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.

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.