JP2002512022A - Human nucleic acid sequence from normal pancreas - Google Patents

Abstract

  (57) [Summary] The present invention relates to human nucleic acids-mRNA, cDNA, genomic sequences-from normal pancreatic tissue, encoding gene products or parts thereof. The invention also relates to the use of this sequence, to the polypeptide obtained by said sequence, and to its use.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to human pancreatic tissue-derived human nucleic acid sequences encoding gene products or parts thereof, their functional genes encoding at least one biologically active polypeptide and their uses. Things. Furthermore, the present invention relates to polypeptides obtained by the sequences and their uses. One of the leading causes of death is pancreatic tumors, and new treatments are needed to control them. Once used therapy, such as chemotherapy, hormonal therapy or surgical removal of tumor tissue, often does not result in complete healing.

[0002] The phenomenon of cancer often involves over- or under-expression of certain genes in degenerated cells, and it remains unclear whether changes in these rates are the cause or consequence of malignant transformation. Not. Identification of these genes will be a key step in the development of new treatments for cancer. The natural formation of cancer is often preceded by a mutant host. They can have the most versatile effects on the expression pattern of the affected tissue, such as underexpression or overexpression, as well as shortened gene expression. Some such changes attributable to these mutation cascades are:
Eventually, it can lead to malignant degeneration. The complexity of these relationships makes experimental approaches extremely difficult.

[0003] Databases composed of so-called ESTs are used to search for candidate genes, ie genes that are more strongly expressed in normal tissues compared to tumor tissues. An EST (expressed sequence tag) is a cDNA, a sequence of reverse transcribed mRNA, and thus a molecule that reflects gene expression. The EST sequence is determined for normal and degenerated tissues. These databases are supplied to a certain extent by various companies. The EST of the LifeSeq database used in the present invention is generally
It is 150 and 350 nucleotides long.

[0004] These represent an undeniable pattern for a gene, which is usually much longer (> 2000 nucleotides). Comparison of the expression patterns of normal and tumor tissues can identify ESTs that are important for tumor formation and growth. However, there are problems: The EST sequences found may belong to different regions of the unknown gene due to differently assembled cDNA libraries, and in this case the ESTs in each tissue Will be completely wrong. This will only be recognized if the complete gene is known and therefore EST is assigned to it.

[0005] It has now been found that the possibility of this error can be reduced if all ESTs are assembled from each tissue type before the expression patterns are compared with each other. . Thus, overlapping ESTs of the same gene were combined to make longer sequences (see FIGS. 1, 2a and 3). This extension, and thus the coverage of essentially longer gene regions in each base, is intended to largely avoid the above errors. Since there was no software product for this purpose, a program for assembling the genomic compartment was used, which was modified and used, to which our own proprietary program was added. The flowchart of the assembling method is shown in FIGS. 2b1-2b4.

[0006] The nucleic acid sequences of SEQ ID NOs: 2-37 and 67 have now been discovered that play a role for candidate genes in pancreatic tumors. Of particular interest are the nucleic acid sequences of SEQ ID NOs: 14, 24, 25, 27-31, 35-37 and 67. Accordingly, the present invention provides: a) a nucleic acid sequence selected from the group consisting of nucleic acid SEQ ID NOs: 14, 24, 25, 27-31, 35-37 and 67; b) an allelic variant of the nucleic acid sequence shown in a) above; Or c) a nucleic acid sequence that encodes a gene product or a part thereof, comprising: a nucleic acid sequence complementary to the nucleic acid sequence shown in a) or b) above.

[0007] In addition, the present invention relates to one of the nucleic acid sequences of SEQ ID NOs: 14, 24, 25, 27-31, 35-37, 67, or a complementary or allelic variant thereof and a nucleic acid sequence thereof. And a sequence having 90% to 95% homology to a human nucleic acid sequence. Further, the present invention relates to SEQ ID NOs: 2-37, which are enhanced and expressed in normal pancreatic tissue.
, And 67 nucleic acid sequences. The present invention also relates to nucleic acid sequences, wherein the nucleic acid sequences comprise a portion of said nucleic acid sequences in an amount sufficient to hybridize with the nucleic acid sequences of SEQ ID NOs: 14, 24, 25, 27-31, 35-37, and 67. It is.

[0008] The nucleic acid sequences according to the invention generally have a length of at least 50 to 4500 bp, preferably at least 150 to 4000 bp, in particular 450 to 3500 bp. Using the subsequences of SEQ ID NOs: 2-37 and 67 according to the invention, an expression cassette can also be assembled by performing the current steps, whereby at least one of the nucleic acid sequences according to the invention is On this cassette, at least one control or regulatory sequence widely known to those skilled in the art, for example a suitable promoter, can be linked. The sequences according to the invention can be inserted in the sense or antisense direction.

[0009] Numerous expression cassettes or vectors and promoters that can be used are known in the literature. The expression cassette or vector comprises: Bacterial, for example phagescript, pBs, φX174, pBluescript SK, pBs K
S, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene), pTrc99A, pKK223-3, pKK233-
3, pDR540, pRIT5 (Pharmacia), 2. Eukaryotic, e.g. pWLneo, pSV2cat, pOG4
4. Defined as pXT1, pSG (Stratagene), pSVK3, pBPV, PMSG, pSVL (Pharmacia).

[0010] A control or regulatory sequence is defined as a suitable promoter. In the present invention, two preferred vectors are pKK232-8 and PCM7 vector. Particularly contemplated are the following promoters: lacI, lacZ, T3, T7 , gpt, lambda P _R, trc, CMV, H
SV thymidine kinase, SV40, LTR from retrovirus and metallothionein-I from mouse. The DNA sequence located on the expression cassette can encode a fusion protein comprising a known protein and a biologically active polypeptide fragment. Expression cassettes are also a subject of the present invention.

The full-length gene can be produced using the nucleic acid fragment according to the present invention. The resulting gene is also a subject of the present invention. The invention further relates to the uses of the nucleic acid sequences according to the invention and to gene fragments obtained from the uses. The nucleic acid sequence according to the invention can be transferred into a host cell together with a suitable vector, in which case the genetic information contained and expressed on the nucleic acid fragment is present as a heterologous part. Host cells containing the nucleic acid fragments are also a subject of the present invention.

A suitable host cell is, for example, a prokaryotic cell line such as E. coli, or an eukaryotic cell line such as animal or human cells or yeast. The nucleic acid sequences according to the invention can be used in sense or antisense form. The production of polypeptides or fragments thereof is carried out by culturing host cells according to current culture methods, and then separating and purifying the peptides or fragments also using current methods. The present invention further relates to nucleic acid sequences encoding at least some of the sequences of the biologically active polypeptide.

The present invention further relates to polypeptide subsequences according to the sequence protocols of SEQ ID NOs: 39-63 and 68-71, so-called ORF (open reading frame) peptides. The present invention further relates to polypeptide sequences having at least 80% homology, especially 90% homology to the polypeptide partial sequences of SEQ ID NOs: 39-63 and 68-71 according to the present invention. The present invention further relates to antibodies against the polypeptide or a fragment thereof, wherein the antibodies are encoded by the nucleic acids of SEQ ID NOs: 2-37 and 67. Antibodies are specifically defined as monoclonal antibodies.

The antibody according to the present invention can be identified by a phage display method.
These antibodies are also the subject of the present invention. The polypeptide partial sequence according to the present invention can be used for a phage display method. Polypeptides which are identified using this method and which bind to a polypeptide subsequence according to the invention are also a subject of the invention. The nucleic acid sequence according to the present invention can also be used for the phage display method.

[0015] The polypeptide sequences of SEQ ID NOs: 39-63 and 68-71 according to the invention can also be used as a means for the discovery of active ingredients against pancreatic tumors, which is also a subject of the invention . Similarly, the use of the nucleic acid sequences shown in SEQ ID NOs: 2-37 and 67 for the expression of polypeptides which can be used as a means of finding active ingredients against pancreatic tumors is also a subject of the present invention. The invention further relates to an agent in gene therapy for the treatment of pancreatic tumors,
Or SEQ ID NOs: 39-63 and for the production of a medicament for the treatment of pancreatic tumors
68-71 polypeptide subsequences.

The present invention further relates to an agent comprising at least one polypeptide subsequence of SEQ ID NOs: 39-63 and 68-71. The nucleic acids found according to the present invention may be genomic or mRNA sequences. Furthermore, the present invention relates to genomic genes, their exon and intron structures and their splice variants, obtained from the cDNAs of SEQ ID NOs: 2-37 and 67,
And the use of them with suitable regulatory elements, such as suitable promoters and / or enhancers.

Using the nucleic acids (cDNA sequences) of SEQ ID NOs: 2-37 and 67 according to the present invention,
BAC, PAC and cosmid libraries were screened and human clones were
It is specifically separated by complementary base pairing (hybridization). The BAC, PAC and cosmid clones isolated by this method are hybridized on the metaphase chromosome using fluorescence in situ hybridization to identify the corresponding chromosomal site where the corresponding genomic gene is present. BAC, PAC and cosmid clones are sequenced (promoter, enhancer, silencer, exon and intron) to reveal the corresponding genomic gene in their complete structure. BAC, PAC
And cosmid clones can be used for gene transfer as independent molecules (see FIG. 5).

The invention further relates to BAC, PAC and cosmid clones containing functional genes, and their chromosomal localization by the sequences of SEQ ID NOs: 2-37 and 67 for use as a vehicle for gene transfer. Things.

Meaning of technical terms and abbreviations Nucleic acid = Nucleic acid in the present invention is defined as mRNA, partial cDNA, full-length cDNA and genomic gene (chromosome). ORF = open reading frame. A constant sequence of amino acids derivable from a cDNA sequence.

Contig = a set of DNA sequences that can be combined into one sequence (consensus) as a result of very similarity. Singleton = Contig containing only one sequence. Module = a domain of a protein with a defined sequence that represents one structural unit and is present in various proteins. N = optionally nucleotides A, T, G or C. X = Optionally, one of the 20 naturally occurring amino acids.

Description of Alignment Parameters Minimum Initial Match = Minimum Initial Identical Region Maximum Pads Per Read = Maximum Number of Inserts Maximum Non-Match Percentage = Maximum Deviance%.

Embodiment 1 Search for tumor-related candidate genes First, all ESTs (from October 1997) of the corresponding tissues were extracted from the LifeSeq database. These were then assembled by the GAP4 program in the Staden package using parameters of 0% non-match, 8 pads per read and 20 minimum matches. Sequences (fail) that were not recorded in the GAP4 database were first copied to this database.
Reassembled with% mismatch and then 2% mismatch. The consensus sequence was calculated from a contig of a database composed of one or more sequences.

A singleton of the database, consisting of only one sequence, was reassembled with a 2% mismatch with sequences not recorded in the GAP4 database. Meanwhile, a consensus sequence was determined for this contig. All other ESTs were reassembled with 4% mismatch. The consensus sequence was re-extracted and finally assembled with the previous consensus sequence and the singleton and sequences that were not recorded in the database with a 4% mismatch. A consensus sequence was generated and used with singletons and fail as an initial reference for tissue comparison. This method ensured that among the parameters used, all sequences represented gene regions independent of one another.

FIGS. 2 b 1-2 b 4 are diagrams showing elongation of normal pancreatic tissue EST. The sequences of each tissue thus assembled were then compared by the same program (FIG. 3). To do this, first the entire sequence of the first tissue was entered into the database. (It was therefore important that they were independent of each other.) Next, the entire sequence of the second tissue was compared to the entire sequence of the first tissue. As a result, a sequence specific to the first tissue or the second tissue, and a sequence present in both were obtained. In the latter case, the ratio of the frequency of appearance in each tissue was evaluated. All programs related to the evaluation of assembled sequences developed themselves.

[0025] All sequences that occurred four or more times in each of the compared tissues, and all sequences that occurred at least five times, as often occur in one of the two tissues,
Further consideration was given. These sequences were subjected to electronic northern (see Example 2.1), which considered their distribution in all tumor and normal tissues (FIG.
4a and FIG. 4b). The relevant candidate sequences were then extended using all Incyte ESTs and all ESTs from the public database (see Example 3).
. The sequences and their translation into potential proteins were then compared to all nucleotide and protein databases to consider potential protein coding regions.

Embodiment 2 FIG. The algorithm for finding algorithms over- or under-expressed genes Me other identification and extension of the partial cDNA sequences with altered expression patterns will be described below.
The individual steps are also summarized in a flowchart for clarity (see FIG. 4b).

2.1. Electronic Northern Blot Standard programs for homology searches, such as BLAST (SFAltschul; W. Gish; W. Mi
ller; EW Myers and DJ Lipman (1990) J. Mol. Biol. 215, 403-410), BLAST2 (
SFAltschul; TLMadden; AASchaffer; J.Zhang; Z.Zhang; W.Miller and DJL
ipman (1997) Nucleic Acids Research 25 3389-3402) or FASTA (WRPearson
USA 85 2444-2448) and the homologous sequences in various EST libraries (private or public) prepared by the tissue were partially DNA sequence S, For example, make decisions on individual ESTs or EST contigs. The frequency of the tissue-specific (relative or absolute) occurrence of this partial sequence S determined in this way is referred to as electronic Northern blot.

2.1.1. In a manner similar to that described in 2.1, the sequence of SEQ ID NO: 2 was found, which appears in normal pancreatic tissue 9 times as frequently as tumor tissue. The results are as follows.

[0029]

[Table 1]

[0030]

[Table 2]

[0031]

[Table 3]

[0032]

[Table 4]

[0033]

[Table 5]

[0034]

[Table 6]

[0035]

[Table 7]

[0036]

[Table 8]

[0037]

[Table 9]

[0038]

[Table 10]

[0039]

[Table 11]

[0040]

[Table 12]

[0041]

[Table 13]

[0042]

[Table 14]

[0043]

[Table 15]

[0044]

[Table 16]

[0045]

[Table 17]

[0046]

[Table 18]

[0047]

[Table 19]

[0048]

[Table 20]

[0049]

[Table 21]

[0050]

[Table 22]

[0051]

[Table 23]

[0052]

[Table 24]

[0053]

[Table 25]

2.2. Fisher test To determine whether a subsequence S of a gene appears significantly more frequently or less frequently in a library for normal tissues than for a degenerated tissue. Perform Fisher's exact test, standard statistical methods (WLHays (1991) Statistics, Harcourt Brace College Publishers, Fort Worth).

The null hypothesis is that the two libraries cannot be distinguished with respect to the frequency of sequences homologous to S. If this null hypothesis can be rejected with sufficient certainty, the gene belonging to S will be recognized as a favorable candidate gene for an oncogene and in the next step attempts to achieve its sequence extension will be made. Done.

Embodiment 3 FIG. Automatic Extension of Subsequence Automatic extension of subsequence S is completed in three steps: Using BLAST, all sequences homologous to S are determined from the entire set of available sequences. 2. These sequences are assembled by the standard program GAP4 (JKBonfield;
KFSmith and R. Staden (1995) Nucleic Acids Research 23 4992-4999) (Generation of contigs).

[0057] 3. The consensus sequence C is calculated from the assembled sequence. The consensus sequence C is generally longer than the initial sequence S. Thus, the electronic Northern blot deviates from that of S. Repeated Fisher tests determine whether another hypothesis about deviation from uniform expression in the two libraries can be maintained. If so, an attempt is made to extend C in the same manner as S. This repetition is repeated until the other hypothesis is rejected (exit if H ₀ ; truncation criterion I) or until automatic extension is no longer possible (while C _i > C _i-1 ; truncation criterion II) ), And continue using the consensus sequence C _i (i: repetition index) obtained in each case.

In the case of truncation criterion II, where the consensus sequence is present after the last repeat,
A complete or nearly complete sequence of the gene associated with cancer is obtained with high statistical certainty. In the same manner as in the above example, it is possible to discover the nucleic acid sequences shown in Table I from normal pancreatic tissue.

Furthermore, for individual nucleic acid sequences, it was possible to determine the peptide sequences (ORFs) listed in Table II, but here there are no peptides assigned to a few nucleic acids, And some nucleic acid sequences can be assigned one or more peptides. As already mentioned, both the determined nucleic acid sequence and the peptide sequence assigned to that nucleic acid sequence are the subject of the present invention.

Embodiment 4 FIG. Mapping Nucleic Acids on the Human Genome The human gene has been mapped using the Stanford G3 Hybrid Panel (Stewart et al., 1997), which is marketed by Research Genetics (Huntsville, Alabama). This panel is composed of 83 different genomic DNAs of the human-hamster hybrid cell line, enabling a 500 kilobase analysis. This hybrid cell line was obtained by fusing Chinese hamster cells to irradiated diploid human cells.

The retention pattern of the human chromosome fragment was determined by polymerase chain reaction using gene-specific primers and analyzed by using the Stanford RH server (http://www.stanford.edu/
Analyze using software available from RH / rhserver # form2.html). This program determines the STS marker closest to the desired gene. The corresponding cytogenetic band was determined using the "Mapview" program of the Genome Database (GDB) (http://gdbwww.dkfz-heidelberg.de).

In addition to mapping genes on human chromosome sets by various experimental methods, it is possible to determine the location of genes on human chromosomes by biological computer methods. To do this, the known program e-PCR was used (Schuler GD (1998) Electronic PCR: bridging the gap between genomic mapping and genomic sequencing. Trends Biotechnol 16: 456-459, Schuler GD (1997) Sequence mapping by electronic PCR, Genome Res. 7: 541-550).

The database used in the present invention no longer corresponds to that cited in this document and is a further development that includes data from the public database RHdb (http: // www .ebi.ac.uk / RHdb / -index.html). As with the hybrid panel mapping, the software above and the Whitehead Institut
e software (http://carbon.wi.mit.edu:8000/cgi-bin/contig/rhmapper.pl
) Was used to evaluate the results.

Embodiment 5 FIG. Acquisition of genomic DNA sequence (BAC clone) Genomic BAC clone containing the corresponding cDNA (http://www.tree.caltech.edu/; H.Sh
izuya; B.Birren, UJ.Kim, V.Mancino, T.Slepak, Y.Tachiiri, M.Simon (1992)
Proc. Natl. Acad. Sci., USA 89: 8794-8797), `` Down to the Well ''
By the method described above. In this method, a library consisting of BAC clones, which covers approximately three times the human genome, is moved into a raster, whereby the DNA of these clones with specific PCR is transferred. Can study.

In doing this, a “pool” of DNA of different BAC clones occurs. Combinatorial analysis allows determination of clones containing the desired DNA. By determining the clone, the address of that clone in the library can be determined. This address and the name of the library used determine the clones and thus the DNA sequence of these clones.

[0066]

[Table 26]

[0067]

[Table 27]

[0068]

[Table 28]

The determined candidate genes SEQ ID NO: 1 to SEQ ID NO: 37 and 67 according to the present invention
And the determined amino acid sequences of SEQ ID NO: 39 to SEQ ID NO: 63 and 68-71 are described in the following sequence protocol.

[0070]

[Sequence list]

 (1) General information: (i) Applicant: (A) NAME: metaGen-Gesellschaft fur Genomforschung mbH (ii) Title of the invention: Human nucleic acid sequence from normal uterine tissue (iii) Number of sequences: 51 (2) Sequence Information of No. 2: (i) Sequence characteristics: (A) Length: 836 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: linear (ii) molecule Type: partial cDNA generated from single-stranded EST by assembly and editing (iii) hypothetical: NO (iii) antisense: NO (vi) origin: (A) organism: human (C) organ: (vii ) Other sources: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 2

[0071]

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(2) Information of SEQ ID NO: 4: (i) Sequence characteristics: (A) Length: 871 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 4

[0073]

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(2) Information of SEQ ID NO: 6: (i) Sequence characteristics: (A) Length: 644 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organs: (vii) Other sources: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 6

[0075]

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(2) Information of SEQ ID NO: 7: (i) Sequence characteristics: (A) Length: 723 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Sequence description: SEQ ID NO: 7

[0077]

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(2) Information of SEQ ID NO: 9: (i) Sequence characteristics: (A) Length: 801 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organs: (vii) Other sources: (A) Library: cDNA library (xi) Sequence description: SEQ ID NO: 9

[0079]

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(2) Information of SEQ ID NO: 11: (i) Sequence characteristics: (A) Length: 608 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organs: (vii) Other sources: (A) Library: cDNA library (xi) Sequence description: SEQ ID NO: 11

[0081]

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(2) Information of SEQ ID NO: 12: (i) Sequence characteristics: (A) Length: 892 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 12

[0083]

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(2) Information of SEQ ID NO: 14: (i) Sequence characteristics: (A) Length: 229 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 14

[0085]

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(2) Information of SEQ ID NO: 15: (i) Sequence characteristics: (A) Length: 885 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Sequence description: SEQ ID NO: 15

[0087]

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(2) Information of SEQ ID NO: 16: (i) Sequence characteristics: (A) Length: 656 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 16

[0089]

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(2) Information of SEQ ID NO: 17: (i) Sequence characteristics: (A) Length: 105 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 17

[0091]

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(2) Information of SEQ ID NO: 18: (i) Sequence characteristics: (A) Length: 1746 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 18

[0093]

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(2) Information of SEQ ID NO: 19: (i) Sequence characteristics: (A) Length: 785 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organs: (vii) Other sources: (A) Library: cDNA library (xi) Sequence description: SEQ ID NO: 19

[0095]

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(2) Information of SEQ ID NO: 21: (i) Sequence characteristics: (A) Length: 901 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organs: (vii) Other sources: (A) Library: cDNA library (xi) Sequence description: SEQ ID NO: 21

[0097]

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(2) Information of SEQ ID NO: 24: (i) Sequence characteristics: (A) Length: 560 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organs: (vii) Other sources: (A) Library: cDNA library (xi) Sequence description: SEQ ID NO: 24

[0099]

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(2) Information of SEQ ID NO: 25: (i) Sequence characteristics: (A) Length: 565 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Sequence description: SEQ ID NO: 25

[0101]

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(2) Information of SEQ ID NO: 27: (i) Sequence characteristics: (A) Length: 553 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 27

[0103]

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(2) Information of SEQ ID NO: 28: (i) Sequence characteristics: (A) Length: 220 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 28

[0105]

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(2) Information of SEQ ID NO: 29: (i) Sequence characteristics: (A) Length: 500 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) molecular form: partial cDNA generated from single-stranded ESTs by assembly and editing (iii) hypothetical: NO (iii) antisense: NO (vi) origin: (A) organism: human (C) Organs: (vii) Other sources: (A) Library: cDNA library (xi) Sequence description: SEQ ID NO: 29

[0107]

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(2) Information of SEQ ID NO: 30: (i) Sequence characteristics: (A) Length: 298 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 30

[0109]

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(2) Information of SEQ ID NO: 31: (i) Sequence characteristics: (A) Length: 970 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 31

[0111]

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(2) Information of SEQ ID NO: 35: (i) Sequence characteristics: (A) Length: 1032 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 35

[0113]

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(2) Information of SEQ ID NO: 36: (i) Sequence characteristics: (A) Length: 1400 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organs: (vii) Other sources: (A) Library: cDNA library (xi) Sequence description: SEQ ID NO: 36

[0115]

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(2) Information of SEQ ID NO: 37: (i) Sequence characteristics: (A) Length: 366 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other sources: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 37

[0117]

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(2) Information of SEQ ID NO: 39: (i) Sequence characteristics: (A) Length: 130 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 39

[0119]

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(2) Information of SEQ ID NO: 40: (i) Sequence characteristics: (A) Length: 127 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 40

[0121]

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(2) Information of SEQ ID NO: 41: (i) Sequence characteristics: (A) Length: 139 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 41

[0123]

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(2) Information of SEQ ID NO: 42: (i) Sequence characteristics: (A) Length: 133 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 42

[0125]

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(2) Information of SEQ ID NO: 43: (i) Sequence characteristics: (A) Length: 117 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 43

[0127]

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(2) Information of SEQ ID NO: 44: (i) Sequence characteristics: (A) Length: 160 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular form: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 44

[0129]

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(2) Information of SEQ ID NO: 45: (i) Sequence characteristics: (A) Length: 73 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 45

[0131]

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(2) Information of SEQ ID NO: 46: (i) Sequence characteristics: (A) Length: 78 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 46

[0133]

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(2) Information of SEQ ID NO: 47: (i) Sequence characteristics: (A) Length: 50 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: straight Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 47

[0135]

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(2) Information of SEQ ID NO: 48: (i) Sequence characteristics: (A) Length: 70 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: straight Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 48

[0137]

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(2) Information of SEQ ID NO: 49: (i) Sequence characteristics: (A) Length: 51 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 49

[0139]

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(2) Information of SEQ ID NO: 50: (i) Sequence characteristics: (A) Length: 161 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 50

[0141]

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(2) Information of SEQ ID NO: 51: (i) Sequence characteristics: (A) Length: 107 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 51

[0143]

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(2) Information of SEQ ID NO: 52: (i) Sequence characteristics: (A) Length: 118 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 52

[0145]

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(2) Information of SEQ ID NO: 56: (i) Sequence characteristics: (A) Length: 76 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: straight Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 56

[0147]

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(2) Information of SEQ ID NO: 57: (i) Sequence characteristics: (A) Length: 78 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 57

[0149]

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(2) Information of SEQ ID NO: 58: (i) Sequence characteristics: (A) Length: 136 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 58

[0151]

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(2) Information of SEQ ID NO: 59: (i) Sequence characteristics: (A) Length: 115 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: straight Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 59

[0153]

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(2) Information of SEQ ID NO: 60: (i) Sequence characteristics: (A) Length: 70 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 60

[0155]

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(2) Information of SEQ ID NO: 61: (i) Sequence characteristics: (A) Length: 95 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 61

[0157]

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(2) Information of SEQ ID NO: 62: (i) Sequence characteristics: (A) Length: 62 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 62

[0159]

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(2) Information of SEQ ID NO: 63: (i) Sequence characteristics: (A) Length: 69 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 63

[0161]

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(2) Information of SEQ ID NO: 67: (i) Sequence characteristics: (A) Length: 1850 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: Linear (ii) Molecular form: partial cDNA generated from single-stranded EST by assembly and editing (iii) Hypothetical: NO (iii) Antisense: NO (vi) Origin: (A) Organism: human (C) Organ: (vii) Other origin: (A) Library: cDNA library (xi) Description of sequence: SEQ ID NO: 67

[0163]

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(2) Information of SEQ ID NO: 68: (i) Sequence characteristics: (A) Length: 74 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 68

[0165]

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(2) Information of SEQ ID NO: 69: (i) Sequence characteristics: (A) Length: 63 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 69

[0167]

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(2) Information of SEQ ID NO: 70: (i) Sequence characteristics: (A) Length: 530 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 70

[0169]

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(2) Information of SEQ ID NO: 71: (i) Sequence characteristics: (A) Length: 82 amino acids (B) Type: protein (C) Number of chains: single chain (D) Topology: direct Linear (ii) molecular type: ORF (iii) hypothetical: yes (vi) origin (A) organism: human (xi) description of sequence: SEQ ID NO: 71

[0171]

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[Sequence list]

[Brief description of the drawings]

FIG. 1 is a diagram showing a systematic gene search in the Incyte LifeSeq database.

FIG. 2a is a diagram showing the principle of EST assembly.

FIG. 2b1 is a diagram showing the overall principle of EST assembly.

FIG. 2b2 is a diagram showing the overall principle of EST assembly.

FIG. 2b3 is a diagram showing the overall principle of EST assembly.

FIG. 2b4 is a diagram showing the overall principle of EST assembly.

FIG. 3 shows the intra-silica subtraction of gene expression in various tissues.

FIG. 4a shows the determination of tissue-specific expression by electronic Northern.

FIG. 4b is a diagram showing electronic northern.

FIG. 5 is a diagram showing the separation of genomic BAC and PAC clones.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12P 21/02 C 5/10 21/08 C12P 21/02 C12N 15/00 ZNAA 21/08 5/00 A (72) Inventor Schmidt, Alumin Germany, Day 14197 Berlin, Laubacher Strasse 6 / tubite (72) Inventor Pilarski, Christian Germany, Day 01474 Schönfeld-Bishik, Heinrich Lange -Strasse 13 Tse (72) Inventor Darl, Edgar Germany, Day 14480 Potzdam, Eleonore-Projeska-Strasse 6 (72) Inventor Rosenthal, Andre Germany, Day 10115 Bell Down, Coppens Platz 10

Claims (38)

[Claims] 1. a) a nucleic acid sequence selected from the group of SEQ ID NOs: 14, 24, 25, 27-31, 35-37 and 67; b) an allelic variant of the nucleic acid sequence named in a) above; Or c) a nucleic acid sequence encoding a gene product or a portion thereof, comprising: a nucleic acid sequence complementary to the nucleic acid sequence named in a) or b) above. 2. A nucleic acid sequence according to one of the sequences SEQ ID NO: 14, 24, 25, 27-31, 35-37, 67 or a complementary or allelic variant thereof. 3. The nucleic acid sequence of SEQ ID NOs: 2-37 and 67, wherein the nucleic acid sequence is enhanced and expressed in normal pancreatic tissue. 4. BACs, PACs containing functional genes and their chromosomal localization according to the sequences of SEQ ID NOs: 2-37 and 67 for use as a vehicle for gene transfer.
And cosmid clones. 5. The method according to claim 1, which has 90% homology with a human nucleic acid sequence.
The nucleic acid sequence according to 1. 6. The method according to claim 1, which has 95% homology with a human nucleic acid sequence.
The nucleic acid sequence according to 1. 7. A nucleic acid sequence comprising a part of the nucleic acid sequence according to claim 1 in an amount sufficient to hybridize with the sequence according to claim 1 to 6. 8. The nucleic acid sequence according to claim 1, wherein the fragment has a length of at least 50 to 4500 bp. 9. The nucleic acid sequence according to claim 1, wherein the fragment has a length of at least 50 to 4000 bp. 10. The nucleic acid sequence according to claim 1, which encodes at least one partial sequence of a biologically active polypeptide. 11. An expression cassette comprising a sequence or nucleic acid fragment according to any one of claims 1 to 9 together with at least one control or regulatory sequence. 12. The control or regulatory sequence is a suitable promoter.
An expression cassette comprising the sequence or nucleic acid fragment of claim 11. 13. The expression cassette according to claim 11, wherein the DNA sequence located on the cassette encodes a fusion protein comprising a known protein and a biologically active polypeptide fragment. 14. Use of the nucleic acid sequence according to claims 1 to 10 for producing a full-length gene. 15. A DNA fragment comprising the gene obtained from the use according to claim 14. 16. A host cell comprising the nucleic acid fragment according to any one of claims 1 to 10 as a heterologous portion of gene information that can be expressed. 17. The host cell according to claim 16, which is a prokaryotic or eukaryotic cell line. 18. The host cell according to claim 16, wherein the prokaryotic cell line is E. coli and the eukaryotic cell line is an animal, human or yeast cell line. 19. A method for producing a polypeptide or fragment, wherein the host cell according to claim 16 is cultured. 20. An antibody against the polypeptide or fragment encoded by the nucleic acids of SEQ ID NOs: 2-37 and 67, obtainable according to claim 19. 21. The antibody of claim 20, which is monoclonal. 22. The antibody according to claim 20, which is a phage display antibody. 23. A partial polypeptide sequence represented by SEQ ID NOs: 39 to 63 and 68 to 71. 24. A sequence having at least 80% homology to these sequences.
24. The polypeptide partial sequence according to 23. 25. A polypeptide known from phage display and capable of binding to the polypeptide-subsequence of claim 23. 26. The sequence having at least 90% homology to these sequences.
24. The polypeptide partial sequence according to 23. 27. Use of the polypeptide subsequences set forth in SEQ ID NOs: 39-63 and 68-71 as a means of discovering active ingredients against pancreatic tumors. 28. Use of the nucleic acid sequences shown in SEQ ID NOs: 2-37 and 67 for the expression of a polypeptide that can be used as a means to discover active ingredients against pancreatic tumors. 29. SEQ ID NOs: 2-37 and 67 in sense or antisense form
Use of the nucleic acid sequence of 30. As an agent in gene therapy for the treatment of pancreatic tumor,
Use of the polypeptide subsequences of SEQ ID NOs: 39-63 and 68-71. 31. Use of a polypeptide subsequence of SEQ ID NOs: 39-63 and 68-71 for the manufacture of a medicament for treating a pancreatic tumor. 32. An agent comprising at least one of the polypeptide subsequences of SEQ ID NOs: 39-63 and 68-71. 33. The nucleic acid sequence according to claim 1, which is a genomic sequence. 34. The nucleic acid sequence according to claim 1, which is an mRNA sequence. 35. Genomic genes, their promoters, enhancers, silencers, exon structures, intron structures and splice variants thereof, obtainable from the cDNAs of SEQ ID NOs: 2-37 and 67. 36. Use of a genomic gene according to claim 33 with appropriate regulatory elements. 37. Use according to claim 36, wherein the regulatory element is a suitable promoter and / or enhancer. 38. The nucleic acid sequence of claim 1, wherein the size of the fragment is at least 300 to 3500 bp in length.