WO2002053589A2

WO2002053589A2 - Nucleic acid molecules encoding dim interactors and uses therefor

Info

Publication number: WO2002053589A2
Application number: PCT/EP2002/000073
Authority: WO
Inventors: Lieven De Veylder; Veronique Katelijne Cecile Kristien Boudolf; Dirk Inze; Valérie Marie-Noëlle FRANKARD; Franky Terras
Original assignee: Cropdesign N.V.
Priority date: 2001-01-05
Filing date: 2002-01-07
Publication date: 2002-07-11
Also published as: WO2002053589A3

Abstract

The invention provides isolated nucleic acids molecules, designated DIM1 interacting molecules (DIMIC) molecules, which encode novel cell cycle associated polypeptides. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing DIMIC nucleic acid molecules, host cells into which the expression vectors have been introduced, and transgenic plants in which a DIMIC gene has been introduced or disrupted. The invention still further provides isolated DIMIC proteins, fusion proteins, antigenic peptides and anti-DIMIC antibodies. Agricultural, and screening methods utilizing molecules and compositions of the invention are also provided. The invention further provides methods for modulating overall growth and yield in a plant, enhancing stress tolerance of a plant, conferring enhanced resistance to pathogens to a plant, modulating pre-mRNA splicing in a plant cell, or modulating vesicle transport/processing in a plant cell, comprising introducing into the plant or plant cell a DIMIC molecule or DIMIC modulator, alone or in combination with a DIM1 molecule.

Description

NUCLEIC ACID MOLECULES ENCODING DIM INTERACTORS AND USES

THEREFOR

Background of the Invention When eukaryotic cells and, thus, also plant cells divide they go through a highly ordered sequence of events collectively termed as the "cell cycle." Briefly, DNA replication or synthesis (S) and mitotic segregation of the chromosomes (M) occur with intervening gap phases (G1 and G2) and the phases follow the sequence G1 -S-G2-M. Cell division is completed after cytokinesis, the last step of the M-phase. Cells that have exited the cell cycle and have become quiescent are said to be in the GO phase. Cells at the GO stage can be stimulated to re-enter the cell cycle at the G1 phase.

The transition between the different phases of the cell cycle is basically driven by the sequential activation/inactivation of a kinase, termed cyclin-dependent kinase or Cdk (e.g., Cdc2 in Schizosaccharomyces pombe and in plants, Cdc18 in Saccharomyces cerevisiae), by different agonists. Also required for kinase activation are proteins called cyclins which are important for targeting the kinase activity to a given subset of substrate(s). Other factors regulating Cdk activity include Cdk inhibitors (CKls or ICKs, Kips, Cips, Inks), Cdk activating kinase (CAK), Cdk phosphatase (Cdc25) and Cdk subunit (CKS) (Mironov et al. (1999) Plant Cell 11 , 509-522 and Reed (1996) Prog Cell Cycle Res 2, 15-27 for reviews).

The dim1+ gene was first isolated in the yeast S. pombe during a screen for second site mutations capable of reducing the restrictive temperature of the fission yeast mutant cdc2-D127N (Berry and Gould (1997) J Cell Biol 137, 1337-1354). When shifted to restrictive temperature, dim1-35 mutant cells arrest before entry into mitosis or proceed through mitosis in the absence of nuclear division, demonstrating an uncoupling of proper DNA segregation from other cell cycle events. Deletion of diml from the S. pombe genome produces a lethal G2 arrest phenotype. Lethality is rescued by overexpression of the mouse diml homologue, mdimh Likewise, deletion of the S. cerevisiae diml homologue, DIB1, is lethal. Both mdiml and dim1+ are capable of rescuing lethality in the dib1::H\S3 mutant. DIB1 was also termed CDH1 ('Saccharomyces cerevisiae DIM1 homologue) by Berry and Gould (1997), J Cell Biol 137, 1337-1354. This alternative terminology is, however, confusing because of the existence of another S. cerevisiae CDH1 gene which is a CDC20/Fizzy-homolog.

Although dim1-35 displays no striking genetic interactions with various other G2/M or mitotic mutants, dim 1-35 cells incubated at a restrictive temperature arrest with low histone H1 kinase activity. Moreover, dim 1-35 displays sensitivity to the microtubule destabilizing drug, thiabendazole (TBZ). Those results suggest that Diml p plays a fundamental, evolutionarily conserved role in the entry of cells into mitosis and in chromosome segregation during mitosis. Based on TBZ sensitivity and failed chromosome segregation in dim1-35, it can also be presumed that Dimlp may play a role in mitotic spindle formation and/or function.

To further understand dimlp function, Berry ef al. (1999), Mol Cell Biol 19, 2535- 2546, undertook a synthetic lethal screen with the temperature- sensitive dim1-35 mutant and isolated lid (for lethal in dim 1-35) mutants. One of the lid mutants is the temperature sensitive Hd1-6 mutant. At the restrictive temperature of 36°C, lid1-6 mutant cells arrest with a "cuf phenotype similar to that of cut4 and cut9 mutants, that are components of the anaphase promoting complex/cyclosome (APC/C; Tyers and Jorgensen (2000) Curr Opin Genet Dev 10, 54-64). The S. pombe genes cut4 and cutθ have known homologues in S. cerevisiae (apd and cdc16, respectively) and at least for cutθ, a metazoan homologue exists (APC6). An epitope tagged version of lidlp is a component of a multiprotein ~20S complex; the presence of lidlp in this complex depends upon the presence of a functional cutif. Lid1 p-myc coimmunoprecipitates with several other proteins, including the APC/C members cut9p and nuc2p, and the presence of cut9p in a 20S complex depends upon the activity of Iid1⁺. Further, Iid1⁺ function is required for the multi-ubiquitination of cut2p, an anaphase-promoting complex (APC/C) target. Thus, lidlp is a component of the S. pombe APC/C. In diml mutants, the abundance of lidlp and the APC/C complex decline significantly, and the ubiquitination of an APC/C target is abolished. These data suggest that at least one role of diml p is to maintain or establish the steady state level of the APC/C.

Human HEF1 is a member of a family of multidomain docking proteins implicated in the regulation of cell adhesion. Expression of HEF1 is cell cycle regulated. The differentially phosphorylated p105^HEF1 and p115^HEF1 proteins are produced upon induction of cell growth and accumulate predominantly in the cytoplasm and to focal adhesions. The p55^HEF1 protein, however, appears at mitosis as the result of processing by a caspase and localizes to the mitotic spindle. The human homolog of the S. pombe diml p protein, hDI 1 , was identified in a two-hybrid library screen as an interactor of p55^HEF1 (Law et al. (1998) Mol Cell Biol 18, 3540-3551 ).

Upon purification of the S. cerevisiae U4/U6-U5 small nuclear ribonucleoprotein (snRNP) particle and subsequent identification of its protein constituents, it was found that Dib1 , the yeast homolog of the S. pombe diml p and human hDIM1 , is an integral component of the U4/U6-U5 snRNP. It was further argued that the previously described cell cycle defects associated with dimlp mutations may be secondary effects arising from defective pre-mRNA splicing (Stevens and Abelson (1999) Proc Natl Acad Sci USA 96, 7226-7231 ). The identification of an FKBP-type peptidyl-prolyl cis-trans isomerase motif in the different DIM1-homologues (Zhang et al. (1999) Physiol Genomics 1 , 109-118) further strengthens the possible involvement of the DIM1 protein in pre-mRNA splicing as prolyl isomerases contribute to this process (Teigelkamp ef al. (1998) RNA 4, 127-141).

At least the human D1M1 protein belongs to the superfamily of proteins adopting a thioredoxin fold. However, none of the DIM1 members contain the CGPC amino acid motif which is required for thioredoxin activity. Therefore, DIM1 proteins are most likely not active as thioredoxins (Zhang ef al. (1999) Physiol Genomics 1 , 109-118). Known dominant-negative mutants include C-terminal truncated DIM1 (deletion of the C-terminal 13 or 14 amino acids; Zhang ef al. (1999) Physiol Genomics 1 , 109-118). A temperature- sensitive mutant , dim 1-35, is known in S. pombe. In dim1-35, a single amino acid is changed relative to wild-type diml, namely the wild-type glycine at position 126 that is changed into an aspartate in dim 1-35 (Berry and Gould (1997) J Cell Biol 137, 1337- 1354). The S. cerevisiae Dib1 protein should not be confused with the S. cerevisiae Diml protein. Whereas Dib1 is the yeast homolog of the S. pombe diml p and the human hDIM1 , the yeast Diml protein is an 18s rRNA dimethylase (Lafontaine et al (1994) J. Mol. Biol. 241 , 492-497). The Arabidopsis homolog of the yeast DIM1 rRNA methylase gene is known as PFC1 (PALEFACE1; Tokuhisa et al. (1998) Plant Ce// 10, 699-711).

Summary of the Invention

The present invention is based, at least in part, on the discovery of novel plant nucleic acid molecules and polypeptides encoded by such nucleic acid molecules, referred to herein as "DIM1 -interacting molecules" or "DIMIC." The DIMIC nucleic acid and polypeptide molecules of the present invention are useful as modulating agents in regulating cell cycle progression in, for example, plants. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding DIMIC polypeptides, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of DIMIC-encoding nucleic acids.

According to a first embodiment the present invention relates to an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising the nucleotide sequence as given in any of SEQ ID NOs 36, 35, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48 or 94, or the complement thereof,

(b) a nucleic acid molecule comprising the RNA sequence corresponding to any of SEQ ID NOs 36, 35, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48 or 94, or the complement thereof,

(c) a nucleic acid molecule specifically hybridizing with the nucleotide sequence as defined in (a) or (b),

(d) a nucleic acid molecule which is at least 60% identical to the nucleotide sequence as given in any of SEQ ID NOs 36, 35, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47,

48 or 94, or the complement thereof,

(e) a nucleic acid molecule encoding a protein comprising an amino acid sequence as given in any of SEQ ID NOs 49 to 53 or 95,

(f) a nucleic acid molecule encoding a protein comprising at least one or at least two or at least three of the amino acid sequences represented in SEQ ID NOs 55, 56 or 96,

(g) a nucleic acid molecule encoding a protein comprising an amino acid sequence which is at least 42 % identical to the amino acid sequence as given in SEQ ID NO 50, (h) a nucleic acid molecule encoding a protein comprising at least one or at least two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve, or thirteen, or fourteen of the amino acid sequences represented in SEQ

ID NOs 59 to 63, or 97 to 105,

(i) a nucleic acid molecule encoding a protein comprising at least one or at least two of the amino acid sequences represented in SEQ ID NOs 64 to 69, 106, 107 or

1 1 1 , (j) a nucleic acid molecule encoding a protein comprising at least one or two or three of the amino acid sequences represented in any of SEQ ID NOs 108, 109 or 110, (k) a nucleic acid molecule encoding a protein comprising an amino acid sequence which is at least 50 % identical to the amino acid sequence as given in any of

SEQ ID NOs 49, 50, 51 , 52, 53 or 95, (I) a nucleic acid molecule which is degenerated to a nucleic acid as defined in any of

(a) to (k) as a result of the genetic code, (m) a nucleic acid molecule which is diverging from a nucleic acid as defined in any of (a) to (k) as a result of differences in codon usage between organisms, (n) a nucleic acid molecule which is diverging from a nucleic acid as defined in any of

(a) to (k) as a result of differences between alleles, and (o) a nucleic acid molecule as defined in any one of (a) to (n) characterized in that said nucleic acid is DNA, cDNA, genomic DNA or synthetic DNA, characterized in that said nucleic acid molecule encodes a DIM1 -interacting molecule

(DIMIC molecule), or a homologue or a derivative thereof and further provided that said nucleic acid is not one of the nucleic acids as deposited under the GenBank Accession numbers AC004261 , AC008148, AB023039 or AC007583.

According to another embodiment, the invention relates to an isolated nucleic acid molecule encoding an immunologically active and/or functional fragment of a DIM1 - interacting molecule encoded by a nucleic acid of claim 1 , or an immunologically active and/or functional fragment of a homologue or a derivative of such a DIM1 -interacting molecule, provided that said nucleic acid is not one of the nucleic acids as deposited under the GenBank Accession number T3K9.20 or T3K9.21. For instance, said isolated nucleic acid molecule are selected from the group consisting of consisting of:

(a) a nucleic acid encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 49, wherein said fragment comprises at least one or two or three of the sequences as represented in any of SEQ ID NOs 55, 56, or 96, (b) a nucleic acid molecule encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 49, wherein, said fragment comprises at least 326 contiguous amino acid residues of the amino acid sequence of SEQ ID NO 49,

(c) a nucleic acid encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 50, wherein said fragment comprises at least one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve, or thirteen, or fourteen of the sequences as represented in any of SEQ ID NOs 59, 60, 61 , 62, 63, 97, 98, 99, 100, 101 , 102, 103, 104, or 105,

(d) a nucleic acid encoding a functional fragment of polypeptide comprising the amino acid sequence of SEQ ID NO 51 , wherein said fragment comprises at least one, or two, or three, or four, or five, or six, or seven, or eight, or nine of the sequences as represented in any of SEQ ID NOs 64, 65, 66, 67, 68, 69, 106, 107 or 111 , (e) a nucleic acid encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 52, wherein said fragment comprises at least one or two of the sequences as represented in SEQ ID NO 108 or 110,

(f) a nucleic acid molecule encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 53, wherein said fragment comprises at least one or two of the sequences as represented in SEQ ID NO 109 or 110,

(g) a nucleic acid encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 95, wherein said fragment comprises at least one or two of the sequences as represented in SEQ ID NO 109 or 110, and

(h) a nucleic acid molecule encoding a functional fragment of a polypeptide comprising the amino acid sequence of any SEQ ID NOs 52, 53 or 95, wherein the fragment comprises at least 178 contiguous amino acid residues of any of the amino acid sequences of SEQ ID NOs 52, 53 or 95.

In one embodiment, a DIMIC nucleic acid molecule of the invention is at least

50%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%,

90%, 92%, 95%, 97%, 98%, 99% or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) of SEQ ID NOs 35-48 or 94, or a complement thereof.

In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NOs 35-48 or 94, or a complement thereof. In another preferred embodiment, an isolated nucleic acid molecule of the invention encodes the amino acid sequence of a plant DIMIC polypeptide. According to a further embodiment the present invention relates to an isolated nucleic acid molecule consisting of the nucleotide sequence as given in any of SEQ ID NOs 36, 35, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48 or 94, or the complement thereof.

Another embodiment of the invention features nucleic acid molecules, preferably DIMIC nucleic acid molecules, which specifically detect DIMIC nucleic acid molecules relative to nucleic acid molecules encoding non-DIMIC polypeptides. For example, in one embodiment, such a nucleic acid molecule is at least 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 532, 550, 600, 650, 700, 750, 800, 850, 900, 950, 976 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NOs 35-48 or 94, or a complement thereof.

In another embodiment, the present invention features fragments of the nucleic acid molecule of SEQ ID NOs 35, 36, or 37, wherein the fragments do not comprise nucleotides 1-975 (SEQ ID NO 85), nucleotides 1087-1236 (SEQ ID NO 86), nucleotides

1237-1326 (SEQ ID NO 87), or nucleotides 1330-1599 (SEQ ID NO 88) of SEQ ID NOs

35, 36, or 37.

In another embodiment, the present invention features fragments of the nucleic acid molecule of SEQ ID NOs 44, 45, 46, 47, or 48, wherein the fragments do not comprise nucleotides 1 -531 of SEQ ID NOs 44, 45, 46, 47, or 48 (SEQ ID NO 91 ), nucleotides 643-948 of SEQ ID NO 45 or 47 (SEQ ID NO 92), or nucleotides 646-810 of

SEQ ID NO 46 or 48 (SEQ ID NO 93).

In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a plant DIMIC polypeptide, wherein the nucleic acid molecule hybridizes to the nucleic acid molecule of SEQ ID NOs 35-48 or 94 under stringent conditions.

Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a DIMIC nucleic acid molecule, e.g., the coding strand of a DIMIC nucleic acid molecule. Therefore the invention also relates to an antisense nucleic acid molecule corresponding to at least one of the DIMIC nucleic acids as described earlier.

The invention also relates to an isolated nucleic acid molecule comprising at least one of the DIMIC nucleic acids as described earlier and a nucleotide sequence encoding a heterologous polypeptide. The invention also relates to a polypeptide encodable by such an isolated nucleic acid.

The invention also relates to a nucleic acid molecule of at least 15 contiguous nucleotides in length specifically hybridizing with or specifically amplifying DIMIC nucleic acids as described earlier.

Another aspect of the invention provides a vector comprising a DIMIC nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector.

The invention therefore relates to a vector comprising any of the nucleic acid molecules of the invention and as described earlier. The invention further relates to an expression vector wherein said nucleic acid sequence of the invention is operably linked to one or more control sequences allowing the expression of said sequence in prokaryotic and/or eukaryotic host cells. In another embodiment, the invention provides a host cell containing a vector of the invention. Therefore the invention relates to host cell comprising a nucleic acid molecule of the invention or a vector as described above, for instance a host cell chosen from a bacterial, insect, fungal, yeast, plant or animal cell. The invention also provides a method for producing a DIMIC polypeptide, by culturing in a suitable medium a host cell of the invention, e.g., a plant host cell such as a host monocot plant cell (e.g., rice, wheat or corn) or a dicot host cell (e.g., Arabidopsis thaliana, oilseed rape, or soybeans) containing a recombinant expression vector, such that the polypeptide is produced. The invention thus relates to a method for producing a polypeptide comprising culturing a host cell as described above under conditions allowing the expression of the polypeptide and recovering the produced polypeptide from the culture.

Another aspect of this invention features isolated or recombinant DIMIC polypeptides. The invention relates to an isolated polypeptide encodable by any of the nucleic acids of the invention, or a homologue or a derivative thereof, or an immunologically active and/or functional fragment thereof. For instance, the invention relates to a polypeptide having an amino acid sequence as given in any of SEQ ID NOs 49 to 53 or 95, or a homologue or a derivative thereof, or an immunologically active and/or functional fragment thereof.

In one embodiment, an isolated DIMIC polypeptide has one or more of the following domains: a "WW or WWP domain", a "non-classical C₂-domain", a "FAB1 activation loop", a "DIMIC5 internal repeat domain", a "DIMIC7 internal repeat domain", a "DIMIC26 internal repeat domain", a "DIMIC26 di-amino acid motif", a "thioredoxin-like domain" and/or a "PEST sequence."

In a preferred embodiment, a DIMIC polypeptide includes at least one or more of the following domains: a "WW or WWP domain", a "non-classical C₂-domain", a "FAB1 activation loop", a "DIMIC5 internal repeat domain", a "DIMIC7 internal repeat domain", a "DIMIC26 internal repeat domain", a "DIM1C26 di-amino acid motif", a "thioredoxin-like domain" and/or a "PEST sequence", and has an amino acid sequence at least about 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NOs 49-53 or 95.

In another preferred embodiment, a DIMIC polypeptide includes at least one or more of the following domains: a "WW or WWP domain", a "non-classical C₂-domain", a "FAB1 activation loop", a "DIM1C5 internal repeat domain", a "DIMIC7 internal repeat domain", a "DIMIC26 internal repeat domain", a "DIMIC26 di-amino acid motif", a "thioredoxin-like domain" and/or a "PEST sequence" and has a DIMIC activity (as described herein). In yet another preferred embodiment, a DIMIC polypeptide includes one or more of the following domains: a "WW or WWP domain", a "non-classical C₂-domain", a "FAB1 activation loop", a "DIMIC5 internal repeat domain", a "DIMIC7 internal repeat domain", a "DIMIC26 internal repeat domain", a "DIMIC26 di-amino acid motif", a "thioredoxin-like domain" and/or a "PEST sequence" and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs 35-48 or 94.

In another embodiment, the invention features fragments of the polypeptide having the amino acid sequence of SEQ ID NOs 49-53 or 95, wherein the fragment comprises at least 178, 200, 250, 300, 326, 350, or more amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NOs 49-53 or 95. In another embodiment, a DIMIC polypeptide has the amino acid sequence of SEQ ID NOs 49-53 or 95.

In a further embodiment, the invention features fragments of the polypeptide having the amino acid sequence of SEQ ID NO 49, wherein the fragments do not comprise amino acid residues 1-325 (SEQ ID NO 81 ), amino acid residues 363-412 (SEQ ID NO 82), amino acid residues 413-442 (SEQ ID NO 83), or amino acid residues 444- 463 (SEQ ID NO 84) of SEQ ID NO 49.

In another embodiment, the invention features fragments of the polypeptide having the amino acid sequence of SEQ ID NO 52 or 53, wherein the fragments do not comprise amino acid residues 1-177 of SEQ ID NO 52 (SEQ ID NO 89), amino acid residues 215-268 of SEQ ID NO 52, or amino acid residues 216-269 of SEQ ID NO 53 (SEQ ID NO 90).

In another embodiment, the invention features a DIMIC protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 98%, 99% or more identical to a nucleotide sequence of SEQ ID NOs 35-48 or 95, or a complement thereof. This invention further features a DIMIC polypeptide, which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs 35-48 or 95, or a- complement thereof.

In another embodiment the invention provides transgenic plants (e.g., monocot or dicot plants) containing an isolated nucleic acid molecule of the present invention. For example, the invention provides transgenic plants containing a recombinant expression cassette including a plant promoter operably linked to an isolated nucleic acid molecule of the present invention. The present invention also provides transgenic seed from the transgenic plants. In another embodiment the invention provides methods of modulating, in a transgenic plant, the expression of the nucleic acids of the invention. The invention thus relates to a method for the production of altered plant cells, plant tissues or plants comprising the introduction of a polypeptide as defined earlier directly into said plant cell or tissue or in an organ of said plant.

The invention also relates to a method for effecting the expression of a polypeptide as defined earlier in plant cells, tissues or plants comprising the introduction of any of the nucleic acid molecules of the invention operably linked to one or more control sequences or a vector of the invention stably into the genome of a plant cell.

The invention also relates to a method for the production of transgenic plant cells, plant tissues or plants comprising the introduction of a nucleic acid of the invention in an expressible format or a vector of the invention in said plant cell, plant tissue or plant. The invention also relates to a method as described above further comprising regenerating a plant from said plant cell.

The invention further relates to a transgenic plant cell comprising any of the nucleic acids of the invention which is operably linked to regulatory elements allowing transcription and/or expression of said nucleic acid in plant cells or a transgenic plant cell obtainable by any of the methods described above. The invention relates to said transgenic plant cell wherein said nucleic acid is stably integrated into the genome of said plant cell.

The invention also relates to a transgenic plant or plant tissue comprising transgenic plant cells as described above or a transgenic plant obtainable by the method described above. The invention also relates to a harvestable part of said transgenic plant, for instance a harvestable part which is selected from the group consisting of seeds, leaves, fruits, stem cultures, rhizomes and bulbs. The invention also relates to the progeny derived from any of the transgenic plants or plant parts described above. The proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-DIMIC polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins.

The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind polypeptide of the invention, preferably DIMIC polypeptide.

The invention thus relates to an antibody specifically recognizing a polypeptide of the invention or a specific epitope of said polypeptide.

In addition, the DIMIC polypeptide or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detecting the presence of a DIMIC nucleic acid molecule or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting a DIMIC nucleic acid molecule or polypeptide such that the presence of a DIMIC nucleic acid molecule or polypeptide is detected in the biological sample.

Therefore, the invention further relates to a method for detecting the presence of a polypeptide of the invention in a sample comprising:

(a) contacting the sample with a compound which selectively binds to said polypeptide, for instance an antibody; and

(b) determining whether the compound binds to the polypeptide in the sample to thereby detect the presence of a polypeptide of the invention in the sample.

The invention also relates to a method for detecting the presence of any of the nucleic acid molecules of the invention in a sample comprising: (a) contacting the sample with a nucleic acid probe or primer as described earlier which selectively hybridizes to or amplifies one of the nucleic acid molecules of the invention, and (b) determining whether said nucleic acid probe or primer binds to a nucleic acid molecule in the sample to thereby detect the presence of said one nucleic acid molecule in the sample.

The invention further relates to the method described above, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.

The invention further relates to a diagnostic kit comprising at least one of the nucleic acid molecules of the invention, at least one of the polypeptides of the invention, at least one of the antibodies described above, at leat one of the compounds obtainable by any of the methods described further.

In another aspect, the present invention provides a method for detecting the presence of DIMIC activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of DIMIC activity such that the presence of DIMIC activity is detected in the biological sample.

In another aspect, the invention provides a method for modulating DIMIC activity comprising contacting a cell capable of expressing DIMIC with an agent that modulates DIMIC activity such that DIMIC activity in the cell is modulated. In one embodiment, the agent inhibits DIMIC activity. In another embodiment, the agent stimulates DIMIC activity. In one embodiment, the agent is an antibody that specifically binds to a DIMIC polypeptide. In another embodiment, the agent modulates expression of DIMIC by modulating transcription of a DIMIC gene or translation of a DIMIC mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a DIMIC mRNA or a DIMIC gene.

In one embodiment, the methods of the present invention are used to increase crop yield, improve the growth characteristics of a plant (such as growth rate or size of specific tissues or organs in the plant), modify the architecture or morphology of a plant, improve tolerance to environmental stress conditions (such as drought, salt, temperature, nutrient or deprivation), or improve tolerance to plant pathogens (e.g., pathogens that abuse the cell cycle) by modulating DIMIC activity in a cell. In one embodiment, the DIMIC activity is modulated by modulating the expression of a DIMIC nucleic acid molecule. In yet another embodiment, the DIMIC activity is modulated by modulating the activity of a DIMIC polypeptide. Modulators of the expression of DIMIC nucleic acids or DIMIC activity include, for example, a DIMIC nucleic acid such as an antisense version of a DIMIC nucleic acid molecule or a DIMIC polypeptide molecule. Other DIMIC modulators comprise antibodies to DIM1 or DIMIC molecules, small molecular weight compounds interacting with or modulating the activity of DIM1 or DIMIC molecules, ribozymes and the like. Modulation of DIMIC activity can be achieved for instance by introducing a DIMIC nucleic acid molecule in a cell. This may lead to overexpression of the exogenous DIMIC molecule in said cell. Alternatively, this may lead to downregulation of expression of the endogenous DIMIC molecule in the cell, a phenomenon known under the term "silencing".

One example of such a DIMIC modulator is a nucleic acid molecule comprising at least part of the nucleotide sequence of a DIMIC molecule, for instance as represented in any of SEQ ID NOs 35 to 48 or 94, and at least part of the corresponding antisense version of said part, seperated by at least a short stretch of nucleotides, in an inverted repeat confirmation.

Another example of such a DIMIC modulator is a fragment of said DIMIC polypeptide that, for instance, contains a destruction box, for instance a PEST sequence, which saturates the specific proteolytic machinery of the plant cell so that the endogenous polypeptide can survive longer in the plant cell. Other fragments of DIMIC polypeptides which can be used herein as a DIMIC modulator (or to modulate the activity of DIMIC molecules) are described further and comprise the specific polypeptide fragments of the DIMIC molecules of the invention (for instance SEQ ID NOs 81 , 82, 83, 84, 89, 90, 96, 97, 108, 109) FKBP domains (for instance SEQ ID NO 54), WW or WWP domains (for instance SEQ ID NO 55), Non-classical C₂ domains (for instance SEQ ID NO 56), DIM1C5 internal repeat domains (for instance SEQ ID NO 57), FAB1 activation loops (for instance SEQ ID NO 58), DIMIC7 internal repeat domains (for instance any of SEQ ID NOs 59 to 63), DIMIC26 internal repeat domains (for instance any of SEQ ID NOs 64 to 69), DIMIC 26 di-amino acid motifs, thioredocin-like domains, PEST sequences (for instance any of SEQ ID NOs 98 to 105 or 107 or 110) and PHD fingers (for instance SEQ ID NO 111 ).

According to yet another embodiment the invention relates to a method for modulating the growth of a plant, comprising introducing into the plant a DIM1 interacting (DIMIC) molecule or a DIMIC modulator in an amount sufficient to modulate the growth of the plant, thereby modulating the growth of the plant.

It should be understood herein that the DIM1 interacting molecule as used in the context for use in any of the methods described herein, comprises any DIM1 interacting molecule from prokaryotic or eukaryotic origin. In interesting embodiments, a plant DIM1 interacting molecule is used, in other interesting embodiments, at least one of the DIM1 interacting molecules identified herein is used.

The invention also relates to a method for modulating the cell cycle in a plant, comprising introducing into the plant a DIM1 interacting (DIMIC) molecule or a DIMIC modulator in an amount sufficient to modulate the cell cycle in the plant, thereby modulating the cell cycle in the plant.

The invention further relates to a method for enhancing overall growth and yield of a plant comprising introducing into the plant a DIM1 interacting (DIMIC) molecule or a DIMIC modulator in an amount sufficient to modulate the growth of the plant, thereby enhancing overall growth and yield of said plant. The invention also relates to a method for increasing yield of a plant comprising introducing into the plant a DIM1 interacting (DIMIC) molecule or a DIMIC modulator in an amount sufficient to modulate the growth of the plant, thereby increasing yield of said plant. The present invention also relates to a method for enhancing stress tolerance, for instance osmotolerance or temperature tolerance, in a plant comprising introducing into the plant a DIM1 interacting (DIMIC) molecule or a DIMIC modulator in an amount sufficient to modulate the growth of the plant, thereby enhancing stress tolerance of said plant. Also according to the invention is the use of a stress inducible promoter herein, to drive the expression of the DIMIC molecule or DIMIC modulator, with the aim to produce the osmoprotectant as it is necessary.

As used herein, "stress tolerance" refers to the capacity to grow and produce biomass during stress, the capacity to reinitiate growth and biomass production after stress, and the capacity to survive stress. The term "stress tolerance" also covers the capacity of the plant to undergo its developmental program during stress similarly to under non-stressed conditions, e.g. to switch from dormancy to germination and from vegetative to reproductive phase under stressed conditions similarly as under non- stressed conditions. Methodologies to determine plant growth or response to stress include, but are not limited to height measurements, leaf area, plant water relations, ability to flower, ability to generate progeny and yield or any other methodology known to those skilled in the art.

The expression "stress tolerance" as used herein preferably relates to tolerance against osmotic stress, caused by salt or drought and/or temperature stress, caused by cold, chilling and freezing stress. The invention also relates to a method for conferring enhanced resistance to pathogens of a plant comprising introducing into the plant a DIM1 interacting (DIMIC) molecule or a DIMIC modulator in an amount sufficient to confer resistance to pathogens of the plant, thereby conferring enhanced resistance to pathogens of said plant.

The invention further relates to any of the above described methods wherein at least one nucleic acid encoding a plant DIM1 interacting (DIMIC) molecule, a homologue or a derivative thereof or an enzy atically active fragment thereof is expressed in specific cells or tissues of said plant.

The invention further relates to the above method furhter comprising stably integrating into the genome of said plant or in specific plant cells or tissues of said plant at least one expressible nucleic acid encoding a D1M1 interacting (DIMIC) molecule, a homologue or a derivative thereof or an enzymatically active fragment thereof

The invention further relates to any of the above methods wherein said expression of said nucleic acid leads to overexpresion of a DIM1 interacting (DIMIC) molecule in said plant or alternatively wherein said expression of said nucleic acid leads to downregulation of expression of a DIM1 interacting (DIMIC) molecule.

The invention furhte relates to any of the methods as described above wherein said DIM1 interacting (DIMIC) molecule is selected from any of the following nucleic acids: (a) a nucleic acid molecule comprising the nucleotide sequence as given in any of

SEQ ID NOs 36, 35, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48 or 94, or the complement thereof,

(d) a nucleic acid molecule which is at least 60% identical to the nucleotide sequence as given in any of SEQ ID NOs 36, 35, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48 or 94, or the complement thereof,

(g) a nucleic acid molecule encoding a protein comprising an amino acid sequence which is at least 42 % identical to the amino acid sequence as given in SEQ ID NO 50,

(h) a nucleic acid molecule encoding a protein comprising at least one or at least two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve, or thirteen, or fourteen of the amino acid sequences represented in SEQ

ID NOs 59 to 63, or 97 to 105,

(i) a nucleic acid molecule encoding a protein comprising at least one or at least two, or three, or four, or five, or six, or seven, or eight, or nine of the amino acid sequences represented in SEQ ID NOs 64 to 69, 106, 107 or 111 , (j) a nucleic acid molecule encoding a protein comprising at least one, or two or three of the amino acid sequences represented in any of SEQ ID NOs 108, 109 or 110, (k) a nucleic acid molecule encoding a protein comprising an amino acid sequence which is at least 50 % identical to the amino acid sequence as given in any of SEQ ID NOs 49, 50, 51 , 52, 53 or 95,

(I) a nucleic acid molecule which is degenerated to a nucleic acid as defined in any of

(a) to (k) as a result of differences between alleles, and (o) a nucleic acid molecule as defined in any one of (a) to (n) characterized in that said nucleic acid is DNA, cDNA, genomic DNA or synthetic DNA, or to an isolated nucleic acid molecule encoding an immunologically active and/or functional fragment of a DIM1 -interacting molecule encoded by a nucleic acid of any of (a) to (o), or an immunologically active and/or functional fragment of a homologue or a derivative of such a DIM1 -interacting molecule selected from one of the following:

(a) a nucleic acid encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 49, wherein said fragment comprises at least one of the sequences as represented in any of SEQ ID NOs 55, 56, or 96,

(b) a nucleic acid molecule encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 49, wherein, said fragment comprises at least 326 contiguous amino acid residues of the amino acid sequence of SEQ ID NO 49, (c) a nucleic acid encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 50, wherein said fragment comprises at least one of or at least two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve, or thirteen, or fourteen of the sequences as represented in any of SEQ ID NOs 59, 60, 61 , 62, 63, 97, 98, 99, 100, 101 , 102, 103, 104, or 105,

(d) a nucleic acid encoding a functional fragment of polypeptide comprising the amino acid sequence of SEQ ID NO 51 , wherein said fragment comprises at least one of the sequences as represented in any of SEQ ID NOs 64, 65, 66, 67, 68, 69, 106, 107 or 111 , (e) a nucleic acid encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 52, wherein said fragment comprises at least one of the sequences as represented in SEQ ID NO 108 or 110,

(f) a nucleic acid molecule encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 53, wherein said fragment comprises at least one of the sequences as represented in SEQ ID NO 109 or 110,

(g) a nucleic acid encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 95, wherein said fragment comprises at least one of the sequences as represented in SEQ ID NO 109 or 110, and

The invention further relates to any of the methods described above wherein the expression or activity of a nucleic acid encoding a plant DIM1 interacting (DIMIC) molecule or a homologue thereof is modulated by a DIMIC modulator, for instance a DIMIC modulator selected from the group consisting of any of the described antibodies, antisense molecules, ribozymes, or compounds obtainable by any of the methods described further.

The invention further relates to the method described above wherein said DIMIC modulator is capable of modulating DIMIC nucleic acid expression or wherein said DIMIC modulator is capable of modulating DIMIC polypeptide activity. According to yet another embodiment the invention also relates to any of the methods described earlier comprising co-expression of a DIM1 interacting (DIMIC) molecule or a DIMIC modulator and a DIM1 molecule in said plant.

The present inventors have performed a two hybrid screening with the Arabidopsis thaliana DIM1 (AtDIMI ) as a bait to define a number of plant interacting proteins with AtDIMI in yeast cells, for instance the DIM1 interacting (DIMIC) molecules as described earlier. These physical interactions are evidence that they occur in plant cells. Therefore the DIMIC molecules are the preferred partners to coexpress with DIML A number of DIM1 molecules are described herein and are represented in SEQ ID NOs 1 to 34. In one example, co-expression of DIM1 and DIMIC5 in plants is performed. An effect on pre-mRNA splicing is expected, such as a more rapid and efficient intron splicing. More messenger can be translated into proteins, with a direct effect on cell growth, and thereafter on cell cycle progression. Other DIMIC molecules to be coexpressed with a DIM1 molecule comprise for instance any of the DIMIC molecules described earlier, or a functional fragment thereof.

The invention also extends to the use of homologues, orthologues, paralogues or derivatives of the DIMIC molecules described herein and to functional fragments thereof. Futhermore the invention also relates to new two hybrid screening methods which can be performed with any of the DIMIC molecules of the invention, for instance DIMIC5, DIMIC7, DIMIC26 or DIMIC70A/B/C, for instance to identify other interacting proteins of the spliceosome. Combined expression of these new interactors with either DIM1 or DIMIC5 or both in transgenic plants is yet another example to promote enhanced growth in plants. With "co-expression" is meant the expression or overexpression of two or more genes or proteins. The same or, more preferably, different promoters are used to confer expression of said genes or proteins.

The present invention also provides diagnostic assays for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a DIMIC polypeptide; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of a DIMIC polypeptide, wherein a wild-type form of the gene encodes a protein with a DIMIC activity.

In another aspect the invention provides methods for identifying a compound that binds to or modulates the activity of a DIMIC polypeptide, by providing an indicator composition comprising a DIMIC polypeptide having DIMIC activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on DIMIC activity in the indicator composition to identify a compound that modulates the activity of a DIMIC polypeptide. The identified compounds may be used as herbicides or plant growth regulators. According to yet another embodiment the invention relates to a method for identifying compounds or mixtures of compounds which specifically bind to a polypeptide of the invention, comprising the steps of

(a) combining a polypeptide of the invention or a cell expressing said polypeptide with said compound or mixtures of compounds under conditions suitable to allow complex formation, and, (b) detecting complex formation, wherein the presence of a complex identifies a compound or mixture of compounds which specifically binds said polypeptide.

The invention further relates to a method as described above, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of:

(a) detection of binding by direct detection of test compound/polypeptide binding;

(b) detection of binding using a competition binding assay; and

(c) detection of binding using an assay for testing the activity of the DIM1- interacting molecule.

According to a further embodiment the invention relates to a method for identifying and obtaining compounds interacting with or modulating the activity of a polypeptide of the invention comprising the steps of: (a) providing a two-hybrid system wherein a polypeptide of the invention and an interacting protein partner, preferably a DIM1 molecule are expressed,

(b) interacting said compound with the complex formed by the expressed polypeptides as defined in a), and,

(c) performing measurement of interaction of said compound with said polypeptide or the complex formed by the expressed polypeptides as defined in (a).

The invention further relates to a method for modulating the activity of a polypeptide of the invention comprising contacting a polypeptide of the invention or a host cell of the invention expressing said polypeptide with a compound which binds to the polypeptide or obtainable by any of the methods described above, in a sufficient concentration to modulate the activity of the polypeptide.

The invention further relates to a method for preparing a DIMIC modulator composition using a compound identifiable by any of the methods described above.

In another aspect, the present invention features methods for modulating pre- mRNA splicing in a cell, e.g., a plant cell, by introducing into the cell a DIMIC modulator in an amount sufficient to modulate pre-mRNA splicing in the cell, thereby modulating pre- mRNA splicing in the cell. In one embodiment, the DIMIC modulator comprises the nucleotide sequence of SEQ ID NOs 35-48, or a fragment thereof. In another embodiment, the DIMIC modulator is a DIMIC polypeptide comprising the amino acid sequence of SEQ ID NOs 49-53, or a fragment thereof. In another aspect, the present invention features methods for modulating vesicle transport/processing in a cell, e.g., a plant cell, by introducing into the cell a DIMIC modulator in an amount sufficient to modulate vesicle transport/processing in the cell, thereby modulating vesicle transport processing in the cell. In one embodiment, the DIMIC modulator comprises the nucleotide sequence of SEQ ID NOs 1-17 or 35-48, or a fragment thereof. In another embodiment, the DIMIC modulator is a DIM or a DIMIC polypeptide comprising the amino acid sequence of SEQ ID NOs 18-34 or 49-53, or a fragment thereof. Examples of such DIMIC modulators are described earlier.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

Brief Description of the Drawings

Figure 1 depicts an alignment of DIM1 protein sequences from various organisms. The amino acid residues differing from the consensus are shaded in a black box. Plants: At: Arabidopsis thaliana; Gm: Glycine max; Mt: Medicago truncatula; Le: Lycopersicon esculentum; Ga: Gossypium arboreum; Ly. Lotus japonica; Zm: Zea mays; Os: Oryza sativa; Pp: hybrid aspen (Populus tremula x Populus tremuloides); Pt: Pinus taeda; Hv: Hordeυm vulgare; Ts: Thellungiella salsuginea; Cj: Cryptomeria japonica; Mc: Mesembryanthemum crystallinυm; Ta: Triticum aestivum. Fungi: Sp: Schizosaccharomyces pombe. Animals: Dm: Drosophila melanogaster. Figure 2 depicts the amino acid sequence of the DIMIC5 protein (SEQ ID NO 49).

Indicated in the figure are the tandem WW WWP domains (boxed, tryptophane and proline residues marked with an asterisk) separated by an 18-amino acid residue spacer. The C₂-domain is underscored by a rounded bracket. The DIMIC5 internal repeat domains are aligned as indicated by vertical lines connecting the conserved amino acid residues. The double underlined amino acid sequences represent the sequences of the GenBank entries with accession numbers AC004261 (protein ID AAD12009)/T02117. Amino acid residues not present in DIMIC5 are marked by a grey shaded box and are double underlined. The single underlined amino acid sequence corresponds to parts of the GenBank entry with accession number T02116. Extra amino acid residues present in T02116 but not in D1MIC5 or amino acid residues different between T02166 and DIMIC5 are indicated in a box. When amino acid sequence alignments are made identical residues are marked by a black box whereas similar residues (according to the groups (F.W.Y), (M.I.L.V), (R,K,H), (D,E), (N,Q), (S,T)) are marked by a grey shaded box. Gaps ('-') are introduced to obtain an optimal alignment. Figure 3 depicts the genomic region of Arabidopsis thaliana (GenBank entry with accession number AC004261) comprising the DIMIC5 open reading frame (nucleotides 17241 to 20717 SEQ ID NO 37). Intron/exon positioning was modified (relative to the ORF predicted for the protein with ID AAD12009) to be in line with the experimentally determined partial DIMIC5 cDNA sequence (SEQ ID NO 35). Nucleotide residues marked by grey shaded boxes correspond to intron sequences. Bold-faced and underlined nucleotide residues correspond to the 5' extension added to complete the partial DIMIC5 cDNA. The 3' underlined nucleotide residues correspond to the 3' UTR of the partial DIMIC5 cDNA which are also part of AC004261 (nucleotides 20718 to 20924). The poly A⁺ tail of the DIMIC5 cDNA is indicated between brackets purely for illustrative reasons. Combination of the exon sequences yield the DIMIC5 ORF as partially present in the DIMIC5 cDNA (SEQ ID NO 36).

Figure 4 depicts the genomic region (SEQ ID NO 40) of Arabidopsis thaliana (GenBank entry with accession number AC008148) comprising the DIMIC7=DIMIC40 open reading frame (nucleotides 100439 to 106312: SEQ ID NO 39). Nucleotide residues marked by grey shaded boxes correspond to intron sequences. The 3' underlined nucleotide residues correspond to the partial DIMIC7 cDNA (SEQ ID NO 38) including the 3'UTR (the latter comprising nucleotides 106313 to 106564 which are also part of AC008148). The poly A⁺ tail of the DIMIC7 cDNA is indicated between brackets purely for illustrative reasons. Combination of the exon sequences yield the DIMIC7=40 ORF as partially present in the DIMIC7=40 cDNA.

Figure 5 shows the amino acid sequence of the DIMIC7=DIMIC40 protein (SEQ ID NO 50) aligned with the Arabidopsis thaliana FAB1 -like protein (AtFABI ; GenBank entry AL035525; protein ID CAB36798) and with homologous parts of the Saccharomyces cerevisiae (yeast) FAB1 kinase (GenBank entry P34756) as well as with the homologous part of the mouse CCTd protein (GenBank entry Z31554). Amino acid residues not present in mouse CCTd and not present in DIMIC7, AtFABI or yeast FAB1 are inserted were appropriate in block arrows. When amino acid sequence alignments are made identical residues are marked by a black box whereas similar residues (according to the groups (F,W,Y), (M,I,LN), (R,K,H), (D,E), (Ν,Q), (S,T)) are marked by a grey shaded box. Gaps ('-') are introduced to obtain an optimal alignment. The amino acid residues corresponding to the partial DIMIC7=40 protein are underlined (SEQ ID NO 97). The activation loop of FAB1-type kinases is surrounded by a grey shaded box. Residues conserved in the C-terminal catalytic domain of Fab1 -type kinases are marked with an asterisk. Among these are the invariant residues K2059, D2196 and D2216 (numbering relative to yeast FAB1) which are further marked by a surrounding box.

Figure 6 depicts the internal repeat domains found in the DIMIC7=DIMIC40 protein. Shown are the five different motifs (DIMIC7/1 to DIMIC7/5 corresponding with SEQ ID NOs 59 to 63) with indication of their position in the DIMIC7=40 protein sequence and the corresponding consensus sequence. When amino acid sequence alignments are made identical residues are marked by a black box whereas similar residues (according to the groups (F.W.Y), (M,I,L,V), (R,K,H), (D,E), (N,Q), (S,T)) are marked by a grey shaded box. Figure 7 depicts the genomic region of Arabidopsis thaliana (GenBank entry with accession number AB023039) comprising the DIMIC26 open reading frame (nucleotides 19634 to 21435). Nucleotide residues marked by grey shaded boxes correspond to intron sequences. The 3' underlined nucleotide residues correspond to the partial DIMIC26 cDNA including the 3'UTR (the latter comprising nucleotides 19553 to 19633) which are also part of AB023039. The poly A⁺ tail of the DIMIC26 cDNA is indicated between brackets purely for illustrative reasons. Combination of the exon sequences yield the DIMIC26 ORF as partially present in the DIMIC26 cDNA.

Figure 8 (A) depicts the amino acid sequence of the DIMIC26 protein (SEQ ID NO 51). The amino acid residues corresponding to the partial DIMIC26 protein (SEQ ID NO 106) are underlined. Figure 8 (B) depicts the '[M/I/L/V][R/K/Hj" amino acid pair (double underlined) and the '[R/K H][M/I/LΛ/]' amino acid pair (single underlined) which are repeated multiple times in the DIMIC26 protein. Note that both pairs can overlap.

Figure 9 depicts an alignment of the homologous amino acid regions of the D1MIC26 protein (SEQ ID NO 51) and the human centrosome protein E (CENP-E; GenBank accession number NM001813). When amino acid sequence alignments are made identical residues are marked by a black box whereas similar residues (according to the groups (F.W.Y), (M,I,LN), (R,K,H), (D,E), (Ν,Q), (S,T)) are marked by a grey shaded box. Gaps ('-') are introduced to ensure optimal alignment. Further indicated in this figure are the '[M/I/LΛ ][R/K/H]' amino acid pair (double underlined) and the '[R/K/H][M/I/L V]' amino acid pair (single underlined) which are repeated in both protein parts. Note that both pairs can overlap.

Figure 10 depicts an alignment of the homologous amino acid regions of the DIMIC26 protein (SEQ ID NO 51 ) and the human nonmuscle type B myosin heavy chain (NMMHC-B; GenBank accession number P35580). When amino acid sequence alignments are made identical residues are marked by a black box whereas similar residues (according to the groups (F,W,Y), (M,I,LN), (R,K,H), (D,E), (Ν,Q), (S,T)) are marked by a grey shaded box. Gaps ('-') are introduced to ensure optimal alignment.

Figure 11 depicts the internal repeat domains found in the DIMIC26 protein. Shown are the six different motifs (DIMIC26/1 to DIMIC26/6 corresponding to SEQ ID NOs 64 to 69) with indication of their position in the DIMIC26 protein sequence and the corresponding consensus sequence. When amino acid sequence alignments are made identical residues are marked by a black box whereas similar residues (according to the groups (F,W,Y), (M,I,LN), (R,K,H), (D,E), (Ν,Q), (S,T)) are marked by a grey shaded box. Figure 12 represents the genomic region of Arabidopsis thaliana (GenBank entry with accession number AC007583) comprising the DIMIC70B and DIMIC70C open reading frame (nucleotides 64105 to 65587). Intron/exon positioning was modified (relative to the ORF predicted for the protein with ID AAF75085) to be in line with the experimentally determined partial DIMIC70B (SEQ ID NO 46) (and DIMIC70C (SEQ I D NO 94) cDNA sequence). (A) Nucleotide residues marked by grey shaded boxes correspond to intron sequences in respect of DIMIC70B. The 3' underlined nucleotide residues correspond to the partial DIMIC70B cDNA (SEQ ID NO 46) including the 3'UTR (the latter comprising nucleotides 63964 to 64104 which are also part of AC007583). The poly A⁺ tail of the DIMIC70B cDNA is indicated between brackets purely for illustrative reasons. The bold-faced 'tga' nucleotide-triplet (nucleotides 64486-64484) is not present in the DIMIC70A cDNA (SEQ ID NO 45). Omission of the triplet thus results in the genomic sequence of the DIMIC70A allele (SEQ ID NO 47). (B) Nucleotide residues marked by grey shaded boxes correspond to intron sequences in respect of DIMIC70C. Figure 13 shows the DIMIC70A cDNA sequence (A) (SEQ ID NO 44)as well as the DIMIC70A protein sequence (B) (SEQ ID NO 52). The underlined N-terminal extension added to complete the DIMIC70A protein is derived from GenBank entry AC007583/protein ID AAF75085. Amino acid residues not present in the protein with ID AAF75085 are bold-faced and indicated between brackets. (SEQ ID NO 108) Figure 14 shows the DIMIC70B cDNA sequence (A) (SEQ ID NO 46) as well as the DIMIC70B protein sequence (B) (SEQ ID NO 53). The underlined N-terminal extension added to complete the DIMIC70B protein is derived from GenBank entry AC007583/protein ID AAF75085. Amino acid residues not present in said protein with ID AAF75085 are bold-faced and indicated between brackets (SEQ ID NO 109). Figure 15 depicts an alignment of the C-terminal part of the DIMIC70A protein

(SEQ ID NO 52) with the PRODOM family PD12637 consensus sequence which comprises the redox-active center of thioredoxins and thioredoxin-like proteins. The amino acid residues of DIMIC70A marked with an asterisk correspond to amino acid residues which are, according to the PD12637 consensus domain, preferably occurring at that given position. Amino acid residues of DIMIC70A marked with a '+' deviate from the preferably occurring amino acid residues. The N-terminal asterisks overlie the conserved thioredoxin-like 'CXXC consensus motif. Further indicated are the redox-active centers of a number of proteins from different organisms including yeast TRG1 (Gϋnther ef al. (1991 ) J Biol Chem 266, 24557-24563), a number of protein disulfide isomerases (Wang and Chang (1999) EMBO J 18, 5972-5982) and a number of thioredoxins and thioredoxin-like proteins as indicated with their GenBank accession and protein ID numbers. S The redox-active centers are aligned with the redox-active center of DIMIC70A and of the PRODOM PD12637 consensus sequence. When amino acid sequence alignments are made identical residues are marked by a black box whereas similar residues (according to the groups (F,WN), (M,I,LN), (R,K,H), (D,E), (Ν,Q), (S,T)) are marked by a grey shaded box.

Figure 16 shows the DIMIC70C cDNA sequence (A) (SEQ ID NO 94) as well as the DIMIC70C protein sequence (B) (SEQ ID NO 95). The start and stop codons are represented in bold in the nucleotide sequence. The first 17 amino acids of the deduced protein sequence indicated in bold differ from the DIMIC70B amino acid sequence.

Detailed Description of the Invention

The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as "DIM1 -interacting molecules" or "DIMIC" nucleic acid and polypeptide molecules. The DIMIC molecules of the present invention were identified based on their ability, as determined using yeast two-hybrid assays (described in detail in Example 2), to interact with the protein AtDIMI , the DIM1 homolog from Arabidopsis thaliana as well as with other DIM1 homologs such as those of other plants (described in detail in Example 1).

DIM1 is involved in one or more of the following processes: (a) Cell cycle processes including, but not limited to, processes associated with G2/M transition or chromosome movement and segregation, spindle formation and elongation, cytokinesis, and regulation of the APC/C (Berry and Gould (1997) J Cell Biol 137, 1337-1354; Berry et al. (1999), Mol Cell Biol 19, 2535-2546; Law et al. (1998) Mol Cell Biol 18, 3540-3551 ; and see Example 5); (b) Pre-mRNA splicing (Stevens and Abelson (1999) Proc Natl Acad Sci USA 96, 7226-7231 ; Teigelkamp et al. (1998) RNA 4, 127-141 ; Zhang et al. (1999) Physiol Genomics 1 , 109-118); (c) Vesicle transport or processing (see Examples 3 and 4).

Because of their ability to interact with (e.g., bind to) AtDIMI and possibly AtDIMI homologues (see Example 1), the DIMIC molecules of the present invention may modulate, e.g., upregulate or downregulate, the activity of DIM1. Furthermore, because of their ability to interact with (e.g., bind to) AtDIMI and possibly AtDIMI homologues which are proteins involved in cell cycle regulation and/or pre-mRNA splicing and/or vesicle transport/processing, the DIMIC molecules of the present invention may also play a role in cell cycle regulation and/or pre-mRNA splicing and/or vesicle transport processing in, for example, plant or animal cells.

As used herein, the terms "DIM1 -interacting protein" or "DIMIC" include a polypeptide which interacts with, e.g., binds to a DIM1 protein, and which is involved in controlling or regulating the cell cycle and/or pre-mRNA splicing and/or vesicle transport/processing, or part of any of these processes, in a cell, tissue, organ or in a whole organism. DIMIC molecules of the present invention may also be capable of binding to, regulating, or being regulated by cyclin-dependent kinases, such as plant cyclin dependent kinases, e.g., CDC2a or CDC2b, or their subunits. The term DIMIC also includes fragments, variants, homologs, alleles or precursors (e.g., pre-proteins, pre-pro- proteins or pro-proteins) of DIMIC polypeptides. As used herein, the term "cell cycle" includes the cyclic biochemical and structural events associated with the growth, division and proliferation of cells, and in particular with the regulation of the replication of DNA and mitosis. The cell cycle is divided into periods or phases called: GO, Gap1 (G1 ), DNA synthesis (S), Gap2 (G2), and mitosis (M). Normally these four phases occur sequentially, however, the term "cell cycle" also includes modified cycles wherein one or more phases are absent resulting in modified cell cycle such as endomitosis, acytokinesis, polyploidy, polyteny, and endoreduplication.

As used herein, the term "pre-mRNA splicing" includes the biochemical events associated with the nuclear processing of eukaryotic pre-mRNA leading to their conversion into mature mRNA species competent for translation into a protein. "Pre- mRNA splicing" is effectuated by small ribonucleoprotein (snRNPs) particles in association with several non-snRNPs (Staley and Guthrie (1998) Cell 92, 315-326), including DIM1 (Stevens and Abelson (1999) Proc Natl Acad Sci USA 96, 7226-7231 ).

As used herein, the term "vesicle transport/processing" includes all processes leading to the formation, transport, processing and fusion of cellular organelles surrounded by a phospholipid membrane as well as to whole cells surrounded by a phospholipid membrane. The term'Vesicle transport/processing" further includes the biosynthesis, transport, processing and degradation of components of the phospholipid membranes as well as of non-phospholipid components, e.g., membrane proteins or lipoproteins, carried within said phospholipid membranes or within the lumen of vesicles surrounded by said membranes.

As used herein, the term "plant" includes whole plants, plant organs (e.g., leaves, stems, or roots), plant tissue, plant seeds, and plant cells and progeny thereof. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. Particularly preferred plants are Arabidopsis thaliana, rice, wheat, barley, sorghum, maize, tomato, potato, cotton, alfalfa, oilseed rape, soybean, cotton, sunflower or canola. The term plant also includes monocotyledonous (monocot) plants and dicotyledonous (dicot) plants including a fodder or forage legume, ornamental plants, food crops, trees, or shrubs selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chaenomeles spp.,Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Diheteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia villosa, Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flemingia spp, Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemarthia altissima, Heteropogon contortus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hyperthelia dissoluta, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago sativa, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthria fleckii, Pogonarthria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys verticillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, brussel sprout, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugarbeet, sugar cane, sunflower, tomato, squash, and tea, amongst others, or the seeds of any plant specifically named above or a tissue, cell or organ culture of any of the above species.

The term "plant cell", as used herein includes seeds, seed suspension cultures, embryos, cells from meristematic regions, cells from callus tissue, cells from leaves, cells from roots, cells from shoots, gametophytes, sporophytes, pollen, and microspores The DIMIC molecules of the present invention are involved in the regulation of cell cycle and/or pre-mRNA splicing and/or vesicle transport/processing, or part of any of these processes in plants, fungi and animals. Accordingly, the DIMIC molecules of the present invention, or derivatives thereof, may be used to modulate the cell cycle and/or pre-mRNA splicing and/or vesicle transport processing, or part of any of these processes in an organism by, for example, modulating the activity or level of expression of a DIMIC molecule of the present invention. In plants, the DIMIC molecules of the present invention may be used in agriculture to, for example, improve the growth characteristics of a plant such as the growth rate of a plant; the size of specific tissues or organs in a plant; or the architecture or morphology of a plant. The DIMIC molecules of the present invention may also be used in agriculture to increase crop yield, improve tolerance to environmental stress conditions (such as drought, salt, temperature, or nutrient deprivation), improve tolerance to plant pathogens that abuse the cell cycle, or as targets to facilitate the identification of inhibitors or activators of DIMs or DIMICs that may be useful as phytopharmaceuticals, herbicides or plant growth regulators. The DIMIC molecules of the present invention may also be used, e.g., in agriculture, to treat a cell cycle associated disorder.

As used herein, the term "cell cycle associated disorder" includes a disorder, disease or condition which is caused or characterized by a misregulation (e.g., downregulation or upregulation), abuse, arrest, or modification of the cell cycle. In plants cell cycle associated disorders include endomitosis, acytokinesis, polyploidy, polyteny, and endoreduplication which may be caused by external factors such as pathogens (nematodes, viruses, fungi, or insects), chemicals, environmental stress (e.g., drought, temperature, nutrients, or UV light) resulting in, for example, neoplastic tissue (e.g., galls, root knots) or inhibition of cell division/proliferation (e.g., stunted growth). Cell cycle associated disorders in animals include proliferative disorders or differentiative disorders, such as cancer, e.g., melanoma, prostate cancer, servical cancer, breast cancer, colon cancer, or sarcoma.

The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as DIMIC protein and nucleic acid molecules, which comprise a family of molecules having certain conserved structural and functional features. The term "family" when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non- naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of plant, e.g. Arabidopsis, origin, as well as other, distinct proteins of plant, e.g., Arabidopsis, origin or alternatively, can contain homologues of other plants, e.g., rice, or of non-plant origin. Members of a family may also have common functional characteristics.

In one embodiment of the invention, a DIMIC protein of the present invention, is identified based on the presence of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fiveteen, at least sixteen, at least seventeen, at least eightteen, at least nineteen, at least twenty, or at least more more of the following motifs:

A. FKBP domain

As used herein, the term "FKBP domain" includes a domain of about 16-20 amino acid residues in length and which has the following consensus pattern:

[L7I/V/M/C] X [Y/F] X [G V/L] X^ [LJF/T] X₂ G X₃ [D/E] [S T/A/E/Q/K] [S/T/A/N] (SEQ ID NO 54), with 'X' being any amino acid residue, "X_n" being a stretch of "n" random amino acid residues and, e.g., '[Y/F]' meaning either a tyrosine or phenylalanine residue occurring at that position.

B. WW or WWP domain

As used herein, the term "WW domain" or "WWP domain" includes a domain of about 27-30 amino acid residues in length and which has the following consensus pattern: WX₂₂WX₂P (SEQ ID NO 55), with "X" being any amino acid and "X_n" being a stretch of n of Xs. WW domains are, typically, small and compact globular structures that interact with proline-rich ligands (Bedford et al. (1997) EMBO J 16, 2376-2383; Chan et al. (1996) EMBO J 15, 1045-1054; Einbond and Sudol (1996) FEBS Lett 384, 1 -8).

C. Non-classical Cp-domain

As used herein, the term "non-classical C₂-domain" includes a C₂-domain as present in human and mouse polyglutamine tract-binding protein (PQBP-1 ) and includes a domain of about 30-35, preferably about 32-33, amino acid residues in length and which has the following consensus pattern: KKX₅D[D/E]ELDPMDPSSYSDAPRGXWX₂GLX₀-ιK (SEQ ID NO 56) with X being any amino acid and Xn being a stretch of n of Xs and [D/E] being either an aspartate or glutamate residue at that position.

Most proteins containing C₂-domains are functional in signal transduction or membrane traffic. Phospholipid binding to many C₂-domains is regulated by Ca²⁺ and, therefore, C₂-domain proteins are implicated in Ca²⁺-dependent phospholipid signalling (Rizo and Sϋdhof (1998) J Biol Chem 273, 15879-15882).

P. DIMIC5 internal repeat domain

As used herein, the term "DIMIC5 internal repeat domain" includes a domain of about 5-10, preferably 7, amino acid residues in length and which has the following consensus pattern: GGWXVGL (SEQ ID NO 57) with X being any amino acid.

E. FAB1 activation loop

As used herein, the term "FAB1 activation loop" includes a domain of about 18-22, preferably 19, amino acid residues in length and which has the following consensus pattern: T[F/Y]T[W/L]DKKLE[S/T/M]WVKXXG[I/L][V/L]G (SEQ ID NO 58) with the with X being any amino acid.

This motif may be involved in defining Ptdlns3P as the substrate for 5- phosphorylation (McEwen et al. (1999) J. Biol. Chem. 274, 33905-33912).

F. DIMIC7 internal repeat domains

As used herein, the term "DIM1C7 internal repeat domain" or "motif DIMIC7/N" includes domains (numbered by 'N' in DIMIC7/N annotation) of about 6-8 amino acid residues in length and which have one of the following consensus patterns: Motif DIMIC7/1 : PLGR[F/W/Y][M/I/L/V] (SEQ ID NO 59); Motif DIMIC7/2: EXXG[R/K H]IW (SEQ ID NO 60); Motif DIMIC7/3: DLXXPT[M/I/L/V] (SEQ ID NO 61 ); Motif DIMIC7/4: DDXXSXYF (SEQ ID NO 62);and Motif DIMIC7/5: TEXSDXLN (SEQ ID NO 63); with X being any amino acid and, e.g., [D/E] being either an aspartate or glutamate residue at that position.

G. DIMIC26 internal repeat domains

As used herein, the term "DIMIC26 internal repeat domain" or "motif DIMIC26/N" includes domains (numbered by 'N' in DIMIC26/N annotation) of about 6-9 amino acid residues in length and which have one of the following consensus patterns: Motif DIMIC26/1 : CXCXIC (SEQ ID NO 64); Motif DIMIC26/2: ACNRXXE[M/I/L V][M/I/LΛ ](SEQ ID NO 65); Motif DIMIC26/3: QXSGGG (SEQ ID NO 66); Motif DIMIC26/4: [M/I/1JV]DX[M/I/LW]KXGL (SEQ ID NO 67); Motif DIMIC26/5: SEXXAEKQ(SEQ ID NO 68); and

Motif DIMIC26/6: RLXXAEA[D/E](SEQ ID NO 69); with X being any amino acid and, e.g., [D/E] being either an aspartate or glutamate residue at that position.

H. DIMIC26 di-amino acid motifs As used herein, the term "DIMIC26 di-amino acid motifs" includes domains of 2 amino acid residues in length and which have the following consensus patterns: [M/l/L V] [R/K/H]; and [R/K/H] [M/I/L V] with, e.g., [R/K/H] being either an arginine, lysine or histidine residue at that position.

I. Thioredoxin-like domain

As used herein, the term "thioredoxin-like domain" includes a domain of about 4 amino acid residues in length and which has the following consensus patterns: CXXC (SEQ ID NO 70) (Wang and Chang (1999) EMBO J 18, 5972-5982) with X being any amino acid.

J. PEST sequence

As used herein, the term "PEST sequence" includes an amino acid domain of variable length which is enriched in the amino acid residues proline, glutamate, serine and/or threonine. Potential PEST sequences can be identified using the PESTFIND software (can be downloaded from http://www.ebi.ac.uk). The presence of a PEST sequence in a protein is indicative of a high turnover rate, i.e., low stability or short half- life, of said protein (Rogers et a/ (1986) Science 234, 364-368).

K. PHD finger The PHD finger is a C4HC3 zinc-finger-like motif found in nuclear proteins thought to be involved in chromatin-mediated transcriptional regulation (Gibson et al (1995) Trends Biochem. Sci. 20: 56-59). The PHD finger motif is reminiscent of, but distinct from the C3HC4 type RING finger. The function of this domain is not yet known but in analogy with the LIM domain it could be involved in protein-protein interaction and be important for the assembly or activity of multicomponent complexes involved in transcriptional activation or repression. In similarity to the RING finger and the LIM domain, the PHD finger is thought to bind two zinc ions.

Isolated DIMIC proteins of the present invention have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NOs 49-53 and 95 or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NOs 35-48 or 94.

As used herein, the term "sufficiently identical" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains have at least 30%, 40%, or 50% identity, preferably 60% identity, more preferably 70%-80%, and even more preferably 90-95% identity across the amino acid sequences of the domains and contain at least one, or at least two or three or four structural domains or motifs, are defined herein as sufficiently identical. Furthermore, amino acid or nucleotide sequences which share at least 30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or 90-95% identity and share a common functional activity are defined herein as sufficiently identical.

As used interchangeably herein, a "DIMIC activity", "biological activity of DIMIC", or "functional activity of DIMIC", refers to an activity exerted by a DIMIC protein, polypeptide or nucleic acid molecule on a DIMIC responsive cell or tissue, or on a DIMIC protein substrate, as determined in vivo, or in vitro, according to standard techniques. In one embodiment, a DIMIC activity is a direct activity, such as an association with DIMIC- target molecule, e.g., DIML As used herein, a "target molecule" or "binding partner" is a molecule with which a DIMIC protein binds or interacts in nature, such that DIMIC- mediated function is achieved. A DIMIC target molecule can be a non-DIMIC molecule, or a DIMIC protein or polypeptide of the present invention. In an exemplary embodiment, a DIMIC target molecule is a DIMIC ligand. Alternatively, a DIMIC activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the DIMIC protein with a DIMIC ligand. The biological activities of DIMICs are described herein.

For example, the DIMIC proteins of the present invention can have one or more of the following functions: (1) they may act in the cell cycle, more specifically in cell cycle processes including but not limited to G2/M transition or chromosome movement and segregation, spindle formation and elongation, cytokinesis, or regulation of the APC/C; (2) they may modulate pre-mRNA splicing; and (3) they may modulate vesicle transport or processing.

Accordingly, another embodiment of the present invention features isolated DIMIC proteins and polypeptides having a DIMIC activity. Preferred proteins are DIMIC proteins, e.g., DIMIC proteins from plants, having at least one or more of the following domains: a "WW or WWP domain", a "non-classical C₂-domain", a "FAB1 activation loop", a "DIMIC5 internal repeat domain", a "DIMIC7 internal repeat domain", a "DIMIC26 internal repeat domain", a "DIMIC26 di-amino acid motif", a "thioredoxin-like domain" and/or a "PEST sequence", and, preferably, a DIMIC activity activity.

Additional preferred proteins, e.g., DIMIC proteins from plants, have at least one or more of the following domains: a "WW or WWP domain", a "non-classical C₂-domain", a "FAB1 activation loop", a "DIMIC5 internal repeat domain", a "DIMIC7 internal repeat domain", a "DIMIC26 internal repeat domain", a "DIMIC26 di-amino acid motif", a "thioredoxin-like domain" and/or a "PEST sequence" and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs 35-48.

The sequences of the present invention are summarized below, in Table I.

TABLE I

Various aspects of the invention are described in further detail in the following subsections:

I. Isolated Nucleic Acid Molecules One aspect of the invention pertains to isolated nucleic acid molecules that encode DIMIC proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify DIMIC-encoding nucleic acids (e.g., DIMIC mRNA) and fragments for use as PCR primers for the amplification or mutation of DIMIC nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term "isolated" includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated DIMIC nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOs 35-48 or 94, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, using all or portion of the nucleic acid sequence of SEQ ID NOs 35-48 or 94, as a hybridization probe, DIMIC nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NOS 35-48 or 94can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NOS 35-48, respectively.

A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to DIMIC nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NOs 35-48 or 94.

In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NOs35-48, or a portion of any of these nucleotide sequences. A nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NOs 35-48 or 94, is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOs 35-48 or 94, respectively, such that it can hybridize to the nucleotide sequence shown in SEQ ID NOs 35-48 or 94, respectively, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NOs 35-48 or 94, or a portion of any of these nucleotide sequences.

Moreover, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NOs 35-48 or 94, for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a DIMIC protein. The nucleotide sequence determined from the cloning of the DIMIC gene allows for the generation of probes and primers designed for use in identifying and/or cloning other DIMIC family members, as well as DIMIC homologues from other species. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NOs 35-48 or 94, or of a naturally occurring allelic variant or mutant of SEQ ID NOs 35-48. In an exemplary embodiment, a nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NOs 35-48 or 94. Probes based on the DIMIC nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which misexpress a DIMIC protein, such as by measuring a level of a DIMIC-encoding nucleic acid in a sample of cells from a subject e.g., detecting DIMIC mRNA levels or determining whether a genomic DIMIC gene has been mutated or deleted.

A nucleic acid fragment encoding a "biologically active portion of a DIMIC protein" can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NOs 35-48 or 94, which encodes a polypeptide having a DIMIC biological activity (the biological activities of the DIMIC proteins are described herein), expressing the encoded portion of the DIMIC protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the DIMIC protein. The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NOs 35-48 or 94, due to the degeneracy of the genetic code and, thus, encode the same DIMIC proteins as those encoded by the nucleotide sequence shown in SEQ ID NOs 35-48 or 94. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a DIMIC protein.

In addition to the DIMIC nucleotide sequences shown in SEQ ID NOs 35-48 or 94, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the DIMIC proteins may exist within a population (e.g., an Arabidopsis or rice plant population). Such genetic polymorphism in the DIMIC genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules which include an open reading frame encoding an DIMIC protein, preferably a plant DIMIC protein, and can further include non-coding regulatory sequences, and introns. Such natural allelic variations include both functional and non-functional DIMIC proteins and can typically result in 1-5% variance in the nucleotide sequence of a DIMIC gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in DIMIC genes that are the result of natural allelic variation and that do not alter the functional activity of a DIMIC protein are intended to be within the scope of the invention. Natural allelic variants are further include molecules that comprise single nucleotide polymorphisms (SNPs) as well as small insertion/deletion polymorphisms (INDELs; the size of INDELs is usually less than about 100 bp). SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms. They are helpful in mapping genes and in discovery of genes and gene functions. They are furthermore helpful in the identification of genetic loci, e.g., plant genes, involved in determining processes such as growth rate, plant size and plant yield, plant vigor, disease resistance, stress tolerance and the like. Many techniques are nowadays available to identify SNPs and/or INDELs including (i) PCR followed by denaturing high performance liquid chromatography (DHPLC; e.g., Cho ef al. (1999) Nature Genet 23, 203-207); (ii) constant denaturant capillary electrophoresis (CDCE) combined with high-fidelity PCR (e.g., Li-Sucholeiki ef al. (1999) Electrophoresis 20, 1224-1232); (iii) denaturing gradient gel electrophoresis (Fischer and Lerman (1983) Proc. Natl. Acad. Sci. USA 80, 1579-1583); (iv) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; e.g., Ross ef al (2000) Biotechniques 29, 620-629); (v) real-time fluorescence monitoring PCR assays (Tapp et al (2000) Biotechniques 28, 732-738); (vi) AcryditeTM gel technology (Kenney et al (1998) Biotechniques 25, 516-521); (vii) cycle dideoxy fingerprinting (CddF; Langemeier ef al (1994) Biotechniques 17, 484-490); (viii) single-strand conformation polymorphism (SSCP) analysis (Vidal-Puig and Moller (1994) Biotechniques 17, 490-496) and (ix) mini-sequencing primer extension reaction (Syvanen (1999) Hum Mutat 13, 1- 10). The technique of Targeting Induced Local Lesions in Genomes' (TILLING; McCallum et al. (2000) Nat. Biotechnol Λ3, 455-457; Plant Physiol 123, 439-442), which is a variant of (i) supra, can also be applied to rapidly identify an altered gene in, e.g., chemically mutagenized plant individuals showing interesting phenotypes.

Differences in preferred codon usage are illustrated below for Agrobacterium tumefaciens (a bacterium), Arabidopsis thaliana, Medicago sativa (two dicotyledonous plants) and Oryza sativa (a monocotyledonous plant). These examples were extracted from http://www.kazusa.or.ip/codon. For example, the codon GGC (for glycine) is the most frequently used codon in A. tumefaciens (36.2 %₀), is the second most frequently used codon in O. sativa but is used at much lower frequencies in A. thaliana and M. sativa (9 %o and 8.4 %o , respectively). Of the four possible codons encoding glycine the GGC codon is most preferably used in A. tumefaciens and O. sativa. However, in A. thaliana the GGA (and GGU) codon is most preferably used, whereas in M. sativa the GGU (and GGA) codon is most preferably used.

Moreover, nucleic acid molecules encoding other DIMIC family members and, thus, which have a nucleotide sequence which differs from the DIMIC sequences of SEQ ID NOs 35-48 or 94 are intended to be within the scope of the invention. For example, another DIMIC cDNA can be identified based on the nucleotide sequence of the plant DIMIC moleculels described herein. Moreover, nucleic acid molecules encoding DIMIC proteins from different species, and thus which have a nucleotide sequence which differs from the DIMIC sequences of SEQ ID NOs 35-48 or 94 are intended to be within the scope of the invention. For example, a human DIMIC cDNA can be identified based on the nucleotide sequence of a plant DIMIC.

Nucleic acid molecules corresponding to natural allelic variants and homologues of the DIMIC cDNAs of the invention can be isolated based on their homology to the DIMIC nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.

Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs 35-48. In other embodiment, the nucleic acid is at least 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 nucleotides in length. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 30%, 40%, 50%, or 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1 % SDS at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1 % SDS at 50°C, preferably at 55°C, more preferably at 60°C, and even more preferably at 65°C. Ranges intermediate to the above-recited values, e.g., at 60-65 °C or at 55-60 °C are also intended to be encompassed by the present invention. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NOs 35-48 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of the DIMIC sequences that may exist in nature, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NOs 35-48 or 94, thereby leading to changes in the amino acid sequence of the encoded DIMIC proteins, without altering the functional ability of the DIMIC proteins. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of a DIMIC protein. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of DIMIC without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the DIMIC proteins of the present invention, are predicted to be particularly unamenable to alteration. Furthermore, additional amino acid residues that are conserved between the DIMIC proteins of the present invention and other DIMIC family members are not likely to be amenable to alteration. Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding DIMIC proteins that contain changes in amino acid residues that are not essential for activity.

An isolated nucleic acid molecule encoding a DIMIC protein homologous to the DIMIC proteins of the present invention can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOs 35-48 or 94, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NOs 35-48 or 94 by standard techniques, such as site-directed mutagenesis and PCR- mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a DIMIC protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a DIMIC coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for DIMIC biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NOs 35-48 or 94, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

In a preferred embodiment, a mutant DIMIC protein can be assayed for the ability to: (1 ) modulate cell cycle processes including but not limited to G2/M transition or chromosome movement and segregation, spindle formation and elongation, cytokinesis, or regulation of the APC/C; (2) modulate pre-mRNA splicing; (3) modulate vesicle transport or processing; or (4) interact with DIM1 in, e.g., a yeast two hybrid assay.

In addition to the nucleic acid molecules encoding DIMIC proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire DIMIC coding strand, or only to a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding DIMIC. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding DIMIC. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).

Given the coding strand sequences encoding DIMIC disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of DIMIC mRNA, but more, preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of DIMIC mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of DIMIC mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino- 3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection). Preferably, production of antisense nucleic acids in plants occurs by means of a stably integrated transgene comprising a promoter operative in plants, an antisense oligonucleotide, and a terminator. Other known nucleotide modifications include methylation, cyclization and 'caps' and substitution of one or more of the naturally occurring nucleotides with an analog such as inosine. Modifications of nucleotides include the addition of acridine, amine, biotin, cascade blue, cholesterol, Cy3^®, Cy5^®, Cy5.5^® Dabcyl, digoxigenin, dinitrophenyl, Edans, 6-FAM, fluorescein, 3'-glyceryl, HEX, IRD-700, IRD-800, JOE, phosphate psoralen, rhodamine, ROX, thiol (SH), spacers, TAMRA, TET, AMCA-S^®, SE, BODIPY^®, Marina Blue^®, Pacific Blue^®, Oregon Green^®, Rhodamine Green^®, Rhodamine Red^®, Rhodol Green^® and Texas Red^®. Polynucleotide backbone modifications include methylphosphonate, 2'-OMe-methylphosphonate RNA, phosphorothiorate, RNA, 2'-OMeRNA. Base modifications include 2-amino-dA, 2- aminopurine, 3'-(ddA), 3'dA(cordycepin), 7-deaza-dA, 8-Br-dA, 8-oxo-dA, N⁶-Me-dA, abasic site (dSpacer), biotin dT, 2'-OMe-5Me-C, 2'-OMe-propynyl-C, 3'-(5-Me-dC), 3'- (ddC), 5-Br-dC, 5-l-dC, 5-Me-dC, 5-F-dC, carboxy-dT, convertible dA, convertible dC, convertible dG, convertible dT, convertible dU, 7-deaza-dG, 8-Br-dG, 8-oxo-dG, 0⁶-Me- dG, S6-DNP-dG, 4-methyi-indole, 5-nitroindole, 2'-OMe-inosine, 2'-dl, 0⁶-phenyl-dl, 4- methyl-indole, 2'-deoxynebularine, 5-nitroindole, 2-aminopurine, dP(purine analogue), dK(pyrimidine analogue), 3-nitropyrrole, 2-thio-dT, 4-thio-dT, biotin-dT, carboxy-dT, O⁴- Me-dT, 0 -triazol dT, 2'-OMe-propynyl-U, 5-Br-dU, 2'-dU, 5-F-dU, 5-l-dU, 0⁴-triazol dU.

The antisense nucleic acid molecules of the invention are typically introduced into a plant or administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a DIMIC protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of introduction or administration of antisense nucleic acid molecules of the invention include transformation in a plant or direct injection at a tissue site in a subject. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a constitutive promoter or a strong pol II or pol III promoter are preferred. In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625- 6641 ). The antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

In another embodiment, the antisense nucleic acid molecule further comprises a sense nucleic acid molecule complementary to the antisense nucleic acid molecule. Gene silencing methods based on such nucleic acid molecules are well known to the skilled artisan (e.g., Grierson et al. (1998) WO 98/53083; Waterhouse et al. (1999) WO 99/53050).

In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591 )) can be used to catalytically cleave DIMIC mRNA transcripts to thereby inhibit translation of DIMIC mRNA. A ribozyme having specificity for a DIMIC-encoding nucleic acid can be designed based upon the nucleotide sequence of a DIMIC cDNA disclosed herein (i.e., SEQ ID NOs 35-48). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a DIMIC-encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071 ; and Cech ef al. U.S. Patent No. 5,116,742. Alternatively, DIMIC mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261 -.1411-1418.

The use of ribozymes for gene silencing in plants is known in the art (e.g., Atkins et al. (1994) WO 94/00012; Lenne et al. (1995) WO 95/03404; Lutziger et al. (2000) WO 00/00619; Prinsen ef al. (1997) WO 97/13865 and Scott ef al. (1997) WO/ 97/381 16). Alternatively, DIMIC gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the DIMIC (e.g., the DIMIC promoter and/or enhancers) to form triple helical structures that prevent transcription of the DIMIC gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. ef al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14(12):807-15.

In yet another embodiment, the DIMIC nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675. PNAs of DIMIC nucleic acid molecules can be used for increasing crop yield in plants or in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of DIMIC nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

In another embodiment, PNAs of DIMIC can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of DIMIC nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P.J. ef al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P.J. ef al. (1996) supra). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).

In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad.

^■ Sci. US. 86:6553-6556; Lemaitre ef al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652;

PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication

No. W089/10134). In addition, oligonucleotides can be modified with hybridization- triggered cleavage agents (See, e.g., Krol ef al. (1988) Bio-Techniques 6:958-976) or intercalating agents (see, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

II. Isolated DIMIC Proteins and Anti-DIMIC Antibodies

One aspect of the invention pertains to isolated DIMIC proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-DIMIC antibodies. In one embodiment, native DIMIC proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, DIMIC proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a DIMIC protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the DIMIC protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of DIMIC protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of DIMIC protein having less than about 30% (by dry weight) of non-DIMIC protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-DIMIC protein, still more preferably less than about 10% of non-DIMIC protein, and most preferably less than about 5% non-DIMIC protein. When the DIMIC protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

The language "substantially free of chemical precursors or other chemicals" includes preparations of DIMIC protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of DIMIC protein having less than about 30% (by dry weight) of chemical precursors or non-DIMIC chemicals, more preferably less than about 20% chemical precursors or non-DIMIC chemicals, still more preferably less than about 10% chemical precursors or non-DIMIC chemicals, and most preferably less than about 5% chemical precursors or non-DIMIC chemicals.

Biologically active portions of a DIMIC protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the DIMIC protein, which include less amino acids than the full length DIMIC proteins, and exhibit at least one activity of a DIMIC protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the DIMIC protein. A biologically active portion of a DIMIC protein can be a polypeptide which is, for example, at least 10, 25, 50, 100 or more amino acids in length.

To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, ef al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to DIMIC nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to DIMIC protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul ef al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

The invention also provides DIMIC chimeric or fusion proteins. As used herein, a DIMIC "chimeric protein" or "fusion protein" comprises a DIMIC polypeptide operatively linked to a non-DIMIC polypeptide. An "DIMIC polypeptide" refers to a polypeptide having an amino acid sequence corresponding to DIMIC, whereas a "non-DIMIC polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the DIMIC protein, e.g., a protein which is different from the DIMIC protein and which is derived from the same or a different organism. The non- DIMIC polypeptide can, for example, be (histidine)₆-tag, glutathione S-transferase, protein A, maltose-binding protein, dihydrofolate reductase, Tag«100 epitope (EETARFQPGYRS; SEQ ID NO 75), c-myc epitope (EQKLISEEDL; SEQ ID NO 76), FLAG^®-epitope (DYKDDDK; SEQ ID NO 77), lacZ, CMP (calmodulin-binding peptide), HA epitope (YPYDVPDYA; SEQ ID NO 78), protein C epitope (EDQVDPRLIDGK; SEQ ID NO 79) or VSV epitope (YTDIEMNRLGK; SEQ ID NO 80).

Within a DIMIC fusion protein the DIMIC polypeptide can correspond to all or a portion of a DIMIC protein. In a preferred embodiment, a DIMIC fusion protein comprises at least one biologically active portion of a DIMIC protein. In another preferred embodiment, a DIMIC fusion protein comprises at least two biologically active portions of a DIMIC protein. Within the fusion protein, the term "operatively linked" is intended to indicate that the DIMIC polypeptide and the non-DIMIC polypeptide are fused in-frame to each other. The non-DIMIC polypeptide can be fused to the N-terminus or C-terminus of the DIMIC polypeptide.

For example, in one embodiment, the fusion protein is a GST-DIMIC fusion protein in which the DIMIC sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant DIMIC.

In another embodiment, the fusion protein is a DIMIC protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., plant or mammalian host cells), expression and/or secretion of DIMIC can be increased through use of a heterologous signal sequence.

The DIMIC fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a plant or a subject in vivo. The DIMIC fusion proteins can be used to affect the bioavailability of a DIMIC substrate. Use of DIMIC fusion proteins may be useful agriculturally for the increase of crop yields or therapeutically for the treatment of cellular growth related disorders, e.g., cancer. Moreover, the DIMIC-fusion proteins of the invention can be used as immunogens to produce anti-DIMIC antibodies in a subject, to purify DIMIC ligands and in screening assays to identify molecules which inhibit the interaction of DIMIC with a DIMIC substrate, e.g., a kinase such as CDC2b. Preferably, a DIMIC chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A DIMIC- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the DIMIC protein.

The present invention also pertains to variants of the DIMIC proteins which function as either DIMIC agonists (mimetics) or as DIMIC antagonists. Variants of the DIMIC proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of a DIMIC protein. An agonist of the DIMIC proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a DIMIC protein. An antagonist of a DIMIC protein can inhibit one or more of the activities of the naturally occurring form of the DIMIC protein by, for example, competitively modulating a cellular activity of a DIMIC protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the DIMIC protein.

In one embodiment, variants of a DIMIC protein which function as either DIMIC agonists (mimetics) or as DIMIC antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a DIMIC protein for DIMIC protein agonist or antagonist, activity. In one embodiment, a variegated library of DIMIC variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of DIMIC variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential DIMIC sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of DIMIC sequences therein. There are a variety of methods which can be used to produce libraries of potential DIMIC variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential DIMIC sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura ef al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477.

In addition, libraries of fragments of a DIMIC protein coding sequence can be used to generate a variegated population of DIMIC fragments for screening and subsequent selection of variants of a DIMIC protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a DIMIC coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the DIMIC protein.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of DIMIC proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recrusive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify DIMIC variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze a variegated DIMIC library. For example, a library of expression vectors can be transfected into a cell line which ordinarily synthesizes and secretes DIMIC. The transfected cells are then cultured such that DIMIC and a particular mutant DIMIC are secreted and the effect of expression of the mutant on DIMIC activity in cell supernatants can be detected, e.g., by any of a number of enzymatic assays. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of DIMIC activity, and the individual clones further characterized.

An isolated DIMIC protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind DIMIC using standard techniques for polyclonal and monoclonal antibody preparation. A full-length DIMIC protein can be used or, alternatively, the invention provides antigenic peptide fragments of DIMIC for use as immunogens. The antigenic peptide of DIMIC comprises at least 8 amino acid residues and encompasses an epitope of DIMIC such that an antibody raised against the peptide forms a specific immune complex with DIMIC. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions of DIMIC that are located on the surface of the protein, e.g., hydrophilic regions.

A DIMIC immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed DIMIC protein or a chemically synthesized DIMIC polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic DIMIC preparation induces a polyclonal anti-DIMIC antibody response. Accordingly, another aspect of the invention pertains to anti-DIMIC antibodies.

The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as DIMIC. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind DIMIC. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of DIMIC. A monoclonal antibody composition thus typically displays a single binding affinity for a particular DIMIC protein with which it immunoreacts.

Polyclonal anti-DIMIC antibodies can be prepared as described above by immunizing a suitable subject with a DIMIC immunogen. The anti-DIMIC antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized DIMIC. If desired, the antibody molecules directed against DIMIC can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-DIMIC antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown ef al. (1980) J. Biol. Chem .255:4980-83; Yeh ef al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31 ; and Yeh et al. (1982) /nf. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor ef al. (1983) Immunol Today 4:72), the EBV- hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter ef al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a DIMIC immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds DIMIC.

Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-DIMIC monoclonal antibody (see, e.g., G. Galfre ef al. (1977) Nature 266:55052; Gefter et al. Somatic Cell Genet, cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1 -Ag4-1 , P3-x63-Ag8.653 or Sp2/0-Ag14 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind DIMIC, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-DIMIC antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with DIMIC to thereby isolate immunoglobulin library members that bind DIMIC. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01 ; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang ef al. PCT International Publication No. WO 92/18619; Dower ef al. PCT International Publication No. WO 91/17271 ; Winter et al. PCT International Publication WO 92/20791 ; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty ef al. PCT International Publication No. WO 92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner ef al. PCT International Publication No. WO 90/02809; Fuchs ef al. (1991 ) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81 -85; Huse et al. (1989) Science 246: 1275-1281 ; Griffiths et al. (1993) EMBO d 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991 ) Nature 352:624-628; Gram ef al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad ef al. (1991 ) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137; Barbas ef al. (1991) Proc. Natl. Acad. Sci.^' USA 88:7978-7982; and McCafferty ef al. Nature (1990) 348:552-554.

Additionally, recombinant anti-DIMIC antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, ef al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171 ,496; Morrison ef al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly ef al. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura ef al. (1987) Cane. Res. 47:999-1005; Wood ef al. (1985) Nature 314:446-449; and Shaw ef al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi ef al. (1986) BioTechniques 4:214; Winter U.S. Patent 5,225,539; Jones ef al. (1986) Nature 321 :552-525; Verhoeyan ef al. (1988) Science 239:1534; and Beidler ef al. (1988) J. Immunol. 141 :4053-4060.

An anti-DIMIC antibody (e.g., monoclonal antibody) can be used to isolate DIMIC by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-DIMIC antibody can facilitate the purification of natural DIMIC from cells and of recombinantly produced DIMIC expressed in host cells. Moreover, an anti-DIMIC antibody can be used to detect DIMIC protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the DIMIC protein. These antibodies can also be used, for example, for the immunoprecipitation and immunolocalization of proteins according to the invention as well as for the monitoring of the synthesis of such proteins, for example, in recombinant organisms, and for the identification of compounds interacting with the protein according to the invention.

Anti-DIMIC antibodies can be used diagnosticaily to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ' 1, ° I, S or °H. "Homologues" or "Homologs" of a protein of the invention are those peptides, oligopeptides, polypeptides, proteins and enzymes which contain amino acid substitutions, deletions and/or additions relative to the said protein with respect to which they are a homologue without altering one or more of its functional properties, in particular without reducing the activity of the resulting product. For example, a homologue of said protein will consist of a bioactive amino acid sequence variant of said protein. To produce such homologues, amino acids present in the said protein can be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity, propensity to form or break α-helical structures or β- sheet structures, and so on. Two special forms of homology, orthologous and paralogous, are evolutionary concepts used to describe ancestral relationships of genes. The term "paralogous" relates to gene-duplications within the genome of a species leading to paralogous genes. The term "orthologous" relates to homologous genes in different organisms due to ancestral relationship. The present invention thus also relates to homologues, paralogues and orthologues of the genes and proteins of the invention. The paralogues or orthologues of the genes and proteins of the invention may have a lesser percentage of sequence identity with the sequences or proteins of the invention than the strictly interpreted "homologues" as defined earlier.

"Derivatives" of a protein of the invention are those peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise additional naturally-occurring, altered glycosylated, acylated or non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally-occurring form of said polypeptide. Alternatively or in addition, a derivative may comprise one or more non-amino acid substituents compared to the amino acid sequence of a naturally-occurring form of said polypeptide, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence such as, for example, a reporter molecule which is bound thereto to facilitate its detection. A derivative of a protein retains the biological or enzymatical activity of the protein where it is derived from.

III. Computer Readable Means The nucleotide or amino acid sequences of the invention are also provided in a variety of mediums to facilitate use thereof. As used herein, "provided" refers to a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a nucleotide or amino acid sequences of the present invention. Such a manufacture provides the nucleotide or amino acid sequences, or a subset thereof (e.g., a subset of open reading frames (ORI's)) in a form which allows a skilled artisan to examine the manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exist in nature or in purified form.

In one application of this embodiment, a nucleotide or amino acid sequence of the present invention can be recorded on computer readable media. As used herein "computer readable media" includes any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such a CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. The skilled artisan will readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention.

As used herein "recorded" refers to a process of storing information on computer readable medium. The skilled artisan can readily adopt any of the presently known methods for recording information on a computer readable medium to generate manufactures comprising the nucleotide or amino acid sequence information of the present invention.

A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially- available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase Oracle, or the like. The skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention. By providing the nucleotide or amino acid sequences of the invention in computer readable form, the skilled artisan can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the nucleotide or amino acid sequences of the invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identity fragments or regions of the sequences of the invention which match a particular target sequence or target motif.

As used herein, a "target sequence" can be any DNA or amino acid sequence of six or more nucleotide or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. The most preferred sequence length of a target sequence is from about 10 to 100 amino acids or form about 30 to 300 nucleotide residues. However, it is well recognized that commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be shorter length. As used herein, "a target structural motif," or "target motif," refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).

Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences. A variety of known algorithms are disclosed publicly and a variety of commercially available software of conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software include, but are not limited to, MacPatter (EMBL), BLASTN and BASTX (NCBIA).

For example, software which implements the BLAST (Altschul ef al. (1990J J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al. (1993) Comp. Chem. 17:203-207) search algorithms on a Sybase system can be used to identify open reading frames (ORFs) of the sequences of the invention which contain homology to ORFs or proteins from other libraries. Such ORFs are protein encoding fragments and are useful in producing commercially important proteins such as enzyme used in various reactions and in the production of commercially useful metabolites.

IV. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a DIMIC protein (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, e.g., a plant cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., DIMIC proteins, mutant forms of DIMIC proteins, fusion proteins, and the like).

The vectors of the invention comprise a selectable and/or scorable marker. Selectable marker genes useful for the selection of transformed plant cells, callus, plant tissue and plants are well known to those skilled in the art and comprise, for example, antimetabolite resistance as the basis of selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149); npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera- Estrella, EMBO J. 2 (1983), 987-995) and hygro, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481 -485). Additional selectable genes have been described, namely trpB, which allow cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate isomerase which allows cells to utilize mannose (WO 94/20627) and ODC (omithine decarboxylase) which confers resistance to the omithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-omithine, DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.) or deaminase from Aspergillus terreus which confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338). Useful scorable markers are also known to those skilled in the art and are commercially available. Advantageously, the marker is a gene encoding luciferase (Giacomin, PI. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44-47) or β-glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907). This embodiment is particularly useful for simple and rapid screening of cells, tissues and organisms containing a vector of the invention.

A "plant promoter" is a promoter capable of initiating transcription in plant cells. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria. Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression and/or to alter the spatial expression and/or temporal expression of a nucleic acid molecule to which it is operably connected. For example, copper-responsive, glucocorticoid-responsive or dexamethasone-responsive regulatory elements may be placed adjacent to a heterologous promoter sequence driving expression of a nucleic acid molecule to confer copper inducible, glucocorticoid-inducible, or dexamethasone-inducible expression respectively, on said nucleic acid molecule. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, endosperm, embryos, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as "tissue preferred." Promoters which initiate transcription only in certain tissue are referred to as "tissue specific." A "cell type" specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter which is active under most environmental conditions.

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, a DIMIC protein can be expressed in plant cells, bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or elecfroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, ef al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals. Means for introducing a recombinant expression vector of this invention into plant tissue or cells include, but are not limited to, transformation using CaCI₂ and variations thereof, in particular the method described by Hanahan (J. Mol.Biol. 166, 557-560, 1983), direct DNA uptake into protoplasts (Krens et al, Nature 296: 72-74, 1982; Paszkowski ef al, EMBO J. 3:2717-2722, 1984), PEG-mediated uptake to protoplasts (Armstrong et al, Plant Cell Reports 9: 335-339, 1990) microparticle bombardment, elecfroporation (Fromm et al., Proc. Natl. Acad. Sci. (USA) 82:5824-5828, 1985), microinjection of DNA (Crossway ef al., Mol. Gen. Genet. 202:179-185, 1986), microparticle bombardment of tissue explants or cells (Christou ef al, Plant Physiol 87: 671-674, 1988; Sanford, Particulate Science and Technology 5: 27-37, 1987), vacuum-infiltration of tissue with nucleic acid, or in the case of plants, T-DNA-mediated transfer from Agrobacterium to the plant tissue as described essentially by An ef al.( EMBO J 4:277-284, 1985), Herrera- Estrella ef al. (Nature 303: 209-213, 1983a; EMBO J. 2: 987-995, 1983b; In: Plant Genetic Engineering, Cambridge University Press, N.Y., pp 63-93, 1985), or in planta method using Agrobacterium tumefaciens such as that described by Bechtold ef al., (CR. Acad. Sci. (Paris, Sciences de la vie/ Life Sciences)316: 1194-1199, 1993) or Clough ef al (Plant J. 16: 735-743, 1998). The vector DNA may further comprise a selectable marker gene to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct. Suitable selectable marker genes contemplated herein include the ampicillin resistance (Amp^r), tetracycline resistance gene Tc^r), bacterial kanamycin resistance gene (Kan^r), phosphinothricin resistance gene, neomycin phosphotransferase gene (npfll), hygromycin resistance gene, D-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein (gfp) gene (Haseloff et al, 1997), and luciferase gene.

Methods for transformation of monocotyledonous plants are well known in the art and include Agrobacfer/um-mediated transformation (Cheng ef al. (1997) WO 97/48814; Hansen (1998) WO 98/54961 ; Hiei et al. (1994) WO 94/00977; Hiei ef al. (1998) WO 98/17813; Rikiishi et al. (1999) WO 99/04618; Saito ef al. (1995) WO 95/06722), microprojectile bombardment (Adams et al. (1999) US 5,969,213; Bowen et al. (1998) US 5,736,369; Chang ef al. (1994) WO 94/13822; Lundquist et al. (1999) US 5,874,265/US 5,990,390; Vasil and Vasil (1995) US 5,405,765; Walker ef al. (1999) US 5,955,362), DNA uptake (Eval et al. (1993) WO 93/181 ,168), microinjection of Agrobacterium cells (von Holt 1994 DE 4309203) and sonication (Finer et al. (1997) US 5,693,512).

For microparticle bombardment of cells, a microparticle is propelled into a cell to produce a transformed cell. Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp ef al. (U.S. Patent No. 5,122,466) and Sanford and Wolf (U.S. Patent No. 4,945,050). When using ballistic transformation procedures, the gene construct may incorporate a plasmid capable of replicating in the cell to be transformed. Examples of microparticles suitable for use in such systems include 1 to 5 μm gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.

A whole plant may be regenerated from the transformed or transfected cell, in accordance with procedures well known in the art. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a gene construct of the present invention and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).

The term "organogenesis", as used herein, includes a process by which shoots and roots are developed sequentially from meristematic centres.

The term "embryogenesis", as used herein, includes a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.

Preferably, the plant is produced according to the methods of the invention by transfecting or transforming the plant with a genetic sequence, or by introducing to the plant a protein, by any art-recognized means, such as microprojectile bombardment, microinjection, >Agro acfer/um-mediated transformation (including in planta transformation), protoplast fusion, or elecfroporation, amongst others. Most preferably the plant is produced by Agrobacterium-mediated transformation. Agrobacfer/um-mediated transformation or agrolistic transformation of plants, yeast, moulds or filamentous fungi is based on the transfer of part of the transformation vector sequences, called the T-DNA, to the nucleus and on integration of said T-DNA in the genome of said eukaryote.

The term "Agrobacterium" as used herein, includes a member of the Agrobacteriaceae, more preferably Agrobacterium or Rhizobacterium and most preferably Agrobacterium tumefaciens. The term "T-DNA", or "transferred DNA", as used herein, includes the transformation vector flanked by T-DNA borders which is, after activation of the Agrobacterium vir genes, nicked at the T-DNA borders and is transferred as a single stranded DNA to the nucleus of an eukaryotic cell.

As used herein, the terms "T-DNA borders", "T-DNA border region", or "border region" include either right T-DNA borders (RB) or left T-DNA borders (LB), which comprise a core sequence flanked by a border inner region as part of the T-DNA flanking the border and/or a border outer region as part of the vector backbone flanking the border. The core sequences comprise 22 bp in case of octopine-type vectors and 25 bp in case of nopaline-type vectors. The core sequences in the right border region and left border region form imperfect repeats. Border core sequences are indispensable for recognition and processing by the Agrobacterium nicking complex consisting of at least VirD1 and VirD2. Core sequences flanking a T-DNA are sufficient to promote transfer of the T-DNA. However, efficiency of transformation using transformation vectors carrying the T-DNA solely flanked by the core sequences is low. Border inner and outer regions are known to modulate efficiency of T-DNA transfer (Wang ef al. 1987). One element enhancing T-DNA transfer has been characterized and resides in the right border outer region and is called overdrive (Peralta ef al. 1986, van Haaren ef al. 1987).

As used herein, the term "T-DNA transformation vector" or "T-DNA vector" includes any vector encompassing a T-DNA sequence flanked by a right and left T-DNA border consisting of at least the right and left border core sequences, respectively, and used for transformation of any eukaryotic cell.

As used herein, the term "T-DNA vector backbone sequence" or "T-DNA vector backbone sequences" includes all DNA of a T-DNA containing vector that lies outside of the T-DNA borders and, more specifically, outside the nicking sites of the border core imperfect repeats. The present invention includes optimized T-DNA vectors such that vector backbone integration in the genome of a eukaryotic cell is minimized or absent. The term "optimized T-DNA vector" as used herein includes a T-DNA vector designed either to decrease or abolish transfer of vector backbone sequences to the genome of a eukaryotic cell. Such T-DNA vectors are known to the one of skill in the art and include those described by Hanson et al. (1999) and by Stuiver etal. (1999 - WO9901563).

The current invention clearly considers the inclusion of a DNA sequence encoding a DIMIC, homologue, analogue, derivative or immunologically active fragment thereof as defined supra, in any T-DNA vector comprising binary transformation vectors, super- binary transformation vectors, co-integrate transformation vectors, Ri-derived transformation vectors as well as in T-DNA carrying vectors used in agrolistic transformation.

As used herein, the term "binary transformation vector" includes a T-DNA transformation vector comprising: a T-DNA region comprising at least one gene of interest and/or at least one selectable marker active in the eukaryotic cell to be transformed; and a vector backbone region comprising at least origins of replication active in E. coli and Agrobacterium and markers for selection in E. coli and Agrobacterium. Alternatively, replication of the binary transformation vector in Agrobacterium is dependent on the presence of a separate helper plasmid. The binary vector pGreen and the helper plasmid pSoup form an example of such a system (Hellens et al. (2000) Plant Mol. Biol. 42, 819- 832; http://www.pgreen.ac.uk).

The T-DNA borders of a binary transformation vector can be derived from octopine-type or nopaline-type Ti plasmids or from both. The T-DNA of a binary vector is only transferred to a eukaryotic cell in conjunction with a helper plasmid. As used herein, the term "helper plasmid" includes a plasmid that is stably maintained in Agrobacterium and is at least carrying the set of vir genes necessary for enabling transfer of the T-DNA. The set of vir genes can be derived from either octopine-type or nopaline-type Ti plasmids or from both. As used herein, the term "super-binary transformation vector" includes a binary transformation vector additionally carrying in the vector backbone region a vir region of the Ti plasmid pTiBo542 of the super-virulent A. tumefaciens strain A281 (EP0604662, EP0687730). Super-binary transformation vectors are used in conjunction with a helper plasmid. As used herein, the term "co-integrate transformation vector" includes a T-DNA vector at least comprising: a T-DNA region comprising at least one gene of interest and/or at least one selectable marker active in plants; and a vector backbone region comprising at least origins of replication active in Escherichia coli and Agrobacterium, and markers for selection in E. coli and Agrobacterium, and a set of vir genes necessary for enabling transfer of the T-DNA. The T-DNA borders and the set of vir genes of the T- DNA vector can be derived from either octopine-type or nopaline-type Ti plasmids or from both.

The term "Ri-derived plant transformation vector" includes a binary transformation vector in which the T-DNA borders are derived from a Ti plasmid and the binary transformation vector being used in conjunction with a 'helper' Ri-plasmid carrying the necessary set of vir genes.

The terms "agrolistics", "agrolistic transformation" or "agrolistic transfer" include a transformation method combining features of /.grajbacferutn-mediated transformation and of biolistic DNA delivery. As such, a T-DNA containing target plasmid is co-delivered with DNA/RNA enabling in planta production of VirD1 and VirD2 with or without VirE2

(Hansen and Chilton 1996; Hansen et al. 1997; Hansen and Chilton 1997 - WO9712046).

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a DIMIC protein. Accordingly, the invention further provides methods for producing a DIMIC protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a DIMIC protein has been introduced) in a suitable medium such that a DIMIC protein is produced. In another embodiment, the method further comprises isolating a DIMIC protein from the medium or the host cell. The host cells of the invention can also be used to produce transgenic plant or non-human transgenic animals in which exogenous DIMIC sequences have been introduced into their genome or homologous recombinant plants or animals in which endogenous DIMIC sequences have been altered. Such plants and animals are useful for studying the function and/or activity of a DIMIC and for identifying and/or evaluating modulators of DIMIC activity.

Trangenic Plants

As used herein, "transgenic plant" includes a plant which comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heteroglogous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses as asexual propagation from the initial transgenic. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring event such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

A transgenic plant of the invention can be created by introducing a DIMIC- encoding nucleic acid into the plant by placing it under the control of regulatory elements which ensure the expression in plant cells. These regulatory elements may be heterologous or homologous with respect to the nucleic acid molecule to be expressed as well with respect to the plant species to be transformed. In general, such regulatory elements comprise a promoter active in plant cells. These promoters can be used to modulate (e.g. increase or decrease) DIMIC content and/or composition in a desired tissue. To obtain expression in all tissues of a transgenic plant, preferably constitutive promoters are used, such as the 35 S promoter of CaMV (Odell, Nature 313 (1985), 810- 812) or promoters from such genes as rice actin (McElroy ef al. (1990) Plant Cell 2:163- 171) maize H3 histone (Lepetit ef al. (1992) Mol. Gen. Genet 231 :276-285) or promoters of the polyubiquitin genes of maize (Christensen, Plant Mol. Biol. 18 (1982), 675-689). In order to achieve expression in specific tissues of a transgenic plant it is possible to use tissue specific promoters (see, e.g., Stockhaus, EMBO J. 8 (1989), 2245-2251 or Table II, below).

Table II:

The promoters listed in the foregoing table are provided for the purposes of exemplification only and the present invention is not to be limited by the list provided therein. Those skilled in the art will readily be in a position to provide additional promoters that are useful in performing the present invention. The promoters listed may also be modified to provide specificity of expression as required.

Known are also promoters which are specifically active in tubers of potatoes or in seeds of different plants species, such as maize, Vicia, wheat, barley and the like. Inducible promoters may be used in order to be able to exactly control expression under certain environmental or developmental conditions such as pathogens, anaerobia, or light. Examples of inducible promoters include the promoters of genes encoding heat shock proteins or microspore-specific regulatory elements (W096/16182). Furthermore, the chemically inducible Tet-system may be employed (Gatz, Mol. Gen. Genet. 227 (1991 ); 229-237). Further suitable promoters are known to the person skilled in the art and are described, e.g., in Ward (Plant Mol. Biol. 22 (1993), 361-366). The regulatory elements may further comprise transcriptional and/or translational enhancers functional in plants cells. Furthermore, the regulatory elements may include transcription termination signals, such as a poly-A signal, which lead to the addition of a poly A tail to the transcript which may improve its stability.

In the case that a nucleic acid molecule according to the invention is expressed in the sense orientation, the coding sequence can be modified such that the protein is located in any desired compartment of the plant cell, e.g., the nucleus, endoplasmatic reticulum, the vacuole, the mitochondria, the plastids, the apoplast, or the cytoplasm.

Methods for the introduction of foreign DNA into plants are also well known in the art. These include, for example, the transformation of plant cells or tissues with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes, the fusion of protoplasts, direct gene transfer (see, e.g., EP-A 164 575), injection, electroporation, biolistic methods like particle bombardment, pollen-mediated transformation, plant RNA virus-mediated transformation, liposome-mediated transformation, transformation using wounded or enzyme-degraded immature embryos, or wounded or enzyme-degraded embryogenic callus and other methods known in the art. The vectors used in the method of the invention may contain further functional elements, for example "left border"- and "right border"-sequences of the T-DNA of Agrobacterium which allow for stably integration into the plant genome. Furthermore, methods and vectors are known to the person skilled in the art which permit the generation of marker free transgenic plants, i.e., the selectable or scorable marker gene is lost at a certain stage of plant development or plant breeding. This can be achieved by, for example, cotransformation (Lyznik, Plant Mol. Biol. 13 (1989), 151-161 ; Peng, Plant Mol. Biol. 27 (1995), 91 -104) and/or by using systems which utilize enzymes capable of promoting homologous recombination in plants (see, e.g., WO97/08331 ; Bayley, Plant Mol. Biol. 18 (1992), 353-361); Lloyd, Mol. Gen. Genet. 242 (1994), 653-657; Maeser, Mol. Gen. Genet. 230 (1991), 170-176; Onouchi, Nucl. Acids Res. 19 (1991), 6373-6378). Methods for the preparation of appropriate vectors are described by, e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

Suitable strains of Agrobacterium tumefaciens and vectors, as well as transformation of Agrobacteria, and appropriate growth and selection media are described in, for example, GV3101 (pMK90RK), Koncz, Mol. Gen. Genet. 204 (1986), 383-396; C58C1 (pGV 3850kan), Deblaere, Nucl. Acid Res. 13 (1985), 4777; Bevan, Nucleic. Acid Res. 12(1984), 8711 ; Koncz, Proc. Natl. Acad. Sci. USA 86 (1989), 8467- 8471 ; Koncz, Plant Mol. Biol. 20 (1992), 963-976; Koncz, Specialized vectors for gene tagging and expression studies. In: Plant Molecular Biology Manual Vol 2, Gelvin and Schilperoort (Eds.), Dordrecht, The Netherlands: Kluwer Academic Publ. (1994), 1-22; EP- A-120 516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V, Fraley, Grit. Rev. Plant. Sci., 4, 1-46; An, EMBO J. 4 (1985), 277-287). Although the use of Agrobacterium tumefaciens is preferred in the method of the invention, other Agrobacterium strains, such as Agrobacterium rhizogenes, may be used, for example, if a phenotype conferred by said strain is desired.

Methods for the transformation using biolistic methods are known to the person skilled in the art; see, e.g., Wan, Plant Physiol. 104 (1994), 37-48; Vasil, Bio/Technology 11 (1993), 1553-1558 and Christou (1996) Trends in Plant Science 1 , 423-431. Microinjection can be performed as described in Potrykus and Spangenberg (eds.), Gene Transfer To Plants. Springer Verlag, Berlin, NY (1995).

The transformation of most dicotyledonous plants may be performed using the methods described above or using transformation via biolistic methods as, e.g., described above as well as protoplast transformation, electroporation of partially permeabilized cells, or introduction of DNA using glass fibers. In general, the plants which are modified according to the invention may be derived from any desired plant species. They can be monocotyledonous plants or dicotyledonous plants, preferably they belong to plant species of interest in agriculture, wood culture or horticulture interest, such as crop plants (e.g., maize, rice, barley, wheat, rye, oats), potatoes, oil producing plants (e.g., oilseed rape, sunflower, pea nut, soy bean), cotton, sugar beet, sugar cane, leguminous plants (e.g., beans, peas), or wood producing plants, preferably trees.

The present invention also relates to a transgenic plant cell which contains (preferably stably integrated into its genome) a nucleic acid molecule of the present invention linked to regulatory elements which allow expression of the nucleic acid molecule in plant cells. The presence and expression of the nucleic acid molecule in the transgenic plant cells leads to the synthesis of a DIMIC protein and may lead to physiological and phenotypic changes in plants containing such cells.

Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype. Such regeneration techniques often rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced with a polynucleotide of the present invention.

Plant cells transformed with a plant expression vector can be regenerated, e.g., from single cells, callus tissue or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, Macmillilan Publishing Company, New York, pp. 124-176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985).

Transformed plant cells, calli or explant can be cultured on regeneration medium in the dark for several weeks, generally about 1 to 3 weeks to allow the somatic embryos to mature. Preferred regeneration media include media containing MS salts, such as PHI-E and PHI-F media. The plant cells, calli or explant are then typically cultured on rooting medium in a light/dark cycle until shoots and roots develop. Methods for plant regeneration are known in the art and preferred methods are provided by Kamo ef al., (Bot. Gaz. 146(3):324-334, 1985), West et al., (The Plant Cell 5:1361 -1369. 1993), and Duncan et al. (Planta 165:322-332, 1985). Small plantlets can then be transferred to tubes containing rooting medium and allowed to grow and develop more roots for approximately another week. The plants can then be transplanted to soil mixture in pots in the greenhouse.

The regeneration of plants containing the foreign gene introduced by Agrobacterium from leaft explants can be achieved as described by Horsch ef al., Science,. 227:1229-1231 (1985). In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed as described by Fraley et al., Proc. Natl. Acad. Sci, U.S.A. 80:4803 (1983). This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transgenic plants of the present invention may be fertile or sterile.

Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al., Ann. Rev. of Plant Phys., 38:467-486(1987). The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, from example, Methods for Plant Molecular Biology, A. Weissbach and H. Weissback, eds., Academic Press, Inc., San Diego, Calif. (1988). This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting ht transformant shoots and growth of the plantlets in soil. For maize cell culture and regeneration see generally, The Maize Handbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3^rd edition, Sprague and Dudley Eds., American Society of Agronomy, Madison, Wisconsin (1988).

One of skill will recognize that after the recombinant expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

In vegetatively propagated crops, mature transgenic plants can be propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use. In seed propagated crops, mature transgenic plants can be self crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced heterologous nucleic acid. These seeds can be grown to produce plants that would produce the selected phenotype, (e.g., altered cell cycle content or composition). Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit and the like are included in the invention, provided that these parts comprise cells comprising the isolated nucleic acid of the present invention. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences. Transgenic plants expressing the selectable marker can be screened for transmission of the nucleic acid of the present invention by, for example, standard immunoblot and DNA detection techniques. Transgenic lines are also typically evaluated on levels of expression of the heterologous nucleic acid. Expression at the RNA level can be determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA templates and solution hybridization assays using heterologous nucleic acid-specific probes. The RNA-positive plants can then analyzed for protein expression by Western immunoblot analysis using the specifically reactive antibodies of the present invention. In addition, in situ hybridization and immunocytochemistry according to standard protocols can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue. Generally, a number of transgenic lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles. A preferred embodiment of the invention is a transgenic plant that is homozygous for the added heterologous nucleic acid; i.e., a transgenic plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced for altered cell division relative to a control plant (i.e., native, non-transgenic). Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated. The present invention also relates to transgenic plants and plant tissue comprising transgenic plant cells according to the invention. Due to the (over)expression of a DIMIC molecule, e.g., at developmental stages and/or in plant tissue in which they do not naturally occur, these transgenic plants may show various physiological, developmental and/or morphological modifications in comparison to wild-type plants. Therefore, part of this invention is the use of the DIMIC molecules to modulate the cell cycle and/or plant cell division and/or growth in plant cells, plant tissues, plant organs and/or whole plants. To the scope of the invention also belongs a method for influencing the activity of CDKs such as CDC2a, or CDC2b, CKSs, CKIs, PLPs and KLPNTs in a plant cell by transforming the plant cell with a nucleic acid molecule according to the invention and/or manipulation of the expression of the molecule.

Furthermore, the invention also relates to a transgenic plant cell which contains (preferably stably integrated into its genome) a nucleic acid molecule of the invention or part thereof, wherein the transcription and or expression of the nucleic acid molecule or part thereof leads to reduction of the synthesis of a DIMIC. In a preferred embodiment, the reduction is achieved by an anti-sense, sense, ribozyme, co-suppression and/or dominant mutant effect. The reduction of the synthesis of a protein according to the invention in the transgenic plant cells can result in an alteration in, e.g., cell division. In transgenic plants comprising such cells this can lead to various physiological, developmental and/or morphological changes. In yet another aspect, the invention relates to harvestable parts and to propagation material of the transgenic plants of the invention which either contain transgenic plant cells expressing a nucleic acid molecule according to the invention or which contain cells which show a reduced level of the described protein. Harvestable parts can be in principle any useful parts of a plant, for example, flowers, pollen, seedlings, tubers, leaves, stems, fruit, seeds, roots etc. Propagation material includes, for example, seeds, fruits, cuttings, seedlings, tubers, rootstocks, and the like.

Transgenic Animals As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non- human animal, preferably a mammal, more preferably a mouse, in which an endogenous DIMIC gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing a DIMIC- encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The DIMIC cDNA sequence of SEQ ID NOs 35-48 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a nonhuman homologue of a human DIMIC gene, such as a mouse or rat DIMIC gene, can be used as a transgene. Alternatively, a DIMIC gene homologue, such as another DIMIC family member, can be isolated based on hybridization to the DIMIC cDNA sequences of SEQ ID NOs 35-48 (described further in subsection I above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a DIMIC transgene to direct expression of a DIMIC protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Patent No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of a DIMIC transgene in its genome and/or expression of DIMIC mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a DIMIC protein can further be bred to other transgenic animals carrying other transgenes.

V. Agricultural. Phvtopharmaceutical. and Pharmaceutical Compositions

The DIMIC nucleic acid molecules, DIMIC proteins, and anti-DIMIC antibodies (also referred to herein as "active compounds") of the invention can be incorporated into compositions useful in agriculture and in plant cell and tissue culture. Plant protection compositions can be prepared by conventional means commonly used for the application of, for example, herbicides and pesticides. For example, certain additives known to those skilled in the art stabilizers or substances which facilitate the uptake by the plant cell, plant tissue or plant may be used. The DIMIC nucleic acid molecules, DIMIC proteins, and anti-DIMIC antibodies

(also referred to herein as "active compounds") of the invention can also be incorporated into pharmaceutical compositions suitable for administration into animals. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a plant or subject by, for example, injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054-3057). The agricultural or pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the agricultural or pharmaceutical preparation can include one or more cells which produce the gene delivery system.

The agricultural and pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

VI. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) agricultural uses (e.g., to increase plant yield and to develop phytopharmaceuticals); b) screening assays; c) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials); d) methods of treatment (e.g., phytotherapeutic, therapeutic and prophylactic); e) transcriptomics; f) proteomics; g) metabolomics; h) ligandomics; and i) pharmacogenetics or pharmacogenomics. The isolated nucleic acid molecules of the invention can be used, for example, to express a DIMIC protein (e.g., via a recombinant expression vector in a host cell or in gene therapy applications), to detect DIMIC mRNA (e.g., in a biological sample) or a genetic alteration in a DIMIC gene, and to modulate DIM or DIMIC activity, as described further below. The DIMIC proteins can be used to treat disorders characterized by insufficient or excessive production of a DIMIC substrate or production of DIMIC inhibitors. In addition, the DIMIC proteins can be used to screen for naturally occurring DIMIC substrates, to screen for drugs or compounds which modulate DIMIC activity, as well as to treat disorders characterized by insufficient or excessive production of DIMIC protein or production of DIMIC protein forms which have decreased or aberrant activity compared to DIMIC wild type protein. Moreover, the anti-DIMIC antibodies of the invention can be used to detect and isolate DIMIC proteins, regulate the bioavailability of DIMIC proteins, and modulate DIMIC activity.

A. Agricultural Uses:

In another embodiment of the invention, a method is provided for modifying cell fate and/or plant development and/or plant morphology and/or plant biochemistry and/or plant physiology by modifying the expression of a DIMIC molecule of the present invention in particular cells, tissues or organs of a plant.

Modulation of the expression of a DIMIC molecule of the present invention in a plant can produce a range of desirable phenotypes in plants, such as, for example, the modification of one or more morphological, biochemical, or physiological characteristics including: (i) modification of the length of the G1 and/or the S and/or the G2 and/or the M phase of the cell cycle of a plant; (ii) modification of the G1/S and/or S/G2 and/or G2/M and/or M/G1 phase transition of a plant cell; (iii) modification of the initiation, promotion, stimulation or enhancement of cell division; (iv) modification of the initiation, promotion, stimulation or enhancement of DNA replication;(v) modification of the initiation, promotion, stimulation or enhancement of seed set and/or seed size and/or seed development; (vi) modification of the initiation, promotion, stimulation or enhancement of tuber formation; (vii) modification of the initiation, promotion, stimulation or enhancement of fruit formation; (viii) modification of the initiation, promotion, stimulation or enhancement of leaf formation; (ix) modification of the initiation, promotion, stimulation or enhancement of shoot initiation and/or development; (x) modification of the initiation, promotion, stimulation or enhancement of root initiation and/or development; (xi) modification of the initiation, promotion, stimulation or enhancement of lateral root initiation and/or development; (xii) modification of the initiation, promotion, stimulation or enhancement of nodule formation and/or nodule function; (xiii) modification of the initiation, promotion, stimulation or enhancement of the bushiness of the plant; (xiv) modification of the initiation, promotion, stimulation or enhancement of dwarfism in the plant; (xv) modification of the initiation, promotion, stimulation or enhancement of senescence; (xvi) modification of stem thickness and/or strength characteristics and/or wind-resistance of the stem and/or stem length; (xvii) modification of tolerance and/or resistance to biotic stresses such as pathogen infection; (xviii) modification of tolerance and/or resistance to abiotic stresses such as drought stress or salt stress; (xviv) modification of the initiation, promotion, stimulation or enhancement of pre-mRNA processing; and (xx) modification of the initiation, promotion, stimulation or enhancement of vesicle trafficking/processing.

Methods to effect expression of a DIM and/or DIMIC or a homologue, analogue or derivative thereof as defined in the present invention in a plant cell, tissue or organ, include either the introduction of the protein directly to a cell, tissue or organ such as by microinjection of ballistic means or, alternatively, introduction of an isolated nucleic acid molecule encoding the protein into the cell, tissue or organ in an expressible format. Methods to effect expression of a DIM and/or DIMIC or a homologue, analogue or derivative thereof as defined in the current invention in whole plants include regeneration of whole plants from the transformed cells in which an isolated nucleic acid molecule encoding the protein was introduced in an expressible format.

The present invention clearly extends to any plant produced by the inventive method described herein, and any and all plant parts and propagules thereof. The present invention extends further to encompass the progeny derived from a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by the inventive method, the only requirement being that the progeny exhibits the same genotypic and/or phenotypic characteristic(s) as those characteristic(s) that (have) been produced in the parent by the performance of the inventive method. Exploiting plant DIM and/or DIMIC functions to regulate plant growth and development can depend on methods comprising enhancing a DIM and/or DIMIC gene expression or ectopic expression of a DIM and/or DIMIC genes.

As used herein, the terms "ectopic expression" or "ectopic overexpression" of a gene or a protein refer to expression patterns and/or expression levels of the gene or protein normally not occurring under natural conditions.

By "cell fate and/or plant development and/or plant morphology and/or biochemistry and/or physiology" is meant that one or more developmental and/or morphological and/or biochemical and/or physiological characteristics of a plant is altered by the performance of one or more steps pertaining to the invention described herein. "Cell fate" includes the cell-type or cellular characteristics of a particular cell that are produced during plant development or a cellular process therefor, in particular durincj the cell cycle or as a consequence of a cell cycle process.

The term "plant development" or the term "plant developmental characteristic" or similar terms shall, when used herein, be taken to mean any cellular process of a plant that is involved in determining the developmental fate of a plant cell, in particular the specific tissue or organ type into which a progenitor cell will develop. Cellular processes relevant to plant development will be known to those skilled in the art. Such processes include, for example, morphogenesis, photomorphogenesis, shoot development, root development, vegetative development, reproductive development, stem elongation, flowering, and regulatory mechanisms involved in determining cell fate, in particular a process or regulatory process involving the cell cycle.

The term "plant morphology" or the term "plant morphological characteristic" or similar term will, when used herein, be understood by those skilled in the art to include the external appearance of a plant, including any one or more structural features or combination of structural features thereof. Such structural features include the shape, size, number, position, color, texture, arrangement, and patternation of any cell, tissue or organ or groups of cells, tissues or organs of a plant, including the root, stem, leaf, shoot, petiole, trichome, flower, petal, stigma, style, stamen, pollen, ovule, seed, embryo, endosperm, seed coat, aleurone, fiber, fruit, cambium, wood, heartwood, parenchyma, aerenchyma, sieve element, phloem or vascular tissue. The term "plant biochemistry" or the term "plant biochemical characteristic" or similar term will, when used herein, be understood by those skilled in the art to include the metabolic and catalytic processes of a plant, including primary and secondary metabolism and the products thereof, including any small molecules, macromolecules or chemical compounds, such as but not limited to starches, sugars, proteins, peptides, enzymes, hormones, growth factors, nucleic acid molecules, celluloses, hemicelluloses, calloses, lectins, fibers, pigments such as anthocyanins, vitamins, minerals, micronutrients, or macronutrients, that are produced by plants.

The term "plant physiology" or the term "plant physiological characteristic" or similar term will, when used herein, be understood to include the functional processes of a plant, including developmental processes such as growth, expansion and differentiation, sexual development, sexual reproduction, seed set, seed development, grain filling, asexual reproduction, cell division, dormancy, germination, light adaptation, photosynthesis, leaf expansion, fiber production, secondary growth or wood production, amongst others; responses of a plant to externally-applied factors such as metals, chemicals, hormones, growth factors, environment and environmental stress factors (e.g., anoxia, hypoxia, high temperature, low temperature, dehydration, light, day length, flooding, salt, heavy metals, amongst others), including adaptive responses of plants to said externally-applied factors. The DIM and DIMIC molecules of the present invention are useful in agriculture.

The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used to modulate the protein levels or activity of a protein involved in the cell cycle, pre-mRNA processing or vesicle transport/processing.

Thus, the DIM and/or DIMIC molecules of the present invention may be used to modulate, e.g., enhance, crop yields; modulate, e.g., attenuate, stress, e.g., heat or nutrient deprivation, tolerance; modulate tolerance to pests and diseases; modulate plant architecture; modulate plant quality traits; or modulate plant reproduction and seed development.

The DIM and/or DIMIC molecules of the present invention may also be used to modulate endoreduplication in storage cells, storage tissues, and/or storage organs of plants or parts thereof. The term "endoreduplication" includes recurrent DNA replication without consequent mitosis and cytokinesis. Preferred target storage organs and parts thereof for the modulation of endoreduplication are, for example, seeds (such as from cereals, oilseed crops), roots (such as in sugar beet), tubers (such as in potatoes) and fruits (such as in vegetables and fruit species). Increased endoreduplication in storage organs, and parts thereof, correlates with enhanced storage capacity and, thus, with improved yield. In another embodiment of the invention, the endoreduplication of a whole plant is modulated. Grain yield in crop plants is largely a function of the amount of starch produced in the endosperm of the seed. The amount of protein produced in the endosperm is also a contributing factor to grain yield (Traas et al. (1998) Current Opin. Plant Biol. 1 , 498-503). In contrast, the embryo and aleurone layers contribute little in terms of the total weight of the mature grain. By virtue of being linked to cell expansion and metabolic activity, endoreduplication is generally considered to be an important factor for increasing yield . As grain endosperm development initially includes extensive endoreduplication (Olsen et al. (1999) Trends Plant Sci. 4, 253-257), enhancing, promoting or stimulating this process is likely to result in increased grain yield. Enhancing, promoting or stimulating cell division during seed development as described supra is an alternative way to increase grain yield. In another aspect, the present invention also features a method for the production of Si0₂ from the peels or husks of larger rice seeds. Methods for extraction and/or production of pure Si0₂ from rice seed peels or husks are known in the art (e.g. Gorthy and Pudukottah 1999) and units for production of Si0₂ from rice seed peels are being set up (visit e.g. http://bisnis.doc.gov/bisnis/leads/990604sp.htm). Si0₂ has many applications including electronics, perfume industry and pharmacology and silicone production. Ectopic expression, preferably downregulation of expression, of DIM and/or

DIMICs may also confer enhanced resistance to pathogens causing neoplastic plant growth, such as plant pathogenic bacteria including Agrobacterium tumefaciens, Rhodococcus fascians, Pseudomonas savastanoi, Xanthomonas campestris pv citri and Erwinia herbicola, plant pathogenic fungi including Plasmodiophora brassicae, Crinipellis perniciosa, Pucciniastrum geoppertianum, Taphrina wiesneri, Ustilaga maydis, Exobasidium vaccinii, E. camelliae, Entorrhiza casparyana and Apiosporina morbosum and plant pathogenic gall-inducing insects including the midge Mayetiola poae.

Ectopic expression, preferably downregulation of expression, of a DIM and/or DIMIC molecule may also confer enhanced resistance or tolerance against pathogens which rely on endoreduplication events in the infected host cells to survive. The ectopic expression, preferably downregulation of expression, of a DIM and/or DIMIC molecule is expected to inhibit endoreduplication events. Pathogens relying on host cell endoreduplication to, for example, establish a feeding structure, include nematodes such as Heterodera species and Meloidogyne species. B. Screening Assays:

The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to DIMIC proteins, have a stimulatory or inhibitory effect on, for example, DIMIC expression or DIMIC activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a DIMIC substrate.

In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a DIMIC protein or polypeptide or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a DIMIC protein or polypeptide or biologically active portion thereof, e.g., modulate the ability of DIMIC to interact with its cognate ligand. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al.

(1994) Proc. Natl. Acad. Sci. USA 91 :11422; Zuckermann et al. (1994). J. Med. Chem.

37:2678; Cho et al. (1993) Science 261 :1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061 ; and in Gallop ef al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten (1992)

Biotechniques 13:412-421 ), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor

(1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner USP '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage

(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406);

(Cwiria et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991 ) J. Mol. Biol.

222:301 -310); (Ladner supra.). In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a DIMIC target molecule (e.g., a plant cyclin dependent kinase) with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the DIMIC target molecule. Determining the ability of the test compound to modulate the activity of a DIMIC target molecule can be accomplished, for example, by determining the ability of the DIMIC protein to bind to or interact with the DIMIC target molecule, or by determining the ability of the target molecule, e.g., the plant cyclin dependent kinase, to phosphorylate a protein.

The ability of the target molecule, e.g., the plant cyclin dependent kinase, to phosphorylate a protein can be determined by, for example, an in vitro kinase assay. Briefly, a protein can be incubated with the target molecule, e.g., the plant cyclin dependent kinase, and radioactive ATP, e.g., [D-³²P] ATP, in a buffer containing MgCl2 and MnCl2, e.g., 10 mM MgCl2 and 5 mM MnCl2. Following the incubation, the immunoprecipitated protein can be separated by SDS-polyacrylamide gel electrophoresis under reducing conditions, transferred to a membrane, e.g., a PVDF membrane, and autoradiographed. The appearance of detectable bands on the autoradiograph indicates that the protein has been phosphorylated. Phosphoaminoacid analysis of the phosphorylated substrate can also be performed in order to determine which residues on the protein are phosphorylated. Briefly, the radiophosphorylated protein band can be excised from the SDS gel and subjected to partial acid hydrolysis. The products can then be separated by one-dimensional electrophoresis and analyzed on, for example, a phosphoimager and compared to ninhydrin-stained phosphoaminoacid standards.

Determining the ability of the DIMIC protein to bind to or interact with a DIMIC target molecule can be accomplished by determining direct binding. Determining the ability of the DIMIC protein to bind to or interact with a DIMIC target molecule can be accomplished, for example, by coupling the DIMIC protein with a radioisotope or enzymatic label such that binding of the DIMIC protein to a DIMIC target molecule can be determined by detecting the labeled DIMIC protein in a complex. For example, DIMIC molecules, e.g., DIMIC proteins, can be labeled with ¹²⁵l, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, DIMIC molecules can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. It is also within the scope of this invention to determine the ability of a compound to modulate the interaction between DIMIC and its target molecule, without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of DIMIC with its target molecule without the labeling of either DIMIC or the target molecule. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between compound and receptor. In a preferred embodiment, determining the ability of the DIMIC protein to bind to or interact with a DIMIC target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca²⁺, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., chloramphenicol acetyl transferase), or detecting a target- regulated cellular response.

In yet another embodiment, an assay of the present invention is a cell-free assay in which a DIMIC protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the DIMIC protein or biologically active portion thereof is determined. Binding of the test compound to the DIMIC protein can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the DIMIC protein or biologically active portion thereof with a known compound which binds DIMIC to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a DIMIC protein, wherein determining the ability of the test compound to interact with a DIMIC protein comprises determining the ability of the test compound to preferentially bind to DIMIC or biologically active portion thereof as compared to the known compound.

In another embodiment, the assay is a cell-free assay in which a DIMIC protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the DIMIC protein or biologically active portion thereof is determined. Determining the ability of the test compound to modulate the activity of a DIMIC protein can be accomplished, for example, by determining the ability of the DIMIC protein to bind to a DIMIC target molecule by one of the methods described above for determining direct binding. Determining the ability of the DIMIC protein to bind to a DIMIC target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, "BIA" is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules. In an alternative embodiment, determining the ability of the test compound to modulate the activity of a DIMIC protein can be accomplished by determining the ability of the DIMIC protein to further modulate the activity of a DIMIC target molecule (e.g., a DIMIC mediated signal transduction pathway component). For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined as previously described.

In yet another embodiment, the cell-free assay involves contacting a DIMIC protein or biologically active portion thereof with a known compound which binds the DIMIC protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the DIMIC protein, wherein determining the ability of the test compound to interact with the DIMIC protein comprises determining the ability of the DIMIC protein to preferentially bind to or modulate the activity of a DIMIC target molecule.

The cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of proteins (e.g., DIMIC proteins or biologically active portions thereof). In the case of cell-free assays in which a membrane-bound form a protein is used it may be desirable to utilize a solubilizing agent such that the membrane- bound form of the protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n- dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X- 100, Triton® X-114, Thesit®, lsotridecypoly(ethylene glycol ether)_n, 3-[(3- cholamidopropyl)dimethylamminio]-1 -propane sulfonate (CHAPS), 3-[(3- cholamidopropyl)dimethylamminio]-2-hydroxy-1 -propane sulfonate (CHAPSO), or N- dodecyl=N,N-dimethyl-3-ammonio-1 -propane sulfonate.

In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either DIMIC or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a DIMIC protein, or interaction of a DIMIC protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/ DIMIC fusion proteins or glutathione-S- transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or DIMIC protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of DIMIC binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a DIMIC protein or a DIMIC target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated DIMIC protein or target molecules can be prepared from biotin-NHS (N-hydroxy- succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with DIMIC protein or target molecules but which do not interfere with binding of the DIMIC protein to its target molecule can be derivatized to the wells of the plate, and unbound target or DIMIC protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the DIMIC protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the DIMIC protein or target molecule.

In another embodiment, modulators of DIMIC expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of DIMIC mRNA or protein in the cell is determined. The level of expression of DIMIC mRNA or protein in the presence of the candidate compound is compared to the level of expression of DIMIC mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of DIMIC expression based on this comparison. For example, when expression of DIMIC mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of DIMIC mRNA or protein expression. Alternatively, when expression of DIMIC mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of DIMIC mRNA or protein expression. The level of DIMIC mRNA or protein expression in the cells can be determined by methods described herein for detecting DIMIC mRNA or protein.

In yet another aspect of the invention, the DIMIC proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with DIMIC ("DIMIC-binding proteins" or "DIMIC-bp") and are involved in DIMIC activity. Such DIMIC-binding proteins are also likely to be involved in the propagation of signals by the DIMIC proteins or DIMIC targets as, for example, downstream elements of a DIMIC-mediated signaling pathway. Alternatively, such DIMIC-binding proteins are likely to be DIMIC inhibitors.

Alternatively, a mammalian two-hybrid system can be used which includes e.g. a chimeric green fluorescent protein encoding reporter gene (Shioda et al. 2000, Proc. Natl. Acad. Sci. USA 97, 5520-5224). Yet another alternative consists of a bacterial two-hybrid system using e.g. HIS as reporter gene (Joung et al. 2000, Proc. Natl. Acad. Sci. USA 97, 7382-7387).

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a DIMIC protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a DIMIC-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the DIMIC protein.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate plant or animal model. For example, an agent identified as described herein (e.g., a DIMIC modulating agent, an antisense DIMIC nucleic acid molecule, a DIMIC-specific antibody, or a DIMIC-binding partner) can be used in a plant or animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in a plant or animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for the agricultutal and therapeutic uses described herein.

C Detection Assays

Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; identify an individual from a minute biological sample (tissue typing); and aid in forensic identification of a biological sample. Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the DIMIC nucleotide sequences, described herein, can be used to map the location of the DIMIC genes on a chromosome. The mapping of the DIMIC sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.

Briefly, DIMIC genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the DIMIC nucleotide sequences. Computer analysis of the DIMIC sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of cell hybrids containing individual plant or human chromosomes. Only those hybrids containing the plant or human gene corresponding to the DIMIC sequences will yield an amplified fragment.

Other mapping strategies which can similarly be used to map a DIMIC sequence to its chromosome include in situ hybridization (described in Fan, Y. ef al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1 ,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1 ,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York 1988). Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.

Moreover, differences in the DNA sequences between plants affected and unaffected with a disease associated with the DIMIC gene, can be determined. If a mutation is observed in some or all of the affected plants but not in any unaffected plants, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected plants generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several plants can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

D. Predictive Medicine:

The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.

Accordingly, one aspect of the present invention relates to diagnostic assays for determining DIMIC protein and/or nucleic acid expression as well as DIMIC activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant DIMIC expression or activity. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with DIMIC protein, nucleic acid expression or activity. For example, mutations in a DIMIC gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby phophylactically treat an individual prior to the onset of a disorder characterized by or associated with

DIMIC protein, nucleic acid expression or activity.

Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of DIMIC in clinical trials.

E. Methods of Treatment:

The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant DIMIC expression or activity. With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. "Pharmacogenomics", as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype".) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the DIMIC molecules of the present invention or DIMIC modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing are incorporated herein by reference.

EXAMPLES

EXAMPLE 1: IDENTIFICATION OF PLANT DIM1 PROTEINS

The amino acid sequence of the Schizosaccharomyces pombe dimlp protein (GenBank accession number AF001 14; protein ID AAC49744.1) was used to perform a blastp search in the publicly available GenBank database. The nucleotide sequence corresponding to the protein hit found in Arabidopsis thaliana (GenBank accession number AL392174; protein ID CAC08329.1) was subsequently used to perform an additional blastn search in the publicly available GenBank database. A number of plant homologues of the S. pombe dimlp protein were identified as summarized in Table III. The amino acid sequences of these proteins are aligned in Figure 1. For comparison purposes, the S. pombe and Drosophila melanogaster DMI sequences are also included in Table III and Figure 1.

The DIM1 homologues invariably carry at their amino-termini the motif characteristic for the FKBP-type peptidyl-prolyl cis-trans isomerases:

[ l/V/M/C] X [Y/F] X [G/V/L] X^ [IJF/T] X₂ G X₃ [D/E] [S/T/A/E/Q/K] [S/T/A/N] (SEQ ID NO 54), with 'X' being any amino acid residue, 'Xn' being a stretch of 'n' random amino acid residues and, e.g., '[Y/F]' meaning either a tyrosine or phenylalanine residue occurring at that position. The nearly perfect conservation of the identified plant DIM1 proteins implies that the DIMIC proteins of the present invention interact not only with the Arabidopsis DIM1 , but almost certainly also with the DIM1 proteins of all other plants.

Table III. Overview of the identified plant, fission yeast and Drosophila DIM homologs. Indicated are the GenBank accesion numbers of DNA and protein as well as the SEQ ID NO defining said DNA and protein sequences.

EXAMPLE 2: IDENTIFICATION OF DIM AND DIM-INTERACTING

CLONES

A two-hybrid screening was performed using as a bait a fusion between the GAL4 DNA-binding domain and DIM1AL Vectors and strains used were provided with the Matchmaker Two-Hybrid System (Clontech, Palo Alto, CA). The bait was constructed by inserting the DIMIAt PCR fragment into the pGBT9 vector. The PCR fragment was created from the cDNA using a sense primer incorporating an EcoRI restriction enzyme site (5'-GGGAAJTCATGTCGTATCTTCTTCCACATCTGC-3', EcoRI-site underlined; SEQ ID NO 71) and an antisense primer incorporating a BamYW restricition enzyme site (5'-GGGGATCCAAATTTCATTTCACTGAATCATGTTCG-3', BamHI-site underlined; SEQ ID NO 72). The PCR fragment was cut with EcoRI and BamYW and cloned into the EcoRI and SamHI sites of pGBT9, resulting in the plasmid pGBTDIML The GAL4 activation domain cDNA fusion library was constructed using mRNA of Arabidopsis thaliana cell suspension cultures harvested at various growing stages: early exponential, exponential, early stationary, and stationary phase.

For the two-hybrid screening a 1 -liter culture of the Saccharomyces cerevisiae strain PJ69-4A containing the pGBTDIMI plasmid was transformed with 120 μg DNA of the library, and 1000 μg herring testes carrier DNA using the lithium acetate method (Gietz et al., 1992). To estimate the number of independent cotransformants, 1/1000 of the transformation mix was plated on Leu- and Trp- medium. The rest of the transformation mix was plated on the medium to select for histidine prototrophy (Trp-, Leu-, His-, +10mM 3AT). After 5 days of growth at 30°C, colonies larger than 2 mm were streaked on adenine lacking medium.

A total of 10⁶ independent cotransformants were screened for their ability to grow on adenine free medium. A 5-day incubation at 30°C yielded 28 colonies growing on adenine free medium.

Of the Ade⁺ colonies the activation domain plasmids were isolated as described (Hoffman and Winston, 1987). The pGAD10 inserts were PCR amplified using the primers 5'-ATACCACTACAATGGATG-3' (SEQ ID NO 73) and 5'- AGTTGAAGTGAACTTGCGGG-3' (SEQ ID NO 74). PCR fragments were digested with Alu\ and fractionized on a 2% agarose gel. Plasmid DNA, the inserts of which gave rise to different restriction patterns, was electroporated into Escherichia co/ XL1 -Blue, and the DNA sequence of the inserts was determined. Extracted DNA was also used to retransform PJ69-4A to test the specificity of the interaction. After the sequential selection rounds four true positive clones were identified and are defined herein as "DIM-lnteracting Clones" or "DIMICs."

EXAMPLE 3: CHARACTERIZATION OF DIMIC5 The partial nucleotide sequence of the DIMIC5 cDNA was determined (SEQ ID NO

35) and a full-length sequence reconstituted based on GenBank entries AC004261 (ORF corresponding to protein ID AAD12009), T021 17 (hypothetical protein T3K9.21 which is identical to AAD12009) and T02116. None of these entries, however, fully matches with DIMIC5. The protein AAD12009/T02117 extends the partial DIMIC5 protein derived from the partial DIMIC5 cDNA by 88 amino acids. The further 237 amino acids of AAD12009/T021 17 are identical to the 237 N-terminal amino acids of the partial DIMIC5 protein. The partial DIMIC5 protein has, however, relatively to AAD12009/T02117 an additional C-terminal region consisting of 138 amino acids. The C-terminal region is partially covered by T02116. However, T02116 comprises an internal stretch of 31 amino acids not present in the partial DIMIC5 protein. Based on these observations, the full- length DIMIC5 amino acid and nucleotide sequences were reconstituted by adding the 88 N-terminal amino acids of AAD12009 and the corresponding nucleotides of AC004261 , respectively. The full-length DIMIC5 protein sequence is set forth in Figure 2 and in SEQ ID NO 49. The region of the Arabidopsis genome covering the DIMIC5 open reading frame is shown in Figure 3, defined in SEQ ID NO 37 and corresponds to nucleotides 17241 to 20717 of AC004261. Also indicated in Figure 3 is the full-length DIMIC5 cDNA sequence (SEQ ID NO 36) interrupted by the intron sequences. Both the DIMIC5 protein and cDNA thus are novel molecules as such not present in the GenBank database. A closer analysis of the DIMIC5 protein revealed the presence of two WW-domains

(consensus sequence [W X₂₂ W X₂ P] (SEQ ID NO 55) with X_n being a stretch of n random amino acid residues) organized in tandem followed by a C-terminal domain comprising a non-classical C₂-domain (SEQ ID NO 56). The C-terminal domain is highly homologous to the C-terminal domain of human PQBP-1 (polyglutamine tract-binding protein) proteins (Waragai et al. (1999) Human Mol Genet 8, 977-987; GenBank accession number AJ242829) and mouse PQBP-1 (GenBank accession number NM019478) both of which contain a C₂-domain. WW domains represent small and compact globular structures that interact with proline-rich ligands (Bedford et al. (1997) EMBO J 16, 2376-2383; Chan et al. (1996) EMBO J 15, 1045-1054; Einbond and Sudol (1996) FEBS Lett 384, 1-8). Most proteins containing C₂-domains are functional in signal transduction or membrane trafficking. Phospholipid binding to many C₂-domains is regulated by Ca²⁺ and C₂-domain proteins are, therefore, implicated in Ca²⁺-dependent phospholipid signalling (Rizo and Sϋdhof (1998) J Biol Chem 273, 15879-15882). An additional intramolecularly repeated motif, termed DIMIC5 internal repeat domainl , was furthermore discerned by dot plot analysis of the DIMICS protein sequence (Omiga 2.0 software; scoring matrix: Blosum 62; stringency: 60%; window: 8; hash size: 2). This motif is indicated in Figure 2 and consists of the amino acid sequence GGWXVGL (SEQ ID NO 57) wherein X is any amino acid. Using the PESTFIND software (downloadable from http://www.ebi.ac.uk). a potential PEST sequence with a PEST-FIND score 14 was identified in the DIMIC5 protein with sequence ΗAEDDELDPMDPSSYSDAPR' (residues 373-392 of SEQ ID NO 49).

EXAMPLE 4: CHARACTERIZATION OF DIMIC7=DIMIC40

The partial nucleotide sequence of the DIMIC7 cDNA was determined (SEQ ID NO 38) and shown to be identical to a second DIMIC clone, DIMIC40, that was identified independently of DIMIC7. The full-length DIMIC7 sequences were reconstituted based on GenBank entry AC008148 (ORF corresponding to protein ID AAD55502; unknown protein). The region of the Arabidopsis genome covering the DIMIC7=DIMIC40 ORF is shown in Figure 4 and defined by SEQ ID NO 40. This region corresponds to nucleotides 100439-106312 of AC008148. Also indicated in Figure 4 is the full-length DIMIC7=DIMIC40 cDNA sequence (SEQ ID NO 39) interrupted by intron sequences. The full-length DIMIC7=DIMIC40 protein is shown in Figure 5 (and defined in SEQ ID NO 50) with an indication of the partial amino acid sequence as derived from the partial DIMIC7=DIMIC40 cDNA.

The full-length DIMIC7=DIMIC40 protein displays 41% identity / 51% similarity to the A. thaliana protein AtFABI (GenBank accession number AL035525, protein ID CAB36798). The AtFABI protein is aligned with D1MIC7 in Figure 5. AtFABI is known in the art as a type III phosphatidylinositol 3-phosphate 5-kinase (Ptdlns3P 5-kinase; Cooke ef al. (1998) Current Biol 8, 1219-1222; McEwen ef al. (1999) J Biol Chem 274, 33905- 33912). Characteristic for FAB1 proteins is the conservation of three domains: (i) the FYVE zinc-finger domain (consensus sequence [R/K][R/K]HHCR); (ii) a CCT-homology domain; and (iii) the catalytic domain (McEwen ef al. (1999) J Biol Chem 274, 33905- 33912). The FYVE-domain specifically binds to Ptdlns3P in vitro (Odorizzi et al. (200O) TIBS 25, 229-235). CCT or 'Chaperonine Containg TCP-1 ' is a cytosolic hetero- oligomeric chaperone acting on a limited number of substrates. The CCTδ , CCTβ , and CCTε subunits have, for example, been shown to interact with α-actin (Llorca et al. (1999) Nature 402, 693-696) whereas TCP-1 activity is required for growth of microtubules off the centrosome (Brown ef al. (1996) J Biol Chem 271 , 824-832). The FYVE-domain is present in AtFABI (see Figure 5) but is lacking in DIMIC7=DIMIC40. The CCT-homology domain is conserved in DIMIC7=DIMIC40 as is illustrated in Figure 5 where the corresponding regions of the AtFABI , the S. cerevisiae Fabl p (GenBank accession number P34756) and of the mouse CCTδ (GenBank accession number Z31554) proteins are aligned with DIMIC7=DIMIC40. The C-terminal catalytic domain characteristic of the FAB1 kinases is also present and conserved in DIMIC7=DIMIC40 as is obvious from Figure 5 (alignment of AtFABI and yeast Fab1 p catalytic domains with the corresponding region of DIMIC7). Residues invariant in the catalytic domains of these proteins are indicated by an asterisk in Figure 5 and these include the residues invariant in all FAB1 kinases known to date: (i) K2059; (ii) D2196; and (iii) D2216 (numbering of all three residues relative to the yeast Fabl p protein. The FAB1 activation loop with consensus sequence T[F/Y]T[W/L]DKKLE[S 7M]WVKXXG[l/L][V/L]G (SEQ ID NO 58) is, with the exception of the first threonine residue (which is a glutamine in DIMIC7), completely conserved in DIMIC7 (see Figure 5). This motif may be involved in defining Ptdlns3P as the substrate for 5-phosphorylation (McEwen ef al. (1999) J Biol Chem 274, 33905- 33912).

Five different intramolecularly repeated motifs, termed DIMIC7 internal repeat domains 1-5, were furthermore discerned by dot plot analysis of the DIMIC7 protein sequence (Omiga 2.0 software; scoring matrix: Blosum62; stringency: 60%; window: 8; hash size: 2). These motifs are depicted in Figure 6 and are: Motif DIMIC7/1 : PLGR[F/W/Y][M/I/L V] (SEQ ID NO 59); Motif DIMIC7/2: EXXG[R/K/H]IW (SEQ ID NO 60); Motif DIMIC7/3: DLXXPT[M/I/L/V] (SEQ ID NO 61 ); Motif DIMIC7/4: DDXXSXYF (SEQ ID NO 62);and

Motif DIMIC7/5: TEXSDXLN (SEQ ID NO 63); with X being any amino acid and, e.g., [D/E] being either an aspartate or glutamate residue at that position.

Phosphoinositides are generally known as key regulators of vesicle-mediated protein trafficking. Ptdlns(3,5)P₂, in particular, was shown to be required for normal vacuolar morphology and function. In the absence of active Fabl p, yeast cells accumulate large vacuoles with a reduced hydrolytic activity as the result of poor acidification. The number of multivesicular bodies (MVBs) is also strongly reduced. MVBs target membrane proteins for vacuolar degradation by fusing with the vacuole. In the absence of functional Fabl p, however, the membrane proteins are delivered to the vacuolar outer membrane instead of into the vacuolar lumen. This process could explain the increase in vacuole size (Odorizzi ef al. (1998) Cell 95, 847-858; Odorizzi et al. (2000) TIBS 25, 229-235). The vacuolar surface area increases up to 2.5-fold and is probably the cause of inappropriate nuclear segregation during mitosis (Gary ef al. (1998) J Cell Biol 143, 65-79). Interestingly, Fabl p is also likely to be involved, via Ptdlns(3,5)P₂, in the acute osmoprotective response which is accompanied by or requires enhanced Ptdlns(3,5)P₂- dependent vesicle trafficking. Ptdlns(3,5)P₂ is present in yeast as well as in mammalian and plant cells. Hyperosmotically stressed yeast and mammalian cells display a rapid increase and decrease, respectively, in the levels of Ptdlns(3,5)P₂ whereas hypo- osmotically stressed mammalian cells rapidly accumulate high levels of Ptdlns(3,5)P₂ (Dove et al. (1997) Nature 390, 187-192). Fablp might furthermore be involved in retrograde transport from the vacuole via Ptdlns(3,5)P₂ synthesis and the recruitment of Ptdlns(3,5)P₂-binding proteins (Cooke et al. (1998) Current Biol 8, 1219-1222). FAB1 homologues are found in plants other than Arabidopsis thaliana, e.g., in rice (GenBank accession number C28212) and date palm (Corniquel and Mercier (1997) Int J Plant Sci 158, 152-156).

Using the PESTFIND software (downloadable from http://www.ebi.ac.uk). eight potential PEST sequences were identified in the DIMIC7 protein. These PEST sequences are given in Table IV.

Table IV. Overview of the potential PEST sequences identified in the DIMIC7 protein.

EXAMPLE 5: CHARACTERIZATION OF DIMIC26

The partial nucleotide of the DIMIC26 cDNA was determined (SEQ ID NO 41) and a full-length sequence was reconstituted based on GenBank entry AB023039 (ORF corresponding to protein ID BAA96996). The region of the Arabidopsis genome covering the DIMIC26 ORF is shown in Figure 7 and defined in SEQ ID NO 43. This region corresponds to the inverse complement of nucleotides 19634-21435 of AB023039. Also indicated in Figure 7 is the full-length DIMIC26 cDNA sequence (SEQ ID NO 42) interrupted by one intron sequence. The full-length DIMIC26 protein is shown in Figure 8 and is defined in SEQ ID NO 51 with an indication of the partial amino acid sequence as derived from the partial DIMIC26 cDNA. The function of DIMIC26 is currently not known. The presence, however, of DIMIC26 regions with weak homology to parts of the human CENP-E (centrosome protein E; GenBank accession number NM0018 3) and NMMHC-B (nonmuscle type B myosin heavy chain; GenBank accession number P35580) point at a role of DIMIC26 in cell cycle processes such as chromosome movement and/or spindle elongation and/or cytokinesis. Such a role is in line with the reported function of DIM1 in chromosome segregation and the localization of DIM1 to the mitotic spindle (supra). The alignment of the homologous regions of D1M1C26 and CENP-E (37% identity, 53% similiarity) is given in Figure 9, and the alignment of the homologous regions of DIMIC26 and NMMHC-B (29% identity, 41 % similarity) is given in Figure 10.

Six different intramolecularly repeated motifs, termed DIMIC26 internal repeat domains 1-6, were furthermore discerned by dot plot analysis of the DIM1C26 protein sequence (Omiga 2.0 software; scoring matrix: Blosum62; stringency: 60%; window: 8; hash size: 2). These motifs are depicted in Figure 11 and are:

Motif DIMIC26/1 : CXCXIC (SEQ ID NO 64);

Motif DIMIC26/2: ACNRXXE[M/I/L/V][M/I/L/V](SEQ ID NO 65); Motif DIMIC26/3: QXSGGG (SEQ ID NO 66);

Motif DIMIC26/4: [M/I/LΛ ]DX[M/I/L V]KXGL (SEQ ID NO 67);

Motif DIMIC26/5: SEXXAEKQ(SEQ ID NO 68); and

Motif DIMIC26/6: RLXXAEA[D/E](SEQ ID NO 69); with X being any amino acid and, e.g.,

[D/E] being either an aspartate or glutamate residue at that position. Further present in DIMIC26 are the '[M/I/LΛ ][R/K/H]' amino acid pair and the

'[R/K/H][M/I/L/V]' amino acid pair which are repeated multiple times. Note that both pairs can overlap.

Using the PESTFIND software (downloadable from http://www.ebi.ac.uk). one potential PEST sequence was identified in the DIMIC26 protein. Said PEST sequences comprises amino acid residues 41-63 of SEQ ID NO 51, has a PEST-FIND score of 6.3 and comprises the sequence 'RESPAESASSQETWPLGDTVAGK' (SEQ ID NO 107).

Within the DIMIC26 protein sequence (SEQ ID Nr. 51 ), a PHD finger motif

(InterPro Accession number IPR001965 and IPR001841) was found with the software program InterPro ( <http://www.ebi.ac.uk/interpro>). This domain is positioned from amino acid 218 till 291 with conserved cystein residues in between: (SEQ ID NO 111 :

NRKGFCNLCMCTICNKFDFSVNTCRWIGCDLCSHWTHTDCAIRDGQITTGSSAKNNTSG

PGEIVFKCRACNRT).This domain has implications for chromatin-mediated transcriptional regulation and therefor constitutes an important active domain of the

DIMID26 protein.

EXAMPLE 6: CHARACTERIZATION OF DIMIC70

The partial nucleotide sequence of the DIM1C70 cDNA was determined (SEQ ID NO 44) and a full-length sequence was reconstituted based on GenBank entry AC007583 (ORF corresponding to protein ID AAF75085). Thus, the partial DIMIC70 protein sequence as derived from the partial DIMIC70 cDNA was N-terminally extended with the N-terminal 126 amino acids of AAF75085. The region of the Arabidopsis genome covering the DIMIC70 ORF is shown in Figure 12 and defined by SEQ ID NO 47. This region corresponds to the inverse complement of nucleotides 64105-65587 of AC007583. The predicted protein AAF75085 is, however, lacking a stretch of 37 amino acids that is present in the partial DIMIC70 sequence, most likely as the result of wrong intron-exon prediction in the ORF corresponding to AAF75085. In addition, the DNA region encoding the 37-amino acid stretch of DIMIC70 is shorter with 3 nucleotides compared to the corresponding region of the genomic DNA fragment (nucleotides "tga" at positions 64486- 64484 in SEQ ID NO 47). Thus, probably as a result of allelic variation, it can be concluded that two DIMIC70 gene, cDNA and protein forms exist which are referred to as DIMIC70A (full-length protein shown in Figure 13B and defined by SEQ ID NO 52; the full-length DIMIC70A cDNA sequence is given in Figure 13A and defined by SEQ ID NO 45; the gene defined by SEQ ID NO 47) and DIMIC70B (full-length protein shown in Figure 14B and defined by SEQ ID NO 53; the full-length DIMIC70B cDNA sequence is given in Figure 14A and defined by SEQ ID NO 46; the gene defined by SEQ ID NO 48). Compared to the DIMIC70A protein the DIMIC70B protein contains an additional amino acid "D" or aspartate at position 203 of DIMIC70B (SEQ ID NO 53; Figure 14B). Both the DIMIC70A and DIMIC70B proteins and cDNAs thus are novel molecules as such not present in the GenBank database.

A third DIMIC70 molecule is represented in figure 16 (A: nucleic acid sequence; B; amino acid sequence) and represents a third variant cDNA sequence. The DIMIC 70C protein (SEQ ID NO 95) is shorter than the DIMIC70B protein and differs in the first 17 amino acids. The (thioredoxin -like protein) motif described is present in the DIMIC70C protein sequence (SEQ ID NO 95);

The C-terminal part of DIMIC70 displays a 34% identity and a 45% similarity to the PRODOM family PD012637 consensus sequence which comprises the redox-active center of thioredoxins and thioredoxin-like proteins. The consensus thioredoxin-like domain 'CXXC (SEQ ID NO 70) wherein 'X' is any amino acid (Wang and Chang (1999) EMBO J 18, 5972-5982) together with the alignment of PD012637 with the homologous DIMIC70 region is indicated in Figure 15. Thus, unlike DIM1 , DIMIC70 does contain a candidate thioredoxin active center and is, thus, a putative active thioredoxin. Without being intenting to be bound by any theory or mode of action, it is postulated that DIMIC70 could be involved in the process leading to the proper protein folding of DIM1. Using the PESTFIND software (downloadable from http://www.ebi.ac.uk). one potential PEST sequence was identified in the DIMIC70A protein as well as in the DIMIC70B and DIMIC70C protein. These PEST sequences comprise amino acid residues 129-156 of SEQ ID NO 52 and of SEQ ID NO 53, have a PEST-FIND score of 1.0 and comprise the sequence 'KSLSQENLVELSDENDDLCPVECVTEFK' (SEQ ID MO 110).

EXAMPLE 7: EXPRESSION OF RECOMBINANT DIMIC PROTEINS IN

TRANSGENIC PLANTS In this example, the DIMIC molecules of the present invention were expressed in a

³⁵S expression vector in transgenic plants. The DIMIC molecules of this invention were cloned using standard cloning procedures between the CaMV ³⁵S promoter and the NOS 3' untranslated region in the Nco\ and BamYW sites of PH³⁵S (Hemerly et al, EMBO J.14 (1995), 3925-3936), resulting in the PH³⁵SDIMIC vector. This construct was cloned in the binary vector PSV4 (Herouart et al., Plant Physiol. 104 (1994), 873-880) and in Agrobacterium tumefaciens. The constructs were introduced in Nicotiana tabacum cv. Petit havana (SR1) plants by the leaf disk protocol (Horsh, Science 227 (1985), 1229- 1231).

For tissue-specific expression, the DIMIC gene is expressed under control of the minimal ³⁵S promoter containing UAS elements. These UAS elements are sites for transcriptional activation by the GAL4-VP16 fusion protein. The GAL4-VP16 fusion protein in turn is expressed under control of a tissue-specific promoter. The UAS-DIMIC construct and the GAL4-VP16 construct are combined by co-transformation of both constructs, subsequent transformation of single constructs or by sexual cross of lines that contain the single constructs. The advantage of this two-component system is that a wide array of tissue-specific expression patterns can be generated for a specific transgene, by simply crossing selected parent lines expressing the UAS-DIMIC construct with various tissue-specific GAL4-VP16 lines. A tissue-specific promoter/DIMIC combination that gives a desired phenotype can subsequently be recloned in a single expression vector, to avoid stacking of transgene constructs in commercial lines.

Primary transformants were selfed and characterized by Northern and Western blotting using 3 week old plantlets. Expression levels were compared with those of non- transformed (control) plants. EXAMPLE 8: DOWNREGULATION OF TARGET DIMIC GENES IN

TRANSGENIC PLANTS

Plant genes can be specifically downregulated by antisense and co-suppression technologies. These technologies are based on the synthesis of antisense transcripts, complementary to the mRNA of a given DIMIC gene. There are several methods described in literature, that increase the efficiency of this downregulation, for example to express the sense strand with introduced inverted repeats, rather than the antisense strand. The constructs for downregulation of target genes are made similarly as those for expression of recombinant proteins, i.e., they are fused to promoter sequences and transcription termination sequences. Promoters used for this purpose are constitutive promoters as well as tissue-specific promoters.

EXAMPLE 9: AG/?Oft4C7^"E/?/t/M-MEDIATED RICE TRANSFORMATION

Mature dry seeds of the rice japonica cultivars Nipponbare or Taipei 309 are dehusked, sterilised and germinated on a medium containing 2,4-D (2,4- dichlorophenoxyacetic acid). After incubation in the dark for four weeks, embryogenic, scutellum-derived calli are excised and propagated on the same medium. Selected embryogenic callus is then co-cultivated with Agrobacterium. Widely used Agrobacterium strains such as LBA4404 or C58 harbouring binary T-DNA vectors can be used. The hpt gene in combination with hygromycin is suitable as a selectable marker system but other systems can be used. Co-cultivated callus is grown on 2,4-D-containing medium for 4 to 5 weeks in the dark in the presence of a suitable concentration of the selective agent. During this period, rapidly growing resistant callus islands develop. After transfer of this material to a medium with a reduced concentration of 2,4-D and incubation in the light, the embryogenic potential is released and shoots develop in the next four to five weeks. Shoots are excised from the callus and incubated for one week on an auxin-containing medium from which they can be transferred to the soil. Hardened shoots are grown under high humidity and short days in a phytotron. Seeds can be harvested three to five months after transplanting. The method yields single locus transformants at a rate of over 50 % (Aldemita and Hodges (1996) Planta 199, 612-617 ; Chan et al. (1993) Plant Mol Biol 22, 491-506, Hiei et al. (1994), Plant J 6, 271-282). Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. An isolated nucleic acid molecule selected from the group consisting of:

(a) a nucleic acid molecule comprising the nucleotide sequence as given in any of SEQ ID NOs 36, 35, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48 or 94, or the complement thereof,

(b) a nucleic acid molecule comprising the RNA sequence corresponding to any of SEQ ID NOs 36, 35, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48 or 94, or the complement thereof, (c) a nucleic acid molecule specifically hybridizing with the nucleotide sequence as defined in (a) or (b), (d) a nucleic acid molecule which is at least 60% identical to the nucleotide sequence as given in any of SEQ ID NOs 36, 35, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47,

48 or 94, or the complement thereof, (e) a nucleic acid molecule encoding a protein comprising an amino acid sequence as given in any of SEQ ID NOs 49 to 53 or 95,

(f) a nucleic acid molecule encoding a protein comprising at least one of the amino acid sequences represented in SEQ ID NOs 55, 56 or 96,

(g) a nucleic acid molecule encoding a protein comprising an amino acid sequence which is at least 42 % identical to the amino acid sequence as given in SEQ ID

NO 50, (h) a nucleic acid molecule encoding a protein comprising at least one of the amino acid sequences represented in SEQ ID NOs 59 to 63, or 97 to 105, (i) a nucleic acid molecule encoding a protein comprising at least one of the amino acid sequences represented in SEQ ID NOs 64 to 69, 106, 107 or 111 ,

(j) a nucleic acid molecule encoding a protein comprising at least one of the amino acid sequences represented in any of SEQ ID NOs 108, 109 or 110, (k) a nucleic acid molecule encoding a protein comprising an amino acid sequence which is at least 50 % identical to the amino acid sequence as given in any of SEQ ID NOs 49, 50, 51 , 52, 53 or 95,

(a) to (k) as a result of the genetic code, (m) a nucleic acid molecule which is diverging from a nucleic acid as defined in any of (a) to (k) as a result of differences in codon usage between organisms, ill

(n) a nucleic acid molecule which is diverging from a nucleic acid as defined in any of

(a) to (k) as a result of differences between alleles, and (o) a nucleic acid molecule as defined in any one of (a) to (n) characterized in that said nucleic acid is DNA, cDNA, genomic DNA or synthetic DNA, characterized in that said nucleic acid molecule encodes a DIM1 -interacting molecule (DIMIC molecule), or a homologue or a derivative thereof and further provided that said nucleic acid is not one of the nucleic acids as deposited under the GenBank Accession numbers AC004261 , AC008148, AB023039 or AC007583.

2. An isolated nucleic acid molecule encoding an immunologically active and/or functional fragment of a DIM1 -interacting molecule encoded by a nucleic acid of claim 1 , or an immunologically active and/or functional fragment of a homologue or a derivative of such a DIM1 -interacting molecule, provided that said nucleic acid is not one of the nucleic acids as deposited under the GenBank Accession number T3K9.20 or T3K9.21.

3. An isolated nucleic acid molecule according to claim 2 selected from the group consisting of consisting of

(a) a nucleic acid encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 49, wherein said fragment comprises at least one of the sequences as represented in any of SEQ ID NOs 55, 56, or 96, (b) a nucleic acid molecule encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 49, wherein, said fragment comprises at least 326 contiguous amino acid residues of the amino acid sequence of SEQ ID NO 49,

(c) a nucleic acid encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 50, wherein said fragment comprises at least one of the sequences as represented in any of SEQ ID NOs 59, 60, 61 , 62, 63, 97, 98, 99, 100, 101 , 02, 103, 104, or 105,

(d) a nucleic acid encoding a functional fragment of polypeptide comprising the amino acid sequence of SEQ ID NO 51 , wherein said fragment comprises the sequence as represented in any of SEQ ID NOs 64, 65, 66, 67, 68, 69, 106, 107 or 111 ,

(e) a nucleic acid encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 52, wherein said fragment comprises at least one of the sequences as represented in SEQ ID NO 108 or 1 10, (f) a nucleic acid molecule encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 53, wherein said fragment comprises at least one of the sequences as represented in SEQ ID NO 109 or 110, (g) a nucleic acid encoding a functional fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO 95, wherein said fragment comprises at least one of the sequences as represented in SEQ ID NO 109 or 1 10, and

4. An antisense nucleic acid molecule corresponding to at least one of the nucleic acids as defined in any of claims 1 to 3.

5. An isolated nucleic acid molecule comprising the nucleic acid molecule of any one of claims 1 to 4 and a nucleotide sequence encoding a heterologous polypeptide.

6. A nucleic acid molecule of at least 15 contiguous nucleotides in length specifically hybridizing with a nucleic acid as defined in any of claims 1 to 3.

7. A nucleic acid molecule of at least 15 contiguous nucleotides in length specifically amplifying a nucleic acid as defined in any of claims 1 to 3.

8. A vector comprising a nucleic acid sequence as defined in any of claims 1 to 5.

9. A vector according to claim 8 which is an expression vector wherein said nucleic acid sequence is operably linked to one or more control sequences allowing the expression of said sequence in prokaryotic and/or eukaryotic host cells.

10. A host cell comprising a nucleic acid molecule as defined in any of claims 1 to 5 or a vector according to claim 8 or 9.

11. A host cell according to claim 10, wherein the host cell is a bacterial, insect, fungal, yeast, plant or animal cell.

12. An isolated polypeptide encodable by a nucleic acid as defined in any of claims 1 to 3, or a homologue or a derivative thereof, or an immunologically active and/or functional fragment thereof.

13. A polypeptide of claim 12 having an amino acid sequence as given in any of SEQ I D NOs 49 to 53 or 95, or a homologue or a derivative thereof, or an immunologically active and/or functional fragment thereof.

14. A polypeptide of claim 12 or 13 further comprising heterologous amino acid sequences or a polypeptide encodable by a nucleic acid of claim 5.

15. A method for producing a polypeptide as defined in any of claims 12 to 14 comprising culturing a host cell of claim 10 or 11 under conditions allowing the expression of the polypeptide and recovering the produced polypeptide from the culture.

16. An antibody specifically recognizing a polypeptide of claim 12 or 13 or a specific epitope of said polypeptide.

17. A method for the production of altered plant cells, plant tissues or plants comprising the introduction of a polypeptide as defined in any of claims 12 to 14 directly into said plant cell or tissue or in an organ of said plant.

18. A method for effecting the expression of a polypeptide as defined in any of claims 12 to 14 in plant cells, tissues or plants comprising the introduction of a nucleic acid molecule as defined in any of claims to 1 to 3 or 5 operably linked to one or more control sequences or a vector of claim 8 or 9 stably into the genome of a plant cell.

19. A method for the production of transgenic plant cells, plant tissues or plants comprising the introduction of a nucleic acid as defined in any of claims 1 to 5 in an expressible format or a vector of claim 8 or 9 in said plant cell, plant tissue or plant.

20. A method according to claims 18 or 19 further comprising regenerating a plant from said plant cell.

21. A transgenic plant cell comprising a nucleic acid as defined in any of claims 1 to 5 which is operably linked to regulatory elements allowing transcription and/or expression of said nucleic acid in plant cells or a transgenic plant cell obtainable by a method of claim 19.

22. A transgenic plant cell of claim 21 wherein said nucleic acid as defined in any of claims 1 to 5 is stably integrated into the genome of said plant cell.

23. A transgenic plant or plant tissue comprising plant cells of claim 21 or 22 or a transgenic plant obtainable by the method of claim 20.

24. A harvestable part of a plant of claim 23.

25. The harvestable part of a plant of claim 24 which is selected from the group consisting of seeds, leaves, fruits, stem cultures, rhizomes and bulbs.

26. The progeny derived from any of the plants or plant parts of any of claims 23 to 25.

27. A method for identifying compounds or mixtures of compounds which specifically bind to a polypeptide of claim 12 or 13, comprising the steps of

(a) combining a polypeptide of claim 12 or 13 or a cell expressing said polypeptide with said compound or mixtures of compounds under conditions suitable to allow complex formation, and,

(b) detecting complex formation, wherein the presence of a complex identifies a compound or mixture of compounds which specifically binds said polypeptide.

28. The method of claim 27, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of:

(b) detection of binding using a competition binding assay; and (c) detection of binding using an assay for testing the activity of the DIM1- interacting molecule.

29. A method for identifying and obtaining compounds interacting with or modulating the activity of a polypeptide of claim 12 or 13 comprising the steps of:

(a) providing a two-hybrid system wherein a polypeptide of claim 12 or 13 and an interacting protein partner, preferably a DIM1 molecule are expressed,

30. A method for modulating the activity of a polypeptide of claim 12 or 13 comprising contacting a polypeptide of claim 12 or 13 or a host cell of claim 10 or 11 expressing said polypeptide with a compound which binds to the polypeptide or obtainable by any of the methods of claims 27 to 29, in a sufficient concentration to modulate the activity of the polypeptide.

31. A method for modulating the growth of a plant, comprising introducing into the plant a DIM1 interacting (DIMIC) molecule or a DIMIC modulator in an amount sufficient to modulate the growth of the plant, thereby modulating the growth of the plant.

32. A method for modulating the cell cycle in a plant, comprising introducing into the plant a DIM1 interacting (DIMIC) molecule or a DIMIC modulator in an amount sufficient to modulate the cell cycle in the plant, thereby modulating the cell cycle in the plant.

33. A method for enhancing overall growth and yield of a plant comprising introducing into the plant a DIM1 interacting (DIMIC) molecule or a DIMIC modulator in an amount sufficient to modulate the growth of the plant, thereby enhancing overall growth and yield of said plant.

34. A method for modulating pre-mRNA splicing in a plant cell comprising introducing into the cell a DIM1 interacting (DIMIC) molecule or a DIMIC modulator in an amount sufficient to modulate pre-mRNA splicing in the cell, thereby modulating pre-mRNA splicing in the cell.

35. A method for modulating vesicle transport processing in a plant cell comprising introducing into the cell a DIM1 interacting (DIMIC) molecule or a DIMIC modulator in an amount sufficient to modulate vesicle transport processing in the cell, thereby modulating vesicle transport/processing in the cell.

36. A method for increasing yield of a plant comprising introducing into the plant a DIM1 interacting (DIMIC) molecule or a DIMIC modulator in an amount sufficient to modulate the growth of the plant, thereby increasing yield of said plant.

37. A method for enhancing stress tolerance in a plant comprising introducing into the plant a DIM1 interacting (DIMIC) molecule or a DIMIC modulator in an amount sufficient to modulate the growth of the plant, thereby enhancing stress tolerance of said plant.

38. A method for conferring enhanced resistance to pathogens of a plant comprising introducing into the plant a DIM1 interacting (DIMIC) molecule or a DIMIC modulator in an amount sufficient to confer resistance to pathogens of the plant, thereby conferring enhanced resistance to pathogens of said plant.

39. A method according to any of claims 31 to 38 wherein at least one nucleic acid encoding a plant DIM1 interacting (DIMIC) molecule, a homologue or a derivative thereof or an enzymatically active fragment thereof is expressed in specific cells or tissues of said plant.

40. A method according to claim 39 comprising stably integrating into the genome of said plant or in specific plant cells or tissues of said plant at least one expressible nucleic acid encoding a DIM1 interacting (DIMIC) molecule, a homologue or a derivative thereof or an enzymatically active fragment thereof

41. A method according to claim 39 or 40 wherein said expression of said nucleic acid leads to overexpresion of a DIM1 interacting (DIMIC) molecule in said plant.

42. A method according to claim 39 or 40 wherein said expression of said nucleic acid leads to downregulation of expression of a DIM1 interacting (DIMIC) molecule.

43. A method according to any of claims 31 to 42 wherein said DIM1 interacting (DIMIC) molecule is selected from any of the nucleic acids as defined in any of claims 1 to 4.

44. A method according to any of claims 31 to 42 wherein said DIM1 interacting (DIMIC) molecule is selected from the group of nucleic acids as given in any of SEQ ID NOs

35 to 48 or 94, or a homologue or a derivative thereof, or a functional fragment thereof.

45. A method according to any of claims 31 to 44 wherein the expression or activity of a nucleic acid encoding a plant DIM1 interacting (DIMIC) molecule or a homologue thereof is modulated by a DIMIC modulator.

46. A method according to claim 45 wherein said DIMIC modulator is selected from the group consisting of an antibody of claim 16, an antisense molecule of claim 4, a ribozyme, or a compound obtainable by any of the methods of claims 27 to 29.

47. A method of claim 46 wherein said DIMIC modulator is capable of modulating DIMIC nucleic acid expression.

48. A method of claim 46 wherein said DIMIC modulator is capable of modulating DIMIC polypeptide activity.

49. A method according to any of claims 31 to 48 comprising co-expression of a DIM1 interacting (DIMIC) molecule or a DIMIC modulator and a DIM1 molecule in said plant.

50. A method for detecting the presence of a polypeptide of claim 12 or 13 in a sample comprising:

(a) contacting the sample with a compound which selectively binds to said polypeptide; and

(b) determining whether the compound binds to the polypeptide in the sample to thereby detect the presence of a polypeptide of claim 12 or 13 n the sample.

51 . A method of claim 50, wherein the compound which binds to the polypeptide is an antibody.

52. A method for detecting the presence of a nucleic acid molecule as defined in any of claims 1 to 4 in a sample comprising:

(a) contacting the sample with a nucleic acid probe or primer of claim 6 or 7 which selectively hybridizes to or amplifies the nucleic acid molecule of any of claims 1 to 4, and

(b) determining whether said nucleic acid probe or primer binds to a nucleic acid molecule in the sample to thereby detect the presence of a nucleic acid molecule of any of claims 1 to 4 in the sample.

53. The method of claim 52, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.

54. A diagnostic kit comprising at least one of the nucleic acid molecules of claims 1 to 5, the polypeptides of claims 12 to 14, the antibodies of claim 16, the compounds obtainable by the method of any of claims 27 to 29.