1297
Gln Pro Ile Lys Gly Leu Leu Asp Pro Lys Ala Met Ala Arg Leu Ala
420 425 430
gtc gga gaa gca ctc aca aat ctt gtt tgg gcg aaa att aca tct ctt 1345
Val Gly Glu Ala Leu Thr Asn Leu Val Trp Ala Lys Ile Thr Ser Leu
435 440 445
tct gat gtt aaa gca agt ggg aat tgg atg tat get gca aag cta.gat 1393
Ser Asp Val Lys Ala Ser Gly Asn Trp Met Tyr Ala Ala Lys Leu Asp
450 455 460
ggt gaa gga get gca atg tat gac get get gtt get ctt tct gaa get 1441
Gly Glu Gly Ala Ala Met Tyr Asp Ala Ala Val Ala Leu Ser Glu Ala
465 470 475 480
atg att gaa ctt gga att gca att gat ggg ggg aaa gac agc ctt tcc 1489
Met Ile Glu Leu Gly Ile Ala Ile Asp Gly Gly Lys Asp Ser Leu Ser
485 490 495
atg gca gcc cac tcg tct ggg gaa gtt gtt aaa gcc cca ggg aat cta 1537
Met Ala Ala His Ser Ser Gly Glu Val Val Lys Ala Pro Gly Asn Leu
500 505 510
gtc atc agt acc tat gtg aca tgt cct gat ata acc aag aca gtt acg 1585
Val Ile 5er Thr Tyr Val Thr Cys Pro Asp Ile Thr Lys Thr Val Thr
515 520 525
cca gac ttg aag ctt gga gat gat ggt gta ctg ctt cat att gac ttg 1633
Pro Asp Leu Lys Leu Gly Asp Asp Gly Val Leu Leu His Ile Asp Leu
530 535 540
get aaa gga aaa cga cga ctt ggt gga tct get ctt gcc cag gtt ttt 1681
Ala Lys Gly Lys Arg Arg Leu Gly Gly Ser Ala Leu Ala Gln Val Phe
545 550 555 560
gat caa att ggg gac gaa agt cct gat ctg gat gac gta tct tat ctt 1729
Asp Gln Ile GIy Asp Glu Sex Pro Asp Leu Asp Asp Val Ser Tyr Leu
565 570 575
17
fl
CA 02388851 2002-04-23
aag act gtt ttt aat gag gtt cag aat cta atc tct gat gag ctg ata 1777
Lys Thr Val Phe Asn Glu Val Gln Asn Leu Ile Ser Asp Glu Leu Ile
580 585 590
tct get ggt cat gat atc agt gat ggg gga ctt tta gtg aat get ctg 1825
Ser Ala Gly His Asp Ile Ser Asp Gly Gly Leu Leu Val Asn Ala Leu
595 600 605
gaa atg gca ttc gca ggg aac tgt ggc att cac ttg gat tta act tct 1873
Glu Met Ala Phe Ala Gly Asn Cys Gly Ile His Leu Asp Leu Thr Ser
610 615 620
tta ggg agt agt gta ccc caa aca ctt ttt gca gag gag ctt ggc ctg 1921
Leu Gly Ser Ser Val Pro Gln Thr Leu Phe Ala Glu Glu Leu Gly Leu
625 630 635 640
ctc att gaa gtt agc agg aag aac ttg gat tta gtt ctg gaa aag ctc 1969
Leu IIe Glu Val Ser Arg Lys Asn Leu Asp Leu Val Leu Glu Lys Leu
645 650 655
tgc agt ggt get gtt tca get aat att att ggt caa gtt act tca tct 2017
Cys Ser Gly Ala Val Ser Ala Asn Ile Ile Gly Gln Val Thr Ser Ser
660 665 670
cca ata gtt gaa ttg agg gtt gac ggg gtt act cat ttg aat gac aaa 2065
Pro Ile Val Glu Leu Arg Val Asp Gly Val Thr His Leu Asn Asp Lys
675 680 685
act tct gtg ctc agg gat atg tgg gaa gaa acc agc ttt caa ttg gas ~ 2113
Thr Ser Val Leu Arg Asp Met Trp Glu Glu Thr Ser Phe Gln Leu Glu
690 695 700
aag ctc caa aga ctg get tcg tgt gta gaa tta gaa aaa gaa gga ttg 2161
Lys Leu Gln Arg Leu Ala Ser Cys Val Glu Leu Glu Lys Glu Gly Leu
705 710 715 72p
aag aat cgg cat gaa cca tcc tgg aaa cta tcc ttc aca cca aca ttt 2209
Lys Asn Arg His Glu Pro Ser Trp Lys Leu Ser Phe Thr Pro Thr Phe
?25 730 735
act gat gat aag tat atg act gcc gtt tca aag cca aag gtc gca att 2257
Thr Asp Asp Lys Tyr Met Thr Ala Val Ser Lys Pro Lys Val Ala Ile
740 745 750
att cgc gag gaa ggc agc aat ggt gat aga gaa atg act gca get ttt 2305
Ile Arg Glu Glu Gly Ser Asn Gly Asp Arg Glu Met Thr Ala A1a Phe
755 760 765
18
r
CA 02388851 2002-04-23
tat get get gga ttt gag cca tgg gat gtt gca atg tca gac ctt ctc 2353
Tyr Ala Ala Gly Phe Glu Pro Trp Asp Val Ala Met Ser Asp Leu Leu
770 775 780
aat gga gtc atc atg ctt gat gaa ttt aga gga att gtg ttt gtt gga 2401
Asn Gly Val Ile Met Leu Asp Glu Phe Arg Gly Ile Val Phe Val Gly
785 790 795 800
ggt ttt agt tat get gac gtg ctt gat tct gca aaa ggc tgg gca gcg 2449
Gly Phe Ser Tyr Ala Asp Val Leu Asp Ser Ala Lys Gly Trp Ala Ala
805 810 815
tcc att cgc ttt aat caa cct ctt tta aac caa ttt cag gca ttt tat 2497
Ser Ile Arg Phe Asn Gln Pro Leu Leu Asn Gln Phe G1n Ala Phe Tyr
820 825 830
aac cgt cca gac act ttc agc ctt gga gtt tgc aat ggg tgc caa ctt 2545
Asn Arg Pro Asp Thr Phe Ser Leu Gly Val Cys Asn Gly Cys Gln Leu
835 840 845
atg get ctg ttg ggt tgg gtt ccg ggg ccc caa gtg gga ggt gtt ttc 2593
Met Ala Leu Leu Gly Trp Val Pro Gly Pro Gln Val Gly Gly Val Phe
850 855 860
ggt gcc ggc ggg gac cca tca cag cct agg ttt gta cat aat gag tct 2641
G.ly Ala Gly Gly Asp Pro Ser Gln Pro Arg Phe Val His Asn Glu Ser
865 870 875 880
gga aga ttt gaa tgc cgc ttc acg agt gtg aca ata gaa gaa tca ccg 2689
Gly Arg Phe Glu Cys Arg Phe Thr Ser Val Thr Ile Glu Glu Ser Pro
885 890 895
gcc ata atg ttc aaa ggt atg gaa ggt agt aca ctg ggc gtt tgg get 2737
Ala Ile Met Phe Lys Gly Met Glu Gly Ser Thr Leu Gly Val Trp Ala
900 905 910
get cat ggt gaa gga aga get tat ttc cct gat gat agt gtt ttc aat 2785
Ala His Gly Glu Gly Arg Ala Tyr Phe Pro Asp Asp Ser Val Phe Asn
9I5 920 925
cat att gtt ggc tcc aac ttg gca cca gtg aaa tat tgc gat gat gat 2833
His Ile Val Gly Ser Asn Leu Ala Pro Val Lys Tyr Cys Asp Asp Asp
930 935 940
ggc aga cca aca gat ata tat cct ttc aat ctt aat ggt tct ccc ttg 2881
Gly Arg Pro Thr Asp Ile Tyr Pro Phe Asn Leu Asn Gly Ser Pro Leu
945 950 955 960
19
' CA 02388851 2002-04-23
ggt gtg gcg gca att tgt tct cca gat ggg cga cat ctt gcg ata atg 2929
Gly Val Ala Ala Ile Cys Ser Pro Asp Gly Arg His Leu Ala Ile Met
965 970 975
cct cat cca gaa cgc tgt ttc ttg atg tgg cag ttc cca tgg tat cct 2977
Pro His Pro Glu Arg Cys Phe Leu Met Trp Gln Phe Pro Trp Tyr Pro
980 985 990
aaa aat tgg gat gtt gaa aag aaa ggt cca agt ccc tgg ttg cgc atg 3025
Lys Asn Trp Asp Val Glu Lys Lys Gly Pro Ser Pro Trp Leu Arg Met
995 1000 1005
ttc caa aat gcc aga gaa tgg tgc tca tgagattttc tgaaaattct 3072
Phe Gln Asn Ala Arg Glu Trp Cys Ser
1010 1015
gaagtcatga cattgtttga cagacaaata ttattagagg ggcaaagctg agaggcaggt 3132
gctgttcttt atcacataga ggttaaagtg caaagattgg acagaagcga cacagtttaa 3192
tagtcagtgc tgaaggtgca gagattaaga tcagtgcaga gcttgagatc tctgcaattt 3252
tgtttctgat accatggcac aaagcagatg aatttttcaa tttaactggc tgattgtcgt 3312
ttctttcgat actgatggac tttttcattg cctcttagct gtttcgcttg tttcttgtat 3372
accataattg gtttttctta tgtcttacat atctgatcaa ctgattttgc ggccgcgaat 3432
tc
3434
<210> 4
<211> 1017
<212> PRT
<213> Nicotiana tabacum
<400> 4
Asn Ser Arg Pro Arg Gly Arg Glu Phe Ala Ala Ala Pro Tyr Pro Gly
1 5 10 15
Ala Glu Thr Gly Ala Gly Gly Arg Ile Arg Asp Thr His Ala Thr Gly
20 25 30
Arg Gly Ser Phe Val Val Ala Ser Thr Ala Gly Tyr Cys Val Gly Asn
35 40 45
Leu His Ile Glu Gly Ser Tyr Ala Pro Trp G1u Asp Pro Ser Phe Thr
i
CA 02388851 2002-04-23
_ 50 55 60
Tyr Pro Ala Asn Leu Ala Ser Pro Leu Gln Ile Leu Ile Asp Ala Ser
65 70 75 80
Asn Gly Ala Ser Asp Tyr Gly Asn Lys Phe Gly Glu Pro Leu Ile Gln
85 90 95
Gly Tyr Cys Arg Thr Phe Gly Met Arg Leu Pro Ser Gly Glu Arg Arg
100 105 110
Glu Trp Leu Lys Pro Ile Met Phe Ser Ala Gly Ile Gly Gln Ile Asp
115 120 125
His Leu His Leu Ser Lys Gly Glu Pro Glu Ile Gly Met Leu Val Val
130 135 140
Lys Ile Gly Gly Pro Ala Tyr Arg Ile Gly Met Gly Gly Gly Ala Ala
145 150 155 160
Ser Ser Met Val Ser Gly Gln Asn Asp Ala Glu Leu Asp Phe Asn Ala
165 170 175
Val Gln Arg Gly Asp Ala Glu Met Ala Gln Lys Leu Tyr Arg Val Val
180 185 190
Arg Ala Cys Ile Glu Met Gly Asp Asn Asn Pro Ile Ile Ser Ile His
195 200 205
Asp Gln Gly Ala Gly Gly Asn Cys Asn Val Val Lys Glu Ile Ile His
21o z15 2zo
Pro Gln Gly Ala Lys Ile Asp Ile Arg Ala Ile Val Val Gly Asp His
225 230 235 240
Thr Met Ser Val Leu Glu Ile Trp Gly Ala Glu Tyr Gln Glu Gln Asp
245 250 255
Ala Ile Leu Val Lys Pro Glu Ser Arg Asp Leu Leu Gln Ala Ile Cys
260 265 270
Ala Arg Glu Arg Val Ser Met Ala Val Ile Gly Thr Ile Asn Gly Glu
275 280 285
Gly Arg Ile Val Leu Glu Asp Ser Val Ala Ile Glu Lys Thr Arg Ser
290 295 300
Ser Gly Leu Pro Pro Pro Pro Pro Ala Val Asp Leu Glu Leu Glu Lys
21
~ CA 02388851 2002-04-23
305 310 315 320
Val Leu Gly Asp Met Pro Lys Lys Thr Phe Glu Phe Arg Arg Met Asn
325 330 335
Tyr Leu Arg Glu Pro Leu Asp Ile Ala Pro Ala Thr Thr Val Leu Asp
340 345 350
Ser Leu Lys Arg Val Leu Arg Leu Pro Ser Val Cys Ser Lys Arg Phe
355 360 365
Leu Thr Thr Lys Val Asp Arg Cys Val Thr Gly Leu Val Ala Gln Gln
370 375 380
Gln Thr Val Gly Pro Leu Gln Ile Pro Leu Ala Asp Val Ala Val Ile
385 390 395 400
Ala Gln Thr Tyr Thr Asp Leu Thr Gly Gly Ala Cys Ser Ile Gly Glu
405 410 415
Gln Pro Ile Lys Gly Leu Leu Asp Pro Lys Ala Met Ala Arg Leu Ala
420 425 430
Val Gly Glu Ala Leu Thr Asn Leu Val Trp Ala Lys Ile Thr Ser Leu
435 440 445
Ser Asp Val Lys Ala Ser Gly Asn Trp Met Tyr Ala Ala Lys Leu Asp
450 455 460
Gly Glu Gly Ala Ala Met Tyr Asp Ala Ala Val Ala Leu Ser Glu Ala
465 470 475 480
Met Ile Glu Leu Gly Ile Ala Ile Asp Gly Gly Lys Asp Ser Leu Ser
485 490 495
Met Ala Ala His Ser Ser Gly Glu Val Val Lys Ala Pro Gly Asn Leu
500 505 510
Val Ile Ser Thr Tyr Val Thr Cys Pro Asp Ile Thr Lys Thr Val Thr
515 520 525
Pro Asp Leu Lys Leu Gly Asp Asp Gly Val Leu Leu His Ile Asp Leu
530 535 540
Ala Lys Gly Lys Arg Arg Leu Gly Gly Ser Ala Leu Ala Gln Val Phe
545 550 555 560
Asp Gln Ile Gly Asp Glu Ser Pro Asp Leu Asp Asp Val Ser Tyr Leu
22
f.
CA 02388851 2002-04-23
565 570 575
Lys Thr Val Phe Asn Glu Val Gln Asn Leu Ile 5er Asp Glu Leu Ile
580 585 590
Ser Ala Gly His Asp Ile Ser Asp Gly Gly Leu Leu Val Asn Ala Leu
595 600 605
Glu Met Ala Phe Ala Gly Asn Cys Gly Ile His Leu Asp Leu Thr Ser
610 615 620
Leu Gly Ser Ser Val Pro Gln Thr Leu Phe Ala Glu Glu Leu Gly Leu
625 630 635 640
Leu Ile Glu Val Ser Arg Lys Asn Leu Asp Leu Val Leu Glu Lys Leu
645 650 655
Cys Ser Gly Ala Val Ser Ala Asn Ile Ile Gly Gln Val Thr Ser Ser
660 665 6?0
Pro Ile Val Glu Leu Arg Val Asp Gly Val Thr His Leu Asn Asp Lys
675 680 685
Thr Ser Val Leu Arg Asp Met Trp Glu Glu Thr Ser Phe Gln Leu Glu
690 695 700
Lys Leu Gln Arg Leu Ala Ser Cys Val Glu Leu Glu Lys Glu Gly Leu
705 710 715 720
Lys Asn Arg His Glu Pro Ser Trp Lys Leu Ser Phe Thr Pro Thr Phe
725 730 735
Thr Asp Asp Lys Tyr Met Thr Ala Val Ser Lys Pro Lys Val Ala Ile
740 745 750
Ile Arg Glu Glu Gly Ser Asn Gly Asp Arg Glu Met Thr Ala Ala Phe
755 760 765
Tyr Ala Ala Gly Phe Glu Pro Trp Asp Val Ala Met Ser Asp Leu Leu
770 775 780
Asn Gly Val Ile Met Leu Asp Glu Phe Arg Gly Ile Val Phe Val Gly
785 790 795 800
Gly Phe Ser Tyr Ala Asp Val Leu Asp Ser Ala Lys Gly Trp Ala Ala
805 810 815
Ser Ile Arg Phe Asn Gln Pro Leu Leu Asn Gln Phe Gln Ala Phe Tyr
23
s,
CA 02388851 2002-04-23
.. a
820 825 830
Asn Arg Pro Asp Thr Phe Ser Leu Gly Val Cys Asn Gly Cys Gln Leu
835 840 845
Met Ala Leu Leu Gly Trp Val Pro Gly Pro Gln Val Gly Gly Val Phe
850 855 860
Gly Ala Gly Gly Asp Pro Ser Gln Pro Arg Phe Val His Asn Glu Ser
865 870 875 880
Gly Arg Phe Glu Cys Arg Phe Thr Ser Val Thr Ile Glu Glu Ser Pro
885 890 895
Ala Ile Met Phe Lys Gly Met Glu Gly Ser Thr Leu Gly Val Trp Ala
900 905 910
Ala His Gly Glu Gly Arg Ala Tyr Phe Pro Asp Asp Ser Val Phe Asn
915 920 925
His Ile Val Gly Ser Asn Leu Ala Pro Val Lys Tyr Cys Asp Asp Asp
930 935 940
Gly Arg Pro Thr Asp Ile Tyr Pro Phe Asn Leu Asn Gly Ser Pro Leu
945 950 955 960
Gly Val Ala Ala Ile Cys Ser Pro Asp Gly Arg His Leu AIa Ile Met
965 970 975
Pro His Pro Glu Arg Cys Phe Leu Met Trp Gln Phe Prv Trp Tyr Pro
980 985 990
Lys Asn Trp Asp Val Glu Lys Lys Gly Pro Ser Pro Trp Leu Arg Met
995 1000 1005
Phe Gln Asn Ala Arg Glu Trp Cys Ser
1010 1015
<210> 5
<211> 47B
<212> DNA
<213> Chilopsis linearis
<220>
<221> CDS
<222> (3)..(293)
24
i
CA 02388851 2002-04-23
<400> 5
ga att cgg cac gag gat gtt ttt ggc agt ata ctg gag tcg aac ttg 47
Ile Arg His Glu Asp Val Phe Gly Ser Ile Leu Glu Ser Asn Leu
1 5 10 15
get cct gtg aga tat tgt gat gat gtt ggg aat cct aca gaa gtc tat 95
Ala Pro Val Arg Tyr Cys Asp Asp Val Gly Asn Pro Thr Glu Val Tyr
20 25 30
cca ttc aac ctt aat ggt tct cct tta ggt gtt gca get att tgt tcc 143
Pro Phe Asn Leu Asn Gly Ser Pro Leu Gly Val Ala Ala Ile Cys Ser
35 40 45
cca gat ggg agg cat ctt gcc atg atg cct cat cca gag cgt tgc ttc 191
Pro Asp Gly Arg His Leu Ala Met Met Pro His Pro GIu Arg Cys Phe
50 55 60
ttg atg tgg cag tat ccg tgg tat ccc aag aac tgg aag gtg gag aag 239
Leu Met Trp Gln Tyr Pro Trp Tyr Pro Lys Asn Trp Lys Val Glu Lys
65 70 75
aaa ggt cct agt ccg tgg tta cgc atg ttt cag aat get agg gag tgg 287
Lys Gly Pro Ser Pro Trp Leu Arg Met Phe Gln Asn Ala Arg Glu Trp
80 85 90 95
tgt tca tgaggaaatg aaggcctaga agaataagaa ggataatact tggagaggac 343
Cys Ser
nactgaaaag ggatgttagt agctttttat gatttgaatt ggttttctga acttttgtag 403
aaagccctga gctaattttg gtgtttagtt gagattttca gaaaaaaaaa aaaaaaaaaa 463
aaaaaaaaac tcgag 478
<210> 6
<211> 97
<212> PRT
<213> Chilopsis linearis
<400> 6
Ile Arg His Glu Asp Val Phe Gly Ser Ile Leu Glu Ser Asn Leu Ala
1 5 10 15
Pro Val Arg Tyr Cys Asp Asp Val Gly Asn Pro Thr Glu Val Tyr Pro
20 25 30
Phe Asn Leu Asn Gly Ser Pro Leu Gly Val Ala Ala Ile Cys Ser Pro
CA 02388851 2002-04-23
35 40 45
Asp Gly Arg His Leu Ala Met Met Pro His Pro Glu Arg Cys Phe Leu
50 55 60
Met Trp Gln Tyr Pro Trp Tyr Pro Lys Asn Trp Lys Val Glu Lys Lys
65 70 75 80
Gly Pro Ser Pro Trp Leu Arg Met Phe Gln Asn Ala Arg Glu Trp Cys
85 90 95
Ser
26
