Note: Descriptions are shown in the official language in which they were submitted.
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
_1_
URACIL PERMEASE FROM ARABIDOPSIS AS HERBICIDAL TARGET GENE
The invention relates to a gene isolated from Arabidopsis that codes for a
protein essential
for seedling growth. The invention also includes the methods of using this
protein as an
herbicide target, based on the essentiality of the gene for normal growth and
development.
The invention is also useful as a screening assay to identify inhibitors that
are potential
herbicides. The invention may also be applied to the development of herbicide
tolerant
plants, plant tissues, plant seeds, and plant cells.
The use of herbicides to control undesirable vegetation such as weeds in crop
fields has
become almost a universal practice. The herbicide market exceeds 15 billion
dollars
annually. Despite this extensive use, weed control remains a significant and
costly problem
for farmers.
Effective use of herbicides requires sound management. For instance, the time
and
method of application and stage of weed plant development are critical to
getting good
weed control with herbicides. Since various weed species are resistant to
herbicides, the
production of effective new herbicides becomes increasingly important. Novel
herbicides
can now be discovered using high-throughput screens that implement recombinant
DNA
technology. Metabolic enzymes found to be essential to plant growth and
development can
be recombinantly produced through standard molecular biological techniques and
utilized
as herbicide targets in screens for novel inhibitors of the enzyme activity.
The novel
inhibitors discovered through such screens may then be used as herbicides to
control
undesirable vegetation.
Herbicides that exhibit greater potency, broader weed spectrum, and more rapid
degradation in soil can also, unfortunately, have greater crop phytotoxicity.
One solution
applied to this problem has been to develop crops that are resistant or
tolerant to
herbicides. Crop hybrids or varieties tolerant to the herbicides allow for the
use of the
herbicides to kill weeds without attendant risk of damage to the crop.
Development of
tolerance can allow application of a herbicide to a crop where its use was
previously
precluded or limited (e.g. to pre-emergence use) due to sensitivity of the
crop to the
herbicide. For example, U.S. Patent No. 4,761,373 to Anderson ef al. is
directed to plants
resistant to various imidazolinone or sulfonamide herbicides. The resistance
is conferred by
an altered acetohydroxyacid synthase (AHAS) enzyme. U.S. Patent No. 4,975,374
to
Goodman et al. relates to plant cells and plants containing a gene encoding a
mutant
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-2-
glutamine synthetase (GS) resistant to inhibition by herbicides that were
known to inhibit
GS, e.g. phosphinothricin and methionine sulfoximine. U.S. Patent No.
5,013,659 to
Bedbrook et al. is directed to plants expressing a mutant acetolactate
synthase that renders
the plants resistant to inhibition by sulfonylurea herbicides. U.S. Patent No.
5,162,602 to
Somers et al. discloses plants tolerant to inhibition by cyclohexanedione and
aryloxyphenoxypropanoic acid herbicides. The tolerance is conferred by an
altered acetyl
coenzyme A carboxylase (ACCase}.
Notwithstanding the above described advancements, there remains persistent and
ongoing problems with unwanted or detrimental vegetation growth (e.g. weeds).
Furthermore, as the population continues to grow, there will be increasing
food shortages.
Therefore, there exists a long felt, yet unfulfilled need, to find new,
effective, and economic
herbicides.
DEFINITIONS
For clarity, certain terms used in the specification are defined and presented
as follows:
Chimeric: is used to indicate that a DNA sequence, such as a vector or a gene,
is
comprised of more than one DNA sequences of distinct origin which are fused
together by
recombinant DNA techniques resulting in a DNA sequence, which does not occur
naturally,
Co-factor: natural reactant, such as an organic molecule or a metal ion,
required in
an enzyme-catalyzed reaction. A co-factor is e.g. NAD(P}, riboflavin
(including FAD and
FMN), folate, molybdopterin, thiamin, biotin, lipoic acid, pantothenic acid
and coenzyme A,
S-adenosylmethionine, pyridoxal phosphate, ubiquinone, menaquinone.
Optionally, a co-
factor can be regenerated and reused.
DNA shufflin4: DNA shuffling is a method to introduce mutations or
rearrangements,
preferably randomly, in a DNA molecule or to generate exchanges of DNA
sequences
between two or more DNA molecules, preferably randomly. The DNA molecule
resulting
from DNA shuffling is a shuffled DNA molecule that is a non-naturally
occurring DNA
molecule derived from at least one template DNA molecule. The shuffled DNA
encodes an
enzyme modified with respect to the enzyme encoded by the template DNA, and
preferably
has an altered biological activity with respect to the enzyme encoded by the
template DNA.
Enzyme activity: means herein the ability of an enzyme to catalyze the
conversion of
a substrate into a product. A substrate for the enzyme comprises the natural
substrate of
the enzyme but also comprises analogues of the natural substrate which can
also be
CA 02340332 2001-02-21
WO 00/15809 PGT/EP99/06766
-3-
converted by the enzyme into a product or into an analogue of a product. The
activity of the
enzyme is measured for example by determining the amount of product in the
reaction after
a certain period of time, or by determining the amount of substrate remaining
in the reaction
mixture after a certain period of time. The activity of the enzyme is also
measured by
determining the amount of an unused co-factor of the reaction remaining in the
reaction
mixture after a certain period of time or by determining the amount of used co-
factor in the
reaction mixture after a certain period of time. The activity of the enzyme is
also measured
by determining the amount of a donor of free energy or energy-rich molecule
(e.g. ATP,
phosphoenolpyruvate, acetyl phosphate or phosphocreatine) remaining in the
reaction
mixture after a certain period of time or by determining the amount of a used
donor of free
energy or energy-rich molecule (e.g. ADP, pyruvate, acetate or creatine) in
the reaction
mixture after a certain period of time.
Expression: refers to the transcription and/or translation of an endogenous
gene or a
transgene in plants. In the case of antisense constructs, for example,
expression may refer
to the transcription of the antisense DNA only.
Gene: refers to a coding sequence and associated regulatory sequences wherein
the coding sequence is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA,
sense
RNA or antisense RNA. Examples of regulatory sequences are promoter sequences,
5' and
3' untransfated sequences. Further elements that may be present are, for
example, introns.
..gene of interest: refers to any gene which, when transferred to a plant,
confers
upon the plant a desired characteristic such as antibiotic resistance, virus
resistance, insect
resistance, disease resistance, or resistance to other pests, herbicide
tolerance, improved
nutritional value, improved performance in an industrial process or altered
reproductive
capability. The "gene of interest" may also be one that is transferred to
plants for the
production of commercially valuable enzymes or metabolites in the plant.
Herbicide: a chemical substance used to kill or suppress the grawth of plants,
plant
cells, plant seeds, or plant tissues.
Heteroloqous DNA Sequence: a DNA sequence not naturally associated with a host
cell into which it is introduced, including non-naturally occurring multiple
copies of a
naturally occurring DNA sequence.
Homologous DNA Sequence: a DNA sequence naturally associated with a host cell
into which it is introduced.
Identit : The percentage of sequence identity is determined using computer
programs that are based on dynamic programming algorithms. Computer programs
that are
CA 02340332 2001-02-21
WO 00/15809 PC'f/EP99/06766
-4-
preferred within the scope of the present invention include the BLAST (Basic
Local
Alignment Search Tool) search programs designed to explore all of the
available sequence
databases regardless of whether the query is protein or DNA. Version BLAST 2.0
(Gapped
BLAST) of this search tool has been made publicly available on the Internet
(currently
http://www.ncbi.nlm.nih.gov/BLAST/). It uses a heuristic algorithm which seeks
local as
opposed to global alignments and is therefore able to detect relationships
among
sequences which share only isolated regions. The scores assigned in a BLAST
search have
a well-defined statistical.
Inhibitor: a chemical substance that inactivates the enzymatic activity of a
protein
such as a biosynthetic enzyme, receptor, signal transduction protein,
structural gene
product, or transport protein that is essential to the growth or survival of
the plant. In the
context of the instant invention, an inhibitor is a chemical substance that
inactivates the
enzymatic activity of 4788 from a plant.
Isogenic: plants which are genetically identical, except that they may differ
by the
presence or absence of a transgene.
Isolated: in the context of the present invention, an isolated DNA molecule or
an
isolated enzyme is a DNA molecule or enzyme that, by the hand of man, exists
apart from
its native environment and is therefore not a product of nature. An isolated
DNA molecule
or enzyme may exist in a purified form or may exist in a non-native
environment such as, for
example, a transgenic host cell.
Marker gene: a gene encoding a selectable or screenable trait.
Mature protein: protein which is normally targeted to a cellular organelle,
such as a
chloroplast, and from which the transit peptide has been removed.
Minimal Promoter: promoter elements, particularly a TATA element, that are
inactive
or that have greatly reduced promoter activity in the absence of upstream
activation. In the
presence of a suitable transcription factor, the minimal promoter functions to
permit
transcription.
Modified Enzyme Activity: enzyme activity different from that which naturally
occurs
in a plant (i.e. enzyme activity that occurs naturally in the absence of
direct or indirect
manipulation of such activity by man), which is tolerant to inhibitors that
inhibit the naturally
occurring enzyme activity.
Operable linked to/ associated with: a regulatory DNA sequence is said to be
"operably linked to" or "associated with" a DNA sequence that codes for an RNA
or a
CA 02340332 2001-02-21
WO OOI15809 PCT/EP99/06766
-5-
protein if the two sequences are situated such that the regulatory DNA
sequence affects
expression of the coding DNA sequence.
Plant: refers to any plant, particularly to seed plants
Plant cell: structural and physiological unit of the plant, comprising a
protoplast and
a cell wall. The plant cell may be in form of an isolated single cell or a
cultured cell, or as a
part of higher organized unit such as, for example, a plant tissue, or a plant
organ.
Plant material: refers to leaves, stems, roots, flowers or flower parts,
fruits, pollen,
pollen tubes, ovules, embryo sacs, egg cells, zygotes, embryos, seeds,
cuttings, cell or
tissue cultures, or any other part or product of a plant.
Pre-protein: protein which is normally targeted to a cellular organelle, such
as a
chloroplast, and still comprising its transit peptide.
Recombinant DNA molecule: a combination of DNA sequences that are joined
together using recombinant DNA technology
recombinant DNA technoloay: procedures used to join together DNA sequences as
described, for example, in Sambrook et al., 1989, Cold Spring Harbor, NY: Cold
Spring
Harbor Laboratory Press
Selectable marker: a gene whose expression in a plant cell gives the cell a
selective
advantage. The selective advantage possessed by the cells transformed with the
selectable
marker gene may be due to their ability to grow in the presence of a negative
selective
agent, such as an antibiotic or a herbicide, compared to the growth of non-
transformed
cells. The selective advantage possessed by the transformed cells, compared to
non-
transformed cells, may also be due to their enhanced or novel capacity to
utilize an added
compound as a nutrient, growth factor or energy source. Selectable marker gene
also refers
to a gene or a combination of genes whose expression in a plant cell gives the
cell both, a
negative and a positive selective advantageSianificant Increase: an increase
in enzymatic
activity that is larger than the margin of error inherent in the measurement
technique,
preferably an increase by about 2-fold or greater of the activity of the wild-
type enzyme in
the presence of the inhibitor, more preferably an increase by about 5-fold or
greater, and
most preferably an increase by about 10-fold or greater.
Siq_nificantly less: means that the amount of a product of an enzymatic
reaction is
larger than the margin of error inherent in the measurement technique,
preferably a
decrease by about 2-fold or greater of the activity of the wild-type enzyme in
the absence of
the inhibitor, more preferably an decrease by about 5-fold or greater, and
most preferably
an decrease by about 10-fold or greater.
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-6-
In its broadest sense, the term "substantially similar", when used herein with
respect
to a nucleotide sequence, means a nucleotide sequence corresponding to a
reference
nucleotide sequence, wherein the corresponding sequence encodes a pofypeptide
having
substantially the same structure and function as the polypeptide encoded by
the reference
nucleotide sequence, e.g. where only changes in amino acids not affecting the
polypeptide
function occur. Desirably the substantially similar nucleotide sequence
encodes the
polypeptide encoded by the reference nucleotide sequence. The term
"substantially similar"
is specifically intended to include nucleotide sequences wherein the sequence
has been
modified to optimize expression in particular cells. The percentage of
identity between the
substantially similar nucleotide sequence and the reference nucleotide
sequence desirably
is at least 65%, more desirably at least 75%, preferably at least 85%, more
preferably at
least 90%, still more preferably at least 95%, yet still more preferably at
least 99%.
Sequence comparisons are carried out using a Smith-Waterman sequence alignment
algorithm (see e.g. Waterman, M.S. Introduction to Computational Biology:
Maps,
sequences and genomes. Chapman & Hall. London: 1995. ISBN 0-412-99391-0, or at
http://www-hto.usc.edu/software/seqaln/index.html). The IocaIS program,
version 1.16, is
used with following parameters: match: 1, mismatch penalty: 0.33, open-gap
penalty: 2,
extended-gap penalty: 2. A nucleotide sequence "substantially similar" to
reference
nucleotide sequence hybridizes to the reference nucleotide sequence in 7%
sodium
dodecyl sulfate (SDS), 0.5 M NaPOa, 1 mM EDTA at 50°C with washing in
2X SSC, 0.1
SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaP04, 1 mM
EDTA at 50°C with washing in 1 X SSC, 0.1 % SDS at 50°C, more
desirably still in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with
washing in 0.5X
SSC, 0.1 % SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS),
0.5 M NaP04, 1
mM EDTA at 50°C with washing in 0.1 X SSC, 0.1 % SDS at 50°C,
more preferably in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with
washing in 0.1 X
SSC, 0.1 % SDS at 65°C.
The term "substantially similar", when used herein with respect to a protein,
means a
protein corresponding to a reference protein, wherein the protein has
substantially the same
structure and function as the reference protein, e.g. where only changes in
amino acids
sequence not affecting the polypeptide function occur. When used for a protein
or an amino
acid sequence the percentage of identity between the substantially similar and
the
reference protein or amino acid sequence desirably is at least 65%, more
desirably at least
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
7_
75%, preferably at least 85%, more preferably at least 90%, still more
preferably at least
95%, yet still more preferably at least 99%.
Substrate: a substrate is the molecule that the enzyme naturally recognizes
and
converts to a product in the biochemical pathway in which the enzyme naturally
carries out
its function, or is a modified version of the molecule, which is also
recognized by the
enzyme and is converted by the enzyme to a product in an enzymatic reaction
similar to the
naturally-occurring reaction.
Tolerance: the ability to continue normal growth or function when exposed to
an
inhibitor or herbicide in an amount sufficient to suppress the normal growth
or function of
native, unmodified plants.
Transformation: a process for introducing heterologous DNA into a cell,
tissue, or
plant. Transformed cells, tissues, or plants are understood to encompass not
only the end
product of a transformation process, but also transgenic progeny thereof.
Transgenic: stably transformed with a recombinant DNA molecule that preferably
comprises a suitable promoter operatively linked to a DNA sequence of
interest.
BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING
SEQ ID N0:1 genomic DNA sequence for the Arabidopsis 4788 gene
SEQ ID N0:2 cDNA sequence for the Arabidopsis 4788 gene
SEQ ID N0:3 amino acid sequence of the Arabidopsis 4788 protein
SEQ ID N0:4 oligonucleotide SLP346for
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
_g_
It is an object of the invention to provide an effective and beneficial method
to identify novel
herbicides. A feature of the invention is the identification of a putative
permease gene in
Arabidopsis. Another feature of the invention is the discovery that the
putative permease
gene is essential for seedling growth and development. An advantage of the
present
invention is that the newly discovered essential gene containing a novel
herbicidal mode of
action enables one skilled in the art to easily and rapidly identify novel
herbicides.
One object of the present invention is to provide an essential gene in plants
for
assay development for inhibitory compounds with herbicidal activity. Genetic
results show
that when the putative permease gene is mutated in Arabidopsis, the resulting
phenotype is
seedling lethal in the homozygous state. This suggests a critical role for the
gene product
encoded by the mutated gene.
Using T-DNA insertion mutagenesis, the inventors of the present invention have
demonstrated that the activity is essential in Arabidopsis seedlings. This
implies that
chemicals which inhibit the function of the protein in plants are likely to
have detrimental
effects on plants and are potentially good herbicide candidates. The present
invention
therefore provides methods of using a purified protein encoded by the gene
sequence
described below to identify inhibitors thereof, which can then be used as
herbicides to
suppress the growth of undesirable vegetation, e.g. in fields where crops are
grown,
particularly agronomically important crops such as maize and other cereal
crops such as
wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and
the like, as well
as cotton, sugar cane, sugar beet, oilseed rape, and soybeans.
The present invention discloses a novel nucleotide sequence derived from
Arabidopsis, designated the 4788 gene. The nucleotide sequence of the genomic
clone is
set forth in SEQ ID N0:1, the nucleotide sequence of the corresponding cDNA
clone is set
forth in SEQ ID N0:2, and the amino acid sequence of the Arabidopsis 4788
protein is set
forth in SEQ ID N0:3. The present invention also includes nucleotide sequences
substantially similar to those set forth in SEQ ID N0:1 and SEQ ID NO: 2.
The present invention also encompasses nucleotide sequences substantially
similar to
those set forth in SEO ID N0:1 and SEQ ID NO: 2, wherein said nucleotide
sequence is a
plant nucleotide sequence. Preferred is a nucleotide sequences substantially
similar to
those set forth in SEQ ID N0:1 and SEO ID NO: 2, wherein said nucleotide
sequence is an
Arabidopsis thaliana nucleotide sequence.
Further encompassed is a nucleotide sequence substantially similar to those
set forth in
SEQ ID N0:1 and SEO ID NO: 2, wherein the encoded protein has permease
activity. More
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
_g_
preferred is a nucleotide sequence substantially similar to those set forth in
SECZ ID N0:1
and SEQ ID NO: 2, wherein the encoded protein has purine or pyrimidine
permease activity.
Particularly preferred is a nucleotide sequence substantially similar to those
set forth in SEQ
ID N0:1 and SEQ ID NO: 2, wherein said encoded protein has uracil permease
activity.
Further encompassed is an amino acid sequence comprising an amino acid
sequence
encoded by a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ
1D NO: 2.
Also encomassed is an amino acid sequence comprising an amino acid sequence
encoded
by SEQ ID NO: 1 or SEQ ID NO: 2.
The present invention also encompasses proteins whose amino acid sequence are
substantially similar to the amino acid sequences set forth in SEQ ID N0:3.
Also encompassed is an amino acid sequence comprising and amino acid sequence
substantially similar to SEGO ID N0:3. Preferred is an amino acid sequence
comprising and
amino acid sequence which is SEGO ID N0:3.
Encompassed is an amino acid sequence comprising amino acid sequence encoded
by a
nucleotide sequence substantially similar to SECT ID NO:1 or SEQ ID N0:2.,
wherein the
protein has permease activity. More preferred is an amino acid sequence
comprising amino
acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID
N0:1 or
SEQ ID N0:2, wherein the protein has purine or pyrimidine permease activity.
Particularly
preferred is an amino acid sequence comprising amino acid sequence encoded by
a
nucleotide sequence substantially similar to SEQ ID N0:1 or SEQ ID N0:2.,
wherein the
protein has uracil permease.
Further encompassed is an amino acid sequence comprising at least 20
consecutive amino
acid residues of the amino acid sequence encoded by SEQ ID NO: 1 or SEO ID NO:
2.
Further encompassed is an amino acid sequence comprising at least 20
consecutive amino
acid residues of the amino acid sequence of SEQ ID NO: 3.
An further embodiment is a chimeric gene comprising a promoter operatively
linked to a
nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID N0:2.
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-10-
Further encompassed is a recombinant vector comprising a chimeric gene
comprising a
promoter operatively linked to a nucleotide sequence substantially similar to
SEQ ID NO: 1
or SEQ ID N0:2, wherein said vector is capable of being stably transformed
into a host cell.
Further encompassed is a host cell comprising a vector comprising a chimeric
gene
comprising a promoter operatively linked to a nucleotide sequence
substantially similar to
SEQ ID NO: 1 or SEQ ID N0:2, wherein said vector is capable of being stably
transformed
into a host cell and wherein said nucleotide sequence is expressible in said
cell.
A preferred host cell according to the invention is an eukaryotic cell, more
preferred is a
host cell selected from the group consisting of an insect cell, a yeast cell,
and a plant cell.
A further preferred host cell is a prokaryotic cell, more preferred is a
bacterial cell.
Also encompassed is a plant comprising a vector comprising a chimeric gene
comprising a
promoter operatively finked to a nucleotide sequence substantially similar to
SEO ID NO: 1
or SEQ ID N0:2, wherein said vector is capable of being stably transformed
into a plant cell;
also including the progeny and seed for such a plant, which seed is optionally
treated (e.g.,
primed or coated) and/or packaged, e.g. placed in a bag or other container
with instructions
for use. More preferred is a plant according to the invention wherin said
plant being tolerant
to an inhibitor of permease activity; also including the progeny and seed for
such a plant,
which seed is optionally treated (e.g., primed or coated) and/or packaged,
e.g. placed in a
bag or other container with instructions for use.
A further embodiment of the invention is a process for making nucleotides
sequences
encoding gene products having altered permease activity comprising,
a) shuffling a nucleotide sequence substantially similar to SEQ ID NO: 1 or
SEQ ID
N0:2,
b) expressing the resulting shuffled nucleotide sequences and
c) selecting for altered permease activity as compared to the permease
activity of the
gene product of said unmodified nucleotide sequence.
Preferred is a process according to the invention, wherein the nucleotide
sequence is SECT
ID NO: 1 or SEQ ID NO: 2. More preferred is a process according to the
invention, wherein
said permease activity is purine or pyrimidine permease activity. Particularly
preferred is a
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-11-
process according to the invention, wherein said permease activity is uracil
permease
activity.
Further encompassed by the invention is a shuffled DNA molecule obtainable by
the
process for making nucleotides sequences encoding gene products having altered
permease activity comprising,
a) shuffling a nucleotide sequence substantially similar to SEQ ID NO: 1 or
SEQ ID
N0:2,
b) expressing the resulting shuffled nucleotide sequences and
c) selecting for altered permease activity as compared to the permease
activity of the
gene product of said unmodified nucleotide sequence.
A further embodiment of the invention is a shuffled DNA molecule produced by a
process
according to the invention.
Also comprised by the invention is a shuffled DNA molecule obtained a process
according
to the invention, wherein said shuffled DNA molecule encodes a gene product
having
enhanced tolerance to an inhibitor of permease activity.
A further embodiment of the invention is a chimeric gene comprising a promoter
operatively
linked to a shuffled DNA molecule produced by a process according to the
invention.
A further embodiment of the invention is a recombinant vector comprising a
chimeric gene
comprising a promoter operatively linked to a shuffled DNA molecule produced
by a
process according to the invention, wherein said vector is capable of being
stably
transformed into a host cell.
Further encompassed is a host cell comprising a vector according to claim
comprising a
chimeric gene comprising a promoter operatively linked to a shuffled DNA
molecule
produced by said process, wherein said vector is capable of being stably
transformed into a
host cell and wherein said nucleotide sequence is expressible in said cell.
Preferred is a
host cell according to the invention which is an eukaryotic cell, more
preferred wherein said
host cell is selected from the group consisting of an insect cell, a yeast
cell, and a plant cell.
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-12-
Also preferred is a host cell according to the invention which is a
prokaryotic cell, more
preferred is a host cell according to the invention which is a bacterial cell.
A further embodiment is a plant comprising a plant cell comprising a vector
according
according to the invention comprising a chimeric gene comprising a promoter
operatively
linked to a shuffled DNA molecule produced by the process according to the
invention,
wherein said vector is capable of being stably transformed into a plant cell
and wherein said
nucleotide sequence is expressible in said cell; also including the progeny
and seed for
such a plant, which seed is optionally treated (e.g., primed or coated) and/or
packaged, e.g.
placed in a bag or other container with instructions for use.
A further embodiment is a plant according to the invention which is tolerant
to an inhibitor of
permease activity; also including the progeny and seed for such a plant, which
seed is
optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a
bag or other
container with instructions for use.
Further embodied is a process of identifying compounds having herbicidal
activity
comprising:
a) combining a protein comprising an amino acid sequence encoded by a
nucleotide
sequence substantially similar to SEQ ID NO: 1 or SEO ID NO: 2.
and a compound to be tested for the ability to bind to said protein, under
conditions
conducive to binding,
b) selecting a compound identified in step (a) that is capable of binding said
protein,
c) applying identified compound in step (b) to a plant to test for herbicidal
activity,
and d) selecting compounds having herbicidal activity.
Further encompassed is a compound having herbicidal activity identifiable
according to the
process according to the invention.
Further encompassed is a process of identifying an inhibitor of permease
activity having
herbicidal activity comprising:
a) combining a permease and a compound to be tested for the ability to inhibit
the
activity of said permease, under conditions conducive to such inhibition,
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-13-
b) selecting a compound identified in step (a) that is capable of inhibiting
said permease
activity,
c) applying identified compound in step (b) to a plant to test for herbicidal
activity,
and d) selecting compounds having herbicidal activity.
Encompassed by the invention is a process according to the invention, wherein
said
permease is a purine or pyrimidine permease, more preferred, wherein said
permease is a
uracil permease.
A further embodiment of the invention is a compound having herbicidal activity
identifiable
according to the process according to the invnetion.
A further embodiment is a process of identifying an inhibitor of permease
activity
comprising,
a} introducing SEQ ID NO: 1 or SEQ lD N0:2, or nucleotide sequences
substantially
similar thereto into a permease-deficient host cell, such as E. coli uraA such
that said
sequence is functionally expressable;
b) combining said host cell containing said nucleotide sequence, with a
minimal
inhibitory concentration of 5-fluorouracil, and with a compound to be tested
for the ability
to inhibit the activity of said permease, under conditions conducive to such
inhibition,
c) measure host cell growth under the conditions of step (b); and
d) selecting said compound that inhibits host cell growth in step (c).
Further encompassed is a compound having herbicidal activity identifiable
according to the
process according to the invention.
Further encompassed is a method for suppressing the growth of a plant
comprising,
applying to said plant a compound that inhibits the activity of the amino acid
sequence
comprising an amino acid sequence encoded by a nucleotide sequence
substantially similar
to SEQ ID NO: 1 or SEQ ID NO: 2 in an amount sufficient to suppress the growth
of said
plant.
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-14-
Further encompassed is a process of identifying compounds having herbicidal
activity
comprising:
a) combining a protein of claim 10 and a compound to be tested for the ability
to bind to
said protein, under conditions conducive to binding,
b) selecting a compound identified in step (a) that is capable of binding said
protein,
c) applying identified compound in step (b) to a plant to test for herbicidal
activity,
and d) selecting compounds having herbicidal activity.
and the compounds having herbicidal activity identifiable according to the
process
according to the invention.
Further encompassed is a method of improving crops comprising, applying to a
herbicide
tolerant plant or seed selected from the group consisting of the plant or seed
according to
the invention wherein said plant is tolerant to an inhibitor of permease, a
compound
according to the invention having herbicidal activity in an amount that
inhibits the growth of
undesired vegetation without significantly suppressing the growth of the
herbicide tolerant
plant or seed.
The present invention also includes methods of using the 4788 gene product as
an
herbicide target, based on the essentiality of the gene for normal growth and
development.
Furthermore, the invention can be used in a screening assay to identify
inhibitors that are
potential herbicides.
In another preferred embodiment, the present invention describes a method for
identifying chemicals having the ability to inhibit 4788 activity in plants
preferably comprising
the steps of: a) obtaining transgenic plants, plant tissue, plant seeds or
plant cells,
preferably stably transformed, comprising a non-native nucleotide sequence
encoding an
enzyme having 4788 activity and capable of overexpressing an enzymatically
active 4788
gene product; b) applying a chemical to the transgenic plants, plant cells,
tissues or parts
and to the isogenic non-transformed plants, plant cells, tissues or parts; c)
determining the
growth or viability of the transgenic and non-transformed plants, plant cells,
tissues after
application of the chemical; d) comparing the growth or viability of the
transgenic and non-
transformed plants, plant cells, tissues after application of the chemical;
and e) selecting
chemicals suppress the viability or growth of the non-transgenic plants, plant
cells, tissues
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-15-
or parts, without significantly suppressing the growth of the viability or
growth of the
isogenic transgenic plants, plant cells, tissues or parts. In a preferred
embodiment, the
enzyme having 4788 activity is encoded by a nucleotide sequence derived from a
plant,
preferably Arabidopsis thaliana, desirably identical or substantially similar
to the nucleotide
sequence set forth in SEQ ID N0:1 or SEQ ID N0:2. In another embodiment, the
enzyme
having 4788 activity is encoded by a nucleotide sequence capable of encoding
the amino
acid sequence of SEQ ID N0:3. In yet another embodiment, the enzyme having
4788
activity has an amino acid sequence identical or substantially similar to the
amino acid
sequence set forth in SEQ ID N0:3.
The present invention further embodies plants, plant tissues, and plant cells
that
have modified 4788 activity and that are therefore tolerant to inhibition by a
herbicide at
levels normally inhibitory to naturally occurring 4788 activity; also
including the progeny and
seed for such a plant, which seed is optionally treated (e.g., primed or
coated) and/or
packaged, e.g. placed in a bag or other container with instructions for use.
Herbicide
tolerant plants encompassed by the invention include those that would
otherwise be
potential targets for normally inhibiting herbicides, particularly the
agronomically important
crops mentioned above. According to this embodiment, plants, plant tissue,
plant seeds, or
plant cells are transformed, preferably stably transformed, with a recombinant
DNA
molecule comprising a suitable promoter functional in plants operatively
linked to a
nucleotide coding sequence that encodes a modified 4788 gene that is tolerant
to inhibition
by a herbicide at a concentration that would normally inhibit the activity of
wild-type,
unmodified 4788 gene product. Modified 4788 activity may also be conferred
upon a plant
by increasing expression of wild-type herbicide-sensitive 4788 protein by
providing multiple
copies of wild-type 4788 genes to the plant or by overexpression of wild-type
4788 genes
under control of a stronger-than-wild-type promoter. The transgenic plants,
plant tissue,
plant seeds, or plant cells thus created are then selected by conventional
selection
techniques, whereby herbicide tolerant lines are isolated, characterized, and
developed.
Alternately, random or site-specific mutagenesis may be used to generate
herbicide tolerant
lines.
Therefore, the present invention provides a plant, plant cell, or plant tissue
transformed with a DNA molecule comprising a nucleotide sequence isolated from
a plant
that encodes an enzyme having 4788 activity, also including the progeny and
seed for such
a plant, which seed is optionally treated (e.g., primed or coated) and/or
packaged, e.g.
placed in a bag or other container with instructions for use, wherein the
enzyme has 4788
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-16-
activity and wherein the DNA molecule confers upon the plant, plant cell,
plant seed, or
plant tissue tolerance to a herbicide in amounts that normally inhibits
naturally occurring
4788 activity. According to one example of this embodiment, the enzyme having
4788
activity is encoded by a nucleotide sequence identical or substantially
similar to the
nucleotide sequence set forth in SEO ID N0:1 or SEQ ID N0:2, or has an amino
acid
sequence identical or substantially similar to the amino acid sequence set
forth in SEQ ID
N0:3.
The invention also provides a method for suppressing the growth of a plant
comprising the step of applying to the plant a chemical that inhibits the
naturally occurring
4788 activity in the plant. In a related aspect, the present invention is
directed to a method
for selectively suppressing the growth of undesired vegetation in a field
containing a crop of
planted crop seeds or plants, comprising the steps of: (a) optionally planting
herbicide
tolerant crops or crop seeds, which are plants or plant seeds that are
tolerant to a herbicide
that inhibits the naturally occurring 4788 activity; and (b) applying to the
crops or crop seeds
and the undesired vegetation in the field a herbicide in amounts that inhibit
naturally
occurring 4788 activity, wherein the herbicide suppresses the growth of the
weeds without
significantly suppressing the growth of the crops.
Other objects and advantages of the present invention will become apparent to
those
skilled in the art from a study of the following description of the invention
and non-limiting
examples.
Essentiality of the 4788 Gene in Arabidopsis Demonstrated by T-DNA Insertion
Mutagenesis
As shown in the examples below, the identification of a novel gene structure,
as well as the
essentiality of the 4788 gene for normal plant growth and development, have
been
demonstrated for the first time in Arabidopsis using T-DNA insertion
mutagenesis. Having
established the essentiality of 4788 function in plants and having identified
the gene
encoding this essential activity, the inventors thereby provide an important
and sought after
tool for new herbicide development.
Arabidopsis insertional mutant lines segregating for seedling lethal mutations
are identified
as a first step in the identification of essential proteins. Starting with T2
seeds collected
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-17-
from single T1 plants containing T-DNA insertions in their genomes, those
lines segregating
homozygous seedling lethal seedlings are identified. These lines are found by
placing
seeds onto minimal plant growth media, which contain the fungicides benomyl
and maxim,
and screening for inviable seedlings after 7 and 14 days in the light at room
temperature.
Inviable phenotypes include altered pigmentation or altered morphology. These
phenotypes are observed either on plates directly or in soil following
transplantation of
seedlings.
When a line is identified as segregating a seedling lethal, it is determined
if the resistance
marker in the T-DNA co-segregates with the lethality (Errampalli et al. (1991
) The Plant
Cell, 3:149-157). Co-segregation analysis is done by placing the seeds on
media
containing the selective agent and scoring the seedlings for resistance or
sensitivity to the
agent. Examples of selective agents used are hygromycin or phosphinothricin.
About 35
resistant seedlings are transplanted to soil and their progeny are examined
for the
segregation of the seedling lethal. In the case in which the T-DNA insertion
disrupts an
essential gene, there is co-segregation of the resistance phenotype and the
seedling lethal
phenotype in every plant. Therefore, in such a case, all resistant plants
segregate seedling
lethals in the next generation; this result indicates that each of the
resistant plants are
heterozygous for the DNA causing both phenotypes.
For those lines showing co-segregation of the T-DNA resistance marker and the
seedling
lethal phenotype, Southern analysis is performed as an initial step in the
characterization of
the molecular nature of each insertion. Southerns are done with genomic DNA
isolated
from heterozygotes and using probes capable of hybridizing with the T-DNA
vector DNA.
Often, the T-DNA insertion in a given plant is shown to contain multiple
copies of the T-DNA
vector that insert at a single genetic locus. Using the results of the
Southern, appropriate
restriction enzymes are chosen to perform plasmid rescue in order to
molecularly clone
Arabidopsis genomic DNA flanking one or both sides of the T-DNA insertion.
Plasmids
obtained in this manner are analyzed by restriction enzyme digestion to sort
the plasmids
into classes based on their digestion pattern. For each class of plasmid
clone, the DNA
sequence is determined. The resulting sequences are analyzed for the presence
of non-T-
DNA vector sequences. When such sequences are found, they are used to search
DNA
and protein databases using the BLAST and BLAST2 programs (Altschul et al.
(1990) J
Mol. Biol. 215: 403-410; Altschul et al (1997) Nucleic Acid Res. 25:3389-
3402}. Additional
genomic and cDNA sequences for each gene are identified by standard molecular
biology
procedures.
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-18-
II. Sequence of the Arabidopsis 4788 Gene
The Arabidopsis 4788 gene is sequenced by isolating DNA flanking the T-DNA
border from
the tagged seedling-lethal line #4788. Arabidopsis DNA flanking the T-DNA
border is
identical to a internal region of a sequenced BAC of Arabidopsis (BAC T9J22,
chromosome
2). This BAC clone contains 115,851 by of sequence, of which a very small
portion
corresponds to the genomic region that contains the 4788 gene.
Nothwithstanding the BAC
information, the inventors are the first to establish definitively the entire
gene sequence, and
to demonstrate for the first time that the 4788 gene product is essential for
normal growth
and development, as well as defining the function of the 4788 gene product.
The present
invention discloses the nucleotide sequence of the Arabidopsis 4788 gene as
well as the
amino acid sequence of the Arabidopsis 4788 protein. The nucleotide sequence
corresponding to the genomic clone is set forth in SEQ ID N0:1, the
corresponding cDNA
clone is set forth in SEQ lD N0:2, and the amino acid sequence encoding the
mature
protein is set forth in SECT ID N0:3. The present invention also encompasses
an isolated
amino acid sequence derived from a plant, wherein said amino acid sequence is
identical or
substantially similar to the amino acid sequence encoded by the nucleotide
sequence set
forth in SEO ID NO: 1 or SEQ ID N0:2, wherein said amino acid sequence has
4788
activity. Using BLAST and BLAST2 programs with the default settings, the
sequence of the
4788 gene shows similarity to uracil permeases.
The 4788 gene is also a member of a gene family in Arabidopsis. This gene
family
consists of at least six members (Genbank Acc. #s: AC002505 (2739376), BAC
T9J22;
AC004481 (3337350), BAC F13P17; AC001229 (2190545), BAC F5114; AB009053 (n/a),
clone MQB2 (13'" ORF); 083501 (1791307); and AA712474 (EST clone 194H6T7) as
well
as AA605567 (EST clone 205J16XP).
111. Recombinant Production of 4788 and Uses Thereof
For recombinant production of 4788 in a host organism, a nucleotide sequence
encoding
4788 protein is inserted into an expression cassette designed for the chosen
host and
introduced into the host where it is recombinantly produced. The choice of
specific
regulatory sequences such as promoter, signal sequence, 5' and 3' untranslated
sequences, and enhancer appropriate for the chosen host is within the level of
skill of the
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99106766
_19_
routineer in the art. The resultant molecule, containing the individual
elements operably
linked in proper reading frame, may be inserted into a vector capable of being
transformed
into the host cell. Suitable expression vectors and methods for recombinant
production of
proteins are well known for host organisms such as E. coli, yeast, and insect
cells (see,
e.g., Luckow and Summers, 8iolTechnol. 6: 47 (1988), and baculovirus
expression vectors,
e.g., those derived from the genome of Autographica californica nuclear
polyhedrosis virus
(AcMNPV). A preferred baculovirus/insect system is pAcHLT (Pharmingen, San
Diego, CA)
used to transfect Spodoptera frugiperda Sf9 cells (ATCC) in the presence of
linear
Autographa californica baculovirus DNA (Pharmigen, San Diego, CA). The
resulting virus is
used to infect HighFive Tricoplusia ni cells (Invitrogen, La Jolla, CA).
In a preferred embodiment, the nucleotide sequence encoding a protein having
4788 activity is derived from an eukaryote, such as a mammal, a fly or a
yeast, but is
preferably derived from a plant. In a further preferred embodiment, the
nucleotide sequence
is identical or substantially similar to the nucleotide sequence set forth in
SEQ ID N0:1 or
SEQ ID N0:2, or encodes a protein having 4788 activity, whose amino acid
sequence is
identical or substantially similar to the amino acid sequence set forth in SEQ
ID N0:3. The
nucleotide sequence set forth in SEO ID N0:2 encodes the Arabidopsis 4788
protein,
whose amino acid sequence is set forth in SEO ID N0:3. In another preferred
embodiment,
the nucleotide sequence is derived from a prokaryote, preferably a bacteria,
e.g. the uraA
gene in E. coli (Andersen et al. {1995) J. Bacteriol. 177: 2008-2013).
Recombinantly
produced 4788 is isolated and.purified using a variety of standard techniques.
The actual
techniques that may be used will vary depending upon the host organism used,
whether the
protein is designed for secretion, and other such factors familiar to the
skilled artisan (see,
e.g. chapter 16 of Ausubel, F. et al., "Current Protocols in Molecular
Biology", pub. by John
Wiley & Sons, Inc. (1994).
Assays for Characterizing the 4788 Protein
Recombinantly produced 4788 proteins are useful for a variety of purposes. For
example, they can be used in in vitro assays to screen known herbicidal
chemicals whose
target has not been identified to determine if they inhibit 4788. Such in
vitro assays may
also be used as more general screens to identify chemicals that inhibit such
enzymatic
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-20-
activity and that are therefore novel herbicide candidates. Alternatively,
recombinantly
produced 4788 proteins may be used to elucidate the complex structure of these
molecules
and to further characterize their association with known inhibitors in order
to rationally
design new inhibitory herbicides as well as herbicide tolerant forms of the
enzymes.
Nucleotide sequences substantially similar to SEQ lD N0:1 or SEQ ID N0:2 and
proteins
substantially similar to SEQ ID N0:3 from any source, including microbial
sources, can be
used in the assays exemplified herein. Desirably such nucleotide sequences and
proteins
are derived from plants. More desirably, they are derived from dicot plants.
Alternatively,
such nucleotide sequences and proteins are derived from non-maize sources,
alternatively
from non-monocot sources.
Assay for Permease Activity
The 4788 gene product is believed to function as a permease, more specifically
as a uracil
permease similar to the maize Lpe1 protein (Schultes et al. (1996) The Plant
Cell, 8: 463-
475). The leaf permease 1 gene product of maize has similarity to purine and
pyrimidine
permeases from bacteria and fungi, and mutations in Ipe1 are known to
adversely affect
chloroplast development. Therefore, the Ipe 1-encoded protein is a potential
herbicide
target, which is further supported by the demonstration herein of the
essentiality of the
Arabidopsis 4788 gene product. A novel assay can be developed based on
expression of
the plant protein in a bacterial host lacking the corresponding activity
(Andersen et al.
(1995) J. Bacteriol. 177: 2008-2013).
A simple assay can be developed to screen for compounds that affect normal
functioning of the plant-encoded activity. Such compounds are promising in
vitro leads that
can be tested for in vivo herbicidal activity. One assay consists of growing
E. coli uraA
harboring and functionally expressing the 4788 gene in minimal medium in the
presence of
a minimal inhibitory concentration of 5-fluorouracil. This is accomplished in
a 96-well format
for automated high-throughput screening. Compounds that are effective in
blocking
function of the 4788 protein inhibits the ability of the cells to take up 5-
FU, and bacterial
growth results. This growth is measured by simple turbidometric means.
Other assays based on expression of plant genes in corresponding bacterial
mutants have been described. However, in addition to being a novel herbicide
target, a
particular advantage of this assay is that because uracil permease is
expressed on the cell
surface, compounds that are effective in inhibiting its function need not
penetrate the cell in
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
_21 _
order to exhibit their activity. This can be a major problem with using
bacterial systems as
models for finding compounds that will be effective in plants, because many
potent
herbicides are known to lack activity on bacteria due to poor uptake of the
compound by the
bacterium.
In Vitro Inhibitor Assays: Discovery of Small Molecule Ligand that Interacts
with Protein of
Unknown Function
Once a protein has been identified as a potential herbicide target, the next
step is to
develop an assay that allows screening large number of chemicals to determine
which
ones interact with the protein. Although it is straightforward to develop
assays for proteins of
known function, developing assays with proteins of unknown functions is more
difficult.
To address this issue, novel technologies are being examined that can detect
interactions between a protein and a ligand without knowing the biological
function of the
protein. A short description of three methods is presented, including
fluorescence
correlation spectroscopy, surface-enhanced laser desorption/ionization, and
biacore
technologies. Many more of these methods are currently being discovered, and
some may
be amenable to automated, large scale screening in light of this disclosure.
Fluorescence Correlation Spectroscopy (FCS) theory was developed in 1972 but
it is
only in recent years that the technology to perform FCS became available
(Madge et al.
(1972) Phys. Rev. Lett., 29: 705-708; Maiti et al. (1997) Proc. Natl. Acad.
Sci. USA, 94:
11753-11757). FCS measures the average diffusion rate of a fluorescent
molecule within a
small sample volume. The sample size can be as low as 103 fluorescent
molecules and the
sample volume as low as a the cytoplasm of a single bacterium. The diffusion
rate is a
function of the mass of the molecule and decreases as the mass increases. FCS
can
therefore be applied to protein-ligand interaction analysis by measuring the
change in mass
and therefore in diffusion rate of a molecule upon binding.
Surface-Enhanced Laser Desorption/lonization (SELDI) was invented by Hutchens
and
Yip during the late 1980's (Hutchens and Yip (1993) Rapid Commun. Mass
Spectrom. 7:
576-580). When coupled to a time-of-flight mass spectrometer (TOF), SELDI
provides a
mean to rapidly analyze molecules retained on a chip. It can be applied to
ligand-protein
interaction analysis by covalently binding the target protein on the chip and
analyze by MS
the small molecules retained by this protein (Worrall et al. (1998) Anal.
Biochem. 70: 750-
756}.
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-22-
Biacore relies on changes in the refractive index at the surface layer upon
binding of a
ligand to a protein immobilized on the layer. In this system, a collection of
small ligands is
injected sequentially in a 2-5 ul cell with the immobilized protein. Binding
is detected by
surface plasmon resonance (SPR) by recording laser light refracting from the
surface. In
general, the refractive index change for a given change of mass concentration
at the
surface layer, is practically the same for all proteins and peptides, allowing
a single method
to be applicable for any protein (Liedberg et al. (1983) Sensors Actuators 4:
299-304;
Malmquist (1993) Nature, 361: 186-187).
IV. In Vivo Inhibitor Assay
In one embodiment, a suspected herbicide, for example identified by in vitro
screening, is
applied to plants at various concentrations. The suspected herbicide is
preferably sprayed
on the plants. After application of the suspected herbicide, its effect on the
plants, for
example death or suppression of growth is recorded.
In another embodiment, an in vivo screening assay for inhibitors of the 4788
activity
uses transgenic plants, plant tissue, plant seeds or plant cells capable of
overexpressing a
nucleotide sequence having 4788 activity, wherein the 4788 gene product is
enzymatically
active in the transgenic plants, plant tissue, plant seeds or plant cells. The
nucleotide
sequence is preferably derived from an eukaryote, such as a yeast, but is
preferably
derived from a plant. In a further preferred embodiment, the nucleotide
sequence is
identical or substantially similar to the nucleotide sequence set forth in SEQ
ID N0:1 or
SEQ ID N0:2, or encodes an enzyme having 4788 activity, whose amino acid
sequence is
identical or substantially similar to the amino acid sequence set forth in SEQ
ID N0:3. In
another preferred embodiment, the nucleotide sequence is derived from a
prokaryote,
preferably a bacteria, e.g. the uraA gene of E. coli.
A chemical is then applied to the transgenic plants, plant tissue, plant seeds
or plant
cells and to the isogenic non-transgenic plants, plant tissue, plant seeds or
plant cells, and
the growth or viability of the transgenic and non-transformed plants, plant
tissue, plant
seeds or plant cells are determined after application of the chemical and
compared.
Compounds capable of inhibiting the growth of the non-transgenic plants, but
not affecting
the growth of the transgenic plants are selected as specific inhibitors of
4788 activity.
CA 02340332 2001-02-21
WO OO/I5809 PCT/EP99/06766
-23-
V. Herbicide Tolerant Plants
The present invention is further directed to plants, plant tissue, plant
seeds, and plant cells
tolerant to herbicides that inhibit the naturally occurring 4788 activity in
these plants,
wherein the tolerance is conferred by an altered 4788 activity. Altered 4788
activity may be
conferred upon a plant according to the invention by increasing expression of
wild-type
herbicide-sensitive 4788 by providing additional wild-type 4788 genes to the
plant, by
expressing modified herbicide-tolerant 4788 genes in the plant, or by a
combination of
these techniques. Representative plants include any plants to which these
herbicides are
applied for their normally intended purpose. Preferred are agronomically
important crops
such as cotton, soybean, oilseed rape, sugar beet, maize, rice, wheat, barley,
oats, rye,
sorghum, millet, turf, forage, turf grasses, and the like.
A. Increased Expression of Wild-Type 4788
Achieving altered 4788 activity through increased expression results in a
level of a
4788 in the plant cell at least sufficient to overcome growth inhibition
caused by the
herbicide. The level of expressed enzyme generally is at least two times,
preferably at least
five times, and more preferably at least ten times the natively expressed
amount. Increased
expression may be due to multiple copies of a wild-type 4788 gene; multiple
occurrences of
the coding sequence within the gene (i.e. gene amplification) or a mutation in
the
non-coding, regulatory sequence of the endogenous gene in the plant cell.
Plants having
such altered gene activity can be obtained by direct selection in plants by
methods known in
the art (see, e.g. U.S. Patent No. 5,162,602, and U.S. Patent No. 4,761,373,
and
references cited therein). These plants also may be obtained by genetic
engineering
techniques known in the art. Increased expression of a herbicide-sensitive
4788 gene can
also be accomplished by transforming a plant cell with a recombinant or
chimeric DNA
molecule comprising a promoter capable of driving expression of an associated
structural
gene in a plant cell operatively finked to a homologous or heterologous
structural gene
encoding the 4788 protein. Preferably, the transformation is stable, thereby
providing a
heritable transgenic trait.
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-24-
B. Expression of Modified Herbicide-Tolerant 4788 Proteins
According to this embodiment, plants, plant tissue, plant seeds, or plant
cells are
stably transformed with a recombinant DNA molecule comprising a suitable
promoter
functional in plants operatively linked to a coding sequence encoding a
herbicide tolerant
form of 4788. A herbicide tolerant form of the enzyme has at least one amino
acid
substitution, addition or deletion that confers tolerance to a herbicide that
inhibits the
unmodified, naturally occurring form of the enzyme. The transgenic plants,
plant tissue,
plant seeds, or plant cells thus created are then selected by conventional
selection
techniques, whereby herbicide tolerant lines are isolated, characterized, and
developed.
Below are described methods for obtaining genes that encode herbicide tolerant
forms of
4788:
One general strategy involves direct or indirect mutagenesis procedures on
microbes. For instance, a genetically manipulatable microbe such as E. coli or
S. cerevisiae
may be subjected to random mutagenesis in vivo with mutagens such as UV light
or ethyl or
methyl methane sulfonate. Mutagenesis procedures are described, for example,
in Miller,
Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY
(1972); Davis et al., Advanced Bacterial Genetics, Cold Spring Harbor
Laboratory, Cold
Spring Harbor, NY (1980); Sherman et al., Methods in Yeast Genetics, Cold
Spring Harbor
Laboratory, Cold Spring Harbor, NY (1983); and U.S. Patent No. 4,975,374. The
microbe
selected for mutagenesis contains a normal, inhibitor-sensitive 4788 gene and
is dependent
upon the activity conferred by this gene. The mutagenized cells are grown in
the presence
of the inhibitor at concentrations that inhibit the unmodified gene. Colonies
of the
mutagenized microbe that grow better than the unmutagenized microbe in the
presence of
the inhibitor (i.e. exhibit resistance to the inhibitor) are selected for
further analysis. 4788
genes from these colonies are isolated, either by cloning or by PCR
amplification, and their
sequences are elucidated. Sequences encoding altered gene products are then
cloned
back into the microbe to confirm their ability to confer inhibitor tolerance.
A method of obtaining mutant herbicide-tolerant alleles of a plant 4788 gene
involves direct selection in plants. For example, the effect of a mutagenized
4788 gene on
the growth inhibition of plants such as Arabidopsis, soybean, or maize is
determined by
plating seeds sterilized by art-recognized methods on plates on a simple
minimal salts
medium containing increasing concentrations of the inhibitor. Such
concentrations are in
the range of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 110, 300, 1000
and 3000 parts
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-25-
per million (ppm). The lowest dose at which significant growth inhibition can
be reproducibly
detected is used for subsequent experiments. Determination of the lowest dose
is routine in
the art.
Mutagenesis of plant material is utilized to increase the frequency at which
resistant
alleles occur in the selected population. Mutagenized seed material is derived
from a
variety of sources, including chemical or physical mutagenesis or seeds, or
chemical or
physical mutagenesis or pollen (Neuffer, In Maize for Biological Research
Sheridan, ed.
Univ. Press, Grand Forks, ND., pp. 61-64 (1982)), which is then used to
fertilize plants and
the resulting M1 mutant seeds collected. Typically for Arabidopsis, M2 seeds
(Lehle Seeds,
Tucson, AZ), which are progeny seeds of plants grown from seeds mutagenized
with
chemicals, such as ethyl methane sulfonate, or with physical agents, such as
gamma rays
or fast neutrons, are plated at densities of up to 10,000 seeds/plate (10 cm
diameter) on
minimal salts medium containing an appropriate concentration of inhibitor to
select for
tolerance. Seedlings that continue to grow and remain green 7-21 days after
plating are
transplanted to soil and grown to maturity and seed set. Progeny of these
seeds are tested
for tolerance to the herbicide. If the tolerance trait is dominant, plants
whose seed
segregate 3:1 / resistantaensitive are presumed to have been heterozygous for
the
resistance at the M2 generation. Plants that give rise to all resistant seed
are presumed to
have been homozygous for the resistance at the M2 generation. Such mutagenesis
on
intact seeds and screening of their M2 progeny seed can also be carried out on
other
species, for instance soybean (see, e.g. U.S. Pat. No. 5,084,082}.
Alternatively, mutant
seeds to be screened for herbicide tolerance are obtained as a result of
fertilization with
pollen mutagenized by chemical or physical means:
Confirmation that the genetic basis of the herbicide tolerance is a 4788 gene
is
ascertained as exemplified below. First, alleles of the 4788 gene from plants
exhibiting
resistance to the inhibitor are isolated using PCR with primers based either
upon the
Arabidopsis cDNA coding sequences shown in SECT ID N0:2 or, more preferably,
based
upon the unaltered 4788 gene sequence from the plant used to generate tolerant
alleles.
After sequencing the alleles to determine the presence of mutations in the
coding
sequence, the alleles are tested for their ability to confer tolerance to the
inhibitor on plants
into which the putative tolerance-conferring alleles have been transformed.
These plants
can be either Arabidopsis plants or any other plant whose growth is
susceptible to the 4788
inhibitors. Second, the inserted 4788 genes are mapped relative to known
restriction
fragment length polymorphisms (RFLPs} (See, for example, Chang et al. Proc.
Natl. Acad,
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-26-
Sci, USA 85: 6856-6860 (1988); Nam et al., Plant Cell 1: 699-705 (1989),
cleaved amplified
polymorphic sequences (CAPS) (Konieczny and Ausubel (1993) The Plant Journal,
4(2):
403-410), or SSLPs (Bell and Ecker {1994) Genomics, 19: 137-144). The 4788
inhibitor
tolerance trait is independently mapped using the same markers. When tolerance
is due to
a mutation in that 4788 gene, the tolerance trait maps to a position
indistinguishable from
the position of the 4788 gene.
Another method of obtaining herbicide-tolerant alleles of a 4788 gene is by
selection
in plant cell cultures. Explants of plant tissue, e.g. embryos, leaf disks,
etc. or actively
growing callus or suspension cultures of a plant of interest are grown on
medium in the
presence of increasing concentrations of the inhibitory herbicide or an
analogous inhibitor
suitable for use in a laboratory environment. Varying degrees of growth are
recorded in
different cultures. In certain cultures, fast-growing variant colonies arise
that continue to
grow even in the presence of normally inhibitory concentrations of inhibitor.
The frequency
with which such faster-growing variants occur can be increased by treatment
with a
chemical or physical mutagen before exposing the tissues or cells to the
inhibitor. Putative
tolerance-conferring alleles of the 4788 gene are isolated and tested as
described in the
foregoing paragraphs. Those alleles identified as conferring herbicide
tolerance may then
be engineered for optimal expression and transformed into the plant.
Alternatively, plants
can be regenerated from the tissue or cell cultures containing these alleles.
Still another method involves mutagenesis of wild-type, herbicide sensitive
plant
4788 genes in bacteria or yeast, followed by culturing the microbe on medium
that contains
inhibitory concentrations of the inhibitor and then selecting those colonies
that grow in the
presence of the inhibitor. More specifically, a plant cDNA, such as the
Arabidopsis cDNA
encoding the 4788 is cloned into a microbe that otherwise lacks the selected
gene's activity.
The transformed microbe is then subjected to in vivo mutagenesis or to in
vitro mutagenesis
by any of several chemical or enzymatic methods known in the art, e.g. sodium
bisulfite
(Shortle et al., Methods Enzymol. 100:457-468 (1983); methoxylamine (Kadonaga
et al.,
Nucleic Acids Res. 13:1733-1745 (1985); oligonucleotide-directed saturation
mutagenesis
(Hutchinson et al., Proc. Natl. Acad. Sci. USA, 83:710-714 (1986); or various
polymerase
misincorporation strategies {see, e.g. Shortle et al., Proc. Natl. Acad. Sci.
USA,
79:1588-1592 (1982); Shiraishi et al., Gene 64:313-319 (1988); and Leung et
al.,
Technique 1:11-15 (1989). Colonies that grow in the presence of normally
inhibitory
concentrations of inhibitor are picked and purified by repeated restreaking.
Their plasmids
are purified and tested for the ability to confer tolerance to the inhibitor
by retransforming
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-27-
them into the microbe lacking 4788 gene activity. The DNA sequences of cDNA
inserts
from plasmids that pass this test are then determined.
Herbicide resistant 4788 proteins are also obtained using methods involving in
vitro
recombination, also called DNA shuffling. By DNA shuffling, mutations,
preferably random
mutations, are introduced in 4788 genes. DNA shuffling also leads to the
recombination and
rearrangement of sequences within an 4788 gene or to recombination and
exchange of
sequences between two or more different of 4788 genes. These methods allows
for the
production of millions of mutated 4788 genes. The mutated genes, or shuffled
genes, are
screened for desirable properties, e.g. improved tolerance to herbicides and
for mutations
that provide broad spectrum tolerance to the different classes of inhibitor
chemistry. Such
screens are well within the skills of a routineer in the art.
In a preferred embodiment, a mutagenized 4788 gene is formed from at least one
template 4788 gene, wherein the template 4788 gene has been cleaved into
double-
stranded random fragments of a desired size, and comprising the steps of
adding to the
resultant population of double-stranded random fragments one or more single or
double-
stranded oligonucleotides, wherein said oligonucleotides comprise an area of
identity and
an area of heterology to the double-stranded random fragments; denaturing the
resultant
mixture of double-stranded random fragments and oligonucleotides into single-
stranded
fragments; incubating the resultant population of single-stranded fragments
with a
polymerase under conditions which result in the annealing of said single-
stranded
fragments at said areas of identity to form pairs of annealed fragments, said
areas of
identity being sufficient for one member of a pair to prime replication of the
other, thereby
forming a mutagenized double-stranded polynucleotide; and repeating the second
and third
steps for at least two further cycles, wherein the resultant mixture in the
second step of a
further cycle includes the mutagenized double-stranded polynucleotide from the
third step
of the previous cycle, and the further cycle forms a further mutagenized
double-stranded
polynucleotide, wherein the mutagenized polynucleotide is a mutated 4788 gene
having
enhanced tolerance to a herbicide which inhibits naturally occurring 4788
activity. In a
preferred embodiment, the concentration of a single species of double-stranded
random
fragment in the population of double-stranded random fragments is less than 1
% by weight
of the total DNA. In a further preferred embodiment, the template double-
stranded
polynucleotide comprises at least about 100 species of polynucfeotides. In
another
preferred embodiment, the size of the double-stranded random fragments is from
about 5
by to 5 kb. In a further preferred embodiment, the fourth step of the method
comprises
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-28-
repeating the second and the third steps for at least 10 cycles. Such method
is described
e.g. in Stemmer et al. (1994) Nature 370: 389-391, in US Patent 5,605,793 and
in Crameri
et al. (1998) Nature 391: 288-291, Cherry et al. (1999) Nature Biotechnology,
17: 379-384,
as well as in WO 97/20078, and these references are incorporated herein by
reference.
In another preferred embodiment, any combination of two or more different 4788
genes are
mutagenized in vitro by a staggered extension process (StEP), as described
e.g. in Zhao et
al. (1998) Nature Biotechnology 16: 258-261. The two or more 4788 genes are
used as
template for PCR amplification with the extension cycles of the PCR reaction
preferably
carried out at a lower temperature than the optimal polymerization temperature
of the
polymerase. For example, when a thermostable polymerase with an optimal
temperature of
approximately 72°C is used, the temperature for the extension reaction
is desirably below
72°C, more desirably below 65°C, preferably below 60°C,
more preferably the temperature
for the extension reaction is 55°C. Additionally, the duration of the
extension reaction of the
PCR cycles is desirably shorter than usually carried out in the art, more
desirably it is less
than 30 seconds, preferably it is less than 15 seconds, more preferably the
duration of the
extension reaction is 5 seconds. Only a short DNA fragment is polymerized in
each
extension reaction, allowing template switch of the extension products between
the starting
DNA molecules after each cycle of denaturation and annealing, thereby
generating diversity
among the extension products. The optimal number of cycles in the PCR reaction
depends
on the length of the 4788 coding regions to be mutagenized but desirably over
40 cycles,
more desirably over 60 cycles, preferably over 80 cycles are used. Optimal
extension
conditions and the optimal number of PCR cycles for every combination of 4788
genes are
determined as described in using procedures well-known in the art. The other
parameters
for the PCR reaction are essentially the same as commonly used in the art. The
primers for
the amplification reaction are preferably designed to anneal to DNA sequences
located
outside of the coding sequence of the 4788 genes, e.g. to DNA sequences of a
vector
comprising the 4788 genes, whereby the different 4788 genes used in the PCR
reaction are
preferably comprised in separate vectors. The primers desirably anneal to
sequences
located less than 500 by away from 4788 coding sequences, preferably less than
200 by
away from the 4788 coding sequences, more preferably less than 120 by away
from the
4788 coding sequences. Preferably, the 4788 coding sequences are surrounded by
restriction sites, which are included in the DNA sequence amplified during the
PCR reaction,
thereby facilitating the cloning of the amplified products into a suitable
vector.
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
_29_
In another preferred embodiment, fragments of 4788 genes having cohesive ends
are produced as described in WO 98/05765. The cohesive ends are produced by
ligating a
first oligonucleotide corresponding to a part of a 4788 gene to a second
oligonucleotide not
present in the gene or corresponding to a part of the gene not adjoining to
the part of the
gene corresponding to the first oligonucleotide, wherein the second
oligonucleotide contains
at least one ribonucleotide. A double-stranded DNA is produced using the first
oligonucleotide as template and the second oligonucleotide as primer. The
ribonucleotide is
cleaved and removed. The nucleotides) located 5' to the ribonucleotide is also
removed,
resulting in double-stranded fragments having cohesive ends. Such fragments
are randomly
reassembled by ligation to obtain novel combinations of gene sequences.
Herbicide resistant proteins are also obtained using methods involving in situ
modification of a target gene. A technology for targeting and mutating genes
in vivo can be
used, based on self-complementary chimeric oligonucleotides. This approach is
being
developed for the modification of endogenous genes in a site-specific and
genetically
inheritable manner (Beetham et al. (1999) Proc. Natl. Acad. Sci. 96: 8774-
8778; U.S. Patent
No. 5,756,325; U.S. Patent No. 5,871,984; U.S. Patent 5,731,181 ), and these
references
are incorporated herein by reference. Furthermore, methods for producing
plants exhibiting
agronomically desirable traits comprising mutating or modifying genes in situ,
in a plant cell,
are described (W098/54330), and this reference is incorporated herein by
reference. Such
modifications can be made via directed mutagenesis techniques such as
homologous
recombination and selected for based on the resulting herbicide-resistance
phenotype (see,
e.g. Pazkowski et al., EMBO J. 7: 4021-4026 (1988), and U.S. Patent No.
5,487,992,
particularly columns 18-19 and Example 8), and these references are
incorporated herein
by reference.
Any 4788 gene or any combination of 4788 genes is used for in vitro
recombination
in the context of the present invention, for example, an 4788 gene derived
from a plant,
such as, e.g. Arabidopsis fhaliana, e.g. an 4788 gene set forth in SEQ ID N0:1
or SEQ ID
N0:2, an 4788 gene from a bacteria, such as Bacillus caldolyticus (Ghim and
Neuhard
(1994) J. Bacteriol. 176: 3698-3707) or E. coli (Andersen et al. (1995) J.
Bacteriol. 177:
2008-2013), a 4788 gene from Zea mays (Schultes et al. (1996) The Plant Cell,
8: 463-
475), and all of which are incorporated herein by reference. Whole 4788 genes
or portions
thereof are used in the context of the present invention. The library of
mutated 4788 genes
obtained by the methods described above are cloned into appropriate expression
vectors
and the resulting vectors are transformed into an appropriate host, for
example an algae
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-30-
like Chlamydomonas, a yeast or a bacteria. An appropriate host is preferably a
host that
otherwise lacks 4788 gene activity, for example E. coli uraA mutant (Andersen
et al. (1995)
J. Bacteriol. 177: 2008-2013). Host cells transformed with the vectors
comprising the library
of mutated 4788 genes are cultured on medium that contains inhibitory
concentrations of
the inhibitor and those colonies that grow in the presence of the inhibitor
are selected.
Colonies that grow in the presence of normally inhibitory concentrations of
inhibitor are
picked and purified by repeated restreaking. Their plasmids are purified and
the DNA
sequences of cDNA inserts from plasmids that pass this test are then
determined.
An assay for identifying a modified 4788 gene that is tolerant to an inhibitor
may be
performed in the same manner as the assay to identify inhibitors of the 4788
activity
(Inhibitor Assay, above) with the following modifications: First, a mutant
4788 is substituted
in one of the reaction mixtures for the wild-type 4788 of the inhibitor assay.
Second, an
inhibitor of wild-type enzyme is present in both reaction mixtures. Third,
mutated activity
(activity in the presence of inhibitor and mutated enzyme) and unmutated
activity (activity in
the presence of inhibitor and wild-type enzyme) are compared to determine
whether a
significant increase in enzymatic activity is observed in the mutated activity
when compared
to the unmutated activity. Mutated activity is any measure of activity of the
mutated enzyme
while in the presence of a suitable substrate and the inhibitor. Unmutated
activity is any
measure of activity of the wild-type enzyme while in the presence of a
suitable substrate
and the inhibitor. A significant increase is defined as an increase in
enzymatic activity that
is larger than the margin of error inherent in the measurement technique,
preferably an
increase by at least 2-fold or greater of the activity of the wild-type enzyme
in the presence
of the inhibitor, more preferably an increase by at feast 5-fold or greater,
most preferably an
increase by at least 10-fold or greater.
In addition to being used to create herbicide-tolerant plants, genes encoding
herbicide tolerant 4788 can also be used as selectable markers in plant cell
transformation
methods. For example, plants, plant tissue, plant seeds, or plant cells
transformed with a
transgene can also be transformed with a gene encoding an altered 4788 capable
of being
expressed by the plant. The transformed cells are transferred to medium
containing an
inhibitor of the enzyme in an amount sufficient to inhibit the survivability
of plant cells not
expressing the modified gene, wherein only the transformed cells will survive.
The method
is applicable to any plant cell capable of being transformed with a modified
4788-encoding
gene, and can be used with any transgene of interest. Expression of the
transgene and the
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-31 -
modified gene can be driven by the same promoter functional in plant cells, or
by separate
promoters.
VI. Plant Transformation Technology
A wild-type or herbicide-tolerant form of the 4788 gene can be incorporated in
plant or
bacterial cells using conventional recombinant DNA technology. Generally, this
involves
inserting a DNA molecule encoding the 4788 into an expression system to which
the DNA
molecule is heterologous (i.e., not normally present) using standard cloning
procedures
known in the art. The vector contains the necessary elements for the
transcription and
translation of the inserted protein-coding sequences in a host cell containing
the vector. A
large number of vector systems known in the art can be used, such as plasmids,
bacteriophage viruses and other modified viruses. The components of the
expression
system may also be modified to increase expression. For example, truncated
sequences,
nucleotide substitutions or other modifications may be employed. Expression
systems
known in the art can be used to transform virtually any crop plant cell under
suitable
conditions. A transgene comprising a wild-type or herbicide-tolerant form of
the 4788 gene
is preferably stably transformed and integrated into the genome of the host
cells. In another
preferred embodiment, the transgene comprising a wild-type or herbicide-
tolerant form of
the 4788 gene located on a self-replicating vector. Examples of self-
replicating vectors are
viruses, in particular gemini viruses. Transformed cells can be regenerated
into whole plants
such that the chosen form of the 4788 gene confers herbicide tolerance in the
transgenic
plants.
A. Requirements for Construction of Plant Expression Cassettes
Gene sequences intended for expression in transgenic plants are first
assembled in
expression cassettes behind a suitable promoter expressible in plants. The
expression
cassettes may also comprise any further sequences required or selected for the
expression
of the transgene. Such sequences include, but are not restricted to,
transcription
terminators, extraneous sequences to enhance expression such as introns, vital
sequences,
and sequences intended for the targeting of the gene product to specific
organelles and cell
compartments. These expression cassettes can then be easily transferred to the
plant
transformation vectors described infra. The following is a description of
various
components of typical expression cassettes.
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-32-
1. Promoters
The selection of the promoter used in expression cassettes will determine the
spatial and
temporal expression pattern of the transgene in the transgenic plant. Selected
promoters
will express transgenes in specific cell types (such as leaf epidermal cells,
mesophyll cells,
root cortex cells) or in specific tissues or organs (roots, leaves or flowers,
for example) and
the selection will reflect the desired location of accumulation of the gene
product.
Alternatively, the selected promoter may drive expression of the gene under
various
inducing conditions. Promoters vary in their strength, i.e., ability to
promote transcription.
Depending upon the host cell system utilized, any one of a number of suitable
promoters
known in the art can be used. For example, for constitutive expression, the
CaMV 35S
promoter, the rice actin promoter, or the ubiquitin promoter may be used. For
regulatable
expression, the chemically inducible PR-1 promoter from tobacco or Arabidopsis
may be
used (see, e.g., U.S. Patent No. 5,689,044).
2. Transcriptional Terminators
A variety of transcriptional terminators are available for use in expression
cassettes. These
are responsible for the termination of transcription beyond the transgene and
its correct
polyadenylation. Appropriate transcriptional terminators are those that are
known to
function in plants and include the CaMV 35S terminator, the tml terminator,
the nopaline
synthase terminator and the pea rbcS E9 terminator. These can be used in both
monocotyledonous and dicotyledonous plants.
3. Sequences for the Enhancement or Regulation of Expression
Numerous sequences have been found to enhance gene expression from within the
transcriptional unit and these sequences can be used in conjunction with the
genes of this
invention to increase their expression in transgenic plants. For example,
various intron
sequences such as introns of the maize Adhl gene have been shown to enhance
expression, particularly in monocotyledonous cells. In addition, a number of
non-translated
leader sequences derived from viruses are also known to enhance expression,
and these
are particularly effective in dicotyledonous cells.
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-33-
4. Coding Sequence Optimization
The coding sequence of the selected gene may be genetically engineered by
altering the
coding sequence for optimal expression in the crop species of interest.
Methods for
modifying coding sequences to achieve optimal expression in a particular crop
species are
weal known (see, e.g. Perlak ef aG, Proc. Natl. Acad. Sci. USA 88: 3324 (1991
); and Koziel
et al., Bioltechnol. 11: 194 (1993)).
5. Targeting of the Gene Product Within the Cell
Various mechanisms for targeting gene products are known to exist in plants
and the
sequences controlling the functioning of these mechanisms have been
characterized in
some detail. For example, the targeting of gene products to the chloroplast is
controlled by
a signal sequence found at the amino terminal end of various proteins which is
cleaved
during chloroplast import to yield the mature protein (e.g. Comai et al. J.
Biol. Chem. 263:
15104-15109 (1988)). Other gene products are localized to other organelles
such as the
mitochondrion and the peroxisome (e.g. Unger ef at. Plant Molec. Biol. 13: 411-
418 (1989)).
The cDNAs encoding these products can also be manipulated to effect the
targeting of
heterologous gene products to these organelles. In addition, sequences have
been
characterized which cause the targeting of gene products to other cell
compartments.
Amino terminal sequences are responsible for targeting to the ER, the
apoplast, and
extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-
783 (1990)).
Additionally, amino terminal sequences in conjunction with carboxy terminal
sequences are
responsible for vacuolar targeting of gene products (Shinshi et al. Plant
Molec. Biol. 14:
357-368 (1990)). By the fusion of the appropriate targeting sequences
described above to
transgene sequences of interest it is possible to direct the transgene product
to any
organelle or cell compartment.
B. Construction of Plant Transformation Vectors
Numerous transformation vectors available for plant transformation are known
to those of
ordinary skill in the plant transformation arts, and the genes pertinent to
this invention can
be used in conjunction with any such vectors. The selection of vector wilt
depend upon the
preferred transformation technique and the target species for transformation.
For certain
target species, different antibiotic or herbicide selection markers may be
preferred.
Selection markers used routinely in transformation include the nptll gene,
which confers
resistance to kanamycin and related antibiotics (Messing & Vierra. Gene 19:
259-268
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-34-
(1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, which confers
resistance to
the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990),
Spencer et al.
Theor. Appl. Genet 79: 625-631 (1990)), the hph gene, which confers resistance
to the
antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931 ),
and the dhfr
gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. 2 7 :
1099-1104
(1983)), and the EPSPS gene, which confers resistance to glyphosate (U.S.
Patent Nos.
4,940,935 and 5,188,642).
1. Vectors Suitable for Agrobacterium Transformation
Many vectors are available for transformation using Agrobacterium tumefaciens.
These
typically carry at least one T-DNA border sequence and include vectors such as
pBINl9
(Bevan, Nucl. Acids Res. (1984)) and pXYZ. Typical vectors suitable for
Agrobacterium
transformation include the binary vectors pCIB200 and pCIB2001, as well as the
binary
vector pCIBlO and hygromycin selection derivatives thereof. (See, for example,
U.S.
Patent No. 5,639,949).
2. Vectors Suitable for non-Agrobacterium Transformation
Transformation without the use of Agrobacterium tumefaciens circumvents the
requirement
for T-DNA sequences in the chosen transformation vector and consequently
vectors lacking
these sequences can be utilized in addition to vectors such as the ones
described above
which contain T-DNA sequences. Transformation techniques that do not rely on
Agrobacterium include transformation via particle bombardment, protoplast
uptake (e.g.
PEG and electroporation) and microinjection. The choice of vector depends
largely on the
preferred selection for the species being transformed. Typical vectors
suitable for non-
Agrobacterium transformation include pCIB3064, pSOG19, and pSOG35. (See, for
example, U.S. Patent No. 5,639,949).
C. Transformation Techniques
Once the coding sequence of interest has been cloned into an expression
system, it is
transformed into a plant cell. Methods for transformation and regeneration of
plants are well
known in the art. For example, Ti plasmid vectors have been utilized for the
delivery of
foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro-
injection, and
CA 02340332 2001-02-21
WO 00/I5809 PCT/EP99/06766
-35-
microprojectiles. In addition, bacteria from the genus Agrobacterium can be
utilized to
transform plant cells.
Transformation techniques for dicotyledons are well known in the art and
include
Agrobacterium-based techniques and techniques that do not require
Agrobacterium. Non-
Agrobacterium techniques involve the uptake of exogenous genetic material
directly by
protoplasts or cells. This can be accomplished by PEG or electroporation
mediated uptake,
particle bombardment-mediated delivery, or microinjection. In each case the
transformed
cells are regenerated to whole plants using standard techniques known in the
art.
Transformation of most monocotyledon species has now also become routine.
Preferred
techniques include direct gene transfer into protoplasts using PEG or
electroporation
techniques, particle bombardment into callus tissue, as well as Agrobacterium-
mediated
transformation.
VII. Breeding
The wild-type or altered form of a 4788 gene of the present invention can be
utilized to
confer herbicide tolerance to a wide variety of plant cells, including those
of gymnosperms,
monocots, and dicots. Although the gene can be inserted into any plant cell
falling within
these broad classes, it is particularly useful in crop plant cells, such as
rice, wheat, barley,
rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory,
lettuce, cabbage,
cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic,
eggplant, pepper,
celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince,
melon, plum,
cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry,
pineapple,
avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and
sugarcane.
The high-level expression of a wild-type 4788 gene and/or the expression of
herbicide-
tolerant forms of a 4788 gene conferring herbicide tolerance in plants, in
combination with
other characteristics important for production and quality, can be
incorporated into plant
lines through breeding approaches and techniques known in the art.
Where a herbicide tolerant 4788 gene allele is obtained by direct selection in
a crop plant or
plant cell culture from which a crop plant can be regenerated, it is moved
into commercial
varieties using traditional breeding techniques to develop a herbicide
tolerant crop without
the need for genetically engineering the allele and transforming it into the
plant.
CA 02340332 2001-02-21
WO 00/15809 PCTlEP99/06766
-36-
The invention will be further described by reference to the following detailed
examples.
These examples are provided for purposes of illustration only, and are not
intended to be
limiting unless otherwise specified.
CA 02340332 2001-02-21
WO 00/15809 PCC/EP99106766
-37-
EXAMPLES
Standard recombinant DNA and molecular cloning techniques used here are well
known in
the art and are described by Sambrook, ef al., Molecular Cloning, eds., Cold
Spring Harbor
Laboratory Press, Cold Spring Harbor, NY (1989) and by T.J. Silhavy, M.L.
Berman, and
L.W. Enquist, Experiments with Gene Fusions, Cofd Spring Harbor Laboratory,
Cold Spring
Harbor, NY (1984) and by Ausubel, F.M. et al., Current Protocols in Molecular
Bioloay, pub.
by Greene Publishing Assoc. and Wiley-Interscience (1987), Reiter, et al.,
Methods in
Arabidopsis Research, World Scientific Press (1992), and Schultz et al., Plant
Molecular
Biolog~r Manual, Kluwer Academic Publishers (1998}. These references describe
the
standard techniques used for all steps in tagging and cloning genes from T-DNA
mutagenized populations of Arabidopsis: plant infection and transformation;
screening for
the identification of seedling mutants; cosegregation analysis; and plasmid
rescue.
Example 1: Sequence Analysis of Tagged Seedling - Lethal Line #4788 From the T-
DNA
Mutagenized Population of Arabidopsis
The plasmid rescue technique is used to molecularly clone Arabidopsis genomic
DNA
flanking one or both sides of T-DNA insertions resulting from T-DNA
mutagenesis.
Plasmids obtained in this manner are analyzed by restriction enzyme digestion
to sort the
plasmids into classes based on their digestion pattern. For each class of
plasmid clone, the
DNA sequence is determined. The resulting sequences are analyzed for the
presence of
non-T-DNA vector sequences. The plasmids recovered from the plasmid rescue
protocol
are sequenced using the s1p346 primer. Primer s1p346 provides information on
the flanking
sequence immediately adjacent to the left T-DNA border. Plasmid rescue is
validated by
PCR of genomic DNA from a heterozygote for the 4788 mutation. This PCR
experiment
uses a primer anchored in the predicted flanking sequence and the s1p346
primer
(anchored in the T-DNA insertion). Finding a PCR product of the size expected
based on
the sequence of the plasmid rescued clone confirms a valid rescue.
The sequence obtained from the above clone is used in an NCBI WWW blastn
search
against nucleotide sequence databases (Altschul et al. (1990) J Mol. Biol.
215: 403-410;
Altschul et al (1997) Nucleic Acids Res. 25: 3389-3402). The search results
show that the
recovered sequence is identical to genomic DNA from Arabidopsis chromosome II
BAC
T9J22 (Genbank Acc. AC002505 (2739376)). The region of genomic DNA where the
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-38-
insertion event occurred is annotated as encoding a putative permease
(ACCESSION #
2739376). Primers are then designed to the 5' and 3' ends of predicted mRNA,
and PCR is
performed using DNA from an Arabidopsis cDNA library as the template. The
resulting
PCR product is TA-ligated and cloned (Original TA Cloning Kit, fnvitrogen),
and sequenced.
The cDNA sequence is the same as the sequence predicted in the Genbank
annotation,
thus validating for the first time the putative open reading frame annotation.
Example 2: Expression of Recombinant 4788 Protein in E. colt
The coding region of the putative mature protein, corresponding to the cDNA
clone, is
subcloned into previously described expression vectors, and transformed into
E. colt using
the manufacturer's conditions. Specific examples include plasmids such as
pBluescript
(Stratagene, La Jolla, CA), pFLAG (International Biotechnologies, Inc., New
Haven, CT),
and pTrcHis (Invitrogen, La Jolla, CA).
Example 3:In vitro Recombination of 4788 Genes by DNA Shuffling
The A. thaliana 4788 gene encoding the 4788 protein is amplified by PCR. The
resulting
DNA fragment is digested by DNasel treatment essentially as described (Stemmer
et al.
(1994) PNAS 91: 10747-10751 ) and the PCR primers are removed from the
reaction
mixture. A PCR reaction is carried out without primers and is followed by a
PCR reaction
with the primers, both as described (Stemmer et al. (1994) PNAS 91: 10747-
10751 ). The
resulting DNA fragments are cloned into pTRC99a (Pharmacia, Cat no: 27-5007-
01) and
transformed into E.coli uraA strain (Andersen et al. (1995) Journal of
Bacteriol. 177(8):
2008-2013) by electroporation using the Biorad Gene Pulser and the
manufacturer's
conditions. The transformed bacteria are grown on medium that contains
inhibitory
concentrations of the inhibitor and those colonies that grow in the presence
of the inhibitor
are selected. Colonies that grow in the presence of normally inhibitory
concentrations of
inhibitor are picked and purified by repeated restreaking. Their plasmids are
purified and
the DNA sequences of cDNA inserts from plasmids that pass this test are then
determined.
In a similar reaction, PCR-amplified DNA fragments comprising the A. thaliana
4788 gene
encoding the protein and PCR-amplified DNA fragments comprising the E.coli
uraA gene
are recombined in vitro and resulting variants with improved tolerance to the
inhibitor are
recovered as described above.
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-39-
Example 4:In vitro Recombination of 4788 Genes by Staggered Extension Process
The A. thaliana 4788 gene encoding the 4788 protein and the E.coli uraA gene
are each
cloned into the polylinker of a pBluescript vector. A PCR reaction is carried
out essentially
as described {Zhao et al. (1998) Nature Biotechnology 16: 258-261 ) using the
"reverse
primer" and the "M13 -20 primer" (Stratagene Catalog). Amplified PCR fragments
are
digested with appropriate restriction enzymes and cloned into pTRC99a and
mutated 4788
genes are screened as described in Example 3.
Example 5: InVitro Binding Assays
Recombinant 4788 protein is obtained, for example according to Example 2. The
protein is
immobilized on chips appropriate for ligand binding assays. The protein
immobilized on the
chip is exposed to sample compound in solution according to methods well know
in the art.
While the sample compound is in contact with the immobilized protein
measurements
capable of detecting protein-ligand interactions are conducted. Examples of
such
measurements are SELDI, biacore and FCS, described above. Compounds found to
bind
the protein are readily discovered in this fashion and are subjected to
further
characterization, for e.g. accord'ing to Example 6, below.
Example 6: Assay for Uracil Permease Activity
A simple assay is developed to screen for compounds that affect normal
functioning of the
4788 activity. Such compounds are promising in vitro leads that can be tested
for in vivo
herbicidal activity. The assay consists of growing E. coli uraA harboring and
functionally
expressing the 4788 gene in minimal medium in the presence of a minimal
inhibitory
concentration of 5-fluorouracil. This is accomplished in a 96-well format for
automated high-
throughput screening. Compounds that are effective in blocking function of the
4788
protein inhibit the ability of the cells to take up 5-FU, and bacterial growth
results. This
growth is measured by simple turbidometric means. More specifically, the
invention relates
to a process of identifying an inhibitor of permease activity comprising:
a) introduce SECT ID NO: 1 or SEQ ID N0:2, or nucleotide sequences
substantially
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
-40-
similar thereto into a permease-deficient host cell, such as E. coh uraA such
that
said sequence is functionally expressable;
b) combining said host cell containing said nucleotide sequence, with a
minimal
inhibitory concentration of 5-fluorouracil, and with a compound to be tested
for the
ability to inhibit the activity of said permease, under conditions conducive
to such
inhibition,
c) measure host cell growth under the conditions of step (b); and
d) selecting said compound that inhibits host cell growth in step (c), and
optionally
e) applying identified compound in step (d) to a plant to test for herbicidal
activity,
and
f) selecting compounds having herbicidal activity in step (e).
The above disclosed embodiments are illustrative. This disclosure of the
invention will
place one skilled in the art in possession of many variations of the
invention. All such
obvious and foreseeable variations are intended to be encompassed by the
appended
claims.
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
1
SEQUENCE LISTING
<110> Novartis AG
<120> Herbicide Target Gene and Methods
<130> PB/5-30638/CGC 2031
<140>
<141>
<150> Application No. 09/153,278
<151> 1998-09-15
<160> 4
<170> PatentIn Ver. 2.0
<210> 1
<211> 3710
<212> DNA
<213> Arabidopsis thaliana
<400> 1
tcaaacaagt gactttttat atttatctta ttaattctgt agtctcgaga ttcgcttttt 60
ctcctgctta cttcttttta tcatcttctc tttgtcggtt tgatttggaa aaactttctg 120
caaaaaaaag agagattctt aaacttttta tctcaagatc ctttcaaaaa atctgaaaag 180
agaagatttt taataaaaaa gaatggttga aactggtcac catcatcaac atccaccggc 240
accggctgca gccggtcatc cgccggttcc atccatggcg atggcgcgta acatgggaac 300
cacttggcct ccggcggagc aacttcatca tcttcaatat tgcatccact ctaacccttc 360
ttggcgttag tctctctctt tctgcttata ctactttctt tatttttgtt tcagatgtaa 420
aaaataaaaa ttataatctt ttcagtcaaa agttgtatta gttcagttct gtctctctct 480
ctctttatct gctttatgtt gtgtcttaat ttgtttacca aaagattagt ttagatctca 540
gtatctgatt cgattcttta tatttttcct ctgtgttgac tatattttag cttgtgttgg 600
ggactaatta gctttaaatc taatgataaa tgttgtataa ttgttcttga gacacactaa 660
ttgattaaga ttaaaataaa ctgtttcgtt attacttgtt tatgtagttg gtgaatccaa 720
aatgctataa gaattgaatc cttagcagtt aaaagtgtgg ggttgggttt atgctaacac 780
tggtcatgga aaatgaaatg caattagtgt tttaccatta caagaaatgc catttttaag 840
ggattttgta acaaaaggat tgtattattg tttcctttta agaattgatt tgtctcattt 900
ccattaacaa tgaatcattt tacagatgag acggttgtac tggctttcca gcattacatc 960
gttatgctcg ggactactgt tttgattgcc aacacactag tgtcaccaat gggtggagat 1020
cctgtatgtg atgtttttgg tttttggatc cggttgtcta atatttatgt tattatatga 1080
ttctgaaatc gaccgtttgg tattttgaac gataataggg tgataaagcg cgggttatcc 1140
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
2
agactatatt gtttatgtcg ggcataaaca cgttgctaca aacgcttatc gggacaaggc 1200
ttcccacggt tatgggagta tcgttcgcgt atgttcttcc cgtattgtct ataatcagag 1260
attacaacaa tggtcaattc gattccgaga aacaggttaa agctcgtttt atacttgcgc 1320
ggtacggttg ctatatatat attttttttt ttacttgaca aatgccattt tccttgtaga 1380
gattccgaca tactatgaga acggtacaag gatcattaat catttcttca ttcgtcaaca 1440
tcataatcgg atatggtcag gcatggggga acttaataag gtaatataaa agatcaatat 1500
ataacattac tagtttagaa gctgatggaa gttttaacat ttaatgatta tggtttcttt 1560
aatgcagaat ctttagtccc atcattgttg tgcccgttgt ttctgttgtg agccttggcc 1620
tattccttag aggcttccca ctggtaacaa tttcaactaa agtattcagc tttaacaaat 1680
tttaactcct tcactaagtc attatgtgat ctcttcggat tttaacagct tgcaaactgt 1740
gtggaaatcg gtctaccaat gctgattctg ttgatcatca cacagcaagt cgggcttttt 1800
attattttcc gcgctatata tggtggtgat tgtttagata tgtaatatta aatcttgcta 1860
ctgttcatct tgcagtatct taaacatgca ttctcaagga tttctatgat tcttgaaaga 1920
tatgctttac ttgtttgcct ggctatcata tgggcttttg ctgctatcct tactgtttct 1980
ggtgcttata ataatgtttc taccgcaaca aaacaaagtt gtcgaactga tcgtgccttt 2040
cttatgtcat cagctccctg gtaggttgat tactatttga cttgattctt cttttcttct 2100
aaggtctaaa tatctttata ctcagtcact caaactcatc tttcaggatt agaatcccat 2160
atccattcca gtgggggact ccgatattca aagcgagtca tgtttttgga atgtttggtg 2220
ctgcaattgt cgcatctgca gaggttgtta ttcatcatat tagtttctag cttatgttaa 2280
atattcttca tgttacatgc tacaacttgt tcttatgtta tgtatatcct tattttcttt 2340
agtctaccgg agtatttttc gctgcatcta gactagcagg agcaacagcg cctccagcac 2400
atgtcgtctc tcgtagtatc ggtctacagg ttttatttcc agacactaag aaagtttttt 2460
ttaatctttt cgttttctgt tctctctgct aatgttccga ataagtctat aagctgttat 2520
attttccttt aagggtattg gtgtgctcct tgaaggaata tttggttcca ttactggcaa 2580
taccgcgtcc gtgtaagttc taaatatctc ttgctatatg tgctactatc ctttcagaat 2640
ttatacaaag aaactaggta tataattcat cttgatgata tatacaggga aaatgtcggt 2700
ctccttggcc tcacacgaat agggagtaga cgagtggtgc aggtttcaac gtttttcatg 2760
atatttttct ccatatttgg tttgtctttc aaccctctaa tcagtcatct tgactaagta 2820
tagaaagtag ccgttcatgg gttttaaatc cgcgtgtttt caatgatctt caggaaaatt 2880
tggcgcgttc tttgcgtcta ttccgcttcc aatctttgca ggcgtatact gtatactact 2940
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
3
tggaatcgtt ggtgagtagc attttgtata tgaacctaca ctacctgatt tcttgtaaaa 3000
gccggggctg cttatgatac ttttgttatg tgattggccc agttgctgtt ggaatatcgt 3060
ttatacagtt taccgacact aattcgatga gaaacatgta cgtcattggt gtctctctct 3120
tcttaagtct ttccatcgct cagtactttc ttgccaacac ttcaagagca ggatatggac 3180
cagttaggac agcaggagga tgggtaagcc tttcaaagaa ccattgtttg aaacaccatt 3240
ttacggtagt atagggagtg atataatatt tctactatat agtgtttctt tttcttaaat 3300
gtgatgtcgc ggtgaattgt ggtgcagttc aacgatatac ttaatacgat atttgcttcg 3360
gctccgttgg tggcgaccat tcttgcgacc atactagata acacgttgga agcaagacat 3420
gcaagtgacg acgcaagagg aatcccgtgg tggaagccct tccagcacag gaacggagac 3480
ggcaggaacg atgagttcta tagtatgccc cttagaatca acgagttaat gccgacacgg 3540
ttcctttgaa gactgcccct gaacgtttct tctgtatttg gaaatgtaag atatgattat 3600
gtgcatacct tgtagcttca ttggggaaaa attgagtcca gtggatacaa atgaacatag 3660
gcctttgatg gaaaaagcta tttttttgca aactatataa cttgtgttac 3710
<210> 2
<211> 1674
<212> DNA
<213> Arabidopsi s aliana
th
<220>
<221> CDS
<222> (4)..(1659)
<400> 2
aga atg gaaact ggtcaccat catcaacat ccaccggca ccgget 48
gtt
Met Val GluThr GlyHisHis HisGlnHis ProProAla ProAla
1 5 10 15
gca gcc catccg ccggttcca tccatggcg atggcgcgt aacatg 96
ggt
Ala Ala HisPro ProValPro SerMetAla MetAlaArg AsnMet
Gly
20 25 30
gga acc tggcct ccggcggag caacttcat catcttcaa tattgc 144
act
Gly Thr TrpPro ProAlaGlu GlnLeuHis HisLeuGln TyrCys
Thr
35 40 45
atc cac aaccct tcttggcat gagacggtt gtactgget ttccag 192
tct
Ile His AsnPro SerTrpHis GluThrVal ValLeuAla PheGln
Ser
50 55 60
cat tac gttatg ctcgggact actgttttg attgccaac acacta 240
atc
His Tyr ValMet LeuGlyThr ThrValLeu IleAlaAsn ThrLeu
Ile
65 70 75
gtg tca atgggt ggagatcct ggtgataaa gcgcgggtt atccag 288
cca
Val Ser MetGly GlyAspPro GlyAspLys AlaArgVal IleGln
Pro
g0 85 90 95
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
4
act ata ttg ttt atg tcg ggc ata aac acg ttg cta caa acg ctt atc 336
Thr Ile Leu Phe Met Ser Gly Ile Asn Thr Leu Leu Gln Thr Leu Ile
100 105 110
ggg aca agg ctt ccc acg gtt atg gga gta tcg ttc gcg tat gtt ctt 384
Gly Thr Arg Leu Pro Thr Val Met Gly Val Ser Phe Ala Tyr Val Leu
115 120 125
cct gta ttg tct ata atc aga gat tac aac aat ggt caa tcc gat tcc 432
Pro Val Leu Ser Ile Ile Arg Asp Tyr Asn Asn Gly Gln Ser Asp Ser
130 135 140
gag aaa cag aga ttc cga cat act atg aga acg gta caa gga tca tta 480
Glu Lys Gln Arg Phe Arg His Thr Met Arg Thr Val Gln Gly Ser Leu
145 150 155
atc att tct tca ttc gtc aac atc ata atc gga tat ggt cag gca tgg 528
Ile Ile Ser Ser Phe Val Asn Ile Ile Ile Gly Tyr Gly Gln Ala Trp
160 165 170 175
ggg aac tta ata aga atc ttt agt ccc atc att gtt gtg ccc gtt gtt 576
Gly Asn Leu Ile Arg Ile Phe Ser Pro Ile Ile Val Val Pro Val Val
180 185 190
tct gtt gtg agc ctt ggc cta ttc ctt aga ggc ttc cca ctg ctt gca 624
Ser Val Val Ser Leu Gly Leu Phe Leu Arg Gly Phe Pro Leu Leu Ala
195 200 205
aac tgt gtg gaa atc ggt cta cca atg ctg att ctg ttg atc atc aca 672
Asn Cys Val Glu Ile Gly Leu Pro Met Leu Ile Leu Leu Ile Ile Thr
210 215 220
cag caa tat ctt aaa cat gca ttc tca agg att tct atg att ctt gaa 720
Gln Gln Tyr Leu Lys His Ala Phe Ser Arg Ile Ser Met Ile Leu Glu
225 230 235
aga tat get tta ctt gtt tgc ccg get atc ata tgg get ttt get get 768
Arg Tyr Ala Leu Leu Val Cys Pro Ala Ile Ile Trp Ala Phe Ala Ala
240 245 250 255
atc ctt act gtt tct ggt get tat aat aat gtt tct acc gca aca aaa 816
Ile Leu Thr Val Ser Gly Ala Tyr Asn Asn Val Ser Thr Ala Thr Lys
260 265 270
caa agt tgt cga acg gat cgt gcc ttt ctt atg tca tca get ccc tgg 864
Gln Ser Cys Arg Thr Asp Arg Ala Phe Leu Met Ser Ser Ala Pro Trp
275 280 285
att aga atc cca tat cca ttc cag tgg ggg act ccg ata ttc aaa gcg 912
Ile Arg Ile Pro Tyr Pro Phe Gln Trp Gly Thr Pro Ile Phe Lys Ala
290 295 300
agt cat gtt ttt gga atg ttt ggt get gca att gtc gca tct gca gag 960
Ser His Val Phe Gly Met Phe Gly Ala Ala Ile Val Ala Ser Ala Glu
305 310 315
tct acc gga gta ttt ttc get gca tct aga tta gca gga gca aca gcg 1008
Ser Thr Gly Val Phe Phe Ala Ala Ser Arg Leu Ala Gly Ala Thr Ala
320 325 330 335
cct cca gca cat gtc gtc tct cgt agt atc ggt cta cag ggt att ggt 1056
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99106766
Pro Pro Ala His Val Val Ser Arg Ser Ile Gly Leu Gln Gly Ile Gly
340 345 350
gtg ctc ctt gaa gga ata ttt ggt tcc att act ggc aat acc gcg tcc 1104
Val Leu Leu Glu Gly Ile Phe Gly Ser Ile Thr Gly Asn Thr Ala Ser
355 360 365
gtg gaa aat gtc ggt ctc ctt ggc ctc gca cga ata ggg agt aga cga 1152
Val Glu Asn Val Gly Leu Leu Gly Leu Ala Arg Ile Gly Ser Arg Arg
370 375 380
gtg gtg cag gtt tca acg ttt ttc atg ata ttt ttc tcc ata ttt gga 1200
Val Val Gln Val Ser Thr Phe Phe Met Ile Phe Phe Ser Ile Phe Gly
385 390 395
aaa ttt ggc gcg ttc ttt gcg tct att ccg ctt cca atc ttt gca ggc 1248
Lys Phe Gly Ala Phe Phe Ala Ser Ile Pro Leu Pro Ile Phe Ala Gly
400 405 410 415
ata tac tgt ata cta ctt gga atc gtt gtt get gtt gga ata tcg ttt 1296
Ile Tyr Cys Ile Leu Leu Gly Ile Val Val Ala Val Gly Ile Ser Phe
420 425 430
ata cag ttt acc gac act aat tcg atg aga aac atg tac gtc att ggt 1344
Ile Gln Phe Thr Asp Thr Asn Ser Met Arg Asn Met Tyr Val Ile Gly
435 440 445
gtc tct ctc ttc tta agt ctt tcc atc get cag tac ttt ctt gcc aac 1392
Val Ser Leu Phe Leu Ser Leu Ser Ile Ala Gln Tyr Phe Leu Ala Asn
450 455 460
act tca aga gca gga tat gga cca gtt agg aca gca gga gga tgg ttc 1440
Thr Ser Arg Ala Gly Tyr Gly Pro Val Arg Thr Ala Gly Gly Trp Phe
465 470 475
aac gat ata ctt aat acg ata ttt get tcg get ccg ttg gtg gcg acc 1488
Asn Asp Ile Leu Asn Thr Ile Phe Ala Ser Ala Pro Leu Val Ala Thr
480 485 490 495
att ctt gcg acc ata cta gat aac acg ttg gaa gca aga cat gca agt 1536
Ile Leu Ala Thr Ile Leu Asp Asn Thr Leu Glu Ala Arg His Ala Ser
500 505 510
gac gac gca aga gga atc ccg tgg tgg aag ccc ttc cag cac agg aac 1584
Asp Asp Ala Arg Gly Ile Pro Trp Trp Lys Pro Phe Gln His Arg Asn
515 520 525
gga gac ggc agg aac gat gag ttc tat agt atg ccc ctt aga atc aac 1632
Gly Asp Gly Arg Asn Asp Glu Phe Tyr Ser Met Pro Leu Arg Ile Asn
530 535 540
gag tta atg ccg aca cgg ttc ctt tga agactgcccc tgaac 1674
Glu Leu Met Pro Thr Arg Phe Leu
545 550
<210> 3
<211> 551
<212> PRT
<213> Arabidopsis thaliana
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
6
<400> 3
Met Val Glu Thr Gly His His His Gln His Pro Pro Ala Pro Ala Ala
1 5 10 15
Ala Gly His Pro Pro Val Pro Ser Met Ala Met Ala Arg Asn Met Gly
20 25 30
Thr Thr Trp Pro Pro Ala Glu Gln Leu His His Leu Gln Tyr Cys Ile
35 40 45
His Ser Asn Pro Ser Trp His Glu Thr Val Val Leu Ala Phe Gln His
50 55 60
Tyr Ile Val Met Leu Gly Thr Thr Val Leu Ile Ala Asn Thr Leu Val
65 70 75 80
Ser Pro Met Gly Gly Asp Pro Gly Asp Lys Ala Arg Val Ile Gln Thr
g5 90 95
Ile Leu Phe Met Ser Gly Ile Asn Thr Leu Leu Gln Thr Leu Ile Gly
100 105 110
Thr Arg Leu Pro Thr Val Met Gly Val Ser Phe Ala Tyr Val Leu Pro
115 120 125
Val Leu Ser Ile Ile Arg Asp Tyr Asn Asn Gly Gln Ser Asp Ser Glu
130 135 140
Lys Gln Arg Phe Arg His Thr Met Arg Thr Val Gln Gly Ser Leu Ile
145 150 155 160
Ile Ser Ser Phe Val Asn Ile Ile Ile Gly Tyr Gly Gln Ala Trp Gly
165 170 175
Asn Leu Ile Arg Ile Phe Ser Pro Ile Ile Val Val Pro Val Val Ser
180 185 190
Val Val Ser Leu Gly Leu Phe Leu Arg Gly Phe Pro Leu Leu Ala Asn
195 200 205
Cys Val Glu Ile Gly Leu Pro Met Leu Ile Leu Leu Ile Ile Thr Gln
210 215 220
Gln Tyr Leu Lys His Ala Phe Ser Arg Ile Ser Met Ile Leu Glu Arg
225 230 235 240
Tyr Ala Leu Leu Val Cys Pro Ala Ile Ile Trp Ala Phe Ala Ala Ile
245 250 255
Leu Thr Val Ser Gly Ala Tyr Asn Asn Val Ser Thr Ala Thr Lys Gln
260 265 270
Ser Cys Arg Thr Asp Arg Ala Phe Leu Met Ser Ser Ala Pro Trp Ile
275 280 285
Arg Ile Pro Tyr Pro Phe Gln Trp Gly Thr Pro Ile Phe Lys Ala Ser
290 295 300
His Val Phe Gly Met Phe Gly Ala Ala Ile Val Ala Ser Ala Glu Ser
305 310 315 320
CA 02340332 2001-02-21
WO 00/15809 PCT/EP99/06766
7
Thr Gly Val Phe Phe Ala Ala Ser Arg Leu Ala Gly Ala Thr Ala Pro
325 330 335
Pro Ala His Val Val Ser Arg Ser Ile Gly Leu Gln Gly Ile Gly Val
340 345 350
Leu Leu Glu Gly Ile Phe Gly Ser Ile Thr Gly Asn Thr Ala Ser Val
355 360 365
Glu Asn Val Gly Leu Leu Gly Leu Ala Arg Ile Gly Ser Arg Arg Val
370 375 380
Val Gln Val Ser Thr Phe Phe Met Ile Phe Phe Ser Ile Phe Gly Lys
385 390 395 400
Phe Gly Ala Phe Phe Ala Ser Ile Pro Leu Pro Ile Phe Ala Gly Ile
405 410 415
Tyr Cys Ile Leu Leu Gly Ile Val Val Ala Val Gly Ile Ser Phe Ile
420 425 430
Gln Phe Thr Asp Thr Asn Ser Met Arg Asn Met Tyr Val Ile Gly Val
435 440 445
Ser Leu Phe Leu Ser Leu Ser Ile Ala Gln Tyr Phe Leu Ala Asn Thr
450 455 460
Ser Arg Ala Gly Tyr Gly Pro Val Arg Thr Ala Gly Gly Trp Phe Asn
465 470 475 480
Asp Ile Leu Asn Thr Ile Phe Ala Ser Ala Pro Leu Val Ala Thr Ile
485 490 495
Leu Ala Thr Ile Leu Asp Asn Thr Leu Glu Ala Arg His Ala Ser Asp
500 505 510
Asp Ala Arg Gly Ile Pro Trp Trp Lys Pro Phe Gln His Arg Asn Gly
515 520 525
Asp Gly Arg Asn Asp Glu Phe Tyr Ser Met Pro Leu Arg Ile Asn Glu
530 535 540
Leu Met Pro Thr Arg Phe Leu
545 550
<210> 4
<211> 20
<212> DNA
<213> Arabidopsis thaliana
<400> 4
gcggacatct acatttttga 20
