Note: Descriptions are shown in the official language in which they were submitted.
CA 02272608 1999-06-08
CYCLIN-DEPENDENT KINASE INHIBITORS AS PLANT GROWTH
REGULATORS
FIELD OF THE INVENTION
The invention relates to the modification of growth and development of plants
through transgenic sense or anti-sense expression of cyclin-dependent kinase
inhibitor genes.
BACKGROUND OF THE INVENTION
In eucaryotes including plants, the progression of cell cycle events is
regudat~d by a
network of gene products and factors to ensure that this crucial process is
initiated as an
integral part of the growth and the developmental program, and in response to
the external
t0 environment. These factors exert their influences on the cell cycle
machinery via various
pathways. At the center of the machinery lies an enzyme complex consisting of
a catalytic
subunit, cyclin-dependent protein kinase (CDK), and a regulatory subunit,
cyclin. CDKs are a
group of related serinelthreonine kinases and their activity generally depends
on their
association with cyclins (Pines, 1995).
t5 Early work disclosed the existence of CDKs in yeast. A CDK called Cdc2
(p34'°'~, or
CDK1) was identified in fission yeast Schizosaccharomyces pombe (Hindley and
Phear,
1984) and a Cdc2 homolog called CDC28 was identified in budding yeast
Saccharomyces
cerevisiae (Lorincz and Reed, 1984). In yeast, Cdc2 (or CDC28) kinase appears
to be solely
responsible for regulating the progression of the cell cycle.
2o Animal cells have evolved several Cdc2-related CDKs in order to achieve
more
complex regulation at multiple levels. In mammalian cells, seven distinct CDKs
and eight
types of cyclins have been identified (see review by Pines, 1995): Complexes
of these CDKs
and cyclins appear to act sequentially at different checkpoints during the
cell cycle, while
incorporating the input of different developmental and environmental cues.
25 Plants, like higher animals, have multiple CDKs (Francis and Halford, 1995;
Jacobs,
1995) and cyclins (Renaudin et al., 1996). In Arabidopsis lhaliana, at least
two Cdc2
homologues, Cdc2a and Cdc2b (Ferreira et al., 1991; Hirahama et al., 1991) and
as many as
twelve cyclins belonging to three groups (Renaudin et al., 1996) have been
documented. Of
the two Cdc2 homologues in A. rhaliana, Cdc2a resembles more closely Cdc2
homologues
1
CA 02272608 1999-06-08
from other species because it has a conserved PSTAIRE motif a.~d is able to
genetically
complement yeast cdc2 or CDC28 mutants (Ferreira et al., 1991; Hirahama et
al., 1991),
indicating some functional homology of A. thaliana Cdc2a with the yeast Cdc2
kinase.
Expression analyses showed that A. thaliana cdc2a expression was correlated
with the
"competence" of a cell to divide and preceded the re-entry of differentiated
cells into the cell
division cycle (Martinet et al., 1992; Hemerly et al., 1993), and expression
of a dominant
negative cdcla mutant resulted in cell cycle arrest (Hemerly et al., 1995). A.
thaliana Cdc2b
is atypical in that it has a PPTALRE motif in place of the PSTAIRE motif. Like
cdc2a,
cdc2b is also expressed in dividing plant cells. While cdc2a is expressed
constitutively
throughout the cell cycle, cdc2b is reportedly expressed preferably in S and
G2 phases
(Segers et al., 1996).
Relatively little is known about the cyclins and other proteins and factors
which
regulate the activity of CDK-cyclin complexes in plant cells. Results from
yeast and
mammalian studies have demonstrated multiple pathways, both positive and
negative, by
which CDK activity can be modulated (Lees, 1995). In addition to binding by a
cyclin, for
example, activation of CDKs may also involve a CDK-activating kinase (CAK)
which itself
is a CDK, and CDC25 protein phosphatase.
A new aspect of regulating CDK activity was discovered with the identification
of
CDK inhibitors (see reviews by Pines, 1995; Sherr and Roberts, 1995; Harper
and Ellege,
2o 1996). These small proteins are understood to bind stoichiometrically to
negatively regulate
the activity of CDKs. It has been suggested that these inhibitors may be
involved in animal
development and cancer, in addition to their role in cell cycle regulation
(Haiper and Elledge,
1996). A plant CDK inhibitor activity was observed and was suggested to be
involved in
endosperm development in maize (Graft and Larkins, 1995).
The activity of CDK inhibitors has been studied in animals. Transgenic mice
have
been generated lacking p21, p27 and p57 CDK inhibitor genes. The p21 knockout
mice are
reported to develop normally but are deficient in G1 checkpoint control, such
as cell cycle
arrest in response to DNA damage (Deng et al., 1995). Analysis of p27 knockout
mice from
three independent studies show that transgenic mice lacking p27 display larger
body size than
3o control mice (Fern et al., 1996; Kiyokawa et al., 1996; Nakayama et al.,
1996). The enhanced
CA 02272608 1999-06-08
growth is reportedly due to an increase in cell number (Kiyokawa et al., 1996)
and is gene
dose-dependent (Fero et al., 1996). In comparison, none of p21 or p57 knockout
display
enhanced growth. The transgenic mice lacking p57 show a range of developmental
defects
such as defective abdominal muscles, cleft palate and renal medullary
dysplasia (Yan et al.,
1997; Zhang et al., 1997). A few developmental defects were observed in p27-/-
mice. They
include impaired ovarian follicles (thus female sterility), impaired luteal
cell differentiation
and a disordered estrus cycle. These results reflect a disturbance of the
hypothalamic-
pituitary-ovarian axis. In comparison, transgenic mice lacking p21 appear to
develop
normally at both gross anatomic and histologic levels (Deng et al., 1995). In
addition, an
irrrease in apoptosis is observed in mice lacking p57. The CDK inhibitor p27
was over-
expressed in mouse hepatocytes (Wu et al., 1996), resulting in a general a
decrease in overall
number of adult hepatocytes which result in aberrant tissue organization, body
growth and
mortality.
Despite the general conservation of basic cell cycle machinery in eucaryotes,
the role
of plant cell division during plant growth and development is
characteristically different from
other eucaryotic cells. In many respects, the regulation of plant cell
division and growth can
be regarded as distinct from other eucaryotic cells. For example, plant cells
are not mobile
during morphogenesis. Different sets of hormones are involved in modulating
plant growth
and development. Plant cells are remarkable for their ability to re-enter the
cell cycle
following differentiation. Also, cell division in plants is continuous, along
with organ
formation, and plant body size (the number of total cells and size of the
cells) can vary
dramatically under different conditions. Plants also have an inherent ability
to incorporate
additional growth into normal developmental patterns, as is illustrated by a
study showing
that ectopic expression of a mitotic cyclin driven by the cdc2a promoter
resulted in a larger
but normal root system (Doerner et al., 1996). However, relatively little is
known about the
interaction of the regulatory genes controlling cell division patterns in
plants (Meyerowitz,
1997).
A few studies of transgenic expression of cell cycle genes in plants are
documented
using various cell cycle genes other than CDK inhibitors. A heterologous yeast
cdc25, a
mitotic inducer gene, was introduced into tobacco plants under the control of
a constitutive
CA 02272608 1999-06-08
CaMV 35s promoter (Bell et al., 1993). Transgenic tobacco plants showed
abnormal leaves
(lengthened and twisted lamina, pocketed interveinal regions), abnormal
flowers, and also
precocious flowering. Analysis of cell size in the root meristem revealed that
trasngenic
plants expressing the yeast cdc25 had much smaller cells (Bell et al., 1993).
The wild type
cdc2a gene and variants of dominant negative mutations under the control of
CaMV 35s
promoter have been used to transform tobacco and Arabidopsis plants (Hemerly
et al., 1995).
Constitutive expression of wild-type and mutant Cdc2a did not significantly
alter the
development of the transgenic plants. For the dominant negative Cdc2a mutant,
it was not
possible to regenerate Arabidopsis plants. Some tobacco plants expressing this
construct were
obtained and they had considerably fewer but much larger cells. These cells,
however,
underwent normal differentiation. Morphogenesis, histogenesis and
developmental timing
were unaffected (Hemerly et al., 1995). As mentioned above, ectopic expression
of an
Arabidopsis mitotic cyclin gene, cyclAt, under the control of the cdc2a
promoter increases
growth without altering the pattern of lateral root development in Arabidopsis
plants (Doerner
et al., 1996).
The yeast two-hybrid system has been used to identify the cyclin-dependent
kinase
inhibitor gene ICKI from a plant (Wang et al., 1997). ICK1 is different in
sequence, structure
and inhibitory properties from known mammalian CDK inhibitors. It has been
shown that
recombinant protein produced from this gene in bacteria is able to inhibit
plant Cdc2-like
2o kinase activity in vitro (Wang et al., 1997).
Cytotoxin genes, i.e. genes encoding a protein which will cause cell death,
have been
tested in transgenic plants for genetic ablation of specific cells or cell
lines during
development, including RNase (Mariani et al., 1990), DTT chain A (Thorsness et
al., 1991;
Czako et al., 1992), Exotoxin A (Koning et al., 1992) and ribosomal inhibitor
proteins
(United States Patent No. 5,723,765 issued 3 March 1998 to Oliver et al.).
Several
disadvantages may be associated with the use of cytotoxin genes for
modification of
transgenic plants, particularly plants of agronomic importance. The action of
the cytotoxin
may not be specific and may result in non-specific destruction of plant cells.
This effect may
be the result of diffusion of the cytotoxin, or of non-specific expression of
the cytotoxin gene
3o in non-target tissues. Non-specific low-level expression of the cytotoxin
may be a difficult
CA 02272608 1999-06-08
problem to overcome, since most tissue-specific promoters have some levels of
expression in
other tissues in addition to a high level of expression in a particular
tissue. Expression of a
potent cytotoxin gene even at a low concentration may have a negative impact
on growth and
development in non-target tissues. The presence of cytotoxic proteins of
transgenic origin
may also have a negative effect on the marketability of an edible plant, or
plant product, even
if the cytotoxin is demonstrably benign to consumers.
SUMMARY OF THE INVENTION
In one embodiment, the invention provides methods for using CDK inhibitor
genes to
to modify the growth and development of plant cells and organs. In particular,
the invention
provides a method of modifying the development of a plant comprising (i.e.
having or
including, but not limited to.) transforming a plant cell with a nucleic acid
encoding a cyclin
dependent kinase inhibitor to produce a transformed plant cell. A plant may
then be
regenerated from the transformed plant cell under conditions wherein the
cyclin dependent
15 kinase inhibitor is expressed during regeneration of the plant to modify
the development of
the plant. The nucleic acid encoding the cyclin dependent kinase inhibitor may
be
homologous to ICKI, or may be ICKI, respectively encoding a cyclin-dependent
kinase
inhibitor homologous to ICK1 or ICKI itself. In particular embodiments, the
plant may be A.
thaliana, or a member of the Brassica genus, or a canola variety. The nucleic
acid encoding
2o the cyclin-dependent kinase inhibitor may be operably linked to a tissue-
specific promoter,
such as AP3 or a promoter homologous to AP3. In particular embodiments, the
tissue-specific
promoter may mediates expression of the nucleic acid encoding the cyclin-
dependent kinase
inhibitor in petal and stamen primordia, and the development of the plant may
be modified so
that the plant is male sterile.
i5 Another aspect of the invention provides transgenic plants comprising (i.e.
having or
including, but not limited to) an expressible heterologous nucleic acid
encoding a cyclin
dependent kinase inhibitor, wherein the heterologous nucleic acid is
introduced into the plant,
or an ancestor of the plant, by the foregoing method. Alternatively, the
plants may comprise a
nucleic acid encoding a cyclin dependent kinase inhibitor, and the plant cells
may be
3o transformed with an anti-sense nucleic acid complimentary to the nucleic
acid encoding the
CA 02272608 1999-06-08
cyclin dependent kinase inhibitor, to produce a transformed plant cell. So
that regenerating
the plant from the transformed plant cell under conditions wherein the anti-
sense nucleic acid
is transcribed during regeneration of the plant inhibits the expression of the
cyclin dependent
kinase inhibitor and modifies the development of the plant. Plant of the
invention may have a
recombinant genome and the heterologous nucleic acid may be integrated into
the
recombinant genome. The invention encompasses plant tissues, such as seeds,
comprising a
heterologous nucleic acid encoding a cyclin dependent kinase inhibitor, or an
anti-sense
construct, that is expressed during the development of a plant from the tissue
to modify the
development of the plant.
to The invention also provides methods of identifying nucleic acids that
encode.cyclin-
dependent kinase inhibitors, such as nucleic acids homologous to ICKI, that
are active in
plants to modify the development of the plants.
BRIEF DESCRIPTION OF THE DRAVYINGS
is
Figures 1 to 4 show the cDNA sequences of ICK2, ICN2, ICN6 and ICN7,
respectively; and
Figure 5 shows the alignment of deduced amino acid sequences of ICK1,
ICK2, ICN2, ICN6 and ICN7.
DETAILED DESCRIPTION OF THE INVENTION
In the following detailed description, various examples are set out of
particular
embodiments of the invention, together with experimental procedures that may
be used to
implement a wide variety of modifications and variations in the practice of
the present
invention.
In the context of the present invention, "promoter" means a sequence
sufficient to
direct transcription of a gene when the promoter is operably linked to the
gene. The promoter
is accordingly the portion of a gene containing DNA sequences that provide for
the binding of
3o RNA polymerise and initiation of transcription. Promoter sequences are
commonly, but not
6
CA 02272608 1999-06-08
universally, located in the S' non-coding regions of a gene. A promoter and a
gene are
"operably linked" when such sequences are functionally connected so as to
permit gene
expression mediated by the promoter. The term "operably linked" accordingly
indicates that
DNA segments are arranged so that they function in concert for their intended
purposes, such
as initiating transcription in the promoter to proceed through the coding
segment of a gene to
a terminator portion of the gene. Gene expression may occur in some instances
when
appropriate molecules (such as transcriptional activator proteins) are bound
to the promoter.
Expression is the process of conversion of the information of a coding
sequence of a gene
into mRNA by transcription and subsequently into polypeptide (protein) by
translation, as a
to result of which the protein is said to be expressed. As the term is used
herein, a gene or
nucleic acid is "expressible" if it is capable of expression under appropriate
conditions in a
particular host cell. . .
.- For the present invention, promoters may be used that provide for
preferential gene
expression in a specific tissue or during a specific period of development.
For example,
promoters may be used that are specific for leaf (Dunsmuir, et al Nucleic
Acids Res, (1983)
11:4177-4183), root tips (Pokalsky, et al Nucleic Acids Res, (1989) 17:4661-
4673), fruit
(Peat, et al Plant Mol. Biol, (1989) 13:639-651; United States Patent No.
4,943,674 issued 24
July, 1990; International Patent Publication WO-A 8 809 334; United States
Patent No.
5,175,095 issued 29 December, 1992; European Patent Application EP-A 0 409
629; and
2o European Patent Application EP-A 0 409 625) or late embryogenesis (U.S.
Patent No.
5,723,765 issued 3 March 1998 to Oliver er al.). Such promoters may, in some
instances, be
obtained from genomic clones of cDNAs. Depending upon the application of the
present
invention, those skilled in this art may choose a promoter for use,in the
invention which
provides a desired expression pattern. Promoters demonstrating preferential
transcriptional
activity in plant tissues are, for example, described in European Patent
Application EP-A 0
255 378 and International Patent Publication WO-A 9 113 980. Promoters may be
identified
from genes which have a differential pattern of expression in a specific
tissue by screening a
tissue of interest, for example, using methods described in United States
Patent No. 4,943,674
and European Patent Application EP-A 0255378. The disclosure herein includes
examples of
this aspect of the invention, showing that plant tissues and organs can be
modified by
CA 02272608 1999-06-08
transgenic expression of a plant CDK inhibitor.
Non-dividing plant cells may tolerate low level expression of CDK inhibitors,
such as
ICIC~, in non-targeted tissues. Thus, the invention may be practiced in some
embodiments
using tissue specific promoters operably linked to CDK inhibitor encoding
sequences, even
when the promoter mediates a tolerable basal level of expression in other
tissues.
Various aspects of the present invention emcompass nucleic acid or amino acid
sequences that are homologous to other sequences. As the teen is used herein,
an amino acid
or nucleic acid sequence is "homologous" to another sequence if the two
sequences are
substantially identical and the functional activity of the sequences is
conserved (for example,
l0 both sequences function as or encode a cyclin dependent kinase inhibitor;
as used herein,
sequence conservation or identity does not infer evolutionary relatedness).
Nucleic acid
sequences may also be homologous if they encode substantially identical amino
acid
sequences, even if the nucleic acid sequences are not themselves substantially
identical, for
example as a result of the degeneracy of the genetic code.
15 Two amino acid or nucleic acid sequences are considered substantially
identical if,
when optimally aligned, they share at least about 75% sequence identity,
preferably at least
about 90% sequence identity, and more preferably at least about 95% sequence
identity.
Optimal alignment of sequences for comparisons of identity may be conducted
using a
variety of algorithms, such as the local homology algorithm of Smith and
Waterman (1981)
20 Adv. Appl. Math 2: 482, by the homology alignment algorithm of Needleman
and Wunsch
(1970) J. Mol. Biol. 48:443, buy the search for similarity method of Pearson
and Lipman
(1988) Proc. Natl. Acad. Sci. USA 85: 2444, and by computerized
implementations of these
algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group, Madison, WI, U.S.A.). Sequence
identity may
25 also be determined using the BLAST algorithm, described in Altschul et al.
(1990), J. Mol.
Biol. 215:403-10 (using the published default settings). Software for
performing BLAST
analysis may be available through the National Center for Biotechnology
Information
(through the Internet at han:~twww.ncbi.nlm.nih.~o~n. The BLAST algorithm
involves first
identifying high scoring sequence pairs (HSPs) by identifying short words of
length W in the
30 query sequence that either match or satisfy some positive-valued threshold
score T when
CA 02272608 1999-06-08
aligned with a word of the same length in a database sequence. T is referred
to as the
neighborhood word score threshold. Initial neighborhood word hits act as seeds
for initiating
searches to find longer HSPs. The word hits are extended in both directions
along each
sequence for as far as the cumulative alignment score can be increased.
Extension of the word
S hits in each direction is halted when the following parameters are met: the
cumulative
alignment score falls off by the quantity X from its maximum achieved value;
the cumulative
score goes to zero or below, due to the accumulation of one or more negative-
scoring residue
alignments; or the end of either sequence is reached. The BLAST algorithm
parameters W, T
and X determine the sensitivity and speed of the alignment. The BLAST program
may use as
to defaults ~ word length (V~ of 11, the BLOSLTM62 scoring matrix (Henikoff
and Henikoff
(1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50,
expectaion (E) of
10, M=5, N=4, and a compaison of both strands. One measure of the statistical
similarity
between two sequences using the BLAST algorithm is the smallest sum
probability (P(I~),
which provides an indication of the probability by which a match between two
nucleotide or
15 amino acid sequences would occur by chance. In alternative embodiments of
the invention,
nucleotide or amino acid sequences are considered substantially identical if
the smallest sum
probability in a comparison of the test sequences is less than about 1,
preferably less than
about 0.1, more preferably less than about 0.01, and most preferably less than
about 0.001.
An alternative indication that two nucleic acid sequences are substantially
identical is
20 that the two sequences hybridize to each other under moderately stringent,
or preferably
stringent, conditions. Hybridization to filter-bound sequences under
moderately stringent
conditions may, for example, be performed in 0.5 M NaHPO" 7% sodium dodecyl
sulfate
(SDS), 1 mM EDTA at 65 °C, and washing in 0.2 x SSC/0.1 % SDS at 42
°C (see Ausubel, et
al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green
Publishing
25 Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).
Alternatively,
hybridization to filter-bound sequences under stringent conditions may, for
example, be
performed in 0.5 M NaHPO,, 7% SDS, 1 mM EDTA at 65 °C, and washing in
0.1 x
SSC/0.1% SDS at 68°C (see Ausubel, et al. (eds), 1989, supra).
Hybridization conditions
may be modified in accordance with known methods depending on the sequence of
interest
3o (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular
Biology --
9
CA 02272608 1999-06-08
Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of
principles of
hybridization and the strategy of nucleic acid probe assays", Elsevier, New
York). Generally,
stringent conditions are selected to be about 5°C lower than the
thermal melting point for the
specific sequence at a defined ionic strength and pH.
An alternative indication that two amino acid sequences are substantially
identical is
that one peptide is specifically immunologically reactive with antibodies that
are also
specifically immunoreactive against the other peptide. Antibodies are
specifically
immunoreactive to a peptide if the antibodies bind preferentially to the
peptide and do not
bind in a significant amount to other proteins present in the sample, so that
the preferential
to binding of the antibody to the peptide is detectable in an immunoassay and
distinguishable
from non-specific binding to other peptides. Specific immunoreactivity of
antibodies to
peptides may be assessed using a variety of immunoassay formats, such as solid-
phase
ELISA immunoassays for selecting monoclonal antibodies specifically
immunoreactive with
a protein (see Harlow and Lane (1988) Antibodies, A Laboratory Manual, cold
Spring Harbor
15 Publications, New York).
The cyclin dependent kinase inhibitors of the present invention, and the genes
encoding those inhibitors, may include non-naturally occurring sequences, such
as
fimctionally active fi-agments of naturally occurring sequences. For example,
fragments of
ICK2, or amino acid sequences homologous to those fragments, that have cyclin
dependent
2o kinase inhibitory activity may be used in some embodiments of the
invention. The invention
provides methods for identifying such fragments, for example by deletion
mapping of active
cyclin dependent kinase inhibitors. As used herein the term "cyclin dependent
kinase
inhibitor" therefore includes any polypeptide capable of functioning during
plant
development to modify the development of the plant, the invention similarly
encompasses
25 nucleic acid sequences encoding such polypeptides.
As used herein to describe nucleic acid or amino acid sequences the term
"heterologous" refers to molecules or portions of molecules, such as DNA
sequences, that are
artificially introduced into a particular host cell. Heterologous DNA
sequences may for
example be introduced into a host cell by transformation. Such heterologous
molecules may
3o include sequences derived from the host cell. Heterologous DNA sequences
may become
to
CA 02272608 1999-06-08
integrated into the host cell genome, either as a result of the original
transformation of the
host cells, or as the result of subsequent recombination events.
The specificity of a CDK inhibitor may be assayed in vivo. For example, the
ICKI
coding sequence was fused to a known promoter which directed gene expression
in pollen
but not in stamen primordia. The transformants are normal and fertile. This
result indicates
that in specific embodiments of the invention, expression of ICKI is not
generally toxic to
tissues other than the target tissue. Phenotypes may be obtained, for example
with the
exemplified AP3-ICKI transformants, that are due to specific action of the CDK
inhibitor,
such as ICKI protein, on cell division. In such embodiments, the CDK
inhibitor, such as
ICKI, may be used as a specific tool to modify growth and development of
meristematic
tissues without affecting other processes.
In some embodiments, there may be important advantages to using a CDK
inhibitor
gene for genetic engineering in plants, particularly to ablate selected cell
lineages, rather than
using genes encoding cytotoxins. In accordance with the invention, the CDK
inhibitor action
may be made to be specific only to certain cells, avoiding the non-specific
destruction of
plant cells. This specificity may be achieved partly because non-dividing
plant cells in non-
targeted tissues may have better tolerance of low level expression of a CDK
inhibitor than a
cytotoxin. Thus, in accordance with the invention it may be possible to use
tissue specific
promoters for expressing CDK inhibitors when such promoters still have a
tolerable basal
level of expression in other tissues. This may usefully expand the range of
promoters
available for use in the invention, since most tissue-specific promoters have
some levels of
expression in other tissues in addition to a high level of expression in a
particular tissue. In
contrast, expression of a potent cytotoxic gene in one tissue, even at a low
concentrations, can
have a negative impact on growth and development in other tissues.
In an alternative aspect of the invention, the down-regulation of CDK
inhibitors, such
as ICK1, may be used to enhance growth during plant development. Such growth
enhancement may be tissue-specific. For example, anti-sense oligonucleotides
may be
expressed to down-regulate expression of CDK inhibitors. The expression of
such anti-sense
constructs may be made to be tissue-specific by operably linking anti-sense
encoding
sequences to tissue-specific promoters. Anti-sense oligonucleotides, including
anti-sense
11
CA 02272608 1999-06-08
RNA molecules and anti-sense DNA molecules, act to block the translation of
mRNA by
binding to targeted mRNA and inhibiting protein translation from the bound
mRNA. For
example, anti-sense oligonucleotides complementary to regions of a DNA
sequence encoding
a CDK inhibitor, such as ICK1, may be expressed in transformed plant cells
during
development to down-regulate the CDK inhibitor. Alternative methods of down-
regulating
CDK inhibitor gene expression may include the use of ribozymes or other
enzymatic RNA
molecules (such as hammerhead RNA structures) that are capable of catalyzing
the cleavang
of RNA (as disclosed in U.S. Patent Nos. 4,987,071 and 5,591,610). The
mechanism of
ribozyme action generally involves sequence specific hybridization of the
ribozyme molecule
t0 to complementary target RNA , followed by endonucleolytic cleavage.
Arabidopsis thaliana "Columbia" may be used as a convenient model system for
identifying CDK inhibitors that are useful in various embodiments of the
present invention.
Arabidopsis plants are generally grown in pots placed in growth chambers
(20°C 16h/8h of
day/night). Other plants may also of course be used in various embodiments of
the invention
15 in accordance with known transformation and growth techniques.
Yeast two-hybrid cloning and assay techniques may be used to assess and
identify
CDK inhibitors use in the present invention. For example, a cDNA library may
be made
using poly (A) mRNA isolated from whole plants at different stages of
development and
cloned in a suitable vector, such as Gal4 TA- (transcription-activation
domain) vector pBI771
2o (Koholmi et al., 1997). The cDNA of the gene (such as cdcla, cyclin b2 and
cyclin 83) to be
used for screening the library may be cloned in a suitable vector, such as the
Gal4 DB-
(DNA-binding domain) vector. The yeast strain, such as YPB2, harboring the
construct may
be transformed using the library DNA.
In one example, for analysis of Cdc2a interactions, a total of 1.8 X 10' iz-
ansformants
25 were subjected to two-hybrid selection on supplemented synthetic dextrose
medium lacking
leucine, tryptophan and histidine but containing S mM 3-amino-1,2,4-triazole.
The selected
colonies were assayed for ~3-galactosidase activity using standard methods.
DNAs were
isolated from positive clones and used to transform E. coli. Clones harboring
the TA-fusion
cDNAs were identified by PCR and plasmids were then isolated for DNA
sequencing.
3o Interactions in the yeast two-hybrid system may, for example, be analyzed
by either
CA 02272608 1999-06-08
filter assay (Chevray and Nathans, 1992) using X-gal as the substrate or by a
quantification
assay using ONPG (ortho-nitrophenyl-beta-D-galactoside) as the substrate
(Reynolds and
Lundlad, 1994). Three or more independent transformants may be used for each
interaction.
Standard methods are available for the preparation of constructs for use in
identifying
and characterizing CDK inhibitors useful in various embodiments of the
invention. General
molecular techniques may for example be performed by procedures generally
described by
Ausubel et al. ( 1995). Alternative equivalent methods or variations thereof
may be used in
accordance with the general knowledge of those skilled in this art.
In one example, the AP3 promoter was cloned by the polymerase chain reaction
to (PCR) from Arabidopsis thaliana "Columbia" genomic DNA, on the basis of the
published
sequence (lrish and Yamamoto, 1995; GenBank Accession U30729). The promoter
was
cloned in a modified binary vector pBI121 (Clontecla). ICKI cDNA (Wang et al.,
1997) was
similarly amplified by PCR and transcriptionally fused with the AP3 promoter
and the
chimeric gene ends with a nopaline sythase terminator. In addition, a
construct consisting of
15 ICKI fused to an anther-specific promoter of Bgpl (Xu et al., 1993) was
prepared similarly.
The resulting plasmids were introduced into Agrobactrium tumefaciens strain
GV3101
(bearing helper plasmid pMP90; Koncz and Schell 1986).
In accordance with various aspects of the invention, plant cells may be
transformed
with heterologous nucleic acids. Transformation techniques that may be
employed include
2o plant cell membrane disruption by electroporation, microinjection and
polyethylene glycol
based transformation (such as are disclosed in Paszkowski et al. EMBOJ. 3:2717
(1984);
Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824 (1985); Rogers et al.,
Methods Enrymol.
118:627 (1986); and in U.S. Patent Nos. 4,684,611; 4,801,540; 4,743,548 and
5,231,019),
ballistic transformation such as DNA particle bombardment (for example as
disclosed in
25 Klein, et al., Nature 327: 70 (1987); Gordon-Kamm, et al. "The Plant Cell"
2:603 (1990); and
in U.S. Patent Nos. 4,945,050; 5,015,580; 5,149,655 and 5,466,587) and
Agrobacterium
mediated transformation methods (such as those disclosed in Horsch et al.
Science 233: 496
(1984); Fraley et al., Proc. Nat'l Acad. Sci. USA 80:4803 (1983); and U.S.
Patent Nos.
4,940,838 and 5,464,763).
3o Transformed plant cells may be cultured to regenerate whole plants having
the
13
CA 02272608 1999-06-08
transformed genotype and display a desired phenotype, as for example modified
by the
expression of a heterologous CDK inhibitor during development. A variety of
plant culture
techniques may be used to regenerate whole plants, such as are described in
Evans et al.
"Protoplasts Isolation and Culture", Handbook of Plant Cell Culture,
Macmillian Publishing
Company, New York, 1983; or Binding, "Regeneration of Plants, Plant
Protoplasts", CRC
Press, Boca Raton, 1985; or in Klee et al., Ann. Rev. oJPlant Phys. 38:467 (
1987).
Standard techniques may be used for plant transformation, such as
transformation of
Arabidopsis. In one example, the AP3-ICKI and Bgpl-ICKI constructs were tested
in A.
thaliana by in plants transformation techniques. Wild type (WT) A. thaliana
seeds of ecotype
to "Columbia" were planted in soiled 4" pots and plants grew in controlled
growth chamber or
greenhouse. The vacuum infiltration method of in plants transformation
(Bechtold et al.,
1993) was used to transform A. thaliana plants with overnight culture of A.
tumejacian strain
GV3101 bearing both the helper nopline plasmid and the binary construct
containing the
described chimeric gene. pMP90 is a disarmed Ti plasmid with intact vir region
acting in
traps, gentamycin and kanamycin selection markers as described in Koncz and
Schell ( 1986).
Following infiltration, plants were grown to maturity and seeds (T1) were
collected each pod
individually. Seeds were surfaced sterilized and screened on selective medium
containing 50
mg/L kanamycin with or without 200 mg/L timentin. After about four weeks on
selection
medium, the non-transformed seedlings died. The transformed seedlings were
transferred to
2o soil in pots. Leaf DNA was isolated (Edwards et al., 1991 ) and analyzed by
PCR for the
presence of the DNA insertion. Genomic DNA was also isolated and used in
Southern
hybridization (Southern, 1975) to determine the copy number of the inserted
sequence in a
given transformant. To determine the segregation, T2 seeds were collected from
T1 plants.
Wherever the T1 plant was male sterile, crosses was made using the WT A.
thaliana pollen to
i5 obtain seeds. As described, T2 seeds were surface-sterilized and screened
on selective
medium.
Alternative embodiments of the invention may make use of techniques for
transformation of Brassica. Such as transformation of B. napus cv. Westar and
B. carinata
cv. Dorolla or breeding line C90-1088 by co-cultivation of cotyledonary
petioles or hypocotyl
3o explants with A. tumejaciens bearing the plasmids described herein.
Transformation of B.
14
CA 02272608 1999-06-08
napus plants may, for example, be performed according to the method by Moloney
et al.
(1989). Modifications of that method may include the introduction of a 7-day
explant-
recovery period following co-cultivation, on MS medium with the hormone
benzyladenine
(BA), and the antibiotic timentin for the elimination of Agrobacterium.
Transformation of B.
carinata plants may be performed according to the method by Babic et al. (
1998).
Cotyledonary petiole explants may be dipped in suspension of Agrobacterium
bearing the
desired constructs and placed on 7-cm filter paper (Whitman no. 1) on top of
the regeneration
medium for 2 days. After co-cultivation, explants may be transferred onto the
selection
medium containing 50 mg/L kanamycin. Regenerated green shoots may first be
transferred to
to a medium to allow elongation and then to a rooting medium all containing 50
mglL
kanamycin. Putative transformants with roots (TO) may be transferred to soil.
Genomic DNA
may be isolated from developing leaves for PCR and Southern analyses. Seeds
(T1) from
transgenic plants may then be harvested. .
Transgenic plants may be observed and characterized for the presence (absence
of) the
t 5 desired phenotypic trait, such as petals, male sterility and ability to
set seeds after pollination
using wild type pollen. For example, to determine the development of floral
organs, flowers
at different stages of development may be dissected and examined under a
stereomicroscope.
Floral samples may also be examined using scanning electron microscope for
more defined
morphology of floral organ meristems and their development.
2o Genomic clones of sequences encoding putative CDK inhibitors may be cloned
using
standard techniques. For example, to clone a genomic ICK1 encoding sequence,
genomic
DNA may be isolated from two-week old A. ~haliana seedlings according to a
procedure
described (Lohdi et al., 1994). In one example, the genomic sequence spanning
the ICKI
cDNA sequence was amplified by 30 cycles of PCR (polymerise chain reaction)
using
25 sequence-specific primers with incorporated restriction sites. PJu DNA
polymerise
(Stratagene), which has a higher replication fidelity than the Taq DNA
polymerise, may be
used. The amplified DNA fragment may be cloned into a suitable vector, such as
pGEMSZf(+) (Promega). Plasmids may then be purified and sequenced.
In one example, an ICKl cDNA isolated from the two hybrid screening was cloned
in
3o frame into pBI786, a modified His6-tagged vector derived from pRSETB
(Invitrogen) and the
s
is
CA 02272608 1999-06-08
resulting construct used to transform E. colt strain NM522. Recombinant Hisb-
1CK1 was
purified using Ni-NTA agarose resin (QIAGEN) according to manufacturer's
instructions
except that the final washing was with buffer D, pH 6.0 and the protein was
eluted with 2 ml
buffer D, pH 4Ø The eluent was renatured by diluting with lOX volume of a
renaturing
buffer (10 mM Tris pH 7.5, 500 mM NaCI, 400 mM arginine HCI, 20 uM MgCls, 20
pM
ZnAc and 0.1 % Tween 20) and dialysed in the same buffer (500 ml per 2 ml
sample) at 4°C
overnight. The protein samples may be concentrated with Filtron l OK
concentrators. The
sample was then dialyzed at 4°C for 3 h against 1000 volumes of the
kinase assay buffer (see
below) containing 0.4 mM DTT and each (0.4 p.g/ml) of the protease inhibitors
soybean
to trypsin inhibitor, antipain and apotinin. The protein was stored at -
80°C.
Kinase assays may be useful in some aspects of the invention, for example to
assay
the function of CDK inhibitors on particular kinases. For example, kinases may
be purified
from A. thaliana tissues or cultured B. napes cells.plant materials may be
homogenized in 2
ml per gram tissue of ice cold extraction buffer consisting of 25 mM Tris pH
8.0, 100 mM
NaCI, 10 mM DTT, 5 mM NaF, 1 mM Na~VO,, 1 mM [3-glycerophosphate, 2.5 mM EDTA,
400 ~g/ml AEBSF [4-(2-aminoethyl)-enzensulfonyl fluoride], 1 pg/ml leupeptin
and 1 pg/ml
pepstatin. The homogenate was centrifuged at 12,OOOg at 4°C for 30 min.
The supernatants
may be used to purify Cdc2-like protein kinases using p13'"''-conjugated
agarose beads
(Oncogene Sciences). The required amount of supernatant (150 pg protein for
each reaction)
was added to the beads and tumbled at 4°C for 2 h. The beads may be
washed twice in a
washing buffer consisting of 50 mM Tris pH 7.4, 250 mM NaCI, 0.1 % NP-40, 2.5
mM
EDTA, 1 mM DTT and inhibitor cocktail of (in final concentrations) 10 ug/ml
apotinin, 10
pg/ml antipain, 10 pg/ml soybean trypsin inhibitor, 10 mM [3-glycerophosphate,
1 mM NaF
and 0.2 mM Na,VO,. Beads may then be washed twice in the kinase assay buffer
(50 mM
Tris pH 7.4, 10 mM MgCI, 2 mM EGTA, 2 mM DTT and the inhibitor cocktail). For
inhibition assays, the recombinant protein was added to the reactions and
incubated (tumbling
slowly) for 1.5 h at 4°C. The kinase reaction was initiated by adding 1
pg/ul histone H1
(Sigma), 25 pM ATP and 0.05 pCi/ul'~P-Y-ATP (final concentrations), and
stopped after 20
min incubation by adding the sample buffer. Denatured supernatant was resolved
by SDS-
3o PAGE.
16
CA 02272608 1999-06-08
RNA isolation and northern blotting analysis may be useful in various
embodiments
of the invention. For example, to analyze ICKI expression during plant
development, various
tissues may be taken from Arabidopsis plants. To analyse the effects of ABA
and low
temperature, seedlings may be treated as described (Wang et al., 1995).
Briefly, seedlings (12
days) grown in pots may be cleared of soil with water, then floated in 0.1 X
strength MS
medium without sucrose and hormones. Low temperature treatment was at
5°C for 24 h.
ABA treatment was carried out in a solution containing 50 IrM ABA. Seedling
samples may
be removed after various treatment times. Total RNA was isolated using TRIzoI.
reagent
(GIBCO BRL). For northern analysis, the indicated amount of RNA was
fractionated in a
to 1.2% agarose gel and transferred onto Hybond-N+ nylon membrane (Amersham).
The RNA
was crosslinked to the membrane by W-light (Stratalinker, Stratagene) and
hybridized with
'~P-labeled probes. The membranes may be wrapped and used to expose Hyperfilm
MP
(Amersham) film. Membranes may be stripped by treating with a boiling solution
of O.1X
SSC and 0.1 % SDS for S min. Quantitation of hybridized signal was performed
using
15 Molecular Dynamics PhosphorImager and the accompanying software.
In Vitro binding assays may be useful in various aspects of the invention, for
example
to assay the interaction of a CDK inhibitor, or fragments of a CDK inhibitor,
and a particular
kinase. As an example of such an approach, 'SS-Met labeled Cdc2a, CycD3 and
ATMPK2
proteins may be expressed from a T7 promoter construct using an in vitro
coupled rabbit
20 reticulocyte transcription/translation system ('TNT', Promega). Ni'-NTA
beads (Qiagen) may
be equilibrated and blocked in NETN buffer lacking EDTA (NTN) (Bai et al.,
1996), and
supplemented with 2 mglml BSA. Equilibrated beads may be incubated with His6-
ICK1 (5 pg
for each 10 Irl beads) in 1 ml of NTN buffer for 2 h with tumbling at
10°C followed by
washing with 2 X 1 ml NTN buffer. Binding experiments may be carried out in a
total
25 volume of 100 lrl NTN containing 10 pl beads, plus S ul'sS-Met labeled
protein. The binding
reaction was incubated at 10°C for 2 h, followed by washing with 3 X
0.5 ml NTN buffer.
Washed beads may be eluted with 10 ul SDS-containing denaturing buffer at
100°C for 5
min, and bound'sS-Met labeled proteins analyzed by SDS-PAGE. Gels may be
imbibed with
a fluorography enhancer ('Amplify', Amersham) prior to drying and exposure to
X-ray film.
30 Deletion constructs may be useful for domain mapping to determine the
functional
17
CA 02272608 1999-06-08
domains of a CDK inhibitor. For example, N-terminal deletion constructs of
ICK1 were made
using cDNAs with deletions of various lengths from the N-terminal end. The C-
terminal
deletion constructs were prepared by PCR using Pju DNA polymerase with
sequence-specific
primers and the resulting DNA fragments were cloned into the yeast two-hybrid
vector
pBI771. The deletion clones may be verified by DNA sequencing. The constructs
may be
used to transform a suitable yeast strain. In one such example, yeast strain
YPB2 harboring
either cdcla or CycD3 cloned in the BD- (binding domain) vector was
transformed with
deletion constructs. Interactions in the yeast two-hybrid system may then, for
example, be
analyzed by X-gal filter assay (Chevray and Nathans, 1992) and by liquid
culture assays for
to relative (3-galactosidase activity (for example using the modified
procedure of Reynolds and
Lundlad, 1994). Three or more independent transformants may be used for each
interaction.
Sequence Analyses: Sequence analyses, including determination of sequence
IS homology, may be performed using a variety of software, such as LASERGENE
(DNASTAR). Database searches may also use a variety of software tools, such as
the BLAST
program (NCBI).
Analysis ojCDK inhibitor cDNA Clones and Genomic Sequences: The yeast two-
hybrid system (Fields and Song, 1989; Kohalmi et al., 1997) may be used to
identify genes,
20 such as ICKI, that encode inhibitor proteins able to interact with the
plant cyclin dependent
kinases, such as Cdc2 kinase, for use in the present invention. For example,
among the 68
ICK Llnteractors of Cdc2 Kinase) clones identified using Cdc2a as the bait in
a yeast two
hybrid system (Wang et al., 1997), 55 represented various lengths of ICKI , 7
of ICK2 and 6
of ICK3. A contig sequence for homologous clones disclosed by the yeast two
hybrid assay
25 may be used, as was the contig sequence for ICKI cDNA (Wang et al., 1997),
to search
genomic databases, such as an Arabidopsis EST database, for sequences
homologous to those
identified by the two-hybrid screen. Two EST clones homologous to the ICKI
cDNA
sequence have been identified in this way. A clone designated 96D15T7
possessed an extra 5'
sequence to that of the contig assembled from the two-hybrid cDNA clones.
Specific PCR
3o primers may be synthesized and used to clone the genomic sequence spanning
the entire
18
CA 02272608 1999-06-08
coding region of a CDK inhibitor gene. For ICKI, three independent clones
harboring the
genomic sequence were identified in this way, sequenced and found to be
identical.
Alignment of ICKI genomic sequence with the ICKI cDNA sequence (GenBank
U94772,
Wang et al., 1997) reveals three introns. The genomic sequence in the exon
regions is
identical to the contig of cDNA clones except at nucleotide position 318,
which is a T instead
of a G as in the reported cDNA sequence (Wang et al., 1997; a majority of the
cDNA clones
had a G, while other clones had a T at this position). The existence of a T at
this position in
genomic DNA was verified by sequencing additional genomic clones. The longest
open
reading frame in the ICKI cDNA sequence predicts a polypeptide of 191 amino
acids (Wang
to et al., 1997). There is an in-frame translation STOP codon 12 nucleotides
upstream of the first
ATG. In addition, an in-frame translation termination codon was found 30
nucleotides down
stream of the predicted termination codon. Genomic southerns may be performed
to estimate
the copy number of a CDK inhibitor gene and to disclose closely-related genes.
For ICKI in
A. thaliana southern results indicate that ICK1 exists as a single copy gene:
while BamH I
I S and/or EcoR I digested genomic DNA gave a single band, Sal I digestion of
the DNA resulted
in two bands, as was predicted by the presence of an internal Sal I site in
the ICKI sequence.
Hybridization at lower stringency may be used to detect non-identical
homologous sequences.
CDK inhibitor in vitro assays: In vitro kinase assays may be used to
demonstrate that
a recombinant putative CDK inhibitor, such as ICK1 protein, is an effective
inhibitor of plant
2o Cdc2-like kinases. Plant CDK inhibitors may not inhibit CDK from mammalian
and yeast
cells (Wang et al., 1997). For example, recombinant ICK1 is effective in vitro
in inhibiting
the histone H1 kinase activity of pl3f"''-associated kinases from cultwed
cells of
heterologous Brassica napes. In addition, it also inhibits the activity of
such kinases from A.
thaliana seedlings, leavies and floral tissues in vitro.
Expression oJCDK inhibitors: The expression of a CDK inhibitor in particular
plant
tissues may be assayed to determine, for example, whether that CDK inhibitor
will have
utility as a division or growth modulator when expressed in such tissues. For
example, the
expression of ICKI was analyzed in several different plant tissues. In
general, the transcript
abundance of ICKI was relatively low and showed low degrees of variation
compared with
the housekeeping genes such as TUA4 (a tubulin-a gene) and GAPDH
(glyceraldehyde
19
CA 02272608 1999-06-08
phosphate dehydrogenase) of A. thaliana. When leaves from plants of different
ages were
compared, the ICKI level in sample LS (for leaves of 5-week plants) was
slightly higher. To
verify the functional role of a putative CDK inhibitor in such tissues, the
CDK activity may
also be assayed.
Assaying ojCDK inhibitor activity: Putative CDK inhibitors may be assayed for
suitable CDK inhibitor activity for use in the methods of the invention by a
variety of tests.
For example, induction of expression of the putative CDK inhibitor gene by
abscisic acid
(ABA) , a phytohormone known to inhibit plant growth (Evans, 1984), and at low
temperatures. For example, expression of the putative CDK inhibitor gene, such
as ICKI, in
seedlings, such as A. thaliana seedlings, may be analyzed in response to
treatments with
ABA. For ICKI, data from an example assay showed that after 24 h, ABA and low
temperature treatments increased ICKI transcript levels to about 3 times that
of the control
(no ABA and at 22°C) in 2-week seedlings. The expression of the
putative CDK inhibitor
to gene may be quantified. For ICK1, a correlation coefficient was obtained
for the relationship
of cdc2a level, ICKI level and cdc2alICKI ratio with the Cdc2-like kinase
activity. The level
of cdcla expressioci was correlated with the level of Cdc2-like histone H1
kinase activity.
The level of ICKI expression exhibited a weak negative correlation with kinase
activity. The
correlation coefficient for the cdclalICKI ratio with Cdc2-like kinase
activity was similar to
that for cdc2a with Cdc2-like kinase activity. Such results are consistent
with CDK (in this
example Cdc2 kinase) inhibitor activity in plant cells.
Direct Interaction oJICKI with Both Cdc2a and CycD3: CDK inhibitors for use in
vanous aspects of the invention may be identified using a yeast two hybrid
screening protocol
with a variety of bait fusion protein sequences. For example, ICKI was
independently cloned
in a screen using A. thaliana CycD3 as the bait, indicating that ICKI
interacts with CycD3 in
the two hybrid assay. To provide evidence confirming the interaction of a CDK
inhibitor with
a target protein of interest, further binding assays may be conducted. For
example, to test the
interactions of ICK1, cdcla and CycD3 cDNAs were transcribed and translated in
an in vitro
system. In vitro expressed Cdc2a and CycD3 proteins were incubated with
recombinant Hisb-
ICK1 protein expressed in E. coli. Cdc2a and CycD3 bound to Ni-NTA beads only
after they
were incubated with recombinant ICKI. The amount of CycD3 bound to recombinant
ICK1
2o
CA 02272608 1999-06-08
protein was more than the control protein ATMPK2 which showed little binding
despite the
much higher input used. These results demonstrate that ICK1 is able to
interact directly with
both Cdc2a and CycD3. Similar assays may be used to identify CDK inhibitors
capable of
interacting with other cellular targets.
Mapping the Domains for ICKI Interaction with Cdc2a and CycD3: The regions of
a
CDK inhibitor that are functionally involved in interactions with other
proteins may be
mapped by deletion mapping using a variety of techniques, such as the yeast
two hybrid
systems and variations thereof. Such in vitro assay results may be verified by
in vivo tests,
since the persistence of interactions in the two hybrid system may be affected
by possible
alterations in functionality of plant proteins expressed in yeast. As an
example of an in vitro
assay, to determine the functional significance of the C-terminal domain and
other regions of
ICKI, three N-terminal and three C-terminal deletion mutants were assessed for
their
interactions with Cdc2a and CycD3 in the two-hybrid system. Overall, ~i-
galactosidase
marker gene activation in the two hybrid system was stronger for the
interaction of all ICK 1
constructs with CycD3 compared to Cdc2a, indicative of a stronger or more
persistent
interaction between ICK1 and CycD3 in the two-hybrid system. Major shifts in
~3-
galactosidase activity were observed when amino acid regions 3-72, 109-153 and
176-191
were deleted. An increase in activity was observed upon deletion of amino
acids 3-72. In
pairwise comparison, the deletion of amino acid regions 3-72, 73-108, 163-175
or 153-162
had comparable effects on the interactions of ICK1 with Cdc2a versus CycD3, as
reflected by
the marker gene expression, while the deletions of amino acid regions 109-153
and 176-191
had clearly differential effects. The most significant reduction in (3-
galactosidase activity for
the interaction of ICK1 with CycD3 resulted from the deletion of amino acids
109-153,
whereas the deletion of amino acids 176-191 had a more detrimental effect on
the interaction
with Cdc2a. The fimctional importance of a portion of a CDK inhibitor may also
be assayed
by analyzing the portion of cDNA required for the recovery of clones by each
bait construct
in the two hybrid system. For example, the region spanning amino acids 109-153
of ICK1 for
its interaction with CycD3 was supported by the analysis of the minimum cDNA
length
required for the recovery of clones by each bait construct. With CycD3 as the
bait, the
shortest ICKI cDNA was N-terminal deleted for amino acids 1-129, while with
Cdc2a, seven
21
CA 02272608 1999-06-08
clones with further deletions extending to amino acid 154 were also isolated.
Thus, deletions
extending beyond amino acid 130 rendered these clones unrecoverable by the two-
hybrid
screening using CycD3 as the bait. Taken together, the results indicate that,
while the C-
terminal domain (containing the consensus sequence with p27K'°') is
most important for the
interaction with Cdc2a, the amino acid region 109-153 perhaps with the C-
terminal domain is
important for the interaction with CycD3.
Arabidopsis transformation with ICKI constructs: A wide variety of
transformation
techniques may be used in accordance with the invention to introduce CDK
inhibitor genes
into plants. In one aspect, the invention provides methods of assaying
heterologous CDK
inhibitor function in a model plant, such as Arabidopsis. For such assays, in
one embodiment,
transformation may be caned out by infiltration. For example, seeds (T1
generation) collected
from infiltrated Arabidopsis plants may be surface-sterilized and placed onto
MS medium
containing 50 pg/ml kanainycin. The antibiotic timentin may also be included
in the medium
to prevent any bacterial growth, which could occur due to carrier-over from
the infiltration.
The vast majority of germinating seedlings will not be transformed, and will
became pale and
to eventually stop growing, transformed seedlings will be green and display
normal growth due
to the presence of the selectable marker gene. After 4-5 weeks in the
selection medium,
transformants may be transferred to soil in pots. In the exemplary embodiment,
the presence
of the DNA insertion was confirmed by extracting the genomic DNA and then
using it for
PCR amplification. In one example, while the non-transformed wild-type plant
gave a
negative signal, all five plants selected for their resistance to kanamycin
were positive for
transforming DNA.
EJ~'ect ofAP3-ICKI chimericgene on petal and stamen development: Various
aspects
of the invention may be used to obtain a wide variety of phenotypic variations
in plant
morphology or other characteristics. For example, transformed A. thaliana
plants carrying the
AP3-ICKI construct displayed a range of phenotypes with regard to petal and
stamen
morphology (Table 1). Such variation may be due to the insertion in
alternative embodiments
of the invention of the CDK inhibitor gene into different sites of the plant
genome. In the
example of modified petal development using ICK1, the plants may be classed
into three
groups: (1) no visible petals, (2) visible petals but reduced size and (3)
visible petals with no
12
CA 02272608 1999-06-08
apparent difference to those of non-transformed plants (Table 1 ). ~in terms
of fertility, four out
of five plants were sterile. These results demonstrate that tissue-specific
expression of ICKI
may be used to produce plants without petals and/or with male sterility. In
some
embodiments, the transgenic plants with male sterility may set seeds after
pollination, using
pollen from non-transformed plants, indicating that the female reproduction
system is
unaffected in these male sterile plants.
Table 1. Summary of phenotypes ofA. thaliana plants transformed with AP3-ICKI
chimeric gene.
Transformant Petal Sterility Seed setting with
WT
pollen
#1 Reduced size or Sterile yes
number
#2 - No visible petals Sterile yes
~
#3 w Normal Fertile self fertile
#4 Reduced size or Sterile yes
number
#5 Nearly normal Sterile yes
Co-Inheritance ojthe inserted gene and phenotype: T2 plants may be studied to
determine the segregation of the inserted gene and also to verify whether the
particular
phenotype is co-inherited with the inserted gene. For example, T2 seeds of
ICKI
transformants were sterilized and placed onto the selective medium. In one
such assay, T2
seeds of one transformant (#2) showed 1:1 ratio of segregation between
resistant (99) versus
non-resistant (102) seedlings. As transformant #2 was male sterile, the T2
seeds were
obtained by crosses using wild type pollen. This ratio indicates that there is
one insertion in
the genome of this transformant. As expected, T2 plants displayed the same
phenotype as the
corresponding Tl plants.
Transgenic Brassica: In accordance with alternative embodiments of the
invention,
CDK inhibitors may be used to modify a wide variety of plant species. The
division and
growth inhibiting activity of CDK inhibitors in Brassica is exemplified by the
following
13
CA 02272608 1999-06-08
results. The ICK1 cDNA coding region was fused in sense or anti-sense
orientation to the
cauliflower mosaic virus 35S promoter (a constitutive promoter). These
constructs were
tested in Brassica napus transformations. From two independent experiments,
over 60
transformants were regenerated from the anti-sense construct while none was
obtained from
the sense construct, indicating that constitutive expression of the ICK1 CDK
inhibitor
terminated plant growth at a very early stage. [Were there any phenotype
changes in the
anti-sense plants?]
Interaction oJICKI with other proteins: CDK inhibitors may be used in various
aspects of the invention to interact with a variety of regulatory components,
such as other cell
cycle proteins. For example, in some embodiments, it may be desirable to
target a known
regulatory moity with a CDK inhibitor. Accordingly, in one aspect of the
invention, an assay
is provided to determine if a CDK inhibitor interacts with a known protein.
Such interactions
may be analyzed by the yeast two-hybrid assay. For example, the full-length
cDNA of the
gene to be analyzed may be cloned in a GAL4-binding domain vector (Koholmi et
al., 1997)
using PCR and gene specific primers with flanking restriction sites. Such
constructs may be
used to transform the yeast carrying the CDK inhibitor of interest, such as
ICK1 in a GAIA-
activation domain vector. Using this approach, for example, the interactions
of ICK1 with a
number of cell cycle-related genes from A. thaliana were examined in
accordance with the
invention (Table 2). In these examples, the yeast two-hybrid assay results
indicate that in
particular embodiments of the invention, ICK1 protein may interact with Cdc2a
but not with
Cdc2b. Similarly, ICKI may interact with D-class cyclins, CycDl, CycD2 and
CycD3, while
not interacting with A/B-class mitotic cyclins, CycA2, CycBl and CycB2 (Table
2). The
yeast two-hybrid assay results also indicate that ICK1 may not interact in
some embodiments
with PCNA, also a cell cycle protein, and ATMAP2, a kinase sharing some
similarity with
Cdc2 kinase. Results such as these, indicating that ICK1 interacts with the G1
cyclins and
Cdc2a but not the mitotic cyclins and Cdc2b, indicate that a CDK inhibitor,
such as ICKI,
may in some embodiments be used in the regulation of cell cycle initiation
during plant
growth and differentiation.
Table 2. Analyses of ICKl interactions with other proteins in the yeast two-
hybrid
24
CA 02272608 1999-06-08
system
Gene Group Gene in DB- Old Name Interaction
Examined Vector with ICK1
Filter
assay~'~
Quantification~
Control vector alone - 0
Cdc2 kinase cdc2a +++ 2.65
cdc2b - 0
cyclin cycD 1;1 cyclin b +++ 3.13
1
cycD2;1 cyclin 82 +~-++ 14.80
cycD3; l cyclin b3 ++-+-~+ 22.70
cycA2;2 cyc3bAt - 0.03
cycB 1;1 cyc 1 At - 0.06
cycB2;2 cyc2bAt - 0.05
PCNA PCNAAt - 0
MAP kinase ATMAP2 - 0
Other plant CDK inhibitors: Other plant CDK inhibitors and CDK inhibitor genes
sharing fimctional and sequence similarity with ICK1 may be identified using
an approach
similar to the approach used to isolate ICKI, based on their interactions with
either
Arabidospis Cdc2a or a D-class cyclin (cyclin b3 or cyclin S2). The CDK
inhibitors
identified in screens using Cdc2a are designated herein as ICKs (for
Interactors of Cdc2
Kinase) and those identified in screens using cyclins are designated ICNs (for
Interactors of
Cyclin). Some CDK inhibitors may be isolated independently from both types of
screens. The
sequences of ICK2, ICN4, ICN6, and ICN7 are shown in Figs 1 through 4. These
genes share
at least two functional properties with ICK1: First, all of these genes encode
proteins able to
CA 02272608 1999-06-08
interact with either Cdc2a or a D-class cyclin or both. Such interactions may
enable them to
regulate the activity of plant CDKs in alternative embodiments of the
invention. Second,
these ICK/ICN proteins all share some sequence similarity in the region of
ICKI that is
functionally important in some embodiments for its interaction with Cdc2a and
cyclin b3
(discussed above in the section on "domains for ICK1 interactions with Cdc2a
and cyclin
b3"). These homologous genes or proteins may be used in some embodiments, in a
manner
similar to ICK1, to modulate plant growth and development. One or more such
genes or
proteins may be used in some embodiments alone or in combination to provide
temporal and
spatial regulation of cell cycle initiation and progressing during plant
development in
accordance with this invention.
Although various embodiments of the invention are disclosed herein, many
adaptations and modifications may be made within the scope of the invention in
accordance
with the common general: knowledge of those skilled in this art. Such
modifications include
the substitution of known equivalents for any aspect of the invention in order
to achieve the
same result in substantially the same way. For example, additional plant
cyclin-dependent
kinase inhibitor genes useful in regulating morphogenesis may be disclosed
using the
screening methods of the invention, such genes may share functional homology
with ICKI,
while being sequence-divergent from ICKI. The following examples are
illustrative only of
various aspects or embodiments of the invention.
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CA 02272608 1999-06-08
A set of genes and their predicted gene products have been identified in
Arabidopsis thaliana on
the basis of protein:protein interactions using the 2-hybrid system. The
primary structure of these
genes suggest they are components of the cell cycle regulatory machinery in
this model plant
species. These genes bear selected primary sequence similarity to genes and
deduced gene
products from other systems (notably yeast and humans) which are known to have
a role in the
regulation of cell division in these organisms.
A partial primary nucleic acid sequence, and deduced polypeptide sequence of
the genes that have
been discovered are given below:
Nucleotide sequence of ICR2 cDNA
ick2.seq Length: 755 June 7, 1999 13:57 Type: N Check: 1650 ..
1 GTGGAATCTA GGATAATTCT GTCTCCGTGT GTACAGGCGA CGAATCGCGG
51 TGGAATTGTG GCGAGAAATT CAGCAGGAGC GTCGGAGACG AGTGTTGTTA
101 TAGTACGACG GCGAGATTCT CCTCCGGTTG AAGAACAGTG TCAAATCGAA
151 GAAGAAGATT CGTCGGTTTC GTGTTGTTCT ACATCGGAAG AGAAATCGAA
201 ACGGAGAATC GAATTTGTAG ATCTTGAGGA AAATAACGGT GACGATCGTG
251 AAACAGAAAC GTCGTGGATT TACGATGATT TGAATAAGAG TGAGGAATCG
301 ATGAACATGG ATTCTTCTTC GGTGGCTGTT GAAGATGTAG AGTCTCGCCG
351 CAGGTTAAGG AAGAGTCTCC ATGAGACTGT GAAGGAAGCT GAGTTAGAAG
401 ACTTTTTTCA GGTGGCGGAG AAAGATCTTC GGAATAAGTT GTTGGAATGT
451 TCTATGAAGT ATAACTTCGA TTTCGAGAAA GATGAGCCAC TTGGTGGAGG
501 AAGATACGAG TGGGTTAAAT TGAATCCATG AAGAAGACGA TGATGATAAT
551 GATGATCATT GTTTTCACCA AAGTACTTAT TATTTCTCTT CTGTAATAAT
601 CTTTGCTTTG ATTTTTCTTT TAACAAAATC CAAATGTAGA TATCTTTCTC
651 TCGAATAATC AATAACATGT AATTCAACTT TTGTTTGTAC TTCCTTGAGG
701 TAATTAATTA GATTCGTGTT TTTCTCGATT AATAAACTAT AAGTTTATAA
751 CTAAA
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CA 02272608 1999-06-08
Nucleotide sequence of ICN2 cDNA
nucleotide.seq Length: 824 June 7, 1999 13:57 Type: N Check: 7926 ..
1 aaaaaaaagc agagagagag agcacacaaa aatccaagag agaaaaaaat
51 gagcgagaga aagcgagagc ttgcagaaga agcttcaagc acaagcttct
101 caccactgaa gaaaacgaag cttaatgatt cttctgattc atcaccggac
151 tctcatgacg tcatcgtctt cgcggtttca tcttcttccg ttgcttcgtc
201 ggcggcttta gcgtctgatg aatgttccgt taccatcggt ggagaagaaa
251 gtgatcagtc ctcgagtatc agctccggtt gtttcaccag tgaatcgaaa
301 gaaatcgcga agaacagttc gtcgtttggt gtagatctgg aggatcatca
351 aatcgaaacc gaaaccgaaa cctcaacatt catcaccagc aatttcagaa
401 aagagacgag tccagtgagt gagggtttgg gagaaacgac aacagaaatg
451 gaatcatcat cggcaacgaa gagaaaacaa ccgggggtga ggaagactcc
501 aacggcggcg gagattgagg atttgttctc ggagctagag agtcaagacg
551 ataagaagaa gcaattcata gaaaagtaca acttcgatat tgtcaatgac
601 gaaccgcttg aaggtcgcta caagtgggat cgactttaag ccatcaaaaa
651 gcaaatacca tccatgaaga agacaaaaga aaaataggtt ttgtttttcg
701 tggttaacat ttccacttgt acagctctag tctatttctc tttaaaaacc
751 tatgttacta gttcgtacaa aacaaaacaa aaaacacgac ctttataatg
801 aaatttcgga tcttggctac taaa
Nucleotide sequence of ICN6 cDNA
nucleotide.seq Length: 642 June 7, 1999 13:59 Type: N Check: 8341 ..
1 ctctctccag agaaaactat aatgagcttg agagaaatga gcgaaacaaa
51 acccaagaga gattctgagt acgaaggatc aaacatcaag aggatgagac
101 tcgatgatga tgatgacgtt ttacgctcac cgacgagaac tctttcttct
151 tcttcctctt cttctctggc ttactcggtt tcagattccg gaggtttctg
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CA 02272608 1999-06-08
201 ctccgtcgcg ttatctgaag aagaagacga tcatctaagc tcaagcatca
251 gctctggttg ttccagcagc gaaactaacg aaatccctac tcgtcttcca
301 ttttcagatc tggaggctca tgaaatctcc gaaaccgaaa tctcaacgtt
351 actcaccaac aatttcagga aacagggaat ttcatcaagc gagaatctgg
401 gagaaacagc agaaatggac tccccgacga cggagatgag agatcagaga
451 aagacggaga agaagaagaa gatggaaaaa tcaccgacgc aggcagagct
501 tgatgacttt ttctcggcgg cggagagata cgaacagaaa cgattcacag
551 aaaagtacaa ctacgacatc gtcaatgata cgccgcttga aggtcggtac
601 cagtgggtta gtctgaaacc ttagaagcca tggaagaaca as
Nucleotide sequence of ICN7 cDNA
nucleotide.seq Length: 533 June 7, 1999 14:00 Type: N Check: 1632 ..
1 attaaagagt ctggttccag gtctcgcgtt gactcggtta actcggctcc
51 tgtagctcag agctctaatg aagatgaatg ttttgacaat ttcgtgagtg
101 tccaagtttc ttgtggtgaa aacagtctcg gttttgaatc aagacacagc
151 acaagggaga gcacgccttg taactttgtt gacgatatgg agatcatggt
201 tacaccaggg tctagcacga ggtcgatgtg cagagcaacc aaagactaca
251 caagggaaca agataacgtg atcccgacca ctagtgaaat ggaggagttc
301 tttgcatatg cagagcagca gcaacagagg ctattcatgg agaagtacaa
351 cttcgacatt gtgaatgata tccccctcag cggacgttac gaatgggtgc
401 aagtcaaacc atgaagttca aaaggaaaca gctccaaaag acatggtgtg
451 aagttagaga attgtgatgg actttaacag aactaaccaa acatcagaaa
501 tcgtgttaat ccttaagtta ataatgtggg tta
(a) What is the problem?
The problem concerns the discovery of fundamental approaches for intervening
and controlling
plant development using molecular biotechnology. These developmental processes
are known to
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CA 02272608 1999-06-08
involve the coordinated regulation of two fundamental cell biological
processes: cell division, and
the acquisition of cell fate (cell-type).
(b) How may it be accomplished according to the present knowledge?
The directed regulation of cell division could alter the pattern of plants, by
altering the cell-cycle
'decision-making' process during the normal course of development. Such
alternations could
include alteration of the extent, plane or developmental timing of cell
division events, leading to
the development of altered morphologies of plant tissues and organs.
(c) Limitations or drawbacks of present apparatus, product or process.
At this point in time, a predictive capability of the outcome of altering the
expression of specific
cell cycle components cannot be offered. However; the discovery of new genes
and gene
products implicated in the regulation of cell division offers a knowledge base
from which
empirical observations can be made, and predictive models developed.
(d) What is your proposal?
We propose to protect and exploit the genes described here for potential
future applications in the
alteration of plant cell cycle control, with consequences for altering
development.
(e) What is thought to be novel in your proposal?
To our knowledge, these genes have not been previously described at a
functional level (eg.
protein:protein interaction, as described here) which would implicate them in
cell cycle control.
(f) Has the apparatus, product or process been made or tested?
The genes described have been demonstrated to interact at the protein level
with selected
cyclinn and/or cyclin-dependent kinase proteins from Arabidopsis.
Reference List
1. Burssens, S., van Montagu, M., and Inze, D. (1998) The cell cycle in
Arabidopsis.
Plant Physiol.Biochem. 36, 9-19.
2. De Veylder, L., Segers, G., Glab, N., van Montagu, M., and Inze, D. (1997)
Identification of proteins interacting with the Arabidopsis Cdc2aAt protein.
J. Exp. Bot. 48, 2113-2114.
CA 02272608 1999-06-08
Ferreira, P., Hemerly, A., Engler, J.D., Bergounioux, C., Burssens, S., van
Montagu,
M., Engler, G., and Inze, D. ( 1994) Three discrete classes of Arabidopsis
cyclins
are expressed during different intervals of the cell cycle.
Proc.Natl.Acad.Sci.USA
91, 11313-11317.
4. Himmelbach, A., Item M., and Grill, E. ( 1998) Signalling of abscisic acid
to regulate
plant growth. Philos.Trans.R.Soc.Lond.~Biol.J 353, 1439-1444.
S. Liu, C.M. and Meinke, D.W. (1998) The titan mutants of Arabidopsis are
disrupted in
mitosis and cell cycle control during seed development. Plant J. 16, 21-31.
6. Niebel, A., Engler, J.D., Hemerly, A., Ferreira, P., Inze, D., van Montagu,
M., and
Gheysen, G. (1996) Induction of cdc2a and cyclAt expression in Arabidopsis
thaliana during early phases of nematode-induced feeding cell formation. Plant
J.
10, 1037-1043.
7. Rohde, A., van Montagu, M., Inze, D., and Boerjan, W. (1997) Factors
regulating the
expression of cell cycle genes in individual buds of Populus. Planta 201, 43-
52.
8. Segers, G., Gadisseur, L, Bergounioux, C., Engler, J.D., Jacqmard, A., van
Montagu, M., and Inze, D. ( 1996) The Arabidopsis cyclin-dependent kinase gene
cdc2bAt is preferentially expressed during S and GZ phases of the cell cycle.
Plant
J. 10, 601-612.
9. Wang, H., Qi, Q.G., Schorr, P., Cutler, A.J., Crosby, W.L., and Fowke, L.C.
(1998)
ICK1, a cyclin-dependent protein kinase inhibitor from Arabidopsis thaliana
interacts
with both Cdc2a and CycD3, and its expression is induced by abscisic acid.
Plant J.
15, 501-510.
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CA 02272608 1999-06-08
Table 3
ConstructNo. of Green Shoots Seed
Name Explantsrecovered recovered
K1a 500 0 0
K1a 500 0 p
K2a 500 5
K2a 500 ca.60 g
E1a 650 4 2 _
E2a 600 8 6
Table 3 provides results from transgenic Arabidopsis plants
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