Note: Descriptions are shown in the official language in which they were submitted.
CA 02301830 2000-02-24
New PCT Patent Application
CropDesign Nv et al.
Our Ref.: C 2339 PCT
Method and means for modulating plant cell cycle proteins and their use in
controlling plant cell growth
The present invention relates to DNA sequences encoding cell cycle interacting
proteins as well as to methods for obtaining the same. The present invention
also
provides vectors comprising said DNA sequences, wherein the DNA sequences are
operatively linked to regulatory elements allowing expression in prokaryotic
and/or
eukaryotic host cells. In addition, the present invention relates to the
proteins
encoded by said DNA sequences, antibodies to said proteins and methods for
their
production. The present invention also relates to a method for controlling or
altering
growth characteristics of a plant and/or a plant cell comprising introduction
and/or
expression of one or more cell cycle regulatory proteins functional in a plant
or parts
thereof and/or one or more DNA sequences encoding such proteins. Also provided
by the present invention is a process for disruption plant cell division by
interfering in
the expression of a substrate for cyclin-dependent protein kinase using a DNA
sequence according to the invention wherein said plant cell is part of a
transgenic
plant. The present invention further relates to diagnostic compositions
comprising
the aforementioned DNA sequences, proteins and antibodies. The present
invention
also relates to methods for the identification of compounds being capable of
activating or inhibiting the cell cycle. Furthermore, the present invention
relates to
transgenic plant cells, plant tissue and plants containing the above-described
DNA
sequences and vectors as well as to the use of the aforementioned DNA
sequences, vectors, proteins, antibodies and/or compounds identified by the
method
of the invention in plant cell and tissue culture, plant breeding and/or
agriculture.
Cell division is fundamental for growth in humans, animals and plants. Prior
to
dividing in two daughter cells, the mother cell needs to replicate its DNA.
The cell
cycle is traditionally divided into 4 distinct phases:
G1: the gap between mitosis and the onset of DNA synthesis;
CA 02301830 2000-02-24
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S : the phase of DNA synthesis;
G2: the gap between S and mitosis;
M : mitosis, the process of nuclear division leading up to the actual cell
division.
The distinction of these 4 phases provides a convenient way of dividing the
interval
between successive divisions. Although they have served a useful purpose, a
recent
flurry of experimental results, much of it as a consequence of cancer
research, has
resulted in a more intricate picture of the cell cycle's "four seasons"
(Nasmyth,
Science 274, 1643-1645, 1996; Nurse, Nature, 344, 503-508, 1990). The
underlying
mechanism controlling the cell cycle control system has only recently been
studied
in greater detail. In all eukaryotic systems, including plants, this control
mechanism
is based on two key families of proteins which regulate the essential process
of cell
division, namely protein kinases (cyclin dependent kinases or CDKs) and their
activating associated subunits, called cyclins. The activity of these protein
complexes is switched on and off at specific points of the cell cycle.
Particular CDK-
cyclin complexes activated at the G1/S transition trigger the start of DNA
replication.
Different CDK-cyclin complexes are activated at the G2/M transition and induce
mitosis leading to cell division. Each of the CDK-cyclin complexes execute
their
regulatory role via modulating different sets of multiple target proteins.
Furthermore,
the large variety of developmental and environmental signals affecting cell
division
all converge on the regulation of CDK activity. CDKs can therefore be seen as
the
central engine driving cell division.
In animal systems and in yeast, knowledge about cell cycle regulations is now
quite
advanced. The activity of CDK-cyclin complexes is regulated at five levels:
(i)
transcription of the CDK and cyclin genes; (ii) association of specific CDK's
with their
specific cyclin partner; (iii) phosphorylation/dephosphorylation of the CDK
and
cyclins; (iv) interaction with other regulatory proteins such as SUC1/CKS1
homologues and cell cycle kinase inhibitors (CKI); and (v) cell cycle phase-
dependent destruction of the cyclins and CKIs.
The study of cell cycle regulation in plants has lagged behind that in animals
and
yeast. Some basic mechanisms of cell cycle control appear to be conserved
among
eukaryotes, including plants. Plants were shown to also possess CDK's, cyclins
and
CKI's. However plants have unique developmental features which are reflected
in
CA 02301830 2000-02-24
3
specific characteristics of the cell cycle control. These include for instance
the
absence of cell migration, the formation of organs throughout the entire
lifespan
from specialized regions called meristems, the formation of a cell wall and
the
capacity of non-dividing cells to re-enter the cell cycle. Another specific
feature is
that many plant cells, in particular those involved in storage (e.g.
endosperm), are
polyploid due to rounds of DNA synthesis without mitosis. This so-called
endoreduplication is intimately related with cell cycle control.
Due to these fundamental differences, multiple components of the cell cycle of
plants are unique compared to their yeast and animal counterparts. For
example,
plants contain a unique class of CDKs, such as CDC2b in Arabidopsis, which are
both structurally and functionally different from animal and yeast CDKs.
The further elucidation of cell cycle regulation in plants and its differences
and
similarities with other eukaryotic systems is a major research challenge.
Strictly for
the case of comparison, some key elements about yeast and animal systems are
described below in more detail.
As already mentioned above, the control of cell cycle progression in
eukaryotes is
mainly exerted at two transition points: one in late G,, before DNA synthesis,
and
one at the GZ/M boundary. Progression through these control points is mediated
by
cyclin-dependent protein kinase (CDK) complexes, which contain, in more
detail, a
catalytic subunit of approximately 34-kDa encoded by the CDK genes. Both
Saccharomyces cerevisiae and Schizosaccharomyces pombe only utilize one CDK
gene for the regulation of their cell cycle. The kinase activity of their gene
products
p34c~c2 and p34c°cza in Sch. pombe and in S. cerevisiae, respectively,
is dependent
on regulatory proteins, called cyclins. Progression through the different cell
cycle
phases is achieved by the sequential association of p34coc~'cocze with
different
cyclins. Although in higher eukaryotes this regulation mechanism is conserved,
the
situation is more complex since they have evolved to use multiple CDKs to
regulate
the different stages of the cell cycle. In mammals, seven CDKs have been
described, defined as CDK1 to CDK7, each binding a specific subset of cyclins.
In animal systems, CDK activity is not only regulated by its association with
cyclins
but also involves both stimulatory and inhibitory phosphorylations. Kinase
activity is
positively regulated by phosphorylation of a Thr residue located between amino
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4
acids 160-170 (depending on the CDK protein). This phosphorylation is mediated
by
the CDK-activating kinase (CAK) which interestingly is a CDK/cyclin complex
itself.
Inhibitory phosphorylations occur at the ATP-binding site (the Tyr15 residue
together
with Thr14 in higher eukaryotes) and are carried out by at least two protein
kinases.
A specific phosphatase, CDC25, dephosphorylates these residues at the GZIM
checkpoint, thus activating CDK activity and resulting in the onset of
mitosis. CDK
activity is furthermore negatively regulated by a family of mainly low-
molecular
weight proteins, called cyclin-dependent kinase inhibitors (CKIs). Kinase
activity is
inhibited by the tight association of these CKIs with the CDKlcyclin
complexes.
With respect to cell cycle regulation in plants a summary of the state of the
art is
given below. In Arabidopsis, thusfar only two CDK genes have been isolated,
CDC2aAt and CDC2bAt, of which the gene products share 56% amino acid identity.
Both CDKs are distinguished by several features. First, only CDC2aAt is able
to
complement yeast p34~~c~'cocza mutants. Second, CDC2aAt and CDC2bAt bear
different cyclin-binding motifs (PSTAIRE and PPTALRE, respectively),
suggesting
they may bind distinct types of cyclins. Third, although both CDC2aAt and
CDC2bAt
show the same spatial expression pattern, they exhibit a different cell cycle
phase-
specific regulation. The CDC2aAt gene is expressed constitutively throughout
the
whole cell cycle. In contrast, CDC2bAt mRNA levels oscillate, being most
abundant
during the S and GZ phases. In addition, multiple cyclins have been isolated
from
Arabidopsis. The majority displays the strongest sequence similarity with the
animal
A- or B-type class of cyclins, but also D-type cyclins have been identified.
Although
the classification of Arabidopsis cyclins is mainly based upon sequence
similarity,
limited data suggests that this organization corresponds with differential
functions of
each cyclin class. Direct binding of any cyclin with an Arabidopsis CDK
subunit has,
however, not yet been demonstrated.
In order to manage problems related to plant growth, plant architecture and/or
plant
diseases, it is believed to be of utmost importance to identify and isolate
plant genes
and gene products involved in the regulation of the plant cell division, and
more
particularly coding for and interacting with CDK's and/or their interacting
proteins,
responsible for the control of the cell cycle and the completion of the S and
M phase
of the cell cycle. If such novel genes and/or proteins have been isolated and
CA 02301830 2000-02-24
analyzed, the growth of the plant as a whole can be influenced. Also, the
growth of
specific tissues or organs and thus the architecture of the plant can be
modified.
Thus, the technical problem underlying the present invention is to provide
means
and methods for modulating cell cycle proteins that are particular useful in
agriculture and plant cell and tissue culture.
The solution to the technical problem is achieved by providing the embodiments
characterized in the claims.
Accordingly; the invention relates to a DNA sequence encoding a cell cycle
interacting protein or encoding an immunologically active and/or functional
fragment
of such a protein, selected from the group consisting of:
(a) DNA sequences comprising a nucleotide sequence encoding a protein
comprising the amino acid sequence as given in SEQ ID NO: 2;
(b) DNA sequences comprising a nucleotide sequence as given in SEQ ID NO: 1;
(c) DNA sequences hybridizing with the complementary strand of a DNA
sequence as defined in (a) or (b) and encoding an amino acid sequence which
is at least 80% identical to the amino acid sequence encoded by the DNA
sequence of (a) or (b);
(d) DNA sequences, the nucleotide sequence of which is degenerated as a result
of the genetic code to a nucleotide sequence of a DNA sequence as defined in
any one of (a) to (c); and
(e) DNA sequences encoding a fragment of a protein encoded by a DNA
sequence of any one of (a) to (d).
The term "cell cycle interacting protein" as denoted herein means a protein
capable
of binding to cyclin dependent kinases, in particular plant cyclin dependent
kinases.
The term "cell cycle" means the cyclic biochemical and structural events
associated
with growth of cells, and in particular with the regulation of the replication
of DNA
and mitosis. The cycle is divided into periods called: Go, Gap, (G,), DNA
synthesis
(S), Gape (Gz), and mitosis (M).
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6
The terms "gene(s)", "polynucleotide", "nucleic acid sequence", "nucleotide
sequence", "DNA sequence" or "nucleic acid molecule(s)" as used herein refers
to a
polymeric form of nucleotides of any length, either ribonucleotides or
deoxyribonucleotides. This term refers only to the primary structure of the
molecule.
Thus, this term includes double- and single-stranded DNA, and RNA. It also
includes
known types of modifications, for example, methylation, "caps" substitution of
one or
more of the naturally occuring nucleotides with an analog. Preferably, the DNA
sequence of the invention comprises a coding sequence encoding the above
defined cell cycle interacting protein.
A "coding sequence" is a nucleotide sequence which is transcribed into mRNA
and/or translated into a polypeptide when placed under the control of
appropriate
regulatory sequences. The boundaries of the coding sequence are determined by
a
translation start codon at the 5'-terminus and a translation stop codon at the
3'-
terminus. A coding sequence can include, but is not limited to mRNA, cDNA,
recombinant nucleotide sequences or genomic DNA, while introns may be present
as well under certain circumstances.
Studies with a two-hybrid system which had been carried out in accordance with
the
present invention a new gene product interacting with CDC2aAt indicative of a
hitherto unknown plant cell cycle regulatory nucleotide sequence was
identified
(further also called clone th65). The coding nucleotide sequence (reading
frame) for
the isolated clone th65 in SEQ.ID.N0.1 starts at position 3 and terminates at
codon
AGG (position 1184). The N-terminus contained a region rich in glutamine
residues
(SEQ ID NO 2.). Gln-rich domains are often part of the transcriptional
activation
domain of DNA binding factors (Mitchell and Tjian, 1989, Science, 2~4r, 371-
378) and
have also been shown to be involved in protein-protein interactions (Bao et
al., 1996,
PNAS, 9~, 5037-5042). The th65 open reading frame also contains three
consensus
CDK phosphorylation sites. The identification of th65 as a CDC2aAt-associated
protein and the presence of these phosphorylation sites indicates that the
th65 protein
is a substrate for CDKs.
Using a nucleic acid amplification technology, such as the polymerise chain
reaction (PCR), a genomic DNA fragment can be isolated comprising the sequence
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7
defined in SEQ.ID.N0.1. Thus a novel plant nucleotide sequence and polypeptide
sequence, having a molecular weight of about 44 kDa, are provided. A homology
search in databases showed a significant homology to a plant kinesin-related
motor
protein. The homology search was performed with the program BLASTN (version
2.Oa19MP-WashU [build decunix3.2 01:53:29 05-feb-1998] (see Altschul, Nucleic
Acids Res. 25 (1997), 3389-3402) on the Arabidopsis thaliana nucleic acids
database at ATDB at Stanford (http://genome-www2.stanford.edu/cgi-bin/AtDB/nph-
blast2atdb). The mRNA of th65 (AJ001729) was submitted to BLASTN and revealed
homology to the genomic sequence from Arabidopsis thaliana (AB011479)
(P(N)=4.4e-118). The function COMPARE (from the GCG 9.1 package, Genetics
Computer Group Inc., Madison, USA) has been used to quantify the percentage of
homology and similarity. With the parameters Gap weight = 12 and Length weight
=
2 the function COMPARE resulted in an alignment showing 84.638 % similarity
and
79.420 % identity. The genomic sequence (AB011479, clone MNAS) has been
retrieved from the KAOS server with its annotations. A kinesin-like protein c
was
predicted on that sequence (73733..80900) as gene MNA5.12, having 22 exons and
no homologue EST. The protein sequence (as given on the KAOS server) was then
used to perform a BLASTP (version 2Ø4 [feb-24-1998]) with BEAUTY post-
processing provided by the Human Genome Center, Baylor College of Medicine
against the National Center for Biotechnology Information's non-redundant
protein
database (http://dot.imgen.bcm.tmc.edu:9331Iseq-search/protein-search.html)
and
revealed homology to numerous kinesins from Arabidopsis and other organisms.
Kinesins and kinesin-related proteins are microtubule motor proteins involved
in
vesicle transport, spindle assembly and chromosome segregation at meiosis and
mitosis. The present demonstration that CDC2a interacts with a putative
kinesin-
related motor proteins and the presence of consensus sites for CDK
phosphorylation
in the identified th65 clone indicates that CDKs directly modify the
cytoskelet through
phosphorylation of kinesin-related motor proteins. Thus it is expected that
the
nucleic acid molecules of the invention encode proteins that beside their
intrinsic
capability of interacting with cell cycle proteins in addition display the
biological
activity of kinesin-related motor proteins.
CA 02301830 2000-02-24
8
With "kinesin" is meant the superfamily of microtubule-based motor proteins
which
includes both plus- and minus-end-directed varieties and is widely distributed
in
microtubule-containing cells. Functions of kinesins may include membrane-bound
organelle movement and mitosis. Also used specifically for the defining member
of
the superfamily (other members are considered to be kinesin-related proteins).
With "motor proteins" is meant mechanochemical enzymes involved in locomotion
or
transport.
With "mechanochemical enzyme" is meant an enzyme that converts chemical
energy in the form of nucleoside triphosphates to mechanical energy such as
force
or mortility.
The present invention also relates to nucleic acid molecules hybridizing with
the
above-described nucleic acid molecules and differ in one or more positions in
comparison with these as long as they encode a cell cycle interacting protein.
By
"hybridizing" it is meant that such nucleic acid molecules hybridize under
conventional hybridization conditions, preferably under stringent conditions
such as
described by, e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd
Edition,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). An
example
of one such stringent hybridization condition is hybridization at 4XSSC at 65
°C,
followed by a washing in 0.1XSSC at 65 °C for one hour. Alternatively,
an exemplary
stringent hybridization condition is in 50 % formamide, 4XSSC at 42 °C.
Cell cycle
interacting proteins derived from other organisms such as mammals, in
particular
humans, may be encoded by other DNA sequences which hybridize to the
sequences for plant cell cycle interacting proteins under relaxed
hybridization
conditions and which code on expression for peptides having the ability to
interact
with cell cycle proteins. Examples of such non-stringent hybridization
conditions are
4XSSC at 50 °C or hybridization with 30-40 % formamide at 42 °C.
Such molecules
comprise those which are fragments, analogues or derivatives of the cell cycle
interacting protein of the invention and differ, for example, by way of amino
acid
and/or nucleotide deletion(s), insertion(s), substitution(s), addition{s)
and/or
recombination(s) or any other modifications) known in the art either alone or
in
combination from the above-described amino acid sequences or their underlying
CA 02301830 2000-02-24
9
nucleotide sequence(s). Methods for introducing such modifications in the
nucleic
acid molecules according to the invention are well-known to the person skilled
in the
art. The invention also relates to nucleic acid molecules the sequence of
which
differs from the nucleotide sequence of any of the above-described nucleic
acid
molecules due to the degeneracy of the genetic code. All such fragments,
analogues and derivatives of the protein of the invention are included within
the
scope of the present invention, as long as the essential characteristic
immunological
and/or biological properties as defined above remain unaffected in kind, that
is the
novel nucleic acid molecules of the invention include all nucleotide sequences
encoding proteins or peptides which have at least a park of the primary
structural
conformation for one or more epitopes capable of reacting with antibodies to
cell
cycle interacting proteins which are encodable by a nucleic acid molecule as
set
forth above and which have comparable or identical characteristics in terms of
binding to cyclin dependent kinases, in particular plant cyclin dependent
kinases.
Part of the invention is therefore also nucleic acid molecules encoding a
polypeptide
comprising at least a functional part of a cell cycle interacting protein
encoded by a
nucleic acid sequence comprised in a nucleic acid molecule according to the
invention. An example for this is that the polypeptide or a fragment thereof
according
to the invention is embedded in another amino acid sequence.
As is demonstrated in the appended examples a two-hybrid screening assay has
been developed in accordance with the present invention suitable for
identifying cell
cycle interacting proteins. Thus, in another aspect the present invention
relates to a
method for identifying and obtaining cycle interacting proteins comprising a
two-
hybrid screening assay wherein CDC2a as a bait and a cDNA library of
vegetative
plant tissue as prey are used. Preferably, said CDC2a is CDC2aAt. However,
CDC2a from other organisms such as mammals may be employed as well.
The nucleic acid molecules encoding proteins or peptides identified to
interact with
the CDC2a in the above mentioned assay can be easily obtained and sequenced by
methods known in the art; see also the appended examples. Therefore, the
present
CA 02301830 2000-02-24
invention also relates to a DNA sequence encoding a cell cycle interacting
protein
obtainable by the method of the invention.
In a preferred embodiment the nucleic acid molecules according to the
invention are
RNA or DNA molecules, preferably cDNA, genomic DNA or synthetically
synthesized DNA or RNA molecules. Preferably, the nucleic acid molecule of the
invention is derived from a plant, preferably from Arabidopsis thaliana. As
discussed
above, the proteins encoded by the nucleic acid molecules identified according
to
the present invention in Arabidopsis thaliana show some homology to kinesins
from
several organisms. Corresponding proteins displaying similar properties
should,
therefore, be present in other plants as well. Nucleic acid molecules of the
invention
can be obtained, e.g., by hybridization of the above-described nucleic acid
molecules with a (sample of) nucleic acid molecules) of any source. Nucleic
acid
molecules hybridizing with the above-described nucleic acid molecules can in
general be derived from any organism, preferably plant possessing such
molecules,
preferably form monocotyledonous or dicotyledonous plants, in particular from
any
organism, preferably plants of interest in agriculture, horticulture or wood
culture,
such as crop plants, namely those of the family Poaceae, any starch producing
plants, such as potato, maniok, leguminous plants, oil producing plants, such
as
oilseed rape, linenseed, etc., plants using polypeptide as storage substances,
such
as soybean, plants using sucrose as storage substance, such as sugar beet or
sugar cane, trees, ornamental plants etc. Preferably, the nucleic acid
molecules
according to the invention are derived from Arabidopsis thaliana. Nucleic acid
molecules hybridizing to the above-described nucleic acid molecules can be
isolated, e.g., form libraries, such as cDNA or genomic libraries by
techniques well
known in the art. For example, hybridizing nucleic acid molecules can be
identified
and isolated by using the above-described nucleic acid molecules or fragments
thereof or complements thereof as probes to screen libraries by hybridizing
with said
molecules according to standard techniques. Possible is also the isolation of
such
nucleic acid molecules by applying the polymerise chain reaction (PCR) using
as
primers oligonucleotides derived form the above-described nucleic acid
molecules.
CA 02301830 2000-02-24
11
Nucleic acid molecules which hybridize with any of the aforementioned nucleic
acid
molecules also include fragments, derivatives and allelic variants of the
above-
described nucleic acid molecules that encode a cell cycle interacting protein
or an
immunologically or functional fragment thereof. Fragments are understood to be
parts of nucleic acid molecules long enough to encode the described protein or
a
functional or immunologically active fragment thereof as defined above.
Preferably,
the functional fragment contains at least one of the phosphorylation sites
and/or the
Gln-rich domain at the N-terminus of the protein shown in Figure 1; see also
Example 2.
The term "derivative" means in this context that the nucleotide sequence of
these
nucleic acid molecules differs from the sequences of the above-described
nucleic
acid molecules in one or more nucleotide positions and are highly homologous
to
said nucleic acid molecules. Homology is understood to refer to a sequence
identity
of at least 40 %, particularly an identity of at least 60 %, preferably more
than 80
and still more preferably more than 90 %. The term "substantially homologous"
refers to a subject, for instance a nucleic acid, which is at least 50%
identical in
sequence to the reference when the entire ORF (open reading frame) is
compared,
where the sequence identity is preferably at least 70%, more preferably at
least
80%, still more preferably at least 85%, especially more than about 90%, most
preferably 95% or greater, particularly 98% or greater. The deviations from
the
sequences of the nucleic acid molecules described above can, for example, be
the
result of nucleotide substitution(s), deletion(s), addition(s), insertions)
avd/or
recombination(s); see supra.
Homology further means that the respective nucleic acid molecules or encoded
proteins are functionally andlor structurally equivalent. The nucleic acid
molecules
that are homologous to the nucleic acid molecules described above and that are
derivatives of said nucleic acid molecules are, for example, variations of
said nucleic
acid molecules which represent modifications having the same biological
function, in
particular encoding proteins with the same or substantially the same
biological
function. They may be naturally occurring variations, such as sequences from
other
plant varieties or species, or mutations. These mutations may occur naturally
or may
CA 02301830 2000-02-24
12
be obtained by mutagenesis techniques. The allelic variations may be naturally
occurring allelic variants as well as synthetically produced or genetically
engineered
variants; see supra.
The proteins encoded by the various derivatives and variants of the above-
described nucleic acid molecules share specific common characteristics, such
as
biological activity, molecular weight, immunological reactivity, conformation,
etc., as
well as physical properties, such as electrophoretic mobility, chromatographic
behavior, sedimentation coefficients, pH optimum, temperature optimum,
stability,
solubility, spectroscopic properties, etc.
Examples of the different possible applications of the nucleic acid molecules
according to the invention as well as molecules derived from them will be
described
in detail in the following.
Hence, in a further embodiment, the invention relates to nucleic acid
molecules of at
least 15 nucleotides in length hybridizing specifically with a nucleic acid
molecule as
described above or with a complementary strand thereof. Specific hybridization
occurs preferably under stringent conditions and implies no or very little
cross-
hybridization with nucleotide sequences encoding no or substantially different
proteins. Such nucleic acid molecules may be used as probes and/or for the
control
of gene expression. Nucleic acid probe technology is well known to those
skilled in
the art who will readily appreciate that such probes may vary in length.
Preferred are
nucleic acid probes of 16 to 35 nucleotides in length. Of course, it may also
be
appropriate to use nucleic acids of up to 100 and more nucleotides in length.
The
nucleic acid probes of the invention are useful for various applications. On
the one
hand, they may be used as PCR primers for amplification of nucleic acid
sequences
according to the invention. The design and use of said primers is known by the
person skilled in the art. Preferably such amplification primers comprise a
contiguous sequence of at least 6 nucleotides, in particular 13 nucleotides,
preferably 15 to 25 nucleotides or more, identical or complementary to the
nucleotide sequence depicted in SEQ ID NO: 1 or to a nucleotide sequence
CA 02301830 2000-02-24
13
encoding the amino acid sequence of SEQ ID NO: 2. Another application is the
use
as a hybridization probe to identify nucleic acid molecules hybridizing with a
nucleic
acid molecule of the invention by homology screening of genomic DNA or cDNA
libraries. Nucleic acid molecules according to this preferred embodiment of
the
invention which are complementary to a nucleic acid molecule as described
above
may also be used for repression of expression of a cell cycle gene, for
example due
to an antisense or triple helix effect or for the construction of appropriate
ribozymes
(see, e.g., EP-A1 0 291 533, EP-A1 0 321 201, EP-A2 0 360 257) which
specifically
cleave the (pre)-mRNA of a gene comprising a nucleic acid molecule of the
invention or part thereof. Selection of appropriate target sites and
corresponding
ribozymes can be done as described, for example, in Steinecke, Ribozymes,
Methods in Cell Biology 50, Galbraith et al. eds Academic Press, Inc. (1995),
449-
460. Furthermore, the person skilled in the art is well aware that it is also
possible to
label such a nucleic acid probe with an appropriate marker for specific
applications,
such as for the detection of the presence of a nucleic acid molecule of the
invention
in a sample derived from an organism, in particular plants.
The above described nucleic acid molecules may either be DNA or RNA or a
hybrid
thereof. Furthermore, said nucleic acid molecule may contain, for example,
thioester
bonds and/or nucleotide analogues, commonly used in oligonucleotide anti-sense
approaches. Said modifications may be useful for the stabilization of the
nucleic acid
molecule against endo- and/or exonucleases in the cell. Said nucleic acid
molecules
may be transcribed by an appropriate vector containing a chimeric gene which
allows for the transcription of said nucleic acid molecule in the cell.
Furthermore, the so-called "peptide nucleic acid" (PNA) technique can be used
for
the detection or inhibition of the expression of a nucleic acid molecule of
the
invention. For example, the binding of PNAs to complementary as well as
various
single stranded RNA and DNA nucleic acid molecules can be systematically
investigated using thermal denaturation and BIAcore surface-interaction
techniques
(Jensen, Biochemistry 36 (1997), 5072-5077). Furthermore, the nucleic acid
molecules described above as well as PNAs derived therefrom can be used for
CA 02301830 2000-02-24
14
detecting point mutations by hybridization with nucleic acids obtained from a
sample
with an affinity sensor, such as BIAcore; see Gotoh, Rinsho Byori 45 (1997),
224-
228. Hybridization based DNA screening on peptide nucleic acids (PNA) oligomer
arrays are described in the prior art, for example in Weiler, Nucleic Acids
Research
25 (1997), 2792-2799. The synthesis of PNAs can be performed according to
methods known in the art, for example, as described in Koch, J. Pept. Res. 49
(1997), 80-88; Finn, Nucleic Acids Research 24 (1996), 3357-3363. Further
possible
applications of such PNAs, for example as restriction enzymes or as templates
for
the synthesis of nucleic acid oligonucleotides are known to the person skilled
in the
art and are, for example, described in Veselkov, Nature 379 (1996), 214 and
Bohler,
Nature 376 (1995), 578-581.
The present invention also relates to vectors, particularly plasmids, cosmids,
viruses, bacteriophages and other vectors used conventionally in genetic
engineering that contain a nucleic acid molecule according to the invention.
Methods
which are well known to those skilled in the art can be used to construct
various
plasmids and vectors; see, for example, the techniques described in Sambrook,
Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989)
N.Y.
and Ausubel, Current Protocols in Molecular Biology, Green Publishing
Associates
and Wiley Interscience, N.Y. (1989). Alternatively, the nucleic acid molecules
and
vectors of the invention can be reconstituted into liposomes for delivery to
target
cells.
In a preferred embodiment the nucleic acid molecule present in the vector is
linked
to (a) control sequences) which allow the expression of the nucleic acid
molecule in
prokaryotic andlor eukaryotic cells.
The term "control sequence" refers to regulatory DNA sequences which are
necessary to effect the expression of coding sequences to which they are
ligated.
The nature of such control sequences differs depending upon the host organism.
In
prokaryotes, control sequences generally include promoter, ribosomal binding
site,
and terminators. In eukaryotes generally control sequences include promoters,
terminators and, in some instances, enhancers, transactivators or
transcription
CA 02301830 2000-02-24
factors. The term "control sequence" is intended to include, at a minimum, all
components the presence of which are necessary for expression, and may also
include additional advantageous components.
The term "operably linked" refers to a juxtaposition wherein the components so
described are in a relationship permitting them to function in their intended
manner.
A control sequence "operably linked" to a coding sequence is ligated in such a
way
that expression of the coding sequence is achieved under conditions compatible
with the control sequences. In case the control sequence is a promoter, it is
obvious
for a skilled person that double-stranded nucleic acid is used.
Thus, the vector of the invention is preferably an expression vector. An
"expression
vector" is a construct that can be used to transform a selected host cell and
provides
for expression of a coding sequence in the selected host. Expression vectors
can for
instance be cloning vectors, binary vectors or integrating vectors. Expression
comprises transcription of the nucleic acid molecule preferably into a
translatable
mRNA. Regulatory elements ensuring expression in prokaryotic and/or eukaryotic
cells are well known to those skilled in the art. In the case of eukaryotic
cells they
comprise normally promoters ensuring initiation of transcription and
optionally poly-A
signals ensuring termination of transcription and stabilization of the
transcript, for
example, those of the 35S RNA from Cauliflower Mosaic Virus (CaMV). Other
promoters commonly used are the polyubiquitin promoter, and the actin promoter
for
ubiquitous expression. The termination signals usually employed are from the
Nopaline Synthase promoter or from the CAMV 35S promoter. A plant
translational
enhancer often used is the CAMV omega sequences, the inclusion of an intron
(Intron-1 from the Shrunken gene of maize, for example) has been shown to
increase expression levels by up to 100-fold. (Mait, Transgenic Research 6
(1997),
143-156; Ni, Plant Journal 7 (1995), 661-676). Additional regulatory elements
may
include transcriptional as well as translational enhancers. Possible
regulatory
elements permitting expression in prokaryotic host cells comprise, e.g., the
P~, lac,
trp or tac promoter in E. coli, and examples of regulatory elements permitting
expression in eukaryotic host cells are the AOX9 or GALS promoter in yeast or
the
CMV-, SV40- , RSV-promoter (Rows sarcoma virus), CMV-enhancer, SV40-
enhancer or a globin intron in mammalian and other animal cells. In this
context,
CA 02301830 2000-02-24
16
suitable expression vectors are known in the art such as Okayama-Berg cDNA
expression vector pcDV1 (Pharmacia), pCDMB, pRcICMV, pcDNA1, pcDNA3 {In-
vitrogene), pSPORT1 (GIBCO BRL). Advantageously, the above-described vectors
of the invention comprises a selectable andlor scorable marker. Selectable
marker
genes useful for the selection of transformed plant cells, callus, plant
tissue and
plants are well known to those skilled in the art and comprise, for example,
antimetabolite resistance as the basis of selection for dhfr, which confers
resistance
to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149);
npt,
which confers resistance to the aminoglycosides neomycin, kanamycin and
paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995) and hygro, which
confers
resistance to hygromycin (Marsh, Gene 32 {1984), 481-485). Additional
selectable
genes have been described, namely trpB, which allows cells to utilize indole
in place
of tryptophan; hisD, which allows cells to utilize histinol in place of
histidine
(Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate
isomerase which allows cells to utilize mannose (WO 94120627) and ODC
(ornithine
decarboxylase) which confers resistance to the ornithine decarboxylase
inhibitor, 2-
(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: Current
Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.) or
deaminase from Aspergillus terreus which confers resistance to Blasticidin S
(Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338).
Useful scorable marker are also known to those skilled in the art and are
commercially available. Advantageously, said marker is a gene encoding
luciferase
(Giacomin, PI. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121 ),
green
fluorescent protein {Gerdes, FEES Lett. 389 (1996), 44-47) or t3-glucuronidase
(Jefferson, EMBO J. 6 (1987), 3901-3907). This embodiment is particularly
useful for
simple and rapid screening of cells, tissues and organisms containing a vector
of the
invention.
The present invention furthermore relates to host cells comprising a vector as
described above or a nucleic acid molecule according to the invention wherein
the
nucleic acid molecule is foreign to the host cell.
CA 02301830 2000-02-24
17
By "foreign" it is meant that the nucleic acid molecule is either heterologous
with
respect to the host cell, this means derived from a cell or organism with a
different
genomic background, or is homologous with respect to the host cell but located
in a
different genomic environment than the naturally occurring counterpart of said
nucleic acid molecule. This means that, if the nucleic acid molecule is
homologous
with respect to the host cell, it is not located in its natural location in
the genome of
said host cell, in particular it is surrounded by different genes. In this
case the
nucleic acid molecule may be either under the control of its own promoter or
under
the control of a heterologous promoter. The vector or nucleic acid molecule
according to the invention which is present in the host cell may either be
integrated
into the genome of the host cell or it may be maintained in some form
extrachromosomally. In this respect, it is also to be understood that the
nucleic acid
molecule of the invention can be used to restore or create a mutant gene via
homologous recombination (Paszkowski (ed.), Homologous Recombination and
Gene Silencing in Plants. Kluwer Academic Publishers (1994)).
The host cell can be any prokaryotic or eukaryotic cell, such as bacterial,
insect,
fungal, plant or animal cells. Preferred fungal cells are, for example, those
of the
genus Saccharomyces, in particular those of the species S. cerevisiae.
Another subject of the invention is a method for the preparation of cell cycle
interacting proteins which comprises the cultivation of host cells according
to the
invention which, due to the presence of a vector or a nucleic acid molecule
according to the invention, are able to express such a protein, under
conditions
which allow expression of the protein and recovering of the so-produced
protein
from the culture.
The term "expression" means the production of a protein or nucleotide sequence
in
the cell. However, said term also includes expression of the protein in a cell-
free
system. It includes transcription into an RNA product, post-transcriptional
modification and/or translation to a protein product or polypeptide from a DNA
encoding that product, as well as possible post-translational modifications.
Depending on the specific constructs and conditions used, the protein may be
CA 02301830 2000-02-24
18
recovered from.the cells, from the culture medium or from both. For the person
skilled in the art it is well known that it is not only possible to express a
native protein
but also to express the protein as fusion polypeptides or to add signal
sequences
directing the protein to specific compartments of the host cell, e.g.,
ensuring
secretion of the peptide into the culture medium, etc. Furthermore, such a
protein
and fragments thereof can be chemically synthesized and/or modified according
to
standard methods described, for example hereinbelow.
The terms "protein" and "polypeptide" used in this application are
interchangeable.
"Polypeptide" refers to a polymer of amino acids (amino acid sequence) and
does
not refer to a specific length of the molecule. Thus peptides and
oligopeptides are
included within the definition of polypeptide. This term does also refer to or
include
post-translational modifications of the polypeptide, for example,
glycosylations,
acetylations, phosphorylations and the like. Included within the definition
are, for
example, polypeptides containing one or more analogs of an amino acid
(including,
for example, unnatural amino acids, etc.), polypeptides with substituted
linkages, as
well as other modifications known in the art, both naturally occurring and non-
naturally occurring.
The present invention furthermore relates to proteins encoded by the nucleic
acid
molecules according to the invention or produced or obtained by the above-
described methods, and to functional and/or immunologically active fragments
of
such cell cycle interacting proteins. The proteins and polypeptides of the
present
invention are not necessarily translated from a designated nucleic acid
sequence;
the polypeptides may be generated in any manner, including far example,
chemical
synthesis, or expression of a recombinant expression system, or isolation from
a
suitable viral system. The polypeptides may include one or more analogs of
amino
acids, phosphorylated amino acids or unnatural amino acids. Methods of
inserting
analogs of amino acids into a sequence are known in the art. The polypeptides
may
also include one or more labels, which are known to those skilled in the art.
In this
context, it is also understood that the proteins according to the invention
may be
further modified by conventional methods known in the art. By providing the
proteins
according to the present invention it is also possible to determine fragments
which
CA 02301830 2000-02-24
19
retain biological activity, namely the mature, processed form. This allows the
construction of chimeric proteins and peptides comprising an amino sequence
derived from the protein of the invention, which is crucial for its binding
activity and
other functional amino acid sequences, e.g. GUS marker gene (Jefferson, EMBO
J.
6 (1987), 3901-3907). The other functional amino acid sequences may be either
physically linked by, e.g., chemical means to the proteins of the invention or
may be
fused by recombinant DNA techniques well known in the art.
The term "fragment of a sequence" or "part of a sequence" means a truncated
sequence of the original sequence referred to. The truncated sequence (nucleic
acid
or protein sequence) can vary widely in length; the minimum size being a
sequence
of sufficient size to provide a sequence with at least a comparable function
and/or
activity of the original sequence referred to, while the maximum size is not
critical. In
some applications, the maximum size usually is not substantially greater than
that
required to provide the desired activity and/or functions) of the original
sequence.
Typically, the truncated amino acid sequence will range from about 5 to about
60
amino acids in length. More typically, however, the sequence will be a maximum
of
about 50 amino acids in length, preferably a maximum of about 30 amino acids.
It is
usually desirable to select sequences of at least about 10, 12 or 15 amino
acids, up
to a maximum of about 20 or 25 amino acids.
Furthermore, folding simulations and computer redesign of structural motifs of
the
protein of the invention can be performed using appropriate computer programs
(Olszewski, Proteins 25 {1996), 286-299; Hoffman, Comput. Appl. Biosci. 11
(1995),
675-679). Computer modeling of protein folding can be used for the
conformational
and energetic analysis of detailed peptide and protein models (Monge, J. Mol.
Biol.
247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45). In
particular,
the appropriate programs can be used for the identification of interactive
sites of the
protein and cyclin dependent kinases, its receptor, its ligand or other
interacting
proteins by computer assistant searches for complementary peptide sequences
(Fassina, Immunomethods 5 (1994), 114-120. Further appropriate computer
systems
for the design of protein and peptides are described in the prior art, for
example in
Berry, Biochem. Soc. Traps. 22 (1994), 1033-1036; Wodak, Ann. N. Y. Acad. Sci.
501
(1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained
from the
CA 02301830 2000-02-24
above-described computer analysis can be used for, e.g., the preparation of
peptidomimetics of the protein of the invention or fragments thereof. Such
pseudopeptide analogues of the natural amino acid sequence of the protein may
very
efficiently mimic the parent protein (Benkirane, J. Biol. Chem. 271 (1996),
33218-
33224). For example, incorporation of easily available achiral S2-amino acid
residues
into a protein of the invention or a fragment thereof results in the
substitution of amide
bonds by polymethylene units of an aliphatic chain, thereby providing a
convenient
strategy for constructing a peptidomimetic (Banerjee, Biopolymers 39 (1996),
769-
777). Superactive peptidomimetic analogues of small peptide hormones in other
systems are described in the prior art (Zhang, Biochem. Biophys. Res. Commun.
224
(1996), 327-331 ). Appropriate peptidomimetics of the protein of the present
invention
can also be identified by the synthesis of peptidomimetic combinatorial
libraries
through successive amide alkylation and testing the resulting compounds, e.g.,
for
their binding and immunological properties. Methods for the generation and use
of
peptidomimetic combinatorial libraries are described in the prior art, for
example in
Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med.
Chem. 4 (1996), 709-715.
Furthermore, a three-dimensional andlor crystallographic structure of the
protein of the
invention can be used for the design of peptidomimetic inhibitors of the
biological
activity of the protein of the invention (Rose, Biochemistry 35 (1996), 12933-
12944;
Rutenber, Bioorg. Med. Chem. 4 (199fi), 1545-1558).
Furthermore, the present invention relates to antibodies specifically
recognizing a cell
cycle interacting protein according to the invention or parts, i.e. specific
fragments or
epitopes, of such a protein. The antibodies of the invention can be used to
identify
and isolate other cell cycle interacting proteins and genes in any organism,
preferably plants. These antibodies can be monoclonal antibodies, polyclonal
antibodies or synthetic antibodies as well as fragments of antibodies, such as
Fab, Fv
or scFv fragments etc. Monoclonal antibodies can be prepared, for example, by
the
techniques as originally described in Kohler and Milstein, Nature 256 (1975),
495, and
Galfre, Meth. Enzymol. 73 (1981 ), 3, which comprise the fusion of mouse
myeloma
cells to spleen cells derived from immunized mammals. Furthermore, antibodies
or
CA 02301830 2000-02-24
21
fragments thereof to .the aforementioned peptides can be obtained by using
methods which are described, e.g., in Harlow and Lane "Antibodies, A
Laboratory
Manual", CSH Press, Cold Spring Harbor, 1988. These antibodies can be used,
for
example, for the immunoprecipitation and immunolocalization of proteins
according to
the invention as well as for the monitoring of the synthesis of such proteins,
for
example, in recombinant organisms, and for the identification of compounds
interacting with the protein according to the invention. For example, surface
plasmon
resonance as employed in the BIAcore system can be used to increase the
efficiency
of phage antibodies selections, yielding a high increment of affinity from a
single
library of phage antibodies which bind to an epitope of the protein of the
invention
(Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol.
Methods 183 (1995), 7-13). In many cases, the binding phenomena of antibodies
to
antigens is equivalent to other ligand/anti-ligand binding.
Plant cell division can conceptually be influenced in three ways : (i)
inhibiting or
arresting cell division, (ii) maintaining, facilitating or stimulating cell
division or (iii)
uncoupling DNA synthesis from mitosis and cytokinesis. Modulation of the
expression of a polypeptide encoded by a nucleotide sequence according to the
invention has surprisingly an advantageous influence on plant cell division
characteristics, in particular on the disruption of the expression levels of
genes
involved in G1/S and/or G2/M transition and as a result therof on the total
make-up
of the plant concerned or parts thereof. An example is that DNA synthesis or
progression of DNA replication will be negatively influenced by elimination of
specific
substrates for a cyclin-dependent protein kinase complex.
The term "cyclin-dependent protein kinase complex" means the complex formed
when a, preferably functional, cyclin associates with a, preferably,
functional cyclin
dependent kinase. Such complexes may be active in phosphorylating proteins and
may or may not contain additional protein species.
The presence, absence or activity of a substrate for CDK in a plant cell is
influenced
by manipulation of the gene according to the invention. To analyse the
industrial
applicabilities of the invention, transformed plants can be made overproducing
the
CA 02301830 2000-02-24
22
nucleotide sequence according to the invention. Such an overexpression of the
new
gene(s), proteins or inactivated variants thereof will either positively or
negatively
have an effect on cell division. Methods to modify the expression levels
and/or the
activity are known to persons skilled in the art and include for instance
overexpression, co-suppression, the use of ribozymes, sense and anti-sense
strategies, gene silencing approaches. "Sense strand" refers to the strand of
a
double-stranded DNA molecule that is homologous to a mRNA transcript thereof.
The "anti-sense strand" contains an inverted sequence which is complementary
to
that of the "sense strand".
Hence, the nucleic acid molecules according to the invention are in particular
useful
for the genetic manipulation of plant cells in order to modify the
characteristics of
plants and to obtain plants with modified, preferably with improved or useful
phenotypes. Similarly, the invention can also be used to modulate the cell
division
and the growth of cells, preferentially plant cells, in in vitro cultures.
Thus, the present invention provides for a method for the production of
transgenic
plants, plant cells or plant tissue comprising the introduction of a nucleic
acid
molecule or vector of the invention into the genome of said plant, plant cell
or plant
tissue.
For the expression of the nucleic acid molecules according to the invention in
sense or
antisense orientation in plant cells, the molecules are placed under the
control of
regulatory elements which ensure the expression in plant cells. These
regulatory
elements may be heterologous or homologous with respect to the nucleic acid
molecule to be expressed as well with respect to the plant species to be
transformed.
In general, such regulatory elements comprise a promoter active in plant
cells. To
obtain expression in all tissues of a transgenic plant, preferably
constitutive promoters
are used, such as the 35 S promoter of CaMV (Odell, Nature 313 (1985), 810-
812) or
promoters of the polyubiquitin genes of maize (Christensen, Plant Mol. Biol.
18 (1982),
675-689). In order to achieve expression in specific tissues of a transgenic
plant it is
possible to use tissue specific promoters (see, e.g., Stockhaus, EMBO J. 8
(1989),
2245-2251 ). Known are also promoters which are specifically active in tubers
of
CA 02301830 2000-02-24
23
potatoes or in seeds of different plants species, such as maize, Vicia, wheat,
barley
etc. Inducible promoters may be used in order to be able to exactly control
expression.
An example for inducible promoters are the promoters of genes encoding heat
shock
proteins. Also microspore-specific regulatory elements and their uses have
been
described (W096/16182). Furthermore, the chemically inducible Test-system may
be
employed (Gatz, Mol. Gen. Genet. 227 (1991 ); 229-237). Further suitable
promoters
are known to the person skilled in the art and are described, e.g., in Ward
(Plant Mol.
Biol. 22 (1993), 361-366). The regulatory elements may further comprise
transcriptional and/or translational enhancers functional in plants cells.
Furthermore,
the regulatory elements may include transcription termination signals, such as
a poly-
A signal, which lead to the addition of a poly A tail to the transcript which
may improve
its stability.
In the case that a nucleic acid molecule according to the invention is
expressed in
sense orientation it is in principle possible to modify the coding sequence in
such a
way that the protein is located in any desired compartment of the plant cell.
These
include the nucleus, endoplasmatic reticulum, the vacuole, the mitochondria,
the
plastids, the apoplast, the cytoplasm etc. Since CDC2, the interacting
component of
the protein of the invention excerts its effects in the cytoplasm and/or
nucleus,
corresponding signal sequences are preferred to direct the protein of the
invention in
the same compartment. Methods how to carry out this modifications and signal
sequences ensuring localization in a desired compartment are well known to the
person skilled in the art.
Methods for the introduction of foreign DNA into plants are also well known in
the art.
These include, for example, the transformation of plant cells or tissues with
T-DNA
using Agrobacterium tumefaciens or Agrobacterium rhizogenes, the fusion of
protoplasts, direct gene transfer (see, e.g., EP-A 164 575), injection,
electroporation,
biolistic methods like particle bombardment, pollen-mediated transformation,
plant
RNA virus-mediated transformation, liposome-mediated transformation,
transformation using wounded or enzyme-degraded immature embryos, or wounded
or enzyme-degraded embryogenic callus and other methods known in the art. The
CA 02301830 2000-02-24
24
vectors used in the method of the invention may contain further functional
elements,
for example "left border"- and "right border"-sequences of the T-DNA of
Agrobacterium which allow for stably integration into the plant genome.
Furthermore,
methods and vectors are known to the person skilled in the art which permit
the
generation of marker free transgenic plants, i.e. the selectable or scorable
marker
gene is lost at a certain stage of plant development or plant breeding. This
can be
achieved by, for example cotransformation (Lyznik, Plant Mol. Biol. 13 (1989),
151-
161; Peng, Plant Mol. Biol. 27 (1995), 91-104) andlor by using systems which
utilize
enzymes capable of promoting homologous recombination in plants (see, e.g.,
W097/08331; Bayley, Plant Mol. Biol. 18 (1992), 353-361 ); Lloyd, Mol. Gen.
Genet.
242 (1994), 653-657; Maeser, Mol. Gen. Genet. 230 (1991 ), 170-176; Onouchi,
Nucl. Acids Res. 19 (1991 ), 6373-6378). Methods for the preparation of
appropriate
vectors are described by, e.g., Sambrook (Molecular Cloning; A Laboratory
Manual,
2nd Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY).
Suitable strains of Agrobacterium tumefaciens and vectors as well as
transformation
of Agrobacteria and appropriate growth and selection media are well known to
those
skilled in the art and are described in the prior art (GV3101 (pMK90RK),
Koncz, Mol.
Gen. Genet. 204 (1986), 383-396; C58C1 (pGV 3850kan), Deblaere, Nucl. Acid
Res. 13 (1985), 4777; Bevan, Nucleic. Acid Res. 12(1984), 8711; Koncz, Proc.
Natl.
Acad. Sci. USA 86 (1989), 8467-8471; Koncz, Plant Mol. Biol. 20 (1992), 963-
976;
Koncz, Specialized vectors for gene tagging and expression studies. In: Plant
Molecular Biology Manual Vol 2, Gelvin and Schilperoort (Eds.), Dordrecht, The
Netherlands: Kluwer Academic Publ. (1994), 1-22; EP-A-120 516; Hoekema: The
Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam (1985),
Chapter V, Fraley, Crit. Rev. Plant. Sci., 4, 1-46; An, EMBO J. 4 (1985), 277-
287).
Although the use of Agrobacterium tumefaciens is preferred in the method of
the
invention, other Agrobacterium strains, such as Agrobacterium rhizogenes, may
be
used, for example if a phenotype conferred by said strain is desired.
Methods for the transformation using biolistic methods are well known to the
person
skilled in the art; see, e.g., Wan, Plant Physiol. 104 (1994), 37-48; Vasii,
Bio/Technology 11 (1993), 1553-1558 and Christou (1996) Trends in Plant
Science
CA 02301830 2000-02-24
1, 423-431. Microinjection can be performed as described in Potrykus and
Spangenberg (eds.), Gene Transfer To Plants. Springer Verlag, Berlin, NY
(1995).
The transformation of most dicotyledonous plants is possible with the methods
described above. But also for the transformation of monocotyledonous plants
several
successful transformation techniques have been developed. These include the
transformation using biolistic methods as, e.g., described above as well as
protoplast
transformation, electroporation of partially permeabilized cells, introduction
of DNA
using glass fibers, etc.
The term "transformation" as used herein, refers to the transfer of an
exogenous
polynucleotide into a host cell, irrespective of the method used for the
transfer. The
polynucleotide may be transiently or stably introduced into the host cell and
rnay be
maintained non-integrated, for example, as a plasmid, or alternatively, may be
integrated into the host genome. The resulting transformed plant cell can then
be
used to regenerate a transformed plant in a manner known by a skilled person.
In general, the plants which can be modified according to the invention and
which
either show overexpression of a protein according to the invention or a
reduction of
the synthesis of such a protein can be derived from any desired plant species.
They
can be monocotyledonous plants or dicotyledonous plants, preferably they
belong to
plant species of interest in agriculture, wood culture or horticulture
interest, such as
crop plants (e.g. maize, rice, barley, wheat, rye, oats etc.), potatoes, oil
producing
plants (e.g. oilseed rape, sunflower, pea nut, soy bean, etc.), cotton, sugar
beet, sugar
cane, leguminous plants (e.g. beans, peas etc.), wood producing plants,
preferably
trees, etc.
Thus, the present invention relates also to transgenic plant cells which
contain stably
integrated into the genome a nucleic acid molecule according to the invention
linked to
regulatory elements which allow for expression of the nucleic acid molecule in
plant
cells and wherein the nucleic acid molecule is foreign to the transgenic plant
cell. For
the meaning of foreign; see supra.
CA 02301830 2000-02-24
26
The presence and expression of the nucleic acid molecule in the transgenic
plant cells
leads to the synthesis of a cell cycle interacting protein and leads to
physiological and
phenotypic changes in plants containing such cells.
Thus, the present invention also relates to transgenic plants and plant tissue
comprising transgenic plant cells according to the invention. Due to the
(over)expression of a cell cycle interacting protein of the invention, e.g.,
at
developmental stages and/or in plant tissue in which they do not naturally
occur these
transgenic plants may show various physiological, developmental and/or
morphological modifications in comparison to wild-type plants. For example,
these
transgenic plants may display an altered cell elongation.
Therefore, part of this invention is the use of plant cell cycle genes and/or
plant cell
cycle proteins to modulate plant cell division and/or growth in plant cells,
plant
tissues, plant organs and/or whole plants. To the scope of the invention also
belongs a method to influence the activity of cyclin-dependent protein kinase
in a
plant cell by transforming the plant cell with a nucleic acid molecule
according to the
invention and/or manipulation of the expression of said molecule. More in
particular
using a nucleic acid molecule according to the invention, the disruption of
plant cell
division can be accomplished by interfering in the expression of a substrate
for
cyclin-dependent protein kinase. The latter goal may be achieved, for example,
with
methods for reducing the amount of active cell cycle interacting proteins.
Hence, the invention also relates to a transgenic plant cell which contains
(stably
integrated into the genome) a nucleic acid molecule according to the invention
or part
thereof, wherein the transcription and/or expression of the nucleic acid
molecule or
part thereof leads to reduction of the synthesis of a cell cycle interacting
protein.
In a preferred embodiment, the reduction is achieved by an anti-sense, sense,
ribozyme, co-suppression and/or dominant mutant effect.
"Antisense" and "antisense nucleotides" means DNA or RNA constructs which
block
the expression of the naturally occurring gene product. For example, in the
present
CA 02301830 2000-02-24
27
invention use of a DNA construct that produces "th65 antisense RNA" blocks the
expression of "th65" by destroying or inactivating "th65 mRNA".
The provision of the nucleic acid molecules according to the invention opens
up the
possibility to produce transgenic plant cells with a reduced level of the
protein as
described above and, thus, with a defect in the accumulation of a cell cycle
interacting
protein. Techniques how to achieve this are well known to the person skilled
in the art.
These include, for example, the expression of antisense-RNA, ribozymes, of
molecules which combine antisense and ribozyme functions and/or of molecules
which provide for a co-suppression effect; see also supra. When using the
antisense
approach for reduction of the amount of cell cycle interacting proteins in
plant cells, the
nucleic acid molecule encoding the antisense-RNA is preferably of homologous
origin
with respect to the plant species used for transformation. However, it is also
possible
to use nucleic acid molecules which display a high degree of homology to
endogenously occurring nucleic acid molecules encoding a cell cycle
interacting
protein. In this case the homology is preferably higher than 80%, particularly
higher
than 90% and still more preferably higher than 95%.
The reduction of the synthesis of a protein according to the invention in the
transgenic
plant cells can result in an alteration in, e.g., cell division. In transgenic
plants
comprising such cells this can lead to various physiological, developmental
andlor
morphological changes.
Thus, the present invention also relates to transgenic plants comprising the
above-
described transgenic plant cells. These may show, for example, reduced growth
characteristics.
The present invention also relates to cultured plant tissues comprising
transgenic plant
cells as described above which either show overexpression of a protein
according to
the invention or a reduction in synthesis of such a protein.
Any transformed plant obtained according to the invention can be used in a
conventional breeding scheme or in in vitro plant propagation to produce more
transformed plants with the same characteristics and/or can be used to
introduce the
CA 02301830 2000-02-24
28
same characteristic in other varieties of the same or related species. Such
plants are
also part of the invention. Seeds obtained from the transformed plants
genetically
also contain the same characteristic and are part of the invention. As
mentioned
before, the present invention is in principle applicable to any plant and crop
that can
be transformed with any of the transformation method known to those skilled in
the
art and includes for instance corn, wheat, barley, rice, oilseed crops,
cotton, tree
species, sugar beet, cassava, tomato, potato, numerous other vegetables,
fruits.
In yet another aspect, the invention also relates to harvestable parts and to
propagation material of the transgenic plants according to the invention which
either
contain transgenic plant cells expressing a nucleic acid molecule according to
the
invention or which contain cells which show a reduced level of the described
protein.
Harvestable parts can be in principle any useful parts of a plant, for
example, flowers,
pollen, seedlings, tubers, leaves, stems, fruit, seeds, roots etc. Propagation
material
includes, for example, seeds, fruits, cuttings, seedlings, tubers, rootstocks
etc.
The present invention further relates to a method for identifying and
obtaining an
activator or inhibitor of cell cycle proteins comprising the steps of:
(a) combining a compound to be screened with a reaction mixture containing the
protein of the invention and a readout system capable of interacting with the
protein under suitable conditions;
(b) maintaining said reaction mixture in the presence of the compound or a
sample comprising a plurality of compounds under conditions which permit
interaction of the protein with said readout system;
(c) identifying or verifying a sample and compound, respectively, which leads
to
suppression or activation of the readout system.
The term "read out system" in context with the present invention means a DNA
sequence which upon transcription and/or expression in a cell, tissue or
organism
provides for a scorable andlor selectable phenotype. Such read out systems are
well
known to those skilled in the art and comprise, for example, recombinant DNA
molecules and marker genes as described above and in the appended example.
CA 02301830 2000-02-24
29
The term "plurality of compounds" in a method of the invention is to be
understood
as a plurality of substances which may or may not be identical.
Said compound or plurality of compounds may be comprised in, for example,
samples, e.g., cell extracts from, e.g., plants, animals or microorganisms.
Furthermore, said compounds) may be known in the art but hitherto not known to
be capable of suppressing or activating cell cycle interacting proteins. The
reaction
mixture may be a cell free extract or may comprise a cell or tissue culture.
Suitable
set ups for the method of the invention are known to the person skilled in the
art and
are, for example, generally described in Alberts et al., Molecular Biology of
the Cell,
third edition (1994), in particular Chapter 17. The plurality of compounds may
be,
e.g., added to the reaction mixture, culture medium or injected into the cell.
If a sample containing a compound or a plurality of compounds is identified in
the
method of the invention, then it is either possible to isolate the compound
from the
origins! sample identified as containing the compound capable of suppressing
or
activating cell cycle interacting proteins, or one can further subdivide the
original
sample, for example, if it consists of a plurality of different compounds, so
as to
reduce the number of different substances per sample and repeat the method
with
the subdivisions of the original sample. Depending on the complexity of the
samples,
the steps described above can be performed several times, preferably until the
sample identified according to the method of the invention only comprises a
limited
number of or only one substance(s). Preferably said sample comprises
substances
of similar chemical and/or physical properties, and most preferably said
substances
are identical. Preferably, the compound identified according to the above
described
method or its derivative is further formulated in a form suitable for the
application in
plant breeding or plant cell and tissue culture.
The compounds which can be tested and identified according to a method of the
invention may be expression libraries, e.g., cDNA expression libraries,
peptides,
proteins, nucleic acids, antibodies, small organic compounds, hormones,
peptidomimetics, PNAs or the like (Milner, Nature Medicine 1 (1995), 879-880;
Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198 and references
cited
supra). Furthermore, genes encoding a putative regulator of cell cycle
interacting
CA 02301830 2000-02-24
gene and/or which excert their effects up- or downstream the cell cycle
interacting
protein of the invention may be identified using, for example, insertion
mutagenesis
using, for example, gene targeting vectors known in the art (see, e.g.,
Hayashi,
Science 258 (1992), 1350-1353; Fritze and Walden, Gene activation by T-DNA
tagging. In Mefhods in Molecular biology 44 (Gartland, K.M.A. and Davey, M.R.,
eds). Totowa: Human Press (1995), 281-294) or transposon tagging (Chandlee,
Physiologia Plantarum 78 (1990), 105-115). Said compounds can also be
functional
derivatives or analogues of known inhibitors or activators. Methods for the
preparation of chemical derivatives and analogues are well known to those
skilled in
the art and are described in, for example, Beilstein, Handbook of Organic
Chemistry,
Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A.
and
Organic Synthesis, Wiley, New York, USA. Furthermore, said derivatives and
analogues can be tested for their effects according to methods known in the
art.
Furthermore, peptidomimetics andlor computer aided design of appropriate
derivatives and analogues can be used, for example, according to the methods
described above. The cell or tissue that may be employed in the method of the
invention preferably is a host cell, plant cell or plant tissue of the
invention described
in the embodiments hereinbefore.
Determining whether a compound is capable of suppressing or activating cell
cycle
interacting proteins can be done, for example, by monitoring DNA duplication
and
cell division. It can further be done by monitoring the phenotypic
characteristics of
the cell of the invention contacted with the compounds and compare it to that
of
wild-type plants. In an additional embodiment, said characteristics may be
compared
to that of a cell contacted with a compound which is either known to be
capable or
incapable of suppressing or activating cell cycle interacting proteins.
The inhibitor or activator identified by the above-described method may prove
useful
as a herbicide, pesticide and/or as a plant growth regulator. Thus, in a
further
embodiment the invention relates to a compound obtained or identified
according to
the method of the invention said compound being an activator of cell cycle
interacting proteins or an inhibitor of cell cycle interacting proteins. The
above-
CA 02301830 2000-02-24
31
described compounds include, for example, cell cycle kinase inhibitors. "Cell-
cycle
kinase inhibitor" (CKI) is a protein which inhibit CDK/cyclin activity and is
produced
and/or activated when further cell division has to be temporarily or
continuously
prevented.
Such useful compounds can be for example transacting factors which bind to the
cell cycle interacting protein of the invention. Identification of transacting
factors can
be carried out using standard methods in the art (see, e.g., Sambrook, supra,
and
Ausubel, supra). To determine whether a protein binds to the protein of the
invention, standard native gel-shift analyses can be carried out. In order to
identify a
transacting factor which binds to the protein of the invention, the protein of
the
invention can be used as an affinity reagent in standard protein purification
methods,
or as a probe for screening an expression library. Once the transacting factor
is
identified, modulation of its binding to the cell cycle interacting protein of
the
invention can be pursued, beginning with, for example, screening for
inhibitors
against the binding of the transacting factor to the protein of the present
invention.
Activation or repression of cell cycle interacting proteins could then be
achieved in
plants by applying of the transacting factor (or its inhibitor) or the gene
encoding it,
e.g. in a vector for transgenic plants. In addition, if the active form of the
transacting
factor is a dimer, dominant-negative mutants of the transacting factor could
be made
in order to inhibit its activity. Furthermore, upon identification of the
transacting
factor, further components in the pathway leading to activation (e.g. signal
transduction) or repression of a gene involved in the control of cell cycle
then can be
identified. Modulation of the activities of these components can then be
pursued, in
order to develop additional drugs and methods for modulating the cell cycle in
animals and plants.
The invention also relates to a diagnostic composition comprising at least one
of the
aforementioned nucleic acid molecules, vectors, proteins, antibodies or
compounds
and optionally suitable means for detection.
Said diagnostic compositions may be used for methods for detecting expression
of
cell cycle interacting proteins by detecting the presence of the corresponding
mRNA
CA 02301830 2000-02-24
32
which comprises isolation of mRNA from a cell and contacting the mRNA so
obtained with a probe comprising a nucleic acid probe as described above under
hybridizing conditions, detecting the presence of mRNA hybridized to the
probe, and
thereby detecting the expression of the protein in the cell. Further methods
of
detecting the presence of a protein according to the present invention
comprises
immunotechniques well known in the art, for example enzyme linked
immunosorbent
assay. Furthermore, it is possible to use the nucleic acid molecules according
to the
invention as molecular markers in plant breeding.
The person skilled in the art can use proteins according to the invention from
other
organisms such as yeast and animals to influence cell division progression in
those
other organisms such as mammals or insects. In a preferred embodiment one or
more DNA sequences, vectors or proteins of the invention or the above-
described
antibody or compound are, for instance, used to specifically interfere in the
disruption of the expression levels of genes involved in G1/S and/or G2/M
transition
in the cell cycle process in transformed plants, particularly
~ in the complete plant
~ in selected plant organs, tissues or cell types
~ under specific environmental conditions, including abiotic stress such as
cold, heat, drought or salt stress or biotic stress such as pathogen attack
~ during specific developmental stages.
Another aspect of the current invention is that one or more DNA sequences,
vectors
or proteins of the invention or the above-described antibody or compound can
be
used to modulate, for instance, endoreduplication in storage cells, storage
tissues
andlor storage organs of plants or parts thereof. The term "endoreduplication"
means recurrent DNA replication without consequent mitosis and cytokinesis.
Preferred target storage organs and parts thereof for the modulation of
endoreduplication are, for instance, seeds (such as from cereals, oilseed
crops),
roots (such as in sugar beet), tubers (such as in potato) and fruits (such as
in
vegetables and fruit species). Furthermore it is expected that increased
endoreduplication in storage organs and parts thereof correlates with enhanced
storage capacity and as such with improved yield. In yet another embodiment of
the
CA 02301830 2000-02-24
33
invention, a plant with modulated endoreduplication in the whole plant or
parts
thereof can be obtained from a single plant cell by transforming the cell, in
a manner
known to the skilled person, with the above-described means.
In view of the foregoing, the present invention also relates to the use of a
DNA
sequence, vector, protein, antibody or compound of the invention for
modulating
plant cell cycle, plant cell division and/or growth, for influencing the
activity of cyclin-
dependent protein kinase in a plant cell, for disrupting plant cell division
by
influencing the presence or absence or by interfering in the expression of a
substrate for cyclin-dependent protein kinase, for influencing cell division
progression in a host as defined above or for use in a screening method for
the
identification of inhibitors or activators of cell cycle proteins. Beside the
above
described possibilities to use the nucleic acid molecules according to the
invention for
the genetic engineering of plants with modified characteristics and their use
to identify
homologous molecules, the described nucleic acid molecules may also be used
for
several other applications, for example, for the identification of nucleic
acid molecules
which encode proteins which interact with the cell cycle proteins described
above. This
can be achieved by assays well known in the art such as those described above
and
also included, for example, as described in Scofield (Science 274 (1996), 2063-
2065)
by use of the so-called yeast "two-hybrid system"; see also the appended
examples.
In this system the protein encoded by the nucleic acid molecules according to
the
invention or a smaller part thereof is linked to the DNA-binding domain of the
GAL4
transcription factor. A yeast strain expressing this fusion protein and
comprising a IacZ
reporter gene driven by an appropriate promoter, which is recognized by the
GAL4
transcription factor, is transformed with a library of cDNAs which will
express plant
proteins or peptides thereof fused to an activation domain. Thus, if a peptide
encoded
by one of the cDNAs is able to interact with the fusion peptide comprising a
peptide of
a protein of the invention, the complex is able to direct expression of the
reporter
gene. In this way the nucleic acid molecules according to the invention and
the
encoded peptide can be used to identify peptides and proteins interacting with
cell
cycle interacting proteins. It is apparent to the person skilled in the art
that this and
similar systems may then further be exploited for the identification of
inhibitors of the
binding of the interacting proteins.
CA 02301830 2000-02-24
34
Other methods for identifying compounds which interact with the proteins
according to
the invention or nucleic acid molecules encoding such molecules are, for
example, the
in vitro screening with the phage display system as well as filter binding
assays or
"real time" measuring of interaction using, for example, the BIAcore apparatus
(Pharmacia); see references cited supra.
These and other embodiments are disclosed and encompassed by the description
and examples of the present invention. Further literature concerning any one
of the
methods, uses and compounds to be employed in accordance with the present
invention may be retrieved from public libraries, using for example electronic
devices. For example the public database "Medline" may be utilized which is
available on the Internet, for example under
http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Further databases and
addresses, such as http://www.ncbi.nlm.nih.gov/, http://www.infobiogen.fr/,
http://www.fmi.ch/biology/research tools.html, http://www.tigr.org/, are known
to the
person skilled in the art and can also be obtained using, e.g.,
http://www.lycos.com.
An overview of patent information in biotechnology and a survey of relevant
sources
of patent information useful for retrospective searching and for current
awareness is
given in Berks, TIBTECH 12 (1994), 352-364.
The present invention is further described by reference to the following non-
limiting
figures and examples.
The FiQUres show:
Figure 1. Amino acid sequence encoded by the th65 clone. TPNK, SPGR, and SPVR
(in bold type and underlined) in the C-terminal part of the sequence are CDK
consensus phosphorylation sites. In the N-terminal region of the sequence the
amino
acid residue Q (underlined) is repeatedly present.
CA 02301830 2000-02-24
The Examrales illustrate the invention'
Unless stated otherwise in the examples, all recombinant DNA techniques are
performed according to protocols as described in Sambrook et al. (1989),
Molecular
Cloning : A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY or in
Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular
Biology,
Current Protocols. Standard materials and methods for plant molecular work are
described in Plant Molecular Biology Labfase (1993) by R.D.D. Croy, jointly
published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific
Publications (UK).
Example 1: Identification of a cell cycle interacting protein
The used vectors and strains were supplied with the Matchmaker Two-Hybrid
System
(Clontech, Palo Alto, CA). Baits using CDC2aAt were constructed by inserting
PCR
fragments into the pGBT9 vector. The PCR fragments were created from the cDNAs
by using primers to incorporate EcoRl restriction enzyme sites. For CDC2aAt,
the
primers 5'-CGAGATCTGAATTCATGGATCAGTA-3' (SEQ ID NO: 3) and
5'-CGAGATCTGAATTCCTAAGGCATGCC-3' (SEQ ID NO: 4) were used. For
CDC2bAt the primers 5'-CGGATCCGAATTCATGGAGAACGAG-3' (SEQ ID NO: 5)
and 5'-CGGATCCGAATTCTCAGAACTGAGA-3' (SEQ ID NO: 6) were used. The
PCR fragments were cut with EcoRl and cloned into the EcoRl site of pGBT9,
resulting in the plasmids pGBTCDC2A and pGBTCDC2B. The GAL4 activation
domain cDNA fusion library of 3-week-old vegetative Arabidopsis plants was
obtained
from Clontech. For the screening, a 1-liter culture of the Saccharomyces
cerevisiae
strain HF7c (MATa ura3-52 his3-200 ade2-101 lys2-801 trpl-901 ieu2-3,112 gal4-
542
ga180-538 LYS2:: GAL 1 "AS-GAL 1TATa-HiS3 UR,43:: GAL4"me~~3X>-CYC1TATA-LacZ)
was
cotransformed with 590 ~.g pGBTCDC2A, 1100 ~g DNA of the library, and 40 mg
salmon sperm carrier DNA using the lithium acetate method (Gietz ef al., 1992,
Nucleic Acids Research, 2~( , pg.1425). To estimate the number of independent
cotransformants, 1/1000 of the transformation mix was plated on Leu- and Trp-
CA 02301830 2000-02-24
36
medium. The rest of the transformation mix was plated on medium to select for
histidine prototrophy (Trp-, Leu-, His-). After 6 days of growth at
30°C, the colonies
larger than 2 mm were streaked on histidine-lacking medium supplemented with
10
mM 3-amino-1,2,4-triazole (Sigma, St. Louis, MO). Colonies capable of growing
under
these conditions were tested for (3-galactosidase activity. The activation
domain
plasmids were isolated from the His' and LacZ+ colonies. The pGAD10 inserts
were
PCR amplified using the primers 5'-ATACCACTACAATGGATG-3' (SEQ ID NO: 7)
and 5'-AGTTGAAGTGAACTTGCGGG-3' (SEQ ID NO: 8). PCR fragments were
digested with Alul and fractionized on a 2% agarose gel. Plasmids whose PCR
product gave rise to distinct restriction patterns were electroporated into
Escherichia
coli XL1-Blue, and the DNA sequence of the inserts was determined. Extracted
DNA
was also used to retransform HF7c to test the specificity of the interaction.
Example 2: Characterisation of the novel cell cycle gene
Above-mentioned two-hybrid screening was performed using as bait a fusion
protein
between the GAL4 DNA-binding domain and CDC2aAt. For the screening a GAL4
activation domain cDNA fusion library was used, constructed from 3-week-old
vegetative tissue of Arabidopsis thaliana. After sequential selection rounds,
an
interesting clone encoding a CDC2aAt-specific interacting protein was
identified being
designated as fhfi5, and appears to contain a 394-amino-acid-long open reading
frame (figure 1 ) corresponding to SEQ ID NO 2, which had no significant
homology
with any protein in data bases. The N-terminus contained a region rich in
glutamine
residues (SEQ ID NO 2.). Gln-rich domains are often part of the
transcriptional
activation domain of DNA binding factors (Mitchell and Tjian, 1989, Science,
2~, 371-
378) and have also been shown to be involved in protein-protein interactions
(Bao et
al., 1996, PNAS, 93, 5037-5042). The th65 open reading frame also contains
three
consensus CDK phosphorylation sites. The identification of thC5 as a
CDC2aAt-associated protein and the presence of these phosphorylation sites
indicates that the th65 protein is a substrate for CDKs.
CA 02301830 2000-02-24
37
Example 3: Generation of transgenic plants with altered cell cycle
A genomic clone of the TN65 gene was obtained by standard procedures and
entirely sequenced. The full length coding region was subsequently cloned in
sense
and antisense orientation in the binary vector PGSV4 (Herouart et al., 1994,
Plant
Physiol. 104, p 873-886) under the control of the constitutive CaMV 35S
promoter.
Additionally, a construct containing substitutions of the consensus CDK
phosphorylation sites into non-phosphorylatable sites was constructed by point-
mutagenesis and cloned in PGSV4 under the control of the CaMV 35S promoter.
The obtained binary vectors were transformed to Agrobacterium tumefaciens.
These
strains were used to transform Nicotiana tabacum cv. Petit havana using the
leaf
disk protocol (Horsh et al., 1985, Science 227, p 1229-1231 ) and Arabidopsis
thaliana using the root transformation protocol (Valvekens et al., 1988, PNAS
85, p
5536-5540).
CA 02301830 2000-02-24
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
38
(A) NAME: CropDesign NV
(B) STREET: TechnologiePark Zwijnaarde 3
(C) CITY: Gent
(D) STATE: none
(E) COUNTRY: Belgium
(F) POSTAL CODE (ZIP): 9052
(ii) TITLE OF INVENTION: Method and means for modulating plant cell
cycle proteins and their use in controlling plant cell
growth
(iii) NUMBER OF SEQUENCES: 8
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO)
(2) INFORMATION FOR SEQ ID N0: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1184 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
{ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..1184
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
CG GCA AGT GAT GCT CGG AAG GAG CTG TTG GAG AAG GAG AGA GAA AAT 47
Ala Ser Asp Ala Arg Lys Glu Leu Leu Glu Lys Glu Arg Glu Asn
1 5 10 15
CAG AAT CTG AAA CAA GAG GTT GTG GGC TTA AAA AAA GCT CTT AAA GAT 95
Gln Asn Leu Lys Gln Glu Val Val Gly Leu Lys Lys Ala Leu Lys Asp
20 25 30
CA 02301830 2000-02-24
39
GCA AAT GAC CAGTGTGTA TTACTCTACAGT GAA CAGAGAGCGTGG 143
GTG
Ala AsnAsp GlnCysVal LeuLeuTyrSer GluVal GlnArgAlaTrp
35 40 45
AAA GTTTCA TTTACATTG CAATCAGATTTA AAGTCA GAGAATATTATG 191
Lys ValSer PheThrLeu GlnSerAspLeu LysSer GluAsnIleMet
50 55 60
CTT GTAGAC AAACATAGA CTAGAGAAGGAG CAGAAT TCTCAGTTAAGG 239
Leu ValAsp LysHisArg LeuGluLysGlu GlnAsn SerGlnLeuArg
65 70 75
AAT CAAATA GCTCAATTT TTACAGTTGGAT CAGGAA CAGAAGCTGCAA 287
Asn GlnIle AlaGlnPhe LeuGlnLeuAsp GlnGlu GlnLysLeuGln
80 85 90 95
ATG CAACAA CAAGATTCC GCCATTCAAAAT CTCCAG GCTAAAATTACA 335
Met GlnGln GlnAspSer AlaIleGlnAsn LeuGln AlaLysIleThr
100 105 110
GAC TTGGAA TCACAAGTA AGTGAAGCCGTT AGATCT GACACAACAAGA 383
Asp LeuGlu SerGlnVal SerGluAlaVal ArgSer AspThrThrArg
115 120 125
ACA GGAGAT GCCTTGCAA TCTCAGGACATA TTTTCT CCAATACCAAAA 431
Thr GlyAsp AlaLeuGln SerGlnAspIle PheSer ProIleProLys
130 135 140
GCG GTTGAG GGTACAACT GATTCTTCTTCT GTTACC AAGAAACTTGAG 479
Ala ValGlu GlyThrThr AspSerSerSer ValThr LysLysLeuGlu
145 150 155
GAA GAATTG AAAAAACGT GATGCACTGATT GAGAGG TTGCATGAAGAA 527
Glu GluLeu LysLysArg AspAlaLeuIle GluArg LeuHisGluGlu
160 165 170 175
AAC GAAAAG TTGTTTGAC AGATTAACAGAA AGGTCA ATGGCTGTTTCG 575
Asn GluLys LeuPheAsp ArgLeuThrGlu ArgSer MetAlaValSer
180 185 190
ACC CAGGTG TTGAGTCCC TCATTAAGAGCT TCGCCA AACATTCAGCCT 623
Thr GlnVal LeuSerPro SerLeuArgAla SerPro AsnIleGlnPro
195 200 205
GCC AATGTT AACAGGGGT GAAGGATATTCG GCAGAA GCCGTTGCTTTA 671
Ala AsnVal AsnArgGly GluGlyT Ser AlaGl Al
r
y u a ValAlaLeu
210 215 220
CCA TCTACA CCAAATAAG AATAACGGAGCG ATTACG TTAGTAAAATCT 719
Pro SerThr ProAsnLys AsnAsnGlyAla IleThr LeuValLysSer
225 230 235
GGC ACTGAT TTAGTAAAA ACCACTCCAGCT GGAGAA TACTTGACAGCT 767
Gly ThrAsp LeuValLys ThrThrProAla GlyGlu TyrLeuThrAla
240 245 250 255
CA 02301830 2000-02-24
GCATTGAATGAC TTTGAC CCTGAAGAA TATGAA CTT GCTGCCATT 815
GGT
AlaLeuAsnAsp PheAsp ProGluGlu TyrGluGlyLeu AlaAlaIle
260 265 270
GCTGACGGCGCA AACAAG CTACTAATG CTGGTTTTGGCA GCTGTCATC 863
AlaAspGlyAla AsnLys LeuLeuMet LeuValLeuAla AlaValIle
275 280 285
AAGGCTGGTGCT TCCAGA GAGCATGAA ATCCTTGCTGAG ATTAGAGAT 911
LysAlaGlyAla SerArg GluHisGlu IleLeuAlaGlu IleArgAsp
290 295 300
TCTGTCTTTTCA TTTATT CGGAAAATG GAACCAAGAAGA GTAATGGAT 959
SerValPheSer PheIle ArgLysMet GluProArgArg ValMetAsp
305 310 315
ACCATGCTTGTT TCCCGA GTTAGGATA CTATACATAAGG TCCTTACTG 1007
ThrMetLeuVal SerArg ValArgIle LeuTyrIleArg SerLeuLeu
320 325 330 335
GCACGATCACCG GAGCTT CAGACTATC AGGGTCTCTCCT GTCGAGTGC 1055
AlaArgSerPro GluLeu GlnThrIle ArgValSerPro ValGluCys
340 345 350
TTTCTTGAGAAG CCTAAT ACTGGTAGA AGTAAAAGCACT AGCAGGGGT 1103
PheLeuGluLys ProAsn ThrGlyArg SerLysSerThr SerArgGly
355 360 365
AGCAGCCCAGGT AGATCC CCTGTTCGA TATCTTGATACG CAGATCCAT 1151
SerSerProGly ArgSer ProValArg TyrLeuAspThr GlnIleHis
370 375 380
GGCTTTAAAGTA AATATA AAGGCAGAA AGGAGG
1184
GlyPheLysVal AsnIle LysAlaGlu ArgArg
385 390
(2)INFORMATION FORSEQ ID :
N0:
2
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 394 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 2:
Ala Ser Asp Ala Arg Lys Glu Leu Leu Glu Lys Glu Arg Glu Asn Gln
1 5 10 15
Asn Leu Lys Gln Glu Val Val Gly Leu Lys Lys Ala Leu Lys Asp Ala
20 25 30
CA 02301830 2000-02-24
41
Asn Asp Gln Cys Val Leu Leu Tyr Ser Glu Val Gln Arg Ala Trp Lys
35 40 45
Val Ser Phe Thr Leu Gln Ser Asp Leu Lys Ser Glu Asn Ile Met Leu
50 55 60
Val Asp Lys His Arg Leu Glu Lys Glu Gln Asn Ser Gln Leu Arg Asn
65 70 75 80
Gln Ile Ala Gln Phe Leu Gln Leu Asp Gln Glu Gln Lys Leu Gln Met
85 90 95
Gln Gln Gln Asp Ser Ala Ile Gln Asn Leu Gln Ala Lys Ile Thr Asp
100 105 110
Leu Glu Ser Gln Val Ser Glu Ala Val Arg Ser Asp Thr Thr Arg Thr
115 120 125
Gly Asp Ala Leu Gln Ser Gln Asp Ile Phe Ser Pro Ile Pro Lys Ala
130 135 140
Val Glu Gly Thr Thr Asp Ser Ser Ser Val Thr Lys Lys Leu Glu Glu
145 150 155 160
Glu Leu Lys Lys Arg Asp Ala Leu Ile Glu Arg Leu His Glu Glu Asn
165 170 175
Glu Lys Leu Phe Asp Arg Leu Thr Glu Arg Ser Met Ala Val Ser Thr
180 185 190
Gln Val Leu Ser Pro Ser Leu Arg Ala Ser Pro Asn Ile Gln Pro Ala
195 200 205
Asn Val Asn Arg Gly Glu Gly Tyr Ser Ala Glu Ala Val Ala Leu Pro
210 215 220
Ser Thr Pro Asn Lys Asn Asn Gly Ala Ile Thr Leu Val Lys Ser Gly
225 230 235 240
Thr Asp Leu Val Lys Thr Thr Pro Ala Gly Glu Tyr Leu Thr Ala Ala
245 250 255
Leu Asn Asp Phe Asp Pro Glu Glu Tyr Glu Gly Leu Ala Ala Ile Ala
260 265 270
Asp Gly Ala Asn Lys Leu Leu Met Leu Val Leu Ala Ala Val Ile Lys
275 280 285
Ala Gly Ala Ser Arg Glu His Glu Ile Leu Ala Glu Ile Arg Asp Ser
290 295 300
Val Phe Ser Phe Ile Arg Lys Met G1u Pro Arg Arg Val Met Asp Thr
305 310 315 320
Met Leu Val Ser Arg Val Arg Ile Leu Tyr Ile Arg Ser Leu Leu Ala
325 330 335
CA 02301830 2000-02-24
42
Arg Ser Pro Glu Leu Gln Thr Ile Arg Val Ser Pro Val Glu Cys Phe
340 345 350
Leu Glu Lys Pro Asn Thr Gly Arg Ser Lys Ser Thr Ser Arg Gly Ser
355 360 365
Ser Pro Gly Arg Ser Pro Val Arg Tyr Leu Asp Thr Gln Ile His Gly
370 375 380
Phe Lys Val Asn Ile Lys Ala Glu Arg Arg
385 390
(2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
CGAGATCTGA ATTCATGGAT CAGTA 25
(2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE Cu.ARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CGAGATCTGA ATTCCTAAGG CATGCC 26
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CH~~.~CTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
CA 02301830 2000-02-24
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: YES
43
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
CGGATCCGAA TTCATGGAGA ACGAG 25
(2) INFORMATION FOR SEQ ID N0: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CGGATCCGP.A TTCTCAGAAC TGAGA 25
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 7:
ATACCACTAC AATGGATG 18
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02301830 2000-02-24
44
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 8:
AGTTGAAGTG AACTTGCGGG Zp
