Abstract:
The invention concerns a process for producing transgenic eucaryotic cells, particularly plants, which comprises: contacting a culture of untransformed cells with an inhibitor of poly-(ADP-ribose) polymerase for a period of time sufficient to reduce the response of the cultured cells to stress and to reduce their metabolism. The untransformed cells are then contacted with foreign DNA comprising at least one gene of interest under conditions in which the foreign DNA is taken up by the untransformed cells and the gene of interest is stably integrated in the nuclear genome of the untransformed cells to produce the transgenic cells. Optionally, the transgenic cells are recovered from the culture. Preferably, the inhibitor is niacinamide, preferably at a concentration of about 200 mg/l to 500 mg/l and the untransformed cells are cultured in a medium containing the inhibitor for a period of time of approximately 3 to 14 days prior to the contacting with the foreign DNA. The invention also relates to a plant having in the nuclear genome of its cells foreign DNA integrated only in the regions of the nuclear genome that are transcriptionally active in cells of the plant when the cells are treated with an effective amount of a PARP inhibitor for a period of time sufficient to reduce cell metabolism to a state where gene expression is essentially limited to genes expressed irrespective of the differentiated or physiological condition of the cell.

Description:
FIELD OF THE INVENTION  
         [0001]    The invention relates to the use of poly (ADP-ribose) polymerase (PARP) proteins, particularly mutant PARP proteins or parts thereof, and genes encoding the same, to produce eukaryotic cells and organisms, particularly plant cells and plants, with modified programmed cell death. Eukaryotic cells and organism, particularly plant cells and plants, are provided wherein either in at least part of the cells, preferably selected cells, the programmed cell death (PCD) is provoked, or wherein, on the contrary, PCD of the cells or of at least part of the cells in an organism is inhibited, by modulation of the level or activity of PARP proteins in those cells. The invention also relates to eukaryotic cells and organisms, particularly plant cells and plants, expressing such genes.  
         DESCRIPTION OF RELATED ART  
         [0002]    Programmed cell death (PCD) is a physiological cell death process involved in the elimination of selected cells both in animals and in plants during developmental processes or in response to environmental cues (for a review see Ellis et al. 1991; Pennell and Lamb, 1997). The disassembly of cells undergoing PCD is morphologically accompanied by condensation, shrinkage and fragmentation of the cytoplasm and nucleus, often into small sealed packets (Cohen 1993, Wang et al. 1996). Biochemically, PCD is characterized by fragmentation of the nuclear DNA into generally about 50 kb fragments representing oligonucleosomes, as well as the induction of cysteine proteinases and endonucleases. The fragmentation of the DNA can be detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) of DNA 3′-OH groups in sections of cells. (Gavrieli et al. 1992). Cell death by PCD is clearly distinct from cell death by necrosis, the latter involving cell swelling, lysis and leakage of the cell contents.  
           [0003]    In animals, PCD is involved in the elimination or death of unwanted cells such as tadpole tail cells at metamorphosis, cells between developing digits in vertebrates, overproduced vertebrate neurons, cells during cell specialization such as keratocytes etc. Damaged cells, which are no longer able to function properly, can also be eliminated by PCD, preventing them from multiplying and/or spreading. PCD, or the lack thereof, has also been involved in a number of pathological conditions in humans (AIDS, Alzheimer&#39;s disease, Huntington&#39;s disease, Lou Gehring&#39;s disease, cancers).  
           [0004]    In plants, PCD has been demonstrated or is believed to be involved in a number of developmental processes such as e.g., removal of the suspensor cells during the development of an embryo, the elimination of aleurone cells after germination of monocotyledonous seeds; the elimination of the root cap cells after seed germination and seedling growth; cell death during cell specialization as seen in development of xylem tracheary element or trichomes, or floral organ aborting in unisexual flowers. Also the formation of aerochyma in roots under hypoxic conditions and the formation of leaf lobes or perforations in some plants seem to involve PCD. Large scale cell death in plants occurs during upon senescence of leaves or other organs. The hypersensitive response in plants, in other words the rapid cell death occurring at the site of entry of an avirulent pathogen leading to a restricted lesion, is an another example of PCD in response to an environmental cue.  
           [0005]    Animal or plant cells dying in suspension cultures, particularly in low-density cell suspension cultures, also demonstrate the characteristics of PCD.  
           [0006]    An enzyme which has been implied to be involved in PCD or apoptosis is poly(ADP-ribose) polymerase. Poly(ADP-ribose) polymerase (PARP), also known as poly(ADP-ribose) transferase (ADPRT) (EC 2.4.2.30), is a nuclear enzyme found in most eukaryotes, including vertebrates, arthropods, molluscs, slime moulds, dinoflagellates, fungi and other low eukaryotes with the exception of yeast. The enzymatic activity has also been demonstrated in a number of plants (Payne et al., 1976; Willmitzer and Wagner, 1982; Chen et al., 1994; O&#39;Farrell, 1995).  
           [0007]    PARP catalyzes the transfer of an ADP-ribose moiety derived from NAD + , mainly to the carboxyl group of a glutamic acid residue in the target protein, and subsequent ADP-ribose polymerization. The major target protein is PARP itself, but also histones, high mobility group chromosomal proteins, a topoisomerase, endonucleases and DNA polymerases have been shown to be subject to this modification.  
           [0008]    The PARP protein from animals is a nuclear protein of 113-120 kDa, abundant in most cell types, that consist of three major functional domains: an amino-terminal DNA-binding domain containing two Zn-finger domains, a carboxy-terminal catalytic domain, and an internal domain which is automodified (de Murcia and Ménissier de Murcia, 1994; Kameshita et al., 1984; Lindahl et al., 1995). The enzymatic activity in vitro is greatly increased upon binding to single-strand breaks in DNA. The in vivo activity is induced by conditions that eventually result in DNA breaks (Alvarez-Gonzalez and Althaus, 1989; Ikejima et al., 1990). Automodification of the central domain apparently serves as a negative feedback regulation of PARP.  
           [0009]    PARP activity in plant cells was first demonstrated by examining the incorporation of  3 H from labelled NAD +  into the nuclei of root tip cells (Payne et al., 1976; Willmitzer and Wagner, 1982). The enzymatic activity was also partially purified from maize seedlings and found to be associated with a protein of an apparent molecular mass of 113 kDa, suggesting that the plant PARP might be similar to the enzyme from animals (Chen et al., 1994; O&#39;Farrell, 1995).  
           [0010]    cDNAs corresponding to PARP proteins have isolated from several species including mammals, chicken, Xenopus, insects and  Caenorhabditis elegans.  Chen et al. (1994) have reported PARP activity in maize nuclei and associated this enzymatic activity with the presence of an approximately 114 kDa protein present in an extract of maize nuclei. O&#39;Farrel (1995) reported that RT-PCR-amplification on RNA isolated from maize (using degenerate primers based on the most highly conserved sequences) resulted in a 300 bp fragment, showing 60% identity at the amino acid level with the human PARP protein. Lepiniec et al. (1995) have isolated and cloned a full length cDNA from  Arabidopsis thaliana  encoding a 72 kDa protein with high similarity to the catalytic domain of vertebrate PARP. The N-terminal domain of the protein does not reveal any sequence similarity with the corresponding domain of PARP from vertebrates but is composed of four stretches of amino acids (named A1, A2, B and C) showing similarity to the N-terminus of a number of nuclear and DNA binding proteins. The predicted secondary structure of A1 and A2 was a helix-loop-helix structure.  
           [0011]    The Genbank database contains the sequences of two cDNAS from  Zea mays  for which the amino acid sequence of the translation products has either homology to the conventional PARP proteins (AJ222589) or to the non-conventional PARP proteins, as identified in Arabidopsis (AJ222588)  
           [0012]    The function(s) of PARP and poly-ADP ribosylation in eukaryotic cells is (are) not completely clear. PARP is involved or believed to be involved either directly or indirectly in a number of cellular processes such as DNA repair, replication and recombination, in cell division and cell differentiation or in the signalling pathways that sense alterations in the integrity of the genome. As PARP activity may significantly reduce the cellular NAD +  pool, it has also been suggested that the enzyme may play a critical role in programmed cell death (Heller et al., 1995; Zhang et al., 1994). Further, it has been suggested that nicotinamide resulting from NAD +  hydrolysis or the products of the turn-over of poly-ADP-ribose by poly-ADP-ribose glycohydrolase may be stress response signals in eukaryotes.  
           [0013]    The information currently available on the biological function of plant PARP has come from experiments involving PARP inhibitors suggesting an in vivo role in the prevention of homologous recombination at sites of DNA damage as rates of homologous intrachromosomal recombination in tobacco are increased after application of 3-aminobenzamide (3ABA) (Puchta et al., 1995). Furthermore, application of PARP inhibitors, such as 3ABA, nicotinamide, and 6(5H)-phenasthridinone, to differentiating cells of Zinnia or of  Helianthus tuberosum  has been shown to prevent development of tracheary elements (Hawkins and Phillips, 1983; Phillips and Hawkins, 1985; Shoji et al., 1997; Sugiyama et al., 1995), which is considered to be an example of programmed cell death in plants.  
           [0014]    PCT application WO97/06267 describes the use of PARP inhibitors to improve the transformation (qualitatively or quantitatively) of eukaryotic cells, particularly plant cells.  
           [0015]    Lazebnik et al. (1994) identified a protease with properties similar to the interleukin 1-β-converting enzyme capable of cleaving PARP, which is an early event in apoptosis of animal cells.  
           [0016]    Kuepper et al. (1990) and Molinette et aL (1993) have described the overproduction of the 46 kDa human PARP DNA-binding domain and various mutant forms thereof, in transfected CV-1 monkey cells or human fibroblasts and have demonstrated the trans-dominant inhibition of resident PARP activity and the consequent block of base excision DNA repair in these cells.  
           [0017]    Ding et al. (1992), and Smulson et al. (1995) have described depletion of PARP by antisense RNA expression in mammalian cells and observed a delay in DNA strand break joining, and inhibition of differentiation of 3T3-L1 preadipocytes.  
           [0018]    Ménissier de Murcia et al., (1997) and Wang et al. (1995, 1997) have generated transgenic “knock-out” mice mutated in the PARP gene, indicating that PARP is not an essential protein. Cells of PARP-deficient mice are, however, more sensitive to DNA damage and differ from normal cells of animals in some aspects of induced cell death (Heller et al., 1995).  
         SUMMARY AND OBJECTS OF THE INVENTION  
         [0019]    The invention provides a method for modulating programmed cell death in a eukaryotic cell, comprising reducing the functional level of the total PARP activity in a eukaryotic cell 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 to reduce expression of the endogeneous PARP genes, to reduce the apparent activity of the proteins encoded by the endogenous PARP genes or to alter the nucleotide sequence of the endogenous PARP genes.  
           [0020]    The invention also provides a method for modulating programmed cell death in a eukaryotic cell, comprising introducing a first and a second PCD modulating chimeric gene in a eukaryotic cell, preferably a plant cell, wherein the first PCD modulating chimeric gene comprises the following operably linked DNA regions: a promoter, operative in a eukaryotic cell; a DNA region, which when transcribed yields a RNA molecule which is either capable of reducing the functional level of a Zn-finger containing PARP protein of the ZAP class; or is capable of being translated into a peptide or protein which when expressed reduces the functional level of a PARP protein of ZAP class and a DNA region involved in transcription termination and polyadenylation  
           [0021]    and wherein the second PCD modulating chimeric gene comprises the following operably linked DNA regions :a promoter, operative in the eukaryotic cell; a DNA region, which when transcribed yields a RNA molecule which is either capable of reducing the functional level of a PARP protein of the NAP class; or capable of being translated into a peptide or protein which when expressed reduces the functional level of a PARP protein of the NAP class, and a DNA region involved in transcription termination and polyadenylation; and wherein the total apparent PARP activity in the eukaryotic cell is reduced significantly, (preferably the total apparent PARP activity is reduced from about 75% to about 90% of the normal apparent PARP activity in the eukaryotic cell, and the eukaryotic cell is protected against programmed cell death) or almost completely (preferably the total apparent PARP activity is reduced from about 90% to about 100% of the normal apparent PARP activity in the eukaryotic cell, and the cell is killed by programmed cell death).  
           [0022]    Preferably the first transcribed DNA region or the second transcribed DNA region or both, comprise a nucleotide sequence of at least about 100 nucleotides with 75% identity to the sense DNA strand of an endogenous PARP gene of the ZAP or the NAP class, and encode a sense RNA molecule is capable of reducing the expression of the endogenous PARP gene of the ZAP or the NAP class.  
           [0023]    In an alternative method for modulating programmed cell death, provided by the invention, the first transcribed DNA region or the second transcribed DNA region or both, comprise a nucleotide sequence of at least about 100 nucleotides with 75% identity to the complement of the sense DNA strand of an endogenous PARP gene of the ZAP or the NAP class, and encode RNA molecule is capable of reducing the expression of said endogenous PARP gene of the ZAP or the NAP class.  
           [0024]    In yet an alternative method for modulating programmed cell death, provided by the invention, the first and/or second transcribed DNA region encodes a RNA molecule comprising a sense nucleotide sequence of at least about 100 nucleotides with 75% identity to the mRNA resulting from transcription of an endogenous PARP gene of the ZAP or the NAP class and the RNA molecule further comprising an antisense nucleotide sequence of at least about 100 nucleotides with 75% identity to the complement of the mRNA resulting from transcription of the endogenous PARP gene of the ZAP or the NAP class, wherein the sense and antisense nucleotide sequence are capable of forming a double stranded RNA region, and wherein that RNA molecule is capable of reducing the expression of the endogenous PARP gene of the ZAP or the NAP class.  
           [0025]    In a further alternative method for modulating programmed cell death, provided by the invention, the first and/or second transcribed DNA region encodes a dominant negative PARP mutant capable of reducing the apparent activity of the PARP protein encoded by an endogenous PARP gene of the ZAP or the NAP class, preferably comprising amino acid sequence selected from the amino acid sequence of SEQ ID No 4 from amino acid 1 to 159 or the amino acid sequence of SEQ ID No 6 from amino acid 1 to 138 or comprising an amino acid sequence selected from the amino acid sequence of SEQ ID No 2 from amino acid 1 to 370, the amino acid sequence of SEQ ID No 11 from amino acid 1 to 98, or the amino acid sequence of SEQ ID No 2 from amino acid 1 to 370 wherein the amino acid sequence from amino acid 1 to 88 is replaced by the amino acid sequence of SEQ ID No 11.  
           [0026]    The promoter of the first and second chimeric PCD modulating genes, or both, may be a tissue specific or inducible promoter such as a promoter is selected from a fungus-responsive promoter, a nematode-responsive promoter, an anther-selective promoter, a stigma-selective promoter, a dehiscence-zone selective promoter.  
           [0027]    The invention also provides a method for modulating programmed cell death in a plant cell, comprising introduction of a PCD modulating chimeric gene in said plant cell, wherein the PCD modulating chimeric gene comprises the following operably linked DNA regions: a plant-expressible promoter, a DNA region, which when transcribed yields a RNA molecule, which is either capable of reducing the expression of endogenous PARP genes; or is capable of being translated into a peptide or protein which when expressed reduces the apparent PARP activity in the plant cell, and a DNA region involved in transcription termination and polyadenylation, wherein the total apparent PARP activity in the plant cell is reduced from about 75% to about 100% of the normal apparent PARP activity in the plant cell.  
           [0028]    It is another objective of the invention to provide the first and second chimeric PCD modulating gene as well as a eucaryotic cell, particularly a plant cell comprising the first and second chimeric PCD modulating gene and non-human eukaryotic organisms, particularly plants comprising such cells.  
           [0029]    Finally, the invention also provides an isolated DNA sequence comprising the nucleotide sequence of SEQ ID No 1 from the nucleotide at position 113 to the nucleotide at position 3022, an isolated DNA sequence comprising the nucleotide sequence of SEQ ID No 10 from the nucleotide at position 81 to the nucleotide at position 3020 and an isolated DNA sequence comprising the nucleotide sequence of SEQ ID No 3 from the nucleotide at position 107 to the nucleotide at position 2068.  
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0030]    [0030]FIG. 1. The deduced N-terminal amino acid sequences of plant poly(ADP-ribose) polymerases.  
         [0031]    (A) Alignment of the sequences upstream of the NAD + -binding domain found in  Arabidopsis thaliana  APP (A.th. APP; EMBL accession number Z48243; SEQ ID No 6) and the maize homolog NAP (Z.m. NAP; EMBL accession number AJ222588; SEQ ID No 4). The domain division shown is as previously proposed (Lepiniec et al., 1995). The nuclear localization signal (NLS) located in the B domain is indicated by the bracket. The sequence of the B domain is not very well conserved between dicotyledonous and monocotyledonous plants. The C domain is probably comparable in function to the automodification domain of PARP from animals. The imperfect repeats, A1 and A2, are also present in maize NAP. To illustrate the internally imperfect two-fold symmetry within the repeat sequence, the properties of amino acid residues are highlighted below the sequences as follows: filled-in circles, hydrophobic residue; open circle, glycine; (+), positively charged residue; (−), negatively charged residue; wavy line, any residue. The axis of symmetry is indicated by the vertical arrowhead and arrowhead lines mark the regions with the inverted repetition of amino acid side chain properties.  
         [0032]    (B) Alignment of the DNA-binding and auto-catalytic domains of mouse PARP and maize ZAP. Zn-finger-containing maize ZAP1 and ZAP2 (partial cDNA found by the 5′RACE PCR analysis) are indicated as Z.m. ZAP (EMBL accession number AJ222589; SEQ ID No 2) and Z.m. ZAP(race) (SEQ ID No 11 from amino acid at position 1 to amino acid at position 98), respectively, and the mouse PARP, M.m. ADPRT (Swissprot accession number P11103). The Zn-fingers and bipartite NLS of the mouse enzyme are indicated by brackets, the Caspase 3 cleavage site by the asterisk, and the putative NLS in the ZAP protein by the bracket in bold below the maize sequence. The amino acid residues that are conserved in all sequences are boxed; amino acid residues with similar physico-chemical properties are shaded with the uppermost sequence as a reference.  
         [0033]    [0033]FIG. 2. Comparison of the NAD+-binding domain of mouse PARP and plant PARP proteins. The range of the “PARP signature” is indicated above the sequences. Names and sequence alignment are as in FIG. 1.  
         [0034]    [0034]FIG. 3. Estimation of the gene copy number and transcript size for the nap and zap genes.  
         [0035]    (A) and (B) Maize genomic DNA of variety LG2080 digested with the indicated restriction endonucleases, resolved by agarose gel electrophoresis, blotted, and hybridized with radioactively labelled DNA probes prepared from the 5′ domains of the nap and zap cDNA, which do not encode the NAD + -binding domain. The hybridization pattern obtained with the nap probe (A) is simple and indicates a single nap gene in the maize genome. As can be seen from the hybridization pattern (B), there might be at least two zap genes. To determine the size of the transcripts encoded by the zap and nap genes, approximately 1 μg of poly(A) +  RNA extracted from roots (lane  1 ) and shoots (lane  2 ) of 6-day-old seedlings were resolved on an agarose gel after denaturation with glyoxal, blotted, and hybridized with nap (C) and zap (D)  32 P-labelled cDNA.  33 P 5′ end-labelled BstEll fragments of λDNA were used as a molecular weight markers in both DNA and RNA gel blot experiments; their positions are indicated in kb to the left of each panel.  
         [0036]    [0036]FIG. 4. Analysis of APP expression in yeast.  
         [0037]    (A) Schematic drawing of the expression cassette in pV8SPA. The expression of the app cDNA is driven by a chimeric yeast promoter, which consists of the minimal TATA box-containing promoter region of the cycl gene (CYC1) and an upstream activating promoter region of the ga110 gene (GAL10), the latter providing promoter activation by galactose. Downstream regulatory sequences are derived from the gene encoding phosphoglycerol kinase (3PGK) (Kuge and Jones, 1994). The app-coding region is drawn with a division in putative domains as proposed earlier (Lepiniec et al., 1995): A1 and A2 correspond to imperfect 27-amino acid repeats, in between which there is a sequence (B domain), rich in positively charged amino acids and resembling the DNA-binding domains of a number of DNA-binding proteins. The amino acid sequence of the B domain is shown below the map and the stretch of arginine and lysine residues, which may function as an NLS is drawn in bold. Methionine residues (M 1 , M 72 ), which may function as translation initiation codons, are indicated above the map. The C domain is rich in glutamic acid residues, resembling in its composition, but not in its sequence, the auto-modification domain of PARP from animals.  
         [0038]    (B) Immunoblot (Western blot) and Northern blot analyses of the DY (pYeDP1/8-2) and DY(pV8SPA) strains, indicated as (vector) and (app), respectively. Strains were grown in SDC medium supplemented with glucose (GLU), galactose (GAL), galactose and 3 mM of 3ABA (GAL+3ABA), or galactose and 5 mM nicotinamide (GAL+NIC). Total RNA or total protein were extracted from the same cultures. Ten micrograms of total protein were fractionated by electrophoresis on 10% SDS-PAGE, electroblotted, and probed with anti-APP antisera. Five micrograms of total RNA were resolved by electrophoresis on an 1.5% agarose gel, blotted onto nylon membranes, and hybridized with  32 P-labeled DNA fragments derived from the app cDNA. Positions of the molecular weight marker bands are indicated to the left in kilobases (kb) and kilodalton (kDa).  
         [0039]    [0039]FIG. 5. Poly(ADP-ribose) polymerase activity of the APP protein.  
         [0040]    (A) The total protein extracts were prepared from DY(pYeDP1/8-2) grown on SDC with 2% galactose (vector GAL) and DY(pV8SPA) grown either on SDC with 2% glucose (app GLU), on SDC with 2% galactose (app GAL), or on SDC with 2% galactose and 3 mM 3ABA (app GAL+3ABA). To detect the synthesis of the poly(ADP-ribose) in these extracts, samples were incubated with  32 P-NAD +  for 40 min at room temperature. Two control reactions were performed: 100 ng of the purified human PARP were incubated either in a reaction buffer alone (PARP) (lane  5 ), or with protein extract made from DY(pYeDP1/8-2) culture grown on glucose (vector GLU+PARP) (lane  6 ). The autoradiograph obtained after exposure of the dried gel to X-Omat Kodak film is shown. ORi corresponds to the beginning of the sequencing gel.  
         [0041]    (B) Stimulation of poly(ADP-ribose) synthesis by DNA in protein extracts from DY(pV8SPA). Amounts of sonicated salmon sperm DNA added to the nucleic acid depleted yeast extracts are indicated in μg ml −1 . The synthesis of the poly(ADP-ribose) is blocked by 3ABA, which was added in one of the reactions at a concentration of 3 mM (lane  5 ). To ensure the maximal recovery of the poly(ADP-ribose), 20 μg of glycogen were included as a carrier during precipitation steps; this, as can be seen, however resulted in high carry-over of the unincorporated label.  
         [0042]    [0042]FIG. 6. Schematic representation of the T-DNA vectors comprising the PCD modulating chimeric genes of the invention. P35S: CaMV35S promoter; L: cab22 leader; ZAP; coding region of a PARP gene of the ZAP class; 5′ZAP: N-terminal part of the coding regon of a PARP gene of the ZAP class in inverted orientation; 3′ 35S: CaMV35S 3′ end transcription termination signal and polyadenylation signal; pACT2: promoter region of the actin gene; pNOS; nopaline synthase gene promoter; gat: gentamycin acetyl transferase; bar: phosphinotricin acetyl transferase; 3′NOS: 3′ end transcription termination signal and polyadenylation signal of nopaline synthase gene; APP: coding region of a PARP gene of the NAP class; 5′APP: N-terminal part of the coding regon of a PARP gene of the NAP class in inverted orientation; LB: left T-DNA border; RB: right T-DNA border; pTA29: tapetum specific promoter, pNTP303: pollen specific promoter.  
     
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS  
       [0043]    For the purpose of the invention, the term “plant-expressible promoter” means a promoter which is capable of driving transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, e.g., certain promoters of viral or bacterial origin such as the CaMV35S or the T-DNA gene promoters.  
         [0044]    The term “expression of a gene” refers to the process wherein a DNA region under control of regulatory regions, particularly the promoter, is transcribed into an RNA which is biologically active i.e., which is either capable of interaction with another nucleic acid or protein or which is capable of being translated into a biologically active polypeptide or protein. A gene is said to encode an RNA when the end product of the expression of the gene is biologically active RNA, such as e.g. an antisense RNA or a 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.  
         [0045]    The term “gene” means any DNA fragment comprising a DNA region (the “transcribed DNA region”) that is transcribed into a RNA molecule (e.g., a mRNA) in a cell under control of suitable regulatory regions, e.g., a plant-expressible promoter. A gene may thus comprise several operably linked DNA fragments such as a promoter, a 5′ leader sequence, a coding region, and a 3′ region comprising a polyadenylation site. An endogenous plant gene is a gene which is naturally found in a plant species. A chimeric gene is any gene which is not normally found in a plant species or, alternatively, any gene in which the promoter is not associated in nature with part or all of the transcribed DNA region or with at least one other regulatory regions of the gene.  
         [0046]    As used herein “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein. A chimeric gene comprising a DNA region which is functionally or structurally defined, may comprise additional DNA regions etc.  
         [0047]    The invention is based on the one hand on the finding that eukaryotic cells, particularly plant cells, quite particularly Zea mays cells contain simultaneously at least two functional major PARP protein isoforms(classes) which differ in size and amino-acid sequence, yet are both capable of binding DNA, particularly DNA with single stranded breaks, and both have poly-ADP ribosylation activity. On the other hand, the inventors have realized that programmed cell death in eukaryotes, particularly in plants, can be modulated by altering the expression level of the PARP genes or by altering the activity of the encoded proteins genetically, and that in order to achieve this goal, the expression of both genes needs to be altered or in the alternative both classes of proteins need to be altered in their activity.  
         [0048]    Thus, the invention relates to modulation i.e. the enhancement or the inhibition- of programmed cell death or apoptosis in eukaryotic cells, preferably plant cells, by altering the level of expression of PARP genes, or by altering the activity or apparent activity of PARP proteins in that eukaryotic cell. Conveniently, the level of expression of PARP genes or the activity of PARP proteins is controlled genetically by introduction of PCD modulating chimeric genes altering the expression of PARP genes and/or by introduction of PCD modulating chimeric genes altering the apparent activity of the PARP proteins and/or by alteration of the endogenous PARP encoding genes.  
         [0049]    As used herein, “enhanced PCD” with regard to specified cells, refers to the death of those cells, provoked by the methods of the invention, whereby the killed cells were not destined to undergo PCD when compared to similar cells of a normal plant not modified by the methods of the invention, under similar conditions.  
         [0050]    “Inhibited PCD” with regard to specified cells is to be understood as the process whereby a larger fraction of those cells or groups of cells, which would normally (without the intervention by the methods of this invention) undergo programmed cell death under particular conditions, remain alive under those conditions.  
         [0051]    The expression of the introduced PCD modulating chimeric genes or of the modified endogenous genes will thus influence the functional level of PARP protein, and indirectly interfere with programmed cell death. A moderate decrease in the functional level of PARP proteins leads to an inhibition of programmed cell death, particularly to prevention of programmed cell death, while a severe decrease in the functional level of the PARP proteins leads to induction of programmed cell death. In accordance with the invention, it is preferred that in order to inhibit or prevent programmed cell death in a eukaryotic cell, particularly in a plant cell, the combined level of both PARP proteins and/or their activity or apparent activity is decreased significantly, however avoiding that DNA repair (governed directly or indirectly by PARP) is inhibited in such a way that the cells wherein the function of the PARP proteins is inhibited cannot recover from DNA damage or cannot maintain their genome integrity. Preferably, the level and/or activity of the PARP proteins in the target cells, should be decreased about 75%, preferably about 80%, particularly about 90% of the normal level and/or activity in the target cells so that about 25%, preferably about 20%, particularly about 10% of the normal level and/or acttivity of PARP is retained in the target cells. It is further thought that the decrease in level and/or activity of the PARP proteins should not exceed 95%, preferably not exceed 90% of the normal activity and/or level in the target cells. Methods to determine the content of a specific protein such as the PARP proteins are well known to the person skilled in the art and include, but are not limited to (histochemical) quantification of such proteins using specific antibodies. Methods to quantify PARP activity are also available in the art and include the above-mentioned TUNEL assay (in vivo) or the in vitro assay described Collinge and Althaus (1994) for synthesis of poly (ADP-ribose) (see Examples).  
         [0052]    Also in accordance with the invention, it is preferred that in order to trigger programmed cell death in a eukaryotic cell, particularly in a plant cell, the combined level of both PARP proteins and/or their activity or apparent activity is decreased substantially, preferably reduced almost completely such that the DNA repair and maintenance of the genome integrity are no longer possible. Preferably, the combined level and/or activity of the PARP proteins in the target cells, should be decreased at least about 90%, preferably about 95%, more preferably about 99%, of the normal level and/or activity in the target cells, particularly the PARP activity should be inhibited completely. It is particularly preferred that the functional levels of both classes of PARP proteins seperately are reduced to the mentioned levels.  
         [0053]    For the purpose of the invention, PARP proteins are defined as proteins having poly (ADP-ribose) polymerase activity, preferably comprising the so-called “PARP signature”. The PARP signature is an amino acid sequence which is highly conserved between PARP proteins, defined by de Murcia and Menussier de Murcia (1994) as extending from amino acid at position 858 to the amino acid at position 906 from the  Mus musculus  PARP protein. This domain corresponds to the amino acid sequence from position 817 to 865 of the conventional PARP protein of  Zea mays  (ZAP1; SEQ ID No 2) or to the amino acid sequence from position 827 to 875 of the conventional PARP protein of  Zea mays  (ZAP2; SEQ ID No 11) or to the amino acid sequence from position 500 to 547 of the non-conventional PARP protein of  Zea mays  (SEQ ID No 4) or to the amino acid sequence from position 485 to 532 of the non-conventional PARP protein of Arabidopsis thaliana (SEQ ID No 6). This amino sequence is highly conserved between the different PARP proteins (having about 90% to 100% sequence identity). Particularly conserved is the lysine at position 891 (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) of the PARP protein from  Mus musculus,  which is considered to be involved in the catalytic activity of PARP proteins. Particularly the amino acids at position 865, 866, 893, 898 and 899 of the PARP protein of  Mus musculus  or the corresponding positions for the other sequences are variable. PARP proteins may further comprise an N-terminal DNA binding domain and/or a nuclear localization signal (NLS).  
         [0054]    Currently, two classes of PARP proteins have been described. The first class, as defined herein, comprises the so-called classical Zn-finger containing PARP proteins (ZAP). These proteins range in size from 113-120 kDA and are further characterized by the presence of at least one, preferably two Zn-finger domains located in the N-terminal domain of the protein, particularly located within the about 355 to about 375 first amino acids of the protein. The Zn-fingers are defined as peptide sequences having the sequence CxxCx n HxxC (whereby n may vary from 26 to 30) capable of complexing a Zn atom. Examples of amino acid sequences for PARP proteins from the ZAP class include the sequences which can be found in the PIR protein database with accession number P18493 ( Bos taurus ), P26466 ( Gallus gallus ), P35875 ( Drosophila melanogaster ), P09874 ( Homo sapiens ), P11103 ( Mus musculus ), Q08824 ( Oncorynchus masou ), P27008 ( Rattus norvegicus ), Q11208 ( Sarcophaga peregrina ), P31669 ( Xenopus laevis ) and the currently identified sequences of the ZAP1 and ZAP2 protein from  Zea mays  (SEQ ID No 2/SEQ ID No 11).  
         [0055]    The nucleotide sequence of the corresponding cDNAs can be found in the EMBL database under accession numbers D90073 ( Bos taurus ), X52690 ( Gallus gallus ), D13806 ( Drosophila melanogaster ), M32721 ( Homo sapiens ), X14206 ( Mus musculus ), D13809 ( Oncorynchus masou ), X65496 ( Rattus norvegicus ), D16482 ( Sarcophaga peregrina ), D14667 ( Xenopus laevis ) and in SEQ ID No 1 and 10 ( Zea mays ).  
         [0056]    The second class as defined herein, comprises the so-called non-classical PARP proteins (NAP). These proteins are smaller (72-73 kDa) and are further characterized by the absence of a Zn-finger domain at the N-terminus of the protein, and by the presence of an N-terminal domain comprising stretches of amino acids having similarity with DNA binding proteins. Preferably, PARP protein of these class comprise at least one amino acid sequence of about 30 to 32 amino acids which comprise the sequence R G x x x x G x K x x x x x R L (amino acids are represented in the standard one-letter code, whereby x stands for 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 x x R x x L x x R G L x x x G V K x x L V x R L x x A I (SEQ ID No 8) (the so-called A1 domain) or at least 1 amino acid sequence of about 32 amino acids having the sequence G M x x x E L x x x A x x R G x x x x G x K K D x x R L x x (SEQ ID No 9) (the so-called A2 domain) or both. Particularly, the A1 and A2 domain are capable of forming a helix-loop-helix structure. These PARP proteins may further comprise a basic “B” domain (K/R rich amino acid sequence of about 35 to about 56 amino acids, involved in targeting the protein to the nucleus) and/or a an acid “C” domain (D/E rich amino acid sequence of about 36 amino acids). Examples of protein sequences from the NAP class include the APP protein from  Arabidopsis thaliana  (accessible from PIR protein database under accession number Q11207; SEQ ID No 6) and the NAP protein from  Zea  mays (SEQ ID No 4). The sequence of the corresponding cDNAs can be found in the EMBL database under accession number Z48243 (SEQ ID No 5) and in SEQ ID No 3. That the second class of PARP proteins are indeed functional PARP proteins, i.e. are capable of catalyzing DNA dependent poly(ADP-ribose) polymerization has been demonstrated by the inventors (see Example 2).  
         [0057]    The inventors have further demonstrated that eukaryotic cells, particularly plant cells express simultaneously genes encoding PARP proteins from both classes.  
         [0058]    It is clear that for the purpose of the invention, other genes or cDNAs encoding PARP proteins from both classes as defined, or parts thereof, can be isolated from other eukaryotic species or varieties, particularly from other plant species or varieties. These PARP genes or cDNAs can be isolated e.g. by Southern hybridization (either low-stringency or high-stringency hybridization depending on the relation between the species from which one intends to isolate the PARP gene and the species from which the probe was ultimately derived) using as probes DNA fragments with the nucleotide sequence of the above mentioned PARP genes or cDNAs, or parts thereof, preferably parts which are conserved such as a gene fragment comprising the nucleotide sequence encoding the PARP signature mentioned supra. The nucleotide sequences corresponding to the PARP signature from the PARP proteins encoded by plant genes are the nucleotide sequence of SEQ ID No 1 from nucleotide 2558 to 2704 or the nucleotide sequence of SEQ ID No 3 from nucleotide 1595 to 1747 or the nucleotide sequence of SEQ ID No 5 from nucleotide 1575 to 1724. If a discrimination is to be made between the classes of PARP genes, parts of the PARP genes which are specific for the class, such as the N-terminal domains preceding the catalytic domain or parts thereof, should preferably be used.  
         [0059]    Alternatively, the genes or cDNAs encoding PARP proteins or parts thereof, can also be isolated by PCR-amplification using appropriate primers such as the degenerated primers with the nucleotide sequence corresponding to the sequences indicated in SEQ ID No 13, SEQ ID No 14, or primers with the nucleotide sequence corresponding to the sequences indicated in SEQ ID No 15 to 20. However, it is clear that the person skilled in the art can design alternative oligonucleotides for use in PCR or can use oligonucleotides comprising a nucleotide sequence of at least 20, preferably at least about 30, particularly at least about 50, consecutive nucleotides of any of the PARP genes to isolate the genes or part thererof by PCR amplification.  
         [0060]    It is clear that a combination of these techniques, or other techniques (including e.g. RACE-PCR), available to the skilled artisan to isolate genes or cDNAs on the basis of partial fragments and their nucleotide sequence, e.g. obtained by PCR amplification, can be used to isolate PARP genes, or parts thereof, suitable for use in the methods of the invention.  
         [0061]    Moreover, PARP genes, encoding PARP proteins wherein some of the amino acids have been exchanged for other, chemically similar, amino acids (so-called conservative substitutions), or synthetic PARP genes (which encode similar proteins as natural PARP genes but with a different nucleotide sequence, based on the degeneracy of the genetic code) and parts thereof are also suited for the methods of the invention.  
         [0062]    In one aspect of the invention, PCD in eukaryotic cells, particularly in plant cells, is inhibited by a moderate decrease in the functional level of PARP in those eukaryotic cells.  
         [0063]    In one embodiment of this first aspect of the invention, the functional level of PARP in eukaryotic cells, particularly in plant cells is reduced by introduction of at least one PCD modulating chimeric gene in those cells, comprising a promoter capable of directing transcription in these cells, preferably a plant-expressible promoter, and a functional 3′ transcription termination and polyadenylation region, operably linked to a DNA region which when transcribed yields a biologically active RNA molecule which is capable of decreasing the functional level of the endogenous PARP activity encoded by both classes of PARP genes.  
         [0064]    In a preferred embodiment, at least two such PCD modulating chimeric genes are introduced in the cells, whereby the biologically active RNA encoded by the first PCD modulating chimeric gene decreases the functional level of the endogenous PARP activity encoded by the genes of the NAP class, and whereby the biologically active RNA encoded by the second PCD modulating chimeric gene decreases the functional level of the endogenous PARP activity encoded by the genes of the ZAP class, so that the combined PARP activity is moderately decreased.  
         [0065]    In a particularly preferred embodiment, the PCD modulating chimeric genes decrease the functional level of the endogenous PARP activity by reducing the level of expression of the endogenous PARP genes. To this end, the transcribed DNA region encodes a biologically active RNA which decreases the mRNAs encoding NAP and ZAP class PARP proteins, that is available for translation. This can be achieved through techniques such as antisense RNA, co-suppression or ribozyme action.  
         [0066]    As used herein, “co-suppression” refers to the process of transcriptional and/or post-transcriptional suppression of RNA accumulation in a sequence specific manner, resulting in the suppression of expression of homologous endogenous genes or transgenes.  
         [0067]    Suppressing the expression of the endogenous PARP genes can thus be achieved by introduction of a transgene comprising a strong promoter operably linked to a DNA region whereby the resulting transcribed RNA is a sense RNA or an antisense RNA comprising a nucleotide sequence which has at least 75%, preferably at least 80%, particularly at least 85%, more particularly at least 90%, especially at least 95% sequence identity with or is identical to the coding or transcribed DNA sequence (sense) or to the complement (antisense) of part of the PARP gene whose expression is to be suppressed. Preferably, the transcribed DNA region does not code for a functional protein. Particularly, the transcribed region does not code for a protein. Further, the nucleotide sequence of the sense or antisense region should preferably be at least about 100 nucleotides in length, more preferably at least about 250 nucleotides, particularly at least about 500 nucleotides but may extend to the full length of the coding region of the gene whose expression is to be reduced.  
         [0068]    For the purpose of this invention the “sequence identity” of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (×100) divided by the number of positions compared. A gap, i.e. a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues. The alignment of the two sequences is performed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983) using a window-size of 20 nucleotides or amino acids, a word length of 2 amino acids, and a gap penalty of 4. Computer-assisted analysis and interpretation of sequence data, including sequence alignment as described above, can be conveniently performed using commercially available software packages such as the programs of the lntelligenetics˜ Suite (Intelligenetics Inc., Calif.).  
         [0069]    It will be clear to a skilled artisan that one or more sense or antisense PCD modulating chimeric genes can be used to achieve the goals of the first aspect of the invention. When one sense or antisense PCD modulating chimeric gene is used, this gene must be capable of simultaneously reducing the expression of PARP genes of both classes. This can e.g. be achieved by choosing the transcribed region of the chimeric gene in such a way that expression of both classes of genes can be regulated by one sense or antisense RNA, i.e. by choosing target regions corresponding to the highest homology DNA region of the PARP genes of both classes and incorporating a sense or antisense transcribed DNA region corresponding to both target regions, conform to the conditions described above for sense and antisense RNA. Alternatively, different sense or antisense RNA regions, each specific for regulating the expression of one class of PARP genes, can be combined into one RNA molecule, encoded by one transcribed region of one PCD modulating chimeric gene. Obviously, the different sense or antisense RNA regions specific for regulating the expression of one class of PARP genes can be introduced as separate PCD modulating chimeric genes.  
         [0070]    Preferred sense and antisense encoding transcribed regions comprise a nucleotide sequence corresponding (with sequence identity constraints as indicated above) to a sequence of at least about 100 consecutive nucleotides selected from the N-terminal domains of the PARP genes, preferably corresponding to a sequence of at least about 100 consecutive nucleotides selected from the sequence of SEQ ID No 1 from nucleotide position 113 to 1189, the sequence of SEQ ID No 3 from nucleotide position 107 to 583, the sequence of SEQ ID No 5 from nucleotide position 131 to 542 or the sequence of SEQ ID No 10 from nucleotide position 81 to 1180. However, it is clear that sense or antisense encoding transcribed regions can be used comprising a sequence corresponding to the complete sequence of the N-terminal domain of the PARP genes, or even to complete sequence of the PARP genes, particularly the protein-encoding region thereof. Further preferred are sense and antisense encoding transcribed regions which comprise a nucleotide sequence corresponding (with sequence identity constraints as indicated above) to a sequence of at least about 100 consecutive nucleotides selected from the C-terminal catalytic domains of the PARP genes, preferably a sequence of at least 100 nucleotides encompassing the PARP-signature encoding nucleotide sequences, particularly the PARP-signature encoding nucleotide sequences indicated supra. Again, it is clear that sense or antisense encoding transcribed regions can be used comprising a sequence corresponding to the complete sequence of the C-terminal domain of the PARP genes.  
         [0071]    In another particularly preferred embodiment, the PCD modulating chimeric genes decrease the functional level of the endogenous PARP activity by reducing the level of apparent activity of the endogenous PARPs of both classes. To this end, the transcribed DNA region encodes a biologically active RNA which is translated into a protein or inhibiting NAP or ZAP class PARP proteins or both, such as inactivating antibodies or dominant negative PARP mutants.  
         [0072]    “Inactivating antibodies of PARP proteins” are antibodies or parts thereof which specifically bind at least to some epitopes of PARP proteins, such as the epitope covering part of the ZN finger II from position 111-118 in ZAP1 or a corresponding peptide in ZAP2, and which inhibit the activity of the target protein.  
         [0073]    “Dominant negative PARP mutants” as used herein, are proteins or peptides comprising at least part of a PARP protein (or a variant thereof), preferably a PARP protein endogenous to the eukaryotic target host cell, which have no PARP activity, and which have an inhibitory effect on the activity of the endogenous PARP proteins when expressed in that host cell. Preferred dominant negative PARP mutants are proteins comprising or consisting of a functional DNA binding domain (or a variant therof) without a catalytic domain (such as the N-terminal Zn-finger containing domain of about 355 to about 375 amino acids of a PARP of the ZAP class, particularly a DNA binding protein domain comprising the amino acid sequence of SEQ ID No 2 from amino acid 1 to 370 or a DNA binding protein domain comprising the amino acid sequence of SEQ ID No 11 from amino acid 1 to 98, or a DNA binding protein domain comprising the amino acid sequence of SEQ ID No 2 from amino acid 1 to 370 wherein the amino acid sequence from amino acid 1 to 88 is replaced by the amino acid sequence of SEQ ID No 11 from amino acid at position 1 to the amino acid at position 98, or such as the N-terminal DNA binding protein domain of about 135 to 160 amino acids of a PARP of the NAP class, particularly a DNA binding protein domain comprising the amino acid sequence of SEQ ID No 4 from amino acid 1 to 159 or a DNA binding protein domain comprising the amino acid sequence of SEQ ID No 6 from amino acid 1 to 138) or without a functional catalytic domain (such as inactive PARP mutants, mutated in the so-called PARP signature, particularly mutated at the conserved lysine of position 850 of SEQ ID No 2, position 532 of SEQ ID No 4, position 517 of SEQ ID No 6). Preferably, dominant negative PARP mutants should retain their DNA binding activity. Dominant negative PARP mutants can be fused to a carrier protein, such as a β-glucuronidase (SEQ ID No 12).  
         [0074]    Again, one or more PCD modulating genes encoding one or more dominant negative PARP mutants can be used to achieve the goals of the first aspect of the invention. When one PCD modulating chimeric gene is used, this gene must be capable of simultaneously reducing the expression of PARP genes of both classes.  
         [0075]    In another embodiment of the first aspect of the invention, the functional level of PARP in eukaryotic cells, particularly in plant cells is reduced by modification of the nucleotide sequence of the endogenous PARP genes in those cells so that the encoded mutant PARP proteins retain about 10% of their activity. Methods to achieve such a modification of endogenous PARP genes include homologous recombination to exchange the endogenous PARP genes for mutant PARP genes e.g. by the methods described in U.S. Pat. No. 5,527,695. In a preferred embodiment such site-directed modification of the nucleotide sequence of the endogenous PARP genes is achieved by introduction of chimeric DNA/RNA oligonucleotides as described in WO 96/22364 or U.S. Pat. No. 5,565,350.  
         [0076]    In another aspect of the invention, programmed death of eukaryotic cells, preferably selected cells, particularly selected plant cells is enhanced by a severe decrease in the functional level of PARP, preferably reduced almost completely, such that the DNA repair and maintenance of the genome integrity are no longer possible.  
         [0077]    In one embodiment of this aspect of the invention, the functional level of PARP in eukaryotic cells, particularly in plant cells is reduced severely, particularly abolished almost completely, by introduction of at least one PCD modulating chimeric gene in those cells, comprising a promoter capable of directing transcription in these cells, preferably a plant-expressible promoter, and a functional 3′ transcription termination and polyadenylation region, operably linked to a DNA region which when transcribed yields a biologically active RNA molecule which is capable of decreasing the functional level of the endogenous PARP activity encoded by both classes of PARP genes.  
         [0078]    In a preferred embodiment of the second aspect of the invention, at least two such PCD modulating chimeric genes are introduced in the cells, whereby the biologically active RNA encoded by the first PCD modulating chimeric gene decreases the functional level of the endogenous PARP activity encoded by the genes of the NAP class, and whereby the biologically active RNA encoded by the second PCD modulating chimeric gene decreases the functional level of the endogenous PARP activity encoded by the genes of the ZAP class, so that the combined PARP activity is severely decreased, particularly almost completely eliminated.  
         [0079]    As mentioned for the first aspect of this invention, the transcribed regions of the PCD modulating chimeric genes encode biologically active RNA, which can interfere with the expression of the endogenous PARP genes (e.g. through antisense action, co-suppression or ribozyme action) or the biologically active RNA can be further translated into a peptide or protein, capable of inhibiting the PARP proteins of the NAP and ZAP class, such as inactivating antibodies or dominant negative PARP mutants.  
         [0080]    In a particularly preferred embodiment of the second aspect of the invention, the transcribed region of the PCD modulating chimeric genes (PCD enhancing chimeric genes) codes for a biologically active RNA which comprises at least one RNA region (preferably of at least about 100 nucleotides in length) classifying according to the above mentioned criteria as a sense RNA for at least one of the endogenous PARP genes, and at least one other RNA region (preferably of at least about 100 nucleotides in length), classifying according to the above mentioned criteria as an antisense RNA for at least one of the endogenous PARP genes, whereby the antisense and sense RNA region are capable of combining into a double stranded RNA region (preferably over a distance of at least about 100 nucleotides). In an especially 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 an eukaryotic cell or organism, preferably a plant cell or plant.  
         [0081]    It is clear that the different embodiments for the transcribed DNA regions of the chimeric PCD modulating genes of the invention can be used in various combinations to arrive at the goals of the invention. E.g. a first chimeric PCD modulating gene may encode a sense RNA designed to reduce the expression of an endogenous PARP gene of the ZAP class, while the second chimeric PCD modulating gene may encode a dominant negative PARP mutant designed to reduce the expression of an endogenous PARP gene of the NAP class.  
         [0082]    Whether the introduction of PCD modulating chimeric genes into eukaryotic cells will ultimately result in a moderately reduced or a severally reduced functional level of combined PARP in those cells—i.e. in inhibited PCD or enhanced PCD—will usually be determined by the expression level (either on transcriptional level or combined transcriptional/tranlational level) of those PCD modulating genes. A major contributing factor to the expression level of the PCD modulating 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 usage of the transcribed region, mRNA stability, presence of consensus sequence around translation initiation site, choice of 5′ and 3′ untranslated RNA regions, presence of PEST sequences, the influence of chromatin structure surrounding the insertion site of a stabile integrated PCD modulating gene, copy number of the introduced PCD modulating genes, etc.) or combinations thereof will also contribute to the ultimate expression level of the PCD modulating gene. In general, it can be assumed that moderate reduction of functional levels of combined PARP can be achieved by PCD modulating genes comprising a relatively weak promoter, while severe reduction of functional levels of combined PARP can be achieved by PCD modulating genes comprising a relatively strong promoter. However, the expression level of a PCD modulating gene comprising a specific promoter and eventually its effect on PCD, can vary as a function of the other contributing factors, as already mentioned.  
         [0083]    For the purpose of particular embodiments of the invention, the PCD modulating chimeric genes may comprise a constitutive promoter, or a promoter which is expressed in all or the majority of the cell types throughout the organism, particularly throughout the plant, such as the promoter regions derived from the T-DNA genes, particularly the opine synthase genes of Agrobacterium Ti- or Ri-plasmids (e.g. nos, ocs promoters), or the promoter regions of viral genes (such as CaMV35S promoters, or variants thereof).  
         [0084]    It may be further be advantageous to control the expression of the PCD modulating gene at will or in response to environmental cues, e.g. by inclusion of an inducible promoter which can be activated by an external stimuli, such as, but not limited to application of chemical compounds (e.g. safeners, herbicides, glucocorticoids), light conditions, exposure to abiotic stress (e.g. wounding, heavy metals, extreme temperatures, salinity or drought) or biotic stress (e.g. pathogen or pest infection including infection by fungi, viruses, bacteria, insects, nematodes, mycoplasms and mycoplasma like organisms etc.). Examples of plant-expressible inducible promoters suitable for the invention are: nematode inducible promoters (such as disclosed in WO 92/21757), fungus inducible promoters (WO 93/19188, WO 96/28561), promoters inducible after application of glucocorticoids such as dexamethasone (), or promoters repressed or activated after application of tetracyclin (Gatz et al. 1988; Weimann et al. 1994)  
         [0085]    In several embodiments of the invention, particularly for the second aspect of the invention (i.e. enhanced PCD), it may be convenient or required to restrict the effect on programmed cell death to a particular subset of the cells of the organism, particularly of the plant, hence the PCD modulating genes may include tissue-specific or cell type-specific promoters. Examples of suitable plant-expressible promoters selectively expressed in particular tissues or cell types are well known in the art and include but are not limited to seed-specific promoters (e.g. WO89/03887), organ-primordia specific promoters (An et al., 1996), stem-specific promoters (Keller et al., 1988), leaf specific promoters (Hudspeth et al., 1989), mesophyl-specific promoters (such as the light-inducible Rubisco promoters), 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 (such as the promoter of the SHOOTMERISTEMLESS (STM) gene, Long et al., 1996), primordia specific promoter (such as the promoter of the Antirrhinum CycD3a gene, Doonan et al., 1998), anther specific promoters (WO 89/10396, WO9213956, WO9213957) stigma-specific promoters (WO 91/02068), dehiscence-zone specific promoters (WO 97/13865), seed-specific promoters (WO 89/03887) etc.  
         [0086]    Preferably the chimeric PCD modulating genes of the invention are accompanied by a marker gene, preferably a chimeric marker gene comprising a marker DNA that is operably linked at its 5′ end to a plant-expressible promoter, preferably a constitutive promoter, such as the CaMV 35S promoter, or a light inducible promoter such as the promoter of the gene encoding the small subunit of Rubisco; and operably linked at its 3′ end to suitable plant transcription 3′ end formation and polyadenylation signals. It is expected that the choice of the marker DNA is not critical, and any suitable marker DNA can be used. For example, a marker DNA can encode a protein that provides a distinguishable “color” to the transformed plant cell, such as the A1 gene (Meyer et al., 1987) or Green Fluorescent Protein (Sheen et al., 1995), can provide herbicide resistance to the transformed plant cell, such as the bar gene, encoding resistance to phosphinothricin (EP 0,242,246), or can provided antibiotic resistance to the transformed cells, such as the aac(6′) gene, encoding resistance to gentamycin (WO94/01560).  
         [0087]    Methods to introduce PCD modulating chimeric genes into eukaryotic cells, particularly methods to transform plant cells are well known in the art, and are believed not to be critical for the methods of the invention. Transformation results in either transient or stably transformed cells (whereby the PCD modulating chimeric genes are stably inserted in the genome of the cell, particularly in the nuclear genome of the cell).  
         [0088]    It is clear that the methods and means described in this invention to alter the programmed cell death in eukaryotic cells and organisms, particularly in plant cells and plants, has several important application possibilities. Inhibition of PCD by the methods and means of the invention, can be used to relieve the stress imposed upon the cells, particularly the plant cells, during transformation and thus to increase transformation efficiency, as described in WO 97/06267. Inhibition of PCD can also be used to improve cell culture of eukaryotic cells, particularly of plant cells. Triggering of PCD in particular cell types using the means and methods of the invention, can be used for methods which call upon the use of a cytotoxin. Since PCD is the “natural” way for cells to die, the use of PCD enhancing chimeric genes of the invention constitutes an improvement over the use of other cytotoxic genes such as RNAse or diptheria toxin genes which lead to cell lysis. Moreover, low-level expression of PCD enhancing genes in cells different than the targeted cells, will lead to a moderate reduction instead of a severe reduction of PARP activity in those cells, thus actually inhibiting PCD in non-target cells.  
         [0089]    For plants, preferred applications of PCD enhancing chimeric genes include, but are not limited to:  
         [0090]    1. the generation of plants protected against fungus infection, whereby the PCD enhancing chimeric gene or genes comprise a fungus-responsive promoter as described in WO 93/19188 or WO 96/28561.  
         [0091]    2. the generation of nematode resistant plants, whereby the PCD enhancing chimeric gene or genes comprise a nematode inducible promoters such as disclosed in WO 92/21757  
         [0092]    3. the generation of male or female sterile plants, whereby the PCD enhancing chimeric gene or genes comprise anther-specific promoters (such as disclosed in WO 89/10396, WO9213956, WO9213957) or stigma-specific promoters (such as disclosed in WO 91/02068)  
         [0093]    4. the generation of plants with improved seed shatter characteristics whereby the PCD enhancing chimeric gene or genes comprise dehiscence zone-specific promoters (such as disclosed in WO 97/13865).  
         [0094]    Although it is clear that the invention can be applied essentially to all plant species and varieties, the invention will be especially suited to alter programmed cell death in plants with a commercial value. Particularly preferred plants to which the invention can be applied are corn, oil seed rape, linseed, wheat, grasses, alfalfa, legumes, a brassica vegetable, tomato, lettuce, cotton, rice, barley, potato, tobacco, sugar beet, sunflower, and ornamental plants such as carnation, chrysanthemum, roses, tulips and the like.  
         [0095]    The obtained transformed plant can be used in a conventional breeding scheme to produce more transformed plants with the same characteristics or to introduce the chimeric cell-division controlling gene of the invention in 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.  
         [0096]    The following non-limiting Examples describe the construction of chimeric apoptosis controlling genes and the use of such genes for the modulation of the programmed cell death in eukaryotic cells and organisms. Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989)  Molecular Cloning: A Laboratory Manual,  Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994)  Current Protocols in Molecular Biology, Current Protocols,  USA. Standard materials and methods for plant molecular work are described in  Plant Molecular Biology Labfax  (1993) by R. D. D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.  
         [0097]    Throughout the description and Examples, reference is made to the following sequences:  
         [0098]    SEQ ID No 1: DNA sequence of the ZAP gene of  Zea mays  (zap1)  
         [0099]    SEQ ID No 2: protein sequence of the ZAP protein of  Zea mays  (ZAP1)  
         [0100]    SEQ ID No 3: DNA sequence of the NAP gene of  Zea mays  (nap)  
         [0101]    SEQ ID No 4: protein sequence of the NAP protein of  Zea mays  (NAP)  
         [0102]    SEQ ID No 5: DNA sequence of the NAP gene of  Arabidopsis thaliana  (app)  
         [0103]    SEQ ID No 6: protein sequence of the NAP protein of  Arabidopsis thaliana  (APP)  
         [0104]    SEQ ID No 7: consensus sequence for the A domain of non-conventional PARP proteins  
         [0105]    SEQ ID No 8: consensus sequence for the A1 domain of non-conventional PARP proteins  
         [0106]    SEQ ID No 9: consensus sequence for the A2 domain of non-conventional PARP proteins  
         [0107]    SEQ ID No 10: DNA sequence of the second ZAP gene of  Zea mays  (Zap2)  
         [0108]    SEQ ID No 11: protein sequence of the ZAP protein of  Zea mays  (ZAP2)  
         [0109]    SEQ ID No 12: amino acid sequence of a fusion protein between the DNA binding domain of APP and the GUS protein  
         [0110]    SEQ ID No 13: degenerated PCR primer  
         [0111]    SEQ ID No 14: degenerated PCR primer  
         [0112]    SEQ ID No 15: PCR primer  
         [0113]    SEQ ID No 16: PCR primer  
         [0114]    SEQ ID No 17: PCR primer  
         [0115]    SEQ ID No 18: PCR primer  
         [0116]    SEQ ID No 19: PCR primer  
         [0117]    SEQ ID No 20: PCR primer  
         [0118]    SEQ ID No 21: app promoter-gus translational fusion  
         [0119]    Sequence listing free text  
         [0120]    The following free text has been used in the Sequence Listing part of this application  
         [0121]    &lt;223&gt;Description of Artificial Sequence: A domain of non-conventional PARP proteins  
         [0122]    &lt;223&gt;Description of Artificial Sequence: A 1  domain on non conventional PARP protein  
         [0123]    &lt;223&gt;Description of Artificial Sequence: A 2  domain of non-conventional PARP protein  
         [0124]    &lt;223&gt;Description of Artificial Sequence: fusion protein between APP N-terminal domain and GUS protein  
         [0125]    &lt;223&gt;Description of Artificial Sequence: degenerated  
         [0126]    PCR primer  
         [0127]    &lt;223&gt;Description of Artificial Sequence: oligonucleotide for use as PCR primer  
         [0128]    &lt; 223 &gt;Description of Artificial Sequence: APP promoter fusion with beta-glucuronidase gene  
         [0129]    &lt; 223 &gt;traslation initiation codon  
       EXAMPLES  
     Experimental Procedures  
       [0130]    Yeast and bacterial strains  
         [0131]    [0131] Saccharomyces cerevisiae  strain DY (MATa his3 can1-10 ade2 leu2 trp1 ura3::(3xSV40 AP1-lacZ) (Kuge and Jones, 1994) was used for the expression of the APP protein. Yeast transformation was carried out according to Dohmen et al. (1991). Strains were grown on a minimal SDC medium (0.67% yeast nitrogen base, 0.37% casamino acids, 2% glucose, 50 mg l −1  of adenine and 40 mg l −1  of tryptophan). For the induction of the APP expression, glucose in SDC was substituted with 2% galactose.  
         [0132]    [0132] Escherichia coli  strain XL-I (Stratagene, La Jolla, Calif.) was used for the plasmid manipulations and library screenings, which were carried out according to standard procedures (Ausubel et al., 1987; Sambrook et al., 1989).  E. coli  BL21 (Studier and Moffat, 1986) was used for the APP protein expression and  Agrobacterium tumefaciens  C58C1Rif R (pGV2260) (Deblaere et al., 1985) for the stable transformation of plants.  
         [0133]    Poly(ADP-ribose)polymerase activity assay  
         [0134]    Enzymatic activity of the APP was assayed in total protein extracts of yeast strains prepared as follows. DY(pV8SPA) or DY(pYeDP1/8-2) were grown in 50 ml of SDC medium overnight at 30° C. on a gyratory shaker at 150 rpm. Yeast cells were harvested by centrifugation at 1,000×g, washed three times with 150 ml of 0.1M potassium phosphate buffer (pH 6.5), and resuspended in 5 ml of sorbitol buffer (1.2M sorbitol, 0.12M K 2 HPO 4 , 0.033M citric acid, pH 5.9). Lyticase (Boehringer, Mannheim, Germany) was added to the cell suspension to a final concentration of 30 U ml −1  and cells were incubated at 30° C. for 1 h. Yeast spheroplasts were then washed three times with sorbitol buffer and resuspended in 2 ml of ice-cold lysis buffer (100 mM Tris-HCl, pH 7.5, 400 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM DTT). After sonication, the lysate was centrifuged at 20,000×g for 20 min at 4° C. and the supernatant was desalted on a Econo-Pack™ 10 DG column (Bio-Rad, Richmond, Calif.) equilibrated with reaction buffer (100 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 1 mM DTT). To reduce proteolytic degradation of proteins, the lysis and reaction buffers were supplemented with a protease inhibitor cocktail (Boehringer), one tablet per 50 ml. Nucleic acids were removed from the total extracts by adding NaCl and protamine sulfate to a final concentration of 600 mM and 10 mg ml −1 , respectively. After incubation at room temperature for 10 min, the precipitate was removed by centrifugation at 20,000×g for 15 min at 4° C. The buffer of the supernatant was exchanged for the reaction buffer by gel filtration on an Econo-Pack™ 10 DG column.  
         [0135]    The assay for the synthesis of poly(ADP-ribose) was adapted from Collinge and Althaus (1994). Approximately 500 μg of total yeast protein were incubated in a reaction buffer supplemented with 30 μCi of  32 P-NAD +  (500 Ci mmol −1 ), unlabeled NAD +  to a final concentration of 60 μM, and 10 μg ml −1  sonicated salmon sperm DNA. After incubation for 40 min at room temperature, 500 μl of the stop buffer (200 mM Tris-HCl, pH 7.6, 0.1M NaCl, 5 mM EDTA, 1% Na + -N-lauroyl-sarcosine, and 20 μg ml −1  proteinase K) were added and reactions incubated at 37° C. overnight. After phenol and phenol/chloroform extractions, polymers were precipitated with 2.5 volumes of ethanol with 0.1M NaAc (pH 5.2). The pellet was washed with 70% ethanol, dried, and dissolved in 70% formamide, 10 mM EDTA, 0.01% bromophenol blue, and 0.01% xylene cyanol. Samples were heated at 80° C. for 10 min and then loaded onto a 12% polyacrylamide/6M urea sequencing gel. Gels were dried on 3MM paper (Whatman International, Maidstone, UK) and exposed either to Kodak X-Omat X-ray film (Eastman Kodak, Richmond, N.Y.) or scanned using a Phosphorlmager™ 445SI (Molecular Dynamics, Sunnyvale, Calif.).  
         [0136]    Immunological techniques  
         [0137]    A truncated app cDNA encoding an APP polypeptide from amino acids Met 310  to His 637  was expressed as a translation fusion with six histidine residues at the N terminus after induction of a 500-ml culture of the  E. coli  BL21(pETΔNdeSPA) with 1 mM isopropyl-β-D-thiogalactopyranoside. The APP polypeptide was purified to near homogeneity by affinity chromatography under denaturing conditions (in the presence of 6M guanidinium hydrochloride) on a Ni 2+ -NTA-agarose column, according to the manufacturer&#39;s protocol (Qiagen, Chatsworth, Calif.). After dialysis against PBS, a mixture of the soluble and insoluble APP polypeptides was used to immunize two New Zealand White rabbits following a standard immunization protocol (Harlow and Lane, 1988). For the Western blot analysis, proteins were resolved by denaturing SDS-PAGE (Sambrook et al., 1989; Harlow and Lane, 1988) and transferred onto nitrocellulose membranes (Hybond-C; Amersham), using a Semi-Dry Blotter II (Kem-En-Tec, Copenhagen, Denmark).  
         [0138]    In situ antigen localization in yeast cells was carried out as described (Harlow and Lane, 1988). For the localization of the APP protein in yeast spheroplasts, anti-APP serum was diluted 1:3,000 to 1:5,000 in Tris-buffered saline-BSA buffer. 10H monoclonal antibody, which specifically recognizes poly(ADP-ribose) polymer (Ikajima et al., 1990) was used in a 1:100 dilution in PBS buffer. The mouse antibody were detected with the sheep anti-mouse IgG F(ab′) 2  fragment conjugated to fluorescein isothiocyanate (FITC) (Sigma) at a dilution of 1:200. Rabbit IgG was detected with CY-3 conjugated sheep anti-rabbit IgG sheep F(ab′) 2  fragment (Sigma), at a dilution of 1:200. For the visualization of DNA, slides were incubated for 1 min in PBS with 10 μg ml −1  of 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Fluorescence imaging was performed on an Axioskop epifluorescence microscope (Zeiss, Jena, Germany). For observation of FITC and CY-3 fluorochromes, 23 and 15 filter cubes were used, respectively. Cells were photographed with Fuji Color-100 super plus film.  
         [0139]    Plant material and histochemical analysis  
         [0140]    [0140] Nicotiana tabacum  SR1 (Maliga et al., 1975) was used for the generation of stable transformants following the procedure of leaf disc cocultivation (De Block et al., 1987) with  A. tumefaciens  C58C1 Rif R (pGV2260; pGCNSPAGUS).  N. tabacum  SR1 line transformed with authentic GUS under the control of the 35S CaMV was used as a control.  Arabidopsis thaliana  ecotype Columbia was used for the transformation of the app-promoter-GUS fusion following the in situ infiltration procedure.  
         [0141]    For in situ histochemical staining of the GUS activity, plant samples were fixed in ice-cold 90% acetone for 30 min, washed in 0.1M K 2 HPO 4  (pH 7.8), and then incubated in staining buffer (0.1M K 2 HPO 4 , pH 7.8, 2 mM X-Gluc, 20 mM Fe 3+ -EDTA) at 37° C. Stained plant tissues were stored in 70% ethanol at 4° C. When necessary, browning of tissues due to phenolic oxidation was reduced by incubation with lactophenol (Beeckman and Engler, 1994). The GUS staining was examined under a Jenalumar light microscope (Zeiss). Plant tissues were photographed with Fuji Color-100 super plus film.  
         [0142]    Miscellaneous methods  
         [0143]    The plasmid construction steps were routinely verified by DNA sequencing carried out according to protocols provided by USB Biochemicals (Cleveland, Ohio).  32 P-labeled DNA probes for nucleic acid hybridization were synthesized by the Ready-Prime DNA labelling kit (Amersham). For DNA and RNA hybridization experiments, the buffer system of Church and Gilbert (1984) was used (0.25M sodium phosphate, pH 7.2, 7% SDS, 1% BSA, 1 mM EDTA). For Western blot analysis, yeast total proteins were 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). RNA was resolved on 1.5% agarose gels after denaturation with glyoxal (Sambrook et al., 1989). Hybond-N nylon filters (Amersham) were used for the nucleic acid blotting.  
       Example 1  
     Isolation of Genes Encoding PARP Homologues from  Zea mays    
       [0144]    With the purpose of isolating maize cDNA encoding PARP homologue(s) two approaches were followed. First, a maize cDNA library was screened under low-stringency DNA-DNA hybridization conditions using a DNA probe prepared from the Arabidopsis app cDNA. Secondly, PCR amplification of part of the maize PARP was performed, using the first-strand cDNA as a template and two degenerate primers, designed on the basis of the sequence of the “PARP signature”, the most conserved amino acid sequence between all known PARP proteins.  
         [0145]    A λZAP (Stratagene) cDNA library from leaves of maize ( Zea mays  L.), inbred line B734. Plaques (500,000) were screened according to standard procedures (Sambrook et al., 1989). After screening with the Arabidopsis app probe, one non-full-length cDNA of 1.4 kbp was purified. After the initial cDNA library screening with the app probe and a subsequent 5′ rapid amplification of cDNA ends (RACE) PCR analysis, the nap gene, a maize homologue of the Arabidopsis app, was identified. For the 5′RACE PCR, the template was prepared with the Marathon kit (Clontech, Palo Alto, Calif.) and 0.5 μg of maize poly(A) +  RNA isolated from inner sheath, outer sheath, and leaves of 1-week-old maize seedlings. The gene-specific, nested primers for PCR amplification were 5′-GGGACCATGTAGTTTATCTTGACCT-3′ (SEQ ID No 15) and 5′-GACCTCGTACCCCAACTCTTCCCCAT-3′ (SEQ ID No 16) for nap primers. The amplified PCR products were subcloned and sequenced. A fragment of 800 bp was amplified with nap-specific primers which allowed to reconstruct the 2295-bp-long sequence of nap cDNA (SEQ ID No 3).  
         [0146]    The NAP protein was 653 amino acids long (molecular mass ˜73 kDa; SEQ ID No 4) and highly similar (61% sequence identity and 69% similarity) to the APP. Most importantly, NAP had an organization of the N-terminus congruent to APP (FIG. 1A), suggesting a rather strict selection pressure on the structure of APP-like proteins in plants. The nap gene was unique in the maize genome (FIG. 2A) and encoded a transcript of 2.4 kb (FIG. 2C).  
         [0147]    Using degenerate primers based on very highly conserved regions in the “PARP signature” and first-strand cDNA from  Zea mays  as a template, a 310-bp fragment was amplified. For the 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 a template and was synthesized using 5 μg of poly(A) +  RNA from young maize leaves and MuMLV reverse transcriptase. PCR amplifications were performed with Taq DNA polymerase in 100 μl volume using the following conditions: 1 min at 95° C., 2 min at 45° C., 3 min at 72° C., followed by 38 cycles of 1 min at 95° C., 2 min at 45° C., 3 min at 72° C., with a final incubation for 10 min at 72° C.  
         [0148]    The sequence of the 310 bp fragment showed 55% sequence identity and 64% sequence similarity with human PARP over the same region, but was, however, different from the sequence of the nap cDNA. Three zap cDNAs were identified after screening with the 310-bp fragment, which was obtained by PCR with degenerate primers. These three purified cDNA were all derived from the same transcript because they had identical 3′ non-coding regions; the longest clone (#9) was sequenced on both strands (SEQ ID No 1). This cDNA encoded a PARP-homologous polypeptide of 689 amino acids (SEQ ID No 2; molecular mass ˜109 kDa), which we designated as ZAP1 (FIG. 1B). The first Zn-finger of ZAP1 was probably nonfunctional because it had the sequence CKSCxxxHASV, which included no third cysteine residue.  
         [0149]    5′RACE PCR analysis of zap transcripts from the maize line LG2080 (the screened cDNA library was made from the 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). A 450-bp PCR product was obtained after PCR with zap-specific primers. Eight independent, because of their slight differences in lengths at their 5′ ends, 5′RACE PCR fragments generated with zap-specific primers were sequenced. In all the transcripts from the LG2080 maize plants, there was an insertion of additional sequence in the coding region, which made the ZAP protein longer by 11 amino acids (980 amino acids, molecular mass ˜110.4 kDa). The Zn-finger I of ZAP2 was standard and read CKSCxxxHARC (FIG. 1B; SEQ ID No 11). The sequence difference may be due either to differences between maize varieties, to the expression of two homologous genes, or to alternative splicing. In fact, maize may have at least two zap genes (FIG. 2B), which encode a transcript of 3.4-3.5 kb (FIG. 2D). The DNA gel blot experiment with a probe prepared from the zap cDNA showed that homologous genes were present in Arabidopsis.  
         [0150]    Structurally ZAP was very similar to PARP from animals. It had a well conserved DNA-binding domain composed of two Zn-fingers (36% identity and 45% similarity to the DNA-binding domain of mouse PARP). Even higher homology was shown by comparing only the sequences of the Zn-fingers, Ala 1 -Phe 162  in the mouse enzyme (44% identity and 54% similarity), or a subdomain downstream from the nuclear localization signal (NLS), Leu 237 -Ser 360  in mouse PARP (40% identity and 50% similarity). Whereas the bipartite nuclear localization signal characteristic of mammalian PARP could not be identified in ZAP, the sequence KRKK fitted a monopartite NLS (FIG. 1B). The putative automodification domain was poorly conserved and was shorter in ZAP than in mouse PARP. The compilation of the homology of the catalytic dmains between ZAP, NAP, APP and mouse PARP is shown in FIG. 2. It should be noted that the NAD + -binding domain of ZAP was more similar to the mammalian enzyme (48% identity) than to that of APP and NAP (40% and 42% sequence identity, respectively), whereas APP and NAP were 68% identical and 76% similar in their catalytic domain.  
       Example 2  
     Demonstration that Non-conventional PARP Protein has a DNA-dependent Poly(ADP-ribose) Polymerase Activity  
       [0151]    APP is a DNA-dependent poly(ADP-ribose) polymerase  
         [0152]    A more detailed study of the APP protein (expressed in yeast) was performed to understand the activity of PARP-like proteins from the NAP class. The choice of yeast as the organism for the expression and enzymatic analysis of the Arabidopsis APP protein was made for a number of reasons. As an eukaryote,  Saccharomyces cerevisiae  is better suited for the expression of native proteins from other eukaryotic organisms, and unlike most other eukaryotic cells, it does not possess endogenous PARP activity (Lindahl et al., 1995).  
         [0153]    The full-length app cDNA was placed in pYeDP1/8-2 under the control of a galactose-inducible yeast promoter in the following way, the full-length app cDNA was excised from pC3 (Lepiniec et al., 1995) as an Xhol-EcoRl fragment. The ends were filled in with the Klenow fragment of DNA polymerase I, and the fragment was subcloned into the Smal site of the yeast expression vector pYeDP1/8-2 (Cullin and Pompon, 1988). The resulting expression vector pV8SPA (FIG. 4A) was transformed into  S. cerevisiae  strain DY.  
         [0154]    For APP expression in  E. coli,  the complete coding region of the app cDNA was PCR amplified with Pfu DNA polymerase (Stratagene), using the 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.), resulting in pETSPA. The expression of the full-length APP in  E. coli  BL21 from pETSPA was very poor. To obtain better expression, pETSPA was digested with Ncol and Ndel or with Smal, the ends were filled in by the Klenow fragment of DNA polymerase I, and the plasmids were then self-ligated. Of the resulting plasmids pETΔNdeSPA and pETΔSmaSPA, only pETΔNdeSPA gave satisfactory expression of the truncated APP polypeptide (Met 310  to His 637 ) in  E. coli  BL21.  
         [0155]    The expression of the APP in yeast was verified by Northern and Western blot analysis. (FIG. 4) As the promoter in pV8SPA is inactive when cells are grown on glucose and derepressed on galactose-containing media, the expression was expected to be tightly regulated by the carbon source. However, Northern blot analysis of RNA and immunoblot analysis of proteins in DY(pV8SPA) as compared to the control DY strain containing the empty vector, showed that app mRNA and APP protein were expressed in yeast even when grown on glucose-containing media (FIG. 4B, lane  2 ). The peculiarity of the expression observed on glucose-containing medium was that both app mRNA and APP protein were shorter than the ones detected after induction with galactose (compare lanes  2  and  4  in FIG. 4B). The APP polypeptide with the higher molecular weight, (apparently a full-length protein) was only detected on galactose-containing medium, although such cells also expressed the truncated mRNA and protein. The most probable explanation for this finding is that when the DY(pV8SPA) strain is grown on glucose, there is a leaky expression from the expression cassette, with transcription beginning 200-300 bp downstream from the transcription start observed after galactose induction. This shorter mRNA probably does not code for the first methiorine (Met 1 ) of APP and, therefore, translation is initiated at Met 72 . This would explain the observed difference of −5 kDa (calculated difference being 7.5 kDa) in the molecular masses of the APP polypeptides from strains grown on glucose or on galactose. The possibility that the differences in molecular masses may be attributed to self-modification through poly(ADP-ribos)ylation was ruled out by growing strains in the presence of PARP inhibitors, such as 3ABA and nicotinamide (FIG. 4B, compare lanes  6  and  8  to lane  4 ).  
         [0156]    To detect the synthesis of poly(ADP-ribose), total proteins were extracted from yeast strains grown under different conditions and incubated in the presence of radioactively labeled NAD + . To prevent synthesis of poly(ADP-ribose) and possible automodification of the APP in vivo, strains were also grown in the presence of 3ABA, a reversible inhibitor of PARP, which was subsequently removed from the protein extracts 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 a strain containing the empty vector (FIG. 5A, lane  4 ). It can also be seen that Arabidopsis APP could synthesize polymers up to 40 residues in length (FIG. 5A, lane  1 ) with the majority of the radioactivity being incorporated into 10-15-mer. This observation is consistent with the polymer sizes detected by other authors (Chen et al., 1994). More radioactivity was incorporated into polymer when the yeast strain was grown with 3ABA than without (FIG. 5A, lane  1  compared to lane  2 ); the reason might be that either the APP extracted from inhibited cultures was less automodified (it is believed that automodification inhibits the activity of PARP) or the labeled NAD +  was used by the enzyme from the uninhibited culture for the extension of existing polymer, resulting in a lower specific activity overall. Under the same reaction conditions poly(ADP-ribose) synthesized by human PARP, either in reaction buffer alone or in the presence of a yeast total protein extract from DY(pYeDP1/8-2) (FIG. 5A, lanes  5  and  6 , respectively), showed much longer chains, possibly up to 400-mer (de Murcia and Ménissier de Murcia, 1994).  
         [0157]    The stimulation of enzymatic activity by nicked DNA is a well known property of PARP from animals (Alvarez-Gonzalez and Althaus, 1989). We therefore tested whether the activity of the APP protein was DNA dependent. After removal of yeast nucleic acids (DNA, RNA) and some basic proteins from the galactose-grown DY(pV8SPA) protein extract the synthesis of poly(ADP-ribose) was analyzed in the presence of increasing concentrations of sonicated salmon sperm DNA. As can be seen in FIG. 5B, there was a direct correlation between the amount of DNA present in the reaction and the incorporation of  32 P-NAD + . Scanning of the phosphor-images indicated that ˜6-fold more radioactivity was incorporated into poly(ADP-ribose) in the reaction mixture containing 40 μg ml −1  of DNA than into that with 2 μg ml −1  of DNA (FIG. 5B, lanes  4  and  2 , respectively). The synthesis of the polymer was sensitive to 3ABA in the reaction mix (FIG. 5B, lane  5 ).  
         [0158]    APP is a nuclear protein  
         [0159]    In animal cells PARP activity is localized in the nucleus (Schreiber et al., 1992). The intracellular localization, if nuclear, of APP could provide an important additional indication that APP is a bona fide plant PARP. To this end, the localization of the APP polypeptides in yeast cells was analyzed using anti-APP antisera. The APP polypeptide synthesized in yeast grown on galactose was found mainly in the nucleus. This localization was unaffected by the presence in the media of the PARP inhibitors.  
         [0160]    In addition, we tested whether APP was constitutively active in yeast cells, as has been reported for the human PARP (Collinge and Althaus, 1994). Here, fixed yeast spheroplasts were incubated with monoclonal 10H antibody, which specifically recognizes poly(ADP-ribose) polymers (Kawamitsu et al., 1984). A positive yellowish-green fluorescence signal with 10H antibody was localized in the nucleus and was observed only in DY(pV8SPA) cells grown on galactose. Positive staining was greatly reduced in cells grown in the presence of the PARP inhibitors, 3ABA and nicotinamide.  
         [0161]    To identify the intracellular localization of APP in plant cells, a widely adopted approach in plant studies was used, i.e., the examination of the subcellular location of a fusion protein formed between the protein in question and a reporter gene, once the protein fusion was produced in transgenic plants or transfected cells (Citovsky et al., 1994; Sakamoto and Nagatani, 1996; Terzaghi et al., 1997; von Arnim and Deng, 1994). An N-terminal translational fusion of GUS with the part of the APP polypeptide extending from the Met 1  to Pro 407  was made. The translational fusion of APP with bacterial GUS was constructed as follows. Plasmid pETSPA was cut with Smal, treated with alkaline phosphatase, and ligated to a blunted NcoI-Xbal fragment from pGUS1 (Plant Genetic Systems N.V., Gent, Belgium). The ligation mix was transformed into  E. coli  XL-I and cells were plated onto LB medium supplemented with 0.1 mM isopropyl-β-D-thiogalactopyranoside, 40 μg ml −1  5-bromo-4-chloro-3-indolyl-β-D-glucuronide, and 100 μg ml −1  of ampicillin. In this way, pETSPAGUS was selected as blue colonies. The expression in  E. coli  of the ˜110-kDa fusion protein was confirmed by in situ GUS activity gels (Lee et al., 1995). The APP-GUS fusion was placed under the control of the 35S promoter of the CaMV (the Klenow-blunted BamHI fragment from pETSPAGUS was subcloned into Smal-digested pJD330; Gallie and Walbot, 1992) and the resulting expression cassette was subcloned as an Xbal fragment into the Xbal site of the pCGN1547 binary vector (McBride and Summerfelt, 1990) to give pGCNSPAGUS. The pGCNSPAGUS was finally introduced into  A. tumefaciens  C58C1Rif R (pGV2260) by the freezing-thawing transformation procedure.  
         [0162]    Expression of the fusion protein was verified in  E. coli.  The chimeric cDNA under the control of the 35S CaMV promoter was stably integrated into the tobacco genome. Progeny from four independent transgenic tobacco plants were analyzed for the subcellular distribution of the GUS activity after in situ histochemical staining (Jefferson et al., 1987). In 2-day-old seedlings GUS activity could be detected in cotyledons and in roots, but not in hypocotyls or root tips. Because of the transparency of root tissues, GUS staining was clearly localized in the nuclei of root hairs and epidermal cells. Additionally, some diffuse, non-localized staining of other root cells was seen, in particular along the vascular cylinders. This non-nuclear GUS staining was more pronounced in leaf tissues. Whereas young true leaves or cotyledons displayed intense blue staining of the nuclei, there was also some diffuse staining of the cytoplasm. In fully expanded leaves, however, GUS stainig became homogenous and similar to the staining of control plants transformed with GUS under the control of the CaMV 35S promoter, in which GUS was expressed in the cytoplasm. Eventually, older leaves or cotyledons exhibited practically no histochemically detectable GUS activity, with the exception of the vascular bundles, where the GUS staining could not be confined to any particular cell compartment.  
         [0163]    Deficiency in DNA ligase I induces expression of the app gene  
         [0164]    PARP in animal cells is one of the most abundant nuclear proteins and its activity is regulated by allosteric changes in the protein upon 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 might be essential for APP function in vivo. To test this hypothesis, the expression of the app gene was studied during in vivo genome destabilization caused by a DNA ligase I deficiency. A T-DNA insertion mutation, line SK1B2, in the Arabidopsis DNA ligase I gene was isolated previously (Babiychuk et al., 1997). The mutation is lethal in the homozygous state, but the mutant allele shows normal transmission through the gametes. We therefore expected that cells homozygous for the mutation would die due to incomplete DNA synthesis during the S phase of the cell cycle, soon after the fertilization of the mutant embryo sac with mutant pollen.  
         [0165]    An app promoter-GUS translational fusion, in which the coding region of GUS was fused in-frame with the first five amino acids of APP and 2 kb of app 5′ flanking sequences was constructed (SEQ ID No 21). The gene encoding the fusion protein was transformed into Arabidopsis. After two back-crosses to a wild type, heterozygous plants transformed with app promoter-GUS were crossed with Arabidopsis line SK1B2. The inflorescences of the control plants and plants heterozygous for the ligase mutation were stained for the activity of GUS. The GUS staining pattern mostly detected in aging tissues probably reflects the expression of the app gene, although we have no firm evidence that all of the regulatory sequences were present in the constructs used. This pattern was the same both in the inflorescences of control plants, not carrying the mutant ligase gene and plants heterozygous for a mutation. Approximately one-fourth of the ovules in the mutant plants with the fusion protein are GUS positive. Closer microscopical examination showed that in the GUS-positive ovules only the gametophyte was stained. The only difference between the control plants and the mutant plant was a mutation in a DNA ligase gene. We therefore conclude that the app gene is induced because of either the accumulation of DNA breaks, or the death of the mutant embryo sacs fertilized with mutant pollen. GUS staining of embryo sacs was found to appear within 24 h aftepollination, or therefore very soon after fertilization.  
       Example 3  
     Construction of PCD Modulating Chimeric Genes and Introduction of the T-DNA Vectors Comprising such PCD Modulating Genes in an Agrobacterium Strain  
       [0166]    3.1. Construction of the p35S:(dsRNA-APP) and p35S:(dsRNA-ZAP) genes  
         [0167]    Using standard recombinant DNA procedures, the following DNA regions are operably linked, as schematically outlined in FIG. 6 (constructs  1  and  5 ):  
         [0168]    For the p35S:(dsRNA-ZAP) chimeric gene  
         [0169]    a CaMV 35S promoter region (Odell et al., 1985)  
         [0170]    a Cab22 leader region (Harpster et al., 1988)  
         [0171]    a ZAP encoding DNA region (about complete) (the  Arabidopsis thaliana  homologue to SEQ ID No 10, isolated by hybridization)  
         [0172]    about 500 bp of the 5′ end of the ZAP2 encoding DNA region in inverse orientation  
         [0173]    a CaMV35S 3′ end region (Mogen et al., 1990)  
         [0174]    For the p35S:(dsRNA-APP) chimeric gene  
         [0175]    a CaMV 35S promoter region (Odell et al., 1985)  
         [0176]    a Cab22 leader region (Harpster et al., 1988)  
         [0177]    an APP encoding DNA region (about complete) (SEQ ID No 5)  
         [0178]    about 500 bp of the 5′ end of the APP encoding DNA region in inverse orientation  
         [0179]    a CaMV35S 3′ end region (Mogen et al., 1990)  
         [0180]    3.2. Construction of the pNOS:(dsRNA-APP) and pNOS:(dsRNA-ZAP) genes  
         [0181]    Using standard recombinant DNA procedures, the following DNA regions are operably linked, as schematically outlined in FIG. 6 (constructs  2  and  6 ):  
         [0182]    For the pNOS:(dsRNA-ZAP) chimeric gene  
         [0183]    a NOS promoter region (Herrera-Estrella et al., 1983)  
         [0184]    a Cab22 leader region (Harpster et al., 1988)  
         [0185]    a ZAP encoding DNA region (about complete) (the  Arabidopsis thaliana  homologue to SEQ ID No 10, isolated by hybridization)  
         [0186]    about 500 bp of the 5′ end of the ZAP2 encoding DNA region in inverse orientation  
         [0187]    a CaMV35S 3′ end region (Mogen et al., 1990)  
         [0188]    For the pNOS:(dsRNA-APP) chimeric gene  
         [0189]    a NOS promoter region (Herrera-Estrella et al., 1983)  
         [0190]    a Cab22 leader region (Harpster et al., 1988)  
         [0191]    an APP encoding DNA region (about complete) (SEQ ID No 5)  
         [0192]    about 500 bp of the 5′ end of the APP encoding DNA region in inverse orientation  
         [0193]    a CaMV35S 3′ end region (Mogen et al., 1990)  
         [0194]    3.3. Construction of the pTA29:(dsRNA-APP) and pTA29:(dsRNA-ZAP) genes  
         [0195]    Using standard recombinant DNA procedures, the following DNA regions are operably linked, as schematically outlined in FIG. 6 (constructs  3  and  7 ):  
         [0196]    For the pTA29:(dsRNA-ZAP) chimeric gene  
         [0197]    a TA29 promoter region (WO 89/10396)  
         [0198]    a Cab22 leader region (Harpster et al., 1988)  
         [0199]    a ZAP encoding DNA region (about complete) (the  Arabidopsis thaliana  homologue to SEQ ID No 10, isolated by hybridization)  
         [0200]    about 500 bp of the 5′ end of the ZAP2 encoding DNA region in inverse orientation  
         [0201]    a CaMV35S 3′ end region (Mogen et al., 1990)  
         [0202]    For the pTA29:(dsRNA-APP) chimeric gene  
         [0203]    a TA29 promoter region (WO 89/10396)  
         [0204]    a Cab22 leader region (Harpster et al., 1988)  
         [0205]    an APP encoding DNA region (about complete) (SEQ ID No 5)  
         [0206]    about 500 bp of the 5′ end of the APP encoding DNA region in inverse orientation  
         [0207]    a CaMV35S 3′ end region (Mogen et al., 1990)  
         [0208]    3.4. Construction of the pNTP303:(dsRNA-APP) and pNTP303:(dsRNA-ZAP) genes  
         [0209]    Using standard recombinant DNA procedures, the following DNA regions are operably linked, as schematically outlined in FIG. 6 (constructs  4  and  8 ):  
         [0210]    For the pNTP303:(dsRNA-ZAP) chimeric gene  
         [0211]    a NTP303 promoter region (Wetering 1994)  
         [0212]    a Cab22 leader region (Harpster et al., 1988)  
         [0213]    a ZAP encoding DNA region (about complete) (the  Arabidopsis thaliana  homologue to SEQ ID No 10, isolated by hybridization)  
         [0214]    about 500 bp of the 5′ end of the ZAP2 encoding DNA region in inverse orientation  
         [0215]    a CaMV35S 3′ end region (Mogen et al., 1990)  
         [0216]    For the pNTP303:(dsRNA-APP) chimeric gene  
         [0217]    a NTP303 promoter region (Wetering, 1994)  
         [0218]    a Cab22 leader region (Harpster et al., 1988)  
         [0219]    an APP encoding DNA region (about complete) (SEQ ID No 5)  
         [0220]    about 500 bp of the 5′ end of the APP encoding DNA region in inverse orientation  
         [0221]    a CaMV35S 3′ end region (Mogen et al., 1990)  
         [0222]    3.5 Construction of the chimeric marker genes  
         [0223]    Using standard recombinant DNA procedures, the following DNA regions are operably linked, as schematically outlined in FIG. 6:  
         [0224]    For the gat marker gene  
         [0225]    an Act2 promoter region (An et al., 1996)  
         [0226]    a aminoglycoside 6′-acetyltransferase encoding DNA (WO 94/26913)  
         [0227]    a 3′ end region of a nopaline synthase gene (Depicker et al., 1982)  
         [0228]    For the bar marker gene  
         [0229]    an Act2 promoter region (An et al., 1996)  
         [0230]    a phosphinotricin acetyltransferase encoding DNA (U.S. Pat. No. 5,646,024)  
         [0231]    a 3′ end region of a nopaline synthase gene (Depicker et al., 1982)  
         [0232]    3.6. Construction of the T-DNA vectors comprising the PCD modulating chimeric genes  
         [0233]    Using appropriate restriction enzymes, the chimeric PCD modulating genes described under 3.1 to 3.5 are excised and introduced in the polylinker between the T-DNA borders of a T-DNA vector derived from pGSV5 (WO 97/13865) together with either the gat marker gene or the bar marker gene. The resulting T-DNA vectors are schematically represented in FIG. 6.  
         [0234]    3.7. Introduction of the T-DNA vectors in Agrobacterium  
         [0235]    The T-DNA vectors are introduced in  Agrobacterium tumefaciens  C58C1Rif(pGV4000) by electroporation as described by Walkerpeach and Velten (1995) and transformants are selected using spectinomycin and streptomycin.  
       Example 4  
     Agrobacterium-mediated Transformation of  Arabidopsis thaliana  with the T-DNA Vectors of Example 3  
       [0236]    The Agrobacterium strains are used to transform  Arabidopsis thaliana  var. C24 applying the root transformation method as described by Valvekens et al. (1992). The explants are coinfected with the Agrobacteria strains containing the dsRNA-APP respectively the dsRNA-ZAP constructs. The dsRNA-APP constructs are used in combination with the pact:bar gene. The dsRNA-ZAP constructs are used in combination with the pact:gat gene. Transformants are selected for phosphinothricin resistance. The regenerated rooted transgenic lines are tested for the presence of the other T-DNA by screening for kanamycin resistance. Transgenic lines containing both T-DNA&#39;s are transfered to the greenhouse. The phenotype of the T0-transgenic lines is scored and the T1-generations are studied further in more detail.  
       Example 5  
     Agrobacterium-mediated Transformation of  Brassica napus  with the T-DNA Vectors of Example 3  
       [0237]    The Agrobacterium strains are used to transform the  Brassica napus  var. N90-740 applying the hypocotyl transformation method essentially as described by De Block et al. (1989), except for the following modifications:  
         [0238]    hypocotyl explants are precultured for 1 day on A2 medium [MS, 0.5 g/l Mes (pH5.7), 1.2% glucose, 0.5% agarose, 1 mg/l 2,4-D, 0.25 mg/l naphthalene acetic acid (NAA)and 1 mg/l 6-benzylaminopurine (BAP)].  
         [0239]    infection medium A3 is MS, 0.5 g/l Mes (pH5.7), 1.2% glucose, 0.1 mg/l NAA, 0.75 mg/l BAP and 0.01 mg/l gibberellinic acid (GA3).  
         [0240]    selection medium A5G is MS, 0.5 g/l Mes (pH5.7), 1.2% glucose, 40 mg/l adenine.SO 4 , 0.5 g/l polyvinylpyrrolidone (PVP), 0.5% agarose, 0.1 mg/l NAA, 0.75 mg/l BAP, 0.01 mg/l GA3, 250 mg/l carbenicillin, 250 mg/l triacillin, 5 mg/l AgNO 3  for three weeks. After this period selection is continued on A5J medium (similar a A5G but with 3% sucrose)  
         [0241]    regeneration medium A6 is MS, 0.5 g/l Mes (pH5.7), 2% sucrose, 40 mg/l adenine.SO 4 , 0.5 g/l PVP, 0.5% agarose, 0.0025 mg/l BAP and 250 mg/l triacillin.  
         [0242]    healthy shoots are transferred to rooting medium which wa A9: half concentrated MS, 1.5% sucrose (pH5.8), 100 mg/l triacillin, 0.6% agar in 1 liter vessels.  
         [0243]    MS stands for Murashige and Skoog medium (Murashige and Skoog, 1962)  
         [0244]    For introducing both the dsRNA-APP and the dsRNA-ZAP T-DNA constructs into a same plant cell the co-transformation method is applied, essentially as described by De Block and Debrouwer (1991). Transformed plant lines are selected on phosphinothricin containing medium after which the presence of the second T-DNA is screened by testing the regenerated rooted shoots for kanamycin resistance. In the co-transformation experiments, the dsRNA-APP construct are used in combination with the pact:bar gene. The dsRNA-ZAP constructs are used in combination with the pact:gat gene. Transgenic lines containing both T-DNA&#39;s are transfered to the greenhouse. The phenotype of the T0-transgenic lines is scored and the T1-generations are studied further in more detail.  
       Example 6  
     In vitro Assay to Test Vigor of Plant Lines  
       [0245]    6.1. Fitness assay for  Brassica napus    
         [0246]    Media and reaction buffers  
         [0247]    Sowing medium:  
         [0248]    Half concentrated Murashige and Skoog salts  
         [0249]    2% sucrose  
         [0250]    pH 5.8  
         [0251]    0.6% agar  
         [0252]    Callus inducing medium: A2S  
         [0253]    MS medium, 0.5 g/l Mes (pH 5.8), 3% sucrose, 40 mg/l adenine-SO 4 , 0.5% agarose, 1 mg/l 2,4-D, 0.25 mg/l NAA, 1 mg/l BAP  
         [0254]    Incubation medium:  
         [0255]    25 mM K-phosphate buffer pH 5.8  
         [0256]    2% sucrose  
         [0257]    1 drop Tween20 for 25 ml medium  
         [0258]    Reaction buffer:  
         [0259]    50 mM K-phosphate buffer pH7.4  
         [0260]    10 mM 2,3,5-triphenyltetrazoliumchloride (TTC) (=3.35 mg/ml)  
         [0261]    1 drop Tween20 for 25 ml buffer  
         [0262]    Sterilization of seeds and growing of the seedlings  
         [0263]    Seeds are soaked in 70% ethanol for 2 min, then surface-sterilized for 15 min in a sodium hypochlorite solution (with about 6% active chlorine) containing 0.1% Tween20. Finally, the seeds are rinsed with 1 l of sterile destilled water. Put 7 seeds/1 l vessel (Weck) containing about 75 ml of sowing medium. The seeds are germinated at 23° C. and 30 μEinstein/s −1 m −2  with a daylength of 16 h.  
         [0264]    The line N90-740 is always included for standardization between experiments.  
         [0265]    Preculture of the hypocotyl explants  
         [0266]    12-14 days after sowing, the hypocotyls are cut in about 7 mm segments. 25 hypocotyls/Optilux Petridisch (Falcon S1005)  
         [0267]    The hypocotyl explants are cultured for 4 days on medium A2S at 23-25° C. (at 30 μEinstein/s −1 m −2 ).  
         [0268]    [0268]         P.S.: about 150-300 hypocotyl explants/line are needed to cary out the asssay  
         [0269]    Transfer the hypocotyl explants to Optilux Petridishes (Falcon S1005) containing 30 ml of incubation medium.  
         [0270]    Incubate for about 20 hours at 24° C. in the dark.  
         [0271]    TTC-assay  
         [0272]    Transfer 150 hypocotyl explants to a 50 ml Falcon tube.  
         [0273]    Wash with reaction buffer (without TTC).  
         [0274]    Add 25 ml-30 ml of reaction buffer/tube.  
         [0275]    tube 1          no TTC added  
         [0276]    for measuring background absorption  
         [0277]    one line/experiment is sufficient  
         [0278]    tube 2          +10 mM TTC  
         [0279]    (explants have to be submerged, but do not vacuum infiltrate!)  
         [0280]    turn tubes upside down  
         [0281]    Incubate for about 1 hour in the dark at 26° C. (no end reaction!)  
         [0282]    Wash hypocotyls with deionized water  
         [0283]    Remove water  
         [0284]    Freeze at −70° C. for 30 min.  
         [0285]    Thaw at room°t (in the dark)  
         [0286]    Add 50 ml ethanol (technical)  
         [0287]    Extract reduced TTC-H by shaking for 1 hour  
         [0288]    Measure absorptions of extracts at 485 nm  
         [0289]    P.S.: reduced TTC-H is not stable          keep in the dark and measure O.D. 485  as soon as possible  
         [0290]    O.D. 485 (TTC-H) =(O.D. 485 +TTC)−(O.D. 485 −TTC)  
         [0291]    Comparison of the TTC-reducing capacities between samples of different independent experiments can be done by setting the TTC-reducing capacity of N90-740 in the different experiment at 100%.  
         [0292]    Lines with a high TTC-reducing capacity are vigorous, while lines with a low TTC-reducing capacity are weak.  
         [0293]    6.2. Fitness assay Arabidopsis  
         [0294]    Media and reaction buffers  
         [0295]    Plant medium: Half concentrated Murashige and Skoog salts  
         [0296]    1.5% sucrose  
         [0297]    pH 5.8  
         [0298]    0.6% agar  
         [0299]    →autoclave 15 min.  
         [0300]    add filter sterilized −100 mg/l myo-inositol  
         [0301]    −0.5 mg/l pyridoxine  
         [0302]    −0.5 mg/l nicotinic acid  
         [0303]    −1 mg/l thiamine  
         [0304]    Incubation medium: 10 mM K-phosphate buffer pH5.8  
         [0305]    2% sucrose  
         [0306]    1 drop Tween20 for 25 ml medium  
         [0307]    Reaction buffer: 50 mM K-phosphate buffer pH7.4  
         [0308]    10 mM 2,3,5-triphenyltetrazoliumchloride (TTC) (=3.35 mg/ml)  
         [0309]    1 drop Tween20 for 25 ml buffer  
         [0310]    Arabidopsis plants  
         [0311]    Sterilization of Arabidopsis seeds  
         [0312]    2 min. 70% ethanol  
         [0313]    10 min. bleach (6% active chlorine)+1 drop Tween 20 for 20 ml solution  
         [0314]    wash 5 times with sterile water  
         [0315]    P.S.: sterilization is done in 2 ml eppendorf tubes Arabidopsis seeds sink to the bottom of the tube, allowing removal of the liquids by means of a 1 ml pipetman  
         [0316]    Growing of Arabidopsis plants  
         [0317]    Seeds are sown in ‘Intergrid Tissue Culture disks of Falcon’ (nr. 3025) containing ±100 ml of plant medium: 1 seed/grid.  
         [0318]    Plants are grown at 23° C.  
         [0319]    40 μEinstein s −1 m −2    
         [0320]    16 hours light-8 hours dark  
         [0321]    for about 3 weeks (plants start to form flower buds)  
         [0322]    →P.S.: *about 90-110 plants/line are needed to cary out the asssay  
         [0323]    * include control line (C24; Columbia; . . . ) for calibration  
         [0324]    Pre-incubation  
         [0325]    Harvest Arabidopsis shoots by cutting of roots (by means of scissors) Put each shoot immediatly in incubation medium (shoots have to be submerged, but do not vacuum infiltrate)  
         [0326]    Incubation medium: ±150 ml in ‘Intergrid Tissue Culture disks of Falcon’ (nr. 3025)  
         [0327]    a) incubation medium: for quantification of background absorption (see TTC-asssay)  
         [0328]    b) incubation medium  
         [0329]    c) incubation medium+2 mM niacinamide  
         [0330]    30-35 shoots/petridish (but same amount of shoots for all lines and for each condition)  
         [0331]    Incubate at 24° C. in the dark for ±20 hours  
         [0332]    TTC-assay  
         [0333]    Transfer shoots to 50 ml Falcon tubes  
         [0334]    Wash with reaction buffer (without TTC)  
         [0335]    Add 30-35ml of reaction buffer/tube  
         [0336]    a) no TTC added (for measuring background absorption)  
         [0337]    b and c) +10 mM TTC  
         [0338]    (Shoots have to be submerged, but do not vacuum infiltrate!)  
         [0339]    Incubate for about 2 hours in the dark at 26° C. (no end reaction!)  
         [0340]    Wash shoots with deionized water  
         [0341]    Remove water  
         [0342]    Freeze at −70° C. for 30 min.  
         [0343]    Thaw at room°t (in the dark)  
         [0344]    Add 50 ml ethanol (technical)  
         [0345]    Extract reduced TTC-H by shaking for 1 hour  
         [0346]    Measure absorptions of extracts at 485 nm  
         [0347]    P.S.: reduced TTC-H is not stable→keep in the dark and measure O.D. 485  as soon as possible  
         [0348]    Compare reducing profiles of tested lines versus control line (for population of 30 to 35 plants)  
         [0349]    O.D. 485 (TTC-H) =(O.D. 485 +TTC)−(O.D. 485 −TTC) 
         [0350]    [0350] 
         [0351]    Comparison of the TTC-reducing capacities between samples of different independent experiments can be done by setting the TTC-reducing capacity of control line (C24; Columbia; . . . ) in the different experiments at 100%.  
         [0352]    Lines with a high TTC-reducing capacity are vigorous, while lines with a low TTC-reducing capacity are weak.  
         [0353]    If the addition of niacinamide to the incubation medium results in a higher TTC-reducing capacity indicates to a lower fitness (as shown for C24 and Columbia).  
       Example 7  
     Phenotypic Analyses of the Transgenic Lines Containing Both dsRNA-APP and dsRNA-ZAP Constructs  
       [0354]    The flower phenotype and pollen viability (Alexander staining (Alexander, 1969) and germination asssay) of the T0-lines containing dsRNA-APP and dsRNA-ZAP under the control of tapetum or pollen specific promoters were scored. For Arabidopsis, the T1-generation is obtained by selving or if the plants are male sterile by backcrossing using pollen of non-transformed wild type plants. For  Brassica napus,  the T1-generation is always obtained by backcrossing using pollen of non-transformed plants.  
         [0355]    T1-seed is germinated on kanamycin containing medium after which the resistant plants are scored by means of the ammonium-multiwell assay for phosphinothricine resistance (De Block et al., 1995). One half of the plants that contains both T-DNA&#39;s is transfered to the greenhouse to score the male fertility of the plants, while the other half is used to quantify the vigor of the plants by means of the fitness assay.  
         [0356]    For plants comprising combinations (APP/ZAP) of PCD modulating genes under control of 35S or NOS promoter, a high vigor is observed in a number of the transgenic lines.  
         [0357]    For plants comprising combinations (APP/ZAP) of PCD modulating genes under control of TA29 male sterility is observed in a number of the transgenic lines.  
         [0358]    For plants comprising combinations (APP/ZAP) of PCD modulating genes under control of NTP303 sterile pollen is observed in a number the transgenic lines.  
       REFERENCES  
       [0359]    Alexander (1969) Stain Technology 44, 117  
         [0360]    Alvarez-Gonzalez and Althaus (1989)  Mut. Res.  218, 67-74  
         [0361]    An et al. (1996)  The Plant Cell  8, 15-30  
         [0362]    An et al. (1996b)  Plant Journal  10, 107-121  
         [0363]    Ausubel et al. (1994)  Current Protocols in Molecular Biology, Current Protocols,  USA.  
         [0364]    Ausubel et al. (1987)  Current Protocols in Molecular Biology  1987-1988. New York: Greene Publishing Associates &amp; Wiley-Interscience  
         [0365]    Babiychuk et al (1997)  Proc. Natl. Acad. Sci. USA,  94, 12722-12727  
         [0366]    Beeckman and Engler (1994)  Plant Mol. Biol. Rep.  12, 37-42.  
         [0367]    Chen et al. (1994)  Eur. J. Biochem  224, 135-142  
         [0368]    Citovsky et al. (1994)  Proc. Natl. Acad. Sci. USA,  91, 3210-3214.  
         [0369]    Cohen (1993)  Immunol. Today  14, 126-130  
         [0370]    Collinge and Althaus (1994)  Mol. Gen. Genet.  245, 686-693  
         [0371]    Croy (1993)  Plant Molecular Biology Labfax  jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.  
         [0372]    Cullin and Pompon (1988)  Gene,  65, 203-217  
         [0373]    De Block et al. (1987)  EMBO J.  6, 2513-2518  
         [0374]    De Block et al. (1989)  Plant Physiol.  91: 694  
         [0375]    De Block and Debrouwer (1991)  Theor. Appl. Genet  82, 257-233  
         [0376]    De Block et al. (1995)  Planta  197, 619-626  
         [0377]    de Murcia and Ménissier de Murcia (1994)  Trends Biochem. Sci.  19, 172-176.  
         [0378]    Deblaere et al. (1985)  Nucleic Acids Res.  13, 4777-4788  
         [0379]    Depicker et al. (1982)  J. Mol. App. Gen.  1, 561-573  
         [0380]    Ding et al. (1992)  J. Biol. Chem.  267, 12804-12812  
         [0381]    Dohmen et al. (1991)  Yeast,  7, 691-692  
         [0382]    Doonan et al. (1998) in “ Plant Cell Division”  (Francis, Duditz and Inzé, Eds.) Portland Press, London  
         [0383]    Ellis et al. (1991)  Annu. Rev. Cell. Biol.  7, 663-698  
         [0384]    Gallie and Walbot (1992)  Nucleic Acids Res.  20, 4631-4638  
         [0385]    Gatz et al. (1988)  Proc. Natl. Acad. Sc. USA  85,1394-1397  
         [0386]    Gavrieli et al. (1992)  J. Cell. Biol.  119, 493-501  
         [0387]    Harlow and Lane (1988)  Antibodies: A Laboratory Manual.  Cold Spring Harbor: Cold Spring Harbor Laboratory Press  
         [0388]    Harpster et al. (1988)  Mol. Gen. Genet.  212, 182-190.  
         [0389]    Hawkins and Phillips (1983)  Plant Sci. Lett.  32, 221-224  
         [0390]    Heller et al. (1995)  J. Biol. Chem.  270, 11176-11180  
         [0391]    Herrera-Estrella et al. (1983)  EMBO J.  2, 987  
         [0392]    Hudspeth et al. (1989)  Plant Mol Biol  12, 579-589  
         [0393]    Hurkman and Tanaka (1986)  Plant Physiol.  81, 802-806  
         [0394]    Ikajima et al. (1990)  J. Biol. Chem.  265, 21907-21913  
         [0395]    Jefferson et al. (1987)  EMBO J.  6, 3901-3907  
         [0396]    Kameshita et al. (1984)  J. Biol. Chem.  259, 4770-4776  
         [0397]    Kawamitsu et al. (1 984)  Biochemistry,  23, 3771-3777  
         [0398]    Keil et al. (1989)  EMBO J.  8, 1323-1330  
         [0399]    Keller et al. (1988)  EMBO J.  7, 3625-3633  
         [0400]    Keller et al. (1989)  Genes Devel.  3, 1639-1646  
         [0401]    Kuepper et al. (1990)  J. Biol. Chem.  265, 18721-18724  
         [0402]    Kuge and Jones (1994)  EMBO J.  13, 655-664  
         [0403]    Lazebnik et al. (1994)  Nature  371, 346-347  
         [0404]    Lee et al. (1995)  Plant J.  8, 603-612  
         [0405]    Lepiniec et al. (1995)  FEBS Letters  364, 103-108  
         [0406]    Lindahl et al. (1995)  Trends Biochem. Sci.  20, 405-411  
         [0407]    Long et al. (1996)  Nature,  379, 66-69  
         [0408]    Maliga et al. (1975)  Nature,  255, 401-402  
         [0409]    McBride and Summerfelt (1990)  Plant Mol. Biol.  14, 269-276  
         [0410]    Ménissier de Murcia et al. (1997)  Proc. Natl. Acad. Sci. USA,  94, 7303-7307  
         [0411]    Mogen et al. (1990)  The Plant Cell  2, 1261  
         [0412]    Molinette et al. (1993)  EMBO J.  12, 2109-2117  
         [0413]    Odell et al. (1985)  Nature  313, 810  
         [0414]    O&#39;Farrel (1995)  Biochimie  77, 486-491  
         [0415]    Payne et al. (1976)  Exp. Cell Res.  99, 428-432  
         [0416]    Peleman et al. (1989)  Gene  84, 359-369  
         [0417]    Pennell and Lamb (1997)  The Plant Cell  9, 1157-1168  
         [0418]    Phillips and Hawkins (1985)  J. Exp. Bot.  36,119-128  
         [0419]    Puchta et al. (1995)  Plant J.  7, 203-210  
         [0420]    Sakamoto and Nagatani (1996)  Plant J.  10, 859-868  
         [0421]    Sambrook et al. (1989)  Molecular Cloning: A Laboratory Manual,  Second Edition, Cold Spring Harbor Laboratory Press, NY  
         [0422]    Schreiber et al. (1992)  EMBO J.  11, 3263-3269  
         [0423]    Sheen et al. (1995)  The Plant Journal  8, 777-784  
         [0424]    Shoji et al. (1997)  Plant Cell Physiol.  38, 36-43  
         [0425]    Smulson et al. (1995)  J. Biol. Chem.  270, 119-127  
         [0426]    Studier and Moffat (1986)  J. Mol. Biol.  189, 113-130  
         [0427]    Sugiyama et al. (1995)  J. Plant Res.  108, 351-361  
         [0428]    Terzaghi et al. (1997)  Plant J.  11, 967-982  
         [0429]    Valvekens et al. (1988)  PNAS  85, 5536  
         [0430]    von Arnim and Deng (1994)  Cell,  79, 1035-1045  
         [0431]    Wang et al. (1995)  Genes Dev.  9, 509-520  
         [0432]    Wang et al. (1996)  Plant Cell  8, 375-391  
         [0433]    Wang et al. (1997)  Genes Dev.  11, 2347-2358  
         [0434]    Weimann et al. (1994)  Plant J.  5, 559-569  
         [0435]    Wetering (1994) PhD thesis, Katholieke Universiteis Nijmegen  
         [0436]    Walkerpeach and Velten (1995) In: Gelvin SB, Schilperoort RA, Verma DPS (eds) Plant Molecular Biology Manual pp B1/1-B1/19. Kluwer Academic Publishers, Dordrecht  
         [0437]    Wilbur and Lipmann (1983)  Proc. Natl. Acad. Sci. USA  80, 706  
         [0438]    Willmitzer and Wagner (1982) In  ADP - Ribosylation Reactions  (Hayashi, O. and Ueda, K., eds). New York: Academic Press, pp. 241-252  
         [0439]    Zhang et al. (1994)  Science,  263, 687-689  
     
       
       
         1 
         
           
             
21 
 
           
           
             
               3211 BASES  
               NUCLEIC ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              1 

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 

 
           
           
             
               969 RESIDUES  
               AMINO ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              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 
            740                 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 

 
           
           
             
               2295 BASES  
               NUCLEIC ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              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                 115 

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 
                520                 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 

 
           
           
             
               653 RESIDUES  
               AMINO ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              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 

 
           
           
             
               2147 BASES  
               NUCLEIC ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              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 
            210                 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 

 
           
           
             
               637 RESIDUES  
               AMINO ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              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 

 
           
           
             
               16 RESIDUES  
               AMINO ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              7 

Arg Gly Xaa Xaa Xaa Xaa Gly Xaa Lys Xaa Xaa Xaa Xaa Xaa Arg Leu 
  1               5                  10                  15 

 
           
           
             
               33 RESIDUES  
               AMINO ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              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 

 
           
           
             
               30 RESIDUES  
               AMINO ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              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 

 
           
           
             
               3212 BASES  
               NUCLEIC ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              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                 705                 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 

 
           
           
             
               980 RESIDUES  
               AMINO ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              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 

 
           
           
             
               1010 RESIDUES  
               AMINO ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              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 

 
           
           
             
               25 BASES  
               NUCLEIC ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              13 

ccgaattcgg ntayatgtty ggnaa                                           25 

 
           
           
             
               25 BASES  
               NUCLEIC ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              14 

ccgaattcac natrtaytcr ttrta                                           25 

 
           
           
             
               25 BASES  
               NUCLEIC ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              15 

gggaccatgt agtttatctt gacct                                           25 

 
           
           
             
               26 BASES  
               NUCLEIC ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              16 

gacctcgtac cccaactctt ccccat                                          26 

 
           
           
             
               36 BASES  
               NUCLEIC ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              17 

aagtcgacgc ggccgccaca cctagtgcca ggtcag                               36 

 
           
           
             
               24 BASES  
               NUCLEIC ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              18 

atctcaattg tacatttctc agga                                            24 

 
           
           
             
               31 BASES  
               NUCLEIC ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              19 

aggatcccat ggcgaacaag ctcaaagtga c                                    31 

 
           
           
             
               26 BASES  
               NUCLEIC ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              20 

aggatcctta gtgcttgtag ttgaat                                          26 

 
           
           
             
               4947 BASES  
               NUCLEIC ACID  
               SINGLE  
               LINEAR  
             
             
               not provided 
             
              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