EP1080195A1

EP1080195A1 - Endometriosis-associated gene

Info

Publication number: EP1080195A1
Application number: EP99927792A
Authority: EP
Inventors: Anna Starzinski-Powitz; Silvia Kotzian; Heike Handrow-Metzmacher
Original assignee: Individual
Current assignee: Individual
Priority date: 1998-05-29
Filing date: 1999-05-28
Publication date: 2001-03-07
Also published as: US6586569B1; NZ508439A; AU4502499A; JP2002517193A; US20030109018A1; CA2329527A1; CN1307636A; AU765515B2; WO1999063079A1; DE19824230A1

Abstract

The invention relates to a gene associated with invasive processes, e.g. endometriosis, to a polypeptide coded by said gene, to an antibody directed against the polypeptide, and to the pharmaceutical application of the nucleic acid, the polypeptide and the antibody.

Description

ENDOMETRIOSE-ASSOCIATED GEN

description

The present invention relates to an invasive process, e.g. Endometriosis-associated gene, a polypeptide encoded thereof, an antibody directed against the polypeptide and the pharmaceutical application of the nucleic acid, the polypeptide and the antibody.

Endometriosis is the second most common female disease and is defined as the presence of endometrial cells outside the uterus. Endometriosis affects about one in five women of reproductive age, and even one in two in women with fertility problems.

Under normal circumstances, uterine lining (endometrium) is found exclusively in the uterus. With endometriosis, tissue outside of the uterus that histologically looks like uterine mucosa can be found, for example on the outside of the uterus, on the intestine, or even in the pancreas or lungs. Although these foci of endometriosis are located outside the uterus, they also bleed during menstruation, so they are influenced by the hormones of the female cycle. Since the focus of the endometriosis, like the endometrium, changes in volume in the cycle, depending on the location, these changes can cause pain. In addition, the body responds with an inflammatory reaction to the endometriosis cells, which in turn triggers pain. The inflammation also leads to adhesions in the area of the ovaries and fallopian tubes and is therefore responsible for the so-called mechanical sterility of the women concerned. Apparently, messenger substances (e.g. cytokines, prostaglandins) are also used in endometriosis. f that can reduce the fertility of the women affected, even if there are no adhesions.

Due to their patho-biological properties, endometriosis cells could be classified between normal cells and tumor cells: on the one hand, they do not show any neoplastic behavior, on the other hand, however, like metastatic tumor cells, they have the ability to move across organ boundaries and to grow into other organs, i.e. they show invasive behavior. For this reason, endometriosis cells are defined in the literature as "benign tumor cells", although no tumor-specific mutations in proto-oncogenes have been found in such cells.

Since the pathogenesis of endometriosis is still completely unknown, there are no effective treatment or prevention options for endometriosis-related diseases.

The invention was based on the task of identifying new genes which play a role in invasive processes and which may be associated with the pathophysiological phenotype of endometriosis.

This object is achieved according to the invention by identifying, cloning and characterizing a gene which is referred to as an endometriosis-associated gene and which codes for a polypeptide. This gene sequence was discovered using differential display RT-PCR (Liang and Pardee, Science 257 (1992), 967-971). For this purpose, invasive and non-invasive variants of an endometriosis cell line were compared with each other. This led to a cDNA sequence that is specific for the invasive variant of the endometriosis cells. An associated RNA of 4 kb in length was found. A corresponding cDNA isolated from a cDNA phage bank has an open reading frame (ORF) of 302 amino acids. An object of the present invention is a nucleic acid which

(a) the in SEQ ID NO. 1, 3 or / and 5 nucleotide sequences shown, a combination or a protein-coding section thereof,

(b) a nucleotide sequence corresponding to the sequence from (a) as part of the degeneration of the genetic code or

(c) comprises a nucleotide sequence hybridizing with the sequences from (a) and / or (b) under stringent conditions.

These nucleic acids preferably encode a polypeptide associated with invasive processes, or a portion thereof.

The following nucleotide sequences with the following accession numbers are stored in the EMBL EST database: Z98886, Ac003017, AL023586, Aa452993, Aa452856. These sequences do not represent nucleic acids according to the invention. The first two of these sequences are DNAs which have been isolated from the human brain and which, with the sections from nucleotide 970 to approx. 2000 and from 760 to approx. 1450, show a 90% base identity with SEQ ID NO. 1, or with the sections from nucleotide 1054 to 2084 and from 844 to approx. 1534 based on SEQ ID NO. 3, which has 84 additional bases at the 5 'end. ALO23586 is also a human sequence, very similar to Z98885 and also with SEQ ID NO. 1 in the range from 970 to approx. 2000 homology.

The sequences Aa452993 and Aa452856 originate from mouse embryos and show base identity with the nucleotides (nt) from approx. 1060 to approx. 1450 and from approx. 24 to 440 of SEQ ID NO. 1 or from about 1 144 to about 1534 or from about 108 to about 524 according to the nucleotide positions in SEQ ID NO. 3. None of these 4 sequences has been assigned a reading frame or a function. The in SEQ ID NO. 1 nucleotide sequence contains an open reading frame, which corresponds to a polypeptide with a length of 302 amino acids. This polypeptide is in SEQ ID NO. 2 amino acid sequence shown. SEQ ID NO. shows a nucleotide sequence as in SEQ ID NO. 1, but has 84 additional nucleotides at the 5 'end. This shifts the positions of the corresponding nucleotides by 84 nucleotides each. The SEQ ID NO. 3 encoded polypeptides thus has 28 additional amino acids at the N-terminus and, with a total of 330 amino acids, is in SEQ ID NO. 4 shown. SEQ ID NO. 2 and 4 represent a portion of the C-terminal end of the native polypeptide.

For illustration, reference is made to FIG. 1, which shows a schematic representation of the cDNA of the endometriosis-associated gene according to the invention. 5 exons E1 to E5 and the position of fragment 1 (394 nt) used as a probe in the DDRT-PCR can be seen. The positions of the PCR primers (see Example 4, Table 1) which were used for the RT-PCR are also shown.

Another exon 4a, whose nucleotide sequence is shown in SEQ ID NO. 5 is shown. This exon 4a can be present. If it exists, it is located between exon 4 and exon 5. This corresponds to the position between nt1054 and nt 1055 in SEQ ID NO.3. A combination of the sequences SEQ ID NO. 1/3 with SEQ ID NO. 5 is thus e.g. a sequence which contains the sequence of exon 4a at the position mentioned.

In addition to the in SEQ ID NO. 1, 3 and 5 nucleotide sequences shown, as well as combinations thereof, such as the sequence of SEQ ID NO. 3, which between nt1054 and 1055 the sequence of SEQ ID NO. 5, and nucleotide sequences corresponding to one of these sequences in the context of the degeneration of the genetic code also encompasses the present invention Nucleotide sequences that hybridize with one of the aforementioned sequences. The term "hybridization" according to the present invention is used in Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1 989), 1 .101-1 .104). One speaks preferably of a stringent hybridization if, after washing for 1 hour with 1 × SSC and 0.1% SDS at 50 ° C., preferably at 55 ° C., particularly preferably at 62 ° C. and most preferably at 68 ° C, in particular for 1 h in 0.2 X SSC and 0.1% SDS at 55 ° C, preferably at 55 ° C, particularly preferably at 62 ° C and most preferably at 68 ° C, a positive hybridization signal is also observed . One under such washing conditions with one or more of those in SEQ ID NO. 1, 3 and 5 nucleotide sequences shown or a nucleotide sequence hybridizing to these sequences in the context of the degeneration of the genetic code hybridizing nucleotide sequence is a nucleotide sequence according to the invention.

The nucleotide sequence according to the invention is preferably a DNA. However, it can also comprise an RNA or a nucleic acid analog, such as a peptidic nucleic acid. The nucleic acid according to the invention particularly preferably comprises a protein-coding section of the sequence shown in SEQ ID NO. 1, 3 or / and 5 nucleotide sequences shown or a sequence which has a homology of more than 80%, preferably more than 90% and particularly preferably more than 95% to that in SEQ ID NO. 1, 3 or 5 nucleotide sequences shown or a preferably at least 20 nucleotides (nt) and particularly preferably at least 50 nt long section thereof. The same also applies to nucleic acids which, as described above, have the sequence of SEQ ID NO. 5 in addition to those of SEQ ID NO. 1 or 3. The homology is given in percent of identical positions when comparing two nucleic acids (or peptide chains), whereby 100% homology means the complete identity of the compared chain molecules (Herder: Lexicon of Biochemistry and Molecular Biology, Spektrum Akademischer Verlag 1 995). Nucleic acids according to the invention are preferably obtainable from mammals and in particular from humans. They can be made using known techniques using short sections of the SEQ ID NO. 1, 3 or / and 5 nucleotide sequences shown are isolated as hybridization probes and / or as amplification primers. Furthermore, nucleic acids according to the invention can also be produced by chemical synthesis, it being possible, instead of the usual nucleotide building blocks, to use modified nucleotide building blocks, for example 2′-O-alkylated nucleotide building blocks.

The nucleic acids or sections thereof according to the invention can thus be used for the production of primers and probes which are preferably provided with markers or marking groups. Intron-spanning oligonucleotide primers are also preferred, which are particularly suitable for identifying different mRNA species.

Another object of the present invention are the polypeptides encoded by the nucleic acids as defined above. These polypeptides preferably contain (a) those in SEQ ID NO. 2 or 4 amino acid sequence shown or

(b) a homology of more than 70%, preferably more than 80% and particularly preferably more than 90% to the amino acid sequence according to (a).

In addition to the in SEQ ID NO. 2 or 4 polypeptides shown, the invention also relates to muteins, variants and fragments thereof. These are to be understood as sequences which differ from those in SEQ ID NO. By substitution, deletion and / or insertion of individual amino acids or short amino acid segments. Distinguish 2 or 4 amino acid sequences shown.

The term "variant" includes both naturally occurring allelic variations or splice variations of the endometriosis protein, as well as recombinant DNA technology (in particular in w ^' trσ mutagenesis with the help of chemically synthesized oligonucleotides) produced proteins which, in terms of their biological and / or immunological activity, correspond to those described in SEQ IN NO. 2 or 4 shown proteins essentially correspond. This term also includes chemically modified polypeptides. These include polypeptides that are modified on the termini and / or on reactive amino acid side groups by acylation, for example acetylation or amidation. The amino acid sequences according to the invention also include polypeptide fragments (peptides) which have a section of at least 10 amino acids long in the sequence shown in SEQ ID NO. Represent 2 or 4 amino acid sequence shown.

Another object of the present invention is a vector which contains at least one copy of a nucleic acid according to the invention. This vector can be any prokaryotic or eukaryotic vector on which the DNA sequence according to the invention is preferably located in connection with expression signals such as a promoter, operator, enhancer etc. Examples of prokaryotic vectors are chromosomal vectors, such as bacteriophages, and extrachromosomal vectors, such as plasmids, with circular plasmid vectors being particularly preferred. Suitable prokaryotic vectors are e.g. in Sambrook et al., supra, Chapters 1-4. The vector according to the invention is particularly preferably a eukaryotic vector, e.g. a yeast vector, or a higher cell vector, e.g. a plasmid vector, viral vector or plant vector. Such vectors are familiar to the person skilled in the field of molecular biology, so that there is no need to go into them here. In this context, reference is made in particular to Sambrook et al., Supra, Chapter 1 6.

The invention also relates to a vector which has a section of at least 21 nucleotides long in SEQ ID NO. 1, 3 or / and 5 sequences shown or a combination thereof. This preferably has Section of a nucleotide sequence which originates from the protein-coding region of the said sequences or from a region essential for the expression of the protein or polypeptide. These nucleic acids are particularly suitable for the production of therapeutically usable antisense nucleic acids, which are preferably up to 50 nucleotides long.

Another object of the present invention is a cell which is transformed with a nucleic acid according to the invention or a vector according to the invention. The cell can be both a eukaryotic and a prokaryotic cell. Methods for transforming cells with nucleic acids are state of the art and therefore need not be explained in more detail. Examples of preferred cells are eukaryotic cells, in particular animal and particularly preferably mammalian cells.

Another object of the present invention is an antibody or a fragment of such an antibody against the polypeptide (s) encoded by the endometriosis gene or variants thereof. Such antibodies are particularly preferably directed against the entire polypeptides encoded therein or against a peptide sequence which corresponds to the amino acids 1 -330 of the amino acids shown in SEQ ID NO. 4 corresponds to the amino acid sequence shown.

The identification, isolation and expression of a gene according to the invention which is specifically associated with invasive processes and in particular with endometriosis provides the prerequisites for the diagnosis, therapy and prevention of diseases which are based on the above-mentioned disorders.

With the aid of a polypeptide according to the invention or fragments of this polypeptide as an immunogen, it becomes possible to produce antibodies against such polypeptides. Antibodies can be produced in a customary manner by immunizing experimental animals with the complete polypeptide or fragments thereof and subsequently obtaining them of the resulting polyclonal antisera. According to the Köhler and Milstein method and their further developments, monoclonal antibodies can be obtained from the antibody-producing cells of the experimental animals in a known manner by cell fusion. Human monoclonal antibodies can also be produced by known methods. Such antibodies could then be used both for diagnostic tests, in particular of endometriosis cell tissue, or for therapy.

For example, using the ELISA technique, samples such as body fluids, especially human body fluids (eg blood, lymph or cerebrospinal fluid) can be examined for the presence of a polypeptide encoded by the endometriosis gene and for the presence of autoantibodies against such a polypeptide . Polypeptides or fragments thereof encoded by the endometriosis gene can then be detected in such samples with the help of a specific antibody, e.g. of an antibody according to the invention can be detected. For the detection of autoantibodies, recombinant fusion proteins can preferably be used which contain part or a whole or all of the polypeptide which is encoded by the endometriosis gene and which are fused to a protein domain which enables detection, for example the maltose-binding Protein (MBP).

Diagnostic tests can also be carried out using specific nucleic acid probes for detection at the nucleic acid level, e.g. at the gene or transcript level.

Furthermore, the provision of the nucleotide and amino acid sequences and antibodies according to the invention enables a targeted search for effectors of the polypeptides / proteins. Effectors are substances which have an inhibitory or activating effect on the polypeptide according to the invention and which are able to control the cell functions controlled by the polypeptides selectively influence. These can then be used in the therapy of corresponding clinical pictures, such as those based on invasive processes. The invention thus also relates to a method for identifying effectors of the endometriosis proteins, in which cells which express the protein are brought into contact with various potential effector substances, for example low-molecular substances, and the cells are checked for changes, for example cell-activating, cell-inhibiting. cell proliferative and / or cell genetic changes, analyzed. In this way, binding targets of the endometriosis proteins can also be identified.

Since many tumor diseases are associated with invasive processes, the discovery of the gene according to the invention provides additional possibilities for the diagnosis, prevention and therapy of cancerous diseases.

The discovery of a gene which is also responsible for invasive processes not only opens up possibilities for the treatment of diseases which are based on such cell changes, but the sequences according to the invention can also be used to make such processes usable. This could be important, for example, when implanting embryos.

The present invention thus also relates to a pharmaceutical composition which comprises, as active components, nucleic acids, vectors, cells, polypeptides, peptides and / or antibodies, as stated above.

The pharmaceutical composition according to the invention can furthermore contain pharmaceutically customary excipients, auxiliaries and / or additives, and optionally further active components. The pharmaceutical composition can be used in particular for the diagnosis, therapy or prevention of diseases involving invasive Processes are associated. Furthermore, the composition according to the invention can also be used for the diagnosis of a predisposition to such diseases, in particular for the diagnosis of a risk for endometriosis.

The invention is explained in more detail by the following figures, sequence listing and examples.

FIG. 1 shows a schematic representation of the cDNA of the endometriosis-associated gene, only exons E1 to E5 being shown.

SEQ ID NO. 1 represents a nucleotide sequence which contains genetic information coding for the endometriosis-associated gene, an open reading frame ranging from nucleotide 3 to 91 1, and SEQ ID NO. 2 represents the amino acid sequence of the open reading frame of the one in SEQ ID NO. 1 shown nucleotide sequence, wherein the amino acid sequence of the open reading frame ranges from amino acid 1 to 302.

SEQ ID NO. 3 represents a nucleotide sequence like that of SEQ ID NO. 1, but it contains an additional 84 nucleotides at the 5 'end, the open reading frame extends from nucleotide 3 to 995. SEQ ID NO. 4 represents the amino acid sequence of the open reading frame of the one in SEQ ID NO. 3 shows the nucleotide sequence shown, this amino acid sequence having 320 amino acids, of which the C-terminal 302 are identical to those in SEQ ID NO. 2. SEQ ID NO. 5 shows the nucleotide sequence of the additional exon 4a which may be present, consisting of the 21 8 nt shown, with exon 4a between nucleotide 1 054 and 1055 (based on SEQ ID NO. 3), if it exists.

EXAMPLES

Example 1 Cultivation of cells

To identify an endometriosis-associated gene, invasive and non-invasive cells from the epithelial endometriosis cell line EEC1 45T ^{+ were} used. The cells were cultivated in Dulbecco medium (DMEM) with 10% fetal calf serum and diluted 1: 5 twice a week (passage). For the comparison of the expression patterns by means of DDRT-PCR (see below), invasive cells from passage 1 7 and non-invasive cells from passage 33 were used. The cells were transformed with SV40 and analyzed by differential display reverse transcription polymerase chain reaction (DDRT-PCR).

Example 2 DDRT-PCR

This method developed by Liang and Pardee is a method for distinguishing the expression pattern of different cell types or the changing expression pattern of a cell type under different living conditions or during changing developmental stages (Liang and Pardee (1 992), Science 257, 967-971). The basis of the DDRT-PCR technique is based on the consideration that approximately 1,500 genes are expressed in each cell and that in principle each individual mRNA molecule can be represented by means of reverse transcription and amplification with random primers.

In this example, the cellular PolyA ⁺ RNA was first transcribed into cDNA with the aid of several different dT ^ VX primers (downstream primers, anchor primers). The resulting cDNA populations were then PCR-amplified with 4 downstream and 20 upstream primers from the RNA-Map ™ kit from Genhunter, Nashville (1 994) with the addition of a radioactively labeled nucleotide. After the amplification, the reaction mixtures were concentrated in vacuo and the cDNA fragments obtained were separated in a six percent native PAA gel (polyacrylamide). DNA detection was carried out by autoradiography. PCR approaches which showed clear differences in the band pattern for the two cell variants to be examined were repeated twice in order to check the reproducibility. If the differences found previously were confirmed, the bands were eluted from the gel, reamplified, cloned and sequenced according to known methods.

This method resulted in a 394 bp fragment (fragment 1, nucleotides 1 235 to 1 628 of the nucleic acid sequence shown in SEQ ID NO. 1, see also FIG. 1), which was specific for the invasive cell variant. This fragment 1 was used as a probe in Northern blot analysis (see below).

Example 3 Analysis of the Fragment 1 Expression Profile in Human Northern Blot Analyzes

Northern blot analyzes were carried out to check the expression pattern for the DDRT-PCR fragment 1. For this purpose, 20 μg total RNA or 4 μg polyA ⁺ RNA were separated in 1% denaturing agarose gels and transferred to a nylon membrane overnight. The RNA was fixed on the membrane by irradiation with UV light. The hybridization with ³² P-labeled probes (labeling using the RPL kit from Amersham) was carried out overnight in a hybridization solution containing formamide at 42 ° C. The membrane was then washed with increasing stringency until a selective intensity of the radioactive radiation was measurable. The hybridization pattern was displayed by placing an X-ray film (NEF-NEN: DuPont) and exposure for several days. Northern blot analyzes with RNA from the following cells or tissues were carried out to determine the expression pattern for DDRT-PCR fragment 1: invasive cells of the epithelial endometriosis cell line EEC145T ^"1" (passage 17) non-invasive cells of the epithelial endometriosis cell line EEC145T ⁺

(Passage 33)

Cells of the peritoneal cell line EEC143T ⁺

Endometrial tissue - cells of the invasive human bladder carcinoma cell line EJ28

Cells of the non-invasive human bladder carcinoma cell line RT1 1 2

After hybridization with the probe for DDRT-PCR fragment 1, an approximately 4 kb mRNA could be detected, which could only be detected in the invasive variant of the endometriosis cell line EEC 1 45T ⁺ .

Other human tissues were tested. An mRNA of 4 kb in length which clearly hybridized with fragment 1 was found in the spleen and in brain mRNAs of 4 kb and> 9 kb in length.

The Northern blot analyzes were carried out using two human Multiple Tissue Northern (MTN) blots from Clontech according to the manufacturer's protocol. The expression was checked in the following tissues: large intestine, small intestine, heart, brain, testes, liver, lungs, spleen, kidney, ovaries, pancreas, peripheral blood leukocytes, placenta, prostate, skeletal muscle, thymus. The expression pattern obtained with the radioactively labeled 3'-probe "DDRT-PCR-fragment1" is as follows: 4 kb mRNA (expected size): brain spleen

Pancreas 9, 5 kb mRNA: brain No specific hybridization was found in the other tissues.

In situ hybridization

To elucidate the cellular expression pattern, mRNA was carried out in situ hybridizations on 10 μm paraffin sections from different tissues. For this purpose, the "DDRT-PCR fragment1" was used as a digoxigenin-labeled RNA probe. The detection reaction was carried out using a digoxigenin-specific antibody coupled to alkaline phosphatase (A). BM Purple served as the substrate for the AP, from which a blue precipitate is formed after dephosphorylation. The results are shown in the table below and show predominant expression in invasive / migrating cells.

Example 4 RT-PCR

RT-PCR (Reverse Transcription PCR) offers a sensitive method for checking the expression pattern.

For this purpose, 1 μg of the respective PolyA ⁺ RNA was analyzed using 400 U M-MLV reverse transcriptase (Gibco-BRL) in a total volume of 30 μl rewritten in cDNA. 1 μl of it was used for the subsequent PCR with different primer combinations.

The PCR primers P1 to P7 used are shown in Table 1 (see Figure 1).

Table 1

RT-PCR experiments using different primer combinations were carried out with polyA ⁺ RNA from various cell lines and tissues. The results are shown in Table 2.

Table 2

PK = primer combination

P1 7 = endometriosis cell line EEC 1 45T, passage 1 7, invasive P33 = EEC145T endomteriosis cell line, passage 33, non-invasive

Per = Peritoneum cell line Per143T

EM = endometrial tissue

EJ28 = invasive bladder carcinoma cell line

RT1 1 2 = non-invasive bladder carcinoma cell line

E = endometrial tissue

EE = endometrial tissue from an endometriosis patient

PEE = peritoneal endometriosis biopsy n.g. = not tested

The RT-PCR results confirmed the expression specific for fragment 1 in the early passages (passage 17, passage 20) of the endometriosis cell line EEC145T ^"1" . In contrast to the Northern blot analyzes, weak expression in the endometrium was also shown.

RT-PCR analyzes with intron spanning primers

To check possible alternative exons, RT-PCR experiments with intron-spanning primers were carried out. It was shown that in addition to the mRNA described, there is at least one further mRNA species that contains another exon (4a) with a length of 21 8 bp between the 4th and 5th exon. This exon is located in the 3'-UTR (untranslated region), that is, following the coding region. The sequence of exon 4a is shown below.

gcggttgtcc ggaatgccag tggctcctgg gcagatgtgc accccagatt cagcctttgt gatagattcc aacacgttct ggcctcagac cacctttgtg gtggggccag actgctctgg gcaaagtgaa gctggccttt atgctcccaggattcggcggccggccggccg

Nucleotide sequence of the alternative exon 4a Example 5 Preparation of the cDNA phage bank EEC14

The cDNA phage bank EEC14 was developed according to the method of Short, J.M. et al. (1 988) Nucleic Acids Res. 1 6: 7583-7600.

First, the reverse transcription of polyA ⁺ RNA of invasive cells (passage 1 7) of the epithelial endometriosis cell line EEC145T ^{"1" was carried out} . The primer used for this is composed of an X / 7θ / interface and a 1 8 nucleotide long poly (dT) sequence. An adapter containing a £ co /? / Site was ligated to the resulting cDNA fragments. The two restriction sites allow a directed insertion of the cDNA fragments into the ZAP Express ™ vector. Inserts can be cut out of the phage in the form of a kanamycin-resistant pBK CMV phagemid.

Example 6 Phage bank screening

The DDRT-PCR fragment 1 (394 bp) was used as a probe to screen 10 ⁶ pfu (plaque forming units) of the cDNA phage bank EEC14 according to the manufacturer's protocol (company Stratagene). The probe was labeled with digoxigenin (Boehringer Mannheim) using PCR. The plaques formed after infection of the XL Iblue MRF 'bacterial strain were transferred to a nylon membrane and hybridized thereon with the above-mentioned probe. The hybridized, digoxigenin-labeled probe was detected according to the Boehringer Mannheim chemiluminescence protocol.

Positive plaques were selected and rescreened. The plaques positive in the rescreening were used for the excision. By cutting out the vector portion from the phage using ExAssist helper phages, kanamycin-resistant pBK CMV phagemids were created, which after propagation in the bacterial strain XL0LR ™ isolated and sequenced could become. The isolated phagemid clone Q2A with a size of 2.3 kb contained the longest insert, the sequence of which was determined and SEQ ID NO. 1 is shown. The sequence of the DDRT-PCR fragment 1 is found in nucleotides 1 235 to 1 628 based on SEQ ID NO. 1 .

Example 7 Southern Blot Analysis

10μg genomic DNA from female and male subjects was cut with different restriction endonucleases. The fragments were separated in an agarose gel and transferred to a nylon membrane. Hybridization with the digoxigenin-labeled DDRT-PCR fragment1 took place on this membrane.

The hybridization could be demonstrated by chemiluminescence according to the Boehringer protocol. When using different restriction endonucleases, only one band was detected in both female and male DNA samples. This result suggests that the gene on which fragment 1 is based is a singular, non-gender-specific gene. Meanwhile, two genomic clones PAC J 1472 and PAC N 1 977 have been isolated with the DDRT-PCR fragment 1.

Example 8 Fluorescence In Situ Hybridization (FISH)

The genomic clones obtained in Example 7 were localized to chromosome 1 (1 p36) by means of fluorescence in situ hybridization (Lichter et al. (1 990), Science 247: 64-69).

Example 9 Production of Specific Antibodies

The nucleotides 584 to 909 of the cDNA sequence mentioned above were cloned into the expression vector pMAL cRI via suitable restriction sites. to Expression of the sequence, the construct was transformed into E.coli DH5 σ cells. The translated protein fragment was cut out of the SDS polyacrylamide gel and used to immunize rabbits.

Example 10 RACE (rapid amplification of cDNA ends)

Since the length of the cDNA clone Q2A (see Example 6) deviates from the size of the detected mRNA (approx. 4 kb), RACE experiments were carried out to obtain further sequence information. With the help of this method, it is possible to obtain cDNA sequences from an mRNA template between a defined internal sequence and unknown sequences at the 5 'or 3' end. The 3 'end of clone Q2A was confirmed by 3'-RACE experiments from the 5th exon.

For the 5'RACE, the cDNA first strand synthesis was carried out with a gene-specific primer, which Exon hybridizes, and then a homopolymeric nucleotide tail is attached using the enzyme terminal transferase. This attached sequence allowed an amplification of the sequence region, which is between the gene-specific primer and the homopolymeric nucleotide tail. The following additional sequence was obtained, which is 5 'of the Q2A sequence and belongs to the first exon: cc egg ccg cec ega gtg gag egg atc cac ggg cag atg cag atg cet 47

Arg Pro Pro Arg Val Glu Arg Ile His Gly Gin Met Gin Met Pro

1 5 10 15

ega gec aga egg gec cac agg cec egg gac cag geg gec gec etc gtg 95 Arg Ala Arg Arg Ala His Arg Pro Arg Asp Gin Ala Ala Ala Leu Val 20 25 30

The underlined sequence represents the first nucleotides of the Q2A sequence, the sequence before that corresponds to the new one obtained by 5'RACE Sequence. The open reading frame fits into the one already derived for fragment and contains two putative start codons (underlined).

The nucleotide acid sequence, which the previously obtained and in SEQ ID NO. 1 sequence shown and the additional 84 nt at the 5 'end is in SEQ ID NO. 3 shown.

Claims

Expectations

1 . Nucleic acid, characterized in that it

(a) those in SEQ ID NO. 1, 3 and/or 5 shown nucleotide sequences, a combination or a protein-coding section thereof, (b) a nucleotide sequence corresponding to the sequence from (a) in the context of the degeneration of the genetic code or (c) one with the sequences from (a ) and/or (b) under stringent

Conditions hybridizing nucleotide sequence includes, with the proviso that the nucleic acid is different from the sequences given with the accession numbers Z98886, Ac00301 7, Aa452993, AL023586 and Aa452856 in the EMBL EST database.

2. Nucleic acid according to claim 1, characterized in that it has a protein-coding section in SEQ ID NO. 1, 3 and/or 5 nucleotide sequences shown.

3. Nucleic acid according to claim 1, characterized in that it has a homology of more than 80% to that in SEQ ID NO. 1, 3 and/or 5 has the nucleotide sequences shown or a section thereof.

4. Nucleic acid according to one of claims 1 to 3, characterized in that it codes for a polypeptide associated with invasive processes or a section thereof.

5. Modified nucleic acid or nucleic acid analogue comprising a nucleotide sequence according to any one of claims 1 to 4.

6. Polypeptide, characterized in that it is encoded by a nucleic acid according to one of claims 1 to 4, the proviso of claim 1 not being taken into account.

7. Polypeptide according to claim 6, characterized in that it

(a) those in SEQ ID NO. 2 or 4 shown amino acid sequence or (b) has a homology of more than 70% to the amino acid sequence according to (a).

8. Modified polypeptide comprising an amino acid sequence according to claim 6 or 7.

9. Peptide, characterized in that it has a section of at least 10 amino acids long in SEQ ID NO. 2 or 4 represents the amino acid sequence shown.

0. Vector, characterized in that it has at least one copy of a nucleic acid according to one of claims 1 to 4 or a portion thereof.

1 . Vector according to claim 10, characterized in that it enables the expression of the nucleic acid in a suitable host cell.

2. Cell, characterized in that it is transformed with a nucleic acid according to one of claims 1 to 4 or a vector according to claim 10 or 1 1.

?. Antibodies against a polypeptide according to one of claims 6 to 8 or against a peptide according to claim 9.

4. Antibody according to claim 1 3, characterized in that it is against the entire polypeptide or against a fragment thereof selected from a section of amino acids 1 to 330 from SEQ ID NO. 4 is directed.

5. Pharmaceutical composition, characterized in that it comprises as active component:

(a) a nucleic acid according to one of claims 1 to 5, wherein the proviso of claim 1 is not taken into account, (b) a vector according to claim 10 or 1 1,

(c) a cell according to claims 1 2,

(d) a polypeptide according to one of claims 6 to 8, (e) a peptide according to claim 9 and/or

(f) an antibody according to claims 1 3 or 14.

6. Composition according to claim 1 5, characterized in that it further contains pharmaceutically customary carriers, auxiliaries and / or additives.

1 7. Use of a polypeptide according to one of claims 6 to 8 or a fragment of this polypeptide as an immunogen for the production of antibodies.

1 8. Use of a composition according to claim 1 5 or 1 6 for the diagnosis of diseases that are related to invasive processes.

1 9. Use of a composition according to claim 1 5 or 1 6 for diagnosing a predisposition to diseases that are related to invasive processes.

20. Use of a composition according to claim 1 5 or 1 6 for the therapy or prevention of diseases that are related to invasive processes.

21. Use of a composition according to claims 1 5 or 1 6 for the diagnosis, therapy or prevention of endometriosis.

22. Use of a composition according to claim 1 5 or 1 6 for the diagnosis, therapy or prevention of tumor diseases.

23. Use of a composition according to claim 1 5 or 1 6 as an agent for gene therapy.

24. Use of a composition according to claim 15 or 16 as an antisense inhibitor.

25. Use of a composition according to claim 1, 5 or 16 in the implantation of embryos.

26. Use of a composition according to claim 15 or 16 for identifying inhibitors of a polypeptide according to any one of claims 6 to 8 and/or inhibitors of molecules capable of binding to the polypeptide.

27. Use of a polypeptide according to one of claims 6 to 8 or a fragment of this polypeptide for detecting antibodies against an endometriosis-associated protein or fragments thereof in a sample.

28. Use of an antibody according to claim 13 or 14 or a fragment of this antibody for detecting endometriosis-associated proteins or fragments thereof.