U.S. patent application number 10/688291 was filed with the patent office on 2004-04-15 for cyclin-dependent kinase inhibitors and uses thereof.
Invention is credited to Almeida, Janice De, Inze, Dirk, Landrieu, Isabelle, Veylder, Lieven De.
Application Number | 20040073969 10/688291 |
Document ID | / |
Family ID | 31985002 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040073969 |
Kind Code |
A1 |
Inze, Dirk ; et al. |
April 15, 2004 |
Cyclin-dependent kinase inhibitors and uses thereof
Abstract
Provided are DNA sequences encoding cyclin-dependent kinase
inhibitor(s) as well as to methods for obtaining the same.
Furthermore, vectors comprising said DNA sequences are described,
wherein the DNA sequences are operatively linked to regulatory
elements allowing expression in prokaryotic and/or eukaryotic host
cells. In addition, proteins encoded by said DNA sequences,
antibodies to said proteins and methods for their production are
provided. Furthermore, regulatory sequences which naturally
regulate the expression of the above described DNA sequences are
described. Also described is 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 cyclin-dependent
kinase inhibitor(s) functional in a plant or parts thereof and/or
one or more DNA sequences encoding such proteins. Also provided is
a process for disruption plant cell division by interfering in the
expression or activity of a cyclin-dependent protein kinase
inhibitor using a DNA sequence according to the invention wherein
said plant cell is part of a transgenic plant. Further described
are diagnostic compositions comprising the aforementioned DNA
sequences, proteins, antibodies and regulatory sequences. Methods
for the identification of compounds being capable of activating or
inhibiting the cyclin-dependent kinase inhibitors are described as
well. Furthermore, transgenic plant cells, plant tissue and plants
containing the above-described DNA sequences and vectors are
described as well as the use of the aforementioned DNA sequences,
vectors, proteins, antibodies, regulatory sequences and/or
compounds identified by the method of the invention in plant cell
and tissue culture, plant breeding and/or agriculture.
Inventors: |
Inze, Dirk; (Moorsel-Aalst,
BE) ; Veylder, Lieven De; (Aalst, BE) ;
Almeida, Janice De; (Bellem, BE) ; Landrieu,
Isabelle; (Wiers, BE) |
Correspondence
Address: |
Ann R. Pokalsky, Esq.
DILWORTH & BARRESE, LLP
333 Earle Ovington Blvd.
Uniondale
NY
11553
US
|
Family ID: |
31985002 |
Appl. No.: |
10/688291 |
Filed: |
October 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10688291 |
Oct 17, 2003 |
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09526597 |
Mar 16, 2000 |
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09526597 |
Mar 16, 2000 |
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PCT/EP98/05895 |
Sep 16, 1998 |
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Current U.S.
Class: |
800/278 ;
435/184; 435/320.1; 435/419; 435/69.2; 536/23.2 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8261 20130101; A01N 65/08 20130101; Y02A 40/146
20180101 |
Class at
Publication: |
800/278 ;
435/069.2; 435/320.1; 435/419; 435/184; 536/023.2 |
International
Class: |
A01H 001/00; C12N
015/82; C07H 021/04; C12N 009/99; C12N 005/04 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 24, 1997 |
EP |
97 204 111.5 |
Sep 16, 1997 |
EP |
97 202 838.5 |
Claims
1. A DNA sequence encoding a cyclin-dependent kinase inhibitor 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, 4 or
6; (b) DNA sequences comprising a nucleotide sequence as given in
SEQ ID NO: 1, 3 or 5; (c) DNA sequences comprising the nucleotide
sequence encoding a protein comprising the amino acid sequence from
amino acid position 75 to 209 of SEQ ID NO: 2 or from amino acid
position 11 to 216 of SEQ ID NO: 4 or comprising the nucleotide
sequence from nucleotide position 305 to 932 of SEQ ID NO: 1; (d)
DNA sequences hybridizing with the complementary strand of a DNA
sequence as defined in any one of (a) to (c); (e) DNA sequences
encoding an amino acid sequence which is at least 30% identical to
the amino acid sequence encoded by the DNA sequence of any one of
(a) to (c); (f) 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 (e); and
(g) DNA sequences encoding a fragment of a protein encoded by a DNA
sequence of any one of (a) to (f).
2. A method for identifying and obtaining a cyclin-dependent kinase
inhibitor comprising a two-hybrid screening assay wherein CDC2a as
a bait and a cDNA library of a cell suspension as prey are
used.
3. The method of claim 2, wherein said CDC2a is CDC2aAt.
4. A DNA sequence encoding a cyclin-dependent kinase inhibitor
obtainable by the method of claim 2 or 3 and which has an amino
acid sequence at least 30% identical to the amino acid sequence
encoded by the DNA sequence of claim 1(a) or claim 1(b).
5. A nucleic acid molecule of at least 15 nucleotides in length
hybridizing specifically with a DNA sequence of claim 1 or 4 or
with a complementary strand thereof.
6. A vector comprising a DNA sequence of claim 1 or 4.
7. The vector of claim 6 which is an expression vector wherein the
DNA sequence is operatively linked to one or more control sequences
allowing the expression in prokaryotic and/or eukaryotic host
cells.
8. A host cell containing a vector of claim 6 or 7 or a DNA
sequence of claim 1 or 4.
9. The host cell of claim 8 which is a bacterial, insect, fungal,
plant or animal cell.
10. A method for the production of a cyclin-dependent kinase
inhibitor or an immunologically active and/or functional fragment
thereof comprising culturing a host cell of claim 8 or 9 under
conditions allowing the expression of the protein and recovering
the produced protein from the culture.
11. A cyclin-dependent kinase inhibitor or an immunologically
active and/or functional fragment thereof encodable by a DNA
sequence of claim 1 or 4 or obtainable by the method of claim 2, 3
or 10.
12. An antibody specifically recognizing the cyclin-dependent
kinase inhibitor of claim 11 or a fragment or epitope thereof.
13. A method for the production of transgenic plants, plant cells
or plant tissue comprising the introduction of a DNA sequence of
claim 1, 4 or 5 or a vector of claim 6 or 7 into the genome of said
plant, plant cell or plant tissue.
14. The method of claim 13 further comprising regenerating a plant
from said plant tissue or plant cell.
15. A transgenic plant cell comprising a DNA sequence of claim 1 or
4 which is operably linked to regulatory elements allowing
transcription and/or expression of the DNA sequence in plant cells
or obtainable according to the method of claim 13 or 14.
16. The transgenic plant cell of claim 15 wherein said DNA sequence
or said vector is stably integrated into the genome of the plant
cell.
17. A transgenic plant or a plant tissue comprising plant cells of
claim 15 or 16.
18. The transgenic plant of claim 17 in which plant cell division
and/or growth is altered.
19. A transgenic plant cell which contains stably integrated into
the genome a DNA sequence of claim 1, 4 or 5 or part thereof or
obtainable according to the method of claim 13 or 14, wherein the
transcription and/or expression of the DNA sequence or part thereof
leads to reduction of the synthesis of the cyclin-dependent kinase
inhibitor of claim 11 in the cells.
20. The plant cell of claim 19, wherein the reduction is achieved
by an antisense, sense, ribozyme, co-suppression and/or dominant
mutant effect.
21. A transgenic plant or plant tissue comprising the plant cells
of claim 19 or 20.
22. The transgenic plant of claim 21 which displays a deficiency in
plant cell division and/or growth.
23. Harvestable parts or propagation material of plants of any one
of claims 17, 18, 21 or 22 comprising plant cells of claim 15, 16,
19 or 20.
24. A regulatory sequence of a promoter regulating the expression
of a nucleic acid molecule of claim 1 or 4 or of a nucleic acid
molecule homologous to said nucleic acid molecule.
25. The regulatory sequence of claim 24 wherein said regulatory
sequence is capable of conferring expression of a heterologous DNA
sequence in (a) young root meristems, pericycle cells in the
vascular tissue, shoot apical meristem, surface and tip of young
leaves, the epidermis of the stem of young seedlings, tapetal layer
of the anthers in pollen grains, flower buds and mature ovaries,
embryos at the globular, heart and torpedo stages, embryonic root;
(b) root and shoot apical meristems, young differentiating leaves,
flower buds and young flowers, ovary wall, funiculus, ovules and
pollen grains, embryo at the globular stage, embryonic root; or (c)
main and lateral root meristems and shoot apical meristems,
vascular tissue, pericycle, mature ovaries, globular and heart
embryonic root.
26. A recombinant DNA molecule comprising the regulatory sequence
of claim 24 or 25.
27. The recombinant DNA molecule of claim 26, wherein said
regulatory sequence is operatively linked to a heterologous DNA
sequence.
28. The recombinant DNA molecule of claim 27, wherein said
heterologous DNA sequence encodes a peptide, protein, antisense
RNA, sense RNA and/or ribozyme.
29. A nucleic acid molecule of at least 15 nucleotides in length
hybridizing specifically with the regulatory sequence of claim 24
or 25.
30. A vector comprising a regulatory sequence of claim 24 or 25 or
a recombinant DNA molecule of any one of claims 26 to 28.
31. A cell transformed with a regulatory sequence of claim 24 or 25
or a recombinant DNA molecule of any one of claims 26 to 28 or the
vector of claim 30.
32. A method for the production of transgenic plants, plant cells
or plant tissue comprising the introduction of a recombinant DNA
molecule of any one of claims 26 to 28 or the vector of claim 30
into the genome of said plant, plant cell or plant tissue.
33. A transgenic plant cell which contains stably integrated into
the genome a recombinant DNA molecule of any one of claims 26 to 28
or a vector of claim 30 or obtainable according to the method of
claim 32.
34. A transgenic plant or plant tissue comprising plant cells of
claim 33 or obtainable by the method of claim 32.
35. Harvestable parts or propagation material of a plant of claim
34 comprising plant cells of claim 33.
36. A method for the identification of an activator or inhibitor of
genes encoding cyclin-dependent kinase inhibitors comprising the
steps of: (a) providing a plant, plant cell, or plant tissue
comprising a recombinant DNA molecule comprising a readout system
operatively linked to a regulatory sequence of claim 24 or 25; (b)
culturing said plant cell or tissue or maintaining said plant in
the presence of a compound or a sample comprising a plurality of
compounds under conditions which permit expression of said readout
system; (c) identifying or verifying a sample and compound,
respectively, which leads to suppression or activation and/or
enhancement of expression of said readout system in said plant,
plant cell, or plant tissue.
37. A method for identifying and obtaining an activator or
inhibitor of cyclin-dependent kinase inhibitors comprising the
steps of: (a) combining a compound to be screened with a reaction
mixture containing the protein of claim 11 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.
38. A method of producing a plant herbicide comprising the steps of
the method of claim 36 or 37 and formulating the compound obtained
or identified in step (c) or a derivative thereof in a form
suitable for the application in agriculture or plant cell and
tissue culture.
39. A compound obtained or identified by the method of claim 36 or
37, which is an activator or inhibitor of cyclin-dependent kinase
inhibitors.
40. A diagnostic composition comprising a DNA sequence of claim 1,
4 or 5, a vector of claim 6 or 7, a protein of claim 11, an
antibody of claim 12, the regulatory sequence of claim 24 or 25,
the recombinant DNA molecule of any one of claims 26 to 28, the
nucleic acid molecule of claim 29, the vector of claim 30, or the
compound of claim 39, and optionally suitable means for
detection.
41. Use of a DNA sequence of claim 1, 4 or 5, the vector of claim 6
or 7, the protein of claim 11, the antibody of claim 12, the
regulatory sequence of claim 24 or 25, the recombinant DNA molecule
of any one of claims 26 to 28, the nucleic acid molecule of claim
29, the vector of claim 30, or the compound of claim 39 for
modulating plant cell cycle, plant cell division and/or growth, for
influencing the activity of cyclin-dependent protein kinase, 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 modifying growth inhibition of
plants caused by environmental stress conditions, for inducing male
or female sterility, for influencing cell division progression in a
host as defined in claim 9 or for use in a screening method for the
identification of inhibitors or activators of cell cycle
proteins.
42. Use of the compound of claim 39 as growth regulator and/or
herbicide.
Description
[0001] The present invention relates to DNA sequences encoding
cyclin-dependent kinase inhibitors 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.
Furthermore, the present invention relates to regulatory sequences
which naturally regulate the expression of the above described DNA
sequences. 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
cyclin-dependent kinase inhibitors 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, regulatory sequences and/or compounds identified by the
method of the invention in plant cell and tissue culture, plant
breeding and/or agriculture.
[0002] 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:
[0003] G1: the gap between mitosis and the onset of DNA
synthesis;
[0004] S: the phase of DNA synthesis;
[0005] G2: the gap between S and mitosis;
[0006] M: mitosis, the process of nuclear division leading up to
the actual cell division.
[0007] 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.
[0008] 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.
[0009] 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 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.
[0010] 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.
[0011] As already mentioned above, the control of cell cycle
progression in eukaryotes is mainly exerted at two transition
points: one in late G.sub.1, before DNA synthesis, and one at the
G.sub.2/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 p34.sup.CDC2 and p34.sup.CDC28 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 p34.sup.CDC2/CDC28
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 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 G.sub.2/M 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 CDK/cyclin complexes. 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 CDK/cyclin complexes. CKIs are
produced during development when further cell division has to be
prevented. In mammals CKIs have been shown to be involved in many
different aspects of cell division and cell differentiation. First,
CKI expression has been demonstrated to be induced under stress
conditions such as for instance irradiation of cells or the
influence of carcinogenic agents, which both potentially damage
DNA. This arrest allows DNA to be repaired prior to DNA replication
and mitosis. Second, inhibition of CDKs by CKIs has been
demonstrated to correlate with cell differentiation and inhibition
of programmed cell death. Third, the knock-out of certain members
of the CKI family in mice results in an increase of body size and
formation of tumors.
[0012] 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 p.sub.34CDC2/CDC28 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
G.sub.2 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. Recently, a CDK
inhibitor has been identified in Arabidopsis thaliana (ICK1) that
shares some limited similarity with the mammalian p27.sup.klp1
kinase inhibitor (Wang, Nature 386 (1997), 451452). This CDK
inhibitor was predominantly identified when screening a library
with a yeast two-hybrid "bait" construct harboring Arabidopsis
thaliana CDC2aAt cDNA suggesting that only one class of CDK
inhibitors is present in plants. However, the function and
expression of CDK inhibitors in plants still needs to be
determined.
[0013] 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, isolate plant and characterize 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 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.
[0014] 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.
[0015] The solution to the technical problem is achieved by
providing the embodiments characterized in the claims.
[0016] Accordingly, the invention relates to a DNA sequence
encoding a cyclin-dependent kinase inhibitor or encoding an
immunologically active and/or functional fragment of such a
protein, selected from the group consisting of:
[0017] (a) DNA sequences comprising a nucleotide sequence encoding
a protein comprising the amino acid sequence as given in SEQ ID NO:
2, 4 or 6;
[0018] (b) DNA sequences comprising a nucleotide sequence as given
in SEQ ID NO: 1, 3 or 5;
[0019] (c) DNA sequences comprising the nucleotide sequence
encoding a protein comprising the amino acid sequence from amino
acid position 75 to 209 of SEQ ID NO: 2 or from amino acid position
11 to 216 of SEQ ID NO: 4 or comprising the nucleotide sequence
from nucleotide position 305 to 932 of SEQ ID NO: 1;
[0020] (d) DNA sequences hybridizing with the complementary strand
of a DNA sequence as defined in any one of (a) to (c);
[0021] (e) DNA sequences encoding an amino acid sequence which is
at least 30% identical to the amino acid sequence encoded by the
DNA sequence of any one of (a) to (c);
[0022] (f) 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 (e);
and
[0023] (g) DNA sequences encoding a fragment of a protein encoded
by a DNA sequence of any one of (a) to (f).
[0024] The term "cyclin-dependent kinase inhibitor" also designated
CDK inhibitor, CKI or CDKI as denoted herein means a protein which
inhibits CDK/cyclin activity and is produced during development
when further cell division has to be prevented. A CDK inhibitor of
the invention is capable of inhibiting or suppressing the kinase
activity of protein kinases, in particular of cyclin-dependent
kinases. The capability of a inhibiting or suppressing protein
kinase activity can be determined according to methods well known
in the art; see, e.g., Wang, supra and the appended examples. 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: G.sub.0, Gap.sub.1 (G.sub.1), DNA
synthesis (S), Gap.sub.2 (G.sub.2), and mitosis (M).
[0025] 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.
[0026] 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.
[0027] In accordance with the present invention new plant gene
products with a putative CDK inhibitory function were screened by
using the two-hybrid system (Fields, Nature 340 (1989), 245-246).
For this purpose the CDC2aAt protein was exploited as bait.
Previous attempts using the identical bait and a cDNA library
constructed with RNA from 3-week-old Arabidopsis thaliana
vegetative tissues were unsuccessful (De Veylder, Febs Lett. 412
(1997), 446-452; De Veylder, J. Exp. Bot. 48 (1997), 2113-2114). A
new attempt was undertaken using a newly constructed library made
from a RNA mixture of Arabidopsis thaliana cell suspensions
harvested at various growing stages: early exponential,
exponential, early stationary and stationary phase. This library
has the advantage above the previous one to include mainly genes
expressed in cells at the onset of cell division, actively dividing
cells, cells redrawing from the cell cycle, and non-cycling cells.
Surprisingly, using this specific library several positive clones
were identified encoding proteins with a putative CDK inhibitory
function. These clones were designated LDV39, LDV66, and
LDV159.
[0028] A homology search in databases revealed that the last 23
amino-acids showed significant homology to the human CKIs
p21.sup.cip1 and p27.sup.kip1. The LDV39 gene was 622 bp long,
consisting of 423 bp coding region and 199 bp 3' UTR (excluding the
poly-A tail). The LDV66 gene was 611 bp long, consisting of 379 bp
coding region and 232 bp 3' UTR (excluding the poly-A tail). Since
the LDV39 and LDV66 clones encode partial proteins, lacking their
amino-terminal part, a flower cDNA library obtained from the ABRC
stock centre (library stock number CD4-6) was screened. The
positive clones were denominated FL39 and FL66, corresponding to
longer clones of LDV39 and LDV66, respectively.
[0029] The FL39 clone is 932 bp (SEQ ID NO: 1) long and contains an
ORF encoding a protein of 209 amino acids (SEQ ID NO: 2) with a
calculated molecular mass of 24 kDa. In its 3' UTR a
poly-adenylation signal can be recognised. The amino-terminal part
of the FL39 protein contains a repeated motif of 11 amino acids
(VRRRD/ExxxVEE). This motif is not found in any other protein in
the databanks and its significance in unknown. The FL39 protein
also contains a putative nuclear localization signal (amino acids
23-26) and a PEST-rich region (amino acids 71-98; PESTFIND score
+15.5). These sequences, rich in proline, glutamic acid, serine and
proline, are characteristically present in unstable proteins
(Rogers et al., 1986, Science 234, 364-368).
[0030] The FL66 sequence does not contain an in frame stopcodon,
and may therefore not be full length. The FL66 clone is 875 bp long
(SEQ ID NO: 3) and bears an ORF of 216 amino acids (SEQ ID NO: 4),
encoding a protein of 24 kD. No nuclear localization signal or PEST
domains are present. Furthermore, a CDK inhibitor named ALFCDKI
from alfalfa has been identified in accordance with the present
invention using a two-hybrid screening assay. This gene comprises
1202 nucleotides (SEQ ID NO:5) with a coding region from nucleotide
position 94 to 760 encoding a protein of 224 amino acids (SEQ ID
NO:6). The LDV159 clone was identical to ICK1 (GenBank accession
number U94772 as published by Wang, Nature 386 (1997), p451-452).
Surprisingly, the three other clones were novel and encoded
proteins only distantly related to ICK1 (Table 1).
1TABLE 1 Sequence similarity and identity between the different
plant cyclin- dependent kinase inhibitors. CrCKI is the Chenopodium
rubrum CKI (accession number AJ00217). FL39 FL66 FL67 ICK1 CrCKI
ALFCDKI FL39 27.805 33.333 32.292 34.392 N.S. FL66 21.463 39.545
37.017 34.574 34.389 FL67 20.000 30.909 30.220 N.S. N.S. ICK 23.958
30.939 22.527 32.105 25.131 CrCKI 24.868 28.723 N.S. 27.368 26.667
MsCKI N.S. 28.054 N.S. 21.990 20.513
[0031] The percentage similarity (bold) and identity (italic)
between the different Cyclin-dependent kinase inhibitors was
determined using the GAP program (from the GCG 9.1 package) using
the parameters Gap weight=12 and Length weight=4. N.S.: Not
Significant.
[0032] Furthermore, the genomic organisation of the FL39, FL66 and
ICK1 clones was tested by DNA gel blot analysis. The results of the
experiments suggest the presence of an additional FL66 related gene
and, therefore, it can be concluded that there are at least four
different CKI proteins present in A. thaliana. From the foregoing
it is evident that more than one CDK inhibitor in plants exist and
therefore different functions during plant development and/or
expression patterns can be assumed. Further studies that have been
performed in accordance with the present invention revealed that
the CDK inhibitors are expressed at different time points during
the cultivation of the plant cell culture; see Example 8. Moreover,
it could be demonstrated in accordance with the present invention
that the CDK inhibitor FL66 is regulated by NaCl; see Example 9.
The inhibitory function of the CDK inhibitor of the invention is
exemplified with FL66; see Example 6. In addition, in situ
hybridization using antisense probes derived from cDNAs from LDV39,
LDV66 and LDV159 demonstrated that each of these CDK inhibitors
exhibit distinct expression patterns; see Example 13. Thus, the
findings of the present invention establishes that in plants
several CDK inhibitors exist which due to their differential
expression pattern may have different functions during the
development of the plant. It can be expected that similar gene
families encoding CDK inhibitors are present in other plant species
than Arabidopsis and alfalfa as well. These cyclin-dependent
inhibitors are also within the scope of the present invention.
[0033] Accordingly, 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 cyclin-dependent kinase inhibitor.
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, N.Y. (1989)).
Preferably, the hybridization conditions used in the examples are
employed. Cyclin-dependent kinase inhibitor derived from other
organisms such as mammals, in particular humans, may be encoded by
other DNA sequences which hybridize to the sequences for plant
cyclin-dependent kinase inhibitor 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 4.times.SSC at
50.degree. C. or hybridization with 30-40% formamide at 42.degree.
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 modification(s) known in the art
either alone or in combination from the above-described amino acid
sequences or their underlying 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 part of the primary structural
conformation for one or more epitopes capable of reacting with
antibodies to cyclin-dependent kinase inhibitor which are encodable
by a nucleic acid molecule as set forth above and which have
comparable or identical characteristics in terms of inhibiting
cyclin dependent kinases, in particular plant cyclin dependent
kinases. Part of the invention are therefore also nucleic acid
molecules encoding a polypeptide comprising at least a functional
part of cyclin-dependent kinase inhibitor 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.
[0034] As is demonstrated in the appended examples a two-hybrid
screening assay has been developed in accordance with the present
invention suitable for identifying cyclin-dependent kinase
inhibitor. Thus, in another aspect the present invention relates to
a method for identifying and obtaining cyclin-dependent kinase
inhibitors comprising a two-hybrid screening assay wherein CDC2a as
a bait and a cDNA library of cell suspension as prey are used.
Preferably, said CDC2a is CDC2aAt. However, CDC2a from other
organisms such as other plants but also mammals may be employed as
well.
[0035] 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 invention
also relates to a DNA sequence encoding a cyclin-dependent kinase
inhibitor obtainable by the method of the invention. Preferably,
the amino acid sequence of said protein obtainable by the method of
the invention has an identity to the amino acid sequence of any one
of SEQ ID NOS: 2, 4 or 6 of at least 30 %, more preferably 40 to
60% and most preferably 70% to 90%.
[0036] 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, a cyclin-dependent kinase inhibitor could also be
identified in Medicago sativa (Alfalfa). 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 molecule(s) 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 polymerase chain reaction (PCR) using as primers
oligonucleotides derived form the above-described nucleic acid
molecules.
[0037] 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 cyclin-dependent kinase inhibitor 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 a motif of 11 amino acids
(VRRRD/ExxxVEE; SEQ ID NO: 33) present in the amino terminal part
of the FL39 protein. This motif is not found in any other protein
in the databanks and its significance in unknown. Furthermore, the
fragment may contain the putative nuclear localization signal
(amino acids 23-26 of SEQ ID NO: 2) and/or the PEST-rich region
(amino acids 71-98 of SEQ ID NO: 2; see also Example 3).
[0038] 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 30%, 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), insertion(s) and/or recombination(s); see supra.
[0039] Homology further means that the respective nucleic acid
molecules or encoded proteins are functionally and/or 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 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.
[0040] 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.
[0041] 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.
[0042] 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,
3 or 5 or to a nucleotide sequence encoding the amino acid sequence
of SEQ ID NO: 2, 4 or 6. 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 CKI encoding 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.
[0043] 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.
[0044] 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 BlAcore
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 detecting point
mutations by hybridization with nucleic acids obtained from a
sample with an affinity sensor, such as BlAcore; 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.
[0045] 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.
[0046] In a preferred embodiment the nucleic acid molecule present
in the vector is linked to (a) control sequence(s) which allow the
expression of the nucleic acid molecule in prokaryotic and/or
eukaryotic cells.
[0047] 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 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.
[0048] 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.
[0049] 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.sub.L, lac, trp or tac promoter in E. coli,
and examples of regulatory elements permitting expression in
eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the
CMV-, SV40- , RSV-promoter (Rous sarcoma virus), CMV-enhancer,
SV40-enhancer or a globin intron in mammalian and other animal
cells. In this context, suitable expression vectors are known in
the art such as Okayama-Berg cDNA expression vector pcDV1
(Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1
(GIBCO BRL). Advantageously, the above-described vectors of the
invention comprises a selectable and/or 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 94/20627) 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).
[0050] 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, Pl. Sci. 116 (1996),
59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent
protein (Gerdes, FEBS Lett. 389 (1996), 44-47) or
.beta.-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.
[0051] 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.
[0052] 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)).
[0053] 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.
[0054] Another subject of the invention is a method for the
preparation of a cyclin-dependent kinase inhibitor 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.
[0055] 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 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.
[0056] 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.
[0057] The present invention furthermore relates to CKIs 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 cyclin-dependent
kinase inhibitor. 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 for 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 retain biological
activity, for example, 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.
[0058] 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 function(s) 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. Preferably,
the polypeptides according to the invention comprising the amino
acid sequence as defined above and/or a fragment thereof have a
molecular weight of approximately 15-20 kDa.
[0059] 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), 3745). In particular, the
appropriate programs can be used for the identification of
interactive sites of the CKI and cyclin dependent kinases, 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. Trans. 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 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 .OMEGA.-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, kinase inhibitory
and/or 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.
[0060] Furthermore, a three-dimensional and/or 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 (1996), 1545-1558).
[0061] Furthermore, the present invention relates to antibodies
specifically recognizing a cyclin-dependent kinase inhibitor
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 cyclin-dependent kinase
inhibitors 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 Galfr, Meth.
Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma
cells to spleen cells derived from immunized mammals. Furthermore,
antibodies or 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.
[0062] 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 or the biological
activity of the proteins 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 inactivating or
inhibiting cyclin-dependent protein kinase complexes.
[0063] 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.
[0064] The activity of a 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 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".
[0065] 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.
[0066] 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.
[0067] 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). Furthermore, the expression of the nucleic acid
molecules of the invention can be controlled by, e.g., introduction
of high constitutive, tissue specific, cell type specific or
inducible promoters adjacent to said nucleotide sequence or
fragment thereof, multiple gene repeats and other similar
techniques. For instance transgenic plants can thus be obtained
which can not form feeding cells upon nematode infection of the
roots. It is also feasible to generate transgenic plants which are
resistant to certain viral infections such as a gemini viral
infection. 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 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 (WO96/16182). 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.
[0068] 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 cyclin-dependent kinases the interacting component of the
protein of the invention excert their 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.
[0069] 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 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) and/or 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, N.Y.).
[0070] 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.
[0071] 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), 3748; Vasil, Bio/Technology 11 (1993),
1553-1558 and Christou (1996) Trends in Plant Science 1, 423-431.
Microinjection can be performed as described in Potrykus and
Spangenberg (eds.), Gene Transfer To Plants. Springer Verlag,
Berlin, N.Y. (1995).
[0072] 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.
[0073] 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 may 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.
[0074] 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.
[0075] 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.
Alternatively, a plant cell having (a) nucleic acid molecule(s)
encoding a cyclin-dependent kinase inhibitor present in its genome
can be used and modified such that said plant cell expresses the
endogenous gene(s) corresponding to these nucleic acid molecules
under the control of an heterologous promoter and/or enhancer
elements. The introduction of the heterologous promoter and
mentioned elements which do not naturally control the expression of
a nucleic acid molecule encoding the above described protein using,
e.g., gene targeting vectors can be done according to standard
methods, see supra and, e.g., Hayashi, Science 258 (1992),
1350-1353; Fritze and Walden, Gene activation by T-DNA tagging. In
Methods 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).
Suitable promoters and other regulatory elements such as enhancers
include those mentioned hereinbefore.
[0076] The presence and expression of the nucleic acid molecule in
the transgenic plant cells leads to the synthesis of a
cyclin-dependent kinase inhibitor and leads to physiological and
phenotypic changes in plants containing such cells.
[0077] 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 and/or for improved and/or disease resistance.
[0078] Therefore, part of this invention is the use of CKIs and the
encoding DNA sequences 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 activity of
cyclin-dependent protein kinases or their inhibitors. The latter
goal may also be achieved, for example, with methods for reducing
the amount of active cyclin-dependent kinase inhibitor.
[0079] 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
cyclin-dependent kinase inhibitor.
[0080] In a preferred embodiment, the reduction is achieved by an
anti-sense, sense, ribozyme, co-suppression and/or dominant mutant
effect.
[0081] "Antisense" and "antisense nucleotides" means DNA or RNA
constructs which block the expression of the naturally occurring
gene product.
[0082] 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 cyclin-dependent
kinase inhibitor. 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 cyclin-dependent
kinase inhibitor 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 cyclindependent kinase inhibitor. In this case the
homology is preferably higher than 80%, particularly higher than
90% and still more preferably higher than 95%.
[0083] 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 and/or
morphological changes.
[0084] Thus, the present invention also relates to transgenic
plants comprising the above-described transgenic plant cells. These
may show, for example, reduced or enhanced growth
characteristics.
[0085] 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.
[0086] 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 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.
[0087] 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.
[0088] As mentioned above, the cyclin-dependent kinase inhibitors
of the invention display distinct expression patterns in plants and
cell suspension. Thus, the regulatory sequences that naturally
drive the expression of the above described cyclin-dependent kinase
inhibitors may prove useful for the expression of heterologous DNA
sequences in certain plant tissues and/or at different
developmental stages in plant development.
[0089] Accordingly, in a further aspect the present invention
relates to a regulatory sequence of a promoter naturally regulating
the expression of a nucleic acid molecule of the invention
described above or of a nucleic acid molecule homologous to a
nucleic acid molecule of the invention. The expression patter of
CKI genes has been studied in detail in accordance with the present
invention and is summarized in Example 8, 9 and in particular in
Example 13. With methods well known in the art it is possible to
isolate the regulatory sequences of the promoters that naturally
regulate the expression of the above-described DNA sequences. For
example, using the CKI genes as probes a genomic library consisting
of plant genomic DNA cloned into phage or bacterial vectors can be
screened by a person skilled in the art. Such a library consists
e.g. of genomic DNA prepared from seedlings, fractionized in
fragments ranging from 5 kb to 50 kb, cloned into the lambda GEM1 1
(Promega) phages. Phages hybridizing with the probes can be
purified. From the purified phages DNA can be extracted and
sequenced. Having isolated the genomic sequences corresponding to
the genes encoding the above-described cyclin-dependent kinase
inhibitors, it is possible to fuse heterologous DNA sequences to
these promoters or their regulatory sequences via transcriptional
or translational fusions well known to the person skilled in the
art. In order to identify the regulatory sequences and specific
elements of the CKI genes, 5'-upstream genomic fragments can be
cloned in front of marker genes such as luc, gfp or the GUS coding
region and the resulting chimeric genes can be introduced by means
of Agrobacterium tumefaciens mediated gene transfer into plants or
transfected into plant cells or plant tissue for transient
expression. The expression pattern observed in the transgenic
plants or transfected plant cells containing the marker gene under
the control of the regulatory sequences of the invention reveal the
boundaries of the promoter and its regulatory sequences.
Preferably, said regulatory sequence is capable of conferring
expression of a heterologous DNA sequence in
[0090] (a) young root meristems, pericycle cells in the vascular
tissue, shoot apical meristem, surface and tip of young leaves,
epidermis of the stem in young seedlings, tapetal layer of the
anthers in pollen grains, flower buds and mature ovaries, embryos
at the globular, heart and torpedo stages, embryonic root;
[0091] (b) root and shoot apical meristems, young differentiating
leaves, flower buds and young flowers, ovary wall, funiculus,
ovules and pollen grains, embryo at the globular stage, embryonic
root; or
[0092] (c) main and lateral root meristems and shoot apical
meristerns, vascular tissue, pericycle, mature ovaries, globular
and heart embryonic root.
[0093] In context with the present invention, the term "regulatory
sequence" refers to sequences which influence the specificity
and/or level of expression, for example in the sense that they
confer cell and/or tissue specificity; see supra. Such regions can
be located upstream of the transcription initiation site, but can
also be located downstream of it, e.g., in transcribed but not
translated leader sequences.
[0094] The term "promoter", within the meaning of the present
invention refers to nucleotide sequences necessary for
transcription initiation, i.e. RNA polymerase binding, and may also
include, for example, the TATA box.
[0095] The term "nucleic acid molecule homologous to a nucleic acid
molecule of the invention", as used herein includes promoter
regions and regulatory sequences of other CKI genes, such as the
gene encoding the CKI1 protein as well as genes from other species,
for example, maize, alfalfa, potato, sorghum, millet, coix, barley,
wheat and rice which are homologous to the CKI genes and which
display substantially the same expression pattern. Such promoters
are characterized by their capability of conferring expression of a
heterologous DNA sequence in root meristems and other tissues
metnioned above.
[0096] Thus, according to the present invention, regulatory
sequences from any species can be used that are functionally
homologous to the regulatory sequences of the promoter of the above
defined CKI specific nucleic acid molecules, or promoters of genes
that display an identical or similar pattern of expression, in the
sense of being expressed in the above-mentioned tissues and cells.
However, the expression conferred by the regulatory sequences of
the invention may not be limited to, for example, root meristem
cells but can include or be restricted to, for example, subdomains
of meristems. The particular expression pattern may also depend on
the plant/vector system employed. However, expression of
heterologous DNA sequences driven by the regulatory sequences of
the invention predominantly occurs in the root meristem unless
certain elements of the regulatory sequences of the invention, were
taken and designed by the person skilled in the art to control the
expression of a heterologous DNA sequence in other cell types.
[0097] It is also immediately evident to the person skilled in the
art that further regulatory elements may be added to the regulatory
sequences of the invention. For example, transcriptional enhancers
and/or sequences which allow for induced expression of the
regulatory sequences of the invention may be employed. A suitable
inducible system is for example tetracycline-regulated gene
expression as described, e.g., by Gatz, supra.
[0098] The regulatory sequence of the invention may be derived from
the CKI genes of Arabidopsis thaliana or alfalfa although other
plants may be suitable sources for such regulatory sequences as
well.
[0099] Usually, said regulatory sequence is part of a recombinant
DNA molecule. In a preferred embodiment of the present invention,
the regulatory sequence in the recombinant DNA molecule is
operatively linked to a heterologous DNA sequence. The term
heterologous with respect to the DNA sequence being operatively
linked to the regulatory sequence of the invention means that said
DNA sequence is not naturally linked to the regulatory sequence of
the invention. Expression of said heterologous DNA sequence
comprises transcription of the DNA sequence, preferably into a
translatable mRNA. Regulatory elements ensuring expression in
eukaryotic cells, preferably plant cells, are well known to those
skilled in the art. They usually comprise poly-A signals ensuring
termination of transcription and stabilization of the transcript,
see also supra. Additional regulatory elements may include
transcriptional as well as translational enhancers; see supra.
[0100] In a preferred embodiment, the heterologous DNA sequence of
the above-described recombinant DNA molecules encodes a peptide,
protein, antisense RNA, sense RNA and/or ribozyme. The recombinant
DNA molecule of the invention can be used alone or as part of a
vector to express heterologous DNA sequences, which, e.g., encode
proteins for, e.g., the control of disease resistance, modulation
of nutrition value or diagnostics of CKI related gene expression.
The recombinant DNA molecule or vector containing the DNA sequence
encoding a protein of interest is introduced into the cells which
in turn produce the protein of interest. For example, the
regulatory sequences of the invention can be operatively linked to
sequences encoding Barstar and Barnase, respectively, for use in
the production of male and female sterility in plants.
[0101] On the other hand, said protein can be a scorable marker,
e.g., luciferase, green fluorescent protein or
.beta.-galactosidase. This embodiment is particularly useful for
simple and rapid screening methods for compounds and substances
described herein below capable of modulating CKI specific gene
expression. For example, a cell suspension can be cultured in the
presence and absence of a candidate compound in order to determine
whether the compound affects the expression of genes which are
under the control of regulatory sequences of the invention, which
can be measured, e.g., by monitoring the expression of the
above-mentioned marker. It is also immediately evident to those
skilled in the art that other marker genes may be employed as well,
encoding, for example, a selectable marker which provides for the
direct selection of compounds which induce or inhibit the
expression of said marker.
[0102] The regulatory sequences of the invention may also be used
in methods of antisense approaches. The antisense RNA may be a
short (generally at least 10, preferably at least 14 nucleotides,
and optionally up to 100 or more nucleotides) nucleotide sequence
formulated to be complementary to a portion of a specific mRNA
sequence and/or DNA sequence of the gene of interest. Standard
methods relating to antisense technology have been described; see,
e.g., Klann, Plant Physiol. 112 (1996), 1321-1330. Following
transcription of the DNA sequence into antisense RNA, the antisense
RNA binds to its target sequence within a cell, thereby inhibiting
translation of the mRNA and down-regulating expression of the
protein encoded by the mRNA. Thus, in a further embodiment, the
invention relates to nucleic acid molecules of at least 15
nucleotides in length hybridizing specifically with a regulatory
sequence as described above or with a complementary strand thereof.
For the possible applications of such nucleic acid molecules, see
supra.
[0103] The present invention also relates to vectors, particularly
plasmids, cosmids, viruses and bacteriophages used conventionally
in genetic engineering that comprise a recombinant DNA molecule of
the invention. Preferably, said vector is an expression vector
and/or a vector further comprising a selection marker for plants.
For example of suitable selector markers, see supra. Methods which
are well known to those skilled in the art can be used to construct
recombinant 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 recombinant DNA molecules and vectors of
the invention can be reconstituted into liposomes for delivery to
target cells.
[0104] The present invention furthermore relates to host cells
transformed with a regulatory sequence, a DNA molecule or vector of
the invention. Said host cell may be a prokaryotic or eukaryotic
cell; see supra.
[0105] In a further preferred embodiment, 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, recombinant DNA molecule or vector of the
invention into the genome of said plant, plant cell or plant
tissue. For the expression of the heterologous DNA sequence under
the control of the regulatory sequence according to the invention
in plant cells, further regulatory sequences such as poly A tail
may be fused, preferably 3' to the heterologous DNA sequence, see
also supra. Further possibilities might be to add Matrix Attachment
Sites at the borders of the transgene to act as "delimiters" and
insulate against methylation spread from nearby heterochromatic
sequences. Methods for the introduction of foreign DNA into plants,
plant cells and plant tissue are described above.
[0106] Thus, the present invention relates also to transgenic plant
cells which contain stably integrated into the genome a recombinant
DNA molecule or vector according to the invention.
[0107] Furthermore, the present invention also relates to
transgenic plants and plant tissue comprising the above-described
transgenic plant cells. These plants may show, for example,
increased disease resistance.
[0108] In yet another aspect the invention also relates to
harvestable parts and to propagation material of the transgenic
plants according to the invention which contain transgenic plant
cells described above. Harvestable parts and propagation material
can be in principle any useful part of a plant; see supra.
[0109] With the regulatory sequences of the invention, it will be
possible to study in vivo CKI specific gene expression.
Furthermore, since CKI specific gene expression has different
patterns in different stages of physiological and pathological
conditions, it is now possible to determine further regulatory
sequences which may be important for the up- or down-regulation of
CKI gene expression, for example in response to ions or elicitors.
In addition, it is now possible to in vivo study mutations which
affect different functional or regulatory aspects of specific gene
expression in the cell cycle.
[0110] The in vivo studies referred to above will be suitable to
further broaden the knowledge on the mechanisms involved in the
control of the cell cycle. To date nothing is known about the
activity, nature or mode of act ion of CKIs in the cell cycle or
about their role during plant development. Expression of
heterologous genes or antisense RNA under the control of the
regulatory sequence of the present invention in plants and plant
cells may allow the understanding of the function of each of these
proteins in the plant.
[0111] The present invention further relates to a method for the
identification of an activator or inhibitor of genes encoding
cyclin-dependent kinase inhibitors comprising the steps of:
[0112] (a) providing a plant, plant cell, or plant tissue
comprising a recombinant DNA molecule comprising a readout system
operatively linked to a regulatory sequence of the invention;
[0113] (b) culturing said plant cell or tissue or maintaining said
plant in the presence of a compound or a sample comprising a
plurality of compounds under conditions which permit expression of
said readout system;
[0114] (c) identifying or verifying a sample and compound,
respectively, which leads to suppression or activation and/or
enhancement of expression of said readout system in said plant,
plant cell, or plant tissue.
[0115] The present invention further relates to a method for
identifying and obtaining an activator or inhibitor of
cyclin-dependent kinase inhibitors comprising the steps of:
[0116] (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;
[0117] (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;
[0118] (c) identifying or verifying a sample and compound,
respectively, which leads to suppression or activation of the
readout system.
[0119] 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
and/or 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.
[0120] 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.
[0121] 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 compound(s) 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.
[0122] 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 original sample
identified as containing the compound capable of suppressing or
activating cyclin-dependent kinase inhibitors, 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.
[0123] 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 a cyclin-dependent kinase inhibitor 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
Methods 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,
N.Y., USA. Furthermore, said derivatives and analogues can be
tested for their effects according to methods known in the art.
Furthermore, peptidomimetics and/or 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.
[0124] 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.
[0125] 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 a
cyclin-dependent kinase inhibitor or an inhibitor of a
cyclin-dependent kinase inhibitor.
[0126] Such useful compounds can be for example transacting factors
which bind to the cyclin-dependent kinase inhibitor 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 cyclin-dependent kinase inhibitor 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 cyclin-dependent
kinase inhibitor 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.
[0127] The invention also relates to a diagnostic composition
comprising at least one of the aforementioned nucleic acid
molecules, vectors, proteins, antibodies, regulatory sequences,
recombinant DNA molecules, or compounds and optionally suitable
means for detection.
[0128] Said diagnostic compositions may be used for methods for
detecting expression of cyclin-dependent kinase inhibitors by
detecting the presence of the corresponding mRNA 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.
[0129] 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:
[0130] in the complete plant
[0131] in selected plant organs, tissues or cell types
[0132] under specific environmental conditions, including abiotic
stress such as cold, heat, drought or salt stress or biotic stress
such as pathogen attack
[0133] during specific developmental stages.
[0134] Specifically the plant cell division rate and/or the
inhibition of a plant cell division can be influenced by (partial)
elimination of a gene or reducing the expression of a gene encoding
a protein according to the invention. Said plant cell division rate
and/or the inhibition of a plant cell division can also be
influenced by eliminating or inhibiting the activity of the protein
according to the invention by using for instance antibodies
directed against said protein. As a result of said elimination or
reduction greater organisms or specific organs or tissues can be
obtained; greater in volume and in mass too. Furthermore inhibition
of cell division by various adverse environmental conditions such
as drought, high salt content, chilling and the like can be delayed
or prevented by reduction of said expression of a gene according to
the invention. The division rate of a plant cell can also be
influenced in a transformed plant by overexpression of a sequence
according to the invention. Said transformed plant can be obtained
by transforming a plant cell with a gene encoding a polypeptide
concerned or fragment thereof alone or in combination, whereas the
plant cell may belong to a monocotyledonous or dicotyledonous
plant. For this purpose tissue specific promoters, in one construct
or being present as a separate construct in addition to the
sequence concerned, can be used. Therefore an important aspect of
the current invention is a method to modify plant architecture by
overproduction or reduction of expression of a sequence according
to the invention under the control of a tissue, cell or organ
specific promoter. Another aspect of the present invention is a
method to modify the growth inhibition of plants caused by
environmental stress conditions above mentioned by appropriate use
of sequences according to the invention. Surprisingly using a
polypeptide or fragment thereof according to the invention or using
antisense RNA or any method to reduce the expression of the gene
according to the invention, cell division in the meristem of both
main and lateral roots, shoot apical or the vascular tissue of a
plant can be manipulated. Furthermore any of the DNA sequences of
the invention as well as those encoding CDK1 can be used to
manipulate (reduce or enhance) the level of endopolyploidy and
thereby increasing the storage capacity of, for example, endosperm
cells.
[0135] Another aspect of the current invention is that one or more
DNA sequences, vectors or proteins, regulatory sequences or
recombinant DNA molecules of the invention or the above-described
antibody or compound can be used to modulate, for instance,
endoreduplication in storage cells, storage tissues and/or storage
organs of plants or parts thereof. The term "endoreduplication"
means recurrent DNA replication without consequent mitosis and
cytokinesis.
[0136] 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 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.
[0137] In view of the foregoing, the present invention also relates
to the use of a DNA sequence, vector, protein, antibody, regulatory
sequences, recombinant DNA molecule, nucleic acid molecules 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, for disrupting plant cell division
by influencing the presence or absence or by interfering in the
expression of a cyclin-dependent protein kinase inhibitor, for
modifying growth inhibition of plants caused by environmental
stress conditions, for inducing male or female sterility, 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 lacZ 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.
[0138] 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.
[0139] Furthermore, it is possible to use the nucleic acid
molecules according to the invention as molecular markers in plant
breeding. Moreover, the overexpression of nucleic acid molecules
according to the invention may be useful for the alteration or
modification of plantipathogene interaction. The term "pathogene"
includes, for example, bacteria, viruses and fungi as well as
protozoa.
[0140] 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, material, 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.ht- ml. 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.
[0141] The present invention is further described by reference to
the following non-limiting figures and examples.
THE FIGURES SHOW
[0142] FIG. 1: Sequence alignment of the Arabidopsis thaliana
cyclin-dependent kinase inhibitors FL39, FL66, FL67, ICKI
(accession number AC003040); the Medicago sativa cyclin-dependent
kinase inhibitor ALFCDKI, and the Chenopodium rubrum
cyclin-depndent kinase CrCKI (accession number AJ002173). Alignment
was obtained using the PILEUP program (from the GCG 9.1 package)
using the parameters Gap weight=4 and Length weight=0.
THE EXAMPLES ILLUSTRATE THE INVENTION
[0143] 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 Putative Cyclin-Dependent Kinase Inhibitors
[0144] For the identification of CKIs a two hybrid system based on
GAL4 recognition sites to regulate the expression of both his3 and
lacZ reporter genes was used to identify CDC2aAt-interacting of
proteins. The bait used for the two-hybrid screening was
constructed by inserting the CDC2aAt coding region into the pGBT9
vector (Clontech). The insert was created by PCR using the CDC2aAt
cDNA as template. Primers were designed to incorporate EcoRl
restriction enzyme sites. The primers used were
5'-CGAGATCTGMTTCATGGATCAGTA-3' (SEQ ID NO: 7) and
5'-CGAGATCTGMTTCCTAAGGC- ATGCC-3' (SEQ ID NO: 8). The PCR fragment
was cut with EcoRl and cloned into the EcoRl site of pGBT9,
resulting in the pGBTCDC2A plasmid. For the screening a GAL4
activation domain cDNA fusion library was used constructed from
Arabidopsis thaliana cell suspension cultures. This library was
constructed using RNA isolated from cells harvested at 20 hours, 3,
7 and 10 days after dilution of the culture in new medium. These
time point correspondent to cells from the early exponential growth
phase to the late stationary phase. mRNA was prepared using
Dynabeads oligo(dT).sub.25 according to the manufacturer's
instructions (Dynal). The GAL4 activation domain cDNA fusion
library was generated using the HybriZAP.TM. vector purchased with
the HybriZAP.TM. Two-Hybird cDNA Gigapack cloning Kit (Stratagene)
following the manufacturer's instructions. The resulting library
contained approximately 3.10.sup.6 independent plaque-forming
units, with an average insert size of 1 Kb.
[0145] For the screening a 1-liter culture of the Saccharomyces
cerevisiae strain HF7c (MAT.sub.a ura3-52 his3-200 ade2-101
lys2-801 trp1-901 leu2-3,112 gal4-542 gal80-538
LYS2::GAL1.sub.UAS-GAL1.sub.TATA-HIS3
URA3::GAL4.sub.17mers(3x)-CYC1.sub.TATA-LacZ) was cotransformed
with 400 .mu.g pGBTCDC2A, 500 .mu.g DNA of the library, and 40 mg
salmon sperm carrier DNA using the lithium acetate method (Gietz et
al. 1992, Nucleic Acids Res. 20, 1425). To estimate the number of
independent cotransformants, 1/1000 of the transformation mix was
plated on Leu.sup.- and Trp.sup.- medium. The rest of the
transformation mix was plated on medium to select for histidine
prototrophy (Trp.sup.-, Leu.sup.-, His.sup.-). Of a total of
approximately 1.2.times.10.sup.7 independent transformants 1200
colonies grew after 3 days of incubation at 30.degree. C. The
colonies larger than 2 mm were streaked on histidine-lacking medium
supplemented with 10 mM 3-amino-1,2,4-triazole (Sigma).
Two-hundred-fifty colonies capable of growing under these
conditions were tested for .beta.-galactosidase activity as
described (Breedon and Nasmyth 1995, Cold Spring Harbor Symp.
Quant. Biol. 50, p643-650), and 153 turned out to be His.sup.+ and
LacZ.sup.+. Plasmid DNA was prepared from the positive clones and
sequenced.
[0146] The plasmids pGADLDV39, pGADLDV66, and pGADLDV159 contained
a protein (designated LDV39, LDV66, and LDV159, respectively) of
which the last 23 amino-acids showed significant homology to the
human CKIs p21.sup.cip1 and p27.sup.kip1. The LDV159 clone was
identical to ICK1 (GenBank accession number U94772 as published by
Wang in Nature 386 (1997), 451-452). The two other clones were
novel and encoded proteins only distantly related to ICK1 (Table
1). The LDV39 gene was 622 bp long, consisting of 423 bp coding
region and 199 bp 3' UTR (excluding the poly-A tail). The LDV66
gene was 611 bp long, consisting of 379 bp coding region and 232 bp
3' UTR (excluding the poly-A tail).
[0147] The specificity of the interaction between LDV39, LDV66, and
LDV159 was verified by the retransformation of yeast with pGBTCDC2A
and pGADLDV39/pGADLDV66/pGADLDV159. As controls, pGBTCDC2A was
cotransformed with a vector containing only the GAL4 activation
domain (pGAD424); and the pGADLDV39/pGADLDV66/pGADLDV159 vectors
were cotransformed with a plasmid containing only the GAL4 DNA
binding domain (pGBT9). Transformants were plated on medium with or
without histidine. Only transformants containing both pGBTCDC2A and
pGADLDV39, pGADLDV66, ot pGADLDV1 59 were able to grow in the
absence of histidine.
EXAMPLE 2
LDV66, LDV39 and LDV159 Bind CDC2aAt, not CDC2bAt
[0148] The pGBTCDC2B vector encoding a fusion protein between the
C-terminus of the GAL4 DNA-binding domain and CDC2bAt was
constructed by cloning the full length coding region of CDC2bAt
into the pGBT9 vector. pGBTCDC2B was transformed with
pGADLDV66/pGADLDV39/pGADLDV1 59 in the HF7c yeast and
cotransformants were plated on medium with or without histidine. As
control, pGBTCDC2A was transformed with
pGADLDV66/pGADLDV39/pGADLDV159. In contrast to the transformants
containing the pGBTCDC2A vector were cotransformants containing the
pGBTCDC2B vector unable to grow in the absence of histidine. This
demonstrates that the LDV66, LDV39, LDV159 proteins associate with
CDC2aAt but not with CDC2bAt.
EXAMPLE 3
Isolation of FL39 and FL66 Sequences
[0149] Since the LDV39 and LDV66 clones encode partial proteins,
lacking their amino-terminal part, a flower cDNA library obtained
from the ABRC stock centre (library stock number CD4-6) was
screened. In total 50.000 plaque forming units were hybridised
using a fluorescein-labelled LDV39 or LDV66 probe according to the
manufacturer's protocol (Amersham) using a hybridisation
temperature of 60.degree. C. After 16 hours hybridisation the
filters were washed for 15 min using 2.times.SSC; 0.1.times.SDS,
and 15 min using 1.times.SSC; 0.1.times.SDS. The signals were
detected using the CDP-star detection module according to the
manufacturer's protocol (Amersham). The signals were revealed by
autoradiograpy. For both genes only one positive signal was
identified among the 50.000 phages, suggesting low mRNA levels of
LDV39 and LDV66 in flowers. Phages corresponding to the positive
signals were eluted from gel and purified by two additional
hybridisation rounds, using 1.000 and 50 plaque forming units in
the second and third hybridisation round, respectively. The
hybridisation conditions were similar as those described above.
After pure phages were obtained, DNA was extracted and sequenced.
The positive clones were denominated FL39 and FL66, corresponding
to longer clones of LDV39 and LDV66, respectively.
[0150] The FL39 clone is 932 bp long and contains an ORF encoding a
protein of 209 amino acids with a calculated molecular mass of 24
kDa. In its 3' UTR a poly-adenylation signal can be recognised. The
amino-terminal part of the FL39 protein contains a repeated motif
of 11 amino acids (VRRRD/ExxxVEE; SEQ ID NO: 33). This motif is not
found in any other protein in the databanks and its significance in
unknown. The FL39 protein also contains a putative nuclear
localization signal (amino acids 23-26) and a PEST-rich region
(amino acids 71-98; PESTFIND score +15.5). These sequences, rich in
proline, glutamic acid, serine and proline, are characteristically
present in unstable proteins (Rogers et al., 1986, Science 234,
364-368).
[0151] The FL66 sequence does not contain an in frame stopcodon,
and may therefore not be full length. The FL66 clone is 875 bp long
and bears an ORF of 216 amino acids, encoding a protein of 24 kD.
No nuclear localization signal or PEST domains are present.
[0152] The genomic organisation of the FL39, FL66 and LDV159 clones
was tested by DNA gel blot analysis. A. thaliana C24 DNA digested
with three different restriction enzymes was probed with
fluorescein-labelled prepared from the LDV159, FL39, or FL66
sequences according to the manufacturer's protocol (Amersham).
Hybridisations were performed at 60.degree. C. After 16 hours
hybridisation the membranes were washed for 15 min using
2.times.SSC; 0.1.times.SDS, and 15 min using 1.times.SSC;
0.1.times.SDS. The signals were detected using the CDP-star
detection module according to the manufacturer's protocol
(Amersham). The signals were revealed by autoradiography. For
LDV159 and FL39, only one hybridisation band was noticed for every
digest. For FL66 an additional weak band was observed. The low
intensity bands did not corespondent with any of the bands found
for LDV159 or FL39, suggesting the presence of an additional FL66
related gene. We conclude that there are at least four different
CKI proteins present in A. thaliana.
EXAMPLE 4
The Arabidopsis Thaliana CKIs Bind Exclusively to CDC2aAt in
vivo
[0153] The binding specificity of the FL39 and FL66 proteins
towards CDC2aAt and CDC2bAt was studied using the two-hybrid
system. The FL39 and FL66 coding regions were cloned in frame with
the GAL4 activation-domain in the pGAD424 vector (Clontech). The
FL39 coding region was amplified using the
5'-GGGAATCCATGGGCGGCGGTTAGGAGAAG-3' (SEQ ID NO: 9) and
5'-GGCGGATCCCGTCTTCTTCATGGATTC-3' (SEQ ID NO: 10) primers. The FL66
coding region was amplified using the
5'-GGCGMTCCATGGMGTCTCTAAAGCMC-3' (SEQ ID NO: 11) and
5'-GGCGGATCCTTTTGMCTTCATGGTTTGAC-3' (SEQ ID NO: 12) primers. The
FL66 amplified coding sequence encloses a protein starting at the
methionine at amino-acid position 11, therefore not including the
first 10 amino-acids encoded by the FL66 clone. The PCR fragments
were cut with EcoR1 and BamH1 and cloned into the EcoR1 and BamH1
sites of pGAD424, resulting in the pGADFL39 and pGADFL66 clones.
These plasmids were transformed into the HF7c yeast in combination
with pGBTCDC2A or pGBTCDC2B. The pGBTCDC2B plasmid, encoding a
fusion protein between the C-terminus of the GAL4 DNA-binding
domain and CDC2bAt was obtained by cloning the full length coding
of CDC2bAt into the pGBT9 vector (Clontech).
[0154] In contrast to the transformants containing the pGBTCDC2A
vector were the transformants containing the pGBTCDC2B vector
unable to grow in the absence of histidine. This demonstrates that
the FL39 and FL66 proteins exclusively associate with CDC2aAt.
EXAMPLE 5
Generation of FL39 and FL66 Specific Antibodies
[0155] To obtain sufficient amount of FL66 and FL39 proteins for
immunization, the FL39 and FL66 coding sequences were cloned into
pET vectors. The genes cloned in these vectors are expressed under
the control of the strong inducible T7 promoter in Escherichia coli
(Studier et al., 1986, J. Mol. Biol., 189, p113-130). The coding
region of FL39 and FL66 were amplified by PCR technique. The FL66
amplified coding sequence encloses a protein starting at the
methionine at amino-acid position 11, therefore not including the
first 10 amino-acids encoded by the FL66 clone. Primers used to
amplify FL39 were 5'-TAGGAGCATATGGCGGCGG-- 3' (SEQ ID NO: 29) and
5'-ATCATCGMTTCTTCATGGATTC-3' (SEQ ID NO: 30). Primers used to
amplify FL66 were 5'-ATATCAGCGCCATGGMGTC-3' (SEQ ID NO: 31) and
5'-GGAGCTGGATCCTTTTGGMTTCATGG-3' (SEQ ID NO: 32).
[0156] The obtained FL39 PCR fragment was purified, and cut with
Ndel and EcoRl restriction enzymes. This fragment was cloned into
the Ndel and EcoRl sites of pET derivative pRK172 (McLeod et al.,
1987 , EMBO J. 6, p729-736) The obtained FL66 PCR fragment was
purified, cut with Ncol and BamHl and cloned into the Ncol and
BamHl sites of pET21d. FL66pET21d was transformed in E. coli
BL21(DE3). FL39pRK172 was co-transformed in E. coli BL21(DE3) with
pSBETa (Schenk et al., 1995, Biotechniques 19, p 196-200). PSBETa
encoded the tRNA.sup.UCU that is a low abundant tRNA in E. coli,
corresponding to codons AGG and AGA (arginine). Because of the
presence of a AGG AGA AGA sequence (Arg 5, Arg 6, Arg 7) at the
beginning of FL39 coding sequence, an increase of the tRNA.sup.UCU
pool of E. coli is necessary for the translation of FL39.
[0157] The FL66pET21d/BL21(DE3) and FL39pRK172, pSBETa/ BL21(DE3)
E. coli recombinant strains were grown in LB medium, supplemented
respectively with 50 .mu.g/ml ampicilline and 50 .mu.g/ml
ampicilline; 25 .mu.g/ml kanamycine. The cells were grown at
37.degree. C. until the density of the culture reached an A.sub.600
nm=0.7. At this time point, 0.4 mM IPTG was added to induce the
recombinant protein production. Cells were collected 3 hours later
by centrifugation. The bacterial pellet from 250 ml culture was
suspended in 10 ml lysis buffer (Tris.HCl pH7.5, 1 mM DTT, 1 mM
EDTA, 1 mM PMSF and 0.1% Triton X-100) and submitted to three
freeze/thaw cycles before sonication. Cell lysate was clarified by
centrifugation 20 minutes at 8000 rpm. The pellet collected was
suspended again in extraction buffer, the resulting suspension
sonicated, and pellet collected by centrifugation 20 minutes at
8000 rpm. A third wash was performed the same way with Tris
extraction buffer+1M NaCl and a fourth wash with Tris extraction
buffer. After the different washing steps, the pellet contains FL66
or FL39 protein at 90% homogeneity. The pellets were suspended in
Laemli loading buffer (Laemmli, 1970, Nature 277, p680-681) and
FL66 and FL39 were further purified by SDS/12% polyacrylamide gel
electrophoresis. The gel was stained in 0.025% coomassie brillant
blue R250 in water and destained in water. The strong band
co-migrating at the 31 kDa molecular weight marker position was cut
out of the gel with a scalpel. The polyacrylamid fragments
containing FL66 or FL39 were lyophilized and reduced into powder.
The rabbit immunization was performed in complete Freund adjuvand,
subcutaneaous, with this antigen preparations. One injection
corresponds to 100 .mu.g of protein. The boosting injection were
performed with non-complete Freund adjuvant, sub-cutaneaous. The
obtained sera detected bands of the expected size in protein
extracts prepared from 2-day-old actively dividing cell cultures.
No signals were observed using the pr-immune sera.
EXAMPLE 6
Inhibition of Kinase Activity by FL66
[0158] The FL66pET/BL21(DE3) strain was used for the production of
recombinant FL66. The inclusion bodies containing FL66 were
collected and washed as described above. The recombinant FL66
protein was solubilized in 50 mM Tris.HCl pH7.6, 6M urea and kept
on ice for 1 hour. Refolding of the FL66 protein was performed by
removing urea on a sephadex G25 gel filtration column, equilibrated
in 50 mM Tris.HCl pH7.6, 400 mM NaCl. The collected fractions were
centrifuged and the supernatant was used for the inhibition
assay.
[0159] CDK complexes from A. thaliana were purified on p13.sup.suc1
sepharose beads, starting from 100 .mu.g of total protein extract
prepared from a 2-day-old cell suspension culture. The FL66 protein
was added to these purified complexes at a final concentration of
10 nM, 100 nM, 1 .mu.M and 10 .mu.M. After incubation during 1 hour
on ice the CDK activity was measured using histone Hi as substrate,
according to Azzi et al. (1992, Eur. J. Biochem., 203, 353-380).
When compared to a control sample (without addition of FL66), the
activity was found to be 82% of the control after addition of 10 nM
of FL66, 74% after addition of 100 nM, 56% after addition of 1
.mu.M, and 12% after addition of 10 .mu.M of FL66. Addition of 30
.mu.M of bovine serum albumin by comparison gives only a
non-specific decrease to 70% of the control activity.
[0160] The FL66 preparation was also added to A. thaliana CDK
fraction bound to p13.sup.suc1 beads, prior to washing of these
beads. The kinase activity dropped to 81% and 35% of the control
with a concentration of 0.1 .mu.M and 10 .mu.M of FL66,
respectively.
EXAMPLE 7
The Arabidopsis Thaliana CKI FL66 Associates Exclusively with
CDC2aAt in vitro
[0161] Purified recombinant FL66 protein (prepared as described as
in previous Example 6 was coupled to CNBr-activated Sepharose 4B
(Pharmacia) at a concentration of 5 mg/ml of gel according to the
manufacturer's instructions. Protein extracts were prepared from a
2-day-old cell suspension culture of A. thaliana Col-O in
homogenisation buffer (HB) containing 50 mM Tris-HCl (pH 7.2), 60
mM .beta.-glycerophosphate, 15 mM nitrophenyl phosphate, 15 mM
EGTA, 15 mM MgCl.sub.2, 2 mM dithiothreitol, 0.1 mM vanadate, 50 mM
NaF, 20 .mu.g/ml leupeptin, 20 .mu.g/ml aprotenin, 20 .mu.g/ml
soybean trypsin inhibitor (SBTI), 100 .mu.M benzamidine, 1 mM
phenylmethylsulfonylfluoride, and 0.1% Triton X-100. Two-hundred
.mu.g protein extract in a total volume of 100 .mu.l HB was loaded
on 50 .mu.l 50% (v/v) FL66-Sepharose or control Sepharose beads,
and incubated on a rotating wheel for 2 h at 4.degree. C. The
unbound proteins were collected for later analysis. The beads-bound
fractions were washed 3 times with HB. Beads were resuspended in 30
.mu.l SDS-loading buffer and boiled. The supernatants (beads bound
fractions) and 10 .mu.l of the unbound fractions were separated on
a 12.5% SDS-PAGE gel and electroblotted on nitrocellulose membrane
(Hybond-C.sup.+; Amersham). Filters were blocked overnight with 2%
milk in phosphate buffered saline (PBS), washed 3 times with PBS,
probed for 2 h with specific antibodies for CDC2aAt (1/5000
dilution) or CDC2bAt (1/2500 dilution) in PBS containing 0.5%
Tween-20 and 1% albumin, washed for 1 h with PBS with 0.5%
Tween-20, incubated for 2 h with peroxidase-conjugated secondary
antibody (Amersham), and washed for 1 h with PBS containing 0.5%
Tween 20. Protein detection was done by the chemoluminescent
procedure (Pierce).
[0162] Western blotting revealed that the a significant fraction of
CDC2aAt retained on the FL66-Sepharose beads, but not on the
control beads, demonstrating the in vitro interaction between FL66
and CDC2aAt. In contrast, the CDC2bAt protein did not retain on the
FL66-Sepharose beads but was found back in the unbound fraction.
These results demonstrate the specificity of the FL66 protein for
CDC2aAt.
EXAMPLE 8
Expression of CKIs at Different Time-Points in an Asynchronous Cell
Suspension Culture of Arabidopsis Thaliana
[0163] The expression levels of the different A. thaliana CKI genes
(FL39, FL66, and LDV159) at different time-points during the
cultivation of a A. thaliana cell culture were studied by
reverse-transcriptase polymerase chain reaction (RT-PCR)
technology. Four time-points were considered, representing the cell
culture at different growth phases: day 1 (lag phase), day 5
(exponential growth phase), day 8 (beginning of the stationary
phase), and day 12 (late stationary phase). Total RNA of cells
harvested at these time-points was extracted using the Trizol
reagent (Gibco BRL). 75 .mu.g of this total RNA preparation was
used for mRNA extraction using Dynabeads oligoT25 (Dynal). This
mRNA was used to prepare cDNA using the universal riboclone cDNA
synthesis system (Promega). Five ng of cDNA was subsequently used
for RT-PCR, using 300 ng of each of the appropriate forward and
reverse primers, 160 .mu.M of dNTPs, 10 .mu.l of PCR buffer, and
0.8 .mu.l of Taq polymerase (Promega). The used primers were
5'-CGGCTCGAGGAGMCCACMACACGC-3' (SEQ ID NO: 13) and
5'-CGAMCTAGTTMTTACCTCMGGMG-3' (SEQ ID NO: 14) for FL39;
5'-GATCCCGGGCGATATCAGCGTCATGG-3' (SEQ ID NO: 15) and
5'-GATCCCGGGTTAGTCTGTTMCTCC-3' (SEQ ID NO: 16) for FL66;
5'-GCAGCTACGGAGCCGGAGMTTGT-3' (SEQ ID NO: 17) and
5'-TCTCCTTCTCGAAATCGAAA- TTGTACT-3' (SEQ ID NO: 18) and for LDVI59.
The PCR reaction consisted of 4 min preheating at 94.degree. C.,
followed by cycles of 45 sec 94.degree. C., 45 sec 45.degree. C.,
and 45 sec 72.degree. C. After 10, 15, 20, 25, 30 and 35 cycles 10
.mu.l of the amplification mixture was loaded on an agarose gel and
electophoretically separated. After depurination, denaturation, and
neutralisation of the DNA it was transferred to a nitro-cellulose
membrane (Hybond N.sup.+; Amersham). The DNA was fixed on the
membrane by UV crosslinking.
[0164] Membranes were hybridised using fluorescein-labelled probes
prepared of the FL39, FL66, or LDV159 genes according to the
manufacturer's protocol (Amersham). After 16 hours hybridisation at
65.degree. C., the membranes were washed for 15 min using
2.times.SSC; 0.1.times.SDS, and 15 min using 1.times.SSC;
0.1.times.SDS. The signals were detected using the CDP-star
detection module according to the manufacturer's protocol
(Amersham). The signals were revealed by autoradiography.
[0165] FL39 transcripts could be detected at days 1, 5, and 8; but
not in late stationary cells (day 12). The strongest expression was
noticed in cells being in the exponential growth phase (at day 5).
The FL66 and LDV159 genes were most abundantly expressed at day 5
(during the exponential growth phase), although expression was
already substantial high at day 1 during the lag phase. Both genes
were expressed at a strongly reduced level in stationary cultures
(at day 8 and 12).
EXAMPLE 9
FL66 Transcription is Upregulated by NaCl
[0166] Stationary A. thaliana suspension cultures were diluted at
day 1 in fresh medium and cultivated for 48 hours At this
time-point the culture was divided into two subcultures. At one of
these cultures 1% NaCl was added. The cultures were cultivated for
12 hours after which the cells were collected and frozen in liquid
nitrogen. Of these samples RNA was prepared using the Trizol
reagent (Bibco BRL). 100 .mu.g of this total RNA preparation of
both samples was used for mRNA extraction using Dynabeads oligoT25
(Dynal). The poly-A RNA was electophorically separated on an
agarose gel and blotted onto a nitro-cellulose membrane
(Hybond-N.sup.+, Amersham). The membrane was hybridised using a
fluorescein-labelled probe prepared of the FL66 sequence according
to the manufacturer's protocol (Amersham). After 16 hours
hybridisation at 65.degree. C., the membranes were washed for 15
min using 2.times.SSC; 0.1.times.SDS, and 15 min using 1.times.SSC;
0.1.times.SDS. The signals were detected using the CDP-star
detection module (Amersham). The signals were revealed by
autoradiography.
[0167] A weak hybridising band of approximately 1000 bp was
detected in the control sample. Treatment with 1% NaCl clearly
increased the intensity of the hybridisation signal. This
demonstrates that the stress caused by the addition of NaCl results
in the transcriptional activation of the FL66 gene. This induction
could result in a permanent or transient arrest of cell division
activity.
EXAMPLE 10
Production of the CKIs in Plants
[0168] To obtain transgenic plants overexpressing the A. thaliana
CKI genes, the coding regions of FL36, FL66, and LDVI59 were cloned
into the pAT7002 vector (Aoyama and Chua, 1997, Plant J. 11,
p605-612). This vector allows inducible expression of the cloned
inserts by the addition of the glucocorticoid dexamethasone.
Following the polymerase chain reaction (PCR) technology the coding
regions of FL39, FL66, and ICKI were amplified using the
appropriate primer combinations. The primers used were
5'-CGGCTCGAGGAGMCCACAAACACGC-3' (SEQ ID NO: 19) and
5'-CGAAACTAGTTMTTACCTCMGGAAG-3' (SEQ ID NO: 20) for FL39,
GATCCCGGGCGATATCAGCGTCATGG-3' (SEQ ID NO: 21) and
5'-GATCCCGGGTTAGTCTGTTA- ACTCC-3' (SEQ ID NO: 22) for FL66, and
5'-CCCGCTCGAGATGGTGAGAAATATAGAAAAGC- TAAAGGATTTGTAGMGCTG
GAGTTTCGTCMCGTA-3' (SEQ ID NO: 23) and
5'-GGACTAGTTCACTCTAACTTTACCCATTCG-3' (SEQ ID NO: 24) for LDVI59.
The obtained FL39 and LDVI59 PCR fragments were purified and cut
with Xhol and Spel. Subsequently these fragments were used to clone
into the Xhol and Spel sites of pTA7002. The obtained FL66 fragment
was cut with Smal, purified, and cloned blunt into the Xhol and
Spel sites of the pTA7002 vector. The resulted binary vectors were
transferred into Agrobacterium tumefaciens. These stains were used
to transform Nicotiana tabacum cv. Petit havana using the leaf disk
protocol (Horsh et a/., 1985, Science 227, p1229-1231) and
Arabidopsis thaliana using the root transformation protocol
(Valvekens et al., 1988, PNAS 85, p5536-5540).
EXAMPLE 11
Arabidopsis Thaliana CKIs Expression in Fission Yeast
Schizosaccharomyces Pombe
[0169] To obtain heterologous expression of A. thaliana CKI genes
in the fission yeast Schizosaccharomyces pombe, the FL39 and FL66
were cloned into the pREP81 (Basi et al., 1993, Gene 123, p131-136)
and BNRP3 (Hemerly et al., 1995, EMBO J. 14, p3925-3936) vectors.
These vectors contain the thiamine-repressible promoter nmt1 and
allow inducible expression of the FL39 and FL66 genes (Maundrell et
al., 1990, JBC 265, p10857-10864). The expression is inducible to
different levels: strong induction is obtained with BNRP3, low
induction with pREP81. The coding region of FL39 and FL66 were
amplified by PCR technique. The FL66 amplified coding sequence
encloses a protein starting at the methionine at amino-acid
position 11, therefore not including the first 10 amino-acids
encoded by the FL66 clone. Primers used to amplify FL39 were
5'-GATCATCTTMGCATCATCGTCTTCTTCATGG-3' (SEQ ID NO: 25) and
5'-TAGGAGCATATGGCGGCGG-3' (SEQ ID NO: 26). Primers used to amplify
FL66 were 5'-ATATCAGCGCCATGGMGTC-3' (SEQ ID NO: 27) and
5'-GGAGCTGGATCCTTTTGGMTTCATGG-3' (SEQ ID NO: 28). The obtained FL39
PCR fragment was purified, phosphorylated with polynucleotide
kinase (blunt end) and cut with Ndel. This fragment was cloned into
the Ndel and Smal sites of pREP81. The obtained FL66 PCR fragment
was purified, cut with Ncol and BamHl and cloned into the Ncol and
BamHl sites of BNRP3.
[0170] The resulting recombinant plasmids were transformed in 972
leu1-32 h.sup.-Sch. pombe strain (wild type) by electroporation
technique. Transformant were selected on inducing medium
supplemented with 5 .mu.g/ml of thiamine. Phenotypes of
transformants were then compared with the phenotype of wild type
strain, on non-inducing medium. No cell cycle block could be
observed in Sch. pombe transformants expressing FL39 or FL66.
EXAMPLE 12
Identification of a FL66 Related Gene
[0171] By screening the A. thaliana sequence databank a genomic
sequence was identified encoding a protein highly homologous to
FL66. The protein encoded, annotated as `unknown protein`, was
renamed FL67. FL67 shows 39.545% similarity and 30.909% identity
with FL66.
EXAMPLE 13
In Situ Hybridisation Patterns
[0172] Plant material was fixed in 2.5% glutaraldehyde in 0.1M
cacodylate buffer (pH7.2) and dehydrated until 100% ethanol prior
to embedding in paraffin and tissue sectioning. 35S-UTP-labeled
sense and antisense RNAs of cDNA from FL39, FL66 and LDV159
subcloned in PGem2 were generated by run-off transcription using T7
and Sp6 RNA polymerases according to the manufacturer's
instructions (Boehringer Mannheim). Labeled RNA probes were
hydrolysed to an average length of 200 nt according to Cox et al
(1984). Deparaffinized and rehydrated tissue sections were taken
through the mRNA in situ procedure essentially as described by
Angerer and Angerer (1992). Stringencies during washes were
2.times.SSC at room temperature for 60 min and 0.1.times.SSC in 50%
formamide at 45 C for 30 min. RNase treatment, washing steps,
photograph emulsion coating, and the development of slides were
performed as described by Angerer and Angerer (1992). Photographs
were taken with a Diaplan microscope equipped with dark-field
optics (Leitz, Wetzlar, Germany).
[0173] Distinct expression patterns of the FL39, LDV159 and FL66
genes were observed when applying the mRNA in situ hybridization
technique on Arabidopsis thaliana and radish seedlings. Sections of
paraffin embedded roots, shoot apical meristems, flowers and
siliques of Arabidopsis thaliana, and radish roots and shoot apical
meristems were used to hybridize with the three cyclin-dependent
kinase inhibitors. The FL39 gene is expressed in young root
meristems in a homogeneous pattern. Mature root meristems barely
showed any expression of the gene. Some regions along the root
vascular tissue showed alternating zones of expressing and
nonexpressing cells at the periphery of the vascular bundle. A
region of pericycle cells in the vascular tissue, flanking the
region where new lateral roots usually form, presented a very
strong expression of the FL39 gene. In contrast, pericycle cells on
the region where lateral roots form hardly showed any expression.
These results show that higher levels of FL39 mRNA was observed
close to the region where lateral roots emerge possibly preventing
their formation at these regions. On the other hand, the absence of
FL39 gene expression in the poles of the diarch vascular bundle may
allow lateral root formation at these sites. It possibly assures
that lateral roots are formed by division of pericycle cells
adjacent to a protoxylem group. Uniform expression of FL39 gene was
also observed in all cells of the shoot apical meristem. Strong
signals were observed at the surface and tip of young leaves. The
epidermal and palisade layers of the leaves are the first layers to
vacuolize and differentiate, and the oldest part of the leaves are
at the tip. In addition, the expression pattern of CYCBI;1, a
molecular marker of cell division, shows a basipetal pattern of
cessation of cell division. Therefore, FL39 expression at these
sites may inhibit cell division allowing cell differentiation to
occur during early stages of leaf development. A similar pattern of
expression was observed on radish leaves, roots and shoot apical
meristems. In addition, strong expression at the epidermis of the
stem was also observed on young seedlings. The presence of FL39
mRNA in these cells might allow cells to differentiate. In
Arabidopsis flowers, FL39 was mainly expressed in the tapetal layer
of the anthers and in pollen grains.
[0174] Considering that at this stage, tapetum and pollen grains do
not divide, FL39 might be expressed at these sites to inhibit cell
division. Weaker expression was observed in flower buds and mature
ovaries. During embryo development very strong expression was
observed in embryos at the globular, heart and torpedo stages. At
the later stage strongest expression was at the embryonic root.
Weak or no hybridization signal was observed in mature seeds.
[0175] Expression of the LDV159 was also observed in all cells
along the main and lateral root meristems and shoot apical
meristems, but in a more uniform manner. Expression in vascular
tissue was slightly patchy, and stronger at the pericycle. Often a
pacthy pattern was observed in distinct cells of mature leaves. In
flowers, expression was mainly observed in mature ovaries.
Expression in embryos was mainly observed in globular and heart
stages and in the embryonic root at the torpedo stage. Weak
expression was observed in mature embryos. These results suggest a
function of LDV159 in the regulation of correct progression through
the cell cycle. LDV159 might play a role in the checkpoint control,
avoiding the premature activation of the CDK complexes under
unfavorable conditions. Its association with CDKs could inhibit CDK
activity until the cell perceives the correct signals to progress
to the next cell cycle phase.
[0176] FL66 gene expression was observed in the root and shoot
apical meristems. Stronger expression was observed in young
differentiating leaves often in a patchy manner suggesting a cell
cycle phase dependent expression pattern. Hybridization signal was
also observed along the vascular tissue. FL66 expression was as
well observed in flower buds and young flowers. In mature flowers
stronger expression was observed in the ovary wall, funiculus,
ovules and pollen grains. During embryo development strong
expression was observed at the globular stage. Signal gradually
decreases until the embryo maturation. Stronger signals were often
observed in the embryonic root.
EXAMPLE 14
Identification of a CKI in Alfalfa
[0177] The Medicago sativa cdc2-related kinase (CDC2AMs; Magyar et
al., 1997., The Plant Cell, Vol.: 9, 223-235.) cloned in the vector
pBD-GAL4 Cam phagemid (Stratagene) was used as a bait protein in a
yeast two-hybrid screen. mRNA isolated from young alfalfa (Medicago
truncatula) root nodules was converted to cDNA followed by cloning
into HybridZAP phagemids (Stratagene). The library was converted to
pAD-GAL4 plasmid library by mass excision. The yeast strain Y190
(Clontech) was used as a host for the two hybrid analysis. As a
positive clone interacting in this system with the CDC2MsA kinase,
a partial cDNA clone of 613 bp was isolated coding for 128 amino
acids. Sequencing of this clone revealed extensive homology with
the C-terminal region of known CDK inhibitors (CKI). The full
length cDNA clone was isolated with screening an alfalfa root
nodule Lambda ZAP II (Stratagene) cDNA library with the partial
cDNA as probe and using standard procedures. A clone comprising a
full length cDNA designated ALFCDKI was obtained and the
corresponding nucleotide and amino acid sequences of the encoded
CKI are shown in SEQ ID NO: 5 and 6, respectively.
Sequence CWU 1
1
35 1 932 DNA Arabidopsis thaliana CDS (86)..(712) 1 ggcacgagga
gaaccacaaa cacgcacaca taacgagtga ttttagagag agatagagat 60
ctggaaggtg acgtcgtagg agatt atg gcg gcg gtt agg aga aga gaa cga 112
Met Ala Ala Val Arg Arg Arg Glu Arg 1 5 gat gtg gtt gaa gag aat gga
gtt acg acg acg acg gtg aaa cga agg 160 Asp Val Val Glu Glu Asn Gly
Val Thr Thr Thr Thr Val Lys Arg Arg 10 15 20 25 aag atg gag gag gaa
gtg gat tta gtg gaa tct agg ata att ctg tct 208 Lys Met Glu Glu Glu
Val Asp Leu Val Glu Ser Arg Ile Ile Leu Ser 30 35 40 ccg tgt gta
cag gcg acg aat cgc ggt gga att gtg gcg aga aat tca 256 Pro Cys Val
Gln Ala Thr Asn Arg Gly Gly Ile Val Ala Arg Asn Ser 45 50 55 gca
gga gcg tcg gag acg agt gtt gtt ata gta cga cgg cga gat tct 304 Ala
Gly Ala Ser Glu Thr Ser Val Val Ile Val Arg Arg Arg Asp Ser 60 65
70 cct ccg gtt gaa gaa cag tgt caa atc gaa gaa gaa gat tcg tcg gtt
352 Pro Pro Val Glu Glu Gln Cys Gln Ile Glu Glu Glu Asp Ser Ser Val
75 80 85 tcg tgt tgt tct aca tcg gaa gag aaa tcg aaa cgg aga atc
gaa ttt 400 Ser Cys Cys Ser Thr Ser Glu Glu Lys Ser Lys Arg Arg Ile
Glu Phe 90 95 100 105 gta gat ctt gag gaa aat aac ggt gac gat cgt
gaa aca gaa acg tcg 448 Val Asp Leu Glu Glu Asn Asn Gly Asp Asp Arg
Glu Thr Glu Thr Ser 110 115 120 tgg att tac gat gat ttg aat aag agt
gag gaa tcg atg aac atg gat 496 Trp Ile Tyr Asp Asp Leu Asn Lys Ser
Glu Glu Ser Met Asn Met Asp 125 130 135 tct tct tcg gtg gct gtt gaa
gat gta gag tct cgc cgc agg tta agg 544 Ser Ser Ser Val Ala Val Glu
Asp Val Glu Ser Arg Arg Arg Leu Arg 140 145 150 aag agt ctc cat gag
acg gtg aag gaa gct gag tta gaa gat ttt ttt 592 Lys Ser Leu His Glu
Thr Val Lys Glu Ala Glu Leu Glu Asp Phe Phe 155 160 165 cag gtg gcg
gag aaa gat ctt cgg aat aag ttg ttg gaa tgt tct atg 640 Gln Val Ala
Glu Lys Asp Leu Arg Asn Lys Leu Leu Glu Cys Ser Met 170 175 180 185
aag tat aac ttc gat ttc gag aaa gat gag cca ctt ggt gga gga aga 688
Lys Tyr Asn Phe Asp Phe Glu Lys Asp Glu Pro Leu Gly Gly Gly Arg 190
195 200 tac gag tgg gtt aaa ttg aat cca tgaagaagac gatgatgata
atgatgatca 742 Tyr Glu Trp Val Lys Leu Asn Pro 205 ttgttttcac
caaagtactt attatttttc ttctgtaata atctttgctt tgatttttct 802
tttaacaaaa tccaaatgta gatatctttc tctcgaataa tcaataacat gtaattcaac
862 ttttgtttgt acttccttga ggtaattaat tagattcgtg tttttctcga
ttaataaact 922 ataagtttat 932 2 209 PRT Arabidopsis thaliana 2 Met
Ala Ala Val Arg Arg Arg Glu Arg Asp Val Val Glu Glu Asn Gly 1 5 10
15 Val Thr Thr Thr Thr Val Lys Arg Arg Lys Met Glu Glu Glu Val Asp
20 25 30 Leu Val Glu Ser Arg Ile Ile Leu Ser Pro Cys Val Gln Ala
Thr Asn 35 40 45 Arg Gly Gly Ile Val Ala Arg Asn Ser Ala Gly Ala
Ser Glu Thr Ser 50 55 60 Val Val Ile Val Arg Arg Arg Asp Ser Pro
Pro Val Glu Glu Gln Cys 65 70 75 80 Gln Ile Glu Glu Glu Asp Ser Ser
Val Ser Cys Cys Ser Thr Ser Glu 85 90 95 Glu Lys Ser Lys Arg Arg
Ile Glu Phe Val Asp Leu Glu Glu Asn Asn 100 105 110 Gly Asp Asp Arg
Glu Thr Glu Thr Ser Trp Ile Tyr Asp Asp Leu Asn 115 120 125 Lys Ser
Glu Glu Ser Met Asn Met Asp Ser Ser Ser Val Ala Val Glu 130 135 140
Asp Val Glu Ser Arg Arg Arg Leu Arg Lys Ser Leu His Glu Thr Val 145
150 155 160 Lys Glu Ala Glu Leu Glu Asp Phe Phe Gln Val Ala Glu Lys
Asp Leu 165 170 175 Arg Asn Lys Leu Leu Glu Cys Ser Met Lys Tyr Asn
Phe Asp Phe Glu 180 185 190 Lys Asp Glu Pro Leu Gly Gly Gly Arg Tyr
Glu Trp Val Lys Leu Asn 195 200 205 Pro 3 875 DNA Arabidopsis
thaliana CDS (11)..(658) 3 ggcacgagag aaa tca aag ata act ggc gat
atc agc gtc atg gaa gtc 49 Lys Ser Lys Ile Thr Gly Asp Ile Ser Val
Met Glu Val 1 5 10 tct aaa gca aca gct cca agt cca ggt gtt cga acc
aga gcc gct aaa 97 Ser Lys Ala Thr Ala Pro Ser Pro Gly Val Arg Thr
Arg Ala Ala Lys 15 20 25 acc cta gcc ttg aag cgg ctt aat tcc tcc
gcc gct gat tca gct cta 145 Thr Leu Ala Leu Lys Arg Leu Asn Ser Ser
Ala Ala Asp Ser Ala Leu 30 35 40 45 cct aac gac tct tct tgc tat ctt
cag ctc cgt agc cgc cgt ctc gag 193 Pro Asn Asp Ser Ser Cys Tyr Leu
Gln Leu Arg Ser Arg Arg Leu Glu 50 55 60 aaa ccc tct tcg ctg att
gaa ccg aaa cag ccg ccg aga gtt cac aga 241 Lys Pro Ser Ser Leu Ile
Glu Pro Lys Gln Pro Pro Arg Val His Arg 65 70 75 tcg gga att aaa
gag tct ggt tcc agg tct cgc gtt gac tcg gtt aac 289 Ser Gly Ile Lys
Glu Ser Gly Ser Arg Ser Arg Val Asp Ser Val Asn 80 85 90 tcg gtt
cct gta gct cag agc tct aat gaa gat gaa tgt ttt gac aat 337 Ser Val
Pro Val Ala Gln Ser Ser Asn Glu Asp Glu Cys Phe Asp Asn 95 100 105
ttc gtg agt gtc caa gtt tct tgt ggt gaa aac agt ctc ggt ttt gaa 385
Phe Val Ser Val Gln Val Ser Cys Gly Glu Asn Ser Leu Gly Phe Glu 110
115 120 125 tca aga cac agc aca agg gag agc acg cct tgt aac ttt gtt
gag gat 433 Ser Arg His Ser Thr Arg Glu Ser Thr Pro Cys Asn Phe Val
Glu Asp 130 135 140 atg gag atc atg gtt aca cca ggg tct agc acg agg
tcg atg tgc aga 481 Met Glu Ile Met Val Thr Pro Gly Ser Ser Thr Arg
Ser Met Cys Arg 145 150 155 gca acc aaa gag tac aca agg gaa caa gat
aac gtg atc ccg acc act 529 Ala Thr Lys Glu Tyr Thr Arg Glu Gln Asp
Asn Val Ile Pro Thr Thr 160 165 170 agt gaa atg gag gag ttc ttt gca
tat gca gag cag cag caa cag agg 577 Ser Glu Met Glu Glu Phe Phe Ala
Tyr Ala Glu Gln Gln Gln Gln Arg 175 180 185 cta ttc atg gag aag tac
aac ttc gac att gtg aat gat atc ccc ctc 625 Leu Phe Met Glu Lys Tyr
Asn Phe Asp Ile Val Asn Asp Ile Pro Leu 190 195 200 205 agc gga cgt
tac gaa tgg gtg caa gtc aaa cca tgaagttcaa aaggaaacag 678 Ser Gly
Arg Tyr Glu Trp Val Gln Val Lys Pro 210 215 ctccaaaaga catggtgtga
agttagagaa tgtgatggag ttaacagact aaccaaacat 738 cagaaatcgt
gtaatcttaa gtaataatgt ggttagagaa caagtttgag agtagcttag 798
ggaccttaaa acctcacacc atttgtaata ctaatcttct tcagatgctt agtgaaattt
858 tctcatctgt ttctttc 875 4 222 PRT Arabidopsis thaliana 4 Met Gly
Lys Tyr Met Lys Lys Ser Lys Ile Thr Gly Asp Ile Ser Val 1 5 10 15
Met Glu Val Ser Lys Ala Thr Ala Pro Ser Pro Gly Val Arg Thr Arg 20
25 30 Ala Ala Lys Thr Leu Ala Leu Lys Arg Leu Asn Ser Ser Ala Ala
Asp 35 40 45 Ser Ala Leu Pro Asn Asp Ser Ser Cys Tyr Leu Gln Leu
Arg Ser Arg 50 55 60 Arg Leu Glu Lys Pro Ser Ser Leu Ile Glu Pro
Lys Gln Pro Pro Arg 65 70 75 80 Val His Arg Ser Gly Ile Lys Glu Ser
Gly Ser Arg Ser Arg Val Asp 85 90 95 Ser Val Asn Ser Val Pro Val
Ala Gln Ser Ser Asn Glu Asp Glu Cys 100 105 110 Phe Asp Asn Phe Val
Ser Val Gln Val Ser Cys Gly Glu Asn Ser Leu 115 120 125 Gly Phe Glu
Ser Arg His Ser Thr Arg Glu Ser Thr Pro Cys Asn Phe 130 135 140 Val
Glu Asp Met Glu Ile Met Val Thr Pro Gly Ser Ser Thr Arg Ser 145 150
155 160 Met Cys Arg Ala Thr Lys Glu Tyr Thr Arg Glu Gln Asp Asn Val
Ile 165 170 175 Pro Thr Thr Ser Glu Met Glu Glu Phe Phe Ala Tyr Ala
Glu Gln Gln 180 185 190 Gln Gln Arg Leu Phe Met Glu Lys Tyr Asn Phe
Asp Ile Val Asn Asp 195 200 205 Ile Pro Leu Ser Gly Arg Tyr Glu Trp
Val Gln Val Lys Pro 210 215 220 5 1193 DNA Arabidopsis thaliana CDS
(92)..(763) 5 aaaccactct tcaaatcaaa cactttctta cataagattc
ctctgttttt ctgtgtgctt 60 cttcaaattc ttcccctgtt tttcaacttc a atg ggg
aag tac atg aag aaa 112 Met Gly Lys Tyr Met Lys Lys 1 5 ctc aaa tcc
aaa tca gaa tct cct tca ccc aat tca aca cca aca cca 160 Leu Lys Ser
Lys Ser Glu Ser Pro Ser Pro Asn Ser Thr Pro Thr Pro 10 15 20 tca
cca tca cca tca cca aca cca atc acc acc aat tca cca cca cca 208 Ser
Pro Ser Pro Ser Pro Thr Pro Ile Thr Thr Asn Ser Pro Pro Pro 25 30
35 aca aca ccc aat tcc tct gat ggt gtt cga act cgt gct aga acc cta
256 Thr Thr Pro Asn Ser Ser Asp Gly Val Arg Thr Arg Ala Arg Thr Leu
40 45 50 55 gct ttg gag aat tcc aac aat cag aat cag aat ctt tct gtt
tct tct 304 Ala Leu Glu Asn Ser Asn Asn Gln Asn Gln Asn Leu Ser Val
Ser Ser 60 65 70 gat tct tac ctt cag ctg agg aac cgt cgc ctt aag
aga ccc cta att 352 Asp Ser Tyr Leu Gln Leu Arg Asn Arg Arg Leu Lys
Arg Pro Leu Ile 75 80 85 agg caa cat tcc gct aag agg aat aag ggg
cat gat gga aac cct aaa 400 Arg Gln His Ser Ala Lys Arg Asn Lys Gly
His Asp Gly Asn Pro Lys 90 95 100 tcc cca att ggg gat tca att gct
gaa gag aaa act gtt cag aag agt 448 Ser Pro Ile Gly Asp Ser Ile Ala
Glu Glu Lys Thr Val Gln Lys Ser 105 110 115 cct gag cct gaa aat gct
gaa ttc aag gag aat gct gag gat act gag 496 Pro Glu Pro Glu Asn Ala
Glu Phe Lys Glu Asn Ala Glu Asp Thr Glu 120 125 130 135 aga agc gct
agg gaa act aca ccc gtc cat ttg ata atg cga gca gac 544 Arg Ser Ala
Arg Glu Thr Thr Pro Val His Leu Ile Met Arg Ala Asp 140 145 150 gtt
ctc agg cct cct agg cca att acc agg cgt act ttt cca act gaa 592 Val
Leu Arg Pro Pro Arg Pro Ile Thr Arg Arg Thr Phe Pro Thr Glu 155 160
165 gct aat ccc aaa acg gag cag cca act atc cca att tca cgc gaa ttt
640 Ala Asn Pro Lys Thr Glu Gln Pro Thr Ile Pro Ile Ser Arg Glu Phe
170 175 180 gag gaa ttc tgt gct aaa cat gaa gcc gag cag caa agg gag
ttc atg 688 Glu Glu Phe Cys Ala Lys His Glu Ala Glu Gln Gln Arg Glu
Phe Met 185 190 195 gag aag tac aac ttt gat cct gtg aca gag cag cca
ctc cca ggg cgt 736 Glu Lys Tyr Asn Phe Asp Pro Val Thr Glu Gln Pro
Leu Pro Gly Arg 200 205 210 215 tac gaa tgg gaa aaa gtg tcg ccc tag
aaggcaggct agtattaagt 783 Tyr Glu Trp Glu Lys Val Ser Pro 220
gttccatcaa tacatcttta aagtagcagc agggttagaa tttgttgaaa agggtggtgg
843 tgctatttcc attttccatc actttctatt tacttgtaaa gaaagtagga
ctttcaacat 903 atgtagacta atgatctgta actttacaga ggtgttgatt
acacaacaat acaaagtcct 963 ttgtctagca gatcattaaa gaagggtttg
agggaataag ggtctctagt tgtagggttt 1023 agggtataaa atcaaagtag
ggtatgtaag agaggtttta caagaatttc cttttgttct 1083 tgtgttttac
tcttgttttg tctatacttg tactcatgga acttcaacaa actcttaaga 1143
aataaagaac cagatctccc tcaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1193 6 223
PRT Arabidopsis thaliana 6 Met Gly Lys Tyr Met Lys Lys Leu Lys Ser
Lys Ser Glu Ser Pro Ser 1 5 10 15 Pro Asn Ser Thr Pro Thr Pro Ser
Pro Ser Pro Ser Pro Thr Pro Ile 20 25 30 Thr Thr Asn Ser Pro Pro
Pro Thr Thr Pro Asn Ser Ser Asp Gly Val 35 40 45 Arg Thr Arg Ala
Arg Thr Leu Ala Leu Glu Asn Ser Asn Asn Gln Asn 50 55 60 Gln Asn
Leu Ser Val Ser Ser Asp Ser Tyr Leu Gln Leu Arg Asn Arg 65 70 75 80
Arg Leu Lys Arg Pro Leu Ile Arg Gln His Ser Ala Lys Arg Asn Lys 85
90 95 Gly His Asp Gly Asn Pro Lys Ser Pro Ile Gly Asp Ser Ile Ala
Glu 100 105 110 Glu Lys Thr Val Gln Lys Ser Pro Glu Pro Glu Asn Ala
Glu Phe Lys 115 120 125 Glu Asn Ala Glu Asp Thr Glu Arg Ser Ala Arg
Glu Thr Thr Pro Val 130 135 140 His Leu Ile Met Arg Ala Asp Val Leu
Arg Pro Pro Arg Pro Ile Thr 145 150 155 160 Arg Arg Thr Phe Pro Thr
Glu Ala Asn Pro Lys Thr Glu Gln Pro Thr 165 170 175 Ile Pro Ile Ser
Arg Glu Phe Glu Glu Phe Cys Ala Lys His Glu Ala 180 185 190 Glu Gln
Gln Arg Glu Phe Met Glu Lys Tyr Asn Phe Asp Pro Val Thr 195 200 205
Glu Gln Pro Leu Pro Gly Arg Tyr Glu Trp Glu Lys Val Ser Pro 210 215
220 7 25 DNA Artificial sequence probe or primer 7 cgagatctga
attcatggat cagta 25 8 26 DNA Artificial sequence probe or primer 8
cgagatctga attcctaagg catgcc 26 9 29 DNA Artificial sequence probe
or primer 9 gggaatccat gggcggcggt taggagaag 29 10 27 DNA Artificial
sequence probe or primer 10 ggcggatccc gtcttcttca tggattc 27 11 29
DNA Artificial sequence probe or primer 11 ggcgaatcca tggaagtctc
taaagcaac 29 12 30 DNA Artificial sequence probe or primer 12
ggcggatcct tttgaacttc atggtttgac 30 13 26 DNA Artificial sequence
probe or primer 13 cggctcgagg agaaccacaa acacgc 26 14 27 DNA
Artificial sequence probe or primer 14 cgaaactagt taattacctc
aaggaag 27 15 26 DNA Artificial sequence probe or primer 15
gatcccgggc gatatcagcg tcatgg 26 16 25 DNA Artificial sequence probe
or primer 16 gatcccgggt tagtctgtta actcc 25 17 24 DNA Artificial
sequence probe or primer 17 gcagctacgg agccggagaa ttgt 24 18 27 DNA
Artificial sequence probe or primer 18 tctccttctc gaaatcgaaa
ttgtact 27 19 26 DNA Artificial sequence probe or primer 19
cggctcgagg agaaccacaa acacgc 26 20 27 DNA Artificial sequence probe
or primer 20 cgaaactagt taattacctc aaggaag 27 21 26 DNA Artificial
sequence probe or primer 21 gatcccgggc gatatcagcg tcatgg 26 22 25
DNA Artificial sequence probe or primer 22 gatcccgggt tagtctgtta
actcc 25 23 69 DNA Artificial sequence probe or primer 23
cccgctcgag atggtgagaa aatatagaaa agctaaagga tttgtagaag ctggagtttc
60 gtcaacgta 69 24 30 DNA Artificial sequence probe or primer 24
ggactagttc actctaactt tacccattcg 30 25 32 DNA Artificial sequence
probe or primer 25 gatcatctta agcatcatcg tcttcttcat gg 32 26 19 DNA
Artificial sequence probe or primer 26 taggagcata tggcggcgg 19 27
20 DNA Artificial sequence probe or primer 27 atatcagcgc catggaagtc
20 28 27 DNA Artificial sequence probe or primer 28 ggagctggat
ccttttggaa ttcatgg 27 29 19 DNA Artificial sequence probe or primer
29 taggagcata tggcggcgg 19 30 23 DNA Artificial sequence probe or
primer 30 atcatcgaat tcttcatgga ttc 23 31 20 DNA Artificial
sequence probe or primer 31 atatcagcgc catggaagtc 20 32 27 DNA
Artificial sequence probe or primer 32 ggagctggat ccttttggaa
ttcatgg 27 33 11 PRT Arabidopsis thaliana misc_feature (5)...(5)
Xaa at position 5 may be Asp or Glu 33 Val Arg Arg Arg Xaa Xaa Xaa
Xaa Val Glu Glu 1 5 10 34 327 PRT Arabidopsis thaliana 34 Met Gly
Lys Tyr Ile Arg Lys Ser Lys Ile Asp Gly Ala Gly Ala Gly 1 5 10 15
Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu Ser Ser Ile Ala 20
25 30 Leu Met Asp Val Val Ser Pro Ser Ser Ser Ser Ser Leu Gly Val
Leu
35 40 45 Thr Arg Ala Lys Ser Leu Ala Leu Gln Gln Gln Gln Gln Arg
Cys Leu 50 55 60 Leu Gln Lys Pro Ser Ser Pro Ser Ser Leu Pro Pro
Thr Ser Ala Ser 65 70 75 80 Pro Asn Pro Pro Ser Lys Gln Lys Met Lys
Lys Lys Gln Gln Gln Met 85 90 95 Asn Asp Cys Gly Ser Tyr Leu Gln
Leu Arg Ser Arg Arg Leu Gln Lys 100 105 110 Lys Pro Pro Ile Val Val
Ile Arg Ser Thr Lys Arg Arg Lys Gln Gln 115 120 125 Arg Arg Asn Glu
Thr Cys Gly Arg Asn Pro Asn Pro Arg Ser Asn Leu 130 135 140 Asp Ser
Ile Arg Gly Asp Gly Ser Arg Ser Asp Ser Val Ser Glu Ser 145 150 155
160 Val Val Phe Gly Lys Asp Lys Asp Leu Ile Ser Glu Ile Asn Lys Asp
165 170 175 Pro Thr Phe Gly Gln Asn Phe Phe Asp Leu Glu Glu Glu His
Thr Gln 180 185 190 Ser Phe Asn Arg Thr Thr Arg Glu Ser Thr Pro Cys
Ser Leu Ile Arg 195 200 205 Arg Pro Glu Ile Met Thr Thr Pro Gly Ser
Ser Thr Lys Leu Asn Ile 210 215 220 Cys Val Ser Glu Ser Asn Gln Arg
Glu Asp Ser Leu Ser Arg Ser His 225 230 235 240 Arg Arg Arg Pro Thr
Thr Pro Glu Met Asp Glu Phe Phe Ser Gly Ala 245 250 255 Glu Glu Glu
Gln Gln Lys Gln Phe Ile Glu Lys Tyr Val Phe Pro Arg 260 265 270 Phe
Ile Cys Ser Val Leu Leu Val Met Ser Phe Gln Phe Val Leu Phe 275 280
285 Phe Ser Phe Gly Leu Val Ser Leu Met Val Ser Val Asn Ser Phe Phe
290 295 300 Arg Tyr Asn Phe Asp Pro Val Asn Glu Gln Pro Leu Pro Gly
Arg Phe 305 310 315 320 Glu Trp Thr Lys Val Asp Asp 325 35 9 DNA
VSV epitope 35 agg aga aga 9 Arg Arg Arg
* * * * *
References