Note: Descriptions are shown in the official language in which they were submitted.
~~50/489Z4 CA 02325054 2000-09-27
AMP Deaminase
The present invention relates to a DNA coding for a polypeptide
with AMP deaminase (EC 3.5.4.6, adenosine triphosphate [sic]
aminohydrolase) activity. The invention also relates to the use
of a nucleic acid coding for a protein having AMP deaminase
activity of plant origin for producing a test system for
identifying AMP deaminase inhibitors. The invention further
relates to the use of the nucleic acid coding for plant AMP
deaminase for producing plants with increased resistance to AMP
deaminase inhibitors.
Plants are able to synthesize their cellular components from
carbon dioxide, water and inorganic salts.
This process is possible only by utilization of biochemical
reactions to synthesize organic substances. Plants also need to
synthesize de novo the nucleotides as components of the nucleic
acids.
It is to be assumed that efficient formation, utilization and
distribution of nucleotides influence cell division and the
growth of a plant. Since plants depend on a functioning nucleo-
tide metabolism, this metabolism is a possible target for the use
of herbicides. Some known agents (e. g. 5'-phosphohydantocidin,
alanosin or hadacidin) inhibit adenylosuccinate synthetase
(Stayton et al., 1983, Curr. Top. Cell. Regul., 22, 103-141;
Siehl et al., 1996, Plant Physiol., 110, 753-758). The reaction
catalyzed by adenylosuccinate synthetase is depicted in Figure 1.
Hydantocidin acts as a prodrug in this case. The agent is
metabolized in the plant by phosphorylation at the 5'OH group to
give the herbicide (Siehl et al., 1996, Plant Physiol., 110,
753-758).
An important principle for regulating nucleotide metabolism is,
besides the enzyme reactions of de novo purine biosynthesis (e. g.
adenylosuccinate synthetase), also recycling processes and
breakdown mechanisms. However, AMP deaminase occupies a special
position. Thus, it is also possible to deaminate AMP (adenosine
monophospate) again to give IMP (inosine 5'-phosphate). The
enzyme catalyzes the following reaction:
AMp + Hz0 <--> IMP + NH4 [sic]
0050/48924 CA 02325054 2000-09-27
a
2
The protein has been partially purified from various plants, and
its regulatory propert:Les have been investigated, for example
from spinach leaves (Yoshino and Murakami, 198 [sicj, Z.
Pflanzenphysiologie, 99, 331-338), Jerusalem artichokes (Le
Floc'h and Lafleuriel, 1983, Physiologie Vergetale [sic], 21 (1),
15-2), pea seeds (Turner and Turner, 1961, Biochem. J. 79, 143)
and cell cultures of Catharantus roseus, lesser periwinkle
(Yabuki et al. 1992, Phytochemistry, 31 (6), 1905-1909). It
moreover appears that t:he enzyme plays a central part in
regulation of the adenylate pool in a cell (Chapman and Atkinson,
1973, J. Biol. Chem., ~'.48, 8309; Solano and Coffee,1978; Yoshino
et al., 1979).
It has been shown that the known natural substance coformycin has
a herbicidal effect on plants, and that the,agent in its
phosphorylated form (5'-phosphocoformycin) is the actual AMP
deaminase inhibitor (Frieden et al., 1980, Biochem. 5303; Merkler
et al., 1990, Biochem. 29, 8358-8364; Dancer et al., 1997, Plant
Physiol., 114 (1), 119-129). The patent WO 96/01326 describes a
method for looking for herbicides in an AMP deaminase enzyme
assay (see also: Pillmc>or Pestic. Sci., 1998, 52, 75-80).
Genes which code for AMP deaminases have been isolated from many
organisms. In mammals there appear to be families of AMP
deaminase genes (Morisaki et al. 1990, J. Biol. Chem. 265 (20),
11482-11486). Further cading sequences have been isolated from
Schizosaccharomyces pombe (accession P50998) and Saccharomyces
cerevisiae (accession P15274). Only adenine deaminases are known
from bacteria, and it has not been possible to isolate AMP
deaminases. Expressed ~~equence tags, called est sequences, can be
found, by sequence comparisons, in rice (GenBank Acc: C26026) and
Arabidopsis (T21250) hawing similarity with yeast AMP dea,minases.
Complete cDNA sequences of plant AMP deaminases have not been
described as yet.
It is an object of the present invention to isolate a complete
plant cDNA coding for the enzyme AMP deaminase and to carry out
functional expression thereof in bacterial or eukaryotic cells to
obtain the enzyme in a simple and low-cost manner to carry out
inhibitor-enzyme binding studies.
It is a further object of the present invention to overexpress
the AMP deaminase gene in plants to produce plants which are
tolerant of AMP deamina.se inhibitors.
0050/48924 CA 02325054 2000-09-27
3
We have found that these objects have been achieved by isolating
the gene coding for the plant enzyme AMP deaminase, and the
functional expression thereof in bacterial or plant cells or
plants.
The present invention firstly relates to a DNA sequence SEQ ID
N0:1 comprising the coding region of a plant AMP deaminase from
Arabidopsis thaliana (see Figure 2).
The invention further relates to DNA sequences which are derived
from this SEQ ID N0:1 or hybridized with the latter and which
code for a protein which has the biological activity of an AMP
deaminase.
The invention also relates to expression cassettes whose sequence
[sic] code for an AMP df~aminase from Arabidopsis thaliana or the
functional equivalent thereof. The nucleic acid sequence can in
this connection be, for example a DNA or a cDNA sequence.
The expression cassettes according to the invention additionally
comprise regulatory nuc:Leic acid sequences which control the
expression of the coding sequence in the host cell. In a
preferred embodiment, an expression cassette according to the
invention comprises upsi:ream, i.e. at the 5' end of the coding
sequence, a promoter and downstream, i.e. at the 3' end, a
polyadenylation signal, and, where appropriate, further
regulatory elements which are operatively linked to the AMP
deaminase gene coding ss~quence lying between them. Operative
linkage means sequentia:L arrangement of promoter, coding
sequence, terminator and, where appropriate, further regulatory
elements in such a way that each of the regulatory elements can
carry out its function properly on expression of the coding
sequence.
An expression cassette according to the invention is produced by
fusing a suitable promoter to a suitable AMP deaminase DNA
sequence and a polyaden!~lation signal by conventional recom-
bination and cloning techniques as described, for example, in
T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W.
Enquist, Experiments wiith Gene Fusions, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et
al., Current Protocols :in Molecular Biology, Greene Publishing
Assoc. and Wiley-Interscience (1987).
~~5~/48924 CA 02325054 2000-09-27
4
Particularly preferred sequences are those which ensure targeting
in the apoplasts, in plastids, the vacuoles, the mitochondrion,
the endoplasmic reticul.um (ER) or, owing to the absence of
appropriate operative sequences, ensure retention in the
compartment of production, the cytosol (Kermode, Crit. Rev. Plant
Sci. 15, 4 (1996), 285--423).
For example, the plant expression cassette can be incorporated
into the tobacco transformation vector pBinAR-Hyg (see
Example 5).
Suitable in principle as promoter for the expression cassette
according to the invention is every promoter able to control
expression of foreign genes in plants. It is particularly
preferred to use a plant promoter or a promoter derived from a
plant virus. The CaMV 35S-promoter from cauliflower mosaic virus
(Franck et al., Cell 27.(1980) 285-294) is particularly preferred.
This promoter contains various recognition sequences for trans-
criptional effectors which, in their totality, lead to permanent
and constitutive expression of the introduced gene (Benfey et
al., EMBO J. 8 (1989) 2195-2202).
The expression cassette' according to the invention can also
comprise a chemically .i.nducible promoter through which it is
possible to control expression of the exogenous AMP deaminase
gene in the plant at a particular point in time. Promoters of
this type, such as, fox- example, the PRP1 promoter (Ward et al.,
Plant. Mol. Biol. 22(1993), 361-366), a promoter inducible by
salicylic acid (WO 95/7.919443), a benzenesufonamide-inducible
[sic] (EP 388186), a tetracycline-inducible (Gatz et al., (1992)
Plant J. 2,397-404), an abscisic acid-inducible (EP335528) or an
ethanol- or cyclohexanone-inducible (W09321334) promoter are
described in the literature and can, inter alia, be used.
Further particularly preferred promoters are those which ensure
expression in tissues or plant parts in which the biosynthesis of
purines or their precux-sors takes place. Particular mention may
be made of promoters which ensure leaf-specific expression.
Mention should be made of the promoter of the potato cytosolic
FBPase or the potato ST-LSI promoter (Stockhaus et al., EMBO J. 8
(1989) 2445-245 [sic])"
It is possible with the' aid of a seed-specific promoter to
express a foreign protein stably up to a content of 0.67 of the
total soluble seed protein in the seeds of transgenic tobacco
plants (Fiedler and Conrad, Bio/Technology 10(1995), 1090-1094).
The expression cassette: according to the invention can therefore
0050/48924 CA 02325054 2000-09-27
comprise, for example, a seed-specific promoter (preferably the
phaseolin promoter, thcs USP or LEB4 promoter), the LEB4 signal
peptide, the gene to be expressed and an ER retention signal.
5 Many legumes transport fixed nitrogen in the form of ureides from
the nodules to the above-ground tissue (Schubert, 1986, Ann. Rev.
Plant Phys. 37, 539-574). Ureides are formed via purine
biosynthesis. It is possible with the aid of a node-(nodule-)
specific promoter for i3MP deaminase expression and thus ureide
biosynthesis in legumes to be modified specifically. Gene
expression cassettes according to the invention may accordingly
comprise the promoter of the phosphoribosyl-pyrophosphate
amidotransferase from Glycine max (see also Genbank Accession
Number U87999) or another node-specific promoter as described in
EP 249676.
The inserted nucleotide: sequence coding for an AMP deaminase can
be prepared synthetica:Lly or be obtained naturally or comprise a
mixture of synthetic and natural DNA components. In general,
synthetic nucleotide sequences are produced with codons preferred
by plants. These codon:~ preferred by plants can be determined
from codons which have the highest protein frequency and are
expressed in most plant species of interest. To prepare an
expression cassette it is possible to manipulate various DNA
fragments in order to obtain a nucleotide sequence which
expediently reads in the correct direction and is equipped with a
correct reading frame. Adapters or linkers can be attached to the
fragments to connect the DNA fragments together.
The sequence homology between yeast and plant AMP deaminases at
the DNA level is, based on a selected highly homologous region,
57$, for example in the 1345-2715 base region of the sequence
M30449 (Genbank Accession Number), established using the BLAST
program (Altschul et a:L.,(1990), J. Mol. Bio1.:215:403-419; Gish
and States,(1993), Nature Genet. ~:266-272). In the fragment
described, regions of ;ZO-30 nucleotides are so homologous that
there is a sufficient probability of success for an
oligonucleotide-based screening for AMP deaminase from other
plants. Methods of hybridization with short oligonucleotides to
detect DNA sequences which have good homology only in small
regions with respect to the comparison sequence are described in
Sambrook et al. (1989, Cold Spring Harbor Laboratory Press: ISBN
0-87969-309-6).
~~5~/48924 CA 02325054 2000-09-27
6
The invention also relates to functionally equivalent DNA
sequences which code for an AMP deaminase gene [sic) and which,
based on the total length of the gene, have a sequence homology
of from 40 to 100 with the DNA sequence SEQ ID NO: 1.
The invention preferably relates to functionally equivalent DNA
sequences which code for an AMP deaminase gene [sic] and which,
based on the total length of the gene, have a sequence homology
of from 60 to 100 with the DNA sequence SEQ ID NO: 1.
The invention particularly preferably relates to functionally
equivalent DNA sequences which code for an AMP deaminase gene
[sic) and which, based on the total length of the gene, have a
sequence homology of from 80 to 100 with the DNA sequence SEQ ID
N0: 1.
Functionally equivalent sequences coding for an AMP deaminase
gene [sic] are, according to the invention, those sequences
which, despite a differing nucleotide sequence, still have the
required functions. Functional equivalents thus comprise
naturally occurring variants of the sequences described herein,
and artificial artificial [sic] nucleotide sequences, for example
obtained by chemical synthesis, adapted to the codon usage of a
plant.
A functional equivalent also means in particular natural or
artificial mutations of an originally isolated sequence coding
for an AMP deaminase, which still show the required function.
Mutations comprise substitutions, additions, deletions,
transpositions or insertions of one or more nucleotide residues.
This means, for example, that the present invention also
comprises those nucleotide sequences which are obtained by
modifying this nucleotide sequence. The aim of such a modifi-
cation may be, for example, further localization of the coding
sequence present therein or else, for example, insertion of
further restriction enzyme cleavage sites.
Functional equivalents are also those variants whose function is
attenuated or enhanced by comparison with the initial gene or
gene fragment.
Artificial DNA sequences are also suitable as long as they
confer, as described above, the required property of increasing
the IMP content in the plant by overexpression of the AMP
deaminase gene in crop plants. Such artificial DNA sequences can
be found, for example, by translation back from proteins having
AMP deaminase activity and constructed by molecular modeling, or
0050/48924 CA 02325054 2000-09-27
7
by in vitro selection. Particularly suitable coding DNA sequences
are those obtained by translation back from a polypeptide
sequence in accordance with the codon usage specific for the host
plant. The specific codon usage can easily be found by a skilled
worker familiar with methods of plant genetics by computer
analyses of other known genes of the plant to be transformed.
Further suitable equivalent nucleic acid sequences according to
the invention which may be mentioned are sequences coding for
fusion proteins where one constituent of the fusion protein is a
plant AMP deaminase 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 whose aid it is possible to
detect AMP deaminase expression (e. g. myc tag or his tag).
However, this is preferably a regulatory protein sequence such
as, for example, a signal or transit peptide which guides the AMP
deaminase protein to the required site of action.
The promoter regions according to the invention and the
terminator regions ought expediently to 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, usually 1 to 8, preferably 2 to
6, restriction sites. The size of the linker within the
regulatory regions is generally less than 100 bp, frequently less
than 60 bp, but at least 5 bp. The promoter according to the
invention may be both native or homologous and foreign or
heterologous to the hast plant. The expression cassette according
to the invention comprises in the 5'-3' direction of trans-
cription the promoter according to the invention, any suitable
sequence and a region for transcription termination. Different
termination regions can be exchanged for one another as desired.
It is furthermore possible to employ manipulations which provide
suitable restriction cleavage sites or delete the excess DNA or
restriction cleavage sites. Where insertions, deletions or
substitutions such as, for example, transitions and transversions
are considered, it is possible to use in vitro mutagenesis,
primer repair, restriction or ligation. In the case of suitable
manipulations such as, for example, restriction, chewing-back or
filling in of overhangs for blunt ends, complementary ends of the
fragments can be made available for the ligation.
Particularly important for the result according to the invention
is the attachment of th.e specific ER retention signal SEKDEL
~(Schouten, A. et al. Plant Mol. Biol. 30 (1996), 781 - 792),
0050/48924 CA 02325054 2000-09-27
8
which triples or quadruples the average level of expression. It
is also possible to employ other retention signals which
naturally occur in plant and animal proteins located in the ER
for constructing the cassette.
Preferred polyadenylation signals are plant polyadenylation
signals, preferably those which essentially correspond to T-DNA
polyadenylation signals from Agrobacterium tumefaciens,
especially of gene 3 of the T-DNA (octopine synthase) of the Ti
plasmid pTiACHS (Gielen et al., EMBO J. 3 (1984) 835 ff.) or
functional equivalents.
To transform a host plant with a DNA coding for an AMP deaminase,
an expression cassette according to the invention is incorporated
as insert 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), Chapter 6/7, pages 71-119.
The transfer of foreign genes into the genome of a plant is
referred to as transformation. The methods used for this purpose
are those described for the transformation and regeneration of
plants from plant tissues or plant cells for transient or stable
transformation. Suitable methods are protoplast transformation by
polyethylene glycol-induced DNA uptake, the biolistic approach
with the gene gun, electroporation, incubation of dry embryos in
DNA-containing solution, microinjection and gene transfer
mediated by agrobacterium. The methods mentioned are described,
for example, in 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
in 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 transforming
Agrobacterium tumefacie.ns, for example pBinl9 (Bevan et al.,
Nucl. Acids Res. 12 (1984) 8711).
Agrobacteria transformed with an expression cassette according to
the invention can likewise be used in a known manner for
transforming plants, especially crop plants such as cereals,
corn, soybean, rice, cotton, sugarbeet, canola, sunflower, flax,
hemp, potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and
the various tree, nut and vine species, and legumes, for example
by bathing wounded leaves or pieces of leaves in a solution of
agrobacteria and then cultivating in suitable media.
0050/48924 CA 02325054 2000-09-27
9
The site of purine biosynthesis is generally the leaf tissue, so
that leaf-specific expression of the AMP deaminase gene is
sensible. However, it is obvious that purine biosynthesis need
not be confined to leaf tissue but may also take place in all
other parts of the plant, for example in fat-containing seeds,
tissue-specifically.
Many legumes transport fixed nitrogen in the form of ureides from
the nodules to the above-ground tissue (Schubert, 1986, Ann. Rev.
Plant Phys. 37, 539-574). Ureides are formed via purine
biosynthesis. It is possible with the aid of a node-(nodule-)
specific promoter for AMP deaminase expression and thus ureide
biosynthesis in legumes to be modified specifically, for example
using the promoter of the phosphoribosyl-pyrophosphate
amidotransferase from Glycine max (see also Genbank Accession
Number U87999) or using another node-specific promoter (described
for example in EP 249676.
In addition, constitutive expression of the exogenous AMP
deaminase gene is advantageous. However, on the other hand,
inducible expression may also appear desirable.
Using the recombination and cloning techniques quoted above, the
expression cassettes according to the invention can be cloned
into suitable vectors which make it possible to replicate them,
for example in E. coli. Suitable cloning vectors are, inter alia,
pBR332 [sic], the pUC series, the Ml3mp series and pACYC184.
Binary vectors able to replicate both in E. coli and in
agrobacteria are particularly suitable.
35
The invention further relates to the use of an expression
cassette according to the invention for transforming plants,
plant cells, plant tissues or parts of plants. The aim of the use
is preferably to increase the AMP deaminase content in the plant.
This may involve, depending on the chosen promoter, expression
specifically in the leaves, in the seeds or other parts of the
plant. The present invention further relates to such transgenic
plants, their propagation material and their plant cells, tissue
or parts.
The expression cassette according to the invention can in
addition be employed for transforming bacteria, cyanobacteria,
yeasts, filamentous fungi and algae with the aim of producing
adequate amounts of the enzyme AMP deaminase.
0050/48924 CA 02325054 2000-09-27
The invention further relates to a protein from Arabidopsis
thaliana having the amino acid sequence SEQ ID N0:2 or
derivatives or parts of this protein with AMP deaminase activity.
Compared with Saccharomyces cerevisiae, the homology at the amino
5 acid level is 43 - 47~ identity (see Figure 4).
The invention also relates to plant proteins having AMP deaminase
activity with an amino acid sequence homology with the
Arabidopsis thaliana AMP deaminase of 20 - 100 identity.
Plant proteins having AMP deaminase activity with an amino acid
sequence homology with the Arabidopsis thaliana AMP deaminase of
50 - 100 identity are preferred.
Plant proteins having AMP deaminase activity with an amino acid
sequence homology with the Arabidopsis thaliana AMP deaminase of
80 - 100 identity are particularly preferred.
As already mentioned, the AMP deaminase is a suitable target for
herbicides. In order to be able to find even more efficient AMP
deaminase inhibitors, it is necessary to provide suitable test
systems with which inhibitor-enzyme binding studies can be
carried out. For this purpose, for example, the complete cDNA
sequence of the AMP dea:minase from Arabidopsis thaliana is cloned
into an expression vector (pQE, Qiagen) and overexpressed in
E. coli (see Example 3).
The AMP deaminase protein expressed with the aid of the
expression cassette according to the invention is particularly
suitable for finding inhibitors specific for AMP deaminase.
To this end, the AMP deaminase can be employed, for example, in
an enzyme assay in which the activity of the AMP deaminase is
measured in the presence and absence of the agent to be tested.
Comparison of the two activity determinations allows a
qualitative and quantitative statement to be made about the
inhibiting behavior of the agent to be tested (see Example 4).
It is possible with the aid of the test system according to the
invention to check a large number of chemical compounds rapidly
and straightforwardly for herbicidal properties. The method
allows reproducible selection from a large number of substances
specifically those with great potency in order for further tests
in depth, which are familiar to the skilled worker, then to be
carried out with these substances.
0050/48924 CA 02325054 2000-09-27
11
The invention further relates to herbicides identifiable using
the test system described above.
Overexpression of the gene sequence Seq ID N0: 1 coding for an
AMP deaminase in a plant achieves increased resistance to AMP
deaminase inhibitors. The invention likewise relates to the
transgenic plants produced in this way.
The efficiency of expression of the transgenically expressed AMP
deaminase gene can be measured, for example, in vitro by shoot
meristem propagation or by a germination test. In addition, a
change in the nature anal level of the expression of the AMP
deaminase gene and the effect thereof on the resistance to AMP
deaminase inhibitors ca.n be tested on test plants in glasshouse
experiments.
The invention additionally relates to transgenic plants
transformed with an expression cassette according to the
invention, and to trans.genic cells, tissues, parts and
propagation material of such plants. Particular preference is
given in this connection to transgenic crop plants such as, for
example, barley, wheat, rye, corn, soybean, rice, cotton,
sugarbeet, canola, sunflower, flax, hemp, potato, tobacco,
tomato, oilseed rape, alfalfa, lettuce and the various tree, nut
and vine species, and legumes.
The invention further relates to plants which, after expression
of the DNA-SEQ ID N0:1 in the plant, have an increased IMP
content.
An increased inosine 5'-phosphate (IMP) content means for the
purpose of the present invention the artificially acquired
capability of increasedl IMP biosynthesis owing to functional
overexpression of the F~MP deaminase gene in the plant compared
with the plant which ha.s not been genetically manipulated for the
duration of at least one plant generation.
The invention is illustrated by the examples which now follow,
but is not confined to these:
Examples
A. Methods of genetic manipulation on which the examples are
based:
General cloning method~o
~05~/48924 CA 02325054 2000-09-27
12
Cloning methods such as, for example, restriction cleavages,
agarose gel electrophoresis, purification of DNA fragments,
transfer of nucleic acids to nitrocellulose and nylon membranes,
linkage of DNA fragments, transformation of Escherichia coli
cells, cultivation of bacteria and 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).
Sequence analysis of recombinant DNA
Recombinant DNA molecules were sequenced using a laser
fluorescence DNA sequencer supplied by ABI by the method of
Sanger (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA74,
5463-5467). Fragments resulting from a polymerase chain reaction
were sequenced and checked to avoid polymerase errors in
constructs to be expressed.
Analysis of complete RNA from plant tissues
Complete RNA from plant: tissues was isolated as described by
Logemann et x1.((1987) Anal. Biochem. 163, 21). For the analysis,
in each case 20 mg of RNA were fractionated in a
formaldehyde-containing 1.5~ agarose gel and transferred to nylon
membranes (Hybond, Amersham). Specific transcripts were detected
as described by Amasino ((1986) Anal. Biochem. 152, 304). The
cDNA fragments employed as probe were radiolabeled using a random
primed DNA labeling kit: (Boehringer, Mannheim) and hybrized [sic]
by standard methods (see Hybond information for users, Amersham).
Hyridization [sic] signals were visualized by autoradiography
using X-GMAT AR films supplied by Kodak.
The bacterial strains (E. coli, XL-I [sic] Blue) used hereinafter
were purchased from Stratagene or Pharmacia in the case of NP66.
The agrobacterium strain used for plant transformation
(Agrobacterium tumefaciens, C58C1 with the plasmid pGV2260 or
pGV3850kan) has been described by Deblaere et al. (Nucl. Acids
Res. 13 (1985) 4777). Alternatively, it is also possible to use
the agro- bacterium strain LBA4404 (Clontech) or other suitable
strains. Vectors which can be used for cloning are pUCl9
(Vanish-Perron, Gene 3x(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 ;>cience 66 (1990) 221-230).
Example 1
0050/48924 CA 02325054 2000-09-27
13
PCR-Amplification of the AMP deaminase gene using synthetic
oligonucleotides.
The Arabidopsis est clone coding for the AMP deaminase was
purchased from the Arabidopsis Biological Resource Center (Ohio
State University). This is a partial cDNA clone (T21250) which
does not correspond to the full-length transcript of the AMP
deaminase. PCR amplific:ation of the arabidopsis AMP deaminase
fragment was carried out in a DNA thermal cycler supplied by
Perkin Elmer. The oligonucleotides used, 5' primer
CGAGATAGCTCGTAAC and 3'' primer AGCCCACTCATATTATT, were taken from
the established sequence. The reaction mixtures contained 8 ng/~1
genomic DNA from Eschez:ichia coli, 0.5 ~M of the appropriate
oligonucleotides, 200 yM nucleotides (Pharmacia), 50 mM KC1,
10 mM tris-HC1 (pH 8.3 at 25~C, 1.5 mM MgCl2) and 0.02 U/~l Taq
polymerase (Perkin Elmer).
The amplification conditions were set as follows:
Annealing temperature: 52~C, 1 min
Denaturing temperature:. 92~C, 1 min
Elongation temperature:. 72~C, 1.5 min
Number of cycles: 40
The resulting fragment comprises a small part of the est clone
T21250, which was used to carry out a heterologous screening of
an Arabidopsis thaliana cDNA Bank (Stratagene). 3.0 x 105 lambda
phages of the Arabidopsis thaliana cDNA library (Stratagene) were
plated out on agar plal:es with E. coli XLI-Blue [sic] as
bacterial strain. The phage DNA was transferred by standard
methods (Sambrook et a7L. (1989); Cold Spring Harbor Laboratory
Press: ISBN 0=87969-309-6) to nitrocellulose filters (Gelman
Sciences) and fixed on the filters. The hybridization probes
[sic] used was the PCR fragment described above, which were [sic]
radiolabeled using a multiprime DNA labeling system (Amersham
Buchler) in the present:e of a-32P-dCTP (specific activity
3000 Ci/mmol) in accordance with the manufacturer's instructions.
Hybridization of the membranes took place after prehybridization
at 60~C in 3 x SSPE, 0.1~ sodium dodecyl sulfate (w/v), 0.02
polyvinylpyrolidone [sic] (w/v), 0.02 Ficoll 400 (w/v) and 50
mg/ml calf thymus DNA for 12-16 hours (Sambrook et al. (1989);
Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6). The
filters were then washed in 2 x SSPE, 0.1$ sodium dodecyl sulfate
(w/v) at 60~C for 60 minutes. Positively hybridizing phages were
visualized by autoradiography and purified and isolated by
standard techniques.
~~5~/48924 CA 02325054 2000-09-27
14
Example 2
Sequence analysis of the cDNA clones coding for a protein having
AMP deaminase activity..
64 cDNA clones coding for a polypeptide with great homologies
with yeast AMP deaminase resulted (see Figure 4). The two longest
clones are identical and show in the restriction digest an EcoRI
fragment 2880 base pairs long. This longest clone coding for the
AMP deaminase from Arabidopsis thaliana is called AMP1. The
plasmid is referred to as pBS-AMP1. The cDNA (see Figure 2) has
an open reading frame of 2578 base pairs with a stop codon in
position 2579-2581. The' amino acid sequence starts with the third
base after the linker sequences (bold-printed ends of the
sequence in Figure 2) in the third reading frame and can be
translated into a polypeptide 860 amino acids long, or, from the
first methionine start codon, into a polypeptide of 839 amino
acids (see Figure 3). Another alternative is to use the
methionine in position 46, so that a polypeptide of 824 amino
acids would result.
Whereas the protein aci:ivity is located in the cytosol in yeast,
a cytosolic and a potentially mitochondrial activity has been
described in plants (Yoshino and Murakami, 198, Z.
Pflanzenphysiologie, 99, 331-338). On the basis of the reading
frame of the present cDNA sequence, it can be inferred that it
might possibly be the mitochondrial form or the cytosolic form,
either of which can arise by use of different start codons. It is
noteworthy that with the start of the reading frame from base 3
of the coding sequence a large number of hydrophilic serine and
threonine residues alternating with aliphatic amino acid residues
are present in front o:E the methionine codons, which indicates a
transit sequence for mitochondria or plastids. It is generally
assumed that purine biosynthesis takes place in plastids.
However, it has recent:Ly been shown that the enzymes of purine
biosynthesis are also :Located in mitochondria (Atkins et al.,
1997, Plant Physiol. 113, 127-135). On the basis of the reading
frame of the present cIDNA sequence, it is obvious to assume that
it might possibly be t:he mitochondrial, plastidial or cytosolic
form, each of which results by use of different start codons. The
two longest clones show a continuous open reading frame with two
potential start codons at the N terminus. Compared with the yeast
sequence, the second start codon is approximately at the position
where the yeast sequence also starts (see Figure 4). Over the
entire length of the protein, the plant sequence is about 59-63~
similar to the yeast sequence and about 43-47~ identical,
depending on the choice of the comparison parameters (Lasergene
0050/48924 CA 02325054 2000-09-27
Software, MegAlign Program). It is moreover noticeable that the N
terminus of the plant AMP deaminase shows only little homology in
contrast to the C terminus of the yeast sequence.
5 Example 3
Production of overexpression vectors in E. coli
The following oligonucl.eotide sequences were derived from the
10 established sequence and were provided with a BamHI restriction
cleavage site and to protruding bases. The oligonucleotides are
underlined and numbered in Figure 2. Potential methionine start
codons are shown bold.
15 1. 5' Primer aaggatccATGTTACTCTCTCTTCTGAG,
2. 5' Primer aaggatccATGGAACCCAATATTTAC,
3. 5' Primer aaggatccATGCATTTCAAGGCAC,
4. 3' Primer aaggatcc'.CTATGGAACAACTTCATCAG.
The use of the three different 5' primers in combination with the
3' primer lead [sic] to PCR products of different lengths
(fragment I, II, III), which correspond to the complete reading
frame (fragment I, primers 1 + 4), the sequence from the first
Met start codon (fragment II, primers 2 + 4) and from the second
start codon (fragment I:II, [lacuna] 3 + 4).
The PCR reaction [sic] mixtures contained 8 ng/~1 pBS-AMP1 DNA,
0.5 ~M of the appropriate oligonucleotides, 200 ~M nucleotides
(Pharmacia), 50 mM KC1, 10 mM tris-HC1 (pH 8.3 at 25~C, 1.5 mM
MgCl2) and 0.02 U/~1 Ta.q polymerase (Perkin Elmer). The
amplification conditions were set as follows:
Annealing temperature: 52~C, 1 min
Denaturing temperature:. 92~C, 1 min
Elongation temperature:: 72~C, 2.5 min
Number of cycles: 30
The PCR fragments were cloned into the overexpression vectors
pETlSb, pETlla and pQE9 and employed for protein production by
means of IPTG induction by standard methods (see handbook: The
Quiaexpressionist [sic] (1992), Quiagen [sic], Hilden).
X050/48924 CA 02325054 2000-09-27
16
Example 4
Enzyme assay of the plant AMP deaminase from E. coli resulting
from overexpression cultures
E.coli was disrupted by the pressure disruption method in a
French Press under maximum pressure in a 20 ml pressure chamber
or with the aid of a glass bead mill (IMA disintegrator). 10 ml
of buffer (O.1M KHzP04; pH 7.5; 0.4M sucrose, 0.1 mM DTT) are used
per 1 g of cell pellet. The pellet is disrupted by addition of
2.5 times the amount of glass beads (d=0.5 mm) in the glass bead
mill at 4°C and 2500 rpm for 20 min. The disrupted material is .
centrifuged at 4°C and 100,000 g for 20 minutes. The enzyme
activity was determined. in a photometric assay by measurement at
260 nm in a photometer (Uvikon 933, Kontron). The choice of the
overexpression vectors also made it possible to purify the AMP
deaminase to homogeneity in one step via the histidine anchor by
standard methods when D~TT was omitted from the disruption buffer
(compare also the handbook: The Quiaexpressionist [sic], Quiagen
[sic], Hilden).
The homogenate buffer was changed in the following medium by
dialysis in 40 mM citrate, pH 6.5 (adjusted with 5 N NaOH), 0.05%
BSA (w/v), 100 mM KC1.
10-100 ~.1 portions of the enzyme fraction in the changed buffer
were made up to 700 ~1 with buffer and, by addition of 100 ~1 of a
1 mM AMP solution, 0.5 mM ATP solution and 1 ~M diadenosine
pentaphosphate solutior.~, the decrease in extinction was measured
for 2-10 min using a reference cuvette with 700 ~.1 of reaction
buffer and 100 ~1 of a protein homogenate from an untransformed
E. coli culture. Identical amounts of total protein were employed
for the measurements of: the reference relative to the measured
value.
Example 5
Production of plant expression cassettes
A 35S CaMV promoter wa:~ inserted as EcoRI-KpnI fragment
(corresponding to nucleotides 6909-7437 of cauliflower mosaic
virus (Franck et al. (1980) Cell 21, 285)) into the plasmid
pBinl9 (Bevan et al. (7.980) Nucl. Acids Res. 12, 8711). The
polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid
pTiACHS (Gielen et al., (1984) EMBO J. 3, 835), nucleotides
11749-11939, was isolated as PvuII-HindIII fragment and, after
addition of SphI linkers, cloned onto the PvuII cleavage site
~05~~48924 CA 02325054 2000-09-27
17
between the SpHI-HindI:CI (sic) cleavage site of the vector. The
result was the plasmid pBinAR (HSfgen and Willmitzer (1990) Plant
Science 66, 221-230). The PCR fragments I, II and III (see above)
were cloned into the BamHI cleavage site of the pBinAR vector in
both orientations and employed for transforming tobacco plants.
The resulting plasmids are referred to as pBinAMP-20, pBinaAMP-0,
pBinAMP-0, pBinaAMP -0, pBinAMP+26, pBinaAMP+26 and correspond to
the fragments I, II, III, described in Example 3, from the PCR
mixtures described abo~re.
Example 6
Production of transgenic tobacco plants
The plasmids pBinAMP-20, pBinaAMP-20, pBinAMP-0, pBinaAMP-0,
pBinAMP+26, pBinaAMP+2fi were transformed into Agrobacterium
tumefaciens C58C1:pGV2:?60 (Deblaere et al, 1984, Nucl. Acids.
Res. 13, 4777-4788). Tobacco plants (Nicotiana tabacum cv. Samsun
NN) were transformed using a 1:50 dilution of an overnight
culture of a positively transformed agrobacterium colony in
Murashige-Skoog medium {(1962) Physiol. Plant. 15, 473) with 2%
sucrose (2MS medium). Leaf disks from sterile plants (about 1 cm2
each) were incubated in a Petri dish with a 1:50 agrobacterium
dilution for 5-10 minui~es. This was followed by incubation in the
dark at 25°C on 2MS medium with 0.8% Bacto agar for 2 days. The
cultivation was continued after 2 days with 16 hours light/8
hours dark and continued in a weekly rhythm on MS medium 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 with 2%
sucrose, 250 mg/1 Claforan and 0.8% Bacto agar.
Regenerated shoots are obtained on 2MS medium with kanamycin and
Claforan and, after rooting, transferred to soil and, after
cultivation for two wea_ks in a climate chamber with 16 hours
light/8 hours dark rhythm at 60% humidity, investigated for
foreign gene expression or changed metabolite contents and
phenotypical growth features. Changed nucleotide contents can be
determined, for example, by the method of Stitt et al. (1982,
FEBS Letters, 145, 217-222).
Example 7
To demonstrate the tolerance of AMP deaminase-overexpressing
transgenic tobacco plants to AMP deaminase inhibitors, the latter
were treated with various amounts of coformycin or other AMP
0050/48924 CA 02325054 2000-09-27
1$
deaminase inhibitors. :Ct was possible to show in a glasshouse in
all cases that the plants overexpressing an AMP deaminase show
tolerance to the inhibitors employed by comparison with the
control.
10
20
30
40
CA 02325054 2000-09-27
SEQUENZPfZOTOKOLL
<110> BASF AG
<120> AMP-Deaminase
<130> 19814512.8
<140> 48924
<141> 1998-04-O1
<160> 2
<170> PatentIn Vers. 2.0
<210> 1
<211> 2880
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (78)..!2594)
<400> 1
gaattcggca cgaggtctta ctctctcttc tgagttctga cctgagcaca cacacagaga 60
ttttgattgt gtcactc atg gaa ccc aat att tac caa ctt gcc ctc gcg 110
Met Glu Pro Asn Ile Tyr Gln Leu Ala Leu Ala
1 5 10
get cta ttc gga get tcc ttc gtt get gtt tct ggg ttt ttc atg cat 158
Ala Leu Phe Gly Ala Ser Phe Val Ala Val Ser Gly Phe Phe Met His
15 20 25
ttc aag gca ctg aat cta gtc ctt gag cgt ggt aag gag cgt aaa gag 206
Phe Lys Ala Leu Asn Leu Val Leu. Glu Arg Gly Lys Glu Arg Lys Glu
30 35 40
aac cct gat gga gac gag cct caa. aat ccg acc ttg gtg agg cgg cgg 254
Asn Pro Asp Gly Asp Glu Pro Gln. Asn Pro Thr Leu Val Arg Arg Arg
45 50 55
agc caa gtt aga agg aag gtt aat. gac caa tat ggt cgc agt cct get 302
Ser Gln Val Arg Arg Lys Val Asn Asp Gln Tyr Gly Arg Ser Pro Ala
60 65 70 75
tct ctt cca gat gcc act cct ttt. acc gat ggt ggc ggc ggc ggc ggc 350
1.
CA 02325054 2000-09-27
Ser Leu Pro Asp Ala Thr Pro Phe Thr Asp Gly G1y Gly Gly Gly Gly
80 85 90
ggt gat aca cga cgg agc aac ggt: cac gtt tat gtc gat gaa att cct 398
Gly Asp Thr Arg Arg Ser Asn Gly His Val Tyr Val Asp Glu Ile Pro
95 100 105
cct ggt ctc cct agg ctt cat acct cca tct gaa ggg aga get tct gta 446
Pro Gly Leu Pro Arg Leu His Thr Pro Ser Glu Gly Arg Ala Ser Val
110 11 5 120
cat gga get agt agt atc agg aaa act gga agc ttt gtt aga cca ata 494
His Gly Ala Ser Ser Ile Arg Lys Thr Gly Ser Phe Val Arg Pro Ile
125 130 135
tct ccg aaa tcc cct gtt get agt: get agt get ttt gag agt gtg gaa 542
Ser Pro Lys Ser Pro Val Ala Ser Ala Ser Ala Phe G1u Ser Val Glu
140 145 150 155
gaa tca gat gat gat gat aat ttc~ act aat agt gag ggt tta gat get 590
Glu Ser Asp Asp Asp Asp Asn Leu. Thr Asn Ser Glu Gly Leu Asp Ala
160 165 170
tcc tac ttg caa get aat ggt gac aat gag atg cct gca gat get aat 638
Ser Tyr Leu Gln Ala Asn Gly Asp Asn Glu Met Pro Ala Asp Ala Asn
175 180 185
gaa gaa caa ata tct atg get gct. tca agt atg att cga tcc cat agt 686
Glu Glu Gln Ile Ser Met Ala Ala. Ser Ser Met Ile Arg Ser His Ser
190 195 200
gtg tct ggt gac tta cat gga gtt. cag ctg agt cct att get get gac 734
Val Ser Gly Asp Leu His Gly Val Gln Leu Ser Pro Ile Ala Ala Asp
205 210 215
att ctt cgt aag gag cca gag caa. gag acc ttt gtc cgt ctt aat gtt 782
Ile Leu Arg Lys Glu Pro Glu Gln Glu Thr Phe Val Arg Leu Asn Val
220 225 230 235
cct ctt gag gtg cca acg tcc gat. gaa gtt gaa gcc tat aaa tgt ctg 830
Pro Leu Glu Val Pro Thr Ser Asp Glu Val Glu Ala Tyr Lys Cys Leu
240 245 250
caa gaa tgt ctt gaa ctg cgg aac~ agg tat gtc ttc caa gaa aca gtt 878
Gln Glu Cys Leu Glu Leu Arg Lys; Arg Tyr Val Phe Gln-Glu Thr Val
255 260 265
gca cca tgg gaa aaa gaa gtc ata tct gat cct agt act cca aag cct 926
L.
CA 02325054 2000-09-27
Ala Pro Trp Glu Lys Glu Val Ile Ser Asp Pro Ser Thr Pro Lys Pro
270 275 280
aat aca gag cca ttt gca cac tat cct cag gga aaa tct gat cat tgt 974
Asn Thr Glu Pro Phe Ala His Tyr Pro Gln Gly Lys Ser Asp His Cys
285 290 295
ttt gag atg caa gat ggg gtt gtc cac gtg ttt gca aat aaa gat gca 1022
Phe Glu Met Gln Asp Gly Val Val His Val Phe Ala Asn Lys Asp Ala
300 305 310 315
aaa gaa gat ctc ttc ccg gta get gat gcc aca gcg ttt ttc act gac 1070
Lys Glu Asp Leu Phe Pro Val Ala Asp Ala Thr Ala Phe Phe Thr Asp
320 325 330
ttg cat cac gta ctc aaa gtc ata get gca gga aac atc cgg act ttg 1118
Leu His His Val Leu Lys Val Ile A1a Ala Gly Asn Ile Arg Thr Leu
335 340 345
tgc cac cgt cga cta gtg ctc cta gaa cag aaa ttt aat ctc cat ttg 1166
Cys His Arg Arg Leu Val Leu Leu Glu Gln Lys Phe Asn Leu His Leu
350 355 360
atg ctt aat gcg gat aaa gaa ttt ctt get caa aaa agt gca cca cat 1214
Met Leu Asn Ala Asp Lys Glu Phe Leu Ala Gln Lys Ser Ala Pro His
365 370 375
cgt gat ttt tat aac gtt agg aaa gtc gac act cat gtg cat cat tca 1262
Arg Asp Phe Tyr Asn Val Arg Lys Val Asp Thr His Val His His Ser
380 385 390 395
get tgc atg aac cag aaa cac ctt tta agg ttt att aag tca aag ctc 1310
Ala Cys Met Asn Gln Lys His Leu Leu Arg Phe Ile Lys Ser Lys Leu
400 405 410
cgg aaa gaa ccc gat gag gtt gta ata ttc cga gat gga aca tat ttg 1358
Arg Lys Glu Pro Asp Glu Val Val Ile Phe Arg Asp Gly Thr Tyr Leu
415 420 425
acc ttg aga gaa gtt ttt gag agc ctg gat ctg act gga tat gac ctg 1406
Thr Leu Arg Glu Val Phe Glu Ser Leu Asp Leu Thr Gly Tyr Asp Leu
430 435 440
aac gtc gac ctt ttg gat gtt cat gca gac aaa agt acc ttt cat cgt 1454
Asn Val Asp Leu Leu Asp Val His Ala Asp Lys Ser Thr Phe His Arg
445 450 455
ttt gat aag ttc aac cta aag tat aac cct tgt ggt caa agt agg ctt 1502
3
CA 02325054 2000-09-27
Phe Asp Lys Phe Asn Leu Lys Tyr Asn Pro Cys Gly Gln Ser Arg Leu
460 465 470 475
agg gag att ttc ctt aaa cag gat: aat ctc atc caa ggt cga ttt ctt 1550
Arg Glu Ile Phe Leu Lys Gln Asp Asn Leu Ile Gln Gly Arg Phe Leu
480 485 ~ 490
ggt gag ata aca aag eaa gtc ttc; tet gac ctt gaa get agt aaa tat 1598
Gly Glu Ile Thr Lys Gln Val Phe Ser Asp Leu Glu Ala Ser Lys Tyr
495 500 505
eag atg get gaa tac aga ata tet. ata tat gge aga aaa atg age gag 1646
Gln Met Ala Glu Tyr Arg Ile Ser Ile Tyr Gly Arg Lys Met Ser Glu
510 515 520
tgg gac caa ctc get agt tgg att gtg aac aat gat cta tac agt gag 1694
Trp Asp Gln Leu Ala Ser Trp Ilea Val Asn Asn Asp Leu Tyr Ser G1u
525 530 535
aat gtt gtc tgg tta att cag ctc cca cgc ttg tac aac att tac aag 1742
Asn Val Val Trp Leu Ile Gln Leu. Pro Arg Leu Tyr Asn Ile Tyr Lys
540 545 550 555
gac atg ggt att gtg aca tcg ttc cag aat atc ctg gac aat ata ttc 1790
Asp Met Gly Ile Val Thr Ser Phe Gln Asn Ile Leu Asp Asn Ile Phe
560 565 570
att cct ctg ttt gaa gcc acg gta. gat cct gat tcc cat cct cag ctc 1838
Ile Pro Leu Phe Glu Ala Thr Val Asp Pro Asp Ser His Pro Gln Leu
575 580 585
cat gtt ttt ttg aag cag gtt gtt gga ttt gat ttg gtt gat gat gaa 1886
His Val Phe Leu Lys Gln Val Val Gly Phe Asp Leu Val Asp Asp Glu
590 595 600
agc aaa cct gaa aga cgt cce aca. aaa cac atg cce act eca get caa 1934
Ser Lys Pro Glu Arg Arg Pro Thr Lys His Met Pro Thr Pro Ala Gln
605 610 615
tgg act aac gca ttc aat cct gca. ttt tcg tat tat gtc tac tat tgt 1982
Trp Thr Asn Ala Phe Asn Pro Ala. Phe 5er Tyr Tyr Val Tyr Tyr Cys
620 625 630 635
tat get aac ctc tat gtg tta aat aag ctt cga gag tca aag ggc atg 2030
Tyr Ala Asn Leu Tyr Val Leu Asn. Lys Leu Arg Glu Ser Lys Gly Met
640 645 650
act act ate acg eta cga cca cat. tct gga gag get ggt gac att gac 2078
9:
CA 02325054 2000-09-27
Thr Thr Ile Thr Leu Arg Pro His Ser Gly Glu Ala Gly Asp Ile Asp
655 660 665
cac ttg get get acg ttt cta aca tgc cat agc atc gca cat gga atc 2126
His Leu Ala Ala Thr Phe Leu Thr Cys His Ser Ile Ala His Gly Ile
670 67'_i 680
aat ctg cga aag tct cct gtg ctt: cag tat ctg tac tac ctc gcc cag 2174
Asn Leu Arg Lys Ser Pro Val Leu Gln Tyr Leu Tyr Tyr Leu Ala Gln
685 690 695
att ggt ctg gcc atg tca cca ctc~ agc aac aac tct ttg ttt cta gat 2222
Ile Gly Leu Ala Met Ser Pro Leu Ser Asn Asn Ser Leu Phe Leu Asp
700 705 710 715
tac cac cgg aac ccg ttt cct gtg ttt ttc tta aga ggt ctc aat gtt 2270
Tyr His Arg Asn Pro Phe Pro.Val Phe Phe Leu Arg Gly Leu Asn Val
720 725 730
tct ctg tct act gat gac ccc ctt cag att cac tta act aaa gaa cct 2318
Ser Leu Ser Thr Asp Asp Pro Leu. Gln Ile His Leu Thr Lys Glu Pro
735 740 745
ctc gtg gaa gag tat agc ata get gca tca gtt tgg aag ctg agt gcg 2366
Leu Val Glu Glu Tyr Ser Ile A1a Ala Ser Val Trp Lys Leu Ser Ala
750 755 760
tgt gac ctg tgc gag ata get cgt aac tca gtg tac cag tca ggt ttc 2414
Cys Asp Leu Cys Glu Ile Ala Arg Asn Ser Val Tyr Gln Ser Gly Phe
765 770 775
tca cac gcc ctg aag tcg cac tgg att gga aaa gat tac tac aaa aga 2462
Ser His Ala Leu Lys Ser His Trp Ile Gly Lys Asp Tyr Tyr Lys Arg
780 785 790 795
gga cct gat gga aac gac att cac aaa aca aac gtg cca cac ata agg 2510
Gly Pro Asp Gly Asn Asp Ile His Lys Thr Asn Val Pro His Ile Arg
800 805 810
gtg gag ttc cgt gac acg atc tgg aaa gag gag atg caa cag gtt tat 2558
Val Glu Phe Arg Asp Thr Ile Trp Lys Glu Glu Met Gln Gln Val Tyr
815 820 825
ctg ggc aag get gtt atc tct gat gaa gtt gtt cca taaaaaccac 2604
Leu Gly Lys Ala Val Ile Ser Asp Glu Val Val Pro
830 835
aatcagaaat ggcaagacgt aagaatccaa catattgcag gggaacaaag agagcatttt 2664
CA 02325054 2000-09-27
gagaagtata cgaaagcagg aacctagtag atagggtaat aatatgagtg gctctgtgcc 2724
ctgagaaagc gattaggctg tgccaaaatc ttattgtttt ataaagcttt ttagataatg 2784
agatacaaag agaccagttg agaaccggtt ttaatataat ggatttcagt ttttggatta 2844
aaaaaaaaaa aaaaaaaaaa acctcgtgcc gaattc 2880
<210> 2
<211> 839
<212> PRT
<213> Arabidopsis thaliana
<400> 2
Met Glu Pro Asn Ile Tyr Gln Leu Ala Leu Ala Ala Leu Phe Gly Ala
1 5 10 15
Ser Phe Val Ala Val Ser Gly Phe Phe Met His Phe Lys Ala Leu Asn
20 25 30
Leu Val Leu Glu Arg Gly Lys Glu Arg Lys Glu Asn Pro Asp Gly Asp
35 40 45
Glu Pro Gln Asn Pro Thr Leu Val Arg Arg Arg Ser Gln Val Arg Arg
50 55 60
Lys Val Asn Asp Gln Tyr Gly Arg Ser Pro Ala Ser Leu Pro Asp Ala
65 70 75 80
Thr Pro Phe Thr Asp Gly Gly Gly Gly Gly Gly Gly Asp Thr Arg Arg
85 90 95
Ser Asn Gly His Val Tyr Val Asp Glu Ile Pro Pro Gly Leu Pro Arg
100 105 110
Leu His Thr Pro Ser Glu Gly Arg Ala Ser Val His Gly Ala Ser Ser
115 120 125
Ile Arg Lys Thr Gly Ser Phe Val Arg Pro Ile Ser Pro Lys Ser Pro
130 135 140
Val Ala Ser Ala Ser Ala Phe Glu Ser Val Glu Glu Ser Asp Asp Asp
145 150 155 160
Asp Asn Leu Thr Asn Ser Glu Gly Leu Asp Ala Ser Tyr Leu Gln Ala
165 170 ~ 175
6
CA 02325054 2000-09-27
Asn Gly Asp Asn Glu Met Pro Ala. Asp Ala Asn Glu Glu Gln Ile Ser
180 185 190
Met Ala Ala Ser Ser Met Ile Argr Ser His Ser Val Ser Gly Asp Leu
195 20C 205
His Gly Val Gln Leu Ser Pro Ilea Ala Ala Asp Ile Leu Arg Lys Glu
210 215 220
Pro Glu Gln Glu Thr Phe Val Arg~ Leu Asn Val Pro Leu Glu Val Pro
225 230 235 240
Thr Ser Asp Glu Val Glu Ala Tyr Lys Cys Leu Gln Glu Cys Leu Glu
245 250 255
Leu Arg Lys Arg Tyr Val Phe Gln. Glu Thr Val Ala Pro Trp Glu Lys
260 265 270
Glu Val Ile Ser Asp Pro Ser Thr Pro Lys Pro Asn Thr Glu Pro Phe
275 280 285
Ala His Tyr Pro Gln Gly Lys Ser Asp His Cys Phe Glu Met Gln Asp
290 295 300
Gly Val Val His Val Phe Ala Asn. Lys Asp Ala Lys Glu Asp Leu Phe
305 310 315 320
Pro Val Ala Asp Ala Thr Ala Phe Phe Thr Asp Leu His His Val Leu
325 330 335
Lys Val Ile Ala Ala Gly Asn Ile Arg Thr Leu Cys His Arg Arg Leu
340 345 350
Val Leu Leu Glu Gln Lys Phe Asn. Leu His Leu Met Leu Asn Ala Asp
355 360 365
Lys Glu Phe Leu Ala Gln Lys Ser Ala Pro His Arg Asp Phe Tyr Asn
370 375 380
Val Arg Lys Val Asp Thr His Val. His His Ser Ala Cys Met Asn Gln
385 390 395 400
Lys His Leu Leu Arg Phe I1e Lysc Ser Lys Leu Arg Lys Glu Pro Asp
405 410 415
Glu Val Val Ile Phe Arg Asp Gly Thr Tyr Leu Thr Leu Arg Glu Val
420 425 430
CA 02325054 2000-09-27
Phe Glu Ser Leu Asp Leu Thr Gly Tyr Asp Leu Asn Val Asp Leu Leu
435 440 445
Asp Val His Ala Asp Lys Ser Thr Phe His Arg Phe Asp Lys Phe Asn
450 455 460
Leu Lys Tyr Asn Pro Cys Gly Gln Ser Arg Leu Arg Glu Ile Phe Leu
465 470 475 480
Lys Gln Asp Asn Leu Ile Gln Gly Arg Phe Leu Gly Glu Ile Thr Lys
485 490 495
Gln Val Phe Ser Asp Leu Glu Ala Ser Lys Tyr Gln Met Ala Glu Tyr
500 505 510
Arg Ile Ser Ile Tyr Gly Arg Lys Met Ser Glu Trp Asp Gln Leu Ala
515 520 525
Ser Trp Ile Val Asn Asn Asp Leu Tyr Ser Glu Asn Val Val Trp Leu
530 535 540
Ile Gln Leu Pro Arg Leu Tyr Asn Ile Tyr Lys Asp Met Gly Ile Val
545 550 555 560
Thr Ser Phe Gln Asn Ile Leu Asp Asn Ile Phe Ile Pro Leu Phe Glu
565 570 575
Ala Thr Val Asp Pro Asp Ser His Pro Gln Leu His Val Phe Leu Lys
580 585 590
Gln Val Val Gly Phe Asp Leu Val Asp Asp Glu Ser Lys Pro Glu Arg
595 600 605
Arg Pro Thr Lys His Met Pro Thr Pro Ala Gln Trp Thr Asn Ala Phe
610 615 620
Asn Pro Ala Phe Ser Tyr Tyr Val Tyr Tyr Cys Tyr Ala Asn Leu Tyr
625 630 635 640
Val Leu Asn Lys Leu Arg Glu Ser Lys Gly Met Thr Thr Ile Thr Leu
645 650 655
Arg Pro His Ser Gly Glu Ala Gly Asp Ile Asp His Leu Ala Ala Thr
660 665 670
Phe Leu Thr Cys His Ser Ile Ala His Gly Ile Asn Leu Arg Lys Ser
675 680 685
8
CA 02325054 2000-09-27
Pro Val Leu Gln Tyr Leu Tyr Tyr Leu A1a Gln Ile Gly Leu Ala Met
690 695 700
Ser Pro Leu Ser Asn Asn Ser Leu Phe Leu Asp Tyr His Arg Asn Pro
705 710 715 720
Phe Pro Val Phe Phe Leu Arg Gly Leu Asn Val Ser Leu Ser Thr Asp
725 730 735
Asp Pro Leu Gln Ile His Leu Thr Lys Glu Pro Leu Val Glu Glu Tyr
740 745 750
Ser Ile Ala Ala Ser Val Trp Lys Leu Ser Ala Cys Asp Leu Cys Glu
755 760 765
Ile Ala Arg Asn Ser Val Tyr Gln Ser Gly Phe Ser His Ala Leu Lys
770 775 780
Ser His Trp Ile Gly Lys Asp Tyr Tyr Lys Arg Gly Pro Asp Gly Asn
785 790 795 800
Asp Ile His Lys Thr Asn Val Pro His Ile Arg Val Glu Phe Arg Asp
805 810 815
Thr Ile Trp Lys Glu Glu Met Gln Gln Val Tyr Leu Gly Lys Ala Val
820 825 830
Ile Ser Asp Glu Va1 Val Pro
835
9
