AU2013204574A1 - DNA-sequence and recombinant production of group 4 major allergens from cereals - Google Patents

Abstract

C:\NRPorbl\DOCCGRSW48542_1.DOC-2307/2012 DNA sequence, and recombinant preparation of group 4 major allergens from cereals Abstract The invention relates to the preparation of DNA sequences of group 4 major allergens from cereals. The invention also includes fragments, novel combinations of partial sequences and point mutations with hypoallergenic effects. The recombinant DNA molecules and the derived polypeptides, fragments, novel combinations of partial sequences and variants can be used for therapy of pollen-allergy diseases. The proteins produced by recombination can be applied to in-vitro and in-vivo diagnosis of pollen allergies.

Description

WO 2005/059136 PCT/EP2004/013664 DNA sequence, and recombinant preparation of group 4 major allergens from cereals 5 Background of the invention 10 The present invention relates to the provision of DNA sequences of group 4 major allergens from cereals (Triticeae). The invention also encompasses fragments, new combinations of partial sequences and point mutants hav ing a hypoallergenic action. The recombinant DNA molecules and the derived polypeptides, fragments, new combinations of partial sequences 15 and variants can be utilised for the therapy of pollen-allergic diseases. The proteins prepared by recombinant methods can be employed for in vitro and in vivo diagnosis of pollen allergies. 20 Type 1 allergies are of importance worldwide. Up to 20% of the population in industrialised countries suffer from complaints such as allergic rhinitis, conjunctivitis or bronchial asthma. These allergies are caused by allergens present in the air (aeroallergens) which are released by sources of various 25 origin, such as plant pollen, mites, cats or dogs. Up to 40% of these type 1 allergy sufferers in turn exhibit specific IgE reactivity with grass pollen allergens, inter alia cereal pollen allergens (Freidhoff et al., 1986, J. Allergy Clin. Immunol. 78, 1190-2001). Of the cereal pollen allergens, the allergens of rye have particular importance. 30 The substances which trigger type 1 allergy are proteins, glycoproteins or polypeptides. After uptake via the mucous membranes, these allergens react with the IgE molecules bonded to the surface of mast cells in sensi 35 tised individuals. If two IgE molecules are crosslinked to one another by an allergen, this results in the release of mediators (for example histamine, WO 2005/059136 PCT/EP2004/013664 -2 prostaglandins) and cytokines by the effector cell and thus in the corres ponding clinical symptoms. A distinction is made between major and minor allergens, depending on the 5 relative frequency with which the individual allergen molecules react with the IgE antibodies of allergy sufferers. The allergens from the pollen of various species from the family of the 10 grasses (Poaceae) are divided into groups which are homologous amongst one another. In particular, the molecules of major allergen group 4 have high immuno logical cross-reactivity with one another both with monoclonal murine anti 15 bodies and also with human IgE antibodies (Fahlbusch et al., 1993 Clin. Exp. Allergy 23:51-60; Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98:1065-1072; Su et al., 1996, J. Allergy Clin. Immunol. 97:210; Fahlbusch et al., 1998, Clin. Exp. Allergy 28:799-807; Gavrovic-Jankulovic et al., 2000, Invest. Allergol. Clin. Immunol. 10 (6):361-367; Stumvoll et al. 2002, Biol. 20 Chem. 383:1383-1396; Grote et al., 2002, Biol. Chem. 383:1441-1445; Andersson and Lidholm, 2003, Int. Arch. Allergy Immunol. 130:87-107; Mar, 2003, Clin. Exp. Allergy, 33 (1):43-51). 25 A complete DNA sequence is hitherto not known for any of the group 4 major allergens. From the group 4 allergen from Dactylus glomerata, it has hitherto only 30 been possible for peptides to be obtained by enzymatic degradation and sequenced: DIYNYMEPYVSK (SEQ ID NO 13), VDPTDYFGNEQ (SEQ ID NO 14), ARTAWVDSGAQLGELSY (SEQ ID NO 15) 35 and GVLFNIQYVNYWFAP (SEQ ID NO 16, Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98: 1065-1072).

WO 2005/059136 PCT/EP2004/013664 -3 Peptides have also been obtained from the group 4 allergen of sub-tropical Bermuda grass (Cynodon dactylon) by proteolysis and sequenced: KTVKPLYIITP (SEQ ID NO 17), 5 KQVERDFLTSLTKDIPQLYLKS (SEQ ID NO 18), TVKPLYIITPITAAMI (SEQ ID NO 19), LRKYGTAADNVIDAKWDAQGRLL (SEQ ID NO 20), KWQTVAPALPDPNM (SEQ ID NO 21), VTWIESVPYIPMGDK (SEQ ID NO 22), 10 GTVRDLLXRTSNIKAFGKY (SEQ ID NO 23), TSNIKAFGKYKSDYVLEPIPKKS (SEQ ID NO 24), YRDLDLGVNQVVG (SEQ ID NO 25), SATPPTHRSGVLFNI (SEQ ID NO 26) 15 and AAAALPTQVTRDIYAFMTPYVSKNPRQAYVNYRDLD (SEQ ID NO 27, Liaw et al., 2001, Biochem. Biophys. Research Communication 280: 738 743). For Lolium perenne, peptide fragments having the following sequences 20 have been described for the basic group 4 allergen: FLEPVLGLIFPAGV (SEQ ID NO 28) and GLIEFPAGV (SEQ ID NO 29, Jaggi et al., 1989, int. Arch. Allergy Appl. Immunol. 89: 342-348). 25 As the first sequence of a group 4 allergen, the still unpublished sequence of Phi p 4 from Phieum pretense (SEQ ID NO 11) has been elucidated by the inventors of the present patent application and described in Interna tional Application WO 04/000881. 30 Nothing is hitherto known on the sequences of the group 4 major allergens from cereals (Triceae). 35 The object on which the present invention was based therefore consisted in the provision of DNA sequences of group 4 major allergens from cereals, in WO 2005/059136 PCT/EP2004/013664 -4 particular the allergen Sec c 4 from rye (Secale cerale) (SEQ ID NO 1, 3), Hor v 4 from barley (Hordeum vulgare) (SEQ ID NO 5) and Tr a 4 from wheat (Triticum aestivum) (SEQ ID NO 7, 9) and of corresponding recom binant DNA molecules on the basis of which the allergens can be ex 5 pressed as protein and made available, as such or in modified form, for pharmacologically significant exploitation. The sequence of Phi p 4 (SEQ ID NO 11) was the starting point for the present invention. 10 List of sequences according to the invention The DNA and protein sequences of the mature allergens in accordance 15 with SEQ ID NO 1-10 are preceded by a signal sequence. The encoding region ends with the TGA or TAG stop codons in the DNA sequences. - DNA sequence of Sec c 4. (a) Isoform Sec c 4.01 (SEQ ID NO 1), (b) isoform Sec c 4.02 (SEQ ID NO 3). 20 - Protein sequences (SEQ ID NO 2,4) derived from the DNA sequences in accordance with SEQ ID NO 1 and 3. - DNA sequence of Hor v 4 (SEQ ID NO 5). - Protein sequence (SEQ ID NO 6) derived from the DNA sequence in 25 accordance with SEQ ID NO 5. - DNA sequence of Tri a 4. (a) Isoform Tr a 4.01 (SEQ ID NO 7), (b) iso form Tri a 4.02 (SEQ ID NO 9). - Protein sequences (SEQ ID NO 8, 10) derived from the DNA sequences 30 in accordance with SEQ ID NO 7 and 9. - DNA sequence of Phl p 4 (SEQ ID NO 11), in accordance with SEQ ID NO 5 from WO 04/000881. - Protein sequence of PhI p 4 (SEQ ID NO 12), in accordance with SEQ ID NO 6 from WO 04/000881. 35 WO 2005/059136 PCT/IEP2004/013664 -5 Description of the invention The present invention now provides for the first time DNA sequences of the 5 cereal pollen major allergens Sec c 4, Hor v 4 and Tri a 4, in accordance with SEQ ID NO 1, 3, 5, 7, and 9. The present invention therefore relates to DNA molecules selected from the nucleotide sequences in accordance with SEQ ID NO 1, 3, 5, 7, and 9. 10 The invention furthermore relates to sequences homologous to the DNA sequences according to the invention and corresponding DNA molecules of group 4 allergens from other Poaceae, such as, for example, Lolium 15 perenne, Dactylis glomerata, Poa pratensis, Cynodon dactylon and Holcus lanatus, which, owing to the sequence homology that exists, hybridise with the DNA sequences according to the invention under stringent conditions, or have immunological cross-reactivity with respect to the allergens 20 according to the invention. The invention also encompasses fragments, new combinations of partial sequences and point mutants having a hypoallergenic action. 25 The invention therefore furthermore relates to corresponding partial se quences, a combination of partial sequences, or replacement, elimination or addition mutants which encode an immunomodulatory, T-cell-reactive fragment of a group 4 allergen from the Poaceae. 30 With knowledge of the DNA sequence of the naturally occurring allergens, it is now possible to prepare these allergens as recombinant proteins which can be used in the diagnosis and therapy of allergic diseases (Scheiner 35 and Kraft, 1995, Allergy 50: 384-391).

WO 2005/059136 PCT/EP2004/013664 -6 A classical approach to effective therapeutic treatment of allergies is spe cific immunotherapy or hyposensitisation (Fiebig, 1995, Allergo J. 4 (6): 336-339, Bousquet et al., 1998, J. Allergy Clin. Immunol. 102 (4): 558-562). In this method, the patient is injected subcutaneously with natural allergen extracts in increasing doses. However, there is a risk in this method of allergic reactions or even anaphylactic shock. In order to minimise these risks, innovative preparations in the form of allergoids are employed. These are chemically modified allergen extracts which have significantly reduced 10 IgE reactivity, but identical T-cell reactivity compared with the untreated extract (Fiebig, 1995, Allergo J. 4 (7): 377-382). Even more substantial therapy optimisation would be possible with aller gens prepared by recombinant methods. Defined cocktails of high-purity 15 allergens prepared by recombinant methods, optionally matched to the individual sensitisation patterns of the patients, could replace extracts from natural allergen sources since these, in addition to the various allergens, contain a relatively large number of immunogenic, but non-allergenic sec ondary proteins. 20 Realistic perspectives which may result in reliable hyposensitisation with expression products are offered by specifically mutated recombinant aller gens in which IgE epitopes are specifically deleted without impairing the T-cell epitopes which are essential for therapy (Schramm et al., 1999, 25 J. Immunol. 162: 2406-2414). A further possibility for therapeutic influencing of the disturbed TH cell equilibrium in allergy sufferers is immunotherapeutic DNA vaccination, 30 which involves treatment with expressible DNA which encodes the relevant allergens. Initial experimental evidence of allergen-specific influencing of the immune response has been furnished in rodents by injection of aller gen-encoding DNA (Hsu et al., 1996, Nature Medicine 2 (5): 540-544). 35 WO 2005/059136 PCT/EP20041013664 -7 The present invention therefore also relates to a DNA molecule described above or below as medicament and to a corresponding recombinant expression vector as medicament. 5 The corresponding proteins prepared by recombinant methods can be employed for therapy and for in vitro and in vivo diagnosis of pollen aller gies. For preparation of the recombinant allergen, the cloned nucleic acid is 10 ligated into an expression vector, and this construct is expressed in a suit able host organism. After biochemical purification, this recombinant aller gen is available for detection of IgE antibodies by established methods. 15 The present invention therefore furthermore relates to a recombinant expression vector comprising a DNA molecule described above or below, functionally linked to an expression control sequence, and a host organism transformed with said DNA molecule or said expression vector. 20 The invention also relates to the use of at least one DNA molecule de scribed above or at least one expression vector described above for the preparation of a medicament for the immunotherapeutic DNA vaccination of patients with allergies in the triggering of which group 4 allergens from 25 the Poaceae, preferably Triticeae, in particular Sec c 4, Hor v 4, Tri a 4, are involved and/or for the prevention of such allergies. As already stated, the invention can be used as an essential component in 30 a recombinant allergen- or nucleic acid-containing preparation for specific immunotherapy. A number of possibilities arise here. On the one hand, the protein with an unchanged primary structure may be a constituent of the preparation. On the other hand, a hypoallergenic (allergoid) form can be used in accordance with the invention for therapy in order to avoid unde 35 sired side effects by specific deletion of IgE epitopes of the molecule as a whole or the production of individual fragments which encode T-cell epi- WO 2005/059136 PCT/EP2004/013664 topes. Finally, the nucleic acid per se, if ligated with a eukaryotic expres sion vector, gives a preparation which, when applied directly, modifies the allergic immune state in the therapeutic sense. 5 The present invention furthermore relates to the polypeptides encoded by one or more of the DNA molecules described above, preferably in their property as medicament. These are proteins corresponding to an amino acid sequence in accor 10 dance with SEQ ID NO 2, 4, 6, 8 or 10. In particular, these are mature pro teins (without signal sequence component), beginning with amino acid 23 (SEQ ID NO 2, 4 and 6) and with amino acid 22 (SEQ ID NO 8, 10). The invention furthermore relates to proteins which contain these amino acid 15 sequences or parts of these sequences. The invention accordingly also relates to a process for the preparation of such polypeptides by cultivation of a host organism and isolation of the 20 corresponding polypeptide from the culture. The invention likewise relates to the use of at least one polypeptide de scribed above for the preparation of a medicament for the diagnosis and/or treatment of allergies in the triggering of which group 4 allergens from the 25 Poaceae, preferably Triticeae, in particular Sec c 4, Hor v 4, Tri a 4, are involved and for the prevention of such allergies. 30 When determining the protein and DNA sequences according to the inven tion, the following procedure was followed: Sec c 4 from rye 35 1. Starting from the DNA sequence of PhI p 4 (SEQ ID NO 12, WO 04/000881), specific primers (Table 1) derived from the Phl p 4 sequence WO 2005/059136 PCT/EP2004/013664 -9 were generated. Five clones were obtained from rye pollen DNA by PCR with primers #87 and #83. The amplified Sec c 4 gene fragment I corre sponding to these clones encodes a polypeptide corresponding to amino acids 68-401 of Phi p 4 (SEQ ID NO 12). 5 2. An EST database search was carried out with the partial Sec c 4 se quence. However, no homologous sequences were found in EST data bases specialising in rye. Instead, individual, homologous, non-overlapping 10 EST fragments were found in EST databases specialising in barley and wheat. Individual EST fragments extend into the 5'-UTR region and others into the 3'-UTR region (UTR = untranslated region) of the corresponding genes. 15 3. However, a complete group 4 gene from wheat or barley cannot be con structed from the EST sequences found in the databases since these se quences do not overlap and a homologous group 4 gene is not known. However, it was possible to assign these EST sequences with reference to 20 the Phi p 4 sequence (SEQ ID NO 11) and the Sec c 4 fragment obtained in step 1 and these served as template for the preparation of PCR primers. 4. With the aid of primers #195 and #189 prepared in this way, three clones 25 were obtained by PCR. Primer #195 was derived from a barley EST sequence, primer #189 is a Phi p 4-specific primer and overlaps the PhI p 4 stop codon and the codons of the 10 C-terminal PhI p 4 amino acids. The Sec c 4 gene fragment 2 amplified in this way encodes a polypeptide, 30 beginning within the signal sequence and ending with the position corresponding to position 490 of Phi p 4. This polypeptide covers the N terminal of Sec c 4. 5a. Three further clones were obtained by PCR with primers #195 and 35 #202. Both primers were derived from barley EST sequences. The ampli- WO 2005/059136 PCT/EP2004/013664 -10 fied Sec c 4 gene 3 encodes the corresponding amino acids beginning within the signal sequence and ending at the C terminal of Sec c 4. The complete sequence of mature Sec c 4 is thus present in the sequence determined. 5 The next two steps 5b and 5c serve to double-check the result obtained in step 5a: 10 5b. A further clone was obtained by PCR with primers #195 and #203. Primer #195 was derived from a barley EST sequence, primer #203 from a wheat EST sequence. The amplified Sec c 4 gene encodes the corre sponding amino acids beginning within the signal sequence and ending at 15 the C terminal of Sec c 4. The complete sequence of mature Sec c 4 is therefore present in the sequence determined. 5c. A further clone was obtained by PCR with primers #195 and #198. Also primer #198 The amplified Sec c 4 gene encodes the corresponding amino 20 acids beginning within the signal sequence and ending at the C terminal of Sec c 4. The complete sequence of mature Sec c 4 is therefore present in the sequence determined. 25 Two isoforms Sec c 4.01 and 4.02 were found. The mature allergens begin with amino acid 23 of the sequences in accordance with SEQ ID NO 2, 4 and 6. 30 Hor v 4 from barley With the aid of the Sec c 4 sequences obtained as described above, homo logous EST fragments were found in EST databases of Hordeum vulgare. These fragments overlap, but not to give a complete gene. With reference 35 to the EST sequences found, however, it was possible to generate Hor v WO 2005/059136 PCT/EP2004/013664 - 11 4-specific primers, which were used for amplification of the Hor v 4 gene from genomic DNA. In total, 15 clones were analysed. 54 clones were obtained by PCR with primers #195 and #198. 4 clones were obtained by PCR with primers #195 and #202. 3 clones were obtained by PCR with primers #194 and #198. 4 clones were obtained by PCR with primers #194 and #202. 10 The derived protein sequence begins within the signal sequence of Hor v 4 and extends to the C-terminal end of the protein (from amino acid 23 of SEQ ID NO 6). 15 Tri a 4 from wheat With the aid of the Sec c 4 sequences obtained as described above, homo logous EST fragments were found in EST databases of Triticum aestivum. 20 These fragments overlap, but not to give a complete gene. With reference to the EST sequences found, however, it was possible to generate the Tri a 4-specific primers #199, #203, #204 and #206, which were used for ampli fication of the Tri a 4 gene from genomic DNA. 25 In total, 13 clones were analysed. 4 clones were obtained by PCR with primers #204 and #203. 4 clones were obtained by PCR with primers #204 and #199. 30 3 clones were obtained by PCR with primers #206 and #203. 4 clones were obtained by PCR with primers #206 and #199. The derived protein sequences begin within the signal sequence of Tri a 4 and extend to the C-terminal end of the protein. 35 Two variants Tr a 4.01 (from amino acid 22 of SEQ ID NO 8) and Tri a 4.02 (from amino acid 22 of SEQ ID NO 10) were found.

WO 2005/059136 PCT/EP2004/013664 -12 In order to prepare the recombinant allergens according to the invention, the DNA sequences in accordance with SEQ ID NO 1, 3, 5, 7 and 9 were incorporated into expression vectors (for example pProEx, pSE 380). 5 E. coli-optimised codons were used for the N-terminal amino acids known from the protein sequencing. After transformation in E. coli, expression and purification of the recombi 10 nant allergens according to the invention by various separation techniques, the proteins obtained were subjected to a refolding process. Both allergens can be employed for highly specific diagnosis of grass pol len allergies. This diagnosis can be carried out in vitro by detection of spe 15 cific antibodies (IgE, IgG1 - 4, IgA) and reaction with IgE-loaded effector cells (for example basophiles from blood) or in vivo by skin test reactions and provocation at the reaction organ. The reaction of the allergens according to the invention with T-lymphocytes 20 from grass pollen allergy sufferers can be detected by allergen-specific stimulation of the T-lymphocytes for proliferation and cytokine synthesis both with T-cells in freshly prepared blood lymphocytes and also on estab lished nSec c 4, nHor v 4 or nTri a 4-reactive T-cell lines and clones. 25 The triplets encoding the cysteines were modified by site-specific muta genesis in such a way that they encode other amino acids, preferably ser ine. Both variants in which individual cysteines have been replaced and 30 those in which various combinations of 2 cysteine residues or all cysteines have been modified were prepared. The expressed proteins of these cys teine point mutants have greatly reduced or zero reactivity with IgE anti bodies from allergy sufferers, but react with the T-lymphocytes from these 35 patients.

WO 2005/059136 PCTIEP2004/013664 -13 The present invention therefore furthermore relates to a DNA molecule described above or below in which one, a plurality of or all the cysteine resi dues of the corresponding polypeptide have been replaced with another 5 amino acid by site-specific mutagenesis. The immunomodulatory activity of hypoallergenic fragments which corres pond to polypeptides having T-cell epitopes and that of the hypoallergenic point mutants (for example cysteine replacements) can be detected by their 10 reaction with T-cells from grass pollen allergy sufferers. Such hypoallergenic fragments or point mutants of the cysteines can be employed as preparations for hyposensitisation of allergy sufferers since 15 they react with the T-cells with equal effectiveness, but result in reduced IgE-mediated side effects owing to the reduced or entirely absent IgE reac tivity. If the nucleic acids encoding the hypoallergenic allergen variants according 20 to the invention or the unmodified DNA molecules according to the inven tion are ligated with a human expression vector, these constructs can likewise be used as preparations for immunotherapy (DNA vaccination). 25 Finally, the present invention relates to pharmaceutical compositions com prising at least one DNA molecule described above or at least one expres sion vector described above and optionally further active ingredients and/or adjuvants for the immunotherapeutic DNA vaccination of patients with 30 allergies in the triggering of which group 4 allergens from the Poaceae, preferably Triticeae, in particular Sec c 4, Hor v 4, Tr a 4, are involved and/or for the prevention of such allergies. 35 A further group of pharmaceutical compositions according to the invention comprises at least one polypeptide described above instead of the DNA and is suitable for the diagnosis and/or treatment of said allergies.

WO 2005/059136 PCT/EP2004/013664 -14 Pharmaceutical compositions in the sense of the present invention com prise, as active ingredients, a polypeptide according to the invention or an expression vector and/or respective pharmaceutically usable derivatives thereof, including mixtures thereof in all ratios. The active ingredients according to the invention can be brought into a suitable dosage form here together with at least one solid, liquid and/or semi-liquid excipient or adju vant and optionally in combination with one or more further active ingredi 10 ents. Particularly suitable adjuvants are immunostimulatory DNA or oligonucleo tides having CpG motives. 15 These compositions can be used as therapeutic agents or diagnostic agents in human or veterinary medicine. Suitable excipients are organic or inorganic substances which are suitable for parenteral administration and do not adversely affect the action of the active ingredient according to the invention. Suitable for parenteral use are, in particular, solutions, preferably 20 oil-based or aqueous solutions, furthermore suspensions, emulsions or implants. The active ingredient according to the invention may also be lyophilised and the resultant lyophilisates used, for example, for the prepa ration of injection preparations. The compositions indicated may be steril 25 ised and/or comprise adjuvants, such as preservatives, stabilisers and/or wetting agents, emulsifiers, salts for modifying the osmotic pressure, buffer substances and/or a plurality of further active ingredients. Furthermore, sustained-release preparations can be obtained by corres 30 ponding formulation of the active ingredient according to the invention - for example by adsorption on aluminium hydroxide. The invention thus also serves for improving in vitro diagnosis as part of 35 allergen component-triggering identification of the patient-specific sensiti sation spectrum. The invention likewise serves for the preparation of sig- WO 2005/059136 PCTIEP2004/013664 -15 nificantly improved preparations for the specific immunotherapy of grass pollen allergies. Table I Primers used 5 a) Sec c 4 Primer SEQ ID NO Sequence number 10 #0083 30 GGCTCCCGGGGCGAACCAGTAG #0087 31 ACCAACGCCTCCCACATCCAGTC #0189 32 GATAAGCTTCTCGAGTGATTAGTACTTTTTGAT CAGCGGCGGGATGCTC #0195 33 GCTCTCGATCGGCTACAATGGCG #0198 34 CACGCACTACAAATCTCCATGCAAG #0202 35 CATGCTTGATCCTTATTCTACTAGTTGGGC 15 #0203 36 TACGCACGATCCTTATTCTACTAGTTGGGC a) Horv4 Primer SEQ ID NO Sequence 20 number #0194 37 GCCTTGTCCTGCCACCACGCCGCCGCCACC #0195 38 GCTCTCGATCGGCTACAATGGCG #0198 39 CACGCACTACAAATCTCCATGCAAG #0202 40 CATGCTTGATCCTTATTCTACTAGTTGGGC 25 c) Tri a 4 Primer SEQ ID NO Sequence number 30 #0199 41 CACGCACTAAATCTCCATGCAAG #0203 42 TACGCACGATCCTTATTCTACTAGTTGGGC #0204 43 AAGCTCTATCGCCTACAATGGCG #0206 44 GGTGCTCCTCTTCTGCGCCTTGTCC 35 C:NRPcrtblDCO\GRS%4885421 .DOC-23/07/2012 - 15a Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as, an acknowledgement or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (22)

1. A DNA molecule corresponding to a nucleotide sequence of a cereal 5 pollen major allergen selected from one of the sequences in accor dance with SEQ ID NO 1, 3, 5, 7, and 9.

2. A DNA molecule which hybridises with a DNA molecule according to Claim I under stringent conditions and originates from DNA sequences 10 from Poaceae species.

3. A DNA molecule, encoding a polypeptide, which cross-reacts immuno logically with the major allergens Sec c 4, Hor v 4 or Tr a 4 from Secale 15 cerale, Hordeum vulgare or Titicum aestivum and originates from DNA sequences from Poaceae species.

4. A DNA molecule, corresponding to a partial sequence or a combination 20 of partial sequences according to one or more of Claims 1 to 3, which encodes an immunomodulatory, T-cell-reactive fragment of a group 4 allergen from the Poaceae.

5. A DNA molecule, corresponding to a nucleotide sequence according to 25 one or more of Claims I to 4, encoding an immunomodulatory T-cell reactive fragment, characterised in that said nucleotide sequence has been specifically modified by specific mutation of individual codons, elimination or addition. 30

6. A DNA molecule according to Claim 5, characterised in that the said mutation results in the replacement of one, a plurality of or all cysteines of the corresponding polypeptide with another amino acid. 35 WO 2005/059136 PCT/EP2004/013664 -17

7. A recombinant DNA expression vector or a cloning system comprising a DNA molecule according to one or more of Claims 1 to 6, functionally linked to an expression control sequence. 5

8. A host organism transformed with a DNA molecule according to one or more of Claims 1 to 6 or an expression vector according to Claim 7.

9. A process for the preparation of a polypeptide encoded by a DNA se 10 quence according to one or more of Claims 1 to 6 by cultivation of a host organism according to Claim 8 and isolation of the corresponding polypeptide from the culture. 15

10.A polypeptide corresponding to one of the amino acid sequences in accordance with SEQ ID NO 2,4, 6, 8 and 10, which is encoded by a DNA sequence according to one or more of Claims 1 to 6. 20

11.A polypeptide corresponding to the mature allergen of the amino acid sequences according to Claim 10, selected from the following group of amino acid sequences - one of the amino acid sequences in accordance with SEQ ID NO 2, 4, or 6, beginning with amino acid 23, 25 - one of the amino acid sequences in accordance with SEQ ID NO 8 or 10, beginning with amino acid 22.

12.A polypeptide according to Claim 10 or 11 as medicament. 30

13.A pharmaceutical composition comprising at least one polypeptide according to Claim 12 and optionally further active ingredients and/or adjuvants for the diagnosis and/or treatment of allergies in the triggering 35 of which group 4 allergens from the Poaceae are involved. WO 2005/059136 PCT/EP20041013664 -18

14. Use of at least one polypeptide according to Claim 12 for the prepara tion of a medicament for the diagnosis and/or treatment of allergies in the triggering of which group 4 allergens from the Poaceae are involved 5 and/or for the prevention of such allergies.

15.A DNA molecule according to one or more of Claims 1 to 6 as medica ment. 10

16.A recombinant expression vector according to Claim 7 as medicament.

17.A pharmaceutical composition comprising at least one DNA molecule according to Claim 15 or at least one expression vector according to 15 Claim 16 and optionally further active ingredients and/or adjuvants for the immunotherapeutic DNA vaccination of patients with allergies in the triggering of which group 4 allergens from the Poaceae are involved and/or for the prevention of such allergies. 20

18. Use of at least one DNA molecule according to Claim 15 or at least one expression vector according to Claim 16 fore the preparation of a medi cament for the immunotherapeutic DNA vaccination of patients with allergies in the triggering of which group 4 allergens from the Poaceae 25 are involved and/or for the prevention of such allergies. 30 35 WO 2005/059136 PCT/EP2004/013664 -1- Sequence Listing <110> Merck Patent GmbH <120> DNA sequence, and recombinant preparation of group 4 major allergens from cereals <130> P 03/239 <140> DE 10359351.9 <141> 2003-12-16 <160> 44 <170> Patentln version 3.1 <210> 1 <211> 1603 <212> DNA <213> Secale cereale <220> <221> stop_codon <222> (1555)..(1557) <223> <220> <221> signal sequence DNA <222> (1)..(66) <223> 2013204574 12 Apr 2013 2013204574 12 Apr 2013 ) ) WO 2005/059136 PCT/EP2004/013664 -2- <220> <221> signal_sequence_PROT <222> (1)..(22) <223> <220> <221> CDS <222> (1)..(1557) <223> <400> 1 aac tat agg gcc ttc gcg ctg gcg ctc ctc ttc tgc gcc ttg tee tgc 48 Asn Tyr Arg Ala Phe Ala Leu Ala Leu Leu Phe Cys Ala Leu Ser Cys 1 5 10 15 caa gcc gee gcg gcc gcc tac gcg ccc gtg cct gcc aag gcg gac ttc 96 Gln Ala Ala Ala Ala Ala Tyr Ala Pro Val Pro Ala Lys Ala Asp Phe 20 25 30 ctc gga tgc etc atg aag gag ata ccg gcc cgc etc ctc tac gcc aag 144 Leu Gly Cys Leu Met Lys Glu Ile Pro Ala Arg Leu Leu Tyr Ala Lys 35 40 45 agc tog cct gac tac ccc acc gtg ctg gcg cag acc atc agg aac tcg 192 Ser Ser Pro Asp Tyr Pro Thr Val Leu Ala Gln Thr Ile Arg Asn Ser 50 55 60 cgg tgg tcg tcg ccg cag aac gtg aag ccg atc tac atc atc acc ccc 240 Arg Trp Ser Ser Pro Gln Asn Val Lys Pro Ile Tyr Ile Ile Thr Pro 65 70 75 80 acc aac gcc tcg cac ate cag tcc gcg gtg gtg tgc ggc cgc egg cac 288 Thr Asn Ala Ser His Ile Gin Ser Ala Val Val Cys Gly Arg Arg His 85 90 95 ggc atc cgc etc ego gtg cgg agc ggc ggc cac gac tac gag ggc ctg 336 Gly Ile Arg Leu Arg Val Arg Ser Gly Gly His Asp Tyr Glu Gly Leu 100 105 110 'Leg tac cgg tct gag aaa ccc gag acg ttc gcc gtc gtc gac etc aac 384 Ser Tyr Arg Ser Glu Lys Pro Glu Thr Phe Ala Val Val Asp Leu Asn 115 120 125 aag atg cgg gca gtg tcg gtc gac ggc tac gcc cgc acg gcg tgg gtc 432 Lys Met Arg Ala Val Ser Val Asp Gly Tyr Ala Arg Thr Ala Trp Val 130 135 140 gaa tee ggc gcg cag etc ggc gag etc tac tac gcg ate gcc aag aac 480 Glu Ser Gly Ala Gin Leu Gly Glu Leu Tyr Tyr Ala Ile Ala Lys Asn 145 150 155 160 agc ccc gtg etc gcg ttc ccg get ggc gtc tgc ccg tee ate ggc gtc 528 Ser Pro Val Leu Ala Phe Pro Ala Gly Val Cys Pro Ser Ile Gly Val 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PC17/EF2004/013664 -3- 165 170 175 ggc ggc aac ttc gca ggc ggc ggc ttt ggc atg ctg ctg cgc aag tac 576 Gly Gly Asn Phe Ala Gly Gly Gly Phe Gly Met Leu Leu Arg Lys Tyr 180 185 190 ggc atc gcc get gag aac gtc atc gac gtc aag gtg gtc gac ccc aac 624 Gly Ile Ala Ala Glu Asn Val Ile Asp Val Lys Val Val Asp Pro Asn 195 200 205 ggc aag ctg etc gac aag agc tee atg age gcg gac cac ttc tgg gcc 672 Gly Lys Leu Leu Asp Lys Ser Ser Met Ser Ala Asp His Phe Trp Ala 210 215 220 gtt agg ggc ggc ggc gga gag agc ttt ggc atc gtc gtc tcg tgg cag 720 Val Arg Gly Gly Gly Gly Glu Ser Phe Gly Ile Val Val Ser Trp Gin 225 230 235 240 gtg aag ctc ctg ccg gtg cct ccc ace gtg acc gtg etc aag atc ccc 768 Val Lys Leu Leu Pro Val Pro Pro Thr Val Thr Val Leu Lys Ile Pro 245 250 255 aag acg gtg caa gaa ggc gee ata gac etc gtc aac aag tgg cag ctg 816 Lys Thr Val Gin Glu Gly Ala Ile Asp Leu Val Asn Lys Trp Gin Leu 260 265 270 gtc ggg ccg gca ctt ccc ggc gac etc atg atc cgc atc ate ctt gcc 864 Val Gly Pro Ala Leu Pro Gly Asp Leu Met Ile Arg Ile Ile Leu Ala 275 280 285 ggg aac agc gcg acg ttc gag gcc atg tac ctg ggc ace tgc agt ace 912 Gly Asn Ser Ala Thr Phe Glu Ala Met Tyr Leu Gly Thr Cys Ser Thr 290 295 300 ctg acg ccg ctg atg age agc aaa ttc ccc gag ctt ggc atg aac ccc 960 Leu Thr Pro Leu Met Ser Ser Lys Phe Pro Glu Leu Gly Met Asn Pro 305 310 315 320 tag cac tgc aac gag atg tee tgg ate aag tcc ate ccc ttc ate cac 1008 (\_ Ser His Cys Asn Glu Met Ser Trp Ile Lys Ser Ile Pro Phe Ile His 325 330 335 ctc ggc aag cag aac etc gac gac etc etc aac cgg aac aac acc ttc 1056 Zeu Gly Lys Gln Asn Leu Asp Asp Leu Leu Asn Arg Asn Asn Thr Phe 340 345 350 aaa cca ttc gcc gaa tac aag tog gac tac gtg tac cag ccc ttc ccc 1104 Lys Pro Phe Ala Glu Tyr Lys Ser Asp Tyr Val Tyr Gin Pro Phe Pro 355 360 365 aag ccc gtg tgg gag cag ate ttc ggc tgg ctt gtg aag ccc ggc gcg 1152 Lys Pro Val Trp Glu Gin Ile Phe Gly Trp Leu Val Lys Pro Gly Ala 370 375 380 ggg ate atg atc atg gac ccc tat ggc gcc acc atc agc get ace ccc 1200 Gly Ile Met Ile Met Asp Pro Tyr Gly Ala Thr Ile Ser Ala Thr Pro 385 390 395 400 gaa gcg gcg acg ccg ttc cct cac cgc cag ggc gtc ctc ttc aac ate 1248 Glu Ala Ala Thr Pro Phe Pro His Arg Gin Gly Val Leu Phe Asn Ile 405 410 415 cag tac gtc aac tac tgg ttc get gag tea gcc ggc gcg gcg ccg ctg 1296 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 -4- Gin Tyr Val Asn Tyr Trp Phe Ala Glu Ser Ala Gly Ala Ala Pro Leu 420 425 430 cag tgg agc aag gac ata tac aag ttc atg gag ccg tac gtg agc aaa 1344 Gln Trp Ser Lys Asp Ile Tyr Lys Phe Net Glu Pro Tyr Val Ser Lys 435 440 445 aat ccc agg cag gcg tat gcc aac tac agg gac atc gac ctt ggc agg 1392 Asn Pro Arg Gln Ala Tyr Ala Asn Tyr Arg Asp Ile Asp Leu Gly Arg 450 455 460 aat gag gtg gtg aac gac atc too acc tac agc agc ggc aaa gtg tgg 1440 Asn Glu Val Val Asn Asp Ile Ser Thr Tyr Ser Ser . Gly Lys Val Trp 465 470 475 480 ggt gag aag tac ttc aag ggc aac ttc caa agg ctc gcc att ace aag 1488 Gly Glu Lys Tyr Phe Lys Gly Asn Phe Gin Arg Leu Ala Ile Thr Lys 485 490 495 ggc aag gtg gat cct cag gac tac ttc agg aac gag cag agc atc ccg 1536 Gly Lys Val Asp Pro Gln Asp Tyr Phe Arg Asn Glu Gln Ser Ile Pro 500 505 510 cca ctg gtc gag aag tac tga tcgaggacct tgcatggaaa tttagtgcgt 1587 Pro Leu Val Glu Lys Tyr 515 ggttggcgtt tcacat 1603 <210> 2 <211> 518 <212> PRT <213> Secale cereals <400> 2 Asn Tyr Arg Ala Phe Ala Leu Ala Leu Leu Phe Cys Ala Leu Set Cys 1 5 10 15 Gin Ala Ala Ala Ala Ala Tyr Ala Pro Val Pro Ala Lys Ala Asp Phe 20 25 30 Leu Gly Cys Leu Met Lys Glu Ile Pro Ala Arg Leu Leu Tyr Ala Lys 35 40 45 Ser. Ser Pro Asp Tyr Pro Thr Val Leu Ala Gln Thr Ile Arg Asn Ser 50 55 60 Arg Trp Set Ser Pro Gin Asn Val Lys Pro Ile Tyr Ile Ile Thr Pro 65 70 75 80 Thr Asn Ala Ser His Ile Gin Set Ala Val Val Cys Gly Arg Arg His 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 20051059136 PC' EP2004/1013664 -5- 85 90 95 Gly Ile Arg Leu Arg Val Arg Ser Gly Gly His Asp Tyr Glu Gly Leu 100 105 110 Ser Tyr Arg Ser Glu Lys Pro Glu Thr Phe Ala Val Val Asp Leu Asn 115 120 125 Lys Met Arg Ala Val Ser Val Asp Gly Tyr Ala Arg Thr Ala Trp Val 130 135 140 Glu Ser Gly Ala Gln Leu Gly Glu Leu Tyr Tyr Ala Ile Ala Lys Asn 145 150 155 160 Ser Pro Val Leu Ala Phe Pro Ala Gly Val Cys Pro Ser Ile Gly Val 165 170 175 Gly Gly Asn Phe Ala Gly Gly Gly Phe Gly Met Leu Leu Arg Lys Tyr 180 185 190 Gly Ile Ala Ala Glu Asn Val Ile Asp Val Lys Val Val Asp Pro Asn 195 200 205 Gly Lys Leu Leu Asp Lys Ser Ser Met Ser Ala Asp His Phe Trp Ala 210 215 220 Val Arg Gly Gly Gly Gly Glu Ser Phe Gly Ile Val Val Ser Trp Gln 225 230 235 240 Val Lys Leu Leu Pro Val Pro Pro Thr Val Thr Val Leu Lys Ile Pro 245 250 255 Lys Thr Val Gin Glu Gly Ala Ile Asp Leu Val Asn Lys Trp Gin Leu 260 265 270 Val Gly Pro Ala Leu Pro Gly Asp Leu Met lie Arg Ile Ile Leu Ala 275 280 285 Gly Asn Ser Ala Thr Phe Glu Ala Met Tyr Leu Gly Thr Cys Ser Thr 290 295 300 Leu Thr Pro Leu Met Ser Ser Lys Phe Pro Glu Leu Gly Met Asn Pro 305 310 315 320 Ser His Cys Asn Glu Met Ser Trp Ile Lys Ser Ile Pro Phe Ile His 325 330 335 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 -6- Leu Gly Lys Gln Asn Leu Asp Asp Leu Leu Asn Arg Asn Asn Thr Phe 340 345 350 Lys Pro Phe Ala Glu Tyr Lys Ser Asp Tyr Val Tyr Gin Pro Phe Pro 355 360 365 Lys Pro Val Trp Glu Gln Ile Phe Gly Trp Leu Val Lys Pro Gly Ala 370 375 380 Gly Ile Met Ile Met Asp Pro Tyr Gly Ala Thr Ile Ser Ala Thr Pro 385 390 395 400 Glu Ala Ala Thr Pro Phe Pro His Arg Gin Gly Val Leu Phe Asn Ile 405 410 415 Gin Tyr Val Asn Tyr Trp Phe Ala Glu Ser Ala Gly Ala Ala Pro Leu 420 425 430 Gln Trp Ser Lys Asp Ile Tyr Lys Phe Met Glu Pro Tyr Val Ser Lys 435 440 445 Asn Pro Arg Gin Ala Tyr Ala Asn Tyr Arg Asp Ile Asp Leu Gly Arg 450 455 460 Asn Glu Val Val Asn Asp Ile Ser. Thr Tyr Ser Ser Gly Lys Val Tip 465 470 475 480 Gly Glu Lys Tyr Phe Lys Gly Asn Phe Gin Arg Leu Ala Ile Thr Lys 485 490 495 Gly Lys Val Asp Pro Gln Asp Tyr Phe Arg Awl Glu Gin Ser Ile Pro 500 505 510 Pro Leu Val Glu Lys Tyr 515 <210> 3 <211> 1644 <212> DNA <213> Secale cereale <220> <221> stop_codon 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCTIEP2004/013664 -7- <22> (1561)..(1563) <223> <220> <221> signal_sequence_DNA <222> (1)..(66) <223> <220> <221> signal_sequence_PROT <222> (1)..(22) <223> <220> <221> CDS <222> (1)..(1563) <223> <400> 3 aac tcg agg gcc ttt get ctg gtg ccc ctc ctc atc tgc gtc ttg tcc 48 Asn Ser Arg Ala Phe Ala Leu Val Pro Leu Leu Ile Cys Val Leu Ser 1 5 10 15 tgc cac gcc gcc gtc too tac gcg gcg gcg ccg gtg ccg gcc aag gag 96 Cys His Ala Ala Val Ser Tyr Ala Ala Ala Pro Val Pro Ala Lys Glu 20 25 30 gac ttc ttc gga tgc ctg gtg aag gag ata ccg gcc cgc etc ctc tac 144 Asp Phe Phe Gly Cys Leu Val Lys Giu Ile Pro Ala Arg Leu Leu Tyr 35 40 45 gcc aag agc tcg cct gcc ttc ccc acc gtc ctg gcg cag acc ate agg 192 Ala Lys Ser Ser Pro Ala Phe Pro Thr Val Leu Ala Gin Thr Ile Arg 50 55 60 sac tcg egg tgg tcg tcg ccg cag agc gtg aag ccg ctc tac atc atc 240 Asn Ser Arg Trp Ser Ser Pro Gln Ser Val Lys Pro Leu Tyr Ile Ile 65 70 75 80 acc ccc acc aac gcc tee cac ate cag tcc gcg gtg gtg tgc ggc cgc 288 Thr Pro Thr Asn Ala Ser His Ile Gin Ser Ala Val Val Cys Gly Arg 85 90 95 cgg cac ggc gtc cgc atc cgc gtg cgg agc ggc ggc cac gac tac gag 336 Arg His Gly Val Arg Ile Arg Val Arg Ser Gly Gly His Asp Tyr Glu 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 -8- 100 105 110 ggc ctg tcg tac cgg tcc gag cgc Ccc gag gcg ttc gcc gtc gtc gac 384 Gly Leu Ser Tyr Arg Ser Glu Arg Pro Glu Ala Phe Ala Val Val Asp 115 120 125 ctc aac aag atg cgg gcc gtg gtg gtc gac ggc aag gct cgc acg gcg 432 Leu Asn Lys Met Arg Ala Val Val Val Asp Gly Lys Ala Arg Thr Ala 130 135 140 tgg gtg gac tcc ggt gcg cag ctc ggc gag ctc tac tac gcc atc gcc 480 Trp Val Asp Ser Gly Ala Gln Leu Gly Glu Leu Tyr Tyr Ala Ile Ala 145 150 155 160 aag aac agc ccc gtg ctc gcg ttc ccg gcc ggc gtt tgc cog acc att 52B Lys Asn Ser Pro Val Leu Ala Phe Pro Ala Gly Val Cys Pro Thr Ile 165 170 175 ggt gta ggc ggc aac ttc gct ggc ggc ggc ttc ggc atg ctg ctg cgc 576 Gly Val Gly Gly Asn Phe Ala Gly Gly Gly Phe Gly Met Leu Leu Arg 180 185 190 aag tac ggc atc gcc gcc gag aac gtc atc gac gtg aag gtg gtc gac 624 Lys Tyr Gly Ile Ala Ala Glu Asn Val Ile Asp Val Lys Val Val Asp 195 200 205 gcc aac ggc aca ctg ctc gac aag agc tcc atg agc gcg gat cac ttc 672 Ala Asn Gly Thr Leu Leu Asp Lys Ser Ser Met Ser Ala Asp His Phe 210 215 220 tgg gcc gtc agg ggc ggc ggc gga gag agc ttc ggc atc gtc gtg tcg 720 Trp Ala Val Arg Gly Gly Gly Gly Glu Ser Phe Gly Ile Val Val Ser 225 230 235 240 tgg cag gtg aag ctc ctc ccg gtg cot ccc acc gtg acc gtg ttc aag 768 Trp Gin Val Lys Leu Leu Pro Val Pro Pro Thr Val Thr Val Phe Lys 245 250 255 atc ccc aag acg gtg caa gaa ggc gcc gta gag ctc atc aac aag tgg 816 Ile Pro Lys Thr Val Gln Glu Gly Ala Val Glu Leu Ile Asn Lys Trp 260 265 270 cag cta gtc gcg ccg gcc ctc ccc gac gac ctg atg atc cgo atc atc 864 Gin Leu Val Ala Pro Ala Leu Pro Asp Asp Leu Met Ile Arg Ile Ile 275 280 285 get ttc ggc ggc acc gcc aag ttc gag gcc atg tac ctg ggc ace tgc 912 Ala Phe Gly Gly Thr Ala Lys Phe Glu Ala Met Tyr Leu Gly Thr Cys 290 295 300 aaa gcc ctg aca ccg ctg atg ago agc aga ttc ccc gag ctc ggc atg 960 Lys Ala Leu Thr Pro Leu Met Ser Ser Arg Phe Pro Glu Leu Gly Met 305 310 315 320 aac gcc tcg cac tgc aac gag atg ccc tgg atc aag tcc gtc cca ttc 1008 Asn Ala Ser His Cys Asn Glu Met Pro Trp Ile Lys Ser Val Pro Phe 325 330 335 atc cac ctt ggc aag cag gcc acc ctc tcc gac ctc ctc aac cgg aac 1056 Ile His Leu Gly Lys Gin Ala Thr Leu Ser Asp Leu Leu Asn Arg Asn 340 345 350 aac acc ttc aaa ccc ttc gcc gag tac aag tcg gac tac gtc tac cag 1104 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 -9- Asn Thr Phe Lys Pro Phe Ala Glu Tyr Lys Ser Asp Tyr Val Tyr Gln 355 360 365 ccc gtc ccc aag ccc gtc tgg gcg cag atc ttc gtc tgg ctc gtc aaa 1152 Pro Val Pro Lys Pro Val Trp Ala Gin Ile Phe Val Trp Leu Val Lys 370 375 380 ccc ggc gcc ggg atc atg gtc atg gac ccc tac ggc gcc gcc atc agc 1200 Pro Gly Ala Gly Ile Met Val Met Asp Pro Tyr Gly Ala Ala Ile Ser 385 390 395 400 gcc acc ccc gaa gcc gcc acg ccg ttc cct cac cgc aag gac gtc ctc 1248 Ala Thr Pro Glu Ala Ala Thr Pro Phe Pro His Arg Lys Asp Val Leu 405 410 415 ttc aac atc cag tac gtc aac tac tgg ttc gac gag gca ggc ggc gcc 1296 Phe Asn Ile Gln Tyr Val Asn Tyr Trp Phe Asp Glu Ala Gly Gly Ala 420 425 430 gcg ccg ctg cag tgg agc aag gac atg tac agg ttc atg gag ccg tac 1344 Ala Pro Leu Gin Trp Ser Lys Asp Met Tyr Arg Phe Met Glu Pro Tyr 435 440 445 gtc agc aag aac ccc aga cag gcc tac gcc aac tac agg gac atc gac 1392 Val Ser Lys Asn Pro Arg Gln Ala Tyr Ala Asn Tyr Arg Asp Ile Asp 450 455 460 ctc ggc agg aac gag gtg gtc aac gac atc tcc acc tat gcc agc ggc 1440 Leu Gly Arg Asn Glu Val Val Asn Asp Ile Ser Thr Tyr Ala Ser Gly 465 470 475 480 aag gtc tgg ggc gag aag tac ttc aag ggc aac ttc caa agg ctc gcc 1488 Lys Val Trp Gly Glu Lys Tyr Phe Lys Gly Asn Phe Gln Arg Leu Ala 485 490 495 att acc aag ggc aag gtg gat cct cag gac tac ttc agg aac gag cag 1536 Ile Thr Lys Gly Lys Val Asp Pro Gin Asp Tyr Phe Arg Asn Glu Gin 500 505 510 agc atc ccg ccg ctg cta ggg aag tag tagtactctt gcttgcatgg 1583 Ser Ile Pro Pro Leu Leu Gly Lys 515 520 agatttgtag tgcgtctttc gcgtttcaaa tgcccaacta gtagaataag gatcgtgcgt 1643 a 1644 <210> 4 <211> 520 <212> PRT <213> Secale cereale <400> 4 Asn Ser Arg Ala Phe Ala Leu Val Pro Leu Leu Ile Cys Val Leu Ser 1 5 10 15 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 - 10 - Cys His Ala Ala Val Ser Tyr Ala Ala Ala Pro Val Pro Ala Lys Glu 20 25 30 Asp Phe Phe Gly Cys Leu Val Lys Glu Ile Pro Ala Arg Leu Leu Tyr 35 40 45 Ala Lys Ser Ser Pro Ala Phe Pro Thr Val Leu Ala Gin Thr Ile Arg 50 55 60 Asn Ser Arg Trp Ser Ser Pro Gln Ser Val Lys Pro Leu Tyr Ile Ile 65 70 75 80 Thr Pro Thr Asn Ala Ser His Ile Gin Ser Ala Val Val Cys Gly Arg 85 90 95 Arg His Gly Val Arg Tie Arg Val Arg Ser Gly Gly His Asp Tyr Glu 100 105 110 Gly Len Ser Tyr Arg Ser Glu Arg Pro Glu Ala Phe Ala Val Val Asp 115 120 125 Leu Asn Lys Met Arg Ala Val Val Val Asp Gly Lys Ala Arg Thr Ala 130 135 140 Trp Val Asp Ser Gly Ala Gin Leu Gly Glu Leu Tyr Tyr Ala Ile Ala 145 150 155 160 Lys Asn Ser Pro Val Leu Ala Phe Pro Ala Gly Val Cys Pro Thr Ile 165 170 175 Gly Val Gly Gly Asn Phe Ala Gly Gly Gly Phe Gly Met Leu Leu Arg 180 185 190 Lys Tyr Gly Ile Ala Ala Glu Asn Val Ile Asp Val Lys Val Val Asp 195 200 205 Ala Asn Gly Thr Leu Leu Asp Lys Ser Ser Met Ser Ala Asp His Phe 210 215 220 Trp Ala Val Arg Gly Gly Gly Gly Giu Ser Phe Gly Ile Val Val Ser 225 230 235 240 Trp Gin Val Lys Leu lieu Pro Val Pro Pro Thr Val Thr Val Phe Lys 245 250 255 Ile Pro Lys Thr Val Gln Glu Gly Ala Val Glu Leu Ile Asn Lys Trp 260 265 270 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCTIEP2004/013664 - 11 - Gin Leu Val Ala Pro Ala Leu Pro Asp Asp Leu Met Ile Arg Ile Ile 275 280 285 Ala Phe Gly Gly Thr Ala Lys Phe Glu Ala Met Tyr Leu Gly Thr Cys 290 295 300 Lys Ala Leu Thr Pro Leu Met Ser Ser Arg Phe Pro Glu Leu Gly Met 305 310 315 320 Asn Ala Ser His Cys Asn Glu Met Pro Trp Ile Lys Ser Val Pro Phe 325 330 335 Ile His Leu Gly Lys Gin Ala Thr Leu Ser Asp Leu Leu Asn Arg Asn 340 345 350 Asn Thr Phe Lys Pro Phe Ala Glu Tyr Lys Her Asp Tyr Val Tyr Gln 355 360 365 Pro Val Pro Lys Pro Val Trp Ala Gln Ile Phe Val Trp Leu Val Lys 370 375 380 Pro Gly Ala Gly Ile Met Val Met Asp Pro Tyr Gly Ala Ala Ile Ser 385 390 395 400 Ala Thr Pro Glu Ala Ala Thr Pro Phe Pro His Arg Lys Asp Val Leu 405 410 415 Phe Asn Ile Gln Tyr Val Asn Tyr Trp Phe Asp Glu Ala Gly Gly Ala 420 425 430 Ala Pro Leu Gin Trp Ser Lys Asp Met Tyr Arg Phe Met Glu Pro Tyr 435 440 445 Val Ser Lys Asn Pro Arg Gin Ala Tyr Ala Asn Tyr Arg Asp Ile Asp 450 455 460 Leu Gly Arg Asn Glu Val Val Asn Asp Ile Ser Thr Tyr Ala Ser Gly 465 470 475 480 Lys Val Trp Gly Glu Lys Tyr Phe Lys Gly Asn Phe Gln Arg Leu Ala 485 490 495 Ile Thr Lys Gly Lys Val Asp Pro Gln Asp Tyr Phe Arg Asn Glu Gln 500 505 510 Ser Ile Pro Pro Leu Leu Gly Lys 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 -12- 515 520 <210> 5 <211> 1608 <212> DNA <213> Hordeum vulgare <220> <221> stop_codon <222> (1555)..(1557) <223> <220> <221> signal sequence DNA <222> (1)..(66) <223> <220> <221> signal_sequence_PROT <222> (1)..(22) <223> <220> <221> CDS <222> (1)..(1557) <223> <400> 5 agc tcg agg gcc ttc get ctg gtg ctc ctc etc tgc gee ttg tcc tgc 48 Ser Ser Arg Ala Phe Ala Leu Val Leu Leu Leu Cys Ala Leu Ser Cys 1 5 10 15 cac cac get gcc gtc tcc tcc gcg cag gtg ccg gcc aag gac gac ttc 96 His His Ala Ala Val Ser Ser Ala Gin Val Pro Ala Lys Asp Asp Phe 20 25 30 ctg gga tgc ctc gtg aag gag ata ccg gcc cgc ctc ctc ttc gcc aag 144 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 - 13 - Leu Gly Cys Leu Val Lys Glu Ile Pro Ala Arg Leu Lou Phe Ala Lys 35 .40 45 age tag cct gcc ttc ccc gce gtc ctg gag cag acc atc agg aac tcg 192 Ser Ser Pro Ala Phe Pro Ala Val Leu Glu Gin Thr Ile Arg Asn Ser 50 55 60 cgg tgg tcg tcg ccg cag aac gtg aag ccg ctc tac atc atc acc ccc 240 Arg Trp Ser Ser Pro Gln Asn Val Lys Pro Leu Tyr Ile Ile Thr Pro 65 70 75 80 acc aac acc tcc cac atc cag tot get gtg gtg tgc ggc cge egg cac 288 Thr Asn Thr Ser His Ile Gin Ser Ala Val Val Cys Gly Arg Arg His 85 90 95 ggc gtc cgc ctc cgc gtg cgg agc ggc ggc cac gac tac gag ggc ctg 336 Gly Val Arg Leu Arg Val Arg Ser Gly Gly His Asp Tyr Glu Gly Leu 100 105 110 r-, tcg tac cgg tcc gag cgc ccc gag gcg ttc gcc gtc gta gac etc aac 384 ) Ser Tyr Arg Ser Glu Arg Pro Glu Ala Phe Ala Val Val Asp Leu Asn 115 120 125 aag atg cgg acc gtg ttg gtc aac gaa aag gcc cgc acg gcg tgg gtg 432 Lys Met Arg Thr Val Leu Val Asn Glu Lys Ala Arg Thr Ala Trp Val 130 135 140 gac tee ggc gag cag ctc ggc gag etc tac tac gcc ate gcc aag aac 480 Asp Ser Gly Ala Gin Leu Gly Glu Leu Tyr Tyr Ala Ile Ala Lys Asn 145 150 155 160 age ccc gtg etc gcg ttc cca gcc ggc gtt tgc ccg tee att ggt gta 528 Ser Pro Val Leu Ala Phe Pro Ala Gly Val Cys Pro Ser Ile Gly Val 165 170 175 ggt ggc aac ttc get ggc ggc ggc ttc ggc atg ctg ctg cgc aag tae 576 Gly Gly Asn Phe Ala Gly Gly Gly Phe Gly Met Leu Leu Arg Lys Tyr 180 185 190 ggc ate gcc gee gag aac gtc ate gac gtc aag ctg gtc gac gcc aac 624 ( ) Gly IleAla Ala Glu Asn Val Ile Asp Val Lys Leu Val Asp Ala Asn 195 200 205 ggc aag ctg ctc gac aag agc tee atg agc ccg gac cac ttc tgg gcc 672 Gly Lys Leu Leu Asp Lys Ser Ser Met Ser Pro Asp His Phe Trp Ala 210 215 220 gtc agg ggc ggc ggc gga gag age ttc ggc atc gtc gtc tcg tgg cag 720 Val Arg Gly Gly Gly Gly Giu Ser Phe Gly Ile Val Val Ser Trp Gln 225 230 235 240 gtg aag ctt ctc ccg gtg cct ccc acc gtg act gtg ttt cag ate ccc 768 Val Lys Leu Leu Pro Val Pro Pro Thr Val Thr Val Phe Gin Ile Pro 245 250 255 aag aca gtg caa gaa ggc gcc gta gac ctc ate aac aag tgg cag ctg 816 Lys Thr Val Gin Glu Gly Ala Val Asp Leu Ile Asn Lys Trp Gin Leu 260 265 270 gtc gcg ccg gce ctt ccc ggc gac ate atg atc cgc ate ate gcc atg 864 Val Ala Pro Ala Leu Pro Gly Asp Ile Met Ile Arg Ile Ile Ala Met 275 280 285 2013204574 12 Apr 2013 2013204574 12 Apr 2013 (-)/- WO 2005/059136 PCT/EP2004/013664 -1.4- ggg gac aaa gcg acg ttc gag gcc atg tac ctg ggc acc tgc aaa acc 912 Gly Asp Lys. Ala Thr Phe Glu Ala Met Tyr Leu Gly Thr Cys Lys Thr 290 295 300 ctg acg ccg ctg atg agc agc aaa ttc ccg gag ctt ggc atg aac ccc 960 Leu Thr Pro Leu Met Ser Ser Lys Phe Pro Glu Leu Gly Met Asn Pro 305 310 315 320 tcg cac tgc aac gag atg ccc tgg atc aag too atc ccc ttc atc cac 1008 Ser His Cys Asn Glu Met Pro Trp Ile Lys Ser Ile Pro Phe Ile His 325 330 335 ctt ggc aag cag gcc acc ctg gcc gac ctc ctc aac cgg aac aac acc 1056 Leu Gly Lys Gln Ala Thr Leu Ala Asp Leu Leu Asn Arg Asn Asn Thr 340 345 350 ttc aaa ccc ttc gcc gaa tac aag tcg gac tac gtc tac cag ccc gtc 1104 Phe Lys Pro Phe Ala Glu Tyr Lys Ser Asp Tyr Val Tyr Gln Pro Val 355 360 365 ccc aag ccc gtg tgg gag cag ctc ttc ggc tgg ctc acg aaa ccc ggc 1152 Pro Lys Pro Val Trp Glu Gin Leu Phe Gly Trp Leu Thr Lys Pro Gly 370 375 380 gcg ggg atc atg gtc atg gac cca tac ggc gcc acc atc agc gcc acc 1200 Ala Gly Ile Met Val Met Asp Pro Tyr Giy Ala Thr Ile Ser Ala Thr 385 390 395 400 ccc gaa gcg gcg acg ccg ttc cot cac cgc aag ggc gtc ctc ttc aac 1248 Pro Glu Ala Ala Thr Pro Phe Pro His Arg Lys Gly Val Leu Phe Asn 405 410 415 atc cag tac gtc aac tac tgg ttc gcc gag gca gcc ggc gcc gcg ccg 1296 Ile Gln Tyr Val Asn Tyr Trp Phe Ala Glu Ala Ala Gly Ala Ala Pro 420 425 430 ctg cag tgg ago aag gac att tac aaa ttc atg gag ccg ttc gtg agc 1344 Leu Gin Trp Ser Lys Asp Ile Tyr Lys Phe Met Glu Pro Phe Val Ser 435 440 445 aag aac ccc agg cag gcg tac gcc aac tac agg gac atc gac etc ggc 1392 Lys Asn Pro Arg Gln Ala Tyr Ala Asn Tyr Arg Asp Ile Asp Leu Gly 450 455 460 agg aac gag gtg gtg aac gac atc tca acc tac ago agc ggc aag gtg 1440 Arg Asn Glu Val Val Asn Asp Ile Ser Thr Tyr Ser Ser Gly Lys Val 465 470 475 480 tgg ggc gag aag tac ttc aag ggc aac ttc caa agg ctc gcc atc acc 1488 Trp Gly Glu Lys Tyr Phe Lys Gly Asn Phe Gin Arg Leu Ala Ile Thr 485 490 495 aag ggc aag gtg gat ccc cag gac tac ttc agg aac gag cag agc atc 1536 Lys Giy Lys Val Asp Pro Gln Asp Tyr Phe Arg Asn Glu Gin Ser Ile 500 505 510 ccg ccg ctg ctg ggc aag tag tgaccgagag tcttgcatgg agatttgtag 1587 Pro Pro Leu Leu Gly Lys 515 tgcgtgcttg gcgtttctga t 1608 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCMP2004/013664 - 15 - <210> 6 <211> 518 <212> PRT <213> Hordeum vulgare <400> 6 Ser Ser Arg Ala Phe Ala Leu Val Leu Leu Leu Cys Ala Leu Ser Cys 1 5 10 15 His His Ala Ala Val Ser Ser Ala Gin Val Pro Ala Lys Asp Asp Phe 20 25 30 Leu Gly Cys Leu Val Lys Glu Ile Pro Ala Arg Leu Leu Phe Ala Lys 35 40 45 Ser Ser Pro Ala Phe Pro Ala Val Leu Glu Gin Thr Ile Arg Asn Ser 50 55 60 Arg Trp Ser Ser Pro Gin Asn Val Lys Pro Leu Tyr Ile Ile Thr Pro 65 70 75 80 Thr Asn Thr Ser His Ile Gin Ser Ala Val Val Cys Gly Arg Arg His 85 90 95 Gly Val Arg Leu Arg Val Arg Ser Gly Gly His Asp Tyr Glu Gly Leu 100 105 110 Ser Tyr Arg Ser Glu Arg Pro Glu Ala Phe Ala Val Val Asp Leu Asn 115 120 125 Lys Met Arg Thr Val Leu Val Asn Glu Lys Ala Arg Thr Ala Trp Val 130 135 140 Asp Ser Gly Ala Gin Leu Gly Glu Leu Tyr Tyr Ala Ile Ala Lys Asn 145 150 155 160 Ser Pro Val Leu Ala Phe Pro Ala Gly Val Cys Pro Ser Ile Gly Val 165 170 175 Gly Gly Asn Phe Ala Gly Gly Gly Phe Gly Met Leu Leu Arg Lys Tyr 180 185 190 Gly Ile Ala Ala Glu Asn Val Ile Asp Val Lys Leu Val Asp Ala Asn 195 200 205 2013204574 12 Apr 2013 2013204574 12 Apr 2013 11102005/059136 PCMEF2004/013664 -16- Gly Lys Leu Leu Asp Lys Ser Ser Met Ser Pro Asp His Phe Trp Ala 210 215 220 Val Arg Gly Gly Gly Gly Glu Ser Phe Gly Ile Val Val Ser Trp Gin 225 230 235 240 Val Lys Leu Leu Pro Val Pro Pro Thr Val Thr Val Phe Gin Ile Pro 245 250 255 Lys Thr Val Gin Glu Gly Ala Val Asp Leu Ile Asn Lys Trp Gin Leu 260 265 270 Val Ala Pro Ala Leu Pro Gly Asp Ile Met Ile Arg Ile Ile Ala Met rr 275 280 285 ) Gly Asp Lys Ala Thr Phe Glu Ala Met Tyr Leu Gly Thr Cys Lys Thr 290 295 300 Leu Thr Pro Leu Met Ser Ser Lys Phe Pro Glu Leu Gly Met Asn Pro 305 310 315 320 Ser His Cys Asn Glu Met Pro Trp Ile Lys Ser Ile Pro Phe Ile His 325 330 335 Leu Gly Lys Gln Ala Thr Leu Ala Asp Leu Leu Asn Arg Asn Asn Thr 340 345 350 Phe Lys Pro Phe Ala Glu Tyr Lys Ser Asp Tyr Val Tyr Gln Pro Val 355 360 365 Pro Lys Pro Val Trp Glu Gln Leu Phe Gly Trp Leu Thr Lys Pro Gly 370 375 380 Ala Gly Ile Met Val Met Asp Pro Tyr Gly Ala Thr Ile Ser Ala Thr 385 390 395 400 Pro Glu Ala Ala Thr Pro Phe Pro His Arg Lys Gly Val Leu Phe Asn 405 410 415 Ile Gin Tyr Val Asn Tyr Trp Phe Ala Glu Ala Ala Gly Ala Ala Pro 420 425 430 Leu Gin Trp Ser Lys Asp Ile Tyr Lys Phe Met Glu Pro Phe Val Ser 435 440 445 Lys Asn Pro Arg Gln Ala Tyr Ala Asn Tyr Arg Asp Ile Asp Leu Gly 450 455 460 2013204574 12 Apr 2013 2013204574 12 Apr 2013  WO 2005/059136 PCTIEP2004/013664 - 17 - Arg Asn Glu Val Val Asn Asp Ile Ser Thr Tyr Ser Ser Gly Lys Val 465 470 475 480 Trp Gly Glu Lys Tyr Phe Lys Gly Asn Phe Gin Arg Leu Ala Ile Thr 485 490 495 Lys Gly Lys Val Asp Pro Gin Asp Tyr Phe Arg Asn Glu Gln Ser Ile 500 505 510 Pro Pro Leu Leu Gly Lys 515 Cr) <210> 7 <211> 1603 <212> DNA <213> Triticum aestivum <220> <221> stop colon <222> (1555)..(1557) <223> <220> <221> signalsequenceDNA <222> (1)..(63) <223> <220> <221> signal_sequence_PROT <222> (1)..(21) <223> <220> <221> CDS 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 POYET2004/013664 -18- <222> (1)—(1557) <223> <400> 7 aac tat agg gcc ttc acg ctg gtg ctc etc ttc tgc gcc ttg tcc tgt 48 Asn Tyr Arg Ala Phe Thr Leu Val Leu Leu Phe Cys Ala Leu Ser Cys 1 5 10 15 caa gee gcc gcc ace tac gcg ccg gtg cct gcc aag gag gac ttc ctc 96 Gln Ala Ala Ala Thr Tyr Ala Pro Val Pro Ala Lys Glu Asp Phe Leu 20 25 30 gga tgc ctc atg aag gag ata ccg gca cgc etc ctc tac gcc aag agc 144 Gly Cys Leu Met Lys Glu Ile Pro Ala Arg Leu Leu Tyr Ala Lys Ser 35 40 45 tcg cct gac ttc ccc ace gtc ctg gcg cag acc ate agg aac tcg cgg 192 Ser Pro Asp Phe Pro Thr Val Leu Ala Gin Thr Ile Arg Asn Ser Arg 50 55 60 tgg ttg tcg ccg cag aac gtg aag ccg ctc tac atc atc acc ccc acc 240 Trp Leu Ser Pro Gln Asn Val Lys Pro Leu Tyr Ile Ile Thr Pro Thr 65 70 75 80 aac gce tcg cac atc cag tcc gcg gtg gtg tgc gga cgc cgg cac age 288 Asn Ala Ser His Ile Gin Ser Ala Val Val Cys Gly Arg Arg His Ser 85 90 95 gtc cgc ctc cgc gtc cgg age ggc ggc cac gac tac gag ggc ctg tcg 336 Val Arg Leu Arg Val Arg Ser Gly Gly His Asp Tyr Glu Gly Leu Ser 100 105 110 tac egg tcc gag aaa ccc gag acg ttc gcc gtc gtc gac ctc aac aag 384 Tyr Arg Ser Glu Lys Pro Glu Thr Phe Ala Val Val Asp Leu Asn Lys 115 120 125 atg cgg gca gtg ttg atc gac ggc tac gcc cgc acg gcg tgg gtc gaa 432 Met Arg Ala Val Leu Ile Asp Gly Tyr Ala Arg Thr Ala Trp Val Glu (1) 130 135 140 tee ggc gcg cag ctc ggc gag ctc tac tac gcc atc gcg aaa aac agc 480 Ser Gly Ala Gin Leu Gly Glu Leu Tyr Tyr Ala Ile Ala Lys Asn Ser 145 150 155 160 ccc gtg ctc gcg ttc ccg gcc ggc gtc tgc ccg acc atc ggc gtc ggc 528 Pro Val Leu Ala Phe Pro Ala Gly Val Cys Pro Thr Ile Gly Val Gly 165 170 175 ggc aac ttc gca ggc ggc ggc ttt ggc atg ctg ctg cgg aag tac ggc 576 Gly Asn Phe Ala Gly Gly Gly Phe Gly Met Leu Leu Arg Lys Tyr Gly 180 185 190 atc gcc gcc gag aac gtc ate gac gtc aag gtg gtc gac ccc sac ggc 624 Ile Ala Ala Glu Asn Val Ile Asp Val Lys Val Val Asp Pro Asn Gly 195 200 205 aag ctt ctc gac aag agc tee atg agc ccg gac cac ttc tgg gcc gtc 672 Lys Leu Leu Asp Lys Ser Ser Met Ser Pro Asp His Phe Trp Ala Val 210 215 220 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 - 19 - agg ggc ggc ggc gga gag agc ttt ggc atc gtc gtg tcg tgg caa gtg 720 Arg Gly Gly Gly Gly Glu Ser Phe Gly Ile Val Val Ser Trp Gin Val 225 230 235 240 aag ctc ctg ccg gtg cct ccc acc gtg acc gtg ttc aag atc ccc aag 768 Lys Leu Leu Pro Val Pro Pro Thr Val Thr Val Phe Lys Ile Pro Lys 245 250 255 aca gtg caa gaa ggc gcc gta gac ctc gtc aac aag tgg caa ctg gtc 816 Thr Val Gin Glu Gly Ala Val Asp Leu Val Asn Lys Trp Gln Leu Val 260 265 270 ggg ccg gcc ctt ccc ggc gac ctc atg atc cgc gtc atc get gcg ggg 864 Gly Pro Ala Leu Pro Gly Asp Leu Met Ile Arg Val Ile Ala Ala Gly 275 280 285 aac acc gcg aca ttc gag ggc atg tac ctg ggc acc tgc caa acc ctg 912 Asn Thr Ala Thr Phe Glu Gly Met Tyr Leu Gly Thr Cys Gln Thr Leu 290 295 300 acg ccg ttg atg agc agc caa ttc ccc gag ctt ggc atg aac ccc tat 960 Thr Pro Leu Met Ser Ser Gin Phe Pro Glu Leu Gly Met Asn Pro Tyr 305 310 315 320 cac tgc aac gag atg ccc tgg atc aag tcc atc ccc ttc atc cac ctc 1008 His Cys Asn Glu Met Pro Trp Ile Lys Ser Ile Pro Phe Ile His Leu 325 330 335 ggc aaa gag gcc agc ctg gtc gac ctc ctc aac cgg aac aac acc ttc 1056 Gly Lys Glu Ala Ser Leu Val Asp Leu Leu Asn Arg Asn Asn Thr Phe 340 345 350 aag ccc ttc gcc gaa tac aag tcg gac tac gtg tac cag ccc ttc ccc 1104 Lys Pro Phe Ala Glu Tyr Lys Ser Asp Tyr Val Tyr Gln Pro Phe Pro 355 360 365 aag ccc gtg tgg gag cag atc ttc ggc tgg ctc acg aag ccc ggt ggg 1152 Lys Pro Val Trp Giu Gin Ile Phe Gly Trp Leu Thr Lys Pro Gly Gly 370 375 380 ggg atg atg atc atg gac cca tac ggc gcc acc atc agc gcc acc ccc 1200 Gly Met Met Ile Met Asp Pro Tyr Gly Ala Thr Ile Ser Ala Thr Pro 3B5 390 395 400 gaa gcg gcg acg ccg ttc cct cac cgc cag ggc gtt ctc ttc sac atc 1248 Glu Ala Ala Thr Pro Phe Pro His Arg Gin Gly Val Leu Phe Asn Ile 405 410 415 cag tac gtc aac tac tgg ttc gcc gag gca gcc gcc gcc gcg ccg ctg 1296 Gln Tyr Val Asn Tyr Trp Phe Ala Glu Ala Ala Ala Ala Ala Pro Leu 420 425 430 cag tgg agc aag gac atg tac aat ttc atg gag ccg tac gtg agc aag 1344 Gln Trp Ser Lys Asp Met Tyr Asn Phe Met Glu Pro Tyr Val Ser Lys 435 440 445 aac ccc agg cag gcg tac gcc aac tac agg gac att gac ctc ggc agg 1392 Asn Pro Arg Gln Ala Tyr Ala Asn Tyr Arg Asp Ile Asp Leu Gly Arg 450 455 460 aac gag gtg gtg aac gac atc tca acc tat agc ago ggc aag gtt tgg 1440 Asn Giu Val Val Asn Asp Ile Ser Thr Tyr Ser Ser Gly Lys Val Trp 465 470 475 480 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059/36 PCT/EP2004/013664 - 20 - ggc gag aag tac ttc aag ggc aac ttc caa agg ctc get att acc aag 1488 Gly Glu Lys Tyr Phe Lys Gly Asn Phe Gin Arg Leu Ala Ile Thr Lys 485 490 495 ggc aag gtg gat cct cag gac tac ttc agg aac gag cag agc ate ccg 1536 Gly Lys Val Asp Pro Gin Asp Tyr Phe Arg Asn Glu Gin Ser Ile Pro 500 505 510 ccg ctg ctc gag aag tac tga tcgaggacct tgcatggaga tttagtgcgt 1 587 Pro Leu Leu Glu Lys Tyr 515 ggttgccgtt tcacat 1603 <210> 8 (—)- <211> 518 ) <212> PRT <213> Triticum aestivum <400> 8 Asn Tyr Arg Ala Phe Thr Leu Val Leu Leu Phe Cys Ala Leu Ser Cys 1 5 10 15 Gin Ala Ala Ala Thr Tyr Ala Pro Val Pro Ala Lys Glu Asp Phe Leu 20 25 30 Gly Cys Leu Met Lys Glu Ile Pro Ala Arg Leu Leu Tyr Ala Lys Ser 35 40 45 Ser Pro Asp Phe Pro Thr Val Leu Ala Gln Thr Ile Arg Asn Ser Arg 50 55 60 Trp Leu Ser Pro Gin Asn Val Lys Pro Leu Tyr Ile Ile Thr Pro Thr 65 70 75 80 Asn Ala Ser His Ile Gln Ser Ala Val Val Cys Gly Arg Arg His Ser B5 90 95 Val Arg Leu Arg Val Arg Ser Gly Gly His Asp Tyr Glu Gly Leu Ser 100 105 110 Tyr Arg Ser Glu Lys Pro Glu Thr Phe Ala Val Val Asp Leu Asn Lys 115 120 125 Met Arg Ala Val Leu Ile Asp Gly Tyr Ala Arg Thr Ala Trp Val Glu 130 135 140 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 -21- Ser Gly Ala Gin Leu Gly Glu Leu Tyr Tyr Ala Ile Ala Lys Asn Ser 145 150 155 160 Pro Val Leu Ala Phe Pro Ala Gly Val Cys Pro Thr Ile Gly Val Gly 165 170 175 Gly Asn Phe Ala Gly Gly Gly Phe Gly Met Leu Leu Arg Lys Tyr Gly 180 185 190 Ile Ala Ala Glu Asn Val Ile Atp Val Lys Val Val Asp Pro Asn Gly 195 200 205 Lys Leu Leu Asp Lys Ser Ser Met Ser Pro Asp His Phe Trp Ala Val C 210 215 220 Arg Gly Gly Gly Gly Glu Ser Phe Gly Ile Val Val Ser Trp Gin Val 225 230 235 240 Lys Leu Leu Pro Val Pro Pro Thr Val Thr Val Phe Lys Ile Pro Lys 245 250 255 Thr Val Gln Glu Gly Ala Val Asp Leu Val Asn Lys Trp Gin Leu Val 260 265 270 Gly Pro Ala Leu Pro Gly Asp Leu Met Ile Arg Val Ile Ala Ala Gly 275 280 285 Asn Thr Ala Thr Phe Glu Gly Met Tyr Leu Gly Thr Cys Gin Thr Leu 290 295 300 Thr Pro Leu Met Ser Ser Gln Phe Pro Glu Leu Gly Met Asn Pro Tyr 305 310 315 320 His Cys Asn Glu Met Pro Trp Ile Lys Ser Ile Pro Phe Ile His Leu 325 330 335 Giy Lys Glu Ala Ser Leu Val Asp Leu Leu Asn Arg Asn Asn Thr Phe 340 345 350 Lys Pro Phe Ala Glu Tyr Lys Ser Asp Tyr Val Tyr Gin Pro Phe Pro 355 360 365 Lys Pro Val Trp Glu Gln Ile Phe Gly Trp Leu Thr Lys Pro Gly Gly 370 375 380 Gly Met Met Ile Met Asp Pro Tyr Giy Ala Thr Ile Ser Ala Thr Pro 3B5 390 395 400 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/E1P2004/013664 -22- Giu Ala Ala Thr Pro Phe Pro His Arg Gin Gly Val Leu Phe Asn Ile 405 410 415 Gln Tyr Val Asn Tyr Trp Phe Ala Giu Ala Ala Ala Ala Ala Pro Leu 420 425 430 Gin Trp Ser Lys Asp Met Tyr Asn Phe Met Glu Pro Tyr Val Set Lys 435 440 445 Asn Pro Arg Gin Ala Tyr Ala Asn Tyr Arg Asp Ile Asp Leu Gly Arg 450 455 460 Asn Glu Val Val Asn Asp Ile Ser Thr Tyr Ser Ser Gly Lys Val Trp 465 470 475 480 Gly Glu Lys Tyr Phe Lys Gly Asn Phe Gin Arg Leu Ala Ile Thr Lys 485 490 495 Gly Lys Val Asp Pro Gin Asp Tyr Phe Arg Asn Glu Gin Ser Ile Pro 500 505 510 Pro Leu Leu Glu Lys Tyr 515 <210> 9 <211> 1603 <212> DNA <213> Triticum aestivum <220> <221> stop codon <222> (1555)..(1557) <223> <220> <221> CDS <222> (1)..(1557) <223> 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 20051059136 P CIVEP2004/13136 64 -23- <220> <221> signal_sequence_DNA <222> (1)..(63) <223> <220> <221> signal_sequence_PROT <222> (1)..(21) <223> <400> 9 aac tgt agg gcc ttc gcg cag gtg etc ctc ttc ttc gcc ttg tcc tgc 48 Asn Cys Arg Ala Phe Ala Gin Val Leu Leu Phe Phe Ala Leu Ser Cys 1 5 10 15 caa gcc gcc gcc acc tac gcg ccg gtg cct gcc aag gag gac ttc ctc 96 Gin Ala Ala Ala Thr Tyr Ala Pro Val Pro Ala Lys Glu Asp Phe Leu 20 25 30 gga tgc ctc atg aag gag ata ccg gcc cgc ctc ctc tac gcc aag agc 144 Gly Cys Leu Met Lys Glu Ile Pro Ala Arg Leu Leu Tyr Ala Lys Ser 35 40 45 tcg cct gac tac ccc acc gtg ctg gcg cag acc atc agg aac tcg cgg 192 Ser Pro Asp Tyr Pro Thr Val Leu Ala Gin Thr Ile Arg Asn Ser Arg 50 55 60 tgg tog acg cag cag aac gtg aag ccg ctg tac atc atc acc ccc acc 240 Trp Ser Thr Gin Gln Asn Val Lys Pro Leu Tyr Ile Ile Thr Pro Thr 65 70 75 80 aac gcc tcc cac atc caa tcc gcg gtg gtg tgc ggc cgc cgg cac ggc 288 Asn Ala Ser His Ile Gln Ser Ala Val Val Cys Gly Arg Arg His Gly 85 90 95 gtc cgc ctc cgc gtg cgg agc ggc ggc cac gac tac gag ggc ctg tog 336 Val Arg Leu Arg Val Arg Ser Gly Gly His Asp Tyr Glu Gly Leu Ser 100 105 110 tac cgg tcc gag aaa ccc gag acg ttc gcc gtc gtc gac ctc aac aag 384 Tyr Arg Ser Glu Lys Pro Glu Thr Phe Ala Val Val Asp Leu Asn Lys 115 120 125 atg egg gca gtg gtt gtc gac ggc tac gcc cgc acg gcg tgg gtc gaa 432 Met Arg Ala Val Val Val Asp Gly Tyr Ala Arg Thr Ala Trp Val Glu 130 135 140 tee ggc gcg cag ctc ggc gag ctc tac tac gcc atc gcg aag aac agc 480 Ser Gly Ala Gln Leu Gly Glu Lett Tyr Tyr Ala Ile Ala Lys Asn Ser 145 150 155 160 2013204574 12 Apr 2013 2013204574 12 Apr 2013 (--- WO 2005/059136 PCT/EP2004/013664 - 24 - ccc gtg ctc gcg ttc ccg gcc ggc gtc tgc ccg tcc atc ggc gtc ggc . 528 Pro Val Leu Ala Phe Pro Ala Gly Val Cys Pro Ser Ile Gly Val Gly 165 170 175 ggc aac ttc gca ggc ggc ggc ttc ggc atg ctg ctg cgc aag tac ggc 576 Gly Asn Phe Ala Gly Gly Gly Phe Gly Met Leu Leu Arg Lys Tyr Gly 180 185 190 atc gcc gcc gag aac gtc atc gac gtc aag gtg gtc gac ccc gac ggc 624 Ile Ala Ala Glu Asn Val Ile Asp Val Lys Val Val Asp Pro Asp Gly 195 200 205 aag ctg ctc gac aag age tcc atg agc gcg gac cac ttc tgg gcc gtc 672 Lys Leu Leu Asp Lys Ser Ser Met Ser Ala Asp His Phe Trp Ala Val 210 215 220 agg ggc ggc ggc gga gag agc ttc ggc atc gtc gtc tcg tgg cag gtg 720 Arg Gly Gly Gly Gly Glu Ser Phe Gly Ile Val Val Ser Trp Gin Val 225 230 235 240 aag ctc atg cca gtg cct ccc ace gtc acc gtg ttt aag atc ccc aag 768 Lys Leu Met Pro Val Pro Pro Thr Val Thr Val Phe Lys Ile Pro Lys 245 250 255 acg gtg caa gaa ggc gcc gta gac ctc gtc aac aag tgg cag ctg gtc 816 Thr Val Gin Glu Gly Ala Val Asp Leu Val Asn Lys Trp Gln Leu Val 260 265 270 ggg ccg gca ctt ccc ggc gac ctc atg atc cgc gtc atc get gcc ggg B64 Gly Pro Ala Leu Pro Gly Asp Leu Met Ile Arg Val Ile Ala Ala Gly 275 280 285 aac acg gcg acg ttc gag gcc ttg tac ctg ggc acc tgc aaa ace ctg 912 Asn Thr Ala Thr Phe Glu Ala Leu Tyr Leu Giy Thr Cys Lys Thr Leu 290 295 300 acg ccg ctg atg age agc caa ttc ccc gag ctt ggc atg aac ccc tat 960 Thr Pro Leu Met Ser Ser Gin Phe Pro Glu Leu Gly Met Asn Pro Tyr 305 310 315 320 (_( cac tgc aac gag atg ccc tgg ate aag tcc gtc ccc ttc atc cac ctc 1008 His Cys Asn Glu Met Pro Trp Ile Lys Ser Val Pro Phe Ile His Leu 325 330 335 ggc aaa cag get ggc ctg gac gac ctc ctc aac cgg aac aac acc ttc 1056 Gly Lys Gin Ala Gly Leu Asp Asp Leu Leu Asn Arg Asn Asn Thr Phe 340 345 350 aag ccc ttc gcc gaa tac aag tcg gac tac gtg tac cag ccc ttc ccc 1104 Lys Pro Phe Ala Glu Tyr Lys Ser Asp Tyr Val Tyr Gin Pro Phe Pro 355 360 365 aag ccc gtg tgg gag cag ate ttc ggc tgg ctc gcg aag ccc ggc gcg 1152 Lys Pro Val Trp Glu Gin Ile Phe Gly Trp Leu Ala Lys Pro Gly Ala 370 375 380 ggg atc atg atc atg gac ccc tac ggc gcc acc ate agc gcc acc ccc 1200 Giy Ile Met Ile Met Asp Pro Tyr Gly Ala Thr Ile Ser Ala Thr Pro 385 390 395 400 gaa gcg gcg acg ccg ttc cct cac cgc cag ggc gtc ctc ttc aac ate 1248 Glu Ala Ala Thr Pro Phe Pro His Arg Gin Gly Val Leu Phe Asn Ile 405 410 415 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 -25- cag tat gtc aac tac tgg ttc gcc gag cca gcc ggc gcc gcg ccg ctg 1296 Gin Tyr Val Asn Tyr Trp Phe Ala Glu Pro Ala Gly Ala Ala Pro Leu 420 425 430 cag tgg age aag gac att tac aat ttc atg gag ccg tac gtg agc aag 1344 Gln Trp Ser Lys Asp Ile Tyr Asn Phe Met Glu Pro Tyr Val Ser Lys 435 440 445 aac ccc agg cag gcg tac gcc aac tac agg gac atc gac etc ggc agg 1392 Asn Pro Arg Gin Ala Tyr Ala Asn Tyr Arg Asp Ile Asp Leu Gly Arg 450 455 460 aat gag gtg gtg aac gac ate tca acc tac age agc ggc aag gtg tgg 1440 Asn Glu Val Val Asn Asp Ile Ser Thr Tyr Ser Ser Gly Lys Val Trp 465 470 475 480 ggc gag aag tac ttc aag agc aac ttc caa agg ctc gcc att acc aag 1488 Gly Glu Lys Tyr Phe Lys Ser Asn Phe Gin Arg Leu Ala Ile Thr Lys ( 485 490 495 ggc aag gta gat cct cag gac tac ttc agg aat gag caa age atc ccg 1536 Gly Lys Val Asp Pro Gln Asp Tyr Phe Arg Asn Glu Gin Ser Ile Pro 500 505 510 ccg ctg atc gag aag tac tga tcgaggacct tgcatggaga tttagtgcgt 1587 Pro Leu Ile Glu Lys Tyr 515 ggttggcgtt tcacat 1603 <210> 10 <211> 518 <212> PRT <213> Triticum aestivum <400> 10 Asn Cys Arg Ala Phe Ala Gin Val Leu Leu Phe Phe Ala Leu Ser Cys 1 5 10 15 Gln Ala Ala Ala Thr Tyr Ala Pro Val Pro Ala Lys Glu Asp Phe Leu 20 25 30 Gly Cys Leu Net Lys Glu Ile Pro Ala Arg Leu Leu Tyr Ala Lys Ser 35 .40 45 Ser Pro Asp Tyr Pro Thr Val Leu Ala Gin Thr Ile Arg Asn Ser Arg 50 55 60 Trp Ser Thr Gin Gln Asn Val Lys Pro Leu Tyr Ile Ile Thr Pro Thr 65 70 75 80 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 -26- Asn Ala Set His Ile Gln Ser Ala Val Val Cys Gly Arg Arg His Gly 85 90 95 Val Arg Leu Arg Val Arg Ser Gly Gly His Asp Tyr Glu Gly Leu Ser 100 105 110 Tyr Arg Ser Glu Lys Pro Glu Thr Phe Ala Val Val Asp Leu Asn Lys 115 120 125 Met Arg Ala Val Val Val Asp Gly Tyr Ala Arg Thr Ala Trp Val .Glu 130 135 140 Ser Gly Ala Gln Leu Gly Glu Leu Tyr Tyr Ala Ile Ala Lys Asn Ser 145 150 155 160 Pro Val Leu Ala Phe Pro Ala Gly Val Cys Pro Ser Ile Gly Val Gly 165 170 175 Gly Asn Phe Ala Gly Gly Gly Phe Gly Met Leu Leu Arg Lys Tyr Gly 180 185 190 Ile Ala Ala Glu Asn Val Ile Asp Val Lys Val Val Asp Pro Asp Gly 195 200 205 Lys Leu Leu Asp Lys Ser Ser Met Ser Ala Asp His Phe Trp Ala Val 210 215 220 Arg Gly Gly Gly Gly Glu Ser Phe Gly Ile Val Val Ser Trp Gin Val 225 230 235 240 Lys Leu Met Pro Val Pro Pro Thr Val Thr Val Phe Lys Ile Pro Lys 245 250 255 Thr Val Gin Glu Gly Ala Val Asp Leu Val Asn Lys Trp Gln Leu Val 260 265 270 Gly Pro Ala Leu Pro Gly Asp Leu Met Ile Arg Val Ile Ala Ala Gly 275 280 285 Asn Thr Ala Thr Phe Glu Ala Leu Tyr Leu Gly Thr Cys Lys Thr Leu 290 295 300 Thr Pro Leu Met Ser Set Gln Phe Pro Glu Leu Gly Met Asn Pro Tyr 305 310 315 320 His Cys Asn Giu Met Pro Trp Ile Lys Set Val Pro Phe Ile His Leu 325 330 335 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PC UO2004/013664 -27- Gly Lys Gln Ala Gly Leu Asp Asp Leu Leu Asn Arg Asn Asn Thr Phe 340 345 350 Lys Pro Phe Ala Glu Tyr Lys Ser Asp Tyr Val Tyr Gln Pro Phe Pro 355 360 365 Lys Pro Val Trp Glu Gin Ile Phe Gly Trp Leu Ala Lys Pro Gly Ala 370 375 380 Gly Ile Met Ile Met Asp Pro Tyr Gly Ala Thr Ile Ser Ala Thr Pro 385 390 395 400 Glu Ala Ala Thr Pro Phe Pro His Arg Gin Gly Val Leu Phe Asn Ile 405 410 415 Gin Tyr Val Asn Tyr Trp Phe Ala Glu Pro Ala Gly Ala Ala Pro Leu 420 425 430 Gin Trp Ser Lys Asp Ile Tyr Asn Phe Met Glu Pro Tyr Val Ser Lys 435 440 445 Asn Pro Arg Gln Ala Tyr Ala Asn Tyr Arg Asp Ile Asp Leu Gly Arg 450 455 460 Asn Glu Val Val Asn Asp Ile Ser Thr Tyr Ser Ser Gly Lys Val Trp 465 470 475 480 Gly Glu Lys Tyr Phe Lys Ser Asn Phe Gin Arg Leu Ala Ile Thr Lys 485 490 495 Gly Lys Val Asp Pro Gin Asp Tyr Phe Arg Asn Glu Gin Ser Ile Pro 500 505 510 Pro Leu Ile Glu Lys Tyr 515 <210> 11 <211> 1503 <212> DNA <213> Phleum pratense <220> 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 POYEP2004/013664 - 28 - <221> CDS <222> (1)..(1503} <223> <400> 11 tac ttc ccg ccg ccg get get aaa gaa gac ttc ctg ggt tgc ctg gtt 48 Tyr Phe Pro Pro Pro Ala Ala Lys Glu Asp Phe Leu Gly Cys Leu Val 1 5 10 15 aaa gaa atc ccg ccg cgt ctg ttg tac gcg aaa tcg tcg ccg gcg tat 96 Lys Glu Ile Pro Pro Arg Leu Leu Tyr Ala Lys Ser Ser Pro Ala Tyr 20 25 30 ccc tca gtc ctg ggg cag acc atc cgg aac tcg agg tgg tcg tcg cog 144 (--) Pro Ser Val Leu Gly Gln Thr Ile Arg Asn Ser Arg Trp Ser Ser Pro ) J 35 40 45 gac aac gtg aag ccg ctc tac atc atc acc ccc ace aac gtc tcc cac 192 Asp Asn Val Lys Pro Leu Tyr Ile Ile Thr Pro Thr Asn Val Ser His 50 55 60 atc cag tcc gee gtg gtg tgc ggc cgc cgc cac agc gtc cgc ate cgc 240 Ile Gln Ser Ala Val Val Cys Gly Arg Arg His Ser Val Arg Ile Arg 65 70 75 80 gtg cgc agc ggc ggg cac gac tac gag ggc ctc tcg tac cgg tct ttg 288 Val Arg Ser Gly Gly His Asp Tyr Glu Gly Leu Ser Tyr Arg Ser Leu 85 90 95 cag ccc gag acg ttc gcc gtc gtc gac ctc aac aag atg egg gcg gtg 336 Gln Pro Glu Thr Phe Ala Val Val Asp Leu Asn Lys Met Arg Ala Val 100 105 110 tgg gtg gac ggc aag gcc cgc acg gcg tgg gtg gac tee ggc gcg cag 384 Trp Val Asp Gly Lys Ala Arg Thr Ala Trp Val Asp Ser Gly Ala Gin 115 120 125 ctc ggc gag ctc tac tac gcc atc tat aag gcg age ccc acg ctg gcg 432 Leu Gly Glu Leu Tyr Tyr Ala Ile Tyr Lys Ala Ser Pro Thr Leu Ala 130 135 140 ttc cog gcc ggc gtg tgc ccg acg atc gga gtg ggc ggc aac ttc gcg 480 Phe Pro Ala Gly Val Cys. Pro Thr Ile Gly Val Gly Gly Asn Phe Ala 145 150 155 160 ggc ggc ggc ttc ggc atg ctg ctg cgc aag tac ggc atc gcc gcg gag 528 Gly Gly Gly Phe Gly Met Leu Leu Arg Lys Tyr Gly Ile Ala Ala Glu 165 170 175 aac gtc atc gac gtg aag ctc gtc gac gcc aac ggc aag ctg cac gac 576 Asn Val Ile Asp Val Lys Leu Val Asp Ala Asn Gly Lys Leu His Asp 180 185 190 aag aag tcc atg ggc gac gac cat ttc tgg gee gtc agg ggc ggc ggg 624 Lys Lys Ser Met Gly Asp Asp His Phe Trp Ala Val Arg Gly Gly Gly 195 200 205 ggc gag age ttc ggc atc gtg gtc gcg tgg cag gtg aag ctc ctg ccg 672 Gly Glu Ser Phe Gly Ile Val Val Ala Trp Gln Val Lys Leu Leu Pro 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 -29- 210 215 220 gtg ccg ccc ace gtg aca ata ttc aag atc tcc aag aca gtg agc gag 720 Val Pro Pro Thr Val Thr Ile Phe Lys Ile Ser Lys Thr Val Ser Glu 225 230 235 240 ggc gcc gtg gac atc atc aac aag tgg caa gtg gtc gcg ccg cag ctt 768 Gly Ala Val Asp Ile Ile Asn Lys Trp Gln Val Val Ala Pro Gin Leu 245 250 255 ccc gcc gac ctc atg atc cgc atc atc gcg cag ggg ccc aag gcc acg 816 Pro Ala Asp Leu Met Ile Arg Ile Ile Ala Gln Gly Pro Lys Ala Thr 260 265 270 ttc gag gcc atg tac ctc ggc acc tgc aaa acc ctg acg ccg ttg atg 864 Phe Glu Ala Met Tyr Leu Gly Thr Cys Lys Thr Leu Thr Pro Leu Met 275 280 285 (—). agc age aag ttc ccg gag ctc ggc atg aac ccc tcc cac tgc aac gag 912 Ser Ser Lys Phe Pro Glu Leu Gly Met Asn Pro Ser His Cys Asn Glu 290 295 300 atg tea tgg atc cag tee atc ccc ttc gtc cac etc ggc cac agg gac 960 Met Ser Trp Ile Gln Ser Ile Pro Phe Val His Leu Gly His Arg Asp 305 310 315 320 gcc etc gag gac gac ctc ctc aac cgg aac aac tee ttc aag ccc ttc 1006 Ala Leu Glu Asp Asp Leu Leu Asn Arg Asn Asn Ser Phe Lys Pro Phe 325 330 335 gcc gaa tac aag tee gac tac gtc tac cag ccc ttc ccc aag ace gtc 1056 Ala Glu Tyr Lys Ser Asp Tyr Val Tyr Gin Pro Phe Pro Lys Thr Val 340 345 350 tgg gag cag ate etc aac ace tgg etc gtc aag ccc ggc gcc ggg ate 1104 Trp Glu Gin Ile Leu Asn Thr Trp Leu Val Lys Pro Gly Ala Gly Ile 355 360 365 atg atc ttc gac ccc tac ggc gcc acc ate agc gcc acc ccg gag tee 1152 Met Ile Phe Asp Pro Tyr Gly Ala Thr Ile Ser Ala Thr Pro Glu Ser 370 375 380 gcc acg ccc ttc cct cac egc aag ggc gtc etc ttc aac ate cag tac 1200 Ala Thr Pro Phe Pro His Arg Lys Gly Val Leu Phe Asn Ile Gin Tyr 385 390 395 400 gtc aac tae tgg ttc gcc ccg gga gcc gcc gcc gcg ccc etc tcg tgg 1248 Val Asn Tyr Trp Phe Ala Pro Gly Ala Ala Ala Ala Pro Leu Ser Trp 405 410 415 age aag gac atc tac aac tac atg gag ccc tac gtg age aag aac ccc 1296 Ser Lys Asp Ile Tyr Asn Tyr Met Glu Pro Tyr Val Ser Lys Asn Pro 420 425 430 agg cag gcg tac gca aac tac agg gac atc gac ctc ggc agg aac gag 1344 Arg Gln Ala Tyr Ala Asn Tyr Arg Asp Ile Asp Leu Gly Arg Asn Glu 435 440 445 gtg gtc aac gac gtc tee acc tac gcc agc ggc aag gtc tgg ggc cag 1392 Val Val Asn Asp Val Ser Thr Tyr Ala Ser Gly Lys Val Trp Gly Gln 450 455 460 aaa tac ttc aag ggc aac ttc gag agg etc gcc att acc aag ggc aag 1440 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 - 30 - Lys Tyr Phe Lys Gly Asn Phe Glu Arg Leu Ala Ile Thr Lys Gly Lys 465 470 475 480 gtc gat cct acc gac tac ttc agg aac gag cag agc atc ccg ccg ctc 1486 Val Asp Pro Thr Asp Tyr Phe Arg Asn Glu Gin Ser Ile Pro Pro Leu 485 490 495 atc aaa aag tac tga 1503 Ile Lys Lys Tyr 500 <210> 12 <211> 500 <212> PRT <213> Phleum pratense <400> 12 Tyr Phe Pro Pro Pro Ala Ala Lys Glu Asp Phe Leu Gly Cys Leu Val 1 5 10 15 Lys Glu Ile Pro Pro Arg Leu Leu Tyr Ala Lys Ser Ser Pro Ala Tyr 20 25 30 Pro Ser Val Leu Gly Gin Thr Ile Arg Asn Ser Arg Trp Ser Ser Pro 35 40 45 Asp Asn Val Lys Pro Leu Tyr Ile Ile Thr Pro Thr Asn Val Ser His 50 55 60 ) Ile Gln Set Ala Val Val Cys Gly Arg Arg His Ser Val Arg Ile Arg ) 65 70 75 80 Val Arg Ser Gly Gly His Asp Tyr Glu Gly Leu Ser Tyr Arg Ser Leu 85 90 95 Gin Pro Glu Thr Phe Ala Val Val Asp Leu Asn Lys Met Arg Ala Val 100 105 110 Trp Val Asp Gly Lys Ala Arg Thr Ala Trp Val Asp Ser Gly Ala Gln 115 120 125 Leu Gly Glu Leu Tyr Tyr Ala Ile Tyr Lys Ala Sex . Pro Thr Leu Ala 130 135 140 Phe Pro Ala Gly Val Cys Pro Thr Ile Gly Val Gly Gly Asn Phe Ala 145 150 155 160 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 -31- Gly Gly Gly Phe Gly Met Leu Leu Arg Lys Tyr Gly Ile Ala Ala Glu 165 170 175 Asn Val Ile Asp Val Lys Leu Val Asp Ala Asn Gly Lys Leu His Asp 180 185 190 Lys Lys Ser Met Gly Asp Asp His Phe Trp Ala Val Arg Gly Gly Gly 195 200 205 Gly Glu Ser Phe Gly Ile Val Val Ala Trp Gln Val Lys Leu Leu Pro 210 215 220 Val Pro Pro Thr Val Thr Ile Phe Lys He Ser Lys Thr Val Ser Glu 225 230 235 240 Gly Ala Val Asp Ile Ile Asn Lys Trp Gln Val Val Ala Pro Gln Leu 245 250 255 Pro Ala Asp Leu Met Ile Arg Ile Ile Ala Gln Gly Pro Lys Ala Thr 260 265 270 Phe Glu Ala Met Tyr Leu Gly Thr Cys Lys Thr Leu Thr Pro Leu Met 275 280 285 Ser Ser Lys Phe Pro Glu Leu Gly Met Asn Pro Ser His Cys Asn Glu 290 295 300 Met Ser Trp Ile Gln Ser Ile Pro Phe Val His Leu Gly His Arg Asp 305 310 315 320 Ala Leu Glu Asp Asp Leu Leu Asn Arg Asn Asn Ser Phe Lys Pro Phe 325 330 335 Ala Glu Tyr Lys Ser Asp Tyr Val Tyr Gin Pro Phe Pro Lys Thr Val 340 345 350 Trp Glu Gln Ile Leu Asa Thr Trp Leu Val Lys Pro Gly Ala Gly Ile 355 360 365 Met Ile Phe Asp Pro Tyr Gly Ala Thr Ile Ser Ala Thr Pro Glu Ser 370 375 380 Ala Thr Pro Phe Pro His Arg Lys Gly Val Leu Phe Asn Ile Gin Tyr 385 390 395 400 Val Asn Tyr Trp Phe Ala Pro Gly Ala Ala Ala Ala Pro Leu Ser Trp 405 410 415 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 - 32 - Ser Lys Asp Ile Tyr Asn Tyr Met Glu Pro Tyr Val Ser Lys Asn Pro 420 425 430 Arg Gin Ala Tyr Ala Asn Tyr Arg Asp Ile Asp Leu Gly Arg Asn Glu 435 440 445 Val Val Asn Asp Val Ser Thr Tyr Ala Ser Gly Lys Val Trp Gly Gin 450 455 460 Lys Tyr Phe Lys Gly Asn Phe Glu Arg Leu Ala Ile Thr Lys Gly Lys 465 470 475 480 Val Asp Pro Thr Asp Tyr Phe Arg Asn Glu Gln Ser Ile Pro Pro Leu 485 490 495 Ile Lys Lys Tyr 500 <210> 13 <211> 12 <212> PRT <213> Dactylus glomerata <400> 13 Asp Ile Tyr Asn Tyr Met Glu Pro Tyr Val Ser Lys 1 5 10 <210> 14 <211> 11 <212> PRT <213> Dactylus glomerata <400> 14 Val Asp Pro Thr Asp Tyr Phe Gly Asn Glu Gin 1 5 10 <210> 15 <211> 17 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 - 33 <212> PRT <213> Dactylus glomerate <400> 15 Ala Arg Thr Ala Trp Val Asp Ser Gly Ala Gln Leu Gly Glu Leu Ser 1 5 10 15 Tyr <210> 16 <211> 15 <212> PRT <213> Dactylus glomerate <400> 16 Gly Val Leu Phe Asn Ile Gln Tyr Val Asn Tyr Trp Phe Ala Pro 1 5 10 15 <210> 17 <211> 11 <212> PRT <213> Cynodon dactylon <400> 17 Lys Thr Val Lys Pro Leu Tyr Ile Ile Thr Pro 1 5 10 <210> 18 <211> 22 <212> PRT <213> Cynodon dactylon <400> 18 Lys Gin Val Glu Arg Asp Phe Leu Thr Ser Leu Thr Lys Asp Ile Pro 1 5 10 15 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT1E11004/013664 - 34 - Gin Leu Tyr Leu Lys Ser 20 <210> 19 <211> 16 <212> PRT <213> Cynodon dactylon <400>

19 Thr Val Lys Pro Leu Tyr Ile Ile Thr Pro Ile Thr Ala Ala Met Ile 1 5 10 15 <210> 20 <211> 24 <212> PRT <213> Cynodon dactylon <400>

20 Leu Arg Lys Tyr Gly Thr Ala Ala Asp Asn Val Ile Asp Ala Lys Val 1 5 10 15 Val Asp Ala Gln Gly Arg Leu LeU 20 ( ) <210> 21 <211> 14 <212> PRT <213> Cynodon dactylon <400>

21 Lys Trp Gin Thr Val Ala Pro Ala Leu Pro Asp Pro Asn Met 1 5 10 <210> 22 <211> 15 <212> PRT 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 - 35 - <213> Cynodon dactylon <400>

22 Val Thr Trp Ile Glu Ser Val Pro Tyr Ile Pro Met Gly Asp Lys 1 <210> <211> <212> <213> <220> <221> <222> <223> <400> 5 23 19 PRT Cynodon dactylon MISC FEATURE (8)..(8) undetermined amino acid 23 10 15 Gly Thr Val Arg Asp Leu Leu Xaa Arg Thr Ser Asn Ile Lys Ala Phe 1 5 10 15 Gly Lys Tyr <210> 24 <211> 23 <212> PRT <213> Cynodon dactylon <400> 24 Thr Ser Asn Ile Lys Ala Phe Gly Lys Tyr Lys Ser Asp Tyr Val Leu 1 5 10 15 Glu Pro Ile Pro Lys Lys Ser 20 <210> 25 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 -36- <211> 13 <212> PRT <213> Cynodon dactylon <400> 25 Tyr Arg Asp Leu Asp Leu Gly Val Asn Gln Val Val Gly 1 5 10 <210> 26 <211> 15 <212> PRT <213> Cynodon dactylon <400> 26 Ser Ala Thr Pro Pro Thr His Arg Ser Gly Val Leu Phe Asn Ile 1 5 10 15 <210> 27 <211> 36 <212> PRT <213> Cynodon dactylon <400> 27 Ala Ala Ala Ala Leu Pro Thr Gin Val Thr Arg Asp Ile Tyr Ala Phe 1 5 10 15 Met Thr Pro Tyr Val Ser Lys Asn Pro Arg Gin Ala Tyr Val Asn Tyr 20 25 30 Arg Asp Leu Asp 35 <210> 28 <211> 14 <212> PRT <213> Lolium perenne 2013204574 12 Apr 2013 2013204574 12 Apr 2013 ;.■ WO 2005/059136 PCT/EP2004/013664 -37- <400> 28 Phe Leu Glu Pro Val Leu Gly Leu Ile Phe Pro Ala Gly Val 1 5 10 <210> 29 <211> 9 <212> PRT <213> Lolium perenne 7-Y-■ <400> 29 Gly Leu Ile Glu Phe Pro Ala Gly Val 1 5 <210> 30 <211> 22 <212> DNA <213> Secale cereale <400> 30 ggctcccggg gcgaaccagt ag 22 <210> 31 , ( ( ) <211> 23 <212> DNA <213> Secale cereale <400> 31 accaacgcct cccacatcca gtc 23 <210> 32 <211> 49 <212> DNA <213> Secale cereale <400> 32 2013204574 12 Apr 2013 2013204574 12 Apr 2013 (k, WO 2005/059136 PCT/EP2004/013664 -38- gataagcttc tcgagtgatt agtacttttt gatcagcggc gggatgctc 49 <210> 33 <211> 23 <212> DNA <213> Secale cereale <400> 33 gctctcgatc ggctacaatg gcg 23 <210> 34 <211> 25 <212> DNA <213> Secale cereale <400> 34 cacgcactac aaatctccat gcaag 25 <210> 35 <211> 30 <212> DNA <213> Secale cereale <400> 35 catgcttgat ccttattcta ctagttgggc 30 <210> 36 <211> 30 <212> DNA <213> Secale cereale <400> 36 tacgcacgat ccttattcta ctagttgggc 30 <210> 37 <211> 30 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 -39- <212> DNA <213> Hordeum vulgare <400> 37 gccttgtcct gccaccacgc cgccgccacc 30 <210> 38 <211> 23 <212> DNA <213> Hordeum vulgare <400> 38 gctctcgatc ggctacaatg gcg 23 <210> 39 <211> 25 <212> DNA <213> Hordeum vulgare <400> 39 cacgcactac aaatctccat gcaag 25 <210> 40 <211> 30 <212> DNA <213> Hordeum vulgare <400> 40 catgcttgat ccttattcta ctagttgggc 30 <210> 41 <211> 23 <212> DNA <213> Triticum aestivum <400> 41 2013204574 12 Apr 2013 2013204574 12 Apr 2013 WO 2005/059136 PCT/EP2004/013664 -40- cacgcactaa atctccatgc aag 23 <210> 42 <211> 30 <212> DNA <213> Triticum aestivum <400> 42 tacgcacgat ccttattcta ctagttgggc 30 <210> 43 <211> 23 <212> DNA <213> Triticum aestivum <400> 43 aagctctatc gcctacaatg gcg 23 <210> 44 <211> 25 <212> DNA <213> Triticum aestivum <400> 44 ggtgctcctc ttctgcgcct tgtcc 25 2013204574 12 Apr 2013 2013204574 12 Apr 2013