AU4361699A

AU4361699A - Novel angiotensin receptor, production and use thereof

Info

Publication number: AU4361699A
Application number: AU43616/99A
Authority: AU
Inventors: Holger Heitsch; Jorg-Peter Kleim; Gunther Riess; Gertrud Sibenhorn
Original assignee: Aventis Pharma Deutschland GmbH
Current assignee: Sanofi Aventis Deutschland GmbH
Priority date: 1998-06-08
Filing date: 1999-05-12
Publication date: 1999-12-30
Also published as: BR9912183A; CA2330229A1; HUP0102540A3; PL345949A1; WO1999064585A2; TR200003662T2; ID27566A; KR20010052703A; DE19825494A1; AR020086A1; JP2002517237A; CN1304449A; HUP0102540A2; WO1999064585A3; EP1086217A2

Description

WO 99/64585 PCT/EP99/03249 Description Novel angiotensin receptor, its preparation and use 5 The invention relates to a novel angiotensin receptor, its preparation and use. The renin angiotensin system plays an important role in the regulation of the cardiovascular system and of electrolyte and fluid balance. The 10 physiologically active effector hormones, e.g. angiotensin I (AnglI) and angiotensin 1-7 (Ang1-7) are produced by proteolytic cleavage of the precursor angiotensinogen, which is synthesized in the liver, and, respectively, of angiotensin I, which is formed from the angiotensinogen. A large number of proteases are involved in these cleavages (e.g. renin and 15 angiotensin converting enzyme (ACE)). Two human angiotensin receptors, which have been designated AT1 and AT2, have been described to date. Both receptors are angiotensin I (Ang II) receptors, i.e. these receptors preferably bind Ang 1I. The cDNA 20 sequences of these two receptors (Takayanagi, R. et al. (1992). Biochemical and Biophysical Research Communications 183, 910-915; Tsuzuki, S. et al. (1994). Biochemical and Biophysical Research Communications 200, 1449-1454) each possess seven hydrophobic regions which constitute potential transmembrane domains. Because of 25 this characteristic domain structure, it is assumed that angiotensin receptors AT1 and AT2 belong to the family of 7-transmembrane receptors, which mediate the effect of the ligand angiotensin Il by way of intracellular G protein-coupled signal transduction cascades. 30 Because of the pharmacological effects which are mediated by other angiotensins differing from AnglI and/or of the peptides (endogenously formed fragments of Angi and Angli) which are derived from angiotensin I and angiotensin 11, respectively, it has been suspected that further human angiotensin receptors must exist. In this connection, a receptor has recently 35 been demanded for the heptapeptide Ang1 -7 (Ferrario, CM et al. (1997) Hypertension 30, 535-541).

WO 99/64585 PCT/EP99/03249 2 The present invention relates to a novel angiotensin receptor which possesses at least one subunit which contains the amino acid sequence SEQ ID NO. 10, with this amino acid sequence possibly possessing further amino acids at the C-terminal end. 5 The angiotensin receptor is a transmembrane receptor. In contrast to the previously described angiotensin receptors, it preferably possesses fewer than 7 transmembrane domains. The subunit which contains the sequence SEQ ID NO. 10 preferably possesses one transmembrane domain. This 10 transmembrane domain is preferably more than 6, preferably 8 or 9, particularly preferably 7, amino acids in length. The transmembrane domain preferably has the sequence "MSIVIPL" (written in the single letter code for amino acids). 15 The angiotensin receptor can possess one or more subunits. For example, the receptor can be present either as a) a monomer (one subunit which contains the sequence SEQ ID NO. 10), or as b) a dimer, containing an identical subunit or a different subunit, or as 20 c) a tetramer, containing three further identical subunits or containing one identical subunit and two different subunits. However, the angiotensin receptor can also be present as a monomer (one subunit) or as a dimer (two different subunits) until the ligand has been 25 bound and only change into a dimeric or tetrameric form, respectively, as a result of binding the ligand. Receptors which possess a similar structure to that of the novel angiotensin receptor are, inter alia, the insulin receptor, growth factors, such as nerve 30 growth factor (NGF), endothelial growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), T cell antigen receptors, hematopoietic cytokine receptors (e.g. IL1, IL5), and adhesion molecules (integrins and selectins). 35 Where appropriate, the novel angiotensin receptor can be modified post translationally; for example, the receptor can be glycosylated and/or phosphorylated.

WO 99/64585 PCT/EP99/03249 3 The angiotensin receptor can bind its ligand(s), and can mediate the effect of this/these ligand(s) intracellularly. The angiotensin receptor can bind angiotensin 1-7 and/or a derivative of angiotensin 1-7. Derivatives of angiotensin 1-7 are preferably truncated peptides which are derived from 5 Angl-7, e.g. degradation products of Angl-7. Examples of derivatives of Angl-7 are angiotensin Il and angiotensin IV. The receptor preferably binds Ang1 -7. The invention relates to a receptor which possesses or contains the amino 10 acid sequence shown in Table 2, SEQ ID NO. 10. The angiotensin receptor, or the subunit which contains the sequence SEQ ID NO. 10, is at least 175 amino acids in length. The invention furthermore relates to a nucleic acid which possesses a 15 sequence which encodes the novel angiotensin receptor ("receptor"). For example, the receptor can be encoded, entirely or partially, by a nucleic acid which possesses the sequence SEQ ID NO. 9. A nucleic acid which encodes the receptor or a subunit of the receptor can contain the sequence SEQ ID NO. 9 and, possibly, possess further nucleotides, which belong to 20 the coding sequence, at the 3' end. The nucleic acid may, for example, be a DNA, a cDNA or an RNA. The nucleic acid may be a gene, with it then being possible for the sequence SEQ ID NO. 9 to be interrupted by noncoding sequences (introns). 25 Where appropriate, the nucleic acid can be derivatized. A derivative of the nucleic acid can, for example, be a salt or a nucleic acid derivative which contains modified nucleotides in addition to the natural nucleotides or which is composed completely of modified nucleotides. 30 The invention also relates to a process for preparing a nucleic acid which encodes the angiotensin receptor and which contains the sequence SEQ ID NO. 9. For example, such a nucleic acid can be amplified in a PCR which uses oligonucleotides, as primers, whose sequences correspond to 35 homologous regions (conserved regions) in the nucleotide sequences of the angiotensin receptors AT1 and AT2 and cDNA which is preferably prepared from a tissue (or cells) on which angiotensin 1-7, or a derivative thereof, has an effect and/or from tissue (or cells) in which AT1 and/or AT2 WO 99/64585 PCT/EP99/03249 4 are not expressed. In a particular embodiment of the process, use is made of degenerate primers whose sequences are derived from the homologous regions of the sequences of AT1 and AT2. Preference is given to using the primers Ang3A and Ang3B for this purpose. cDNA which has been 5 prepared from endothelial cells can, for example, be used as the template. For example, cDNA prepared from HUVECs (human umbilical vein endeothelial cells) can be used as a template. The invention also relates to a process for preparing the novel receptor. For 10 example, the novel angiotensin receptor can be prepared by integrating a nucleic acid, whose sequence encodes the angiotensin receptor and which preferably contains the sequence SEQ ID NO. 9, into a suitable vector, with the recombinant vector being introduced into a cell and the angiotensin receptor being expressed in this cell. 15 The invention furthermore relates to the use of a nucleic acid, which encodes the novel angiotensin receptor and which preferably contains the sequence SEQ ID NO. 9, for example in a process for preparing a recombinant angiotensin receptor, with the nucleic acid, which is under the 20 control of a suitable promoter, being introduced into a cell and expressed in this cell. The invention furthermore relates to the use of a nucleic acid, which encodes the novel angiotensin receptor and which preferably contains the 25 sequence SEQ ID NO. 9, for preparing a recombinant cell in which the angiotensin receptor can be expressed. The invention relates to a recombinant cell which expresses an angiotensin receptor having the sequence SEQ ID NO. 10. The invention furthermore relates to the use of a nucleic acid, which encodes the angiotensin receptor and which preferably 30 contains the sequence SEQ ID NO. 9, for producing a drug for the treatment and prevention of diseases which are accompanied by faulty regulation of the cardiovascular system, of electrolyte balance or of fluid balance. The invention relates to a drug that comprises a nucleic acid having the sequence [lacuna] ID NO. 9. 35 The invention furthermore relates to the use of a nucleic acid, which encodes the angiotensin receptor and which preferably contains the sequence SEQ ID NO. 9, for producing a transgenic animal which does not WO 99/64585 PCT/EP99/03249 5 contain the corresponding angiotensin receptor gene, or for producing a transgenic animal which overexpresses the corresponding angiotensin receptor gene. Transgenic animals can be used, for example, for characterizing the specificity and biochemical function of the angiotensin 5 receptor. The invention relates to a nonhuman transgenic animal which expresses the angiotensin receptor. The invention furthermore relates to the use of a nucleic acid, which encodes the angiotensin receptor and which preferably contains the 10 sequence SEQ ID NO. 9, in processes for identifying and characterizing substances which can be employed as antagonists and agonists of the angiotensin receptor and/or in processes in which substances which inhibit or activate the functional expression of the encoded angiotensin receptor are identified. The invention relates to a process for identifying and 15 characterizing agonists or antagonists of the angiotensin receptor, with a nucleic acid which possesses the sequence SEQ ID NO. 9 being introduced into a cell and the angiotensin receptor being expressed in the cell, this cell being incubated with a substance to be investigated and the effect of this substance on the angiotensin receptor being determined. 20 The invention also relates the use of a nucleic acid, which encodes the angiotensin receptor and which preferably contains the sequence SEQ ID NO. 9, in gene therapy or for producing a drug which can be employed in gene therapy. 25 In addition to this, the invention relates to the use of a nucleic acid, which encodes the angiotensin receptor and which preferably contains the [lacuna] SEQ ID NO. 9, as a tool in molecular biology (e.g. as a probe). 30 The invention also relates to the use of the novel angiotensin receptor. For example, the angiotensin receptor can be used in processes for identifying and characterizing substances which can be employed as agonists or antagonists of the receptor (e.g. binding assays and functional assays for screening). 35 Furthermore, the angiotensin receptor, or parts thereof, for example an epitope which is predetermined, for example, by the amino acid sequence SEQ ID NO. 10 or a part thereof, can be used for preparing antibodies in WO 99/64585 PCT/EP99/03249 7 -1, -3 and -4 corresponds to the eukaryotic consensus sequence for the start point for initiating mRNA translation. The open reading frame in SEQ ID NO. 9 encodes a protein containing at least 175 amino acids (SEQ ID NO. 10). A hydrophobicity analysis of this 5 amino acid sequence, carried out using the GCG program, showed that the sequence possesses a hydrophobic domain of about 7 amino acids in length at the N terminus. This hydrophobic domain may represent a transmembrane domain which is used to anchor this novel receptor in the membrane. By contrast, the sequence possesses a domain at the 10 C terminus which is more or less hydrophilic. Examples: Example 1: 15 The oligonucleotide primers Ang3A and Ang3B, which, based on the AT1/2 receptor genes, enclose a 0.19 kbp DNA fragment, were used for the first PCR. cDNA from a commercially available HUVEC cDNA library (Stratagene catalog No. 937223, Stratagene GmbH, Heidelberg) was used as a template DNA. 20 Nucleotide sequence of Ang3A: SEQ ID NO. 1: 5'-TTG TKC TKK YCT TYW TCW TTT SCT GGM TTC CC-3' Nucleotide sequence of Ang3B: 25 SEQ ID NO. 2: 5'-TTY CCM ASA AAR CMA TAM ARA ARM GGA TT-3' where M = (A/C) Y =(C/T) R =(A/G) 30 S = (G/C) K =(G/T) W =(A/T). For the PCR reaction, 1 pg of cDNA from the HUVEC library was incubated 35 with recombinant Thermus aquaticus (Taq) DNA polymerase (Boehringer Mannheim, Germany) and 10 pmol each of primers Ang3A and Ang3B per 50 pl reaction assay in a thermocycler (Perkin Elmer 2400 thermo cycler, Perkin Elmer Applied Biosystems, Weiterstadt). The PCR reaction WO 99/64585 PCT/EP99/03249 8 was carried out using the following temperature profile: 20" at 950C, 10" at 380C and 20" at 720C (60 cycles). A DNA fragment of 130 base pairs (bp) in length was amplified. 5 This 130 bp DNA fragment was ligated into the vector pCR 2.1 TOPO@ (Invitrogen BV, NV Leek, Netherlands) and transformed into E. coli ToplOF'. The sequence of the 130 bp DNA fragment present in pCR2.1 was determined using a capillary electrophoresis sequencer (ABI 310, Applied Biosystems, Weiterstadt) (SEQ ID NO. 3). 10 Example 2: The sequence SEQ ID NO. 3 was compared with the sequences contained in the publicly available databases (e.g. EMBL and GenBank@) using the 15 program modules FASTA and TFASTA (University of Wisconsin Genetics Computer Group (GCG), USA, software package). In addition, the sequence was used for a corresponding homology comparison with the sequences which are included in the Incyte company (Palo Alto, USA) Lifeseq@ database. 20 Sequences of expressed sequence tags (ESTs) which overlap with SEQ ID NO. 3 were identified in the Lifeseq@ database (Lifeseq@ clones Nos. 1458429, 3341232, 3440195, 1250818, 2836520, 1310286 and 1362205). The primers Ang4A and Ang4B were synthesized on the basis of 25 a sequence which the inventors assembled by ordering the overlapping expressed sequence tags (ESTs). Nucleotide sequence of Ang4A: SEQ ID NO. 4: 5'-TGG GGG TTG ATA CAG CAG AGA C-3' 30 Nucleotide sequence of Ang4B: SEQ ID NO. 5: 5'-GCA CTG CCC TCT CTT TAT CCA AA-3' PCR reactions were carried out with in each case 0.1 pg of cDNA from the HUVEC library, as a template, and 10 pmol each of the primers Ang4A and 35 Ang4B per 50 pl reaction assay and using the Advantage cDNA polymerase mix (Clontech, Heidelberg). The following temperature profile was used for the PCR reactions: 30" 940C and 2'30" 600C (60 cycles). A DNA fragment of 500 bp in length was obtained. This was cloned and WO 99/64585 PCT/EP99/03249 9 sequenced (SEQ ID NO. 6) as described in Example 1. The sequence possesses an open reading frame (ORF). Example 3: 5 The primers Ang5A and Ang5B were synthesized for elucidating the sequence of the 500 bp DNA fragment in the 5' region. Nucleotide sequence of Ang5A: 10 SEQ ID NO. 7: 5'-TGG GGA TTG ATA GGC AG-3' Nucleotide sequence of Ang5B: SEQ ID NO. 8: 5'-ATA ACT GTT GGA TTT CTC AA-3' 15 The nucleotide sequence of Ang5A and Ang5B in each case corresponds to a part of the sequence of the 500 bp DNA fragment. These two primers are employed in further PCRs using the M13 reverse and M13 forward (-20) primers, respectively. The M13 reverse and M13 forward (-20) primers correspond to sequences of the Uni-ZAP@ bacteriophage vector 20 used for preparing the HUVEC cDNA library. These PCR reactions were carried out using in each case 0.1 pg of cDNA from the HUVEC library, as a template, and 10 pmol each of the primers Ang5A or Ang5B and, respectively, M13 reverse or M13 forward (-20) (from the Topo-TA cloning kit (Clontech)) and Taq polymerase (Boehringer Mannheim) and also 25 Taq StartTM antibody (Clontech). The PCR reactions were carried out using the following temperature profile: 30" 960C, 30" 500C, and 45" 720C (70 cycles). This resulted in a DNA fragment having a total length of 660 bp being 30 obtained. This fragment was cloned and sequenced as described in Example 1. The DNA fragment has the sequence SEQ ID NO. 9. The sequence SEQ ID NO. 9 contains the entire 5' coding region. The GCG modules Assemble, Map, Translate, Plotstructure and Pileup 35 were also used for performing the computer-assisted sequence analyses, in addition to the software modules which have already been mentioned.

WO 99/64585 PCT/EP99/03249 10 Table 1: SEQ ID NO. 9 (Partial) nucleotide sequence of the angiotensin receptor 1 TCGTGTGTGA ACATCACAGG GTTTGTGGAT GCACTTAGAT GTTTGCAATG 51 AGCACTGTGG CTGGCATGCC CCAGTGTTTT GGATACCAAT GCATAGGACT 101 CCATAGTAAT CGAATTTACC AGAGGCGAAC GTCATGAGCA TAGTGATCCC 151 ATTGGGGGTT GATACAGCAG AGACGTCATA CTTGGAAATG GCTGCAGGTT 201 CAGAACCAGA ATCCGTAGAA GCTAGCCCTG TGGTAGTTGA GAAATCCAAC 251 AGTTATCCCC ACCAGTTATA TACCAGCAGC TCACATCATT CACACAGTTA 301 CATTGGTTTG CCCTATGCGG ACCATAATTA TGGTGCTCGT CCTCCTCCGA 351 CACCTCCGGC TTCCCCTCCT CCATCAGTCC TTATTAGCAA AAATGAAGTA 401 GGCATATTTA CCACTCCTAA TTTTGATGAA ACTTCCAGTG CTACTACAAT 451 CAGCACATCT GAGGATGGAA GTTATGGTAC TGATGTAACC AGGTGCATAT 501 GTGGTTTTAC ACATGATGAT GGATACATGA TCTGTTGTGA CAAATGCAGC 551 GTTTGGCAAC ATATTGACTG CATGGGGATT GATAGGCAGC ATATTCCTGA 601 TACATATCTA TGTGAACGTT GTCAGCCTAG GAATTTGGAT AAAGAGAGGG 651 CAGTGCTACT 5 Table 2: SEQ ID NO. 10 (Partial) amino acid sequence of the novel receptor 1 MSIVIPLGVDTAETSYLEMAAGSEPESVEASPVVVEKSNSYPHQLYTSSS 51 HHSHSYIGLPYADHNYGARPPPTPPASPPPSVLISKNEVG IFTTPNFDET 101 SSATTISTSEDGSYGTDVTRCICGFTHDDGYMICCDKCSV WQHIDCMGID 151 RQHIPDTYLCERCQPRNLDKERAVL WO 99/64585 PCT/EP99/03249 11 Table 3: SEQ ID NO. 3 1 CTCCTCCATC AGTCCTTATT AGCAAAAATG AAGTAGGCAT ATTTACCACT 51 CCTAATTTTG ATGAAACTTC CAGTGCTACT ACAACCAGAG CGAG Table 4: SEQ ID NO. 6 1 ATTGGGGGTT GATACAGCAG AGACGTCATA CTTGGAAATG GCTGCAGGTT 51 CAGAACCAGA ATCCGTAGAA GCTAGCCCTG TGGTAGTTGA GAAATCCAAC 101 AGTTATCCCC ACCAGTTATA TACCAGCAGC TCACATCATT CACACAGTTA 151 CATTGGTTTG CCCTATGCGG ACCATAATTA TGGTGCTCGT CCTCCTCCGA 201 CACCTCCGGC TTCCCCTCCT CCATCAGTCC TTATTAGCAA AAATGAAGTA 251 GGCATATTTA CCACTCCTAA TTTTGATGAA ACTTCCAGTG CTACTACAAT 301 CAGCACATCT GAGGATGGAA GTTATGGTAC TGATGTAACC AGGTGCATAT 351 GTGGTTTTAC ACATGATGAT GGATACATGA TCTGTTGTGA CAAATGCAGC 401 GTTTGGCAAC ATATTGACTG CATGGGGATT GATAGGCAGC ATATTCCTGA 451 TACATATCTA TGTGAACGTT GTCAGCCTAG GAATTTGGAT AAAGAGAGGG 501 CAGTGCTACT 5

Claims

1. An angiotensin receptor which possesses at least one subunit which contains the amino acid sequence SEQ ID NO. 10. 5

2. The angiotensin receptor as claimed in claim 1, wherein the amino acid sequence SEQ ID NO. 10 may possibly possess further amino acids at the C-terminal end. 10 3. The angiotensin receptor as claimed in one or more of claims claim 1 and 2, wherein the subunit which contains the sequence SEQ ID NO. 10 possesses a transmembrane domain.

4. The angiotensin receptor as claimed in one or more of claims 1 to 3, 15 wherein the receptor binds angiotensin 1-7 or a derivative of angiotensin 1-7.

5. The angiotensin receptor as claimed in one or more of claims 1 to 4, wherein at least one subunit of the receptor is encoded by a nucleic 20 acid having the sequence SEQ ID NO. 9, and wherein the sequence SEQ ID NO. 9 possibly contains further nucleotides, which belong to the coding sequence, at the 3' end.

6. A process for preparing a nucleic acid having the sequence SEQ ID 25 NO. 9, wherein, in a PCR reaction, oligonucleotides whose sequences correspond to homologous regions in the nucleotide sequences of the angiotensin receptors AT1 and AT2 are employed as primers and cDNA which is prepared from cells in which AT1 and AT2 receptors are not expressed is employed as a template. 30

7. A process for preparing an angiotensin receptor as claimed in one or more of claims 1 to 5, wherein a nucleic acid which contains the sequence SEQ ID NO. 9 is integrated into a suitable vector, the recombinant vector is introduced into a cell and the angiotensin 35 receptor is expressed in this cell.

8. The use of a nucleic acid which contains the sequence SEQ ID NO. 9 in a process for preparing a recombinant angiotensin WO 99/64585 PCT/EP99/03249 13 receptor, with the nucleic acid, which is under the control of a suitable promoter, being introduced into a cell and expressed in this cell. 5 9. The use of a nucleic acid which contains the sequence SEQ ID NO. 9 for preparing a recombinant cell in which the angiotensin receptor can be expressed.

10. The use of a nucleic acid which possesses the sequence SEQ ID 10 NO. 9 for producing a drug for treating and/or preventing diseases which are accompanied by faulty regulation of the cardiovascular system, of electrolyte balance and/or of fluid balance.

11. The use of a nucleic acid which contains the sequence SEQ ID 15 NO. 9 for producing a drug which can be used in gene therapy.

12. The use of a nucleic acid which contains the sequence SEQ ID NO. 9 in a process for identifying and characterizing substances which can be used as agonists or antagonists of the angiotensin 20 receptor. 3. The use of an angiotensin receptor as claimed in one or more of claims 1 to 5 in a process for identifying and characterizing agonists or antagonists of the angiotensin receptor. 25 4. A process for identifying and characterizing agonists or antagonists of the angiotensin receptor, wherein a nucleic acid which possesses the sequence SEQ ID NO. 9 is introduced into a cell and the angiotensin receptor is expressed in this cell, this cell is incubated 30 with a substance to be investigated and the effect of this substance on the angiotensin receptor is determined. 5. A drug which comprises a nucleic acid which possesses the sequence SEQ ID NO. 9. 35 6. A recombinant cell which is prepared by a nucleic acid having the sequence SEQ ID NO. 9 being introduced into this cell and expressed in the cell. WO 99/64585 PCT/EP99/03249 1/7 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) Applicant: (A) NAME: Hoechst Marion Roussel Deutschland GmbH (B) STREET: (C) CITY: Frankfurt (D) FEDERAL STATE: (E) COUNTRY: Germany (F) POSTAL CODE: 65926 (G) TELEPHONE:

069-305-7072 (H) FAX: 069-35-7175 (I) TELEX: (ii) TITLE OF INVENTION: Novel angiotensin receptor, its prepara tion and use (iii) NUMBER OF SEQUENCES: 10 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA REPLACEMENT SHEET (RULE 26) WO 99/64585 PCT/EP99/03249 2/7 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1..32 (D) MISCELLANEOUS INFORMATION:/note = "M = (A/C); Y = (C/T); R = (A/G); S = (G/C); K = (G/T); W=(.A/T) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TTGTKCTKKY CTTYWTCWTT TSCTGGMTTC CC 32 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1..29 (D) MISCELLANEOUS INFORMATION:/note = "M = (A/C); Y = (C/T); R = (A/G); S = (G/C); K = (G/T); W=(A/T)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TTYCCMASAA ARCMATAMAR AARMGGATT 29 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 94 base pairs (B) TYPE: Nucleotide REPLACEMENT SHEET (RULE 26) WO 99/64585 PCT/EP99/03249 3/7 (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1..94 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CTCCTCCATC AGTCCTTATT AGCAAAAATG 30 AAGTAGGCAT ATTTACCACT CCTAATTTTG 60 ATGAAACTTC CAGTGCTACT ACAACCAGAG CGAG 94 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1..22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TGGGGGTTGA TACAGCAGAG AC 22 REPLACEMENT SHEET (RULE 26) WO 99/64585 PCT/EP99/03249 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1..23 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GCACTGCCCT CTCTTTATCC AAA 23 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 510 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1..510 REPLACEMENT SHEET (RULE 26) WO 99/64585 PCT/EP99/03249 5/7 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ATTGGGGGTT GATACAGCAG AGACGTCATA CTTGGAAATG GCTGCAGGTT CAGAACCAGA 60 ATCCGTAGAA GCTAGCCCTG TGGTAGTTGA GAAATCCAAC AGTTATCCCC ACCAGTTATA 120 TACCAGCAGC TCACATCATT CACACAGTTA CATTGGTTTG CCCTATGCGG ACCATAATTA 180 TGGTGCTCGT CCTCCTCCGA CACCTCCGGC TTCCCCTCCT CCATCAGTCC TTATTAGCAA 240 AAATGAAGTA GGCATATTTA CCACTCCTAA TTTTGATGAA ACTTCCAGTG CTACTACAAT 300 CAGCACATCT GAGGATGGAA GTTATGGTAC TGATGTAACC AGGTGCATAT GTGGTTTTAC 360 ACATGATGAT GGATACATGA TCTGTTGTGA CAAATGCAGC GTTTGGCAAC ATATTGACTG 420 CATGGGGATT GATAGGCAGC ATATTCCTGA TACATATCTA TGTGAACGTT GTCAGCCTAG 480 GAATTTGGAT AAAGAGAGGG CAGTGCTACT 510 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1..17 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TGGGGATTGA TAGGCAG 17 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA REPLACEMENT SHEET (RULE 26) WO 99/64585 PCT/EP99/03249 6/7 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1..20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ATAACTGTTG GATTTCTCAA 20 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 660 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1..660 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TCGTGTGTGA ACATCACAGG GTTTGTGGAT GCACTTAGAT GTTTGCAATG AGCACTGTGG 60 CTGGCATGCC CCAGTGTTTT GGATACCAAT GCATAGGACT CCATAGTAAT CGAATTTACC 120 AGAGGCGAAC GTCATGAGCA TAGTGATCCC ATTGGGGGTT GATACAGCAG AGACGTCATA 180 CTTGGAAATG GCTGCAGGTT CAGAACCAGPA ATCCGTAGAA GCTAGCCCTG TGGTAGTTGA 240 GAAATCCAAC AGTTATCCCC ACCAGTTATA TACCAGCAGC TCACATCATT CACACAGTTA 300 CATTGGTTTG CCCTATGCGG ACCATAATTA TGGTGCTCGT CCTCCTCCGA CACCTCCGGC 360 TTCCCCTCCT CCATCAGTCC TTATTAGCAA AAATGAAGTA GGCATATTTA CCACTCCTAA 420 TTTTGATGAA ACTTCCAGTG CTACTACAAT CAGCACATCT GAGGATGGAA GTTATGGTAC 480 TGATGTAACC AGGTGCATAT GTGGTTTTAC ACATGATGAT GGATACATGA TCTGTTGTGA 540 CAAATGCAGC GTTTGGCAAC ATATTGACTG CATGGGGATT GATAGGCAGC ATATTCCTGA 600 TACATATCTA TGTGAACGTT GTCAGCCTAG GAATTTGGAT AAAGAGAGGG CAGTGCTACT 660 REPLACEMENT SHEET (RULE 26) WO 99/64585 PCT/EP99/03249 7/7 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 175 amino acids (B) TYPE: Amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Protein (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..175 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Met Ser Ile Val Ile Pro Leu Gly Val Asp Thr Ala Glu Thr Ser Tyr 1 5 10 15 Leu Glu Met Ala Ala Gly Ser Glu Pro Glu Ser Val Glu Ala Ser Pro 20 25 30 Val Val Val Glu Lys Ser Asn Ser Tyr Pro His Gin Leu Tyr Thr Ser 35 40 45 Ser Ser His His Ser His Ser Tyr Ile Gly Leu Pro Tyr Ala Asp His 50 55 60 Asn Tyr Gly Ala Arg Pro Pro Pro Thr Pro Pro Ala Ser Pro Pro Pro 65 70 75 80 Ser Val Leu Ile Ser Lys Asn Glu Val Gly Ile Phe Thr Thr Pro Asn 85 90 95 Phe Asp Glu Thr Ser Ser Ala Thr Thr Ile Ser Thr Ser Glu Asp Gly 100 105 110 Ser Tyr Gly Thr Asp Val Thr Arg Cys Ile Cys Gly Phe Thr His Asp 115 120 125 Asp Gly Tyr Met Ile Cys Cys Asp Lys Cys Ser Val Trp Gin His Ile 130 135 140 Asp Cys Met Gly Ile Asp Arg Gin His Ile Pro Asp Thr Tyr Leu Cys 145 150 155 160 Glu Arg Cys Gin Pro Arg Asn Leu Asp Lys Glu Arg Ala Val Leu 165 170 175 REPLACEMENT SHEET (RULE 26)