Method and means for modulating plant cell cycle proteins...

Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters plant part growth

Reexamination Certificate

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C800S298000, C435S320100, C435S419000, C435S468000, C536S023600

Reexamination Certificate

active

06465718

ABSTRACT:

The present invention relates to a novel cell cycle gene in plants and to a method for controlling or altering growth characteristics of a plant and/or a plant cell comprising introduction and/or expression of one or more cell cycle regulatory protein functional in a plant or parts thereof and/or one or more nucleic acid sequence encoding such proteins. Optionally, said sequences are placed under the control of a foreign control sequence in said plant and/or plant cell.
Also provided in the present invention is a method for modulating endoreduplication in plants, plant cells or parts thereof, by genetic engineering techniques. In a preferred embodiment endoreduplication in plants, plant cells or parts thereof is modulated by modifying the plant cell cycle.
Cell division is fundamental for growth in humans, animals and plants. Prior to dividing in two daughter cells, the mother cell needs to replicate its DNA. The cell cycle is traditionally divided into 4 distinct phases:
G1: the gap between mitosis and the onset of DNA synthesis;
S: the phase of DNA synthesis;
G2: the gap between S and mitosis.
M: mitosis, the process of nuclear division leading up to the actual cell division.
The distinction of these 4 phases provides a convenient way of dividing the interval between successive divisions. Although they have served a useful purpose, a recent flurry of experimental results, much of it as a consequence of cancer research, has resulted in a more intricate picture of the cell cycle's “four seasons” (K. Nasmyth, Science 274, 1643-1645, 1996; P. Nurse, Nature, 344, 503-508, 1990)
The underlying mechanism controlling the cell cycle control system has only recently been studied in greater detail. In all eukaryotic systems, including plants, this control mechanism is based on two key families of proteins which regulate the essential process of cell division, namely protein kinases (cyclin dependent kinases or CDKs) and their activating associated subunits, called cyclins. The activity of these protein complexes is switched on and off at specific points of the cell cycle. Particular CDK-cyclin complexes activated at the G1/S transition trigger the start of DNA replication. Different CDK-cyclin complexes are activated at the G2/M transition and induce mitosis leading to cell division.
Each of the CDK-cyclin complexes execute their regulatory role via modulating different sets of multiple target proteins. Furthermore, the large variety of developmental and environmental signals affecting cell division all converge on the regulation of CDK activity. CDKs can therefore be seen as the central engine driving cell division.
In animal systems and in yeast, knowledge about cell cycle regulations is now quite advanced. The activity of CDK-cyclin complexes is regulated at five levels: (i) transcription of the CDK and cyclin genes; (ii) association of specific CDK's with their specific cyclin partner; (iii) phosphorylation/dephosphorylation of the CDK and cyclins; (iv) interaction with other regulatory proteins such as SUC1/CKS1 homologues and cell cycle kinase inhibitors (CKI); and (v) cell cycle phase-dependent destruction of the cyclins and CKIs.
The study of cell cycle regulation in plants has lagged behind that in animals and yeast. Some basic mechanisms of cell cycle control appear to be conserved among eukaryotes, including plants. Plants were shown to also possess CDK's, cyclins and CKI's. However plants have unique developmental features which are reflected in specific characteristics of the cell cycle control. These include for instance the absence of cell migration, the formation of organs throughout the entire lifespan from specialized regions called meristems, the formation of a cell wall and the capacity of non-dividing cells to re-enter the cell cycle. Another specific feature is that many plant cells, in particular those involved in storage (e.g. endosperm), are polyploid due to rounds of DNA synthesis without mitosis. This so-called endoreduplication is intimately related with cell cycle control.
Due to these fundamental differences, multiple components of the cell cycle of plants are unique compared to their yeast and animal counterparts. For example, plants contain a unique class of CDKs, such as CDC2b in Arabidopsis, which are both structurally and functionally different from animal and yeast CDKs.
The further elucidation of cell cycle regulation in plants and its differences and similarities with other eukaryotic systems is a major research challenge. Strictly for the case of comparison, some key elements about yeast and animal systems are described below in more detail.
As already mentioned above, the control of cell cycle progression in eukaryotes is mainly exerted at two transition points: one in late G
1
, before DNA synthesis, and one at the G
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 utilise one CDK gene for the regulation of their cell cycle. The kinase activity of their gene products p34
CDC2
and p34
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
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
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.
The SUC1/CKS1 proteins represent another class of components of CDK complexes. The SUC1 and CKS1 genes were originally identified in
Sch. pombe
and
S.cerevisiae,
respectively as suppressors of certain temperature-sensitive CDC2/CDC28 alleles. Mutant p34
CDC2
proteins suppressible by SUC1 overexpression were shown to have a reduced affinity for the SUC1 protein. Homologues of SUC1/CKS1 have since then been identified in a wide range of organisms, including human, Drosophila and Xenopus. The conserved interaction between SUC1/CKS1 proteins with CDKs allows purification of homologous CDKs from other species using affinity chromatography.
More than one decade after their initial discovery, the function of the SUC1/CKS1 genes is still not resolved. In yeasts, both SUC1 and CKS1 are essential genes, as was demonstrated by gene disruption. Cells deleted for SUC1 show mitotic spindles of varying lengths and condensed chromosomes, typical for a late mitotic arrest. The presence of high cyclin levels suggests that this arrest is attributed to the inability to destroy the mitotic cyclins, which is a prerequisite to leave M phase. Mitotic cyclins are normally destroyed by the ubiquitin-dependent proteosomal pathway. An essential component in this destruction pathway is a multiprotein com

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