HK1038590B - Methods and means to modulate programmed cell death in eukaryotic cells - Google Patents

Description

Methods and means for modulating programmed cell death in eukaryotic cells

Technical Field

The present invention relates to the use of poly (ADP-ribose) polymerase (PARP) proteins, in particular mutant PARP proteins or parts thereof, and the genes encoding them in the production of eukaryotic cells and organisms, in particular plant cells and plants, with altered programmed cell death. Eukaryotic cells and organisms, in particular plant cells and plants, are provided wherein either Programmed Cell Death (PCD) is stimulated in at least a part of the cells, preferably selected cells, or conversely PCD is inhibited in cells or at least a part of the cells of the organism by modulating the level or activity of PARP protein in these cells. The invention also relates to eukaryotic cells and organisms, in particular plant cells and plants, expressing these genes.

Description of the related Art

Programmed Cell Death (PCD) is a physiological cell death process that involves the removal of selected cells during development of animals and plants or in response to environmental instructions (see Ellis et al, 1991; Pennell and lamb.1997). Cellular breakdown by PCD is morphologically accompanied by condensation, contraction and rupture of the cytoplasm and nucleus, often into small, sealed capsules (Cohen 1993, Wang et al, 1996). Biochemically, PCD is characterized by breaking down nuclear DNA into generally small nucleosomes representing fragments of approximately 50kb, as well as inducing cysteine proteases and endonucleases. Fragmentation of DNA can be detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) of the 3' -OH group of DNA in cell sections (Gavrili et al, 1992). Cell death by PCD is quite different from cell death by necrosis, the latter involving cell swelling, cell content lysis and leakage.

In animals, PCD involves the removal or death of unwanted cells, such as tadpole tail cells in metamorphosis, cells between fingers developing in vertebrates, overproduced vertebrate neurons, cells in cell specialization processes such as corneal cells, and the like. Damaged cells that no longer function properly can also be removed by the PCD, preventing them from multiplying and/or spreading. PCD, or a deficiency thereof, is also involved in a number of pathological conditions in humans (AIDS, Alzheimer's disease, Huntington's disease, Lou Gehring's disease, cancer).

In plants, PCD has been shown or believed to be involved in a number of developmental processes, such as removal of suspensor cells during embryonic development, aleurone cells after germination of monocotyledonous seeds, root cap cells after germination of seeds and growth of seedlings, cell death during cell specialization seen in development of xylem tubular molecules or cell lines, or floral organ failure in parthenocarpy. The formation of aerated tissue in roots under hypoxic conditions and the formation of leaf splinters or perforations in some plants also appears to involve PCD. Massive cell death occurs in plants as leaves or other organs age. Allergic reactions in plants, in other words rapid cell death at the site of entry of an avirulent pathogen, produce restrictive lesions, another example of PCD that responds to environmental indications.

The death of animal or plant cells in suspension culture, particularly in low density cell suspension culture, also confirmed the characteristics of PCD.

The enzyme involved in PCD or apoptosis has been suggested to be poly (ADP-ribose) polymerase. Poly (ADP-ribose) polymerase (PARP), also known as poly (ADP-ribose) transferase (ADPRT) (ec2.4.2.30), is an enzyme in the nucleus found in most eukaryotic cells, including vertebrates, arthropods, mollusks, slime molds, dinoflagellates, fungi, and other lower eukaryotic cells other than yeast. Its enzymatic activity has also been demonstrated in many plants (Payne et al, 1976; Willmitzer and Wagner, 1982; Chen et al, 1994; O' Farrell, 1995).

PARP catalyzes the conversion of NAD from⁺The ADP-ribose moiety of (A) is mainly transferred to the carboxyl group of glutamic acid residue of the target protein, followed by ADP-ribose polymerization. The major target protein is PARP itself, but histones, high rate of mobility, have been shownFamily chromosomal proteins, topoisomerases, endonucleases and DNA polymerases are subject to this modification.

The PARP protein from animals is a 113-120kDa nuclear protein, which is abundant in most cell types and consists of three major functional domains: an amino-terminal DNA binding domain comprising two Zn-finger domains, a carboxy-terminal catalytic domain and an auto-modified internal domain (de Murcia and Menissier de Murcia, 1994; Kameshita et al, 1984; Lindahl et al, 1995). Its in vitro enzymatic activity is greatly increased when bound to single-stranded breaks in DNA. Its in vivo activity is induced by conditions that ultimately lead to DNA fragmentation (Alvarez-Gonzalez and Althaus, 1989; lkejima et al, 1990). The automatic modification of the central domain apparently serves as a negative feedback regulation of PARP.

By detection from labeled NAD⁺Is/are as follows³Incorporation of H into the nucleus of root tip cells first confirmed PARP activity in plant cells (Payne et al, 1976; Willmitzer and Wagner, 1982). The enzyme activity was also partially purified from maize seedlings and found to be associated with a protein with an apparent molecular weight of 113kDa, suggesting that plant PARP may be similar to that from animals (Chen et al, 1994; O' Farrell, 1995).

cDNAs corresponding to PARP proteins have been isolated from several species including mammals, chickens, Xenopus, insects and Caenorhabditis elegans. PARP activity in maize nuclei has been reported by Chen et al (1994) and has been linked to the presence of approximately 114kDa protein in extracts from maize nuclei. O' Farrel (1995) reported that RT-PCR-amplification (using degenerate primers based on the highest conserved sequence) on RNA isolated from maize yielded a 300bp fragment that showed 60% identity at the amino acid level to human PARP protein. Lepinie et al (1995) have isolated and cloned full-length cDNAs from Arabidopsis thaliana encoding a 72kDa protein with a high degree of similarity to the catalytic domain of vertebrate PARP. The N-terminal domain of this protein does not show any sequence similar to the corresponding domain of PARP from vertebrates, but consists of four amino acid chains (named a1, a2, B and C), showing similarity to the N-terminus of a number of intranuclear and DNA binding proteins. The putative secondary structures of a1 and a2 are helix-loop-helix structures.

The gene bank database comprises two cDNA sequences from maize whose translation products have homology in amino acid sequence to either classical PARP proteins (AJ22589) or non-classical PARP proteins identified in arabidopsis (AJ 222588).

The function of PARP and poly-ADP ribosylation in eukaryotic cells is not fully understood. PARP is involved in or believed to be directly or indirectly involved in a number of cellular processes, such as DNA repair, replication and recombination, cell division and cell differentiation or in signal pathways that are altered in sense during genomic integration. Cellular NAD can be greatly reduced due to PARP activity⁺Library, and thus this also suggests that the enzyme may play a crucial role in programmed cell death (Heller et al, 1995; Zhang et al, 1994). Moreover, it has been suggested to cause NAD⁺Nicotinamide, which is hydrolyzed or a conversion product of poly-ADP-ribose by poly-ADP-ribose glucohydrolase, may be a stress response signal in eukaryotic cells.

The information currently available as biological functions of plant PARP comes from experiments involving PARP inhibitors, which suggest an in vivo effect of PARP inhibitors in preventing homologous recombination at the site of DNA damage due to an increased rate of homologous chromosomal recombination in tobacco after application of 3-aminobenzamide (3ABA) (Puchta et al, 1995). Furthermore, it has been shown that differentiation of cells of Zinnia or Helianthus tubiosum using PARP inhibitors such as 3ABA, nicotinamide and 6(5H) -phenanthridinone prevents tubular molecule development (Hawkins and Phillips, 1983; Phillips and Hawkins, 1985; Shoji et al, 1987; Sugiyama et al, 1995), which is considered to be an example of programmed cell death in plants.

PCT application WO97/06267 describes the use of PARP inhibitors to enhance the transformation (qualitative or quantitative) of eukaryotic cells, particularly plant cells.

Lazebnik et al (1994) identified that proteases with similar properties to interleukin 1- β -convertase are capable of cleaving PARP, an early event in programmed cell death in animals.

Kuepper et al (1990) and Molinette et al (1993) describe overproduction of the 46kDa human PARPDNA-binding domain and different mutant forms thereof in transfected CV-1 monkey cells or human fibroblasts, and it has been demonstrated that a transdominant inhibition of residual PARP activity and subsequent inhibition of base excision DNA repair in these cells is residual.

Ding et al (1992) and Smulson et al (1995) describe the exclusion of PARP by antisense RNA expression in mammalian cells and observed a delay in DNA strand break ligation and inhibition of 3T3-L1 preadipocyte differentiation.

Menissier de Murcia et al (1997) and Wang et al (1995, 1997) have generated transgenic "knockout" mice that are mutated in the PARP gene, suggesting that PARP is not an essential protein. However, the cells of PARP deficient mice are more sensitive to DNA damage and differ from cells of normal animals in some aspects of inducing cell death (Heller et al, 1995).

Summary and objects of the invention

The present invention provides a method of modulating programmed cell death in eukaryotic cells comprising reducing the functional level of total PARP activity in eukaryotic cells using the nucleotide sequence of a PARP gene of the ZAP class and the nucleotide sequence of a PARP gene of the NAP class, preferably reducing the expression of an endogenous PARP gene, to reduce the apparent activity of a protein encoded by the endogenous PARP gene or to alter the nucleotide sequence of the endogenous PARP gene.

The present invention also provides a method of modulating programmed cell death in a eukaryotic cell comprising introducing into a eukaryotic cell, preferably a plant cell, a first and a second PCD modulating chimeric gene, wherein the first PCD modulating chimeric gene comprises the following operably linked DNA regions: a promoter operable in a eukaryotic cell; a DNA region which when transcribed yields an RNA molecule which either reduces the functional level of Zn-finger containing PARP proteins of the ZAP class; or can be translated into a peptide or protein that when expressed reduces the functional level of a ZAP class of PARP proteins; and DNA regions involved in transcription termination and polyadenylation; and wherein the second PCD modulating chimeric gene comprises the following operably linked DAN regions: a promoter operable in a eukaryotic cell; a DNA region that when transcribed yields an RNA molecule that can either reduce the functional level of NAP-like PARP proteins; or may be translated into a peptide or protein that, when expressed, reduces the functional level of NAP-like PARP proteins; and DNA regions involved in transcription termination and polyadenylation; and wherein the total apparent PARP activity in the eukaryotic cell is substantially reduced, (preferably the total apparent PARP activity is reduced from about 75% to about 90% from the normal apparent PARP activity in the eukaryotic cell and prevents programmed cell death in the eukaryotic cell) or is nearly complete (preferably the total apparent PARP activity is reduced from about 90% to about 100% from the normal apparent PARP activity in the eukaryotic cell and the cell is killed by programmed cell death).

Preferably, the first transcribed DNA region or the second transcribed DNA region, or both, comprises a nucleotide sequence of at least about 100 nucleotides which are 75% identical to the sense DNA strand of an endogenous PARP gene of the ZAP or NAP class and which encode a sense RNA molecule capable of reducing the expression of an endogenous PARP gene of the ZAP or NAP class.

In another method of modulating programmed cell death provided by the present invention, the first transcribed DNA region or the second transcribed DNA region, or both, comprises a nucleotide sequence of at least about 100 nucleotides which are 75% identical to the complementary strand of the sense DNA strand of an endogenous PARP gene of the ZAP or NAP class and which region encodes an RNA molecule capable of reducing the expression of said endogenous PARP gene of the ZAP or NAP class.

In yet another method of modulating apoptosis provided by the present invention, the first and/or second transcribed DNA region encodes an RNA molecule comprising a sense nucleotide sequence of at least about 100 nucleotides that are 75% identical to an mRNA transcribed from an endogenous PARP gene of the ZAP or NAP class, and an antisense nucleotide sequence of at least about 100 nucleotides that is 75% identical to the complement of an mRNA transcribed from an endogenous PARP gene of the ZAP or NAP class, wherein the sense and antisense nucleotide sequences are capable of forming a double stranded RNA region, and wherein the RNA molecule is capable of reducing the expression of an endogenous PARP gene of the ZAP or NAP class.

In yet another method of modulating apoptosis provided by the present invention, the first and/or second transcribed DNA region encodes a dominant negative PARP mutant capable of reducing the apparent activity of a PARP protein encoded by an endogenous PARP gene of the ZAP or NAP class, which mutant preferably comprises an amino acid sequence selected from amino acids 1 to 159 of the amino acid sequence of SEQ ID No 4 or amino acids 1 to 138 of the amino acid sequence of SEQ ID No 6 or comprises an amino acid sequence selected from amino acids 1 to 370 of the amino acid sequence of SEQ ID No 2, amino acids 1 to 98 of the amino acid sequence of SEQ ID No 11 or amino acids 1 to 88 of the amino acid sequence of SEQ ID No 2 substituted by the amino acid sequence of SEQ ID No 11.

The promoter of the first and second chimeric PCD regulatory genes, or both, may be a tissue specific or inducible promoter, for example a promoter selected from a fungal responsive promoter, a nematode responsive promoter, an anther-selective promoter, a stigma-selective promoter, a dehiscence zone-selective promoter.

The present invention also provides a method of modulating apoptosis in a plant cell comprising introducing into said plant cell a PCD modulating chimeric gene, wherein the PCD modulating chimeric gene comprises the following operably linked DNA regions: a plant expressed promoter, a DNA region, which when transcribed yields an RNA molecule, which molecule is either capable of reducing the expression of an endogenous PARP gene; or capable of being translated into a peptide or protein that when expressed reduces the apparent PARP activity in a plant cell and a DNA region involved in transcription termination and polyadenylation, wherein the total apparent PARP activity in the plant cell is reduced by about 75% to about 100% compared to the normal apparent PARP activity in the plant cell.

It is another object of the invention to provide primary and secondary chimeric PCD modulator genes as well as eukaryotic cells, in particular plant cells comprising the first and second chimeric PCD modulator genes and non-human eukaryotic organisms, in particular plants comprising these cells.

It is a further object of the present invention to provide a method of modulating apoptosis in a plant cell comprising introducing into the plant cell a PCD modulating chimeric gene comprising the following operably linked DNA regions: a plant-expressed promoter, a DNA region, which when transcribed yields an RNA molecule capable of reducing the expression of an endogenous PARP gene of the ZAP class; and DNA regions involved in transcription termination and polyadenylation.

The present invention also relates to a method of increasing the growth rate of a plant comprising introducing into a plant cell a PCD modulating chimeric gene, wherein the PCD modulating chimeric gene comprises the following operably linked DNA regions: a plant-expressed promoter, a DNA region, which when transcribed yields an RNA molecule capable of reducing the expression of an endogenous PARP gene of the ZAP class; and DNA regions involved in transcription termination and polyadenylation.

It is a further object of the present invention to provide a method of producing a stress tolerant plant cell comprising introducing into a plant cell a PCD modulating chimeric gene comprising the following operably linked DNA regions: a plant-expressed promoter, a DNA region, which when transcribed yields an RNA molecule capable of reducing the expression of an endogenous PARP gene of the ZAP class; and DNA regions involved in transcription termination and polyadenylation.

The present invention also relates to the use of a nucleotide sequence encoding a protein having PARP activity, preferably a PARP protein of the ZAP class, for modulating apoptosis or producing stress tolerant plant cells or plants or increasing the growth rate of plant cells or plants in a plant cell or plant.

Brief Description of Drawings

FIG. 1: deduced N-terminal amino acid sequence of plant poly (ADP-ribose) polymerase.

(A) NAD found in Arabidopsis thaliana APP (A.th.APP; EMBL accession number Z48243; SEQ ID No 6) and maize homolog NAP (Z.m.NAP; EMBL accession number AJ 222588; SEQ ID No 4)⁺-alignment upstream of the binding domain. The domain splitting shown is as previously proposed (Lepinie et al, 1995). The Nuclear Localization Signal (NLS) located in this B domain is shown in parentheses. The sequence of the B domain is not very conserved between dicots and monocots. The C domain is functionally comparable to the automodified domain of PARP from animals. Incompletely repeated A1 and A2 were also present in maize NAP. To illustrate the internal incomplete twofold symmetry in the repeated sequences, the properties of the amino acid residues in the following sequences are highlighted: a flattening ring and lipophilic residues; ring opening, glycine; (+), a positively charged residue; (-) negatively charged residues; wavy lines, arbitrary residues. The axis of symmetry is indicated by the vertical arrows, and the arrowed lines mark regions with reproducibility of amino acid side chain performance transformations.

(B) Sequence alignment of the DNA-binding and autocatalytic domains of mouse PARP and maize ZAP. The Zn-containing maize ZAP1 and ZAP2 (partial cDNAs found by 5' RACE PCR analysis) were designated Z.m.ZAP (EMBL accession number AJ 222589; SEQ ID No 2) and Z.m.ZAP (RACE) (amino acid positions 1-98 in SEQ ID No 11), and mouse PARP, M.m.ADPRT (Swissprot accession number P11103), respectively. The Zn-designation and the duplicate NLS of this mouse enzyme are indicated by brackets, the Caspase 3 cleavage site is shown by asterisk, and the putative NLS in the ZAP protein is shown by bold brackets in the following maize sequence. Amino acid sequences conserved in all sequences are boxed; amino acid residues with similar physico-chemical properties are masked with the uppermost sequence as reference.

FIG. 2: NAD of mouse PARP and plant PARP proteins⁺-comparison of binding domains. The range of "PARP features" is shown above in the sequence. Name andsequence pairs such as fig. 1.

FIG. 3: evaluation of the gene copy number and transcript size of the nap and zap genes.

Maize genomic DNA of variants LG2080 (A) and (B), digested with the indicated restriction endonucleases, separated by agarose gel electrophoresis, blotted, and hybridized with radiolabeled DNA probes prepared from the 5' domain of nap and zap cDNAs, which do not encode NAD⁺-a binding domain. The hybridization pattern obtained with the nap probe (A) is simple and shows a single nap gene in the maize genome. As can be seen from the hybridization pattern (B), there may be at least two zap genes. To examine the size of the transcripts encoded by the zap and nap genes, approximately 1. mu.g of poly (A) extracted from the roots (route 1) and shoots (route 2) of 6-day-old seedlings were separated on an agarose gel after denaturation with glyoxal⁺RNA, blotting with nap (C) and zap (D)³²P-labeled cDNA. Using lambda DNA³³The BstEII fragment marked at the end of P5' is used as a molecular weight marker in DNA and RNA gel blot experiments; their positions are shown in kb to the left of each square.

FIG. 4: APP expression was analyzed in yeast.

(A) Schematic representation of the expression cassette in pV8 SPA. Expression of the appcDNA was driven by a chimeric yeast promoter consisting of a minimal TATA box comprising the promoter region of CYC1 gene (CYC1) and the upstream activating promoter region of ga110 gene (GAL10), which provides promoter activation by galactose. Downstream regulatory sequences were obtained from the gene encoding phosphoglycerate kinase (3PGK) (Kuge and Jones, 1994). The app coding region is obtained from a partition in a hypothetical domain, as proposed earlier (Lepiniec et al, 1995); a1 and A2 correspond to incomplete 27-amino acid repeats in which there is a sequence (B domain) that is rich in positively charged amino acids and resembles the DNA binding domain of a large number of DNA binding proteins. The amino acid sequence of this B domain is shown in the following figure and the chain of arginine and lysine residues that can function as NLS is drawn in bold. Methionine residue (M) which can function as translation initiation codon¹、M⁷²) As shown in the above figures. C-Domain enriched with glutamic acid residuesGenes that are similar in their composition, but not in their sequence, to PARP automodification domains from animals.

(B) Immunoblot (Western blot) and Northern blot analyses of DY (pYeDP1/8-2) and DY (pV8SPA) strains, as indicated (vector) and (app), respectively. The strains were grown in SDC medium supplemented with Glucose (GLU), Galactose (GAL), galactose and 3mM 3ABA (GAL +3ABA), or galactose and 5mM nicotinamide (GAL + NIC). All RNA or all proteins were extracted from the same culture. The total proteins of 10 microorganisms were separated by electrophoresis on 10% SDS-PAGE, electroblotted, and probed with anti-APP antiserum. Total RNA from 5 microorganisms was separated by electrophoresis on a 1.5% agarose gel, blotted onto a nylon membrane and compared to that obtained from app cDNA³²P-labeled DNA fragments. The position of the molecular weight marker band is shown on the left in kilo-pairs of bases (kb) and kilodaltons (kDa).

FIG. 5: poly (ADP-ribose) polymerase activity of APP protein.

(A) The total protein extract was prepared from DY (pYeDP1/8-2) grown on SDC (vector GAL) with 2% galactose and DY (pV8SPA) grown on SDC (app GLU) with 2% glucose, SDC (app GAL) with 2% galactose, or SDC (app GAL +3ABA) with 2% galactose and 3mM 3 ABA. To detect the poly (ADP-ribose) synthesized in these extracts, samples were used at room temperature³²P-NAD⁺Incubate for 40 minutes. Two control reactions were performed: 100ng of purified human PARP were cultured either in reaction buffer (PARP) alone (lane 5) or with protein extract (vector GLU + PARP) prepared from DY (pYeDP1/8-2) culture grown on glucose (lane 6). Shows autoradiographs obtained after exposure of the xerogels to X-Omat Kodak film. ORi corresponds to the start of the sequencing gel.

(B) Poly (ADP-ribose) synthesis is stimulated by DNA in protein extracts from DY (pV8 SPA). In μ g ml^-1The amount of sonosalmon sperm DNA added to the nucleic acid of the deletion yeast extract is shown. Blocking the synthesis of poly (ADP-ribose) by 3ABA, whereEach of these reactions was supplemented with 3ABA at a concentration of 3mM (lane 5). To ensure maximum recovery of poly (ADP-ribose), 20. mu.g glycogen was included as a carrier in the precipitation step; however, as can be seen, this will result in significant entrainment of the unincorporated indicia.

FIG. 6: a T-DNA vector comprising a PCD modulating chimeric gene of the invention is illustrated. P35S: the CaMV35S promoter; l: the cab22 preamble; ZAP; coding region of the ZAP class of PARP genes; 5' ZAP: the N-terminal part of the coding region of the PARP gene of the ZAP class in inverted orientation; 3' 35S: CaMV35S 3' terminal transcriptional termination and polyadenylation signals; pACT 2: the promoter region of the actin gene; pNOS; a nopaline synthase gene promoter; and (g) gat: gentamicin acetyltransferase; bar: phosphinothricin acetyltransferase; 3' NOS: a transcription termination signal and a polyadenylation signal at the 3' end of the nopaline synthase gene; APP: coding region of NAP-like PARP gene; 5' APP: the N-terminal portion of the coding region of the PARP gene of NAP class in reverse orientation; LB: left T-DNA borders; RB: right T-DNA border; pTA 29: anther-specific promoter, pNTP 303: pollen-specific promoters.

Detailed description of the preferred embodiments

For the purposes of the present invention, the term "plant-expressed promoter" means a promoter capable of driving transcription in plant cells. It includes any promoter of plant origin, but also any promoter of non-plant origin capable of directing transcription in plant cells, for example certain promoters of viral or bacterial origin such as the CaMV35S or the T-DNA gene promoter.

The term "expression of a gene" refers to the process by which a DNA region is transcribed into biologically active RNA (i.e., either capable of interacting with another nucleic acid or protein or being translated into a biologically active polypeptide or protein) under the control of a regulatory region, particularly a promoter. A gene is said to encode RNA when the end product of the gene expression is a biologically active RNA such as an antisense RNA or ribozyme. A gene is said to encode a protein when the end product of the expression of the gene is a biologically active protein or polypeptide.

The term "gene" is meant to include any segment of DNA that is transcribed into a DNA region of an RNA molecule (e.g., mRNA) ("transcribed DNA region") in a cell under the control of suitable regulatory regions, such as a plant-expressed promoter. A gene may thus comprise several operably linked DNA segments such as a promoter, a5 'leader sequence, a coding region and a 3' region comprising a polyadenylation site. Endogenous plant genes are genes found naturally in plant species. The chimeric gene is any gene not normally found in plant species, or any gene whose promoter is not associated in nature with part or all of the transcribed DNA region or with at least one other regulatory region of the gene.

The word "comprise", or variations such as "comprises" or "comprising", when used herein, is interpreted to specify the presence of stated features, integers, steps or components, but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. Thus, for example, a nucleic acid or protein comprising a sequence of nucleotides or amino acids may comprise more nucleotides or amino acids than actually indicated, i.e. be comprised in a larger nucleic acid or protein. Chimeric genes comprising DNA regions defined in function or structure may include other DNA regions, and the like.

One aspect of the present invention is based on the discovery that eukaryotic cells, particularly plant cells, more particularly Zeamays cells, contain simultaneously at least two functional major PARP protein isoforms (classes) that differ in both size and amino acid sequence, are capable of binding DNA, particularly DNA with a single-stranded cleft, and have poly-ADP ribosylation activity. On the other hand, the present inventors have realized that apoptosis in eukaryotic cells, in particular plants, can be regulated by altering the expression level of PARP or by genetically altering the activity of the encoded protein, and in particular to achieve this goal, either the expression of both genes needs to be altered or one of the two classes of proteins needs to be altered in its activity.

It is clear that the prior art does not show that eukaryotic cells (especially plant cells) contain two functional isoforms of the PARP protein encoded by different classes of genes, which prevents efficient regulation of PARP activity in these cells by recombinant DNA methods. While the specification, examples and claims describe different embodiments of these methods and means.

Thus, the present invention relates to modulating, i.e. increasing or inhibiting apoptosis in eukaryotic cells, preferably plant cells, by altering the level of PARP gene expression, or by altering the activity or apparent activity of PARP proteins in eukaryotic cells. Conveniently, the level of PARP gene expression or activity of PARP protein is genetically controlled by the addition of a PCD modulating chimeric gene which alters expression of the PARP gene and/or by the addition of a PCD modulating chimeric gene which alters the apparent activity of the PARP protein and/or by the alteration of an endogenous PARP encoding gene.

As used herein, "increased PCD" with respect to a particular cell refers to the death of such cells stimulated by the methods of the invention, whereby the killed cells are not intended to undergo PCD under similar conditions when compared to similar cells of a normal plant not modified by the methods of the invention.

By "inhibiting PCD" with respect to a particular cell is understood that a majority of these cells or cell families that normally (without intervention by the method of the invention) undergo apoptosis under particular conditions remain viable under these conditions.

Thus the introduction of PCD to modulate the expression of a chimeric gene or a modified endogenous gene will affect the functional level of PARP protein and indirectly interfere with apoptosis. A modest reduction in the functional level of PARP protein results in the inhibition of apoptosis, particularly the prevention of apoptosis, while a severe reduction in the functional level of PARP protein will result in the induction of apoptosis.

According to the present invention, it is preferred that for the purpose of inhibiting or preventing apoptosis in eukaryotic cells, in particular plant cells, the combined level of PARP protein and/or its activity or apparent activity is substantially reduced, but the inhibition of DNA repair (dominated directly or indirectly by PARP) is avoided in the following manner: the function of PARP protein is inhibited in cells that cannot recover from DNA damage or maintain their genomic integrity. Preferably, the level and/or activity of PARP protein in the target cell should be reduced by about 75%, preferably about 80%, especially about 90% of the normal level and/or activity in the target cell, so that about 25%, preferably about 20%, especially about 10% of the normal level and/or activity of PARP is retained in the target cell. It is also contemplated that the level and/or activity of the PARP protein should be reduced by no more than 95%, preferably no more than 90% of the normal activity and/or level in the target cell. Methods for determining the content of specific proteins, such as PARP proteins, are well known to those skilled in the art and include, but are not limited to, the quantification of these proteins using specific antibodies (histochemistry). Methods for quantifying PARP activity are also available in the prior art, including the TUNEL assay described above (in vivo) or Collinge and Althaus (1994) describe in vitro assays for the synthesis of poly (ADP-ribose) (see examples).

Also according to the present invention, preferably in order to trigger apoptosis in eukaryotic cells, in particular plant cells, the combined level of PARP protein and/or its activity or apparent activity is greatly reduced, preferably almost completely reduced, so that DNA repair and maintenance of genomic integrity is no longer possible. Preferably, the combined level and/or activity of PARP protein in a target cell should reduce at least about 90%, preferably about 95%, more preferably about 99% of the normal level and/or activity in the target cell, in particular should completely inhibit PARP activity. It is particularly preferred that the functional levels of the two classes of PARP proteins are reduced to said levels, respectively.

For the purposes of the present invention, PARP proteins are defined as proteins having poly (ADP-ribose) polymerase activity, preferably comprising the so-called "PARP feature". This PARP is characterized by an amino acid sequence highly conserved among PARP proteins, defined by de Murcia and Menussier demucinia (1994) as extending from amino acid position 858 to amino acid position 906 of the Mus musculus PARP protein. This domain corresponds to the amino acid sequence at position 817-. This amino acid sequence is highly conserved (with about 90% -100% sequence identity) between different PARP proteins. The lysine in position 891 of the PARP protein from Mus musculus (corresponding to position 850 of SEQ ID No 2, position 861 of SEQ ID No 11, position 532 of SEQ ID No 4, position 517 of SEQ ID No 6) is particularly conserved and is believed to be involved in the catalytic activity of the PARP protein. In particular, amino acids at positions 865, 866, 893, 898 and 899 or corresponding positions of other sequences of the PARP protein of Musmusculus can be obtained. PARP proteins may also include an N-terminal DNA binding domain and/or a Nuclear Localization Signal (NLS).

Currently, two classes of PARP proteins have been described. The first class, as defined herein, includes the so-called canonical PARP proteins (ZAPs) containing Zn-fingers. These proteins range in size from 113-120kDA and are also identified by the presence of at least one, and preferably two Zn-finger domains located in the N-terminal domain of the protein, particularly in the first about 355 to about 375 amino acids of the protein. The Zn-finger is defined as having a sequence CxCx capable of complexing Zn atoms_nPeptide sequence of HxxC (where n may be 26-30). Examples of amino acid sequences of PARP proteins from the ZAP class include sequences that can be found in the PIR protein databases under accession numbers P18493(Bos taurus), P26466(Gallus balloon), P35875(Drosophila melanogaster), P09874(Homo sapiens), P11103(Mus musculus), Q08824(Oncorynchus masou), P27008(Rattus norvegicus), Q11208 (Sarcophagapegregaria), P31669(Xenopus laevis) and the sequences of ZAP1 and ZAP2 (SEQ ID No 2/SEQ ID No 11) currently identified from Zea mays.

The nucleotide sequence of the corresponding cDNA can be found in the EMBL database under the accession numbers D90073(Bos taurus), X52690(Gallus balloon), D13806(Drosophila melanogaster), M32721(Homosapiens), X14206(Mus musculus), D13809(Oncorynchus masou), X65496(Rattus norvegicus), D16482(Sarcophaga peregrina), D14667 (Xenuglaevis) and in SEQ ID Nos 1 and 10 (maize).

The second class, as defined herein, includes the so-called non-classical PARP proteins (NAPs). These proteins are small (72-73kDa) and are otherwise characterized by the absence of a Zn-finger domain at the N-terminus of the protein, but the presence of an N-terminal domain comprising a stretch of amino acids similar to that of a DNA binding protein. Preferably, such PARP proteins comprise at least one amino acid sequence of about 30-32 amino acids comprising the sequence RG XXXXXG XK XXL (amino acids are represented by standard one letter codes, whereby X represents any amino acid; SEQ ID No 7). Even more preferably these PARP proteins comprise at least 1 amino acid sequence of about 32 amino acids having the sequence x L x V x R x L x RGL x GVK x LV x RL x Al (SEQ ID No 8) (so-called a1 domain) or at least 1 amino acid of about 32 amino acids having the sequence GM x EL x a x RG x G x KKD x RL x (SEQ ID No 9) (so-called a2 domain) or both. In particular, the a1 and a2 domains are capable of forming a helix-loop-helix structure. The PARP proteins may also include a basic "B" domain (a K/R-rich amino acid sequence of about 35 to about 56 amino acids, involved in directing the protein to the nucleolus) and/or an acidic "C" domain (a D/E-rich amino acid sequence of about 36 amino acids). Examples of protein sequences from this NAP class include the APP protein from Arabidopsis (available from PIR protein database with accession number Q11207; SEQ ID No 6) and the NAP protein from maize (SEQ ID No 4). The sequence of the corresponding cDNA can be found in the EMBL database with accession number Z48243 (SEQ ID No 5) and SEQ ID No 3. This second class of PARP proteins is truly functional PARP proteins, i.e. capable of catalyzing DNA-dependent poly (ADP-ribose) polymerization, which has been confirmed by the present inventors (see example 2).

The present inventors have also demonstrated that eukaryotic cells, particularly plant cells, express genes encoding PARP proteins from both classes.

It is obvious that for the purposes of the present invention, other genes or cdnas encoding PARP proteins from both of these defined classes, or parts thereof, can be isolated from other eukaryotic species or variants, in particular from other plant species or variants. These PARP genes or cdnas can be isolated, for example, by Southern hybridization (or low stringency or high stringency hybridization depending on the relationship between the species from which the PARP gene is intended to be isolated and the species from which the probe is ultimately obtained) using as a probe a DNA fragment having the above-described PARP gene or cDNA, or a portion thereof, preferably a conserved portion such as a nucleotide sequence of a gene fragment containing a nucleotide sequence encoding the PARP feature as described above. The nucleotide sequence corresponding to the PARP characteristic of the PARP protein encoded from the plant gene is the nucleotide sequence from nucleotides 2558-2704 of SEQ ID No 1 or the nucleotide sequence from nucleotides 1595-1747 of SEQ ID No 3 or the nucleotide sequence from nucleotides 1575-1724 of SEQ ID No 5. If identification is made between these PARP gene classes, it is preferred to use a part of the PARP gene specific for such class, such as the N-terminal domain preceding the catalytic domain or a part thereof.

In addition, the gene or cDNA encoding the PARP protein, or a part thereof, may also be isolated by PCR amplification using suitable primers such as degenerate primers having nucleotide sequences corresponding to the sequences shown in SEQ ID No 13, SEQ ID No 14 or primers having nucleotide sequences corresponding to the sequences shown in SEQ ID Nos 15-20. However, it is clear that the skilled person can design other oligonucleotides for use in PCR, or can use oligonucleotides comprising a nucleotide sequence of at least 20, preferably at least about 30, in particular at least about 50, conserved nucleotides of any PARP gene to isolate these genes or parts thereof by PCR amplification.

It is clear that a person skilled in the art, in combination with these or other techniques (including e.g. RACE-PCR) for isolating genes or cDNAs on the basis of partial fragments and their nucleotide sequences, e.g. obtained by PCR amplification, can be used to isolate PARP genes or parts thereof suitable for use in the method of the invention.

Furthermore, PARP genes or synthetic PARP genes (which, depending on the degeneracy of the genetic code, encode proteins similar to the natural PARP gene but with different nucleotide sequences) and parts thereof, which encode PARP proteins in which some amino acids have been replaced by other chemically similar amino acids (so-called conservative substitutions), are also suitable for the method of the invention.

In one aspect of the invention PCD in eukaryotic cells, in particular plant cells, is inhibited by suitably reducing the level of PARP function in these eukaryotic cells.

In one embodiment of this first aspect of the invention, the functional level of PARP in eukaryotic cells, particularly plant cells, is reduced by adding to these cells at least one PCD modulating chimeric gene comprising a promoter, preferably plant expressed, capable of directing transcription in these cells, and a functional 3' transcribed end and a polyadenylation region operably linked to a DNA region which, when transcribed, produces a biologically active RNA molecule capable of reducing the functional level of endogenous PARP activity encoded by both types of PARP genes.

In a preferred embodiment, at least 2 such PCD modulating chimeric genes are added to the cells, whereby the biologically active RNA encoded by the first PCD modulating chimeric gene reduces the functional level of endogenous PARP activity encoded by the NAP class, whereby the biologically active RNA encoded by the second PCD modulating chimeric gene reduces the functional level of endogenous PARP activity encoded by the ZAP class gene, whereby the bound PARP activity is moderately reduced.

In a particularly preferred embodiment, the PCD modulating chimeric gene reduces the functional level of endogenous PARP activity by reducing the expression level of endogenous PARP genes. To this end, the transcribed DNA region encodes an RNA that is biologically active to reduce the mRNA encoding NAP and ZAP-like PARP proteins, which can be obtained by translation. This can be achieved by techniques such as antisense RNA, co-suppression or ribozyme action.

As used herein, "co-suppression" refers to transcriptional and/or post-transcriptional suppression in which RNA accumulates in a sequence-specific manner such that expression of a homologous endogenous gene or transgene is suppressed.

By introducing a transgene comprising a strong promoter operably linked to a DNA region, whereby the resulting transcribed RNA is a sense RNA or antisense RNA comprising a nucleotide sequence having at least 75%, preferably at least 80%, in particular at least 85%, more in particular at least 90%, in particular at least 95% sequence identity or identical thereto with the coding or transcribed DNA sequence (sense) or the complementary strand of the part of the PARP gene the expression of which is to be inhibited (antisense). Preferably, the transcribed DNA region does not encode a functional protein. In particular, the transcribed region does not encode a protein. Furthermore, the nucleotide sequence of the sense or antisense region should preferably be at least about 100 nucleotides long, more preferably at least about 250 nucleotides, especially at least about 500 nucleotides, but may extend the full length of the coding region of the gene whose expression is to be reduced.

For the purposes of the present invention, "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in two optimally aligned sequences having identical residues divided by the number of positions compared (× 100). Gaps, i.e., positions where residues are aligned in one sequence but not in another, are considered to be positions having non-identical residues. The alignment of the two sequences was performed using the Wilbur and Lipmann algorithms (Wilbur and Lipmann, 1983) with a window size of 20 nucleotides or amino acids, a word length of 2 amino acids and a gap catch of 4. Computer-assisted analysis and interpretation of sequence data, including sequence alignment as described above, can be performed using commercially available software packages such as intelligentics^TMThe Suite procedure (intelligentics inc., CA) is conveniently performed.

It is obvious that one skilled in the art may use one or more sense or antisense PCD modulating chimeric genes to achieve the first object of the invention. When a sense or antisense PCD is used to modulate a chimeric gene, the gene must be capable of simultaneously reducing the expression of both types of PARP genes. This can be achieved, for example, by selecting the transcribed region of the chimeric gene in such a way that the expression of both types of genes is regulated by a sense or antisense RNA, i.e.by selecting the target subregions corresponding to the most homologous DNA regions of both types of PARP genes and adding the synonymous or antisense transcribed DNA regions corresponding to the target subregions of both types, ensuring the conditions as described above for the sense and antisense RNA. Alternatively, different synonymous or antisense RNA regions, each specifically regulating the expression of a class of PARP genes, may be incorporated into one RNA molecule, encoded by one transcribed region of one PCD regulatory chimeric gene. It is apparent that different sense or antisense RNA regions specific for modulating the expression of a class of PARP genes can be added as a single PCD modulating chimeric gene.

Preferred sense and antisense encoding transcribed regions comprise nucleotide sequences corresponding to at least about 100 conserved nucleotides selected from the N-terminal domain of the PARP gene (sequence identity constraints as described above), preferably to at least about 100 conserved nucleotides selected from nucleotide positions 113 and 1189 of SEQ ID No 1, nucleotide position 107 and 583 of SEQ ID No 3, nucleotide position 131 and 542 of SEQ ID No 5 or nucleotide position 81 and 1180 of SEQ ID No 10. However, it is clear that sense or antisense coding transcribed regions containing sequences corresponding to the complete sequence of the N-terminal domain of the PARP gene, and even to the complete sequence of the PARP gene, in particular to the protein coding region thereof, can be used. And preferably comprises sense and antisense encoding transcribed regions corresponding to nucleotide sequences of at least about 100 conserved nucleotides (with sequence identity constraints as described above) selected from the C-terminal catalytic domain of the PARP gene, preferably sequences of at least 100 nucleotides comprising a PARP characteristic encoding nucleotide sequence, in particular a PARP characteristic encoding nucleotide sequence as described above. Furthermore, it is clear that sense or antisense coding transcribed regions containing sequences corresponding to the entire sequence of the C-terminal domain of the PARP gene can be used.

In another particularly preferred embodiment, the PCD modulating chimeric gene reduces the functional level of endogenous PARP activity by reducing the apparent activity level of both classes of endogenous PARP. To this end, these transcribed DNA regions encode biologically active RNA that is translated into a protein or peptide that inhibits NAP or ZAP-like PARP proteins or both, such as inactive antibodies or dominant negative PARP mutants.

An "inactive antibody to a PARP protein" is an antibody or part thereof that specifically binds at least some epitopes of the PARP protein, e.g. the part ZN covering position 111-118 from ZAP1 refers to a peptide of II or the corresponding peptide in ZAP2, and which inhibits the activity of the target protein.

As used herein, a "dominant-negative PARP mutant" is a protein or peptide containing at least a portion of a PARP protein (or variant thereof), preferably a PARP protein endogenous to a eukaryotic target host cell, which has no PARP activity and has an inhibitory effect on the activity of the endogenous PARP protein when expressed in the host cell. Preferred dominant-negative PARP mutants are proteins comprising or consisting of a functional DNA binding domain (or variant thereof) but No catalytic domain (e.g.an N-terminal Zn-finger comprising about 355 to about 375 ZAP-like PARP amino acids, in particular a DNA binding protein domain comprising the amino acid sequence of amino acids 1 to 370 of SEQ ID No 2 or a DNA binding protein domain comprising the amino acid sequence of amino acids 1 to 98 of SEQ ID No 11, or a DNA binding protein domain comprising the amino acid sequence of amino acids 1 to 370 of SEQ ID No 2, wherein the amino acid sequence of amino acids 1 to 88 is replaced by the amino acid sequence of amino acids 1 to 98 of SEQ ID No 11, or an N-terminal DNA binding protein domain comprising about 135 and 160 NAP-like PARP amino acids, in particular a DNA binding protein domain comprising the amino acid sequence of amino acids 1 to 159 of SEQ ID No 4 or a DNA binding protein domain comprising the amino acid sequence of SEQ ID No 4 DNA binding protein domain of the amino acid sequence of amino acids 1 to 138 of ID No 6) or No functional catalytic domain (e.g. inactive PARP mutants, mutated in the so-called PARP feature, in particular conservative lysine mutations at position 850 of SEQ ID No 2, position 532 of SEQ ID No 4, position 517 of SEQ ID No 6). Preferably, the dominant-negative PARP mutant should retain its DNA binding activity. The dominant negative PARP mutant may be fused to a carrier protein such as beta-glucuronidase (SEQ ID No 12).

Furthermore, the first object of the present invention can be achieved using one or more PCD modulating genes encoding one or more dominant negative PARP mutants. When a PCD is used to regulate a chimeric gene, the gene must be able to simultaneously reduce the expression of both classes of PARP genes.

In another embodiment of the first aspect of the invention, the functional level of PARP in eukaryotic cells, in particular plant cells, is reduced by modulating the nucleotide sequence of the endogenous PARP gene in these cells such that the encoded mutant PARP protein retains about 10% of its activity. Methods to achieve such regulation of endogenous PARP genes include homologous recombination to change the endogenous PARP gene into a mutant PARP gene, for example by methods described in US 5527695. In a preferred embodiment this position-mediated regulation of the nucleotides of the endogenous PARP gene is achieved by the addition of chimeric DNA/RNA oligonucleotides as described in WO96/22364 or US 5565350.

For plant cells, it has however been found that the addition of a PCD modulating chimeric gene, preferably encoding a biologically active RNA that acts to reduce the expression of a class of PARP genes, in particular of ZAPs, may be sufficient to reduce the overall PARP activity in these plant cells according to the first aspect of the invention, i.e. to inhibit or prevent programmed cell death in these plant cells.

In this embodiment of the invention, the PCD modulating chimeric gene preferably comprises a transcribed region encoding a biologically active RNA comprising at least one RNA region, preferably at least 100 nucleotides in length, classified according to the criteria mentioned herein as a sense RNA for one of the endogenous PARP genes, and the sense RNA contains at least one other RNA region, preferably at least 100 nucleotides in length, classified according to the criteria mentioned herein as an antisense RNA for one of the endogenous PARP genes, whereby the antisense and sense RNA regions are capable of constituting a double-stranded region, preferably at least about 100 nucleotides in length.

According to a first aspect of the present invention, it is desirable to add a PCD modulating chimeric gene which reduces the functional or apparent level of a class of PARP proteins, in particular those of the ZAP class described herein, and which may also be sufficient to reduce the overall PARP activity in plant cells.

According to a first aspect of the invention, the reduction or inhibition of programmed cell death in plant cells comprising at least one PCD modulating chimeric gene may result in increased resistance to adverse conditions, such as stress resistance to treatment with chemicals, low temperature stress resistance, stress resistance conferred by pathogens and plagues, drought resistance, high temperature stress resistance, and the like.

In another aspect of the invention, programmed death of eukaryotic cells, preferably selected cells, particularly selected plant cells, is enhanced by a severe reduction, preferably a nearly complete reduction, in the functional level of PARP, such that the DNA is no longer repaired and the genomic integrity is no longer maintained.

In one embodiment of this aspect of the invention, the functional level of PARP is severely reduced, in particular almost completely eliminated, in eukaryotic cells, in particular plant cells, by introducing into these cells at least one PCD modulating chimeric gene comprising a promoter capable of mediating transcription in these cells, preferably a plant-expressed promoter, and a functional 3' transcription termination and polyadenylation region, operably linked to the DAN region, which upon transcription yields a biologically active RNA molecule capable of reducing the functional level of endogenous PARP activity encoded by both types of PARP genes.

In a preferred embodiment of the second aspect of the invention, at least two such PCD modulating chimeric genes are introduced into these cells, whereby the biologically active RNA encoded by the first PCD modulating chimeric gene reduces the functional level of endogenous PARP activity encoded by a gene of the NAP class and whereby the biologically active RNA encoded by the second PCD modulating chimeric gene reduces the functional level of endogenous PARP activity encoded by a gene of the ZAP class, such that the combined PARP activity is greatly reduced, in particular almost completely abolished.

As mentioned in the first aspect of the invention, the transcribed region of the PCD modulating chimeric gene encodes a biologically active RNA which is capable of interfering with the expression of the endogenous PARP gene (e.g. by antisense, co-suppression or ribozyme action) or which is also translatable into a peptide or protein which is capable of inhibiting the PARP proteins of the NAP and ZAP classes, such as inactive antibodies or dominant negative PARP mutants.

In a particularly preferred embodiment of the second aspect of the invention, the transcribed region of the PCD modulating chimeric gene (PCD enhancing chimeric gene) encodes a biologically active RNA comprising at least one RNA region (preferably at least about 100 nucleotides in length) classified according to the above criteria as a sense RNA for at least one endogenous PARP gene and at least one other RNA region (preferably at least about 100 nucleotides in length), classified according to the above criteria as an antisense RNA for at least one endogenous PARP gene, whereby the antisense and sense RNA regions are capable of combining into a double stranded RNA region (preferably at a distance of at least about 100 nucleotides). In a particularly preferred embodiment, two such PCD modulating genes, one targeted to reduce the functional level of a PARP protein of the NAP class and the other targeted to reduce the functional level of a PARP protein of the ZAP class, are introduced into a eukaryotic cell or organism, preferably a plant cell or plant.

It is clear that different embodiments of the transcribed DNA region of the chimeric PCD modulating gene of the invention may be used in different combinations to achieve the object of the invention. For example, a first chimeric PCD modulator gene may be designed to encode a sense RNA that reduces the expression of an endogenous PARP gene of the ZAP class, while the second chimeric PCD modulator gene may be designed to encode a dominant negative PARP mutant that reduces the expression of an endogenous PARP gene of the ZAP class.

Whether the PCD modulating chimeric gene is introduced into eukaryotic cells will ultimately result in a modest or severe reduction in the functional level of the combined PARP in these cells, i.e. inhibition of PCD or enhancement of PCD will always be determined by the expression level of these PCD modulating genes (either at the transcriptional level or at the combined transcriptional/translational level). The main factor that has an effect on the expression level of the PCD regulator gene is the choice of the promoter region, although other factors (such as, but not limited to, the choice of the 3 ' end, the presence of introns, codon choice of the transcribed region, mRNA stability, consensus sequences around the translation start position, the choice of 5 ' and 3 ' untranslated RNA regions, the presence of PEST sequences, the effect of chromatin structure around the insertion site of stably integrated PCD regulator gene, the copy number of the PCD regulator gene introduced, etc.) or combinations thereof also have an effect on the final expression level of the PCD regulator gene. In general, it can be assumed that a modest reduction in the functional level of combined PARP can be achieved by PCD regulatory genes containing rather weak promoters, whereas a severe reduction in the functional level of combined PARP can be achieved by PCD regulatory genes containing rather strong promoters. However, the level of expression of the PCD-regulating gene containing a specific promoter and ultimately the effect on PCD may vary with the function of other influencing factors, as already described.

For purposes of particular embodiments of the present invention, the PCD modulating chimeric gene may comprise a constitutive promoter, or a promoter expressed in all or a major cell type throughout an organism, particularly throughout a plant, such as a promoter region from a T-DNA gene, particularly a opine amino acid of the Agrobacterium Ti-or Ri-plasmid (e.g., nos, ocs promoters), or a promoter region of a viral gene (e.g., the CaMV35S promoter or a variant thereof).

It may also be advantageous to control expression of PCD regulatory genes at will or in response to environmental cues, for example by including inducible promoters, which may be activated by external stimuli such as, but not limited to, the use of compounds (e.g. safeners, herbicides, glucocorticoids), light conditions, exposure to abiotic stress (e.g. injury, heavy metals, extremes of temperature, salinity or drought) or biotic stress (e.g. pathogen or plague infection, including by fungi, viruses, bacteria, insects, nematodes, mycoplasmal and mycoplasma organisms, etc.). Examples of inducible promoters suitable for expression by the plants of the invention are: nematode inducible promoters (WO 93/19188, WO 96/28561), promoters induced after the use of glucocorticoids such as dexamethasone, or promoters presented or activated after the use of tetracycline (Gatz et al, 1988; Weimann et al, 1994).

In several embodiments of the invention, and in particular in the second aspect of the invention (i.e. enhanced PCD), it may be convenient or desirable to limit the effect of programmed cell death on a particular subset of cells of an organism, in particular a plant, and therefore the PCD modulating gene may comprise a tissue-specific or cell-class specific promoter. Examples of suitable plant-expressed promoters that are selectively expressed in a particular tissue or cell type are well known in the art and include, but are not limited to, seed-specific promoters (e.g., WO89/03887), organ-primordial-specific promoters (An et al, 1996), stem-specific promoters (Keller et al, 1988), leaf-specific promoters (Hudspeth et al, 1989), mesophyllic-specific promoters (e.g., the light-induced Rubisco promoter), root-specific promoters (Keller et al, 1989), tuber-specific promoters (Keil et al, 1989), vascular tissue-specific promoters (Peleman et al, 1989), meristem-specific promoters (e.g., the promoter of the SHOOTISTETETELESS (TM) gene, Long et al, 1996), primordial-specific promoters (e.g., the promoter of the Antirrhinum CycD3a gene, Doonan et al, 1998), anther-specific promoters (WO 89/10396/03887), organ-specific promoters (see, WO9213956, WO9213957), stigma-specific promoter (WO 91/02068), dehiscence region-specific promoter (WO 97/13865), seed-specific promoter (WO 89/03887), and the like.

Preferably the chimeric PCD modulating gene of the invention is accompanied by a marker gene, preferably a chimeric marker gene comprising a marker DNA operably linked to the 5' end of a plant-expressed promoter, preferably a constitutive promoter such as the CaMV35S promoter, or a light-inducible promoter such as the promoter of a gene encoding the small subunit of Rubisco; and a3 'end operably linked to a suitable plant transcriptional 3' end to form and polyadenylation signal. The choice of the desired marker DNA is not critical and any suitable marker DNA may be used. For example, the marker DNA may encode a protein which confers a distinctive "colour" on transformed plant cells, such as the A1 gene (Meyer et al, 1987) or the green fluorescent protein (Sheen et al, 1995), may confer herbicide resistance on transformed plant cells, for example the bar gene, encodes phosphinothricin resistance (EP 0242246), or may confer antibiotic resistance on transformed cells, such as the aac (6') gene, encodes gentamicin resistance (WO 94/01560).

Methods for introducing PCD modulating chimeric genes into eukaryotic cells, particularly methods for transforming plant cells, are well known in the art and are not believed to be important to the methods of the present invention. Transforming the resulting or transition or stably transformed cell (thereby stably inserting the PCD modulating chimeric gene into the genome of the cell, in particular the nuclear genome of the cell).

It is clear that the methods and means for altering apoptosis in eukaryotic cells and organisms, particularly plant cells and plants, described in the present invention have several important application possibilities. The inhibition of PCD by the methods and means of the present invention may be used to relieve the stress applied to these cells, particularly plant cells, during transformation and thus increase transformation efficiency, as described in WO 97/06267. Inhibition of PCD can also be used to enhance cell culture of eukaryotic cells, particularly plant cells. Triggering of PCD, particularly of cell type, using the means and methods of the invention may be used to call for methods of using cytotoxins. Since PCD is the "natural" route of cell death, the use of the PCD enhancing chimeric genes of the present invention constitutes an improvement over the use of other cytotoxic genes such as RNAse or diphtheria toxin genes which cause cell lysis. Moreover, low expression of PCD enhancing genes in cells other than the committed cells will result in a modest but not severe reduction in PARP activity in these cells, thus in effect inhibiting PCD in non-target cells.

Preferred applications of PCD enhancing chimeric genes for plants include, but are not limited to:

1. producing a plant protected from fungal infection, whereby the PCD enhancing chimeric gene comprises a fungal responsive promoter as described in WO 93/19188 or WO 96/28561.

2. Producing a nematode-resistant plant, whereby said PCD enhancing chimeric gene comprises a nematode-inducible promoter as described in WO 92/21757.

3. Producing a male or female apomicus plant, whereby the PCD enhancing chimeric gene comprises an anther-specific promoter (as described in WO 89/10396, WO9213956, WO9213957) or a stigma-specific promoter (as disclosed in WO 91/02068).

4. Plants are produced which have improved seed set characteristics, whereby the PCD enhancing chimeric gene comprises a dehiscence zone specific promoter (as disclosed in WO 97/13865).

Surprisingly, it has been found that transformed plant cells, plant calli and plants exhibit increased growth by introducing a PCD modulating chimeric gene according to the first aspect of the invention, preferably a chimeric gene which modulates the expression of a PARP gene of the ZAP class, in particular a chimeric gene which modulates the expression of a PARP gene of the ZAP class in which the transcribed region encodes a biologically active RNA comprising both sense and antisense RNA as described herein.

Although it is not intended to limit the invention to a particular mode of operation, it is believed that this improvement in growth is a result of a reduced number of cells that can undergo apoptosis by increasing the threshold for signal-suppressing cell division, thus making the plant more thriving. These plants also have better stress resistance as explained elsewhere in this application.

Thus, in a third aspect, the present invention also relates to a method of increasing growth, preferably vegetative growth, of plant cells, plant tissues and plants containing at least one PCD modulating chimeric gene according to the first aspect of the invention, preferably a chimeric gene which modulates the expression of a PARP gene of the ZAP class in which the transcribed region encodes a biologically active RNA comprising both sense and antisense RNA.

It is clear that although the invention can be used in essentially all plant species and varieties, it should be particularly suitable for modifying programmed cell death in commercially valuable plants. Particularly preferred plants in which the invention may be used are maize, oilseed rape, linseed, wheat, grasses, alfalfa, beans, canola, tomato, lettuce, cotton, rice, barley, potato, tobacco, sugar beet, sunflower and ornamental plants such as carnation, chrysanthemum, roses, tulips and the like.

The resulting transformed plants can be used in traditional breeding programs to produce more transformed plants with the same characteristics or to introduce the chimeric cell-division controlling genes of the invention into other varieties of the same or related plant species. Seeds obtained from the transformed plants contain the PCD modulating gene of the invention as a stable genomic insert.

The following non-limiting examples describe the construction of chimeric apoptosis-controlling genes and the use of these genes in the regulation of programmed cell death in eukaryotic cells and organisms. In these examples, unless otherwise indicated, the molecular cloning in "molecular cloning: the laboratory rules of the second edition, Cold spring harbor laboratory Press, NY and Ausubel et al (1994) "molecular biology modern methods, modern methods" U.S. Vol.1 and Vol.2 all recombinant DNA techniques were carried out. Standard materials and methods for Plant Molecular research are described in "Plant Molecular Biology Labfax" (1993) by R.D.D.D.Croy, Co-published by BIOS scientific publishing Co., Ltd., UK and Blackwell scientific publishing Co., UK.

Throughout the description and examples, reference is made to the following sequences:

SEQ ID No. 1: DNA sequence of ZAP Gene of maize (ZAP 1)

SEQ ID No. 2: protein sequence of ZAP protein of maize (ZAP 1)

SEQ ID No. 3: DNA sequence (NAP) of NAP Gene of maize

SEQ ID No. 4: protein sequence of NAP protein of maize (NAP)

SEQ ID No. 5: DNA sequence (app) of NAP Gene of Arabidopsis thaliana

SEQ ID No. 6: protein sequence of NAP protein of Arabidopsis thaliana (APP)

SEQ ID No. 7: consensus sequences of the A-domains of non-classical PARP proteins

SEQ ID No. 8: consensus sequence of A1 Domain of non-classical PARP protein

SEQ ID No. 9: consensus sequence of A2 Domain of non-classical PARP protein

SEQ ID No. 10: DNA sequence of the second ZAP Gene of maize (Zap 2)

SEQ ID No 11: protein sequence of ZAP protein of maize (ZAP 2)

SEQ ID No 12: amino acid sequence of a fusion protein between the DNA-binding domain of APP and the GUS protein

SEQ ID No 13: degenerate PCR primers

SEQ ID No 14: degenerate PCR primers

SEQ ID No 15: PCR primer

SEQ ID No 16: PCR primer

SEQ ID No 17: PCR primer

SEQ ID No 18: PCR primer

SEQ ID No 19: PCR primer

SEQ ID No 20: PCR primer

SEQ ID No 21: app promoter-gus translational fusion

Sequence Listing free content

The following freeform content is used in the sequence table section of the present application

<223> description of artificial sequences: a-domain of non-classical PARP proteins

<223> description of artificial sequences: a1 domain of non-classical PARP proteins

<223> description of artificial sequences: a2 domain of non-classical PARP proteins

<223> description of artificial sequences: fusion protein between the N-terminal domain of APP and the GUS protein

<223> description of artificial sequences: degenerate PCR primers

<223> description of artificial sequences: oligonucleotides as primers for PCR

<223> description of artificial sequences: fusion of APP promoter and beta-glucuronidase gene

<223> translation initiation codon

Examples

Experimental procedure

Yeast and bacterial strains

Saccharomyces cerevisiae strain DY (MATa his3 can1-10 ade2 leu2 trp1 ura 3: (3 XSV 40 AP1-lacZ) (Kuge and Jones, 1994) was used to express APP protein Yeast transformation was performed according to Dohmen et al (1991) the strain was grown on basic SDC medium (0.67% yeast nitrogen base, 0.37% casamino acids, 2% glucose, 50mg/l adenine and 40mg/l tryptophan). for induction of APP expression, glucose in SDC was replaced with 2% galactose.

The E.coli strain XL-1(Stratagene, La Jolla, Calif.) was used for plasmid manipulation and library screening according to standard methods (Ausubel et al, 1987; Sambrook et al, 1989). Coli BL21(Studier and Moffat, 1986) was used for APP protein expression and Agrobacterium tumefaciens C58C1Rif^R(pGV2260) (Deblaere et al, 1985) was used for stable transformation of plants.

Determination of Poly (ADP-ribose) polymerase Activity

The enzymatic activity of APP was determined in the total protein extract of yeast strains prepared as follows. DY (pV8SPA) or DY (pYeDP1/8-2) was grown overnight in 50ml of SDC medium on a rotary shaker at 150rpm at 30 ℃. Yeast cells were harvested by centrifugation at 1000 Xg, washed 3 times with 150ml of 0.1M potassium phosphate buffer (pH6.5), and resuspended in 5ml of sorbitol buffer (1.2M sorbitol, 0.12M K)₂HPO₄0.033M citric acid,pH 5.9). Adding a cytolytic enzyme (Boehringer, Mannheim, Germany) to the cell suspension to a final concentration of 30Uml^-1And the cells were cultured at 30 ℃ for 1 hour. Yeast spheroplasts were then washed 3 times with sorbitol buffer and resuspended in 2ml of ice-cold lysis buffer (100mM Tris-HCl, pH7.5, 400mM NaCl, 1mM EDTA, 10% glycerol, 1mM DTT). After sonication, the lysate was centrifuged at 20000 Xg for 20 minutes at 4 ℃ and the supernatant was washed with reaction buffer (100mM Tris-HCl, pH8.0, 10mM MgCl)₂1mM DTT) balanced Econo-Pack^TMDesalting on a 10DG column (Bio-Rad, Richmond, CA). To reduce proteolytic degradation of proteins, the lysis and reaction buffers were supplemented with protease inhibitor cocktail (Boehringer) in one piece per 50 ml. By adding NaCl and protamine sulphate to final concentrations of 600mM and 10mg ml, respectively^-1Nucleic acids are removed from the total extract. After incubation at room temperature for 10 minutes, the precipitate was removed by centrifugation at 20000 Xg for 15 minutes at 4 ℃. In Econo-Pack^TMThe buffer of the supernatant was exchanged for the reaction buffer by gel filtration on a 10DG column.

The assay for the synthesis of poly (ADP-ribose) was modified by Collinge and Althaus (1994). About 500. mu.g of total yeast protein supplemented with 30. mu. Ci³²P-NAD⁺(500Ci mmol^-1) To a final concentration of 60. mu.M unlabelled NAD⁺And 10. mu.g ml^-1The sonicated salmon sperm DNA was incubated in reaction buffer. After incubation at room temperature for 40 minutes, 500. mu.l of stop buffer (200mM Tris-HCl, pH7.6, 0.1M NaCl, 5mM EDTA, 1% Na) was added⁺N-lauroyl-sarcosine and 20. mu.g ml^-1Proteinase K) were incubated overnight at 37 ℃. After phenol and phenol/chloroform extraction, the polymer was precipitated with 2.5 volumes of ethanol with 0.1M NaAc (pH 5.2). The particles were washed with 70% ethanol, dried and dissolved in 70% formamide, 10mM EDTA, 0.01% bromophenol blue and 0.01% xylene cyanide blue. Samples were heated at 80 ℃ for 10 minutes and then loaded onto a 12% polyacrylamide/6M urea sequencing gel. The gel was applied to 3MM paper (Whatman International, Maidstone, UK)) Drying, exposing it to either Kodak X-Omat X-ray film (Eastman Kodak, Richmond, NY) or using a Phosphorlmager^TM445SI (molecular dynamics, Sunnyvale, Calif.) scan.

Immunological techniques

After induction of 500ml of a culture broth of E.coli BL21 (pET. DELTA. NdeSPA) with 1mM isopropyl-. beta. -D-thiogalactopyranoside, the coding sequence was derived from the amino acid Met³¹⁰To His⁶³⁷The truncated APP cDNA of APP polypeptide of (a) is expressed as a translational fusion with 6 histidine residues at the N-terminus. The APP polypeptide was purified to near homogeneity by affinity chromatography on a Ni-NTA-agarose column under denaturing conditions (in the presence of 6M guanidine hydrochloride) according to the manufacturer's protocol (Qiagen, Chatsworth, CA). After dialysis with PBS, two new zealand white rabbits were immunized with a mixture of soluble and insoluble APP polypeptides according to standard immunization protocols (Harlow and Lane, 1988). For Western blot analysis, proteins were solubilized by denaturing SDS-PAGE (Sambrook et al, 1989; Harlow and Lane, 1988) and transferred onto nitrocellulose membranes (Hybond-C; Amersham) using Semi-Dry blot II (Kem-En-Tec, Copenhagen, Denmark).

In situ antigen localization within yeast cells was performed as described (Harlow and Lane, 1988). To localize the APP protein in yeast spheroplasts, anti-APP serum was diluted 1: 3000-1: 5000 in Tris buffered saline-BSA buffer. A10H monoclonal antibody specifically recognizing a poly (ADP-ribose) polymer (Ikajima et al, 1990) was used in PBS buffer at a dilution of 1: 100. Goat anti-mouse IgGF (ab') conjugated with fluorescein isothiocyanate at a 1: 200 dilution₂(Sigma) the mouse antibody was detected. Goat anti-rabbit IgG goat F (ab') conjugated with CY-3 at a dilution of 1: 200₂Fragments (Sigma) were tested for rabbit IgG. For DNA observation, the slide glass was filled with 10. mu.g ml^-14', 6-diamidino-2-phenylindole (DAPI; Sigma) in PBS for 1 minute. Fluorescence imaging was performed on an Axioskop epifluorescence microscope (Zeiss, Jena, Germany). To observe FITC and CY-3 fluorescent dyes, 23 and 15 filters were used, respectively. Enrichment of cellsColour-100 high quality positive film photography.

Plant material and histochemical analysis

According to the method, leaves are mixed with Agrobacterium tumefaciens C58C1Rif^R(pGCNSPAGUS) Co-culture (DeBlock et al, 1987) tobacco (Nicotiana tabacum) SR1(Maliga et al, 1975) was used to generate stable transformants. Tobacco SR1 line transformed with authentic GUS under control of 35S CaMV was used as a control. Arabidopsis thaliana ecotype Columbia was used for transformation of app-promoter-GUS fusions according to the in situ diafiltration method.

For in situ histochemical staining for GUS activity, plant samples were fixed in ice-cold 90% acetone for 30 min with 0.1M K₂HPO₄(pH7.8) and then washed in a staining buffer (0.1M K)₂HPO₄，pH7.8，2mM X-Gluc，20mM Fe³⁺EDTA) at 37 ℃. The stained plant tissue was stored in 70% ethanol at 4 ℃. If necessary, darkening of tissues due to phenol oxidation can be reduced by culturing with lactophenol (Beeckman and Engler, 1994). The GUS staining was detected under Jenalumar optical microscope (Zeiss). Plant tissues were photographed using fuji color-100 quality positive film.

Other methods

DNA sequencing according to the protocol provided by USB biochemistry (Cleveland, OH) routinely validated the plasmid construction procedure. Synthesis of DNA marker cassette (Amersham) for nucleic acid hybridization by preparation of primers³²P-labeled DNA probes. For DNA and RNA hybridization experiments, buffer systems from Church and Gilbert (1984) (0.25M sodium phosphate, pH7.2, 7% SDS, 1% BSA, 1mM EDTA) were used. For Western blot analysis, total yeast protein was extracted with phenol essentially as described for plant tissues (Hurkman and Tanaka, 1986). For Northern blot analysis, total yeast RNA was extracted with hot phenol as described (Ausubel et al, 1987). After denaturation with glyoxal, the RNA was dissolved on a 1.5% agarose gel (Sambrook et al, 1989). Hybond-N nylon filters (Amersham) were used for nucleic acid blotting.

Example 1: isolation of genes encoding maize PARP homologs

To isolate maize cDNA encoding PARP homologues, two experiments were performed. First, a maize cDNA library was screened using a DNA probe prepared from arabidopsis app cDNA under low stringency DNA-DNA hybridization conditions. Second, PCR amplification of partial maize PARP was performed using first strand cDNA as a template and two degenerate primers designed based on the sequence of "PARP features" -the most conserved amino acid sequence among all known PARP proteins.

Lambda ZAP (Stratagene) cDNA library from leaves of maize (Zea mays L.) from inbred line B734. Plaques were screened according to standard procedures (Sambrook et al, 1989) (500000). After screening with the Arabidopsis app probe, a 1.4kbp non-full length cDNA was purified. Following screening of the original cDNA library with the app probe and subsequent 5' Rapid Amplification of CDNA Ends (RACE) PCR analysis, the nap gene, a maize homolog of the arabidopsis app, was identified. For 5' RACE PCR, the Marathon kit (Clontech, Palo Alto, Calif.) and 0.5. mu.g of maize poly (A) isolated from the inner sheath, outer sheath and leaves of 1 week old maize seedlings⁺RNA preparation of the template. The gene-specific nested primers used for PCR amplification were the nap primers 5'-GGGACCATGTAGTTTATCTTGACCT-3' (SEQ ID No 15) and 5'-GACCTCGTACCCCAACTCTTCCCCAT-3' (SET ID No 16. the amplified PCR products were subcloned and sequenced. an 800bp fragment was amplified with nap-specific primers that allowed recombination of 2295-bp-long nap cDNA sequences (SEQ ID No 3).

The NAP protein is 653 amino acids long (molecular weight approximately 73 kDa; SEQ ID No 4) and is highly similar to the APP (61% sequence identity and 69% similarity). Most importantly, NAP has a structure with an N-terminal identical to that of APP (FIG. 1A), suggesting a rather stringent selection pressure on the structure of APP-like proteins in plants. The nap gene is unique in the maize genome (FIG. 2A) and encodes a 2.4kb transcript (FIG. 2C).

Degenerate primers based on very highly conserved regions of the "PARP signature" and derived from maizeThe first strand cDNA of (1) was used as a template to amplify a 310bp fragment. For this PCR with degenerate primers 5 '-CCGAATTCGGNTAYATGTTYGGNAA-3' (SEQ ID No 13) and 5 '-CCGAATTCACNATRTAYTCRTTRTA-3' (SEQ ID No 14) with Y ═ C/T, R ═ a/G, N ═ a/G/C/T, the first strand cDNA was used as template and 5 μ G of poly (a) from maize young leaves was used⁺RNA and MuMLV reverse transcriptase synthesis. PCR amplification was performed with Taq DNA polymerase in a volume of 100 μ l using the following conditions: the culture was repeated 38 times at 95 ℃ for 1 minute, 45 ℃ for 2 minutes, and 72 ℃ for 3 minutes, followed by 95 ℃ for 1 minute, 45 ℃ for 2 minutes, and 72 ℃ for 3 minutes, and finally incubated at 72 ℃ for 10 minutes.

The sequence of this 310bp fragment showed 55% sequence identity and 64% sequence similarity to human PARP within the same region, but in any case, differed from the sequence of nap cdnd. Three zapcDNA were identified after screening with 310-bp fragments obtained by PCR with degenerate primers. The three purified cdnas were all derived from the same transcript because they had the same 3' non-coding region; the longest clone (#9) was sequenced on both strands (SEQ ID No 1). This cDNA encodes a 689 amino acid PARP homologous polypeptide (SEQ ID No 2; molecular weight-109 kDa), which was designated ZAP1 (FIG. 1B). Since the first Zn-finger of ZAP1 has the sequence CKSCxxxHASV (without the third cysteine residue), it may be non-functional.

5 ' RACE PCR analysis of zap transcripts from maize line LG2080 (preparation of the screened cDNA library from inbred line B734) was performed as described above using the following zap-specific primers 5'-AAGTCGACGCGGCCGCCACACCTAGTGCCAGGTCAG-3' (SEQ ID No 17) and 5'-ATCTCAATTGTACATTTCTCAGGA-3' (SEQ ID No 18). A450-bp PCR product was obtained after PCR with zap-specific primers. Due to the slight difference in length at their 5 'ends, 8 independent 5' RACEPCR fragments generated with zap-specific primers were sequenced. In all transcripts from LG2080 maize plants, there are insertions of further sequences in the coding region which increase the ZAP protein by 11 amino acids (980 amino acids, molecular weight approximately 110.4 kDa). The Zn-finger I of ZAP2 is standard and can be read as CKSCxxxHARC (FIG. 1B; SEQ ID No 11). The sequence differences may be due to either differences between maize varieties, expression of two homologous genes, or alternative splicing. In fact, maize may have at least two zap genes (FIG. 2B), which encode 3.4-3.5kb transcripts (FIG. 2D). This DNA gel blot test of the probe prepared with this zap cDNA showed that the homologous gene was present in Arabidopsis.

Structural ZAPs are very similar to PARPs from animals. It has a well-conserved DNA-binding domain consisting of two Zn-fingers (36% identity and 45% similarity to the DNA-binding domain of mouse PARP). Even by comparing only the sequences of these Zn-fingers-Ala in the mouse enzyme (44% identity and 54% similarity)¹-Phe¹⁶²Or from the subdomain downstream of the Nuclear Localization Signal (NLS) - -Leu in mouse PARP (40% identity and 50% similarity)²³⁷-Ser³⁶⁰Showing higher homology. Given that the dichotomous nuclear localization signal signature of mammalian PARP cannot be identified in ZAP, the sequence KRKK is equipped with one dichotomous NLS (fig. 1B). The putative automodified domain is poorly conserved than in mouse PARP and short in ZAP. Homology edits of the catalytic domains between ZAP, NAP, APP and mouse PARP are shown in figure 2. Note that NAP of ZAP compares to those in APP and NAP (40% and 42% sequence identity, respectively)⁺Binding domains are more similar to mammalian enzymes (48% identity), whereas APP and NAP are 68% identical and 76% similar in their catalytic domains.

Example 2: demonstration that non-classical PARP proteins possess DNA-dependent poly (ADP-ribose) polymerase activity

APP is DNA-dependent poly (ADP-ribose) polymerase

A more detailed study of APP protein (expressed in yeast) was performed to understand the activity of PARP-like proteins from this NAP class. There are many reasons to select yeast as the organism for expression and enzymatic analysis of the arabidopsis APP protein. Saccharomyces cerevisiae, a eukaryotic cell, is preferably one that is expressed by native proteins from other eukaryotic organisms and is different from most other eukaryotic cells, and therefore does not have endogenous PARP activity (Lindahl et al, 1995).

The full-length app cDNA was introduced into pYeDP1/8-2 under the control of a galactose-inducible yeast promoter in the following manner. The full-length app cDNA was excised as a Xhol-EcoRI fragment from pC3(Lepinie et al, 1995). The ends were filled with the Klenow fragment of DNA polymerase I and the fragment was subcloned into the yeast expression vector pYeDP1/8-2 at the Smal position (Cullin and Pompon, 1988). The resulting expression vector pV8SPA (FIG. 4A) was transformed into Saccharomyces cerevisiae strain DY.

For APP expression in E.coli, the full coding region of the APP cDNA was PCR amplified with Pfu DNA polymerase (Stratagene) using primers 5'-AGGATCCCATGGCGAACAAGCTCAAAGTGAC-3' (SEQ ID No 19) and 5'-AGGATCCTTAGTGCTTGTAGTTGAAT-3' (SEQ ID No 20) and subcloned as a BamHI fragment into pET19b (Novagene, Madison, Wis.) to obtain pETSPA. Expression of full-length APP from pettspa was very poor in e.coli BL 21. To obtain better expression, pETSPA was digested with Ncol and Ndel or with Smal, the ends were filled in by Klenow fragment of DNA polymerase I, and then these plasmids were self-ligated. Of the resulting plasmids pET. DELTA. NdeSPA and pET. DELTA. SmaSPA, only pET. DELTA. NdeSPA expressed a truncated APP polypeptide (Met. DELTA. NdeSPA) satisfactorily in E.coli BL21³¹⁰-His⁶³⁷)。

Expression of this APP in yeast was assessed by Northern and Western blot analysis. (FIG. 4) since the promoter in pV8SPA is inactive when the cells are grown on glucose and de-repressed on galactose containing medium, it is desirable that the expression is tightly regulated by the carbon source. However, Northern blot analysis of RNA and immunoblot analysis of the protein in DY (pV8SPA) compared to the control DY strain containing the blank vector showed that APP mRNA and APP protein were expressed in yeast even when grown on glucose-containing medium (fig. 4B, lane 2). The expression specificity observed on glucose-containing medium is that APP mRNA and APP protein are shorter than those detected after induction with galactose (compare lanes 2 and 4 in fig. 4B). Although these cells also express truncated mRNA and protein, they do soOnly the higher molecular weight APP polypeptide (apparently the full-length protein) was detected on galactose medium. The most reliable explanation for this finding is that when this DY (pV8SPA) strain was grown on glucose, there was leaky expression from the expression cassette, since transcription was observed to start 200-300bp downstream from the transcription start point after galactose induction. This shorter mRNA may not encode the first methionine (Met) of APP¹) Thus at Met⁷²Translation is started. This should account for the observed difference of about 5kDa in molecular weight of APP polypeptides from strains grown on glucose or galactose (calculated difference of 7.5 kDa). By growing the strains in the presence of PARP inhibitors such as 3ABA and nicotinamide, the possibility that differences in molecular weight might be favourable for self-modification by poly (ADP-ribose) is excluded (fig. 4B, compare lanes 6 and 8 with lane 4).

To examine the synthesis of poly (ADP-ribose), the cells were grown under different conditions and radiolabeled with NAD⁺Total protein was extracted from the yeast strain cultured under the conditions. To prevent APP synthesis of poly (ADP-ribose) and possible auto-modification in vivo, the strains were also grown in the presence of reversible inhibitors of 3ABA, PARP, and then they were removed from the protein extract during desalting. FIG. 5 shows that poly (ADP-ribose) is synthesized by protein extracts of DY (pV8SPA) grown on galactose (FIG. 5A, lanes 1 and 2), but not by strains containing blank vector (FIG. 5A, lane 4). It can also be seen that Arabidopsis APP is able to synthesize polymers up to 40 residues in length (FIG. 5A, lane 1), with most of the radioactivity added as 10-15-mers. This observation is consistent with the polymer size detected by other experts (Chen et al, 1994). When the yeast strain was grown with 3ABA more radioactivity was added to the polymer than without 3ABA (FIG. 5A, lane 1 vs lane 2); the reasons may be either a low automodification of the APP extracted from the inhibition culture (which is believed to inhibit PARP activity), or a labeled NAD⁺Use by enzymes from uninhibited cultures for extension of existing polymers results in a decrease in overall specific activity. Under the same reaction conditions either in the reaction buffer only or in the presence of a compound derived from DY (p)YeDP1/8-2) (fig. 5A, lanes 5 and 6, respectively) showed longer chains, possibly up to 400-mers (de Murcia and meniscede Murcia, 1994).

The activity of the enzyme by nicked DNA stimulators is a well-known property of PARP from animals (Alvarez-Gonzalez and Althaus, 1989). We therefore tested whether the activity of the APP protein is DNA dependent. The synthesis of poly (ADP-ribose) was analyzed at increasing concentrations of sonosalmon sperm DNA after removal of some of the alkaline proteins in the yeast nucleic acids (DNA, RNA) and galactose-grown DY (pV8SPA) protein extracts. As can be seen from FIG. 5B, the presence is³²P-NAD⁺There is a direct correlation between the reaction of (a) and the amount of DNA added. Phosphorus image Scan shows that 40. mu.g ml of phosphorus was added^-1The radioactivity ratio of poly (ADP-ribose) in the reaction mixture of DNA was 2. mu.g ml^-1About 6 times more DNA (FIG. 5B, lanes 4 and 2, respectively). The synthesis of the polymer in the reaction mixture was allergic to 3ABA (fig. 5B, lane 5).

APP is nuclear protein

In animal cells PARP activity is localized in the nucleus (Schreiber et al, 1992). The intracellular localization of APP, if nuclear, can provide important additional indicators: APP is real plant PARP. To this end, the localization of the APP polypeptide in yeast cells was analyzed using anti-APP antisera. APP polypeptides synthesized in yeast grown on galactose are found primarily in this core. The presence in the medium of PARP inhibitors had no effect on this localization.

Furthermore, we tested whether APP activity in yeast cells is consistent, as has been reported for human PARP (Collinge and Althaus, 1994). Herein, immobilized yeast spheroplasts were cultured with a monoclonal 10H antibody that specifically recognizes poly (ADP-ribose) polymers (Kawamitsu et al, 1984). A positive yellowish-green fluorescent signal with the 10H antibody is localized in this core and is observed in DY (pV8SPA) grown only on galactose. The positive staining of cells grown in the presence of PARP inhibitors-3 ABA and nicotinamide was greatly reduced.

To identify the intracellular localization of APP in plant cells, a widely used method in plant research was tested, i.e.to examine the subcellular localization of fusion proteins formed between the protein in question and a reporter gene, which were once produced in transgenic plants or transfected cells (Citovsky et al, 1994; Sakamoto and Nagatani, 1996; Terzaghi et al, 1997; von Arnim and Deng, 1994). Preparation of GUS and antibodies with Met¹To Pro⁴⁰⁷An N-terminal translational fusion of an extended partial APP polypeptide. Translational fusions of APP with bacterial GUS were constructed as follows. The plasmid pETSPA was cut with Smal, treated with alkaline phosphatase, and ligated to the blunt-ended Ncol-Xbal fragment from pGUS1(Plant Genetic Systems N.V., Gent, Belgium). The ligation mixture was transformed into E.coli XL-1 and the cells were transplanted in 40. mu.g ml supplemented with 0.1mM isopropyl-. beta. -D-thiogalactopyranoside^-15-bromo-4-chloro-3-indolyl-beta-D-glucuronide hydrochloride and 100. mu.g ml^-1Ampicillin on LB medium. In this manner, pETSPAGUS was selected as the blue colony. Expression of an approximately 110kDa fusion protein in E.coli was demonstrated by in situ GUS activity gel (Lee et al, 1995). The APP-GUS fusion (subcloning the Klenow blunt-ended BamHI fragment from pETSPAGUS into Smal-digested pJD 330; Gallie and Walbot, 1992) was placed under the control of the CaMV35S promoter, and the resulting expression cassette was subcloned as an Xbal fragment into the Xbal position of the pCGN1547 binary vector (McBride and Summerfelt, 1990) to obtain pGCNSPAGUS. Finally, the pGCNSPAGUS is introduced into the Agrobacterium tumefaciens C58C1Rif by a freezing-thawing transformation step^R(pGV 2260).

The expression of the fusion protein was verified in E.coli. The chimeric cDNA was stably integrated into the tobacco genome under the control of the 35S CaMV promoter. Progeny from four independent transgenic tobacco plants were analyzed for subcellular distribution of GUS activity after in situ histochemical staining. GUS activity was detected in cotyledons and roots in 2-day-old shoots, but not in hypocotyls and root tips. Due to the permeability of root tissue, GUS staining was clearly localized to the core of root hair and epidermal cells. In addition, some diffuse, non-localized staining of other root cells was seen, particularly along the vascular column. This non-nuclear GUS staining was more prominent in leaf tissue. Since young true leaves or cotyledons exhibit a strong blue staining of the core, there is also some scatter staining of the cytoplasm. However, throughout the expanded leaves, the staining of GUS was uniform and similar to that of control plants transformed with GUS under the control of the CaMV35S promoter, where GUS was expressed in the cytoplasm. Finally, older leaves and cotyledons actually exhibited GUS activity beyond the vascular bundle that could not be detected by histochemistry, wherein the GUS staining could not be confined to any particular cell chamber.

Defect of DNA ligase I induces expression of the app gene

PARP in animal cells is one of the most abundant nuclear proteins, whose activity is regulated by allosteric changes in the protein due to binding to damaged DNA. We found that the APP gene in arabidopsis had a rather low level of expression, suggesting that transcriptional activation of this gene may be essential for APP function in vivo. To test this hypothesis, expression of the app gene was studied during in vivo genomic destabilization by DNA ligase I deficiency. The T-DNA insertion mutation line SK1B2 in the Arabidopsis DNA ligase I gene was isolated in advance (Babiychuk et al, 1997). The mutation is lethal in the homozygous state, but the mutant allele shows normal transmission through the gamete. Therefore, we hope that cells homozygous for the mutation will die shortly after fertilization of the mutant embryo sac with mutant pollen due to incomplete DNA synthesis during the S phase of the cell cycle.

an APP promoter-GUS translation fusion in which the GUS coding region is fused in frame with the first 5 amino acids of APP and a 2kb APP 5' reading frame sequence (SEQ ID No 21) is constructed. The gene encoding the fusion protein was transferred into Arabidopsis. After 2 backcrosses with wild type, heterozygous plants transformed with app promoter-GUS were crossed with arabidopsis strain SK1B 2. Inflorescences of control plants and plants heterozygous for the ligase mutation were stained for GUS activity. Although we have no strong evidence that all regulatory sequences are present in the constructs used, most of the GUS staining pattern measured in aged tissues may reflect the expression of the app gene. The pattern was identical in the inflorescences of the control plants and the mutant ligase gene and the plants were not transported for mutation shuffling. Ovules of about 1/4 were GUS positive in the mutant plants of the gene fusion protein. Closer microscopic examination revealed only gametophytic staining in GUS positive ovules. The only difference between the control plants and the mutant plants was a mutation in the DNA ligase gene. We therefore concluded that the app gene was induced either by accumulation of DNA breaks or by death of the mutated embryo sac fertilized with mutated pollen. GUS staining of embryo sacs was found to occur within 24 hours after pollination or thus shortly after fertilization.

Example 3: construction of PCD modulatory chimeric genes and introduction of T-DNA vectors containing these PCD modulatory genes into Agrobacterium strains

3.1. Construction of p 35S: (dsRNA-APP) and p 35S: (dsRNA-ZAP) Gene

The following DNA regions were operably linked, as set forth schematically in fig. 6 (constructs 1 and 5), using standard recombinant DNA procedures:

for p 35S: (dsRNA-ZAP) chimeric Gene

● A CaMV35S promoter region (Odell et al, 1985)

● A Cab22 leader (Harpster et al, 1988)

● A ZAP-encoding DNA region (approx. Whole) (Arabidopsis thaliana homologue of SEQ ID No 10, isolated by hybridization)

● about 500bp 5' end of ZAP-encoding DNA region 2 in reverse orientation

● A CaMV35S 3' terminal region (Mogen et al, 1990)

For p 35S: (dsRNA-APP) chimeric Gene

● A CaMV35S promoter region (Odell et al, 1985)

● A Cab22 leader (Harpster et al, 1988)

● A DNA region encoding APP (about the whole) (SEQ ID No 5)

● about 500bp 5' end of DNA region coding for APP in reverse orientation

● A CaMV35S 3' terminal region (Mogen et al, 1990)

3.2. Construction of pNOS: (dsRNA-APP) and pPOS: (dsRNA-ZAP) Gene

The following DNA regions were operably linked, as set forth schematically in fig. 6 (constructs 2 and 6), using standard recombinant DNA procedures:

for pNOS: (dsRNA-ZAP) chimeric Gene

● A NOS promoter region (Herrera-Estrella et al, 1983)

● A Cab22 leader (Harpster et al, 1988)

● A ZAP-encoding DNA region (approx. Whole) (Arabidopsis thaliana homologue of SEQ ID No 10, isolated by hybridization)

● about 500bp 5' end of ZAP-encoding DNA region 2 in reverse orientation

● A CaMV35S 3' terminal region (Mogen et al, 1990)

For pNOS: (dsRNA-APP) chimeric Gene

● A NOS promoter region (Herrera-Estrella et al, 1983)

● A Cab22 leader (Harpster et al, 1988)

● A DNA region encoding APP (about the whole) (SEQ ID No 5)

● about 500bp 5' end of DNA region coding for APP in reverse orientation

● A CaMV35S 3' terminal region (Mogen et al, 1990)

3.3. Construction of pTA 29: (dsRNA-APP) and pTA 29: (dsRNA-ZAP) Gene

The following DNA regions were operably linked, as set forth schematically in fig. 6 (constructs 3 and 7), using standard recombinant DNA procedures:

for pTP 29: (dsRNA-ZAP) chimeric Gene

● A TP29 promoter region (WO 89/10396)

● A Cab22 leader (Harpster et al, 1988)

● A ZAP-encoding DNA region (approx. Whole) (Arabidopsis thaliana homologue of SEQ ID No 10, isolated by hybridization)

● about 500bp 5' end of ZAP-encoding DNA region 2 in reverse orientation

● A CaMV35S 3' terminal region (Mogen et al, 1990)

For pTP 29: (dsRNA-APP) chimeric Gene

● A TA29 promoter region (WO 89/10396)

● A Cab22 leader (Harpster et al, 1988)

● A DNA region encoding APP (about the whole) (SEQ ID No 5)

● about 500bp 5' end of DNA region coding for APP in reverse orientation

● A CaMV35S 3' terminal region (Mogen et al, 1990)

3.4. Construction of pNTP 303: (dsRNA-APP) and pNTP 303: (dsRNA-ZAP) Gene

Using standard recombinant DNA procedures, the following DNA regions were operably linked as listed schematically in fig. 6 (constructs 4 and 8):

for pNTP 303: (dsRNA-ZAP) chimeric Gene

● NTP303 start area (Wetering 1994)

● A Cab22 leader (Harpster et al, 1988)

● A ZAP-encoding DNA region (about the entire) (Arabidopsis thaliana homologue of SEQ ID No 10, isolated by hybridization)

● about 500bp 5' end of ZAP-encoding DNA region 2 in reverse orientation

● A CaMV35S 3' terminal region (Mogen et al, 1990)

For pNTP 303: (dsRNA-APP) chimeric Gene

● NTP303 start area (Wetering, 1994)

● A Cab22 leader (Harpster et al, 1988)

● A DNA region encoding APP (about the whole) (SEQ ID No 5)

● about 500bp 5' end of DNA region coding for APP in reverse orientation

● A CaMV35S 3' terminal region (Mogen et al, 1990)

3.5. Construction of chimeric marker genes

Using standard recombinant DNA procedures, the following DNA regions were operably linked, as listed schematically in fig. 6:

for the gat marker Gene

● An Act2 promoter region (An et al, 1996)

● A DNA encoding aminoglycoside 6' -acetyltransferase (WO 94/26913)

● 3' end region of a nopaline synthetase gene (Depicker et al, 1982)

For the bar marker Gene

● An Act2 promoter region (An et al, 1996)

● A DNA encoding phosphinothricin acetyltransferase (US5646024)

● 3' end region of a nopaline synthetase gene (Depicker et al, 1982)

3.6. Construction of T-DNA vector containing the PCD-regulated chimeric Gene

The chimeric PCD modulatory genes described under 3.1-3.5 were excised and introduced into the polylinker between the T-DNA borders of the T-DNA vector from pGSV5(WO 97/13865) either together with the gat marker gene or the bar marker gene using appropriate restriction enzymes. The resulting T-DNA vector is illustrated in FIG. 6.

3.7. Introduction of T-DNA vectors into Agrobacterium

The T-DNA vector was introduced into Agrobacterium tumefaciens C58C1Rif (pGV4000) by electroporation as described by Walkerpeach and Velten (1995) and transformants were selected using spectinomycin and streptomycin.

Example 4: agrobacterium-mediated transformation of Arabidopsis thaliana with the T-DNA vector of example 3

Arabidopsis thaliana variant C24 was transformed with Agrobacterium strain using the root transformation method described by Valvekens et al (1992). These explants were co-infected with Agrobacterium strains containing dsRNA-APP and dsRNA-ZAP constructs, respectively. The dsRNA-APP construct was used in combination with the pact: bar gene. The dsRNA-ZAP construct was used in combination with the pact: gat gene. Transformants were selected for phosphinothricin resistance. These regenerating root transgenic lines were tested for the presence of other T-DNA by kanamycin resistance screening. Transgenic lines containing both T-DNA were transferred to the greenhouse. The phenotypic T0-transgenic lines were counted and the T1-generation was studied in further detail.

Example 5: agrobacterium-mediated transformation of Brassica napus with the T-DNA vector of example 3

This Brassica napus variant N90-740 was transformed with an Agrobacterium strain using the hypocotyl transformation method described mainly by De Block et al (1989) except for the following modifications:

hypocotyl explants were pre-cultured for 1 day on A2 medium [ MS, 0.5g/l Mes (pH5.7), 1.2% glucose, 0.5% agarose, 1mg/l 2, 4-D, 0.25mg/l Naphthalene Acetic Acid (NAA) and 1mg/l 6-Benzylaminopurine (BAP) ].

Infection Medium A3 was MS, 0.5g/l Mes (pH5.7), 1.2% glucose, 0.1mg/l NAA, 0.75mg/l BAP and 0.01mg/l pseudogibberellic acid (GA 3).

Selection medium A5G is MS, 0.5g/l Mes (pH5.7), 1.2% glucose, 40mg/l adenine sulphate, 0.5g/l polyvinylpyrrolidone (PVP), 0.5% agarose, 0.1mg/l NAA, 0.75mg/l BAP, 0.01mg/l GA3, 250mg/l carbenicillin, 5mg/l silver nitrate for three weeks. Selection on A25J medium (similar to A5G but with 3% sucrose) continued after this period.

Regeneration medium A6 is MS, 0.5g/l Mes (pH5.7), 2% sucrose, 40mg/l adenine sulfate, 0.5g/l PVP, 0.5% agarose, 0.0025mg/l BAP and 250 mg/ltriacillin.

Healthy shoots were transferred to rooting medium of a9 in 1 liter containers: semi-concentrated MS, 1.5% sucrose (pH5.8), 100mg/l triacillin, 0.6% agar.

MS stands for Murashige and Skoog medium (Murashige and Skoog, 1962).

To introduce both dsRNA-APP and dsRNA-ZAP T-DNA constructs into the same plant cell, co-transformation was used, mainly as described by De Block and Debrower (1991). Transformed plant lines were selected on phosphinothricin-containing medium, after which the secondary T-DNA was screened for the presence by detecting kanamycin resistance in regenerated root shoots. In these co-transformation experiments, the dsRNA-APP construct was used in combination with the pact: bar gene. The dsRNA-ZAP construct was used in combination with the pact: gat gene. Transgenic lines containing both T-DNA were transferred to the greenhouse. This T1-generation was studied in further detail using a phenotype T0-transgenic line counting wall hanging.

Example 6: in vitro assay for determining plant line vigor

Health assay of Brassica napus

Culture medium and reaction buffer

Seeding culture medium:

semi-concentrated Murashige and Skoog salts

2% sucrose

pH5.8

0.6% agar

Callus induction medium: A2S

MS medium, 0.5g/l Mes (pH5.8), 3% sucrose, 40mg/l adenine sulfate, 0.5g/l agarose, 1mg/l 2, 4-D, 0.25mg/l NAA, 1mg/l BAP

Culture medium:

25mM K-phosphate buffer pH5.8

2% sucrose

1 drop of Tween 20 per 25ml of culture medium

Reaction buffer:

50mM K-phosphate buffer pH7.4

10mM 2, 3, 5-Triphenyltetrazolium chloride (TTC) (═ 3.35mg/ml)

1 drop of Tween 20 per 25ml of culture medium

Sterilization of seeds and growth of seedlings

The seeds were soaked in 70% ethanol for 2 minutes and then surface sterilized in sodium hypochlorite solution (with about 6% active chlorine) containing 0.1% tween 20 for 15 minutes. Finally, the seeds were rinsed with 1 liter of sterile distilled water. 7 seeds were placed in 1 liter containers (Weck) containing about 75ml of seeding medium. At 23 ℃ and 30. mu. Einstein/s^-1m^-2And these seeds germinated 16 hours a day long.

Line N90-740 always included standardization between trials.

Pre-culture of hypocotyl explants

12-14 days after sowing, these hypocotyls are cut into pieces of about 7 mm.

25 hypocotyls/Optilux Petri dish (Falcon S1005)

Culturing the hypocotyl explants at 23-25 ℃ for 4 days (at 30. mu. Einstein/s) on medium A2S^-1m^-2Below).

-supplementary notes: about 150-300 hypocotyl explants per strain were required for this assay

Transfer of these hypocotyl explants into Optilux petri dishes (Falcon S1005) containing 30ml incubation medium.

Incubation in the dark at 24 ℃ for about 20 hours.

TTC-determination

Transfer 150 hypocotyl explants into a 50ml Falcon tube.

Rinse with reaction buffer (without TTC).

-adding 25ml to 30ml of reaction buffer to each tube.

Tube 1 without TTC addition

For determining background absorption

One strain per test is sufficient

Tube 2+10mM TTC

(explants must be submerged, but not vacuum infiltrated!)

Upside down tube

Incubation in the dark at 26 ℃ for about 1 hour (not ending the reaction!)

Washing the hypocotyls with deionized water

Removal of water

Freezing at-70 ℃ for 30 minutes

Resuscitated at room temperature (in the dark)

Addition of 50ml ethanol (for industrial use)

Reduction of TTC-H by shaking for 1 hour

Determination of the absorbance of the extract at 485nm

And (4) supplementary notes: reduced TTC-H instability remained in the dark and o.d was measured as soon as possible.₄₈₅

O.D._485(TTC-H)＝(O.D.₄₈₅+TTC)-(O.D.₄₈₅-TTC)

Comparison of TTC-reducing power between samples of different independent tests can be made by setting the TTC-reducing power of N90-740 to 100% in different tests.

Strains with high TTC-reducing power flourish, whereas strains with low TTC-reducing power do not.

6.2. Arabidopsis health assay

Culture medium and reaction buffer

Plant culture medium: semi-concentrated Murashige and Skoog salts

1.5% sucrose

pH5.8

0.6% agar

→ autoclaving for 15 minutes.

Adding filtered and sterilized inositol-100 mg/l

0.5mg/l vitamin B6

-0.5mg/l nicotinic acid

1mg/l thiamine

Culture medium: 10mM K-phosphate buffer pH5.8

2% sucrose

1 drop of Tween 20 per 25ml of culture medium

Reaction buffer: 50mM K-phosphate buffer pH7.4

10mM 2, 3, 5-Triphenyltetrazolium chloride (TTC) (═ 3.35mg/ml)

1 drop of Tween 20 per 25ml buffer

Arabidopsis plants

Sterilization of Arabidopsis seeds

2 min 70% ethanol

10 min bleach (6% active chlorine) +1 drop tween 20 per 20ml solution

Washing with sterile water for 5 times

And (4) supplementary notes: sterilization in 2ml eppendorf tubes

Arabidopsis seeds were submerged in the bottom of the tube and liquid was removed by a 1ml automatic pipettor

Growth of Arabidopsis plants

Seeds were sown in 1 seed/grid in "intersrid tissue Culture disks of Falcon" (nr.3025) containing. + -. 100ml of plant medium.

Plants were grown at 23 ℃ in 40. mu. Einstein s^-1m^-216 hours light-8 hours dark for about 3 weeks (plants begin to form flower buds).

→ appendix: about 90-110 plants per strain were required for this assay

Including a control strain for calibration (C24; Columbia; …)

Pre-culture

Harvesting arabidopsis shoots by cutting the root (with scissors)

Immediately placing each shoot into the cultivation medium (shoots must be submerged, but do not vacuum infiltration)

Culture medium: 150ml in "intermediate Tissue Culture disks of falcon" (nr.3025)

a) Culture medium: for quantification of background absorption (see TTC-assay)

b) Culture medium

c) Incubation Medium +2mM Nicotinamide

30-35 shoots per Petri dish (but using the same amount of shoots for all lines and each condition)

Incubation at 24 ℃ in the dark. + -. 20 hours

TTC-determination

Transfer of shoots into 50ml Falcon tubes

Washing with reaction buffer (TTC-free)

-adding 30-35ml of reaction buffer to each tube

a) No TTC addition (for background absorption measurement)

b) And c) +10mM TTC

(shoots must be submerged, but not vacuum-infiltrated!)

Incubation in the dark at 26 ℃ for about 2 hours (not end reaction!)

-rinsing the shoots with deionized water

Removal of water

Freezing at-70 ℃ for 30 minutes

Resuscitated at room temperature (in the dark)

Addition of 50ml ethanol (for industrial use)

Reduction of TTC-H by shaking for 1 hour

Determination of the absorbance of the extract at 485nm

And (4) supplementary notes: reduced TTC-H is unstable → remains in the dark and o.d is measured as soon as possible.₄₈₅

Comparison of the reduction profiles of the test lines with the control lines (vs. a population of 30-35 plants)

O.D._485(TTC-H)＝(O.D.₄₈₅+TTC)-(O.D.₄₈₅-TTC)

Comparison of TTC-reducing power between samples of different independent tests can be carried out by setting the TTC-reducing power of the control strain (C24; Columbia; …) at 100% in the different tests.

Strains with high TTC-reducing power flourish, whereas strains with low TTC-reducing power do not.

Lower health if nicotinamide was added to the incubation medium resulting in higher TTC-reducing capacity (as shown by C24 and Columbia).

Example 7: phenotypic analysis of transgenic lines containing dsRNA-APP and dsRNA-ZAP constructs

The floral phenotype and pollen viability of T0-lines containing dsRNA-APP and dsRNA-ZAP under the control of anther or pollen specific promoters were counted (Alexander staining (Alexander, 1969) and germination assays). In the case of Arabidopsis, T1-progeny are obtained by selfing or by backcrossing using pollen from an untransformed wild-type plant if the plant is male sterile. For Brassica napus, T1-progeny was always obtained by backcrossing using untransformed plants.

T1-seeds were germinated on kanamycin-containing medium, after which resistant plants were counted by the phosphinothricin-resistant ammoniacal well assay (De Block et al, 1995). Half of the plants containing both T-DNAs were transferred to a greenhouse and male fertility of the plants was counted, while the other half was used to quantify the vigor of the plants by a health assay.

High vigour was observed in many transgenic lines for plants containing a combination of PCD-regulated genes (APP/ZAP) under the control of 35S or NOS promoters.

Male fertility was observed in many transgenic lines for plants containing a combination of PCD regulated genes (APP/ZAP) under control of TA 29.

Sterile pollen was observed in many transgenic lines for plants containing a combination of PCD-regulated genes (APP/ZAP) under the control of NTP 303.

Example 8: phenotypic analysis of plants containing PCD-regulated chimeric genes

Another example p 35S: (dsRNA-ZAP) chimeric gene was constructed by operably linking the following DNA regions using standard recombinant DNA procedures:

● A CaMV35S 2 promoter region (Odell et al, 1985)

● A DNA encoding the Cab22 leader (Harpster et al, 1988)

● A ZAP 2-encoding DNA region of maize from the Hincll position to the SnaB1 position having the nucleotide sequence of SEQ ID No 10 from nucleotide 279 to nucleotide 1728

● 5' end of the ZAP2 coding region in reverse orientation from the Hincll position to the EcoRV position (complement of the nucleotide sequence of SEQ ID No 10 from nucleotide No. 279 to nucleotide No. 792)

● A CaMV35S 3' terminal region (Mogen et al, 1990)

This chimeric gene was introduced into the polylinker between the T-DNA borders of the T-DNA vector from pGSV5 (described in WO 97/13865) together with the bar marker gene and yielded the T-DNA vector pTYG33, which was introduced into Agrobacterium C58C1Rif (pGV4000) by the described electroporation.

Another example pNos: (dsRNA-ZAP) chimeric gene was constructed by operably linking the following DNA regions using standard recombinant DNA procedures:

● A nopaline synthase promoter region (Herrera-Estralla et al, 1985)

● A DNA encoding the Cab22 leader (Harpster et al, 1988)

● A ZAP 2-encoding DNA region of maize from the Hincll position to the SnaB1 position having the nucleotide sequence of SEQ ID No 10 from nucleotide 279 to nucleotide 1728

● 5' end of the ZAP2 coding region in reverse orientation from the Hincll position to the EcoRV position (complement of the nucleotide sequence of SEQ ID No 10 from nucleotide No. 279 to nucleotide No. 792)

● A CaMV35S 3' terminal region (Mogen et al, 1990)

This chimeric gene was introduced into the polylinker between the T-DNA borders of the T-DNA vector from pGSV5 (described in WO 97/13865) together with the bar marker gene and yielded the T-DNA vector pTYG34, which was introduced into Agrobacterium C58C1Rif (pGV4000) by the described electroporation.

Another example p 35S: (dsRNA-APP) chimeric gene was constructed using standard recombinant DNA procedures by operably linking the following DNA regions:

● A CaMV35S 2 promoter region (Odell et al, 1985)

● A DNA encoding the Cab22 leader (Harpster et al, 1988)

● A DNA region of Arabidopsis thaliana encoding APP having the nucleotide sequence of SEQ ID No 5 from nucleotide 189 to nucleotide 1349 from Scal position to Smal position

● from the Scal position to the 5' end of the ZAP2 coding region in reverse orientation (complement of the nucleotide sequence having SEQ ID No 5 from position 189 to position 784 of nucleotide)

● A CaMV35S 3' terminal region (Mogen et al, 1990)

This chimeric gene was introduced into the polylinker between the T-DNA borders of the T-DNA vector from pGSV5 (described in WO 97/13865) together with the bar marker gene and yielded the T-DNA vector pTYG29, which was introduced into Agrobacterium C58C1Rif (pGV4000) by the described electroporation.

Another example pNos: (dsRNA-APP) chimeric gene was constructed by operably linking the following DNA regions using standard recombinant DNA procedures:

● A nopaline synthase promoter region (Herrera-Estralla et al, 1985)

● A DNA encoding the Cab22 leader (Harpster et al, 1988)

● A DNA region of Arabidopsis thaliana encoding APP having the nucleotide sequence of SEQ ID No 5 from nucleotide 189 to nucleotide 1349 from Scal to Smal

● from the Scal position to the 5' end of the ZAP2 coding region in reverse orientation (complement of the nucleotide sequence having SEQ ID No 5 from position 189 to position 784 of nucleotide)

● A CaMV35S 3' terminal region (Mogen et al, 1990)

This chimeric gene was introduced into the polylinker between the T-DNA borders of the T-DNA vector from pGSV5 (described in WO 97/13865) together with the bar marker gene and yielded the T-DNA vector pTYG30, which was introduced into Agrobacterium C58C1Rif (pGV4000) by the described electroporation.

The resulting Agrobacterium strains were used to introduce different PCD modulatory genes into Brassica napus and Arabidopsis (Columbia and C24) plants as described in examples 4 and 5, respectively.

Transgenic Arabidopsis plants obtained by self-flowering insemination of the T0 progeny (T1 progeny) were germinated on phosphinothricin-containing medium. And further breeding the resistant transgenic plants.

The transgenic T1 plants (both from Columbia or C24) containing either the pTYG 33-based pNOS: s (dsRNA-ZAP) construct or the pTYG 34-based p 35S: s (dsRNA-ZAP) construct grew significantly faster than the control transgenic plants transformed with T-DNA without the PCD regulated chimeric gene T-DNA vector (see Table 1).

Stress tolerance of arabidopsis T1 transgenic plants (from Columbia) was evaluated by floating the plantlets on either 10 or 50mg/L salicylic acid solution or water as a control. Stress-sensitive plants grow bleached and curly leaves after 1-2 days of cultivation, whereas stress-tolerant plants remain intact for at least 5 days. In addition, transgenic plants containing either the pTYG 33-based pNOS: formula (dsRNA-ZAP) construct or the pTYG 34-based p 35S: formula (dsRNA-ZAP) construct were significantly more stress tolerant than the control transgenic plants (see Table 1).

anti-PPT transgenic calli obtained from Brassica napus transformed with dsRNA-ZAP or dsRNA-APP by pTYG29, pTYG30, pTYG33 or pTYG34 were incubated for 2 days on media containing 50mg/L aspirin. After 2 days, the weight of these calli was determined and these calli were transferred to media without aspirin and incubated for an additional 5 days. After these 5 days, the weight of these calli was determined and the weight gain was expressed as a percentage of the weight after 2 days of incubation. As a control, transgenic calli transformed with T-DNA without the PCD regulated chimeric gene were rendered by the same procedure except that no aspirin was added during the 2 day incubation. The results are summarized in table II and demonstrate that transgenic Brassica napus cells containing the PCD modulating chimeric gene are more stress tolerant than control cells.

Table 1: evaluation of transgenic Arabidopsis plants (T1 progeny)

Chimeric PCD modulatory genes	Growth (Columbia and C24)	Stress tolerance (Columbia)
			pNOS∷(dsRNA-ZAP)	+++	++
p35S∷(dsRNA-ZAP)	++	+
			pNOS∷(dsRNA-APP)	+	+/-
p35S∷(dsRNA-APP)	+	-
			Control	+	+/-(**)

A. thalina Columbia has some degree of natural resistance to aspirin.

Table 2: regrowth of the transgenic Brassica callica calli after incubation on aspirin

Chimeric PCD modulatory genes	Weight gain (%)
		pNOS∷(dsRNA-ZAP)	80
p35S∷(dsRNA-ZAP)	90
		pNOS∷(dsRNA-APP)	75
p35S∷(dsRNA-APP)	85
		Control	70

The average standard error is less than 5%.

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Sequence listing

<110> plant genetic system, n.v.

<120> methods and means for modulating programmed cell death in eukaryotic cells

<130>PCDMOD WO1

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<150>US SN 09/118276

<151>1998-07-17

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acctacctga atacgtcatc cctaagtgtt ccgcttcctc tgtcgtccgg cctccaactc 60

catcgaaggg gctagggaga ggagggaacc cgaaccacag caggccggcg ca atg gcg 118

Met Ala

1

gcg ccg cca aag gcg tgg aag gcg gag tat gcc aag tct ggg cgg gcc 166

Ala Pro Pro Lys Ala Trp Lys Ala Glu Tyr Ala Lys Ser Gly Arg Ala

5 10 15

tcg tgc aag tca tgc cgg tcc cct atc gcc aag gac cag ctc cgt ctt 214

Ser Cys Lys Ser Cys Arg Ser Pro Ile Ala Lys Asp Gln Leu Arg Leu

20 25 30

ggc aag atg gtt cag gcg tca cag ttc gac ggc ttc atg ccg atg tgg 262

Gly Lys Met Val Gln Ala Ser Gln Phe Asp Gly Phe Met Pro Met Trp

35 40 45 50

aac cat gcc agc gtt gac gat gtt gaa ggg ata gat gca ctt aga tgg 310

Asn His Ala Ser Val Asp Asp Val Glu Gly Ile Asp Ala Leu Arg Trp

55 60 65

gat gat caa gag aag ata cga aac tac gtt ggg agt gcc tca gct ggt 358

Asp Asp Gln Glu Lys Ile Arg Asn Tyr Val Gly Ser Ala Ser Ala Gly

70 75 80

aca agt tct aca gct gct cct cct gag aaa tgt aca att gag att gct 406

Thr Ser Ser Thr Ala Ala Pro Pro Glu Lys Cys Thr Ile Glu Ile Ala

85 90 95

cca tct gcc cgt act tca tgt aga cga tgc agt gaa aag att aca aaa 454

Pro Ser Ala Arg Thr Ser Cys Arg Arg Cys Ser Glu Lys Ile Thr Lys

100 105 110

gga tcg gtc cgt ctt tca gct aag ctt gag agt gaa ggt ccc aag ggt 502

Gly Ser Val Arg Leu Ser Ala Lys Leu Glu Ser Glu Gly Pro Lys Gly

115 120 125 130

ata cca tgg tat cat gcc aac tgt ttc ttt gag gta tcc ccg tct gca 550

Ile Pro Trp Tyr His Ala Asn Cys Phe Phe Glu Val Ser Pro Ser Ala

135 140 145

act gtt gag aag ttc tca ggc tgg gat act ttg tcc gat gag gat aag 598

Thr Val Glu Lys Phe Ser Gly Trp Asp Thr Leu Ser Asp Glu Asp Lys

150 155 160

aga acc atg ctc gat ctt gtt aaa aaa gat gtt ggc aac aat gaa caa 646

Arg Thr Met Leu Asp Leu Val Lys Lys Asp Val Gly Asn Asn Glu Gln

165 170 175

aat aag ggt tcc aag cgc aag aaa agt gaa aat gat att gat agc tac 694

Asn Lys Gly Ser Lys Arg Lys Lys Ser Glu Asn Asp Ile Asp Ser Tyr

180 185 190

aaa tcc gcc agg tta gat gaa agt aca tct gaa ggt aca gtg cga aac 742

Lys Ser Ala Arg Leu Asp Glu Ser Thr Ser Glu Gly Thr Val Arg Asn

195 200 205 210

aaa ggg caa ctt gta gac cca cgt ggt tcc aat act agt tca gct gat 790

Lys Gly Gln Leu Val Asp Pro Arg Gly Ser Asn Thr Ser Ser Ala Asp

215 220 225

atc caa cta aag ctt aag gag caa agt gac aca ctt tgg aag tta aag 838

Ile Gln Leu Lys Leu Lys Glu Gln Ser Asp Thr Leu Trp Lys Leu Lys

230 235 240

gat gga ctt aag act cat gta tcg gct gct gaa tta agg gat atg ctt 886

Asp Gly Leu Lys Thr His Val Ser Ala Ala Glu Leu Arg Asp Met Leu

245 250 255

gag gct aat ggg cag gat aca tca gga cca gaa agg cac cta ttg gat 934

Glu Ala Asn Gly Gln Asp Thr Ser Gly Pro Glu Arg His Leu Leu Asp

260 265 270

cgc tgt gcg gat gga atg ata ttt gga gcg ctg ggt cct tgc cca gtc 982

Arg Cys Ala Asp Gly Met Ile Phe Gly Ala Leu Gly Pro Cys Pro Val

275 280 285 290

tgt gct aat ggc atg tac tat tat aat ggt cag tac caa tgc agt ggt 1030

Cys Ala Asn Gly Met Tyr Tyr Tyr Asn Gly Gln Tyr Gln Cys Ser Gly

295 300 305

aat gtg tca gag tgg tcc aag tgt aca tac tct gcc aca gaa cct gtc 1078

Asn Val Ser Glu Trp Ser Lys Cys Thr Tyr Ser Ala Thr Glu Pro Val

310 315 320

cgc gtt aag aag aag tgg caa att cca cat gga aca aag aat gat tac 1126

Arg Val Lys Lys Lys Trp Gln Ile Pro His Gly Thr Lys Asn Asp Tyr

325 330 335

ctt atg aag tgg ttc aaa tct caa aag gtt aag aaa cca gag agg gtt 1174

Leu Met Lys Trp Phe Lys Ser Gln Lys Val Lys Lys Pro Glu Arg Val

340 345 350

ctt cca cca atg tca cct gag aaa tct gga agt aaa gca act cag aga 1222

Leu Pro Pro Met Ser Pro Glu Lys Ser Gly Ser Lys Ala Thr Gln Arg

355 360 365 370

aca tca ttg ctg tct tct aaa ggg ttg gat aaa tta agg ttt tct gtt 1270

Thr Ser Leu Leu Ser Ser Lys Gly Leu Asp Lys Leu Arg Phe Ser Val

375 380 385

gta gga caa tca aaa gaa gca gca aat gag tgg att gag aag ctc aaa 1318

Val Gly Gln Ser Lys Glu Ala Ala Asn Glu Trp Ile Glu Lys Leu Lys

390 395 400

ctt gct ggt gcc aac ttc tat gcc agg gtt gtc aaa gat att gat tgt 1366

Leu Ala Gly Ala Asn Phe Tyr Ala Arg Val Val Lys Asp Ile Asp Cys

405 410 415

tta att gca tgt ggt gag ctc gac aat gaa aat gct gaa gtc agg aaa 1414

Leu Ile Ala Cys Gly Glu Leu Asp Asn Glu Asn Ala Glu Val Arg Lys

420 425 430

gca agg agg ctg aag ata cca att gta agg gag ggt tac att gga gaa 1462

Ala Arg Arg Leu Lys Ile Pro Ile Val Arg Glu Gly Tyr Ile Gly Glu

435 440 445 450

tgt gtt aaa aag aac aaa atg ctg cca ttt gat ttg tat aaa cta gag 1510

Cys Val Lys Lys Asn Lys Met Leu Pro Phe Asp Leu Tyr Lys Leu Glu

455 460 465

aat gcc tta gag tcc tca aaa ggc agt act gtc act gtt aaa gtt aag 1558

Asn Ala Leu Glu Ser Ser Lys Gly Ser Thr Val Thr Val Lys Val Lys

470 475 480

ggc cga agt gct gtt cat gag tcc tct ggt ttg caa gat act gct cac 1606

Gly Arg Ser Ala Val His Glu Ser Ser Gly Leu Gln Asp Thr Ala His

485 490 495

att ctt gaa gat ggg aaa agc ata tac aat gca acc tta aac atg tct 1654

Ile Leu Glu Asp Gly Lys Ser Ile Tyr Asn Ala Thr Leu Asn Met Ser

500 505 510

gac ctg gca cta ggt gtg aac agc tac tat gta ctc cag atc att gaa 1702

Asp Leu Ala Leu Gly Val Asn Ser Tyr Tyr Val Leu Gln Ile Ile Glu

515 520 525 530

cag gat gat ggg tct gag tgc tac gta ttt cgt aag tgg gga cgg gtt 1750

Gln Asp Asp Gly Ser Glu Cys Tyr Val Phe Arg Lys Trp Gly Arg Val

535 540 545

ggg agt gag aaa att gga ggg caa aaa ctg gag gag atg tca aaa act 1798

Gly Ser Glu Lys Ile Gly Gly Gln Lys Leu Glu Glu Met Ser Lys Thr

550 555 560

gag gca atc aag gaa ttc aaa aga tta ttt ctt gag aag act gga aac 1846

Glu Ala Ile Lys Glu Phe Lys Arg Leu Phe Leu Glu Lys Thr Gly Asn

565 570 575

tca tgg gaa gct tgg gaa tgt aaa acc aat ttt cgg aag cag cct ggg 1894

Ser Trp Glu Ala Trp Glu Cys Lys Thr Asn Phe Arg Lys Gln Pro Gly

580 585 590

aga ttt tac cca ctt gat gtt gat tat ggt gtt aag aaa gca cca aaa 1942

Arg Phe Tyr Pro Leu Asp Val Asp Tyr Gly Val Lys Lys Ala Pro Lys

595 600 605 610

cgg aaa gat atc agt gaa atg aaa agt tct ctt gct cct caa ttg cta 1990

Arg Lys Asp Ile Ser Glu Met Lys Ser Ser Leu Ala Pro Gln Leu Leu

615 620 625

gaa ctc atg aag atg ctt ttc aat gtg gag aca tat aga gct gct atg 2038

Glu Leu Met Lys Met Leu Phe Asn Val Glu Thr Tyr Arg Ala Ala Met

630 635 640

atg gaa ttt gaa att aat atg tca gaa atg cct ctt ggg aag cta agc 2086

Met Glu Phe Glu Ile Asn Met Ser Glu Met Pro Leu Gly Lys Leu Ser

645 650 655

aag gaa aat att gag aaa gga ttt gaa gca tta act gag ata cag aat 2134

Lys Glu Asn Ile Glu Lys Gly Phe Glu Ala Leu Thr Glu Ile Gln Asn

660 665 670

tta ttg aag gac acc gct gat caa gca ctg gct gtt aga gaa agc tta 2182

Leu Leu Lys Asp Thr Ala Asp Gln Ala Leu Ala Val Arg Glu Ser Leu

675 680 685 690

att gtt gct gcg agc aat cgc ttt ttc act ctt atc cct tct att cat 2230

Ile Val Ala Ala Ser Asn Arg Phe Phe Thr Leu Ile Pro Ser Ile His

695 700 705

cct cat att ata cgg gat gag gat gat ttg atg atc aaa gcg aaa atg 2278

Pro His Ile Ile Arg Asp Glu Asp Asp Leu Met Ile Lys Ala Lys Met

710 715 720

ctt gaa gct ctg cag gat att gaa att gct tca aag ata gtt ggc ttc 2326

Leu Glu Ala Leu Gln Asp Ile Glu Ile Ala Ser Lys Ile Val Gly Phe

725 730 735

gat agc gac agt gat gaa tct ctt gat gat aaa tat atg aaa ctt cac 2374

Asp Ser Asp Ser Asp Glu Ser Leu Asp Asp Lys Tyr Met Lys Leu His

740 745 750

tgt gac atc acc ccg ctg gct cac gat agt gaa gat tac aag tta att 2422

Cys Asp Ile Thr Pro Leu Ala His Asp Ser Glu Asp Tyr Lys Leu Ile

755 760 765 770

gag cag tat ctc ctc aac aca cat gct cct act cac aag gac tgg tcg 2470

Glu Gln Tyr Leu Leu Asn Thr His Ala Pro Thr His Lys Asp Trp Ser

775 780 785

ctg gaa ctg gag gaa gtt ttt tca ctt gat cga gat gga gaa ctt aat 2518

Leu Glu Leu Glu Glu Val Phe Ser Leu Asp Arg Asp Gly Glu Leu Asn

790 795 800

aag tac tca aga tat aaa aat aat ctg cat aac aag atg cta tta tgg 2566

Lys Tyr Ser Arg Tyr Lys Asn Asn Leu His Asn Lys Met Leu Leu Trp

805 810 815

cac ggt tca agg ttg acg aat ttt gtg gga att ctt agt caa ggg cta 2614

His Gly Ser Arg Leu Thr Asn Phe Val Gly Ile Leu Ser Gln Gly Leu

820 825 830

aga att gca cct cct gag gca cct gtt act ggc tat atg ttc ggc aaa 2662

Arg Ile Ala Pro Pro Glu Ala Pro Val Thr Gly Tyr Met Phe Gly Lys

835 840 845 850

ggc ctc tac ttt gca gat cta gta agc aag agc gca caa tac tgt tat 2710

Gly Leu Tyr Phe Ala Asp Leu Val Ser Lys Ser Ala Gln Tyr Cys Tyr

855 860 865

gtg gat agg aat aat cct gta ggt ttg atg ctt ctt tct gag gtt gct 2758

Val Asp Arg Asn Asn Pro Val Gly Leu Met Leu Leu Ser Glu Val Ala

870 875 880

tta gga gac atg tat gaa cta aag aaa gcc acg tcc atg gac aaa cct 2806

Leu Gly Asp Met Tyr Glu Leu Lys Lys Ala Thr Ser Met Asp Lys Pro

885 890 895

cca aga ggg aag cat tcg acc aag gga tta ggc aaa acc gtg cca ctg 2854

Pro Arg Gly Lys His Ser Thr Lys Gly Leu Gly Lys Thr Val Pro Leu

900 905 910

gag tca gag ttt gtg aag tgg agg gat gat gtc gta gtt ccc tgc ggc 2902

Glu Ser Glu Phe Val Lys Trp Arg Asp Asp Val Val Val Pro Cys Gly

915 920 925 930

aag ccg gtg cca tca tca att agg agc tct gaa ctc atg tac aat gag 2950

Lys Pro Val Pro Ser Ser Ile Arg Ser Ser Glu Leu Met Tyr Asn Glu

935 940 945

tac atc gtc tac aac aca tcc cag gtg aag atg cag ttc ttg ctg aag 2998

Tyr Ile Val Tyr Asn Thr Ser Gln Val Lys Met Gln Phe Leu Leu Lys

950 955 960

gtg cgt ttc cat cac aag agg tag ctgggagact aggcaagtag agttggaagg 3052

Val Arg Phe His His Lys Arg

965 970

tagagaagca gagttaggcg atgcctcttt tggtattatt agtaagcctg gcatgtattt 3112

atgggtgctc gcgcttgatc cattttggta agtgttgctt gggcatcagc gcgaatagca 3172

ccaatcacac acttttacct aatgacgttt tactgtata 3211

<210>2

<211>969

<212>PRT

<213>Zea mays

<400>2

Met Ala Ala Pro Pro Lys Ala Trp Lys Ala Glu Tyr Ala Lys Ser Gly

1 5 10 15

Arg Ala Ser Cys Lys Ser Cys Arg Ser Pro Ile Ala Lys Asp Gln Leu

20 25 30

Arg Leu Gly Lys Met Val Gln Ala Ser Gln Phe Asp Gly Phe Met Pro

35 40 45

Met Trp Asn His Ala Ser Val Asp Asp Val Glu Gly Ile Asp Ala Leu

50 55 60

Arg Trp Asp Asp Gln Glu Lys Ile Arg Asn Tyr Val Gly Ser Ala Ser

65 70 75 80

Ala Gly Thr Ser Ser Thr Ala Ala Pro Pro Glu Lys Cys Thr Ile Glu

85 90 95

Ile Ala Pro Ser Ala Arg Thr Ser Cys Arg Arg Cys Ser Glu Lys Ile

100 105 110

Thr Lys Gly Ser Val Arg Leu Ser Ala Lys Leu Glu Ser Glu Gly Pro

115 120 125

Lys Gly Ile Pro Trp Tyr His Ala Asn Cys Phe Phe Glu Val Ser Pro

130 135 140

Ser Ala Thr Val Glu Lys Phe Ser Gly Trp Asp Thr Leu Ser Asp Glu

145 150 155 160

Asp Lys Arg Thr Met Leu Asp Leu Val Lys Lys Asp Val Gly Asn Asn

165 170 175

Glu Gln Asn Lys Gly Ser Lys Arg Lys Lys Ser Glu Asn Asp Ile Asp

180 185 190

Ser Tyr Lys Ser Ala Arg Leu Asp Glu Ser Thr Ser Glu Gly Thr Val

195 200 205

Arg Asn Lys Gly Gln Leu Val Asp Pro Arg Gly Ser Asn Thr Ser Ser

210 215 220

Ala Asp Ile Gln Leu Lys Leu Lys Glu Gln Ser Asp Thr Leu Trp Lys

225 230 235 240

Leu Lys Asp Gly Leu Lys Thr His Val Ser Ala Ala Glu Leu Arg Asp

245 250 255

Met Leu Glu Ala Asn Gly Gln Asp Thr Ser Gly Pro Glu Arg His Leu

260 265 270

Leu Asp Arg Cys Ala Asp Gly Met Ile Phe Gly Ala Leu Gly Pro Cys

275 280 285

Pro Val Cys Ala Asn Gly Met Tyr Tyr Tyr Asn Gly Gln Tyr Gln Cys

290 295 300

Ser Gly Asn Val Ser Glu Trp Ser Lys Cys Thr Tyr Ser Ala Thr Glu

305 310 315 320

Pro Val Arg Val Lys Lys Lys Trp Gln Ile Pro His Gly Thr Lys Asn

325 330 335

Asp Tyr Leu Met Lys Trp Phe Lys Ser Gln Lys Val Lys Lys Pro Glu

340 345 350

Arg Val Leu Pro Pro Met Ser Pro Glu Lys Ser Gly Ser Lys Ala Thr

355 360 365

Gln Arg Thr Ser Leu Leu Ser Ser Lys Gly Leu Asp Lys Leu Arg Phe

370 375 380

Ser Val Val Gly Gln Ser Lys Glu Ala Ala Asn Glu Trp Ile Glu Lys

385 390 395 400

Leu Lys Leu Ala Gly Ala Asn Phe Tyr Ala Arg Val Val Lys Asp Ile

405 410 415

Asp Cys Leu Ile Ala Cys Gly Glu Leu Asp Asn Glu Asn Ala Glu Val

420 425 430

Arg Lys Ala Arg Arg Leu Lys Ile Pro Ile Val Arg Glu Gly Tyr Ile

435 440 445

Gly Glu Cys Val Lys Lys Asn Lys Met Leu Pro Phe Asp Leu Tyr Lys

450 455 460

Leu Glu Asn Ala Leu Glu Ser Ser Lys Gly Ser Thr Val Thr Val Lys

465 470 475 480

Val Lys Gly Arg Ser Ala Val His Glu Ser Ser Gly Leu Gln Asp Thr

485 490 495

Ala His Ile Leu Glu Asp Gly Lys Ser Ile Tyr Asn Ala Thr Leu Asn

500 505 510

Met Ser Asp Leu Ala Leu Gly Val Asn Ser Tyr Tyr Val Leu Gln Ile

515 520 525

Ile Glu Gln Asp Asp Gly Ser Glu Cys Tyr Val Phe Arg Lys Trp Gly

530 535 540

Arg Val Gly Ser Glu Lys Ile Gly Gly Gln Lys Leu Glu Glu Met Ser

545 550 555 560

Lys Thr Glu Ala Ile Lys Glu Phe Lys Arg Leu Phe Leu Glu Lys Thr

565 570 575

Gly Asn Ser Trp Glu Ala Trp Glu Cys Lys Thr Asn Phe Arg Lys Gln

580 585 590

Pro Gly Arg Phe Tyr Pro Leu Asp Val Asp Tyr Gly Val Lys Lys Ala

595 600 605

Pro Lys Arg Lys Asp Ile Ser Glu Met Lys Ser Ser Leu Ala Pro Gln

610 615 620

Leu Leu Glu Leu Met Lys Met Leu Phe Asn Val Glu Thr Tyr Arg Ala

625 630 635 640

Ala Met Met Glu Phe Glu Ile Asn Met Ser Glu Met Pro Leu Gly Lys

645 650 655

Leu Ser Lys Glu Asn Ile Glu Lys Gly Phe Glu Ala Leu Thr Glu Ile

660 665 670

Gln Asn Leu Leu Lys Asp Thr Ala Asp Gln Ala Leu Ala Val Arg Glu

675 680 685

Ser Leu Ile Val Ala Ala Ser Asn Arg Phe Phe Thr Leu Ile Pro Ser

690 695 700

Ile His Pro His Ile Ile Arg Asp Glu Asp Asp Leu Met Ile Lys Ala

705 710 715 720

Lys Met Leu Glu Ala Leu Gln Asp Ile Glu Ile Ala Ser Lys Ile Val

725 730 735

Gly Phe Asp Ser Asp Ser Asp Glu Ser Leu Asp Asp Lys Tyr Met Lys

741 745 750

Leu His Cys Asp Ile Thr Pro Leu Ala His Asp Ser Glu Asp Tyr Lys

755 760 765

Leu Ile Glu Gln Tyr Leu Leu Asn Thr His Ala Pro Thr His Lys Asp

770 775 780

Trp Ser Leu Glu Leu Glu Glu Val Phe Ser Leu Asp Arg Asp Gly Glu

785 790 795 800

Leu Asn Lys Tyr Ser Arg Tyr Lys Asn Asn Leu His Asn Lys Met Leu

805 810 815

Leu Trp His Gly Ser Arg Leu Thr Asn Phe Val Gly Ile Leu Ser Gln

820 825 830

Gly Leu Arg Ile Ala Pro Pro Glu Ala Pro Val Thr Gly Tyr Met Phe

835 840 845

Gly Lys Gly Leu Tyr Phe Ala Asp Leu Val Ser Lys Ser Ala Gln Tyr

850 855 860

Cys Tyr Val Asp Arg Asn Asn Pro Val Gly Leu Met Leu Leu Ser Glu

865 870 875 880

Val Ala Leu Gly Asp Met Tyr Glu Leu Lys Lys Ala Thr Ser Met Asp

885 890 895

Lys Pro Pro Arg Gly Lys His Ser Thr Lys Gly Leu Gly Lys Thr Val

900 905 910

Pro Leu Glu Ser Glu Phe Val Lys Trp Arg Asp Asp Val Val Val Pro

915 920 925

Cys Gly Lys Pro Val Pro Ser Ser Ile Arg Ser Ser Glu Leu Met Tyr

930 935 940

Asn Glu Tyr Ile Val Tyr Asn Thr Ser Gln Val Lys Met Gln Phe Leu

945 950 955 960

Leu Lys Val Arg Phe His His Lys Arg

965

<210>3

<211>2295

<212>DNA

<213>Zea mays

<220>

<221>CDS

<222>(107)..(2068)

<400>3

tgacctgttc catcccgcca gcccttccgc tcccacgacc caaccccact gcccggagcc 60

cccgagcctt ctcgaatctt gcgagaaccc caggggcgag gagcag atg tcg gcg 115

Met Ser Ala

1

agg cta cgg gtg gcg gac gtc cgc gcg gag ctt cag cgc cgc ggc ctc 163

Arg Leu Arg Val Ala Asp Val Arg Ala Glu Leu Gln Arg Arg Gly Leu

5 10 15

gat gta tcc ggc acc aag cct gct ctc gtg cgg agg ctg gac gcc gca 211

Asp Val Ser Gly Thr Lys Pro Ala Leu Val Arg Arg Leu Asp Ala Ala

20 25 30 35

att tgc gag gcg gag aag gcc gtg gtg gct gct gcg cca acc agt gtg 259

Ile Cys Glu Ala Glu Lys Ala Val Val Ala Ala Ala Pro Thr Ser Val

40 45 50

gca aat ggg tat gac gta gcc gta gat ggc aaa agg aac tgc ggg aat 307

Ala Asn Gly Tyr Asp Val Ala Val Asp Gly Lys Arg Asn Cys Gly Asn

55 60 65

aat aag agg aaa agg tcc ggg gat ggg ggt gaa gag gga aac ggc gat 355

Asn Lys Arg Lys Arg Ser Gly Asp Gly Gly Glu Glu Gly Asn Gly Asp

70 75 80

acg tgt aca gat gtg aca aaa cta gag ggc atg agc tat cgt gag ctg 403

Thr Cys Thr Asp Val Thr Lys Leu Glu Gly Met Ser Tyr Arg Glu Leu

85 90 95

cag gga ttg gcc aag gca cgt gga gtt gcg gca aat ggg ggc aag aaa 451

Gln Gly Leu Ala Lys Ala Arg Gly Val Ala Ala Asn Gly Gly Lys Lys

100 105 110 125

gat gtt atc cag agg ttg ctc tcg gcg act gct ggt cct gct gca gtt 499

Asp Val Ile Gln Arg Leu Leu Ser Ala Thr Ala Gly Pro Ala Ala Val

120 125 130

gca gat ggt ggt cct ctg ggc gcc aag gaa gtc ata aaa ggt ggt gat 547

Ala Asp Gly Gly Pro Leu Gly Ala Lys Glu Val Ile Lys Gly Gly Asp

135 140 145

gag gag gtt gag gtg aaa aag gag aag atg gtt act gcc acg aag aag 595

Glu Glu Val Glu Val Lys Lys Glu Lys Met Val Thr Ala Thr Lys Lys

150 155 160

gga gct gca gtg ctg gat cag cac att ccc gat cac ata aaa gtg aac 643

Gly Ala Ala Val Leu Asp Gln His Ile Pro Asp His Ile Lys Val Asn

165 170 175

tat cat gtc ttg caa gtg ggc gat gaa atc tat gat gcc acc ttg aac 691

Tyr His Val Leu Gln Val Gly Asp Glu Ile Tyr Asp Ala Thr Leu Asn

180 185 190 195

cag act aat gtt gga gac aac aac aat aag ttc tat atc att caa gtt 739

Gln Thr Asn Val Gly Asp Asn Asn Asn Lys Phe Tyr Ile Ile Gln Val

200 205 210

tta gaa tct gat gct ggt gga agc ttt atg gtt tac aat aga tgg gga 787

Leu Glu Ser Asp Ala Gly Gly Ser Phe Met Val Tyr Asn Arg Trp Gly

215 220 225

aga gtt ggg gta cga ggt caa gat aaa cta cat ggt ccc tcc cca aca 835

Arg Val Gly Val Arg Gly Gln Asp Lys Leu His Gly Pro Ser Pro Thr

230 235 240

cga gac caa gca ata tat gaa ttt gag ggg aag ttc cac aac aaa acc 883

Arg Asp Gln Ala Ile Tyr Glu Phe Glu Gly Lys Phe His Asn Lys Thr

245 250 255

aat aat cat tgg tct gat cgc aag aac ttc aaa tgt tat gca aag aaa 931

Asn Asn His Trp Ser Asp Arg Lys Asn Phe Lys Cys Tyr Ala Lys Lys

260 265 270 275

tac act tgg ctt gaa atg gat tat ggt gaa act gag aaa gaa ata gag 979

Tyr Thr Trp Leu Glu Met Asp Tyr Gly Glu Thr Glu Lys Glu Ile Glu

280 285 290

aaa ggt tcc att act gat cag ata aaa gag aca aaa ctt gaa act aga 1027

Lys Gly Ser Ile Thr Asp Gln Ile Lys Glu Thr Lys Leu Glu Thr Arg

295 300 305

att gcg cag ttc ata tcc ctg atc tgc aat att agc atg atg aag caa 1075

Ile Ala Gln Phe Ile Ser Leu Ile Cys Asn Ile Ser Met Met Lys Gln

310 315 320

aga atg gtg gaa ata ggt tat aat gct gaa aag ctt ccc ctt gga aag 1123

Arg Met Val Glu Ile Gly Tyr Asn Ala Glu Lys Leu Pro Leu Gly Lys

325 330 335

cta agg aaa gct aca ata ctt aag ggt tat cat gtt ttg aaa agg ata 1171

Leu Arg Lys Ala Thr Ile Leu Lys Gly Tyr His Val Leu Lys Arg Ile

340 345 350 355

tcc gat gtt att tca aag gcg gac agg aga cat ctt gag caa ttg act 1219

Ser Asp Val Ile Ser Lys Ala Asp Arg Arg His Leu Glu Gln Leu Thr

360 365 370

ggg gaa ttc tac acc gtg att cct cat gac ttt ggt ttc aga aag atg 1267

Gly Glu Phe Tyr Thr Val Ile Pro His Asp Phe Gly Phe Arg Lys Met

375 380 385

cgt gaa ttt att atc gat act cct cag aaa cta aaa gct aag ctg gag 1315

Arg Glu Phe Ile Ile Asp Thr Pro Gln Lys Leu Lys Ala Lys Leu Glu

390 395 400

atg gtt gaa gcc ctt ggt gag att gaa att gca act aaa ctt ttg gag 1363

Met Val Glu Ala Leu Gly Glu Ile Glu Ile Ala Thr Lys Leu Leu Glu

405 410 415

gat gat tca agt gac cag gat gat ccg ttg tat gct cga tac aag caa 1411

Asp Asp Ser Ser Asp Gln Asp Asp Pro Leu Tyr Ala Arg Tyr Lys Gln

420 425 430 435

ctt cat tgt gat ttc aca cct ctt gaa gct gat tca gat gag tac tct 1459

Leu His Cys Asp Phe Thr Pro Leu Glu Ala Asp Ser Asp Glu Tyr Ser

440 445 450

atg ata aaa tca tat ttg aga aat aca cat gga aaa aca cac tct ggt 1507

Met Ile Lys Ser Tyr Leu Arg Asn Thr His Gly Lys Thr His Ser Gly

455 460 465

tat acg gtg gac ata gtg caa ata ttt aag gtt tca agg cat ggt gaa 1555

Tyr Thr Val Asp Ile Val Gln Ile Phe Lys Val Ser Arg His Gly Glu

470 475 480

aca gag cga ttt caa aaa ttt gct agt aca aga aat agg atg ctt ttg 1603

Thr Glu Arg Phe Gln Lys Phe Ala Ser Thr Arg Asn Arg Met Leu Leu

485 490 495

tgg cat ggt tct cgg ttg agc aac tgg gct ggg atc ctt tct cag ggt 1651

Trp His Gly Ser Arg Leu Ser Asn Trp Ala Gly Ile Leu Ser Gln Gly

500 505 510 515

ctg cga atc gct cct cct gaa gca cct gtt act ggt tac atg ttt ggc 1699

Leu Arg Ile Ala Pro Pro Glu Ala Pro Val Thr Gly Tyr Met Phe Gly

521 525 530

aag ggt gtt tac ttt gct gac atg ttt tca aag agt gca aac tat tgc 1747

Lys Gly Val Tyr Phe Ala Asp Met Phe Ser Lys Ser Ala Asn Tyr Cys

535 540 545

tac gcc tct gaa gca tgt aga tct gga gta ctg ctt tta tgt gag gtt 1795

Tyr Ala Ser Glu Ala Cys Arg Ser Gly Val Leu Leu Leu Cys Glu Val

550 555 560

gca ttg ggc gat atg aat gag cta ctg aat gca gat tac gat gct aat 1843

Ala Leu Gly Asp Met Asn Glu Leu Leu Asn Ala Asp Tyr Asp Ala Asn

565 570 575

aac ctg ccc aaa gga aaa tta aga tcc aag gga gtt ggt caa aca gca 1891

Asn Leu Pro Lys Gly Lys Leu Arg Ser Lys Gly Val Gly Gln Thr Ala

580 585 590 595

cct aac atg gtc gag tct aag gtc gct gac gat ggt gtt gtt gtt ccc 1939

Pro Asn Met Val Glu Ser Lys Val Ala Asp Asp Gly Val Val Val Pro

600 605 610

ctt ggc gaa ccc aaa cag gaa cct tcc aaa agg ggt ggc ttg ctt tat 1987

Leu Gly Glu Pro Lys Gln Glu Pro Ser Lys Arg Gly Gly Leu Leu Tyr

615 620 625

aat gag tac ata gtg tac aac gta gac cag ata aga atg cgg tat gtc 2035

Asn Glu Tyr Ile Val Tyr Asn Val Asp Gln Ile Arg Met Arg Tyr Val

630 635 640

tta cat gtt aac ttc aat ttc aag aga cgg tag atgttgcaaa gagctgaaac 2088

Leu His Val Asn Phe Asn Phe Lys Arg Arg

645 650

tgttgctgag atcttagcag aacatatgtg gacttatagc accaggtgcc ctcagcctca 2148

ttttctgagc aaatttggta gcctttgcat ttcgattttg gtttcagctt ctagccccat 2208

tgatgattga tactgagtgt atatatgaac cattgatatc caccttccat gtacttaagt 2268

ttttttaaca tgtcccatgc ataataa 2295

<210>4

<211>653

<212>PRT

<213>Zea mays

<400>4

Met Ser Ala Arg Leu Arg Val Ala Asp Val Arg Ala Glu Leu Gln Arg

1 5 10 15

Arg Gly Leu Asp Val Ser Gly Thr Lys Pro Ala Leu Val Arg Arg Leu

20 25 30

Asp Ala Ala Ile Cys Glu Ala Glu Lys Ala Val Val Ala Ala Ala Pro

35 40 45

Thr Ser Val Ala Asn Gly Tyr Asp Val Ala Val Asp Gly Lys Arg Asn

50 55 60

Cys Gly Asn Asn Lys Arg Lys Arg Ser Gly Asp Gly Gly Glu Glu Gly

65 70 75 80

Asn Gly Asp Thr Cys Thr Asp Val Thr Lys Leu Glu Gly Met Ser Tyr

85 90 95

Arg Glu Leu Gln Gly Leu Ala Lys Ala Arg Gly Val Ala Ala Asn Gly

100 105 110

Gly Lys Lys Asp Val Ile Gln Arg Leu Leu Ser Ala Thr Ala Gly Pro

115 120 125

Ala Ala Val Ala Asp Gly Gly Pro Leu Gly Ala Lys Glu Val Ile Lys

130 135 140

Gly Gly Asp Glu Glu Val Glu Val Lys Lys Glu Lys Met Val Thr Ala

145 150 155 160

Thr Lys Lys Gly Ala Ala Val Leu Asp Gln His Ile Pro Asp His Ile

165 170 175

Lys Val Asn Tyr His Val Leu Gln Val Gly Asp Glu Ile Tyr Asp Ala

180 185 190

Thr Leu Asn Gln Thr Asn Val Gly Asp Asn Asn Asn Lys Phe Tyr Ile

195 200 205

Ile Gln Val Leu Glu Ser Asp Ala Gly Gly Ser Phe Met Val Tyr Asn

210 215 220

Arg Trp Gly Arg Val Gly Val Arg Gly Gln Asp Lys Leu His Gly Pro

225 230 235 240

Ser Pro Thr Arg Asp Gln Ala Ile Tyr Glu Phe Glu Gly Lys Phe His

245 250 255

Asn Lys Thr Asn Asn His Trp Ser Asp Arg Lys Asn Phe Lys Cys Tyr

260 265 270

Ala Lys Lys Tyr Thr Trp Leu Glu Met Asp Tyr Gly Glu Thr Glu Lys

275 280 285

Glu Ile Glu Lys Gly Ser Ile Thr Asp Gln Ile Lys Glu Thr Lys Leu

290 295 300

Glu Thr Arg Ile Ala Gln Phe Ile Ser Leu Ile Cys Asn Ile Ser Met

305 310 315 320

Met Lys Gln Arg Met Val Glu Ile Gly Tyr Asn Ala Glu Lys Leu Pro

325 330 335

Leu Gly Lys Leu Arg Lys Ala Thr Ile Leu Lys Gly Tyr His Val Leu

340 345 350

Lys Arg Ile Ser Asp Val Ile Ser Lys Ala Asp Arg Arg His Leu Glu

355 360 365

Gln Leu Thr Gly Glu Phe Tyr Thr Val Ile Pro His Asp Phe Gly Phe

370 375 380

Arg Lys Met Arg Glu Phe Ile Ile Asp Thr Pro Gln Lys Leu Lys Ala

385 390 395 400

Lys Leu Glu Met Val Glu Ala Leu Gly Glu Ile Glu Ile Ala Thr Lys

405 410 415

Leu Leu Glu Asp Asp Ser Ser Asp Gln Asp Asp Pro Leu Tyr Ala Arg

420 425 430

Tyr Lys Gln Leu His Cys Asp Phe Thr Pro Leu Glu Ala Asp Ser Asp

435 440 445

Glu Tyr Ser Met Ile Lys Ser Tyr Leu Arg Asn Thr His Gly Lys Thr

450 455 460

His Ser Gly Tyr Thr Val Asp Ile Val Gln Ile Phe Lys Val Ser Arg

465 470 475 480

His Gly Glu Thr Glu Arg Phe Gln Lys Phe Ala Ser Thr Arg Asn Arg

485 490 495

Met Leu Leu Trp His Gly Ser Arg Leu Ser Asn Trp Ala Gly Ile Leu

500 505 510

Ser Gln Gly Leu Arg Ile Ala Pro Pro Glu Ala Pro Val Thr Gly Tyr

515 520 525

Met Phe Gly Lys Gly Val Tyr Phe Ala Asp Met Phe Ser Lys Ser Ala

530 535 540

Asn Tyr Cys Tyr Ala Ser Glu Ala Cys Arg Ser Gly Val Leu Leu Leu

545 550 555 560

Cys Glu Val Ala Leu Gly Asp Met Asn Glu Leu Leu Asn Ala Asp Tyr

565 570 575

Asp Ala Asn Asn Leu Pro Lys Gly Lys Leu Arg Ser Lys Gly Val Gly

580 585 590

Gln Thr Ala Pro Asn Met Val Glu Ser Lys Val Ala Asp Asp Gly Val

595 600 605

Val Val Pro Leu Gly Glu Pro Lys Gln Glu Pro Ser Lys Arg Gly Gly

610 615 620

Leu Leu Tyr Asn Glu Tyr Ile Val Tyr Asn Val Asp Gln Ile Arg Met

625 630 635 640

Arg Tyr Val Leu His Val Asn Phe Asn Phe Lys Arg Arg

645 650

<210>5

<211>2147

<212>DNA

<213>Arabidopsis thaliana

<220>

<221>CDS

<222>(129)..(2042)

<400>5

attgatgaag aagaaaacga agaagaagac tcttcaaatg ctcgcgcgaa ctcacttctg 60

acgaaaacca tacttcctca gtctcattcc ctttccgacg aactattctc ctgaagaaga 120

agacgaaa atg gcg aac aag ctc aaa gtc gac gaa ctc cgt tta aaa ctc 170

Met Ala Asn Lys Leu Lys Val Asp Glu Leu Arg Leu Lys Leu

1 5 10

gcc gag cgt gga ctc agt act act gga gtc aaa gcc gtt ctg gtg gag 218

Ala Glu Arg Gly Leu Ser Thr Thr Gly Val Lys Ala Val Leu Val Glu

15 20 25 30

agg ctt gaa gag gct atc gca gaa gac act aag aag gaa gaa tca aag 266

Arg Leu Glu Glu Ala Ile Ala Glu Asp Thr Lys Lys Glu Glu Ser Lys

35 40 45

agc aag agg aaa aga aat tct tct aat gat act tat gaa tcg aac aaa 314

Ser Lys Arg Lys Arg Asn Ser Ser Asn Asp Thr Tyr Glu Ser Asn Lys

50 55 60

ttg att gca att ggc gaa ttt cgt ggg atg att gtg aag gaa ttg cgt 362

Leu Ile Ala Ile Gly Glu Phe Arg Gly Met Ile Val Lys Glu Leu Arg

65 70 75

gag gaa gct att aag aga ggc tta gat aca aca gga acc aaa aag gat 410

Glu Glu Ala Ile Lys Arg Gly Leu Asp Thr Thr Gly Thr Lys Lys Asp

80 85 90

ctt ctt gag agg ctt tgc aat gat gct aat aac gtt tcc aat gca cca 458

Leu Leu Glu Arg Leu Cys Asn Asp Ala Asn Asn Val Ser Asn Ala Pro

95 100 105 110

gtc aaa tcc agt aat ggg aca gat gaa gct gaa gat gac aac aat ggc 506

Val Lys Ser Ser Asn Gly Thr Asp Glu Ala Glu Asp Asp Asn Asn Gly

115 120 125

ttt gaa gaa gaa aag aaa gaa gag aaa atc gta acc gcg aca aag aag 554

Phe Glu Glu Glu Lys Lys Glu Glu Lys Ile Val Thr Ala Thr Lys Lys

130 135 140

ggt gca gcg gtg cta gat cag tgg att cct gat gag ata aag agt cag 602

Gly Ala Ala Val Leu Asp Gln Trp Ile Pro Asp Glu Ile Lys Ser Gln

145 150 155

tac cat gtt cta caa agg ggt gat gat gtt tat gat gct atc tta aat 650

Tyr His Val Leu Gln Arg Gly Asp Asp Val Tyr Asp Ala Ile Leu Asn

160 165 170

cag aca aat gtc agg gat aat aat aac aag ttc ttt gtc cta caa gtc 698

Gln Thr Asn Val Arg Asp Asn Asn Asn Lys Phe Phe Val Leu Gln Val

175 180 185 190

cta gag tcg gat agt aaa aag aca tac atg gtt tac act aga tgg gga 746

Leu Glu Ser Asp Ser Lys Lys Thr Tyr Met Val Tyr Thr Arg Trp Gly

195 200 205

aga gtt ggt gtg aaa gga caa agt aag cta gat ggg cct tat gac tca 794

Arg Val Gly Val Lys Gly Gln Ser Lys Leu Asp Gly Pro Tyr Asp Ser

211 215 220

tgg gat cgt gcg ata gag ata ttt acc aat aag ttc aat gac aag aca 842

Trp Asp Arg Ala Ile Glu Ile Phe Thr Asn Lys Phe Asn Asp Lys Thr

225 230 235

aag aat tat tgg tct gac aga aag gag ttt atc cca cat ccc aag tcc 890

Lys Asn Tyr Trp Ser Asp Arg Lys Glu Phe Ile Pro His Pro Lys Ser

240 245 250

tat aca tgg ctc gaa atg gat tac gga aaa gag gaa aat gat tca ccg 938

Tyr Thr Trp Leu Glu Met Asp Tyr Gly Lys Glu Glu Asn Asp Ser Pro

255 260 265 270

gtc aat aat gat att ccg agt tca tct tcc gaa gtt aaa cct gaa caa 986

Val Asn Asn Asp Ile Pro Ser Ser Ser Ser Glu Val Lys Pro Glu Gln

275 280 285

tca aaa cta gat act cgg gtt gcc aag ttc atc tct ctt ata tgt aat 1034

Ser Lys Leu Asp Thr Arg Val Ala Lys Phe Ile Ser Leu Ile Cys Asn

290 295 300

gtc agc atg atg gca cag cat atg atg gaa ata gga tat aac gct aac 1082

Val Ser Met Met Ala Gln His Met Met Glu Ile Gly Tyr Asn Ala Asn

305 310 315

aaa ttg cca ctc ggc aag ata agc aag tcc aca att tca aag ggt tat 1130

Lys Leu Pro Leu Gly Lys Ile Ser Lys Ser Thr Ile Ser Lys Gly Tyr

320 325 330

gaa gtg ctg aag aga ata tcg gag gtg att gac cgg tat gat aga acg 1178

Glu Val Leu Lys Arg Ile Ser Glu Val Ile Asp Arg Tyr Asp Arg Thr

335 340 345 350

agg ctt gag gaa ctg agt gga gag ttc tac aca gtg ata cct cat gat 1226

Arg Leu Glu Glu Leu Ser Gly Glu Phe Tyr Thr Val Ile Pro His Asp

355 360 365

ttt ggt ttt aag aaa atg agt cag ttt gtt ata gac act cct caa aag 1274

Phe Gly Phe Lys Lys Met Ser Gln Phe Val Ile Asp Thr Pro Gln Lys

370 375 380

ttg aaa cag aaa att gaa atg gtt gaa gca tta ggt gaa att gaa ctc 1322

Leu Lys Gln Lys Ile Glu Met Val Glu Ala Leu Gly Glu Ile Glu Leu

385 390 395

gca aca aag ttg ttg tcc gtc gac ccg gga ttg cag gat gat cct tta 1370

Ala Thr Lys Leu Leu Ser Val Asp Pro Gly Leu Gln Asp Asp Pro Leu

400 405 410

tat tat cac tac cag caa ctt aat tgt ggt ttg acg cca gta gga aat 1418

Tyr Tyr His Tyr Gln Gln Leu Asn Cys Gly Leu Thr Pro Val Gly Asn

415 420 425 430

gat tca gag gag ttc tct atg gtt gct aat tac atg gag aac act cat 1466

Asp Ser Glu Glu Phe Ser Met Val Ala Asn Tyr Met Glu Asn Thr His

435 440 445

gca aag acg cat tcg gga tat acg gtt gag att gcc caa cta ttt aga 1514

Ala Lys Thr His Ser Gly Tyr Thr Val Glu Ile Ala Gln Leu Phe Arg

450 455 460

gct tcg aga gct gtt gaa gct gat cga ttc caa cag ttt tca agt tcg 1562

Ala Ser Arg Ala Val Glu Ala Asp Arg Phe Gln Gln Phe Ser Ser Ser

465 470 475

aag aac agg atg cta ctc tgg cac ggt tca cgt ctc act aac tgg gct 1610

Lys Asn Arg Met Leu Leu Trp His Gly Ser Arg Leu Thr Asn Trp Ala

480 485 490

ggt att tta tct caa ggt ctg cga ata gct cct cct gaa gcg cct gta 1658

Gly Ile Leu Ser Gln Gly Leu Arg Ile Ala Pro Pro Glu Ala Pro Val

495 500 505 510

act ggt tac atg ttt gga aaa ggg gtt tac ttt gcg gat atg ttc tcc 1706

Thr Gly Tyr Met Phe Gly Lys Gly Val Tyr Phe Ala Asp Met Phe Ser

515 520 525

aag agt gcg aac tat tgc tat gcc aac act ggc gct aat gat ggc gtt 1754

Lys Ser Ala Asn Tyr Cys Tyr Ala Asn Thr Gly Ala Asn Asp Gly Val

530 535 540

ctg ctc ctc tgc gag gtt gct ttg gga gac atg aat gaa ctt ctg tat 1802

Leu Leu Leu Cys Glu Val Ala Leu Gly Asp Met Asn Glu Leu Leu Tyr

545 550 555

tca gat tat aac gcg gat aat cta ccc ccg gga aag cta agc aca aaa 1850

Ser Asp Tyr Asn Ala Asp Asn Leu Pro Pro Gly Lys Leu Ser Thr Lys

560 565 570

ggt gtg ggg aaa aca gca cca aac cca tca gag gct caa aca cta gaa 1898

Gly Val Gly Lys Thr Ala Pro Asn Pro Ser Glu Ala Gln Thr Leu Glu

575 580 585 590

gac ggt gtt gtt gtt cca ctt ggc aaa cca gtg gaa cgt tca tgc tcc 1946

Asp Gly Val Val Val Pro Leu Gly Lys Pro Val Glu Arg Ser Cys Ser

595 600 605

aag ggg atg ttg ttg tac aac gaa tat ata gtc tac aat gtg gaa caa 1994

Lys Gly Met Leu Leu Tyr Asn Glu Tyr Ile Val Tyr Asn Val Glu Gln

610 615 620

atc aag atg cgt tat gtg atc caa gtc aaa ttc aac tac aag cac taa 2042

Ile Lys Met Arg Tyr Val Ile Gln Val Lys Phe Asn Tyr Lys His

625 630 635

aacttatgta tattagcttt tgaacatcaa ctaattatcc aaaaatcagc gttttattgt 2102

atttctttca aactccttca tctctgattt tgcacggttc actcg 2147

<210>6

<211>637

<212>PRT

<213>Arabidopsis thaliana

<400>6

Met Ala Asn Lys Leu Lys Val Asp Glu Leu Arg Leu Lys Leu Ala Glu

1 5 10 15

Arg Gly Leu Ser Thr Thr Gly Val Lys Ala Val Leu Val Glu Arg Leu

20 25 30

Glu Glu Ala Ile Ala Glu Asp Thr Lys Lys Glu Glu Ser Lys Ser Lys

35 40 45

Arg Lys Arg Asn Ser Ser Asn Asp Thr Tyr Glu Ser Asn Lys Leu Ile

50 55 60

Ala Ile Gly Glu Phe Arg Gly Met Ile Val Lys Glu Leu Arg Glu Glu

65 70 75 80

Ala Ile Lys Arg Gly Leu Asp Thr Thr Gly Thr Lys Lys Asp Leu Leu

85 90 95

Glu Arg Leu Cys Asn Asp Ala Asn Asn Val Ser Asn Ala Pro Val Lys

100 105 110

Ser Ser Asn Gly Thr Asp Glu Ala Glu Asp Asp Asn Asn Gly Phe Glu

115 120 125

Glu Glu Lys Lys Glu Glu Lys Ile Val Thr Ala Thr Lys Lys Gly Ala

130 135 140

Ala Val Leu Asp Gln Trp Ile Pro Asp Glu Ile Lys Ser Gln Tyr His

145 150 155 160

Val Leu Gln Arg Gly Asp Asp Val Tyr Asp Ala Ile Leu Asn Gln Thr

165 170 175

Asn Val Arg Asp Asn Asn Asn Lys Phe Phe Val Leu Gln Val Leu Glu

180 185 190

Ser Asp Ser Lys Lys Thr Tyr Met Val Tyr Thr Arg Trp Gly Arg Val

195 200 205

Gly Val Lys Gly Gln Ser Lys Leu Asp Gly Pro Tyr Asp Ser Trp Asp

210 215 220

Arg Ala Ile Glu Ile Phe Thr Asn Lys Phe Asn Asp Lys Thr Lys Asn

225 230 235 240

Tyr Trp Ser Asp Arg Lys Glu Phe Ile Pro His Pro Lys Ser Tyr Thr

245 250 255

Trp Leu Glu Met Asp Tyr Gly Lys Glu Glu Asn Asp Ser Pro Val Asn

260 265 270

Asn Asp Ile Pro Ser Ser Ser Ser Glu Val Lys Pro Glu Gln Ser Lys

275 280 285

Leu Asp Thr Arg Val Ala Lys Phe Ile Ser Leu Ile Cys Asn Val Ser

290 295 300

Met Met Ala Gln His Met Met Glu Ile Gly Tyr Asn Ala Asn Lys Leu

305 310 315 320

Pro Leu Gly Lys Ile Ser Lys Ser Thr Ile Ser Lys Gly Tyr Glu Val

325 330 335

Leu Lys Arg Ile Ser Glu Val Ile Asp Arg Tyr Asp Arg Thr Arg Leu

340 345 350

Glu Glu Leu Ser Gly Glu Phe Tyr Thr Val Ile Pro His Asp Phe Gly

355 360 365

Phe Lys Lys Met Ser Gln Phe Val Ile Asp Thr Pro Gln Lys Leu Lys

370 375 380

Gln Lys Ile Glu Met Val Glu Ala Leu Gly Glu Ile Glu Leu Ala Thr

385 390 395 400

Lys Leu Leu Ser Val Asp Pro Gly Leu Gln Asp Asp Pro Leu Tyr Tyr

405 410 415

His Tyr Gln Gln Leu Asn Cys Gly Leu Thr Pro Val Gly Asn Asp Ser

420 425 430

Glu Glu Phe Ser Met Val Ala Asn Tyr Met Glu Asn Thr His Ala Lys

435 440 445

Thr His Ser Gly Tyr Thr Val Glu Ile Ala Gln Leu Phe Arg Ala Ser

450 455 460

Arg Ala Val Glu Ala Asp Arg Phe Gln Gln Phe Ser Ser Ser Lys Asn

465 470 475 480

Arg Met Leu Leu Trp His Gly Ser Arg Leu Thr Asn Trp Ala Gly Ile

485 490 495

Leu Ser Gln Gly Leu Arg Ile Ala Pro Pro Glu Ala Pro Val Thr Gly

500 505 510

Tyr Met Phe Gly Lys Gly Val Tyr Phe Ala Asp Met Phe Ser Lys Ser

515 520 525

Ala Asn Tyr Cys Tyr Ala Asn Thr Gly Ala Asn Asp Gly Val Leu Leu

530 535 540

Leu Cys Glu Val Ala Leu Gly Asp Met Asn Glu Leu Leu Tyr Ser Asp

545 550 555 560

Tyr Asn Ala Asp Asn Leu Pro Pro Gly Lys Leu Ser Thr Lys Gly Val

565 570 575

Gly Lys Thr Ala Pro Asn Pro Ser Glu Ala Gln Thr Leu Glu Asp Gly

580 585 590

Val Val Val Pro Leu Gly Lys Pro Val Glu Arg Ser Cys Ser Lys Gly

595 600 605

Met Leu Leu Tyr Asn Glu Tyr Ile Val Tyr Asn Val Glu Gln Ile Lys

610 615 620

Met Arg Tyr Val Ile Gln Val Lys Phe Asn Tyr Lys His

625 630 635

<210>7

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> name of artificial sequence: a domain of unconventional PARP proteins

<400>7

Arg Gly Xaa Xaa Xaa Xaa Gly Xaa Lys Xaa Xaa Xaa Xaa Xaa Arg Leu

1 5 10 15

<210>8

<211>33

<212>PRT

<213> Artificial sequence

<220>

<223> name of artificial sequence: a1 domain of unconventional PARP proteins

<400>8

Xaa Leu Xaa Val Xaa Xaa Xaa Arg Xaa Xaa Leu Xaa Xaa Arg Gly Leu

1 5 10 15

Xaa Xaa Xaa Gly Val Lys Xaa Xaa Leu Val Xaa Arg Leu Xaa Xaa Ala

20 25 30

Ile

<210>9

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> name of artificial sequence: a2 domain of unconventional PARP proteins

<400>9

Gly Met Xaa Xaa Xaa Glu Leu Xaa Xaa Xaa Ala Xaa Xaa Arg Gly Xaa

1 5 10 15

Xaa Xaa Xaa Gly Xaa Lys Lys Asp Xaa Xaa Arg Leu Xaa Xaa

20 25 30

<210>10

<211>3212

<212>DNA

<213>Zea mays

<220>

<221>CDS

<222>(81)..(3020)

<400>10

gcttcctctg tcgtccggcc tccaactcca tcgaaggggc tagggagagg agggaacccg 60

aaccacagca ggccggcgca atg gcg gcg ccg cca aag gcg tgg aag gcg gag 113

Met Ala Ala Pro Pro Lys Ala Trp Lys Ala Glu

1 5 10

tat gcc aag tct ggg cgg gcc tcg tgc aag tca tgc cgg tcc cct atc 161

Tyr Ala Lys Ser Gly Arg Ala Ser Cys Lys Ser Cys Arg Ser Pro Ile

15 20 25

gcc aag gac cag ctc cgt ctt ggc aag atg gtt cag gcg tca cag ttc 209

Ala Lys Asp Gln Leu Arg Leu Gly Lys Met Val Gln Ala Ser Gln Phe

30 35 40

gac ggc ttc atg ccg atg tgg aac cat gcc agg tgc atc ttc agc aag 257

Asp Gly Phe Met Pro Met Trp Asn His Ala Arg Cys Ile Phe Ser Lys

45 50 55

aag aac cag ata aaa tcc gtt gac gat gtt gaa ggg ata gat gca ctt 305

Lys Asn Gln Ile Lys Ser Val Asp Asp Val Glu Gly Ile Asp Ala Leu

60 65 70 75

aga tgg gat gat caa gag aag ata cga aac tac gtt ggg agt gcc tca 353

Arg Trp Asp Asp Gln Glu Lys Ile Arg Asn Tyr Val Gly Ser Ala Ser

80 85 90

gct ggt aca agt tct aca gct gct cct cct gag aaa tgt aca att gag 401

Ala Gly Thr Ser Ser Thr Ala Ala Pro Pro Glu Lys Cys Thr Ile Glu

95 100 105

att gct cca tct gcc cgt act tca tgt aga cga tgc agt gaa aag att 449

Ile Ala Pro Ser Ala Arg Thr Ser Cys Arg Arg Cys Ser Glu Lys Ile

110 115 120

aca aaa gga tcg gtc cgt ctt tca gct aag ctt gag agt gaa ggt ccc 497

Thr Lys Gly Ser Val Arg Leu Ser Ala Lys Leu Glu Ser Glu Gly Pro

125 130 135

aag ggt ata cca tgg tat cat gcc aac tgt ttc ttt gag gta tcc ccg 545

Lys Gly Ile Pro Trp Tyr His Ala Asn Cys Phe Phe Glu Val Ser Pro

140 145 150 155

tct gca act gtt gag aag ttc tca ggc tgg gat act ttg tcc gat gag 593

Ser Ala Thr Val Glu Lys Phe Ser Gly Trp Asp Thr Leu Ser Asp Glu

160 165 170

gat aag aga acc atg ctc gat ctt gtt aaa aaa gat gtt ggc aac aat 641

Asp Lys Arg Thr Met Leu Asp Leu Val Lys Lys Asp Val Gly Asn Asn

175 180 185

gaa caa aat aag ggt tcc aag cgc aag aaa agt gaa aat gat att gat 689

Glu Gln Asn Lys Gly Ser Lys Arg Lys Lys Ser Glu Asn Asp Ile Asp

190 195 200

agc tac aaa tcc gcc agg tta gat gaa agt aca tct gaa ggt aca gtg 737

Ser Tyr Lys Ser Ala Arg Leu Asp Glu Ser Thr Ser Glu Gly Thr Val

205 210 215

cga aac aaa ggg caa ctt gta gac cca cgt ggt tcc aat act agt tca 785

Arg Asn Lys Gly Gln Leu Val Asp Pro Arg Gly Ser Asn Thr Ser Ser

220 225 230 235

gct gat atc caa cta aag ctt aag gag caa agt gac aca ctt tgg aag 833

Ala Asp Ile Gln Leu Lys Leu Lys Glu Gln Ser Asp Thr Leu Trp Lys

240 245 250

tta aag gat gga ctt aag act cat gta tcg gct gct gaa tta agg gat 881

Leu Lys Asp Gly Leu Lys Thr His Val Ser Ala Ala Glu Leu Arg Asp

255 260 265

atg ctt gag gct aat ggg cag gat aca tca gga cca gaa agg cac cta 929

Met Leu Glu Ala Asn Gly Gln Asp Thr Ser Gly Pro Glu Arg His Leu

270 275 280

ttg gat cgc tgt gcg gat gga atg ata ttt gga gcg ctg ggt cct tgc 977

Leu Asp Arg Cys Ala Asp Gly Met Ile Phe Gly Ala Leu Gly Pro Cys

285 290 295

cca gtc tgt gct aat ggc atg tac tat tat aat ggt cag tac caa tgc 1025

Pro Val Cys Ala Asn Gly Met Tyr Tyr Tyr Asn Gly Gln Tyr Gln Cys

300 305 310 315

agt ggt aat gtg tca gag tgg tcc aag tgt aca tac tct gcc aca gaa 1073

Ser Gly Asn Val Ser Glu Trp Ser Lys Cys Thr Tyr Ser Ala Thr Glu

320 325 330

cct gtc cgc gtt aag aag aag tgg caa att cca cat gga aca aag aat 1121

Pro Val Arg Val Lys Lys Lys Trp Gln Ile Pro His Gly Thr Lys Asn

335 340 345

gat tac ctt atg aag tgg ttc aaa tct caa aag gtt aag aaa cca gag 1169

Asp Tyr Leu Met Lys Trp Phe Lys Ser Gln Lys Val Lys Lys Pro Glu

350 355 360

agg gtt ctt cca cca atg tca cct gag aaa tct gga agt aaa gca act 1217

Arg Val Leu Pro Pro Met Ser Pro Glu Lys Ser Gly Ser Lys Ala Thr

365 370 375

cag aga aca tca ttg ctg tct tct aaa ggg ttg gat aaa tta agg ttt 1265

Gln Arg Thr Ser Leu Leu Ser Ser Lys Gly Leu Asp Lys Leu Arg Phe

380 385 390 395

tct gtt gta gga caa tca aaa gaa gca gca aat gag tgg att gag aag 1313

Ser Val Val Gly Gln Ser Lys Glu Ala Ala Asn Glu Trp Ile Glu Lys

400 405 410

ctc aaa ctt gct ggt gcc aac ttc tat gcc agg gtt gtc aaa gat att 1361

Leu Lys Leu Ala Gly Ala Asn Phe Tyr Ala Arg Val Val Lys Asp Ile

415 420 425

gat tgt tta att gca tgt ggt gag ctc gac aat gaa aat gct gaa gtc 1409

Asp Cys Leu Ile Ala Cys Gly Glu Leu Asp Asn Glu Asn Ala Glu Val

430 435 440

agg aaa gca agg agg ctg aag ata cca att gta agg gag ggt tac att 1457

Arg Lys Ala Arg Arg Leu Lys Ile Pro Ile Val Arg Glu Gly Tyr Ile

445 450 455

gga gaa tgt gtt aaa aag aac aaa atg ctg cca ttt gat ttg tat aaa 1505

Gly Glu Cys Val Lys Lys Asn Lys Met Leu Pro Phe Asp Leu Tyr Lys

460 465 470 475

cta gag aat gcc tta gag tcc tca aaa ggc agt act gtc act gtt aaa 1553

Leu Glu Asn Ala Leu Glu Ser Ser Lys Gly Ser Thr Val Thr Val Lys

480 485 490

gtt aag ggc cga agt gct gtt cat gag tcc tct ggt ttg caa gat act 1601

Val Lys Gly Arg Ser Ala Val His Glu Ser Ser Gly Leu Gln Asp Thr

495 500 505

gct cac att ctt gaa gat ggg aaa agc ata tac aat gca acc tta aac 1649

Ala His Ile Leu Glu Asp Gly Lys Ser Ile Tyr Asn Ala Thr Leu Asn

510 515 520

atg tct gac ctg gca cta ggt gtg aac agc tac tat gta ctc cag atc 1697

Met Ser Asp Leu Ala Leu Gly Val Asn Ser Tyr Tyr Val Leu Gln Ile

525 530 535

att gaa cag gat gat ggg tct gag tgc tac gta ttt cgt aag tgg gga 1745

Ile Glu Gln Asp Asp Gly Ser Glu Cys Tyr Val Phe Arg Lys Trp Gly

540 545 550 555

cgg gtt ggg agt gag aaa att gga ggg caa aaa ctg gag gag atg tca 1793

Arg Val Gly Ser Glu Lys Ile Gly Gly Gln Lys Leu Glu Glu Met Ser

560 565 570

aaa act gag gca atc aag gaa ttc aaa aga tta ttt ctt gag aag act 1841

Lys Thr Glu Ala Ile Lys Glu Phe Lys Arg Leu Phe Leu Glu Lys Thr

575 580 585

gga aac tca tgg gaa gct tgg gaa tgt aaa acc aat ttt cgg aag cag 1889

Gly Asn Ser Trp Glu Ala Trp Glu Cys Lys Thr Asn Phe Arg Lys Gln

590 595 600

cct ggg aga ttt tac cca ctt gat gtt gat tat ggt gtt aag aaa gca 1937

Pro Gly Arg Phe Tyr Pro Leu Asp Val Asp Tyr Gly Val Lys Lys Ala

605 610 615

cca aaa cgg aaa gat atc agt gaa atg aaa agt tct ctt gct cct caa 1985

Pro Lys Arg Lys Asp Ile Ser Glu Met Lys Ser Ser Leu Ala Pro Gln

620 625 630 635

ttg cta gaa ctc atg aag atg ctt ttc aat gtg gag aca tat aga gct 2033

Leu Leu Glu Leu Met Lys Met Leu Phe Asn Val Glu Thr Tyr Arg Ala

640 645 650

gct atg atg gaa ttt gaa att aat atg tca gaa atg cct ctt ggg aag 2081

Ala Met Met Glu Phe Glu Ile Asn Met Ser Glu Met Pro Leu Gly Lys

655 660 665

cta agc aag gaa aat att gag aaa gga ttt gaa gca tta act gag ata 2129

Leu Ser Lys Glu Asn Ile Glu Lys Gly Phe Glu Ala Leu Thr Glu Ile

670 675 680

cag aat tta ttg aag gac acc gct gat caa gca ctg gct gtt aga gaa 2177

Gln Asn Leu Leu Lys Asp Thr Ala Asp Gln Ala Leu Ala Val Arg Glu

685 690 695

agc tta att gtt gct gcg agc aat cgc ttt ttc act ctt atc cct tct 2225

Ser Leu Ile Val Ala Ala Ser Asn Arg Phe Phe Thr Leu Ile Pro Ser

700 715 710 715

att cat cct cat att ata cgg gat gag gat gat ttg atg atc aaa gcg 2273

Ile His Pro His Ile Ile Arg Asp Glu Asp Asp Leu Met Ile Lys Ala

720 725 730

aaa atg ctt gaa gct ctg cag gat att gaa att gct tca aag ata gtt 2321

Lys Met Leu Glu Ala Leu Gln Asp Ile Glu Ile Ala Ser Lys Ile Val

735 740 745

ggc ttc gat agc gac agt gat gaa tct ctt gat gat aaa tat atg aaa 2369

Gly Phe Asp Ser Asp Ser Asp Glu Ser Leu Asp Asp Lys Tyr Met Lys

750 755 760

ctt cac tgt gac atc acc ccg ctg gct cac gat agt gaa gat tac aag 2417

Leu His Cys Asp Ile Thr Pro Leu Ala His Asp Ser Glu Asp Tyr Lys

765 770 775

tta att gag cag tat ctc ctc aac aca cat gct cct act cac aag gac 2465

Leu Ile Glu Gln Tyr Leu Leu Asn Thr His Ala Pro Thr His Lys Asp

780 785 790 795

tgg tcg ctg gaa ctg gag gaa gtt ttt tca ctt gat cga gat gga gaa 2513

Trp Ser Leu Glu Leu Glu Glu Val Phe Ser Leu Asp Arg Asp Gly Glu

800 805 810

ctt aat aag tac tca aga tat aaa aat aat ctg cat aac aag atg cta 2561

Leu Asn Lys Tyr Ser Arg Tyr Lys Asn Asn Leu His Asn Lys Met Leu

815 820 825

tta tgg cac ggt tca agg ttg acg aat ttt gtg gga att ctt agt caa 2609

Leu Trp His Gly Ser Arg Leu Thr Asn Phe Val Gly Ile Leu Ser Gln

830 835 840

ggg cta aga att gca cct cct gag gca cct gtt act ggc tat atg ttc 2657

Gly Leu Arg Ile Ala Pro Pro Glu Ala Pro Val Thr Gly Tyr Met Phe

845 850 855

ggc aaa ggc ctc tac ttt gca gat cta gta agc aag agc gca caa tac 2705

Gly Lys Gly Leu Tyr Phe Ala Asp Leu Val Ser Lys Ser Ala Gln Tyr

860 865 870 875

tgt tat gtg gat agg aat aat cct gta ggt ttg atg ctt ctt tct gag 2753

Cys Tyr Val Asp Arg Asn Asn Pro Val Gly Leu Met Leu Leu Ser Glu

880 885 890

gtt gct tta gga gac atg tat gaa cta aag aaa gcc acg tcc atg gac 2801

Val Ala Leu Gly Asp Met Tyr Glu Leu Lys Lys Ala Thr Ser Met Asp

895 900 905

aaa cct cca aga ggg aag cat tcg acc aag gga tta ggc aaa acc gtg 2849

Lys Pro Pro Arg Gly Lys His Ser Thr Lys Gly Leu Gly Lys Thr Val

910 915 920

cca ctg gag tca gag ttt gtg aag tgg agg gat gat gtc gta gtt ccc 2897

Pro Leu Glu Ser Glu Phe Val Lys Trp Arg Asp Asp Val Val Val Pro

925 930 935

tgc ggc aag ccg gtg cca tca tca att agg agc tct gaa ctc atg tac 2945

Cys Gly Lys Pro Val Pro Ser Ser Ile Arg Ser Ser Glu Leu Met Tyr

940 945 950 955

aat gag tac atc gtc tac aac aca tcc cag gtg aag atg cag ttc ttg 2993

Asn Glu Tyr Ile Val Tyr Asn Thr Ser Gln Val Lys Met Gln Phe Leu

960 965 970

ctg aag gtg cgt ttc cat cac aag agg tagctgggag actaggcaag 3040

Leu Lys Val Arg Phe His His Lys Arg

975 980

tagagttgga aggtagagaa gcagagttag gcgatgcctc ttttggtatt attagtaagc 3100

ctggcatgta tttatgggtg ctcgcgcttg atccattttg gtaagtgttg cttgggcatc 3160

agcgcgaata gcaccaatca cacactttta cctaatgacg ttttactgta ta 3212

<210>11

<211>980

<212>PRT

<213>Zea mays

<400>11

Met Ala Ala Pro Pro Lys Ala Trp Lys Ala Glu Tyr Ala Lys Ser Gly

1 5 10 15

Arg Ala Ser Cys Lys Ser Cys Arg Ser Pro Ile Ala Lys Asp Gln Leu

20 25 30

Arg Leu Gly Lys Met Val Gln Ala Ser Gln Phe Asp Gly Phe Met Pro

35 40 45

Met Trp Asn His Ala Arg Cys Ile Phe Ser Lys Lys Asn Gln Ile Lys

50 55 60

Ser Val Asp Asp Val Glu Gly Ile Asp Ala Leu Arg Trp Asp Asp Gln

65 70 75 80

Glu Lys Ile Arg Asn Tyr Val Gly Ser Ala Ser Ala Gly Thr Ser Ser

85 90 95

Thr Ala Ala Pro Pro Glu Lys Cys Thr Ile Glu Ile Ala Pro Ser Ala

100 105 110

Arg Thr Ser Cys Arg Arg Cys Ser Glu Lys Ile Thr Lys Gly Ser Val

115 120 125

Arg Leu Ser Ala Lys Leu Glu Ser Glu Gly Pro Lys Gly Ile Pro Trp

130 135 140

Tyr His Ala Asn Cys Phe Phe Glu Val Ser Pro Ser Ala Thr Val Glu

145 150 155 160

Lys Phe Ser Gly Trp Asp Thr Leu Ser Asp Glu Asp Lys Arg Thr Met

165 170 175

Leu Asp Leu Val Lys Lys Asp Val Gly Asn Asn Glu Gln Asn Lys Gly

180 185 190

Ser Lys Arg Lys Lys Ser Glu Asn Asp Ile Asp Ser Tyr Lys Ser Ala

195 200 205

Arg Leu Asp Glu Ser Thr Ser Glu Gly Thr Val Arg Asn Lys Gly Gln

210 215 220

Leu Val Asp Pro Arg Gly Ser Asn Thr Ser Ser Ala Asp Ile Gln Leu

225 230 235 240

Lys Leu Lys Glu Gln Ser Asp Thr Leu Trp Lys Leu Lys Asp Gly Leu

245 250 255

Lys Thr His Val Ser Ala Ala Glu Leu Arg Asp Met Leu Glu Ala Asn

260 265 270

Gly Gln Asp Thr Ser Gly Pro Glu Arg His Leu Leu Asp Arg Cys Ala

275 280 285

Asp Gly Met Ile Phe Gly Ala Leu Gly Pro Cys Pro Val Cys Ala Asn

290 295 300

Gly Met Tyr Tyr Tyr Asn Gly Gln Tyr Gln Cys Ser Gly Asn Val Ser

305 310 315 320

Glu Trp Ser Lys Cys Thr Tyr Ser Ala Thr Glu Pro Val Arg Val Lys

325 330 335

Lys Lys Trp Gln Ile Pro His Gly Thr Lys Asn Asp Tyr Leu Met Lys

340 345 350

Trp Phe Lys Ser Gln Lys Val Lys Lys Pro Glu Arg Val Leu Pro Pro

355 360 365

Met Ser Pro Glu Lys Ser Gly Ser Lys Ala Thr Gln Arg Thr Ser Leu

370 375 380

Leu Ser Ser Lys Gly Leu Asp Lys Leu Arg Phe Ser Val Val Gly Gln

385 390 395 400

Ser Lys Glu Ala Ala Asn Glu Trp Ile Glu Lys Leu Lys Leu Ala Gly

405 410 415

Ala Asn Phe Tyr Ala Arg Val Val Lys Asp Ile Asp Cys Leu Ile Ala

420 425 430

Cys Gly Glu Leu Asp Asn Glu Asn Ala Glu Val Arg Lys Ala Arg Arg

435 440 445

Leu Lys Ile Pro Ile Val Arg Glu Gly Tyr Ile Gly Glu Cys Val Lys

450 455 460

Lys Asn Lys Met Leu Pro Phe Asp Leu Tyr Lys Leu Glu Asn Ala Leu

465 470 475 480

Glu Ser Ser Lys Gly Ser Thr Val Thr Val Lys Val Lys Gly Arg Ser

485 490 495

Ala Val His Glu Ser Ser Gly Leu Gln Asp Thr Ala His Ile Leu Glu

500 505 510

Asp Gly Lys Ser Ile Tyr Asn Ala Thr Leu Asn Met Ser Asp Leu Ala

515 520 525

Leu Gly Val Asn Ser Tyr Tyr Val Leu Gln Ile Ile Glu Gln Asp Asp

530 535 540

Gly Ser Glu Cys Tyr Val Phe Arg Lys Trp Gly Arg Val Gly Ser Glu

545 550 555 560

Lys Ile Gly Gly Gln Lys Leu Glu Glu Met Ser Lys Thr Glu Ala Ile

565 570 575

Lys Glu Phe Lys Arg Leu Phe Leu Glu Lys Thr Gly Asn Ser Trp Glu

580 585 590

Ala Trp Glu Cys Lys Thr Asn Phe Arg Lys Gln Pro Gly Arg Phe Tyr

595 600 605

Pro Leu Asp Val Asp Tyr Gly Val Lys Lys Ala Pro Lys Arg Lys Asp

610 615 620

Ile Ser Glu Met Lys Ser Ser Leu Ala Pro Gln Leu Leu Glu Leu Met

625 630 635 640

Lys Met Leu Phe Asn Val Glu Thr Tyr Arg Ala Ala Met Met Glu Phe

645 650 655

Glu Ile Asn Met Ser Glu Met Pro Leu Gly Lys Leu Ser Lys Glu Asn

660 665 670

Ile Glu Lys Gly Phe Glu Ala Leu Thr Glu Ile Gln Asn Leu Leu Lys

675 680 685

Asp Thr Ala Asp Gln Ala Leu Ala Val Arg Glu Ser Leu Ile Val Ala

690 695 700

Ala Ser Asn Arg Phe Phe Thr Leu Ile Pro Ser Ile His Pro His Ile

705 710 715 720

Ile Arg Asp Glu Asp Asp Leu Met Ile Lys Ala Lys Met Leu Glu Ala

725 730 735

Leu Gln Asp Ile Glu Ile Ala Ser Lys Ile Val Gly Phe Asp Ser Asp

740 745 750

Ser Asp Glu Ser Leu Asp Asp Lys Tyr Met Lys Leu His Cys Asp Ile

755 760 765

Thr Pro Leu Ala His Asp Ser Glu Asp Tyr Lys Leu Ile Glu Gln Tyr

770 775 780

Leu Leu Asn Thr His Ala Pro Thr His Lys Asp Trp Ser Leu Glu Leu

785 790 795 800

Glu Glu Val Phe Ser Leu Asp Arg Asp Gly Glu Leu Asn Lys Tyr Ser

805 810 815

Arg Tyr Lys Asn Asn Leu His Asn Lys Met Leu Leu Trp His Gly Ser

820 825 830

Arg Leu Thr Asn Phe Val Gly Ile Leu Ser Gln Gly Leu Arg Ile Ala

835 840 845

Pro Pro Glu Ala Pro Val Thr Gly Tyr Met Phe Gly Lys Gly Leu Tyr

850 855 860

Phe Ala Asp Leu Val Ser Lys Ser Ala Gln Tyr Cys Tyr Val Asp Arg

865 870 875 880

Asn Asn Pro Val Gly Leu Met Leu Leu Ser Glu Val Ala Leu Gly Asp

885 890 895

Met Tyr Glu Leu Lys Lys Ala Thr Ser Met Asp Lys Pro Pro Arg Gly

900 905 910

Lys His Ser Thr Lys Gly Leu Gly Lys Thr Val Pro Leu Glu Ser Glu

915 920 925

Phe Val Lys Trp Arg Asp Asp Val Val Val Pro Cys Gly Lys Pro Val

930 935 940

Pro Ser Ser Ile Arg Ser Ser Glu Leu Met Tyr Asn Glu Tyr Ile Val

945 950 955 960

Tyr Asn Thr Ser Gln Val Lys Met Gln Phe Leu Leu Lys Val Arg Phe

965 970 975

His His Lys Arg

980

<210>12

<211>1010

<212>PRT

<213> Artificial sequence

<220>

<223> name of artificial sequence: fusion protein between APP N-terminal domain and GUS protein

<400>12

Met Ala Asn Lys Leu Lys Val Asp Glu Leu Arg Leu Lys Leu Ala Glu

1 5 10 15

Arg Gly Leu Ser Thr Thr Gly Val Lys Ala Val Leu Val Glu Arg Leu

20 25 30

Glu Glu Ala Ile Ala Glu Asp Thr Lys Lys Glu Glu Ser Lys Ser Lys

35 40 45

Arg Lys Arg Asn Ser Ser Asn Asp Thr Tyr Glu Ser Asn Lys Leu Ile

50 55 60

Ala Ile Gly Glu Phe Arg Gly Met Ile Val Lys Glu Leu Arg Glu Glu

65 70 75 80

Ala Ile Lys Arg Gly Leu Asp Thr Thr Gly Thr Lys Lys Asp Leu Leu

85 90 95

Glu Arg Leu Cys Asn Asp Ala Asn Asn Val Ser Asn Ala Pro Val Lys

100 105 110

Ser Ser Asn Gly Thr Asp Glu Ala Glu Asp Asp Asn Asn Gly Phe Glu

115 120 125

Glu Glu Lys Lys Glu Glu Lys Ile Val Thr Ala Thr Lys Lys Gly Ala

130 135 140

Ala Val Leu Asp Gln Trp Ile Pro Asp Glu Ile Lys Ser Gln Tyr His

145 150 155 160

Val Leu Gln Arg Gly Asp Asp Val Tyr Asp Ala Ile Leu Asn Gln Thr

165 170 175

Asn Val Arg Asp Asn Asn Asn Lys Phe Phe Val Leu Gln Val Leu Glu

180 185 190

Ser Asp Ser Lys Lys Thr Tyr Met Val Tyr Thr Arg Trp Gly Arg Val

195 200 205

Gly Val Lys Gly Gln Ser Lys Leu Asp Gly Pro Tyr Asp Ser Trp Asp

210 215 220

Arg Ala Ile Glu Ile Phe Thr Asn Lys Phe Asn Asp Lys Thr Lys Asn

225 230 235 240

Tyr Trp Ser Asp Arg Lys Glu Phe Ile Pro His Pro Lys Ser Tyr Thr

245 250 255

Trp Leu Glu Met Asp Tyr Gly Lys Glu Glu Asn Asp Ser Pro Val Asn

260 265 270

Asn Asp Ile Pro Ser Ser Ser Ser Glu Val Lys Pro Glu Gln Ser Lys

275 280 285

Leu Asp Thr Arg Val Ala Lys Phe Ile Ser Leu Ile Cys Asn Val Ser

290 295 300

Met Met Ala Gln His Met Met Glu Ile Gly Tyr Asn Ala Asn Lys Leu

305 310 315 320

Pro Leu Gly Lys Ile Ser Lys Ser Thr Ile Ser Lys Gly Tyr Glu Val

325 330 335

Leu Lys Arg Ile Ser Glu Val Ile Asp Arg Tyr Asp Arg Thr Arg Leu

340 345 350

Glu Glu Leu Ser Gly Glu Phe Tyr Thr Val Ile Pro His Asp Phe Gly

355 360 365

Phe Lys Lys Met Ser Gln Phe Val Ile Asp Thr Pro Gln Lys Leu Lys

370 375 380

Gln Lys Ile Glu Met Val Glu Ala Leu Gly Glu Ile Glu Leu Ala Thr

385 390 395 400

Lys Leu Leu Ser Val Asp Pro Met Val Arg Pro Val Glu Thr Pro Thr

405 410 415

Arg Glu Ile Lys Lys Leu Asp Gly Leu Trp Ala Phe Ser Leu Asp Arg

420 425 430

Glu Asn Cys Gly Ile Asp Gln Arg Trp Trp Glu Ser Ala Leu Gln Glu

435 440 445

Ser Arg Ala Ile Ala Val Pro Gly Ser Phe Asn Asp Gln Phe Ala Asp

450 455 460

Ala Asp Ile Arg Asn Tyr Ala Gly Asn Val Trp Tyr Gln Arg Glu Val

465 470 475 480

Phe Ile Pro Lys Gly Trp Ala Gly Gln Arg Ile Val Leu Arg Phe Asp

485 490 495

Ala Val Thr His Tyr Gly Lys Val Trp Val Asn Asn Gln Glu Val Met

500 505 510

Glu His Gln Gly Gly Tyr Thr Pro Phe Glu Ala Asp Val Thr Pro Tyr

515 520 525

Val Ile Ala Gly Lys Ser Val Arg Ile Thr Val Cys Val Asn Asn Glu

530 535 540

Leu Asn Trp Gln Thr Ile Pro Pro Gly Met Val Ile Thr Asp Glu Asn

545 550 555 560

Gly Lys Lys Lys Gln Ser Tyr Phe His Asp Phe Phe Asn Tyr Ala Gly

565 570 575

Ile His Arg Ser Val Met Leu Tyr Thr Thr Pro Asn Thr Trp Val Asp

580 585 590

Asp Ile Thr Val Val Thr His Val Ala Gln Asp Cys Asn His Ala Ser

595 600 605

Val Asp Trp Gln Val Val Ala Asn Gly Asp Val Ser Val Glu Leu Arg

610 615 620

Asp Ala Asp Gln Gln Val Val Ala Thr Gly Gln Gly Thr Ser Gly Thr

625 630 635 640

Leu Gln Val Val Asn Pro His Leu Trp Gln Pro Gly Glu Gly Tyr Leu

645 650 655

Tyr Glu Leu Cys Val Thr Ala Lys Ser Gln Thr Glu Cys Asp Ile Tyr

660 665 670

Pro Leu Arg Val Gly Ile Arg Ser Val Ala Val Lys Gly Glu Gln Phe

675 680 685

Leu Ile Asn His Lys Pro Phe Tyr Phe Thr Gly Phe Gly Arg His Glu

690 695 700

Asp Ala Asp Leu Arg Gly Lys Gly Phe Asp Asn Val Leu Met Val His

705 710 715 720

Asp His Ala Leu Met Asp Trp Ile Gly Ala Asn Ser Tyr Arg Thr Ser

725 730 735

His Tyr Pro Tyr Ala Glu Glu Met Leu Asp Trp Ala Asp Glu His Gly

740 745 750

Ile Val Val Ile Asp Glu Thr Ala Ala Val Gly Phe Asn Leu Ser Leu

755 760 765

Gly Ile Gly Phe Glu Ala Gly Asn Lys Pro Lys Glu Leu Tyr Ser Glu

770 775 780

Glu Ala Val Asn Gly Glu Thr Gln Gln Ala His Leu Gln Ala Ile Lys

785 790 795 800

Glu Leu Ile Ala Arg Asp Lys Asn His Pro Ser Val Val Met Trp Ser

805 810 815

Ile Ala Asn Glu Pro Asp Thr Arg Pro Gln Gly Ala Arg Glu Tyr Phe

820 825 830

Ala Pro Leu Ala Glu Ala Thr Arg Lys Leu Asp Pro Thr Arg Pro Ile

835 840 845

Thr Cys Val Asn Val Met Phe Cys Asp Ala His Thr Asp Thr Ile Ser

850 855 860

Asp Leu Phe Asp Val Leu Cys Leu Asn Arg Tyr Tyr Gly Trp Tyr Val

865 870 875 880

Gln Ser Gly Asp Leu Glu Thr Ala Glu Lys Val Leu Glu Lys Glu Leu

885 890 895

Leu Ala Trp Gln Glu Lys Leu His Gln Pro Ile Ile Ile Thr Glu Tyr

900 905 910

Gly Val Asp Thr Leu Ala Gly Leu His Ser Met Tyr Thr Asp Met Trp

915 920 925

Ser Glu Glu Tyr Gln Cys Ala Trp Leu Asp Met Tyr His Arg Val Phe

930 935 940

Asp Arg Val Ser Ala Val Val Gly Glu Gln Val Trp Asn Phe Ala Asp

945 950 955 960

Phe Ala Thr Ser Gln Gly Ile Leu Arg Val Gly Gly Asn Lys Lys Gly

965 970 975

Ile Phe Thr Arg Asp Arg Lys Pro Lys Ser Ala Ala Phe Leu Leu Gln

980 985 990

Lys Arg Trp Thr Gly Met Asn Phe Gly Glu Lys Pro Gln Gln Gly Gly

995 1000 1005

Lys Gln

1010

<210>13

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> name of artificial sequence: degenerate PCR primers

<400>13

ccgaattcgg ntayatgtty ggnaa 25

<210>14

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> name of artificial sequence: degenerate PCR primers

<400>14

ccgaattcac natrtaytcr ttrta 25

<210>15

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> name of artificial sequence: oligonucleotides as primers for PCR

<400>15

gggaccatgt agtttatctt gacct 25

<210>16

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> name of artificial sequence: oligonucleotides for PCR

<400>16

gacctcgtac cccaactctt ccccat 26

<210>17

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> name of artificial sequence: oligonucleotides for PCR

<400>17

aagtcgacgc ggccgccaca cctagtgcca ggtcag 36

<210>18

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> name of artificial sequence: oligonucleotides for PCR

<400>18

atctcaattg tacatttctc agga 24

<210>19

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> name of artificial sequence: oligonucleotides for PCR

<400>19

aggatcccat ggcgaacaag ctcaaagtga c 31

<210>20

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> name of artificial sequence: oligonucleotides for PCR

<400>20

aggatcctta gtgcttgtag ttgaat 26

<210>21

<211>4947

<212>DNA

<213> Artificial sequence

<220>

<223> name of artificial sequence: APP promoter fusions with beta-glucuronidase gene

<220>

<221> promoter

<222>(1)..(1961)

<220>

<221> misc-signal

<222>(1962)..(1964)

<223> translation initiation codon

<400>21

ctcgagatag tatatttttt agttactatc attacataag tatattttaa aaaactaatt 60

atatgaatta tgtagctaac tagatagata atcgtataac caattcatgt tagtatagta 120

tagtttaagt atgtattttg ggattacaag tgtggttggc atcaagacaa ggatggtgat 180

agcctttctc tgtaatttgg tttaagaaaa gtttttgcat tttatgtata aacgtgtttt 240

ttttttataa tttcaaattt caacaaaaaa caattttttt taataatgat tgaccactat 300

agacaattta aatgataaaa aaaaggggga atttttcaca atgttttgga gattagtcta 360

gattttttgt ccaaattttc cgattgtaag aattaagaag caatgaacat ttgtgttaag 420

cttaatgatt tgtactcaca atatctttta aatttaaaat tgttaaccaa aatatcctat 480

atattgtact tgtaatagaa atataaacta ttaaaaacaa cactttattc atataatata 540

agttaaaaca tatgtttttt ttagtatgtt ctaatcacac ctattaaaaa aagttgaagc 600

taaatgagcc aaaaagaaaa ataaagatag gggatgggga caggctgtaa tgttaggcgg 660

ttggtatatg aactgagaac atgtctgttg gttcggtcca tctacgccac tcaaccattt 720

ggctatgttt tctttttggc ttttgcatgt tctctctact tttcttcttt ggtcaaaatc 780

tctatctcgt cttttacatg gcttacccga atgttagttg tcatgtaaat ttggttatga 840

aaagatattt tatataaact ttatcgtata ttaatatcgt tatcatctaa ccatttttta 900

aaactaaact agaaccatcc agttttacaa gagttttttt tttttttttc taactaaata 960

atatttgaag tgtacaatat taacaatata tgggccaaat aatagtggaa accaaatcgt 1020

tagtcccact ttatgatggg cctgttgatt cttatgtctt cttcgtaagt tgtgattatg 1080

cagattacgg gctaataaac atgcatgttt agtttttact gtccaagtaa cgaaatttta 1140

tcttttgggt tgttggccca tttcatatat tccaaatgcc aaatccagcc cggctcgaca 1200

cagcactgct cggctcaaca ctcgtatgcg gttggtagcc acttaagacc ttggtttgat 1260

taacatgtta cgaataattt gtgtcccttt ttcttcaagg agactaatct cttttaataa 1320

aaaagaattg tgtcattagt caacacaagt cctataatcc gtttacgtaa tttgtatgca 1380

cgtccttgga aaagtgagta gtggcgtacg ttacagccaa aaactatttg tatattttct 1440

ttcgttaaac aaccagcaaa attttcagaa aaatgttctt aaattataaa ttagtagtac 1500

attttaaaac atagagattt tttgtttctt ttaatagaag agttaaacct atgtacaaaa 1560

tttcaactcc ttttcaaagt atttgcctgt tactagattt ttaacctttt tttttttatc 1620

tttcatgatt ttctattgct tgccatcatc aatggtagga aataaatact attttaaaaa 1680

ggtcaggggt ggatttaaga atcaatccaa aagtttgggg tcttttggag attaaaaagt 1740

tatatgggaa atatccacaa atatgaacga gaacttttgt caaaaaaatt taaaataatt 1800

tttcaaaaag ccctaaagct ttcaagggaa gccatcgatg aagaagaaaa cgaagaagaa 1860

gactcttcaa acgttcgcgc gaactcactt ctgacgaaaa ccatacttcc tcagtctcat 1920

tccctttccg acgaactatt ctcctgaaga agaagacgaa aatggcgaac aagctcaaag 1980

tcgacatggt ccgtcctgta gaaaccccaa cccgtgaaat caaaaaactc gacggcctgt 2040

gggcattcag tctggatcgc gaaaactgtg gaattgatca gcgttggtgg gaaagcgcgt 2100

tacaagaaag ccgggcaatt gctgtgccag gcagttttaa cgatcagttc gccgatgcag 2160

atattcgtaa ttatgcgggc aacgtctggt atcagcgcga agtctttata ccgaaaggtt 2220

gggcaggcca gcgtatcgtg ctgcgtttcg atgcggtcac tcattacggc aaagtgtggg 2280

tcaataatca ggaagtgatg gagcatcagg gcggctatac gccatttgaa gccgatgtca 2340

cgccgtatgt tattgccggg aaaagtgtac gtatcaccgt ttgtgtgaac aacgaactga 2400

actggcagac tatcccgccg ggaatggtga ttaccgacga aaacggcaag aaaaagcagt 2460

cttacttcca tgatttcttt aactatgccg gaatccatcg cagcgtaatg ctctacacca 2520

cgccgaacac ctgggtggac gatatcaccg tggtgacgca tgtcgcgcaa gactgtaacc 2580

acgcgtctgt tgactggcag gtggtggcca atggtgatgt cagcgttgaa ctgcgtgatg 2640

cggatcaaca ggtggttgca actggacaag gcactagcgg gactttgcaa gtggtgaatc 2700

cgcacctctg gcaaccgggt gaaggttatc tctatgaact gtgcgtcaca gccaaaagcc 2760

agacagagtg tgatatctac ccgcttcgcg tcggcatccg gtcagtggca gtgaagggcg 2820

aacagttcct gattaaccac aaaccgttct actttactgg ctttggtcgt catgaagatg 2880

cggacttacg tggcaaagga ttcgataacg tgctgatggt gcacgaccac gcattaatgg 2940

actggattgg ggccaactcc taccgtacct cgcattaccc ttacgctgaa gagatgctcg 3000

actgggcaga tgaacatggc atcgtggtga ttgatgaaac tgctgctgtc ggctttaacc 3060

tctctttagg cattggtttc gaagcgggca acaagccgaa agaactgtac agcgaagagg 3120

cagtcaacgg ggaaactcag caagcgcact tacaggcgat taaagagctg atagcgcgtg 3180

acaaaaacca cccaagcgtg gtgatgtgga gtattgccaa cgaaccggat acccgtccgc 3240

aagtgcacgg gaatatttcg ccactggcgg aagcaacgcg taaactcgac ccgacgcgtc 3300

cgatcacctg cgtcaatgta atgttctgcg acgctcacac cgataccatc agcgatctct 3360

ttgatgtgct gtgcctgaac cgttattacg gatggtatgt ccaaagcggc gatttggaaa 3420

cggcagagaa ggtactggaa aaagaacttc tggcctggca ggagaaactg catcagccga 3480

ttatcatcac cgaatacggc gtggatacgt tagccgggct gcactcaatg tacaccgaca 3540

tgtggagtga agagtatcag tgtgcatggc tggatatgta tcaccgcgtc tttgatcgcg 3600

tcagcgccgt cgtcggtgaa caggtatgga atttcgccga ttttgcgacc tcgcaaggca 3660

tattgcgcgt tggcggtaac aagaaaggga tcttcactcg cgaccgcaaa ccgaagtcgg 3720

cggcttttct gctgcaaaaa cgctggactg gcatgaactt cggtgaaaaa ccgcagcagg 3780

gaggcaaaca atgannnnnn gaattggtcc tgctttaatg agatatgcga gacgcctatg 3840

atcgcatgat atttgctttc aattctgttg tgcacgttgt aaaaaacctg agcatgtgta 3900

gctcagatcc ttaccgccgg tttcggttca ttctaatgaa tatatcaccc gttactatcg 3960

tatttttatg aataatattc tccgttcaat ttactgattg taccctacta cttatatgta 4020

caatattaaa atgaaaacaa tatattgtgc tgaataggtt tatagcgaca tctatgatag 4080

agcgccacaa taacaaacaa ttgcgtttta ttattacaaa tccaatttta aaaaaagcgg 4140

cagaaccggt caaacctaaa agactgatta cataaatctt attcaaattt caaaaggccc 4200

caggggctag tatctacgac acaccgagcg gcgaactaat aacgttcact gaagggaact 4260

ccggttcccc gccggcgcgc atgggtgaga ttccttgaag ttgagtattg gccgtccgct 4320

ctaccgaaag ttacgggcac cattcaaccc ggtccagcac ggcggccggg taaccgactt 4380

gctgccccga gaattatgca gcattttttt ggtgtatgtg ggccccaaat gaagtgcagg 4440

tcaaaccttg acagtgacga caaatcgttg ggcgggtcca gggcgaattt tgcgacaaca 4500

tgtcgaggct cagcaggact ctagaggatc cccgggtacc gagctcgaat tcactggccg 4560

tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 4620

cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 4680

aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc 4740

tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 4800

agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 4860

tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 4920

tttcaccgtc atcaccgaaa cgcgcga 4947

Claims (6)

1. A method of increasing the growth rate of a plant, the method comprising introducing a PCD modulating chimeric gene into cells of the plant to produce transgenic plant cells, and regenerating the transgenic plant cells into a plant, wherein the PCD modulating chimeric gene comprises the following operably linked DNA regions:

a) a promoter for plant expression;

b) a DNA region which when transcribed yields an RNA molecule capable of reducing the expression of an endogenous PARP gene of the ZAP class and comprising the sequence:

i) a sense nucleotide sequence comprising a nucleotide sequence having 75-100% sequence identity and 100 nucleotides to a nucleotide sequence selected from the group consisting of: encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2, encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4, encoding a polypeptide having the amino acid sequence of SEQ ID NO: 6 and a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 11; and

ii) an antisense nucleotide sequence comprising a nucleotide sequence having 75-100% sequence identity to the complement of a nucleotide sequence selected from the group consisting of: encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2, encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4, encoding a polypeptide having the amino acid sequence of SEQ ID NO: 6 and a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 11, said sense nucleotide sequence and said antisense nucleotide sequence being capable of combining into a double-stranded RNA region; and

c) DNA regions involved in transcription termination and polyadenylation.

2. A method of producing a plant having enhanced vigour relative to a control plant, the method comprising introducing a PCD modulating chimeric gene into cells of the plant to produce transgenic plant cells, and regenerating the transgenic plant cells into a plant, wherein the PCD modulating chimeric gene comprises the following operably linked DNA regions:

a) a promoter for plant expression;

b) a DNA region which when transcribed yields an RNA molecule capable of reducing the expression of an endogenous PARP gene of the ZAP class and comprising the sequence:

i) a sense nucleotide sequence comprising a nucleotide sequence having 75-100% sequence identity and 100 nucleotides to a nucleotide sequence selected from the group consisting of: encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2, encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4, encoding a polypeptide having the amino acid sequence of SEQ ID NO: 6 and a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 11; and

ii) an antisense nucleotide sequence comprising a nucleotide sequence having 75-100% sequence identity to the complement of a nucleotide sequence selected from the group consisting of: encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2, encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4, encoding a polypeptide having the amino acid sequence of SEQ ID NO: 6 and a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 11, said sense nucleotide sequence and said antisense nucleotide sequence being capable of combining into a double-stranded RNA region; and

c) DNA regions involved in transcription termination and polyadenylation.

3. Use of a nucleotide sequence encoding a protein having PARP activity or a fragment thereof comprising at least 100 contiguous nucleotides in the production of a high-vigor plant or plant cell by introducing said nucleotide sequence or the complement thereof into said plant cell or cells of said plant thereby reducing the expression of endogenous PARP genes, and said protein having PARP activity has an amino acid sequence selected from the group consisting of: amino acid sequence SEQ ID NO: 2. amino acid sequence SEQ ID NO: 4. amino acid sequence seq id NO: 6 and the amino acid sequence SEQ ID NO: 11.

4. use of a nucleotide sequence encoding a protein having PARP activity or a fragment thereof comprising at least 100 contiguous nucleotides for increasing the growth rate of a plant cell or plant, wherein said increasing the growth rate of a plant cell or plant is effected by introducing said nucleotide sequence or the complement thereof into said plant cell or cells of said plant thereby reducing the expression of an endogenous PARP gene, and said protein having PARP activity has an amino acid sequence selected from the group consisting of: amino acid sequence SEQ ID NO: 2. amino acid sequence SEQ ID NO: 4. amino acid sequence SEQ ID NO: 6 and the amino acid sequence SEQ ID NO: 11.

5. a chimeric gene comprising the following operably linked DNA regions:

a) a promoter for plant expression;

b) a DNA region which when transcribed yields an RNA molecule capable of reducing the expression of an endogenous PARP gene of the ZAP class and comprising the sequence:

i) a sense nucleotide sequence comprising a nucleotide sequence having 75-100% sequence identity and 100 nucleotides to a nucleotide sequence selected from the group consisting of: encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2, encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4, encoding a polypeptide having the amino acid sequence of SEQ ID NO: 6 and a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 11; and

ii) an antisense nucleotide sequence comprising a nucleotide sequence having 75-100% sequence identity to the complement of a nucleotide sequence selected from the group consisting of: encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2, encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4, encoding a polypeptide having the amino acid sequence of SEQ ID NO: 6 and a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 11, said sense nucleotide sequence and said antisense nucleotide sequence being capable of combining into a double-stranded RNA region; and

c) DNA regions involved in transcription termination and polyadenylation.

6. A plant cell comprising the chimeric gene of claim 5.