EP1021564A1

EP1021564A1 - Process for detecting, extracting or removing human or mammalian cells with a disturbed cellular cycle regulation or unlimited proliferation or tumour-forming ability

Info

Publication number: EP1021564A1
Application number: EP98954373A
Authority: EP
Inventors: Hinrich Prof.Dr.Med. Abken
Original assignee: Individual
Current assignee: Curacyte AG
Priority date: 1997-10-07
Filing date: 1998-10-07
Publication date: 2000-07-26
Also published as: WO1999018235A1; JP2001519169A

Abstract

For detecting, identifying, extracting or removing human or animal cells with a disturbed cellular cycle regulation or unlimited proliferation or tumour-forming ability, the presence of an association of cdc37 protein with extrachromosomal nucleic acid is detected in cells or tissue fluids. This can be done, for example, by using a detectable substance which can specifically bind to the associate, a nucleic acid or oligonucleotide which hybridises with the nucleic acid of the association or binding substances immobilised on a solid substrate. This latter method also makes it possible to extract or remove such cells.

Description

Methods for the detection, identification, extraction or removal of human or animal cells with disturbed cell cycle control or with the ability for unlimited proliferation or for tumor formation

description

The invention relates to cdc37 protein-nucleic acid heteromers, cdc37 protein, nucleic acids and antibodies which are suitable for the detection of cells with the ability for unlimited proliferation or for tumor formation, methods for the identification and elimination of human and animal cells with the ability for unlimited proliferation or for tumor formation using such heteromers, proteins, nucleic acids or antibodies.

All normal differentiated cells have a limited potential for cell proliferation before they are subject to cell aging (senescence) and cell death after a defined number of cell divisions. The cell biological processes of cell division and cell aging are subject to a complex regulated genetic control, the disruption of which can lead to cell death on the one hand or to unlimited proliferation or tumor formation on the other hand.

Cell proliferation requires cell division, which is carried out in a cascade of processes in temporal and spatial order (cell cycle). The control of this process takes place at two points in time, namely that at which the DNA synthesis (S phase of the cell cycle) and that at which the mitosis (M phase) begins, and is carried out by a small group of protein kinases, which by phosphorylation regulate the activity of numerous proteins involved in the process. These cell cycle-controlling protein kinases are heterodimeric proteins with regulatory subunits (cyclins), the content and association of which with other proteins changes in the course of the cell cycle in a characteristic manner. The catalytic subunits of these protein kinases are called cyclin dependent kinases (cdk) because they only become active as kinase after association with a cyclin. The cyclin-dependent kinases were first discovered in yeast and named for the genes coding for them, e.g. B. cdc2 (cell division cycle-2) from Schizosaccharomyces pombe or cdc28 from Saccharomyces cerevisiae. It soon became clear that many proteins involved in the control of the cell cycle are highly conserved and are present as homologous genes with similar or identical functions in eukaryotes.

The course of the cell cycle in mammalian cells is controlled by numerous cyclin-dependent kinases in combination with various cyclin molecules (review: C.J. Sherr, Mammalian G 1 Cyclins, Cell 73: 1 059-1 065, 1 993). For example, Cdk2, Cdk4 and Cdk5, each associated with the cyclin D 1, D2, D3 subunits, are involved in initiating the G 1 phase of the cell cycle. A cyclin-dependent kinase associated with different cyclins can control different points in the cell cycle. For example, Cdk2 associated with cyclin E is involved in the control of the G 1 -S transition, while Cdc2 associated with cyclin A and B controls the G2-M transition. The state of knowledge for controlling the cell cycle is still far from a uniform model and further Cdk and cyclin molecules are still being discovered that control defined positions of the cell cycle and thus cell proliferation.

While the cell cycle of normal proliferating cells is controlled in its coordinated manner by the mechanisms described above and still other processes which have not yet been clarified, it is believed that tumorigenic transformed cells have one or more defects in one or more of the control mechanisms of the cell cycle. This idea will clear from the following example. The cyclin-dependent protein kinases can both catalyze and inhibit the transition from one phase of the cell cycle to the next subsequent phase. Damage to the DNA caused by radiation or chemical agents, however, stabilizes the tumor suppressor protein p53, which thereby stimulates the synthesis of a cyclin-dependent kinase inhibitor. This kinase inhibitor in turn binds the G 1 -specific Cdk / cyclin complex, whereupon the cell remains in the G 1 phase until the DNA damage has been repaired and the p53 level has dropped. This complex regulation ensures that only the cells that have completely repaired the DNA damage that has occurred are reproduced. If the repair is unsuccessful, the cell is subject to programmed cell death, which prevents the cell from degenerating into uninhibited proliferation in the event of existing DNA damage. If the gene encoding p53 is mutated, however, the p53-induced delay in the G 1 -S transition does not occur, which leads to replication of damaged DNA and an increase in damaged cells without the cell being subjected to programmed cell death.

It is believed that these and other defects in cell cycle control contribute to permanent proliferation and ultimately tumorigenic degeneration of the cell. Since not all of the essential control proteins and their regulators have yet been identified, the current view of the experts in the defects of cell cycle control in tumorigenic degenerated cells is very incomplete. To make matters worse, the phenotype of tumorigenous transformed cells can be extremely diverse and is characterized by an extremely heterogeneous picture of numerous genetic, phenotypic-morphological and physiological changes, which in turn are influenced by numerous other parameters, such as, for. B. degree of differentiation, cell matrix, surrounding cells, local growth factor concentrations or immunological defense, are dependent, without common obligatory properties are molecularly defined can. This problem will be illustrated using the cell proliferation of normal and tumorigenous transformed cells.

While all normal human and animal cells have a limited potential for cell proliferation, tumorigenic transformed cells often show unlimited cell division. They form constantly growing cell groups, which lead to the formation of a tumor and ultimately, together with the changed expression of other properties (e.g. reduced need for growth factors, proliferation without anchoring and contact inhibition, induction of new blood vessels, etc.) to the tumor disease. According to the current understanding of the development of tumors, cells are most likely to be transformed neoplastic (tumorigenic) if (1) the cells proliferate indefinitely in vivo and / or (2) are arrested in their cell death. The tumorigenic transformation is a multi-stage process, at the end of which the tumor cells express properties (1) and / or (2) along with numerous other properties.

An early event in this process is unlimited proliferation ("immortalization"), which is characterized by the fact that the cell has acquired the ability to remain in the cell cycle and to be able to perform continuous cell divisions (Freund et al., Oncogene 7: 1 979 -1 992). With immortalization, however, the cell does not yet acquire the ability to form tumors in a tissue association (Medina and Kttrell, Carcinogenesis 1 4: 25-28, 1 993; Strauss and Griffin, Cancer Cells 2: 360-365, 1 990). This requires a large number of additional "transforming events", which have not yet been characterized in detail.

On the other hand, induction of the neoplastic transformed phenotype does not require prior immortalization (O'Brien et al., Proc. Natl.Acad. Sci. USA 83: 8659-8663, 1,986). An example of this are cells of chronic lymphoblastic leukemia that are no longer able to divide, but through a transforming event in theirs programmed cell death (apoptosis) are arrested (Buschle et al., J. Exp. Med. 1 77, 21 3-21 8, 1 993). Due to the constant differentiation of progenitor cells, new cells are always formed, which are then inhibited in their cell death. As a result, the number of cells increases, which leads to tumor formation.

Tumor cells can be at different stages of neoplastic transformation and have acquired different "tumor-associated" properties or lost properties of differentiated cells. With phenotypic heterogeneity and the various mechanisms leading to neoplastic transformation, it is extremely difficult to identify a cell capable of unlimited proliferation and tumor formation in an environment of normal cells. For diagnostics and therapy and ultimately for the survival of the patient, it is of crucial importance to find those cells which are no longer subject to normal proliferation control and cell cycle regulation and which have acquired the potential for unlimited proliferation and tumor formation.

A particular difficulty is always the differentiation of the proliferation of neoplastic transformed cells from the physiological proliferation of normal cells in the context of the regeneration or differentiation of tissues, such as in blood formation in the bone marrow or in wound healing. Physiological proliferation can take place either briefly or permanently in response to physiological stimulation, the extent of physiological regenerative proliferation being able to far exceed that of tumor cells. This is particularly evident in the proliferation and differentiation of blood-forming cells. For this reason, it is not possible to use the degree of cell proliferation (cell divisions per time) or the progression in the cell cycle as well as the degree of activation (phosphorilization) of the cell cycle control proteins to identify tumor cells. The known methods for the detection of tumor cells are based on clinical, morphological-histological, cytological or physiological parameters or on antibodies which recognize antigens which are preferentially expressed by proliferating (normal and tumor) cells, e.g. B. the Ki-67 antigen or PCN antigen, thus on indirect parameters that are not causally related to the neoplastic transformation of the cells. Given the variety of possible processes that can lead to neoplastic transformation of the cell, it is obvious that mutations in preferred genes. n found in only a few tumor entities. Examples are mutations of membrane-bound or intracellular receptors for intracellular signal transmission (e.g., fms, erb-B / A, src, yes), mutations of protein kinases (e.g. mos, raf, ras) or nuclear transcription factors (e.g. fos, jun, myc ) or of cell cycle-associated control proteins (eg p53, rb).

It is therefore the object of the invention to detect, eliminate or inhibit the growth of a tissue bandage in situ or in a cell suspension, which have a disturbed cell cycle control or which have acquired the ability to proliferate indefinitely or to form tumors. the tumorigenous transformed cells should be distinguished from normal cells with transient or physiological proliferation.

This object is achieved by a method for the detection, identification, extraction or removal of human or animal cells with disturbed cell cycle control or with the ability for unlimited proliferation or for tumor formation which is characterized in that the presence of an association from the cdc37 protein with Detects extrachromosomal nucleic acid in cells or tissue fluids. The polypeptide portion of the polypeptide-nucleic acid associate represents the human or animal homologous protein to the yeast cdc37 protein and its subunits. The nucleic acid portion consists of extrachromosomal DNA or RNA copies of chromosomal DNA sequences of the respective tumor cell (hereinafter referred to as cdc37 nucleic acid heteromer). This cdc37 nucleic acid heteromer is suitable for the detection of human and animal cells with the ability for unlimited proliferation or tumor formation. The invention therefore furthermore relates to a process for obtaining the cdc37 nucleic acid heteromer by isolation from a soluble fraction from permanently divisible human or animal cells, precipitation in the presence of 1.5 M NaCl and isolation by extraction with phenol and precipitation with ethanol or ammonium acetate.

The following unexpected properties of permanently proliferating or tumor-forming cells are used in the invention:

(i) In cells with the ability to form tumors or unlimited proliferation, but not in normal cells with physiological transient or permanent proliferation, molecules of the cdc37 homologous protein are associated together with nucleic acids in a heteromer; (ii) The cdc37 nucleic acid heteromer is suitable for the detection of cells with the ability to form tumors or unlimited proliferation, regardless of the degree of differentiation or level of neoplastic transformation the cells have or the tissue from which the cells originate.

(iii) Molecules that bind to the cdc37 nucleic acid heteromer are useful for eliminating or enriching cells capable of tumor formation or unlimited proliferation, as well as inhibiting the growth or initiation of cell death of these cells.

From proc. Natl.Acad.Sci.USA 90: 651 9-6922 (1 993) and PCT WO 95/02701 are known to be tumor cells or proliferate indefinitely

Cells carry an extrachromosomal DNA that is associated with proteins.

The DNA-protein complexes described are characterized in that the complexes accumulate in fractions with a density of 1.82-1.86 g / cm ³ upon equilibrium centrifugation in a CsCI density gradient, the DNA is associated with the proteins in an SDS and salt-stable bond and the proteins have molecular weights of 52 kD , 62 kD and 64 kD. However, the cdc37 nucleic acid heteromers according to the invention differ from the above-mentioned DNA-protein complexes in the following properties:

- The cdc37 protein has a molecular weight of approx. 40 kD. Polypeptides with molecular weights of 52 kD, 62 kD or 64 kD are not detectable in the cdc37 nucleic acid heteromer according to the invention.

- The binding of the nucleic acid to the cdc37 protein is in the presence of detergents and high temperature, e.g. 0.1% SDS and 90 ° C soluble, but not the binding of the above. DNA-protein complex.

- The cdc37 protein-associated DNA sequences differ from those of the DNA-protein complexes from PCT WO 95/02701.

In order to describe the invention in more detail, the relevant state of knowledge regarding the biochemistry of the cdc37 protein is summarized below.

20 The cdc37 protein is known and was first isolated from yeast due to the fact that mutations of this protein lead to a disturbed cell cycle (Ferguson et al., 1 986). Isolation from Drosophila was only possible much later, in particular by mutating cdc37 and hsp83 with the signaling pathway through the "sevenless" receptor

25 Tyrosine kinase interfere (Cutforth et al., Cell 77: 1027, 1 994). Over the past three years, the Mammalia homologous cdc37 protein has been cloned due to the highly conserved amino acid sequence to the yeast cdc37 sequence and the ability to be part of a multi-protein complex that contains cyclin-dependent kinase-4 (cdk4) and that heat shock protein

30 hsp90 (Dai et al., J. Biol. Chem. 271: 22030, 1 996; Lamphere et al., Oncogene 1 4: 1 999, 1 997). It is also known that the cdc37 protein can interact with numerous other proteins, z. B. with the retinoblastoma protein RB and with virally encoded proteins with transforming activity, e.g. B. with SV40 large T, adenovirus E 1 A, Papillon virus E7, pp60v-src or raf (Ozaki and Sakiyama, DNA Cell Biol. 1 5: 975, 1 996; Perdew et al., Biochem. 36: 3600, 1 997). The cdc37 protein also binds hyaluronic acid (Grammatikakis et al., J. Biol. Chem. 270: 1 61 98, 1 995). The variety of interactions of the cdc37 protein with other molecules in the cell is so far unclear. Hsp90 binds the protein kinase cdk4 and stabilizes the cdc37 / cdk4 complex. In this form, cdk4 is activated by cyclin D 1. After the cell has passed through the G 1 phase into the S phase of the cell cycle, this complex disintegrates and only builds up again when the cell has reached the G 1 transition point of the cycle again (Stepanova et al., Genes Develop. 1 0 : 1 491, 1 996). The cdc37 / hsp90 complex can be destabilized by an antibiotic, geldanamycin, which leads to the disintegration of the complex. The cell can no longer enter the S phase of the cell cycle (Stepanova et al., Genes Develop. 10: 1491, 1 996). The current state of knowledge assumes that cdc37 is involved in controlling the G 1 -S transition of the cell in the cell cycle and is therefore always expressed when the cell reaches this point in the cell cycle (Ozaki et al., DNA Cell Biol. 1 4: 1 01 7, 1 995). This is in agreement with the observation that the gene expression for the cdc37 protein in proliferating cells can only be detected when the cell changes from G 1 to the S phase and is degraded immediately after entering the S phase. The cdc37 expression thus persists for a maximum of 9 hours with a cycle duration of proliferating cells of approx. 24-36 hours and is undetectable during the other phases of the cell cycle.

From Ozaki and Sakiyama (FEBS Lett. 375: 1 55, 1 995) it is known that the cdc37 protein is able to bind DNA in vitro. The authors demonstrated this by incubating cdc37 protein with genomic DNA from rat cells and cloning the DNA bound to cdc37 under in vitro conditions. The authors received a DNA clone of genomic DNA, that binds to cdc37 protein. The authors speculated "cdc37 might play an important role in the control of cell cycle progression via a sequence-specific DNA binding mechanism." In contrast to these in vitro binding studies and the authors' speculation that cdc37 protein could bind to chromosomal DNA, A DNA association of the cdc37 protein in vivo has not been demonstrated in the cell. On the contrary, the cdc37 protein in normal cells with physiological proliferation has no detectable DNA.

It was all the more surprising that a DNA bound to the cdc37 protein was found in neoplastic transformed cells with the ability for unlimited proliferation or tumor formation. However, contrary to the assumption by Ozaki and Sakiyama described above, this is an extrachromosomal DNA. The DNA sequences contained in the heteromer according to the invention are therefore not homologous to the genomic DNA sequence isolated by Ozaki and Sakiyama. The heteromer of cdc37 protein and extrachromosomal nucleic acid is therefore new and an object of the invention.

Since all known observations indicate that the cdc37 protein is only expressed at the G 1 -S phase transition of the cell cycle, but not constitutively during the entire cell cycle, it was all the more surprising that the cdc37 nucleic acid heteromer in cells was also disturbed Cell cycle control or the ability to unlimited proliferation or tumor formation is constitutively expressed regardless of the cell cycle phase. In contrast, the cdc37 nucleic acid heteromer is not expressed in normal cells regardless of whether the cells proliferate, rest, or senesce. The detection method according to the invention is therefore independent of the cell cycle phase in which the cell with these properties is located. Surprisingly, the cdc37 nucleic acid heteromers according to the invention can also be used to detect cells in different stages of neoplastic transformation, in particular in very early stages, regardless of the mechanisms by which the cell was transformed neoplastic. In particular, the cdc37 nucleic acid heteromeric cells according to the invention can be used to detect cells which have a disturbed control function of the cell cycle or have unlimited tumorigenic proliferation capacity, but have not yet formed a tumor. Preferred test material are tissue biopsies or cell mixtures from body fluids or suitable cell or cytoplasmic fractions which are used in specific reactions for the detection of the cdc37 nucleic acid heteromers according to the invention by detecting the nucleic acid, the protein or its special configuration as a heteromer or its reactivity with substrates or antibodies or other binding molecules can be used. Another object of the invention is thus the detection of tumor cells and other cells with unlimited proliferation capacity using a tissue biopsy, a tissue smear, a body fluid or suitable cell disruption, regardless of whether a clinically detectable tumor has already been formed. Preferred uses of the method according to the invention are tumor diagnostics or preventive purposes in health monitoring.

Any human and animal cells which have a disturbed cell cycle control or which have the ability for permanent proliferation or tumor formation, permanently proliferating cells of different tissues or degrees of differentiation or species, and others, can generally be used as the starting material for obtaining the cdc37 nucleic acid heteromers according to the invention. tumor cell lines or tumors or tumor biopsies of the desired tissues, degrees of differentiation or species familiar to the person skilled in the art. It is surprisingly irrelevant which processes (e.g. ionizing radiation), transforming genes (e.g. viral oncogenes), chemical carcinogens (e.g. nitroso-urea) or other mechanisms the corresponding cell was transformed neoplastic. It also has no influence on the isolability of the cdc37 nucleic acid heteromers according to the invention as to what degree of differentiation the cell has (e.g. epithelium, lymphocyte, myelocyte, fibroblast, melanocyte) or from which species (e.g. human, mouse, rat, Drosophila) the cells descend.

According to the invention Cdc37-nucleic acid heteromers from a cytoplasmic fraction are preferably recovered from permanently proliferating human or animal cells, for example using detergents such as NP40 or SDS, or by means of repeated freezing and thawing of the cells, preferably in the presence of Mg ^{+ +} solutions. The mitochondria are then separated from this cytoplasmic fraction, preferably with the aid of a sucrose gradient according to methods known to those skilled in the art. The cytoplasmic fraction with cdc37 nucleic acid heteromers can alternatively also be obtained by obtaining a protein-containing cell fraction which is free of nuclear DNA. Such a fraction is preferably obtained by lysing the cells in the presence of NaCl and SDS and subsequent centrifugation ("Hirt extraction", J. Mol. Biol. 26: 3655-369, 1,967). The cdc37 nucleic acid heteromers according to the invention can be obtained from the supernatant of the “shepherd extraction” or from mitochondrial-free cell lysates by selective precipitation or electrophoretic or chromatographic methods, preferably by precipitation with 1.5 M NaCl and precipitation of the supernatant with ethanol or ammonium sulfate.

The cdc37 nucleic acid heteromers have surprisingly poor dissolution behavior in both organic and aqueous solvents. According to a further isolation process for the cdc37 nucleic acid heteromers according to the invention from a cytoplasmic lysate, the lysate is therefore subjected to an extraction in organic solvents, preferably subjected to phenol and chloroform. The cdc37 nucleic acid heteromers according to the invention collect in the organic aqueous interphase of the extraction and can be separated from other molecules of the lysate, preferably by precipitation with ethanol.

The cdc37 nucleic acid heteromers according to the invention also have a surprisingly high stability towards proteinases and nucleases, while cytoplasmic organelles and membranes or mitochondrial or viral DNA or RNA are rapidly degraded by nucleases. On the basis of these properties, the cdc37 nucleic acid heteromers according to the invention can also, according to a further object of the invention, by incubation in the presence of proteolytic enzymes, preferably proteinase K (100 / vg / ml) for 1 h at 60 ° C., and subsequent precipitation in the presence of Ethanol or ammonium sulfate or chromatographic purification, preferably using HPLC, can be obtained.

While the protein portion of the heteromer always contains the cdc37 protein, it has surprisingly been found that the nucleic acid sequences of the cdc37 nucleic acid heteromers according to the invention differ in cells of different degrees of differentiation and species. The sequence of the associated DNA of the cdc37 nucleic acid heteromers from melanoma cells according to the invention differs from that from mamma carcinoma or from colon carcinoma cells. Most cdc37-associated DNA molecules have a length of approx. 30 bp - 1,000 bp, but there are also molecules of longer length, e.g. B. up to 3,000 bp in cells of acute myeloid leukemia.

The nucleic acid (RNA / DNA) sequences can be isolated from the cdc37 nucleic acid heteromers according to the invention by methods known to the person skilled in the art, preferably using proteolytic enzymes, detergents, high temperature, enzymatic multiplication or molecular cloning. The nucleic acid sequences of the cdc37 nucleic acid heteromers are increased enzymatically, preferably using the reverse transcriptase or Klenow polymerase or using the PCR method using thermostable DNA polymerases. Furthermore, the DNA sequences can be obtained by hybridizing a genomic library or a cDNA library with the cdc37 nucleic acid heteromers according to the invention. Another preferred method is to obtain the nucleic acid by affinity binding to the cdc37 protein, preferably using extra-chromosomal DNA or cDNA and then enriching the cdc37 nucleic acid heteromer formed, preferably by sedimentation or using antigen-antibody reactions.

The DNA sequences obtained can then be used for the detection of human or animal cells with unlimited proliferation or the ability to form tumors. Preferred methods of DNA or RNA hybridization, enzymatic amplification using sequence-specific oligonucleotides, in situ hybridization to cells or tissue biopsies, or binding of the nucleic acids to the cdc37 protein or the protein are preferred to those skilled in the art to the nucleic acids in a lysate.

Another object of the invention is the use of the cdc37 protein from the cdc37 nucleic acid heteromer for the detection of human and animal cells with the ability for unlimited proliferation or for tumor formation. It was shown that these cells express the cdc37 protein and its mRNA in an increased number of molecules compared to normal cells with physiological proliferation. The comparison of the expression of the cdc37 protein molecules is preferably carried out by an antigen-antibody recognition reaction, preferably with the aid of Western blot detection, immunofluorescence or FACS analysis ("fluorescence activated cell sorter") using a cdc37 protein-specific antibody (for example Dai et al., J. Biol. Chem. 271: 22030-22034, 1 996) or an equivalent binding reaction of a binding molecule or a cdc37-binding nucleic acid (South-Western blot) by methods known to the person skilled in the art (Harlow et al., Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor). The mRNA expression is preferably compared using Northern blot hybridization using a suitable hybridization sample, for example the human cdc37 cDNA sequence (for example dbEST data base, GenBank ™ ID clone R87892). Using these analyzes, for example, a 5-fold increase in cdc37 mRNA expression (Northern blot analysis) and an 8-1-fold cdc37 protein expression (Western blot analysis) were shown in MCF-7 breast carcinoma cells compared to that in normal breast epithelial cells and a 7-fold increased cdc37 mRNA expression in L540 Hodgkin lymphoma cells compared to that in normal lymphocytes.

When using the South-Western method or other methods of binding nucleic acids to the cdc37 protein, DNA or RNA or single- or double-stranded deoxy- or oxy-oligonucleotides or labeled (eg with digoxigenin or biotin) or modified nucleic acids, eg PNA "peptide nucleic acid" used. The binding of the nucleic acids to cdc37 protein in vitro requires the presence of Zn ^{2 +} ions (preferably 8 mM) and monovalent ions, for example Na ⁺ (preferably 8 mM), as well as ATP or GTP (preferably 2 mM). The nucleic acid-cdc37 interaction includes the N-terminal part of the cdc37 polypeptide sequence.

The cdc37 protein is obtainable from the cdc37 nucleic acid heteromer according to the invention by treatment with DNase I, preferably matrix-bound DNasel, or by proteolytic cleavage or treatment with detergent or in the presence of high temperature, preferably 90 ° C., and chromatographic or electrophoretic isolation of the released cdc37 Protein or its fragments. The invention furthermore relates to antibodies and antisera with specificity or recombinant molecules with antibody-like binding specificity for the cdc37 protein nucleic acid heteromers according to the invention. These are suitable for the detection of human and animal cells with the ability for unlimited proliferation or for tumor formation. These antibodies or antisera can be obtained in polyclonal form by methods known per se by immunizing immunocompetent animals, such as mice, rabbits or sheep, with the cdc37 nucleic acid heteromers according to the invention or the cdc37 protein or the nucleic acid of the heteromer. Hybridoma clones producing monoclonal antibodies can also be obtained by methods known per se by fusing antibody-forming lymphocytes with suitable myeloma cells, for example Ag8.653 cells. In addition to complete antibodies, it is also possible to use antibody fragments which can be obtained by known protein chemical and molecular genetic processes. With the help of the cDNA sequences of the V _H and / or V _L immunoglobulin chains from immunocompetent donor lymphocytes can be isolated individually or in combination, binding hybrid molecules with antibody-like specificity, preferably recombinant "single-chain" antibodies which are able to bind the cdc37 nucleic acid heteromers according to the invention. Isolation of recombinant antibodies from a "phage display library" is also preferred, in that the suitable recombinant phages are enriched by specific binding ("panning") to the cdc37 nucleic acid heteromers. The antibody-like fusion proteins on the surface of the phage or in soluble form can then be used like an antibody in suitable detection methods. The use of a "combinational library" is also possible for the isolation of such a recombinant antibody with specificity for cdc37 nucleic acid heteromers and is known to the person skilled in the art (Orlandi et al., Proc. Natl. Acad. Sci. USA 86: 3833-3837, 1,989; Chiang et al., Biotechniques 7: 360-366, 1,989; Winter and Milstein, Nature 349: 293-299, 1 991)

Surprisingly, the antibodies obtained after immunization with the cdc37 nucleic acid heteromers according to the invention bind so specifically to cells with unlimited proliferation, disturbed cell cycle control or the ability to form tumors that they can also be used for labeling cells in a tissue association in situ. The cells sought are preferably labeled in the cytoplasm, while normal cells with physiological permanent or transient proliferation are not labeled. This is all the more surprising since proliferating normal cells express the cdc37 protein during the transition from the G 1 to the S phase of the cell cycle. The antibodies according to the invention are able to distinguish between the cdc37 nucleic acid heteromer in tumor cells and the cdc37 protein normal cells.

The invention therefore furthermore relates to antibodies and antisera which are able to bind heteromeres to cdc37 nucleic acid. Preferred use is the use of the antibodies or antisera or recombinant antibody-like molecules to remove or inhibit growth for detection reactions in a biopsy in situ or in a tissue section or in reactions with cell suspensions or body fluids or with cdc37 nucleic acid heteromers of these cells in a lysate or in body fluids or for the elimination or inhibition of growth of these cells in the tissue or in a cell mixture. The antibodies or their fragments or recombinant binding molecules can be coupled with carriers or labeling molecules such as proteins, sugars, Sepharose, enzymes, dyes, haptens or radioactive ions and used for suitable detection methods. A large number of known antigen stands for the detection reaction. Antibody recognition reactions are available, such as ELISA, RIA, fluorescence immunoassay or fluorescence activated cell sorting (FACS).

One embodiment of the invention consists in measuring the content of the cdc37 nucleic acid heteromers according to the invention, the cdc37 protein portion or the nucleic acid of the heteromer in a mitochondrial-free cytoplasmic fraction of the cell to be examined, preferably from a biopsy, a tissue section or cell suspensions or from a soluble fraction of body fluids. Normal cells with physiological proliferation capacity and regulated Zeil cycle do not contain such heteromers in this fraction, while cells with disturbed cell cycle control carry these heteromers in high copy numbers regardless of the cell cycle phase. Antigen-antibody reactions or nucleic acid hybridizations are preferred for measuring the content of the cdc37 nucleic acid heteromers according to the invention. However, a variety of detection methods are available to those skilled in the art, e.g. ELISA, RIA, fluorescence immunoassay, FACS analysis, nucleic acid hybridization in Southern or South-Western blot, PCR etc.

The detection of the heteromer can also be carried out by enzymatic reaction of the heteromer with a substrate, preferably by cleaving an energy-rich phosphate bond, or by binding to hyaluronic acid. The cdc37 protein nucleic acid heteromer of the invention has kinase activity and / or is (auto) kinased and this is the basis for this detection method.

Of particular importance for the invention is the surprising finding that the cdc37 nucleic acid according to the invention can be found on the cell surface of cells with impaired cell cycle control, unlimited proliferation capacity or the ability to form tumors, but not on the surface of normal cells physiological proliferation capacity. A further embodiment of the invention therefore consists in the detection of cells with impaired cell cycle control, unlimited proliferation capacity or the ability to form tumors by using reagents which are able to identify or bind the cdc37 nucleic acid heteromers according to the invention on the surface of the cell . Labeled antibodies, antisera or recombinant polypeptides with binding specificity are preferably used for the cdc37 nucleic acid heteromers according to the invention. Detection of the cells in situ or in a cell mixture using a cdc37 nucleic acid heteromer-binding antibody according to the invention or its derivatives which is coupled to a dye or enzyme is particularly preferred in a known method of immunostaining biopsies or histological preparations, or enrichment of cells by fluorescence or magnet-activated cell sorting.

The antibodies, antisera or recombinant antibody-like molecules according to the invention against the cdc37 nucleic acid heteromers on the surface of the cell membrane can also be used for the detection of cells with the ability for unlimited proliferation or tumor formation in vivo. Preference is given to using the antibodies according to the invention or recombinant antibody-like molecules or their derivatives after suitable labeling, for example with 99mTc or ¹²¹ J, in immunoscintigraphy or other imaging methods. The antibodies accumulate in tissues where cells with impaired cell cycle control, unlimited proliferation capacity or the ability to form tumors are present. Tissue with normal resting or proliferating cells (e.g. brain or bone marrow) and cells in physiological regeneration, e.g. B. in wound healing, are not marked with the inventive method. Furthermore, the antibodies, nucleic acids or their derivatives according to the invention can be coupled with other detectable or reactive molecules or atoms, for example proteins, sugars, enzymes, biologically active mediators, metal complexes, radioactive isotopes or toxins, and for the detection or / and destruction of cells impaired cell cycle control, unlimited proliferation capacity or the ability to form tumors. Preferred is the use of fusion proteins from a cdc37 protein nucleic acid heteromer-specific antibody with a cellular toxin, for example pseudomonas exotoxin, whereby a so-called immune toxin is generated which specifically binds to cells with unlimited proliferation or tumor formation and kills the cells . Furthermore, the coupling of the antibody with an immunologically active protein, for example a cytokine or a chemokine, is possible, whereby a fusion protein is generated which binds to tumor cells and activates immune cells there for tumor cell lysis.

The known immune toxins or immune cytokines are directed via their antibody portion against antigens which are almost exclusively expressed on tumor cells of a certain tissue or degree of differentiation. In contrast, the fusion molecules according to the invention with specificity for cdc37 protein nucleic acid heteromers are directed against a large number of different tumor cells, irrespective of the tissue from which the cells originate or the degree of differentiation of the tumor cell. With increasing malignancy and metastasis, most tumor cells lose a large number of antigens on their surface, as a result of which the known immune conjugates lose their binding to these highly malignant cells and cells in metatases. The fusion molecules according to the invention with specificity for cdc37 protein nucleic acid heteromers, on the other hand, also bind to these highly malignant cells and cells of metastases, since the fusion molecules bind to a molecule which is required by the cell for each further cell division. This leads to an efficacy which is superior to that of the known immune conjugates. It was furthermore surprisingly found that Fc-carrying antibodies according to the invention or their derivatives with binding ability of the cdc37 nucleic acid heteromers according to the invention are able to trigger cell lysis in cells with impaired cell cycle control, unlimited proliferation capacity or the ability to form tumors. The cell lysis is carried out with the help of complement activation by the Fc portion of the antibody molecule.

The invention therefore also relates to the use of molecules with the ability to bind the cdc37 nucleic acid heteromers for the use of the lysis of cells with impaired cell cycle control, unlimited proliferation capacity or the ability to form tumors, with the binding molecules themselves or fusion products of these molecules other pharmacologically active substances are used.

Furthermore, nucleic acids of the cdc37 protein nucleic acid heteromers or derived oligonucleotides of these nucleic acids can be used for the inhibition of the proliferation of cells with unlimited proliferation capacity. For this purpose, partial sequences (oligonucleotides) of these nucleic acids which are homologous to the binding region of the nucleic acid with the cdc37 protein are preferred. These oligonucleotides interfere with the nucleic acid-cdc37 protein binding and thereby destabilize the heteromer. Surprisingly, it was found that the tumor cell stops its unlimited proliferation, leaves the cell cycle and is subject to cell death. A particular embodiment of the invention is thus the use of oligonucleotides and nucleic acid derivatives which bind and destabilize heteromers to the binding region of the nucleic acid with the cdc37 protein of the cdc37 protein nucleic acid to inhibit growth and / or initiate cell death of the tumor cell. Other substances that change the stability of the cdc37 protein nucleic acid heteromer can also be used for this purpose. Surprisingly, such substances induce growth inhibition and / or cell death of cells with impaired cell cycle control, unlimited proliferation and tumor formation. For this purpose, preference is given to substances which interfere with heteromer formation by binding the nucleic acid or the cdc37 protein. For this purpose, peptide sequences are more preferably used which are derived from the nucleic acid binding region of the cdc37 protein, in particular from the cdc37 protein sequence amino acid 62 to 1 20. The peptides of the sequence are particularly preferred

CysGlnArgLysLeuLysGluLeuGIuValAlaGluSerAspGlyGInValGluLeuGluArg LeuArgAlaGluCys and the sequence CysGluLeuGluArgLeuArgAlaGluAlaGInGInLeuArgLysGluGlueuArgluSLTrG. It was found that these peptides inhibit the growth of tumor cells, but not that of normal cells with physiological proliferation. Suitable peptides can also be used for this application, which cyclize via N- and C-terminal cysteine residues and / or whose basic amino acids are exchanged for neutral amino acids.

Furthermore, substances are preferred which block the function of the cdc37 protein nucleic acid heteromer in the control of the G 1 / S transition of the cell cycle, such as e.g. Antibiotics, fungicides, or other physiological or synthetic substances.

According to the invention, cdc37 protein nucleic acid blocking or destabilizing substances are therefore used to inhibit the proliferation of cells with unlimited proliferation and tumor formation and to initiate cell death of these cells. According to the invention, cells with unlimited proliferation capacity or the ability to form tumors from a cell mixture can also be enriched or depleted with the aid of reagents which are able to bind the cdc37 nucleic acid heteromers according to the invention. For this purpose, the antibodies, antisera or recombinant antibody-like molecules against the cdc37 nucleic acid heteromers according to the invention can be used. For this purpose, methods by fluorescence-activated sorting (FACS) or magnet-activated sorting (MACS) are preferred. The antibodies or their derivatives with reactivity to the cdc37 nucleic acid heteromers according to the invention can also be used for the removal of cells with unlimited proliferation capacity or the ability to form tumors from a cell suspension or a tissue, preferably for "purging" grafts, for example of autologous bone marrow transplants. Antibodies or their derivatives with a binding domain for the cdc37 nucleic acid heteromers according to the invention are preferably bound to a matrix, the cell mixture is incubated with this matrix and the unbound cells are eluted chromatographically. It was found that with the aid of this method cells with unlimited proliferation capacity or the ability to form tumors can be removed very efficiently from a cell mixture of normal cells, irrespective of the tissue from which the tumor cell is derived or the degree of differentiation, proliferation status or malijnity of the respective neoplastic transformed cell in this mixture of cells. Peripheral blood, bone marrow or other body fluids are preferably used as cell mixtures. In a preferred application, this method is suitable for the liberation of blood, bone marrow or other body fluids from cells with unlimited proliferation capacity or the ability to form tumors.

It was also surprisingly found that the cdc37 nucleic acid according to the invention is secreted by cells with unlimited proliferation capacity or the ability to form tumors. The cdc37 nucleic acid heteromers can therefore, for example, in the culture supernatant from Tumor cells or also in the blood, bone marrow, serum, plasma, liquor, urine or other body fluids are detected. Therefore, human and animal cells with impaired cell cycle control, unlimited proliferation capacity or the ability to form tumors can be detected by detecting the cdc37 nucleic acid heteromers according to the invention in body fluids or in cell-free supernatants or filtrates.

The cdc37 nucleic acid heteromers are preferably detected with the aid of antibodies which bind the heteromers, the cdc37 nucleic acid heteromers isolated according to the invention being able to be carried as standard. However, combinations of the methods are also possible, preferably an enrichment with the aid of an antibody

Binding specificity for the cdc37 nucleic acid heteromers according to the invention and detection with a second antibody or with the aid of

Nucleic acids that the cdc37 molecule or the nucleic acid of the cdc37

Nucleic acid heteromer is able to bind, or with the help of an enzymatic reaction that of the cdc37 nucleic acid according to the invention

Heteromer itself runs out, e.g. B. the cleavage of an energy-rich phosphate bond of a substrate.

The sensitivity of the method can be increased further by detecting the associated nucleic acid of the cdc37 nucleic acid heteromers by hybridization with suitable nucleic acid probes or enzymatically, preferably by polymerase chain reaction (Mullis and Fallona, Meth. Enzymol. 1 55: 335-350, 1 987; Saiki et al., Science 239: 487-491, 1 988) using sequence-specific or rapom sequence oligonucleotides as primers. In a special embodiment of the method according to the invention, the cdc37 protein nucleic acid heteromers are therefore obtained from a lysate of a biopsy or from body fluids or cell supernatants using a specific antibody and then the associated nucleic acid is increased enzymatically.

The described embodiments of the method of the invention are suitable for the detection of malignant and benign tumors or their precursors or of cells with impaired cell cycle control, the cells being a biopsy, a tissue section or cells or their lysates or secreted complexes of body fluids from humans or animals Sample material can be used. The described methods are also suitable for finding and localizing such cells in vitro and in vivo. Furthermore, these methods can be used in a correspondingly modified form for isolating the cells from a cell mixture or for their enrichment or depletion and elimination from a cell mixture. Finally, antibodies or recombinant molecules with binding specificity for the cdc37 nucleic acid heteromers according to the invention can be used for the elimination of the cells in vivo and in vitro, the antibodies or recombinant molecules being used alone or as fusion products with other bioactive molecules.

The invention is illustrated by the following examples.

example 1

Isolation of cdc37 nucleic acid heteromers from MCF-7 breast carcinoma cells.

MCF-7 breast carcinoma cells (ATTC HTB22) were lysed in the presence of 50 mM Tris-HCl, pH 7.2, 10 mM EDTA, 1.5 mM MgCl ₂ by repeated freezing and thawing and then the cell nuclei by 30 minutes Centrifugation at 6000 xg sedimented. The mitochondria were separated from the supernatant using a sucrose gradient (J. Biol. Chem. 249: 76991-1 995, 1 974). The mitochondrial-free fraction was treated with RNase A (20 / yg / ml) and RNase T1 (1000 U / ml) for 30 min at 37 ° C, then incubated for 20 min at 60 ° C with Proteinase K (100 μg / ml). SDS (ad 0.4% SDS w / v) and NaCl (ad 4 M NaCl) were added to this mixture, cooled on ice for 60 min and then centrifuged at 6000 xg for 20 min. The supernatant was extracted into phenol (neutralized with Tris-HCl, pH 7.2) and then into chloroform / isoamy alcohol (24: 1 v / v). The cdc37 protein nucleic acid heteromers are concentrated in the organic-aqueous interphase. This interphase was removed, the cdc37 nucleic acid heteromers precipitated by adding 2 vol ethanol and resuspended in 0.1 mM EDTA, 1 mM Tris-HCl, pH 7.2.

The cdc37 nucleic acid heteromers were separated in a native agarose (0.7%) or polyacrylamide (4%) gel, the heteromers from MCF-7 cells showing the migration rate of a protein-free DNA of the size 100-500 bp . In a denaturing SDS polyacrylamide gel, these cdc37 nucleic acid heteromers migrate at approximately 37 kD, 48 kD, 82 kD, 90 kD.

Example 2

Molecular cloning of the nucleic acid of the cdc37 protein nucleic acid heteromers from tumor cells.

The cdc37 protein nucleic acid heteromers (from Example 1) were incubated in the presence of Proteinase K (500 // g / ml) and 0.1% SDS for 10 hours at 60 ° C. This approach was subjected to another extraction in phenol and then in chloroform / isoamyl alcohol. The DNA released from the cdc37 protein nucleic acid heteromer collected in the aqueous phase of the extraction. The DNA was precipitated with ethanol and dissolved in 10 mM Tris-HCl, pH 7.2, 1 mM EDTA.

For cloning, the DNA (approx. 20 ng) of the cdc37 protein nucleic acid heteromers was subjected to a "replenishment reaction" by Klenow polymerase (1 0 U) in the presence of 1 0 mM Tris-HCl, pH 7, 6, 6 mM β-mercaptoethanol, 50 mM NaCI, 1 0 mM MgCl ₂ and each 0, 1 mM TTP, dATP, dCTP, dGTP subjected. This was followed by cloning into the DNA of the Smal-cut pUC 19 vector in the presence of 10 U T4 DNA ligase. The recombinant plasmid obtained was transformed into E. coli K1 2 bacteria. Four independent plasmid clones were obtained (pMCF-7). These plasmid clones contain inserted DNA sequences which represent the nucleic acid portion of the cdc37 protein nucleic acid heteromers and are suitable for the detection of neoplastic transformed cells. Analogously to this procedure, the nucleic acid sequences of the cdc37 protein nucleic acid heteromers were obtained from colon carcinoma, Hodgkin lymphoma, melanoma cells and cells from acute myeioic leukemia. The DNA sequences are shown in SEQ ID NO 1.

Example 3

Obtaining antibodies against cdc37 nucleic acid heteromers.

Mouse Ehrlich-Ascites tumor cells (ATCC CCL77) and human MCF-7 (ATCC HTB22) mammary carcinoma cells carry cdoplasmic cdc37 nucleic acid heteromeres in their cytoplasm, whereas in normal embryonic mouse fibroblasts these heteromers could not be detected. We are looking for an antibody that specifically binds both mouse and human cdc37 nucleic acid heteromers from tumor cells, but does not react with normal cells.

The cdc37 nucleic acid heteromers are obtained from mouse Ehrlich-Ascites tumor cells (ATCC CCL 77) according to the method described in Example 1. Cdc37 nucleic acid heteromers with a share of approx. 100 ng associated DNA (dissolved in 50 μ \ PBS and mixed with Freund's adjuvant) are placed in a Balb / c mouse s.c. injected. Second injections with the same amount of antigen ("booster injections") without adjuvant were made on day 32 and on day 54 after the first injection. On day 62, approx. 500 μ \ But were removed for the production of antiserum (“anti-cdc37 nucleic acid heteromer antiserum”) for further analyzes.

On day 62 after the injection, the spleen of the immunized mouse was obtained, the spleen lymphocytes isolated and fused with cells of the myeloma line X3-Ag8.653 with the aid of polyethylene glycol to produce hybridomas (Köhler G. and Milstein C. Nature 256: 495-497, 1 975; Köhler, G. (ed.) Hybridoma techniques. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1 980). The hybridoma cells formed were selected in the presence of HAT selection medium (1 0 ^"4 M hypoxanthine, 4x 1 0 ^{" 7} M aminopterin, 1, 6 x 1 0 ^"5 M thymidine in RPMI 1 640 medium). On day 5 after fusion, the cells were sown in "limiting dilution" cultures in microtiter plates with about 50-100 cells per well, on day 28 and day 35 after fusion the supernatants were about 400 Cultures screened for antibodies that react with cdc37 nucleic acid heteromers in tumor cells.

The procedure for this is as follows: Ehrlich mouse ascites cells, mouse embryonic fibroblasts (as a negative control) and human MCF-7 breast carcinoma cells (approx. 100 cells per well of a "Terasaki" microtiter plate) are each covered with the supernatant Hybridoma culture (approx. 10 μ \ per well, supernatant 1: 100 diluted in PBS) incubated for 1 hour at 37 ° C., then washed twice with PBS and then for 1 hour with a peroxidase-coupled antibody against mouse immunoglobu - line (anti-mouse Ig antibody) incubated at 37 ° C. After washing again and the reaction of the peroxidase with the substrate AEC, antibodies were found in 6 out of approximately 400 supernatants which react with the cytoplasm of mouse Ehrlich ascites tumor and human MCF-7 carcinoma cells, but not with normal embryonic fibroblasts.

The hybridoma cells of these cultures were subcloned by "limiting dilution" and finally by single cell seeding. A total of approximately 300 cultures were created, the supernatants of which were again checked on day 24 using the method described above for reactive antibodies. Hybridoma cells with reactive supernatants were subcloned again. A stable hybridoma clone ("3D6") was obtained which shows monclonal antibodies with specificity against mouse and human cdc37 nucleic acid heteromers from tumor cells, but does not react with normal cells.

Example 4

Use of the cdc37 protein nucleic acid heteromer-specific antibody 3D6 for the detection of tumor cells in a cell lysate.

Tumor cells should be detected in a mixture with normal cells. Since tissue preservation is often insufficient for histological analysis It is appropriate to demonstrate in this example how to detect tumor cells in a cell lysate.

The human breast carcinoma line MCF-7 (ATCC HTB22) and human normal skin fibroblasts are lysed in the presence of 50 mM Tris-HCl, pH 7.2, 10 mM EDTA, 3 mM MgCl ₂ , 0.2% NP40. Cell debris, cell nuclei and other organelles are separated by centrifugation at 8,000 xg for 2 min. The supernatant (corresponding to approximately 1 0 ⁴ cells) and isolated cdc37 protein nucleic acid heteromers (as a positive control) are denatured in the presence of SDS, electrophoretically separated in a 1, 2.5% SDS polyacrylamide gel and transferred electrophoretically to a nitrocellulose membrane. The membrane is incubated in TBS (20 mM Tris, 1 37 mM NaCl, pH 7.6) 3% Tween 20, 5% (w / v) dry milk powder for 1 hour, then the anti-cdc37 protein nucleic acid heteromer-specific antibody 3D6 (1: 1 00 hybridoma supernatant) was added and incubated again for 1 hour at room temperature. The membrane is washed in TBS, 3% Tween 20 and incubated for 1 hour with the anti-mouse Ig antibody coupled to the peroxidase. The membrane is then washed again in TBS, 3% Tween 20, incubated with the detection reagent ECL (Amersham) and autoradiography is carried out.

Result:

The monoclonal antibody 3D6 specifically recognizes the bands of the cdc37 nucleic acid heteromers (approx. 37 kD, 48 kD, 82 kD, 90 kD in human tumor cells) in the cell lysate of the MCF-7 breast carcinoma cells and the isolated cdc37 in the Western blot -Nucleic acid heteromers (positive control), but does not react with the cytoplasmic protein lysate from normal human fibroblasts. Example 5

Labeling of human tumor cells in a tissue section using the 3D6 monoclonal antibody with specificity for cdc37-nucleic acid heteromers.

The task is to show tumor cells of a malignant meianoma in a tissue section of a biopsy. For this purpose, cryostat sections of a tissue biopsy were made and fixed with acetone / methanol. The endogenous peroxidases were blocked by incubation with 1% H ₂ 0 ₂ (v / v) in 40% methanol / 60% PBS for 1 5 min, the sections were then washed and 1 0 min with 0.2% Triton X- Incubated 1 00 in PBS. After washing again, the sections were incubated with bovine serum albumin (3% w / v in PBS) for 1 hour and washed again. The incubation was then carried out with the anti-cdc37 nucleic acid heteromer-specific antibody 3D6 (1: 10 diluted hybridoma supernatant in PBS) for 2 hours at room temperature. "Sham incubations" with buffer without antibody and incubation with an anti-TAG72 antibody (B72-3) with specificity for the TAG72 antigen, which expresses on cells of the gastrointestinal tract, but not on melanocytes or on cells of the epidermis, served as controls is. The sections were then washed four times with PBS and incubated with a peroxidase-coupled goat anti-mouse IgG (H + L) antibody (Dianova, Hamburg) (1:50 in PBS) for 1 hour at room temperature. After washing four times, the peroxidase was reacted with the substrate AEC for 20 min. The cell nuclei were stained with hemalum.

Result:

The cells of the malignant meianoma reacted in their cytoplasm with the cdc37 nucleic acid heteromer-specific antibody 3D6, while the nucleus of the melanoma cells did not react with the 3D6 antibody. The normal cells surrounding the melanoma and the cells of the normal epidermis show no staining with the 3D6 antibody. Infiltrating Tumor cells in the normal tissue are identified by staining with the 3D6 antibody. Control markings without 3D6 antibodies show no specific staining, nor do reactions with the anti-TAG72 antibody.

Example 6

Detection of cdc3 nucleic acid heteromers on the cell surface of tumor cells

The task is to detect cdc37 protein nucleic acid heteromers on living cells on the cell surface. The intact cell membrane of the living cells prevents the antibodies from entering the cell, while dead cells allow the antibody to enter. Dead cells are identified by staining with propidium iodide and excluded in the subsequent measurement procedure.

Human MCF-7 breast carcinoma cells (A) and normal human fibroblasts (B) are washed in PBS, each divided into 4 batches and incubated with the following reagents for 30 min at room temperature: (1) anti-cdc37 nucleic acid heteromer specific antibody 3D6 (hybridoma supernatant 1: 1 00 in PBS)

(2) anti-TAG72 specific antibody B72-3 (hybridoma supernatant 1: 1 00 in PBS)

(3) PBS without antibody (4) PBS without antibody

The cells were washed in PBS and the batches (1), (2) and (3) were then incubated with a FITC-coupled anti-mouse IgG antibody (Dianova, Hamburg) (1: 500) for 30 min at room temperature . The cells were washed again in PBS and analyzed using the fluorescence-activated cell sorting (FACS) (FACscan, from Becton-Dickinson, Mountain View, Cal.). It was through the configuration suitable measuring window ("gates") dead cells excluded from the analysis, so that the signals obtained come exclusively from living cells with an intact surface membrane.

Result:

MCF-7 breast carcinoma cells of approach (1) showed a FITC fluorescence signal. Cells from batches (2) and (3) showed a background fluorescence which was weaker by a factor of 5-1 0, which has the same strength as the control batch (4) without secondary antibodies and indicates the "inherent fluorescence" of the cells. The labeling in approach (1) showed that MCF-7 cells were specifically labeled by the 3D6 antibody, but not by a control antibody (B72-3) that reacts with an antigen that is not expressed on MCF-7 cells . In contrast to MCF-7 cells, normal human fibroblasts only showed background fluorescence in batch (1), as did batches (2), (3) and (4). It can be concluded that 3D6 antibody reactive cdc37 nucleic acid heteromers are expressed on the surface of MCF-7 breast carcinoma cells, but not on the surface of normal fibroblasts.

Example 7

Detection of cdc37 nucleic acid heteromers in the serum of tumor patients.

Blood serum was obtained from two patients with breast cancer and with melanoma as well as from two healthy donors and 2 μ \ of the serum each with DNasel (1 0 U, 1 hour, 37 ° C) or with Proteinase K (1 0 μg, 1 Hrs, 60 ° C). Untreated and DNasel- or proteinase-treated serum was incubated in the presence of SDS (0.1% w / v) for 2 min at 90 ° C., separated electrophoretically in an SDS-polyacrylamide gel and then electrophoretically in the presence of 50 mM Tris, 380 mM glycine, 0.1% (w / v) SDS, 20% methanol on a nitrocellulose Transfer cellulose membrane. The non-specific binding of the membrane was determined by incubation with 5% (w / v) dry milk powder in TBS (20 mM Tris, 1 37 mM NaCl, pH 7.6) 3% (v / v) Tween 20 for 1 hour at room temperature blocked. The membrane was then incubated with the cdc37 protein nucleic acid heteromer-specific antibody 3D6 (1: 1 00 hybridoma supernatant in blocking buffer) for 1 hour at room temperature, washed three times and with a perxidase-coupled sheep anti-mouse Ig (Amersham) (1: 200 ) incubated for 1 hour. After washing four times, the bound antibody was detected by reaction of the peroxidase with the ECL Detection Reagent (Amersham) and subsequent autoradiography on an X-ray film.

Result:

In the sera of the patients with breast cancer and with melanoma, the 3D6 antibody detects bands with the relative Mr approx. 37 kD, 48 kD, 82 kD, 90 kD, but shows no reaction with sera from the healthy volunteers. The Mr detected corresponds to the relative migration rate of the isolated cdc37 protein nucleic acid heteromers. The 3D6 antibody-reactive bands in the serum of the tumor patients can be degraded by preincubation of the serum with both DNasel and Proteinase K. This shows that the detected bands contain material consisting of both protein and DNA.

Example 8 Enrichment of tumor cells from a mixture of normal cells with the aid of the 3D 6 antibody with specificity for cdc37 protein-nucleic acid heteromers.

To demonstrate the accumulation of tumor cells from a mixture with normal cells, SK-Mel5 cells of a malignant meianoma become

1 0 ⁶ normal human lymphocytes from peripheral blood (PBL) in the following mixing ratios: Sk-Mel5: PBL 1: 1 0, 1: 1 00, 1: 1,000, 1: 1, 0,000, 1: 1, 00,000. The Sk-Mel5 melanoma cells express the HMW-MAA ("high molecular weight melanoma associated antigen") on the surface and can be detected with an anti-HMW-MAA antibody. This enables the detection of melanoma cells in the population of normal cells regardless of the method used. Normal human lymphocytes do not express this antigen.

The cell mixture (approx. 10 ⁶ cells in 1 ml PBS) was incubated with the antibody 3D6 (200 μg purified antibody) for 1 hour at room temperature, washed four times with PBS and then with an anti-mouse Ig antibody

(approx. 50 μg), which is paramagnetically marked (Miltenyi, Bergisch-

Gladbach), incubated again for 1 hour. The cell suspension is washed four times, then placed on a 20 ml separating column (Miltenyi) and exposed to a high magnetic field. The column is washed with 10 times the volume of PBS to remove the unlabeled cells (flow-through fraction). The column is then removed from the magnetic field and the labeled cells are eluted. The

The number of HMW-MAA + tumor cells in the respective fractions is determined using the anti-HMW-MAA antibody 763.74. The result of the FACS

Analysis is summarized in Table 1.

Table 1

Number of cells Anicil SK-Mcl5 number Sl.-Mcl5 cells proportion SK-Mel5 enrichment.

in the cell mixture cells in the IGcsamαcIahl of all cells) cells in öer fa or

Cell mixture in the flow-Eluai-Eluai fraction

FBL Sk-Mel5 fraction

ιo ^c ιo- 95 ±. 53 8.5 * 1.7 x I0 0.315 3, 15

0.1

| 2.7 * .0.8 x I0 ⁵ ] ιo ^< 10 9.3 _* 1.9 i I0 ³ 0.216 21. «

10 "0.01

| 4.3 * .ll x I0 ⁴ ] ιo ^l ιo- 10-3 _ 10 7.6i.2.S x I0 ² 0.042 4 2

| IS * 0.4 x 10 ⁴ )

I0 ^ς ιo- 10- * _ ιo 43 * .39 0.019 1 9 0 [2.2-0.3 x I0 ³ )

10 ^c I0 ¹ 10-5 10 <10

12.6 * 0.3 x I0 ³ )

I0 ^C 10 <10

| 2.8 * .0.4 x I0 ³

10- 70 _ 4E 1.9 _ 1.5 x 10 *

The measuring limit of the method is approx. 10 cells with 5000 counts per measurement.

Average values are given ± _ standard deviation of 6 test runs.

Example 9

Liberation of a cell mixture from tumor cells with the aid of the solid-phase-bound antibody 3D6 with specificity for cdc37 protein-nucleic acid heteromers. This example was carried out as in Example 8, but the separation was carried out with the aid of the solid-phase-coupled antibody 3D6.

To demonstrate the purification of tumor cells from a cell mixture, SK-Mel5 cells of a malignant meianoma are added to 1 0 ⁶ normal human lymphocytes from peripheral blood (PBL) in the following mixing ratios: Sk-Mel5: PBL 1: 1 0, 1: 1 00 , 1: 1,000, 1: 1,0,000, 1: 100,000. The Sk-Mel5 melanoma cells express the HMW-MAA ("high molecular weight melanoma associated antigen") on the surface and can be detected with an anti-HMW-MAA antibody. Normal human lymphocytes do not express this antigen.

The antibody 3D6 (2 mg of purified protein) was bound to CNBr-activated Sepharose (0.5 ml) (Pharmacia) and the Sepharose was then washed in PBS, 0.2% (w / v) dry milk powder. The cell mixture (approx. 10 ⁶ cells in 1 ml PBS) was placed on a column with 3D6 antibody-coupled Sepharose (0.5 ml) and incubated for 1 hour at room temperature. The column was then washed four times with 10 times the volume of PBS in order to remove the unbound cells (flow-through fraction). The bound cells were eluted in the presence of 2 M MgCl ₂ , 10 mM NaP0 ₄ , pH 7.2. The number of HMW-MAA ⁺ tumor cells in the respective fractions was determined using the anti-HMW-MAA antibody 763.74 and the efficiency of the purification (depletion factor) was determined. The results of the FACS analysis are summarized in Table 2. Table 2

Number of cells Number of Sk-Mel5 cells Depletion in line mix in the flow fraction factor PBL Sk-Mel5

[M] [N] [M IN]

lθ6 105 345 + 93 289

106 10 ⁴ 114 + 29 87

106 103 <10

106 102 <10

106 101 <10

10 ⁶ 0 <10

0 105 169 + 76

The detection limit of the method is about 10 cells at 5000 counter ^¬ eignissen per measurement.

Are indicated mean values __ standard deviation of 4 experimental ^¬ guides.

Example 10

Detection of cdc37 protein-nucleic acid heteromers with the help of nuc / unic acid in situ hybridization

Human breast carcinoma line MCF7 (ATCC HTB22) (A) cells and primary skin fibroblasts (passage 12) (B) were cultured on slides. More than 90% of the cells of both cultures proliferate, the

MCF-7 cells have unlimited proliferation potential, primary human skin fibroblasts stop their proliferation in vitro after approx. 1 5 -20 passages, age and die. Furthermore, a solid tumor was generated by subcutaneous injection of MCF-7 cells in Balb / c mice, a frozen section of this tumor was made and transferred to a slide (C).

The slides were briefly washed twice with PBS, overlaid with 200 μ \ paraformaldehyde solution (5% v / v) for 5 minutes for fixation and then washed twice in 70% ethanol. The DNA sequence shown in SEQ ID NO 1 was used to detect the cells with unlimited proliferation potential. This DNA sequence is the nucleic acid portion of the cdc37 protein nucleic acid heteromers from MCF-7 cells and was obtained by molecular cloning as described in Example 1a. The DNA sequence is cloned into the S al interface of the vector pUC 19. The 221 bp MCF-7 / sml DNA fragment was released from the vector by restriction cleavage of the DNA with Smal. The fragment was electrophoretically separated from the pUC1 9 vector DNA (2.7 kb) in an agarose gel, eluted and precipitated with ethanol. The MCF-7 / sm 1 DNA (1 _J / g DNA) was denatured and with the aid of Klenow polymerase (1 U) in the presence of 10 mM Tris-HCl, pH 7.6, 6 mM β-mercaptoethanol, 50 mM NaCI, 1 0 mM MgCI ₂ and 0.2 mM digoxigenin-1 1 - dUTP (Dig-dUTP) (Boehringer Mannheim), each 0.1 mM dATP, dCTP, dGTP and hexanucleotides of a random sequence as primer (Boehringer Mannheim) by incubation Marked at 37 ° C for 2 hours. Unincorporated nucleotides were separated by filtration through a Sepharose G50 column (Pharmacia).

For in situ hybridization, the slides with the fixed cells or tissue sections were treated with 200 μl hybridization solution (50% deionized formamide, 4 x SSC, 10% dextran sulfate, 1 x Denhardts solution, 0.25 mg / ml denatured tRNA, 0.5 mg / ml denatured herring sperm DNA). The solution was placed on the slide with coverslips covered and incubated for 1 hour at 42 ° C in a humid chamber. The solution was then removed, the cells were covered with Dig-dUTP-labeled MCF-7 / sm1 DNA (200 ng DNA in 40 μl hybridization solution) and incubated at 42 ° C. overnight. In a control experiment ("sham hybridization"), the hybridization was carried out without a digoxigenin-labeled DNA sample. Finally, the slides were washed twice for 20 min in 2 x SSC at 42 ° C. To detect the hybridized DNA sample, the cells were incubated with a FITC-coupled anti-digoxigenin antibody (Boehringer Mannheim) for 20 min at room temperature. Unbound antibodies were removed by washing twice in PBS. The preparations were evaluated using fluorescence microscopy at an excitation wavelength of 470 nm.

Result: (A) The MCF-7 tumor cells show strong, distinct fluorescence signals in the cytoplasm, while the nucleus shows no fluorescence. No fluorescence signals were detectable in the control experiment ("sham hybridization").

(B) After hybridization of primary human skin fibroblasts, no cell labeling was obtained.

(C) After hybridizing the tissue section of the tumor induced by MCF-7 cells, only the tumor cells show fluorescence, while the surrounding normal connective tissue, the subcutaneous tissue and the epidermis show no signals. The hybridization signals of the tumor cells are also localized exclusively in the cytoplasm. In control hybridizations ("sham hybridization" and hybridization with digoxigenin-labeled pUC 19 DNA) no fluorescence signal was obtained.

The cytoplasmic cdc37 protein nucleic acid heteromers can thus be specifically detected in tumor cells by hybridizing suitable DNA samples with the nucleic acid associated with the heteromers. The

Implementation as in situ hybridization allows the identification of the individual neoplastic transformed cells in a tissue section or in a biopsy.

Example 1 1 Detection of cdc37Preotein-Nucleic Acid Heteromers Using the Antibody and the Polymerase Chain Reaction of the Associated Nucleic Acid.

The cells of acute myeloid leukemia (AML) contain cytoplasmic cdc37 protein nucleic acid heteromers, which can be enriched with the help of the cdc37 protein nucleic acid heteromer-specific antibody from a cell lysate and their nucleic acid with the help of the polymerase chain reaction and sequence-specific oligonucleotides can be amplified.

1 0 ⁴ cells each of an AML (A) and normal peripheral blood lymphocytes (B) or a mixture of 1 00 AML cells in 1 0 ⁴ blood lymphocytes (C) or 1 00 normal human fibroblasts in 1 0 ⁴ blood lymphocytes (D) was produced. While normal human peripheral blood lymphocytes show no proliferation without antigen stimulation, normal fibroblasts proliferate physiologically until they undergo cell aging after about 50-60 passages. The cells of acute myeloid leukemia, on the other hand, show unlimited proliferation and inhibition of entry into physiological cell death. Although the cells only survive in vitro to a limited extent, they induce a malignant tumor in vivo and show unlimited proliferation.

The cells of batches AD were sedimented, resuspended in 100 μl 10 mM Tris-HCl, pH 7.2, 1.5 mM MgCl ₂ , immediately frozen at -20 ° C. and thawed again. The samples were sedimented by centrifugation at 2,000 xg for 10 min and the supernatant was placed in a hole in a microtiter plate, the surface of which was coated with the cdc37 protein nucleic acid heteromer specific antibody 3D6 is coated. After incubation for 1 h at room temperature, the sample was washed four times with PBS, 2% (w / v) bovine serum albumin. The cdc37 protein nucleic acid heteromers from the cells were now enriched on the microtiter plate. This enrichment ensured that in the subsequent step of DNA amplification only the DNA of the cdc37 protein nucleic acid heteromers can serve as a template for the DNA polymerase, but not the nuclear DNA or any other contaminating DNA.

The cdc37 protein nucleic acid heteromers are detected by a specific amplification of the associated DNA. A particular advantage is that this process can also be carried out on the microtiter plate after the enrichment.

The hole with the enriched cdc37 protein nucleic acid heteromers was incubated with proteinase K (100 μg / ml) for 1 hour at 55 ° C. and then heated at 95 ° C. for 10 minutes. The polymerase chain reaction (PCR) was then carried out by a known method (Mullis and Fallona, Methods Enzymol. 1 55: 335-350, 1 987; Saiki et al, Science 239: 487-491, 1 988). The following approach was chosen.

3 ul 1 0x Taq buffer (200mM Tris-HCl, pH 8.4, 250mM KCI, 0.5% Tween 20, 1mg / ml BSA); 0.5 ul 100 mM MgCl ₂ ; 1 ul dNTP solution (2mM dATP, 2mM dCTP, 2mM dGTP, 2mM dTTP); 1 U Taq DNA polymerase (Boehringer Mannheim); 1 00 ng each of primer oligonucleotides A and B for the amplification of the DNA (SEQ-ID NO 1) of the cdc37 protein nucleic acid heteromer from AML cells.

Primer oligonucleotide A:

5 ^' ACCGCCTCTCCCCGCGCG3' Primer oligonucleotide B:

5 ^' GCGTTGCTGGCGTTTTTCATA3'

35 cycles with the following temperature profile were carried out: 1. 1 x [2 min 95 ° C]

2. 35 x [30 sec 55 ° C; 90 sec 72 ° C; 60 sec 95 ° C]

3. 1 x [1 5 min 72 ° C]

The amplified DNA is separated electrophoretically in a 1% agarose gel and, after incubation with ethidium bromide, made visible in UV light.

Result:

When analyzing the cells of acute leukemia (A), a DNA of approximately 221 bp in length was amplified, but not when using normal human lymphocytes (B). Amplification of the DNA was also obtained when using a mixture of AML cells with normal lymphocytes (C), but not when the normal lymphocytes were mixed with normal fibroblasts (D). This mixture test of tumor cells with normal cells shows that with the help of this test approach contamination of tumor cells with unlimited proliferation in normal cells can be detected. The experimental approach is not disturbed by contamination with physiologically proliferating cells, such as normal fibroblasts.

This test procedure also shows that contamination with cells from a malignant tumor can also be detected if (i) the cells in the sample to be analyzed are no longer intact but a lysate of the cells is present, and (ii) the malignant cells exclusively show the ability to form tumors and unlimited proliferation in vivo but not in vitro. Example 12

Inhibition of the growth of tumor cells using synthetic peptides that bind to the nucleic acid of the cdc37 protein-nucleic acid heteromer.

Cells from the human lymphoma line L540 (sample A) and normal human lymphocytes from peripheral blood (sample B) were each in a concentration of 1 0 ⁴ cells per 1 00 μl RPMI 1 640 culture medium, 1 0% (v / v) fetal calf serum, seeded in 4 holes of a 96-well microtiter plate. The normal lymphocytes were also mitogenically stimulated in the presence of 10 μg / μl pokeweed mitogen (PWM) (sample C). The samples were incubated at 37 ° C for 1 2 hours. Peptides 1, 2 and 3 were mixed with 2 μl DOTAP "transfection reagent" (Boehringer Mannheim, Mannheim, Cat. No. 1 81 1 1 77) and in each case in hole 1-3 of the samples AC in the concentration of 40 μM peptide added, in the hole 4 was added buffer / PBS) plus 2 μl DOTAP "transfection reagent".

Peptide 1 and peptide 2 are derived from an N-terminal sequence of the cdc37 protein and contain the nucleic acid binding region of the protein. Peptide 3 is a random peptide sequence of the same length. The peptides are cyclized via their N- and C-terminal Cys residues.

Peptide 1:

CysGlnArgLysLeuLysGluLeuGluValAlaGluSerAspGlyGInValGluLeuGluArg LeuArgAlaGluCys

Peptide 2:

CysGluLeuGluArgLeuArgAlaGiuAlaGInGInLeuArgLysGluGluArgSerTrpGlu

GlnLysLeuGluCys

Peptide 3: CysGluAlaGInValLeuAspAlaLeuValGluGluLysArgGInValSerGluGluLysArg GlnLysGluAlaCys

The cultures were incubated for 3 days at 37 ° C. and then the cell vitality was determined using the XTT test (Boehringer Mannheim, Mannheim, Cat No. 1 46501 5). The tetrazolium salt XTT is added to the cells that cleave XTT to formazan using the succinate tetrazolium reductase system of the mitochondrial respiratory chain. The metabolite can then be determined photometrically. The amount of the metabolite is proportional to the number of living cells and is used to estimate the vitality and proliferation of a cell population.

Result:

L540 lymphoma cells (sample A) in the presence of peptide 1 (hole 1) and peptide 2 (hole 2) show only 1 5-20% of the XTT metabolism compared to cultures in the presence of control peptide 3 (hole 3) and in the "blank" without peptide (hole 4). Normal human lymphocytes from peripheral blood (sample B) show the same XTT metabolism in the presence of the peptides (hole 1 -3) and in the "blank sample", as do the cultures (hole 1 -4) with lymphocytes after mitogenic stimulation (sample C ).

Obviously, L540 lymphoma cells stop growing in the presence of peptide 1 and peptide 2, while a control peptide (peptide 3) is unable to trigger this growth inhibition. However, the proliferation of normal lymphocytes after mitogenic stimulation is not affected by these peptides. SEQUENCE LOG

(1. GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Univ. -Prof. Dr. med. Hinrich Ab en

(B) STREET: Rhoenstrasse la

(C) LOCATION: Meudt

(E) COUNTRY: DE

(F) POSTAL NUMBER: 56414

(ii) DESCRIPTION OF THE INVENTION: Methods for the detection, identification, extraction or removal of human or animal cells with impaired

Cell cycle control or with the ability to unlimited prolifer. .

(iii) NUMBER OF SEQUENCES: 121

(iv) COMPUTER READABLE VERSION:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPA)

(vi) DATA OF THE URN REGISTRATION:

(A) REGISTRATION NUMBER: DE 19744335.4

(B) REGISTRATION DAY: 07-OCT-1997

(vi) DATA OF THE URN REGISTRATION:

(A) REGISTRATION NUMBER: DE 19749118.9

(B) REGISTRATION DATE: 06-NOV-1997

(2) INFORMATION ON SEQ ID NO: 1:

{i) SEQUENCE LABEL:

(A) LENGTH: 221 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGCTGTTCCG CTTCCTCGCT CACTGACTCG 60

CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ACTCAAAGGC GGTAATACGG 120

TTATCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC CAGCAAAAGG 180

CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCAT A 221 (2) INFORMATION ABOUT SEQ ID NO: 2:

(i) SEQUENCE LABEL:

(A) LENGTH: 120 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATATGTCTTG AAGATGCACT TCCAAGGTCA TGAATGGAAC TCCACCAGCC ACAAAAAGAC 60 CTGCCTATAC CTGGAGTATC GTGCAGTCAG AGTGCTCCGA CTCCTGTGGTT GGAGGTAGAT 120

(2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE LABEL:

(A) LENGTH: 110 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AGACCTAAAA CCATAAAAAC CCTAGAGAAA ACCTAAGGCA TTAACCAATT CAGGACATAG 60 GTGGGCAGGA TTAAGTCAAA ACACCAAAAA AATGGCAACA AAAGCCAAAA 110

(2) INFORMATION ON SEQ ID NO: 4:

(i) SEQUENCE LABEL:

(A) LENGTH: 218 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB6

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

AAGACCAATG AGAGACAATG CAGTCGCTGG GACAACCAGA TTGGACCAGG CAGTCAAGGT 60

GGCTTGGAGG CCACAGCGAG AATTAAAATA CACTGAGAGA TTATACTTCA GGATCCTCTA 120

GAGTCGACCT GCAGGCATGC AAGCTTGCTA ATGCATGGTC TCATAGCTGT TTCCTGTGTG 180

AAATTGTTAT TCCTCCTCAC AATTCCACAC AACATACC 218 (2) INFORMATION ON SEQ ID NO: 5:

(i) SEQUENCE LABEL:

(A) LENGTH: 184 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB1

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

GGCAACCTAC AGAATGGGAG AAAATTTTTG CAATCTATCA TCTGACAGAG GTCTAATATC 60

AGAATCTAAG AAAAACTTAA ACAAGTTTAC AAGAAAAAAA CAAACAACCC CATAAAAAAG 120

TGGGTGAAGG ATATGAACAG ACAGTTCTCA AAAGAAGACA TTTATGTGGT CAACGAACAT 180

ATGA 184 (2) INFORMATION ON SEQ ID NO: 6:

(i) SEQUENCE LABEL:

(A) LENGTH: 131 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB5

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

AAGGTAAATT AACTGAAAGT AAAATTGGAA AATGTGGCTA AAAGGGGGGA AAATAATCAA 60

GTTTAAACCT CTAGAGTCGA CCTGCAGGCA TGACAACGTT GGCGTAATCA AGGTCATAGC 120

TGTGTCCTGT G 131 (2) INFORMATION ON SEQ ID NO: 7:

(i) SEQUENCE LABEL:

(A) LENGTH: 323 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB7

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

GTCAATCAAT TTTATGAGAA TAGCATTGAA TCTATAAATT ACTTTGGGCA GTATGGCCAT 60

TTTCATGATA TTGATTCTTC CTATCCATGA GGATGGAATG TTTTTCCATT TGTTTGTGTC 120

CTCTCTTATT TCCTTGAGCA GTGGTTTGTA GTTCTCCTTG AAGAGGTCTT TCACATCTCT 180

TCTTACCTGT GTTCTTAGGT ATTTATTCTC TTTGTAGCAG TGGCAAATGG GAGTTTATTC 240 ATGATTGGCT CTCTGCTTGT TATTGTTGAG TAAGGAGTAC TGTGATTTGC ACATGCTTGC 300 ATCTAGACTT ATGAGTGCTA CAG 323

(2) INFORMATION ON SEQ ID NO: 8:

(i) SEQUENCE LABEL:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB9

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ACTGTCACTC AGGCTGGAGG GTAGTTAAAC 30

(2) INFORMATION ON SEQ ID NO: 9:

(i) SEQUENCE LABEL:

(A) LENGTH: 219 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB11

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

CAGACATGAG CCACCACGCC CGGCCAGGAA TGAATGTTCT ATGCATGAAA CTGTAAACGG 60

CTTATTGCTA TAATTGTCCT AGTTTATTAT TGTTAATTTC TTACTGTACC TAATTTATAA 120

ATTAAGCTTT ATCAGAGGTA TGTATGTATT AGAAAAAATA TAACATATAT AGGATACAGT 180

ACTATCCAGG ATCAGGAATC TACTGGGCAT ACTAGAAAG 219 (2) INFORMATION ABOUT SEQ ID NO: 10:

(i) SEQUENCE LABEL:

(A) LENGTH: 179 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB12

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: AACTGTAAAC GGCTTATTGC TATAATTATC CTAGTTTATT ATTGTTAATT TCTTACTGTA 60 CCTAATTTAT AAATTAAACT TTATCAGAGG TATGTATGTA TTAGAAAAAA TATAACATAT 120 ATAGGATACA GTACTATCCA TGGTTTCAGG AATCTACTGG GCATACTAGA AAGATCCCC 179 (2) INFORMATION ON SEQ ID NO: 11:

(i) SEQUENCE LABEL:

(A) LENGTH: 234 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB14

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

GGAGCTACAA TTCAAGATGA GATTTGGATC GGGACACTGC CAAACCATAT CATGGGGTTT 60

GTCACTGGCC TGGATGGAAC ATCCTTGTCA AGAGCACACA CACACACACA CACACACACA 120

CACACTTAGA GAGACAGACA AAGAAGAGAG AGAGGAGAGG CACGGTTATA ATGAGGATGG 180

ACAGGTGATT TTGGAAGCTG AGAGTCATTA CTGCCACTGC CAGCTAGGTG TAAT 234 (2) INFORMATION ON SEQ ID NO: 12:

(i) SEQUENCE LABEL:

(A) LENGTH: 269 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB16

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

TGTTATTATA ATTATCAGTC CTGAAACCTG TCTCATCTTA GAAACATAGA TTCTATACAA 60

GTGGCCCTGG AGTGCCTTTT AAGATGTTCT CAGGAGATTT TGGTTGTCTT CTAAACTTTC 120

TGGAAACATC TTGTTCATTT CGTATTCTGG TGGAAACATC TTGTTCATTT CGTATTCTGG 180

ATTTAAAATT AGCTATTTCT CCAAGGAGAT GTCAGCCATT GTGTTTATAC AAACTTGAGT 240

TCCTTTAAGG TTTAACCTTC ATTCAAGAA 269 (2) INFORMATION ABOUT SEQ ID NO: 13:

(i) SEQUENCE LABEL:

(A) LENGTH: 256 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (vii) DIRECT ORIGIN: (B) CLON (E): 17

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

GAAACTGATA AGTTGTTTCT CTAGCTTTTA AAAATACATG GAGTGAGTTC TATAATTCCT 60

CAGGAAGTAA GAGCTGTTCC TTTTGAGCTG AAGAACATAT TGCAATTCCT ATTTCATTGC 120

AGAGATATTT CTCTACTTTA AAATGCTACC CGCTTCTCAT CCACACAAAA ATTGTGAAAC 180

AGAGGGGGCT GGGGCAGTAA GGAAGTTATC AAGAAAGTAA TTCACATTAG GTGAGCTGGA 240

GCCAATAGCT TAATTT 256 (2) INFORMATION ABOUT SEQ ID NO: 14:

(i) SEQUENCE LABEL:

(A) LENGTH: 295 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB18

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

TAGGTGGTGC GTGCCTGTAG TCCCAGCTAC TCGGGAGGCT GAGGCACGAG AATCACTTGA 60

ACCCGTGAAG CAGAGGTTGC AGTGAGCCGA GATCACCCAC TGCACTTCAG CCTGGGCAAC 120

AGAGCAAGAC TCCGTCTCAA AAAAAAAAAA CAAAAAACCA AAAACTTCTT TATATGTAAG 180

AAGAGATGTT CCTGACATGA AAAATAGCAC CTTTACAGCA CTTTTCTCTA GTGTGGACAC 240

TGGCGGATTA CCAAGGTTGT CATTTAAATA GAVATGACAG TTCCACTCAG AGGTT 295 (2) INFORMATION ON SEQ ID NO: 15:

(i) SEQUENCE LABEL:

(A) LENGTH: 166 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB19

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

CCGTCACCAT GCCCAGCTAA TTTTTTTTGT ATTTTTAGTA GAGATGGGGT TTCACTATGT 60

TGGCCAGGTG GTCTTGAACT CCTGAACTTG TGATCCTCTA GAGTCGACCT GCAGGCATGC 120

AAGCTTGGCG TAATCATGGT CATAGCTGTT CCTGTGTGA ATGTTA 166 (2) INFORMATION ON SEQ ID NO: 16:

(i) SEQUENCE LABEL:

(A) LENGTH: 244 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB20

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

AGACGCCATC TCAAAAAAAA AAAAAGCTAT GATTGGGTAA GATTATAAGA AGGATAGTTA 60

AAAATAAATA CCACCTAAAT ATGAACCTAA AGAAAGCGAG TATAAAAATG TAAATATGTG 120

ATATGATAAA CTTAGGTTTA AAAATATCAA TAAGGGCTAC TCTGTATCAT AGTATACAAG 180

TGGACATGGC TGGGCCAGTG GCTCACGCCT GTAATCCAAC ACTTTGGGAG GCAGAGGCAG 240

GCGT 244 (2) INFORMATION ABOUT SEQ ID NO: 17:

(i) SEQUENCE LABEL:

(A) LENGTH: 180 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB22

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

GTCAATCTCA CTCTCTCCCC CAGGCTGAAG AGCAGTGGTG CGATCCTCTA GAGTCGACCT 60

GCAGGCATGC AAGCTTGGCG TAATCATGGT CATAGCTGTT TCCTGTGTGA AATTGTTATC 120

GCTCACAATT CCACACAACA TACGAGCGGA AGCATAAGTG TAAAGGCCTG GGTGCCTTAA 180

(2) INFORMATION ON SEQ ID NO: 18:

(i) SEQUENCE LABEL:

(A) LENGTH: 176 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB24 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

AAAATTTTTG GGCATTTCTA TATCATATTA ACCAGAAACT CAGCATACTT TAAAAAGTTA 60

CCACATACAC AAAAAAGCAA GAAATACAGA CTCATAAAAA ATAAATCAAT TATTAAAAAC 120

AAATTCACAG ATAATCCAGA TATTGGAGTT AATAGATAGG GCATCATAAT ATCTAT 176 (2) INFORMATION ON SEQ ID NO: 19:

(i) SEQUENCE LABEL:

(A) LENGTH: 220 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB25

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

GGCACATGTC ACCATACTGC ACTATTTTAA ATTTTTTGTA GGTCTTGCTA TGTTGCCCAG 60

TCTGGTCTCG AACTCCTGAC CTCAAGCAAT CCTCCCACCT TGGCCTCCCA AAGTGCTTGC 120

ATTACAGGCA TGAGCACTGC CTGGCCCACC CAGGCAGTTC AAAAGCATCT TGAGAGTTCA 180

AAAGCATATT GGTGAGACTC ATTCCATGAG GCTGGTGAGG 220 (2) INFORMATION ON SEQ ID NO: 20:

(i) SEQUENCE LABEL:

(A) LENGTH: 192 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB27

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

AGATGTGAGC ACCCTACTTG GCCAGCCACC TGAAATTCTA TCAGTGACAG ATTCCTTTAG 60

CTGAAGTCAT GTTCAAATGT AATCCTCCCA AGAATGGTAA CATTATACAT CAATGCATTA 120

AACAAAACAA CCTGTGCTTA TGAAAAAGTC ATATGTGCAC ACGGTAAAAC AAAATTCAAA 180

TAGTACAAAG GG 192 (2) INFORMATION ABOUT SEQ ID NO: 21:

(i) SEQUENCE LABEL:

(A) LENGTH: 215 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (vii) DIRECT ORIGIN: (B) CLON (E): HB32

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

ACAGAGCAAG ACCCCATCTC AAAAAAAAAA AAAGCTATGA TTGGGTAAGA TTATAAGAGG 60

GATAGTTAAA AATAAATACC ACCTAAATAT GAACCTAAAG AAAGCGAGAT AAAAAGTAAA 120

TATGTGATAT GATAAACTTA GGTTTAAAAA TACAATAAGG GCTACTCTGT ATGTATAGTA 180

TACAGTGGTA CATGGCGGGC CAGTGGCTCA CGCTG 215 (2) INFORMATION ABOUT SEQ ID NO: 22:

(i) SEQUENCE LABEL:

(A) LENGTH: 222 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB34

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

TTTTTCACAT TCCAATGGGA AAATAACATG GAGCACGGCG TGTTCCCCTC AAGGATAGCT 60

GACAAAGGAA AAGGCCCTTC CTTTATCTCC ACTGGGAGTT TTGTTTTCCT TTTATCTAAA 120

AGGGGTTATC AGTGCTGCTC AAATAAAGAA GAAATGTGTG ATTGAAATGG GTGGAGGATA 180

TGCTAGGGAG AAAAAGAAAG CCTGAACAAA TGAAAATTGT AT 222 (2) INFORMATION ABOUT SEQ ID NO: 23:

(i) SEQUENCE LABEL:

(A) LENGTH: 103 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB36

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: AACCCCCAAC CCCCCCTCTC CTCCTTCTTA CTCAACGCCT TCGGGCTACT CAACGCCACT 60 CCCTTCTCAC CCCTCAGCAC CCTCCAAACA GACACATCGA ACA 103

(2) INFORMATION ON SEQ ID NO: 24:

(i) SEQUENCE LABEL:

(A) LENGTH: 253 base pairs

(B) TYPE: nucleotide (C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB37

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

AAGAGTCTGT TGGATTTAAT ATTTTTTAGA AGGTTTGGTG AATTGGTGTT TGCAAAAGCC 60

AATTGCTGTG CATTGAGGAA TGTGTTTGAT TATTCATTCA AGTAATAAAA AACAAAAAAT 120

CAAGCTGACT CTTGAAGAGG CCCCCCCACA CACACACAAA AGAAAGAAAT GTTTGTCTTA 180

TGTGCTAAGC CTGTTCTAGG TTGTTGAGGA TGTACTGGTA AACCAAATGT CCTGTGTTAT 240

GACACTTAGA TCT 253 (2) INFORMATION ABOUT SEQ ID NO: 25:

(i) SEQUENCE LABEL:

(A) LENGTH: 245 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB39

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

GCCAGTTCCT TTAGGTGCAT CATTAGGCTG TTTATTTGAA GTCTTTCTAT CTTTTTGATG 60

AAAGCATTTA TTGCTATAAT CTTCCCTCTT AGAACAGTTT TTGCTGTATC CCATAGGTTT 120

TGATATATTG TGTTTCCATT GCCATTTGTC TTAAATTTTT AAATTTTCTT TTTAATTTCT 180

TCACTGGCTC ATTCATTGTC CAGGAGCATT GCTGTTTAAT TTCATGAGTT TGTGCAGTTT 240

CAACA 245 (2) INFORMATION ABOUT SEQ ID NO: 26:

(i) SEQUENCE LABEL:

(A) LENGTH: 98 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB40

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: TTATCAGAAG ACAGGTAATG TTGACACATG GAGCTCTGAG ATAAATGGTC TCAAGAGAAG 60 GGATCCTCTA GAGTCGACCT GCAGGCATGC AAGCTTGG 98

(2) INFORMATION ON SEQ ID NO: 27:

(i) SEQUENCE LABEL:

(A) LENGTH: 243 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB41

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

AGAAGGTTTG GTGAATTGGT GTTTGCAAAA GCCAATTGCT GTGCATTGAG GAATATGTTT 60

GATTATTCAT TCAAGTAATA AAAAAGAAAA AAGCAAGCTG ACTCTTGAAG AGGCCCCCCC 120

ACACACACAC AAAAGAAAGA AATGTTTGTC TTATGTGCTA AGCACTGTTC TAGGTTGTTG 180

AGGATGTACT GGTAAACCAA AATGTCCTGT GTTTATGACA CTTAGATTCT AATAGTTGGA 240

GAT 243 (2) INFORMATION ABOUT SEQ ID NO: 28:

(i) SEQUENCE LABEL:

(A) LENGTH: 306 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB43

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

CCCTGTCTCT ATTAAAAATA CAAAATTAGC TGTGTGTGGT GGCACACGCC TGTAGTCCCA 60

GCTACTTAGG AGGGGAGGGA GGTGCCAGGC TGTTTTTAAC AATCAGCTCC CACAGGAACT 120

AATAGAGGAA GAACTCACTC ATTACCACAA GGACAGCACC AAGCCATTCA TGACGGATCT 180

ACCTGCATGA CCCCAACACC TCCAGTTAGG CCCAAGCTTA GGGATCATGA TAACTATGGG 240

TGTGGTTATG ACTTTTTAGA TGGTACACCA AAGGCACATA AATGAAAAGA TAACTGAATC 300

CTAGAC 306 (2) INFORMATION ABOUT SEQ ID NO: 29:

(i) SEQUENCE LABEL:

(A) LENGTH: 237 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB44

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

GGAAAAGCAT GGATGGGATT TAAGCTACAC ATACATGCTC CAAGGTTATT TTAATTTCTG 60

CAAATAACAA TGAAATAAGG TATTTCTTTA GCTGTAATAA ACATCCTTTG CAAATCATTA 120

TTAGATGTTT TATCTTAAGA TAAATTTTTC AAACCAAAGA AAGTTTCAAA CTTTGAACTT 180

TAAATGAAAT ATCAAATTCC TTAATATCAC TTTTAATATT CAAATTAAAT CCATGCA 237 (2) INFORMATION ON SEQ ID NO: 30:

(i) SEQUENCE LABEL:

(A) LENGTH: 212 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB45

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

GAGAATTCTT CTGTCTAGCA GAATATGAAG AAATCCCGAA ACCAACGAAG GCCTCAAGGA 60

GGTCTGAATA TCCACTTGCA GACTTTACAA ACAGAGTGTT TCCTAACTGC TCTATGAAAA 120

AGAAAGGTTA AACTCTGTGA GTTGAACGCA CACAAACACA AAAGGAGTTT ATGAGAATAC 180

TTACTGTACT AGTTTCTATA AAGAAGATAT TT 212 (2) INFORMATION ABOUT SEQ ID NO: 31:

(i) SEQUENCE LABEL:

(A) LENGTH: 503 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): AML02

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

CGCTTATGGG CAGGACTCCA CAGTGTGTCA CCACTGATTT GTTTCCACCC CTTATATTTC 60

TCAGTTTCTC TCTCCTGAGG TGAGGGCTCA AAATGTTGGG TAATCAGCCC TTATATGCTT 120

TTCCTGGACA AGATTTTTTT TTTGTCTCTG TCACCCAGGC TGGAGTGCAA TGGCTTGATC 180

TCAGCTCACT GCAACCTCTG CCTCCCGGGT TCAAGTGATT CCTCTGCCTT ACCCTTCCAA 240 ATTGCTGGGA CTACAGGTTT GCCCCATGAC CCCCGACTAA TTTTTGGATG GTATTTTATT 300

TTATTTAGTA GAAAAGGAGT TTCACCATAT TTGCCAGGCT GGTCTCAAAC TCCTGACCTT 360

ATGATCCGCC CCCTCACCTC CCCAAATGCT GGGAATGCGG GTTTAACCAC CTCCCCGGGC 420

TGCCTTTTTT TAAATCAATT TTTGTTGGGG ATTTCCCTGT AGGGCCACTG CATGTTGTGA 480

GGGGATGCCG CAGACCCTGG TGG 503 (2) INFORMATION ABOUT SEQ ID NO: 32:

(i) SEQUENCE LABEL:

(A) LENGTH: 365 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): AML12

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

CCCTCAGAGC GCTCCAAATA TCCACTTGCA CATACTACAA AAAGAGTGCC TCAAAGCTGC 60

TCTCTGAAAC GGAATGTTCA ACTCTATGAG TTGAATGCAA ACATCACAAA GACGTTTCTG 120

AGAATGCTTC TGTCTAGATT TGATATGAAG ATATTCCCGT TTCCAACGAA ATCTTCAAAT 180

CTATCCAAAT GTCCACTTGC AGATTCAACA AAAAGTGTTT TTCAGAACTG CTCTTTCAAA 240

AGAAAGATCC ACCTCTGTTG GCTGAGTTCA CACATCACAA ACAAGTTTAT GAGAATGCTT 300

CTGTCTAGTT TTTATTTGAA GATATTTCCT TTCTCACCAT AGACCTGAAA GCTGTCCTAA 360

TGTTC 365 (2) INFORMATION ABOUT SEQ ID NO: 33:

(i) SEQUENCE LABEL:

(A) LENGTH: 355 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): AML18

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

CAGTATAACC CCAAATAGGA AAGGGTGGGA AATGTCTATG AAATAAGAAC AATCAAGTAA 60

CTTTGGTCAA GTTCTTATTT TAGGATTAAT GAATGATACC CAGGGCCTTC GTTTTTCGTT 120

TTTGACTGTG TTATTCAATG GTTGCTTTTT TTCTGGGGCA ACCTCCAGAT TTGCTGTTTC 180

TTTGATATCT TATAAATGGT CCATTCCACT TTTGATAAAC TGCAACTCTT GATCTAATAA 240

GTTTATAATG ACTGCTTAAG CTTTACACTT TGTTAAGCCT TTGGAAAAAC TCCACTCACC 300 TTGTTTCAAA GTGGGTTGGG TTTTAAGTTA AAAATAGTTT TCATCTTGAA TTGCC 355

(2) INFORMATION ON SEQ ID NO: 34:

(i) SEQUENCE LABEL:

(A) LENGTH: 385 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): AML19

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

CAATTTCTGC TTTTGCTATA ATTGCTTTTG GTGTCTTTGT CAGGAAATCT TTGCCTGTGC 60

CTATGTTTCT AAATGGTATT GACTAAGTTT TCTTCTACAG TTTTTATAGT TTTGGGTTTT 120

ACATTGAAGT CTTTAATCCA TCTTGAAATT GATTTTTGTA TATGAATGCA AGGAAGGGGT 180

CTGGGTTTCA AGTTTCCTGC ATATGGCTAG CCAGTTCTCC TAGTACCATT TATTGAAACG 240

GGGAACCTTT CCCCCTTGCT TGTTTTTGGT CAGTTTGTTT AAAAAAAAAA ATAGCGGTTA 300

TTTGTGCAGT CTAATTTCTG GGTTCTCTAT TCTGTTCCAT TGTTCTGTGT GTCTGTTCTT 360

ATACCAGTAC CACGCTGTTT CGGTC 385 (2) INFORMATION ON SEQ ID NO: 35:

(i) SEQUENCE LABEL:

(A) LENGTH: 406 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): AML20

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

CAACATTTCT CTTCTCACCT CAGTACACCT TTCCAAGAAG ATTGCAAAGC CAAACTCTAA 60

GATGGTCCAA GTCAAGCCCT ACCAATAAGT GTGCAGAAGA CAGAATCTTC TTAAGACTGG 120

GTATTTGAGA GAGCTTCTAA AGCTCTTAAC TATACTCATT CCTTAGGTGG AGGAAAATCC 180

TAACACCTGG CAGGGAACTT TCTTTCCTTT TCTTAGTTTT ATGCAACTTT GTCCTCTAAT 240

ACGTACACTG AAGTTAGGAA TTGAACTAAA AGCTAGCCTT TGTCAAGAAT TGAAATTTGG 300

TTCTGGTGGT CTGTTCATCA ACTCTCTCAA ACAGTCTCTT TCCATACTAT CTCAACTCTC 360

ATGCTCTCTT TTCCTTAGTT TCAAAAATGC CCTTGGGCCG AAAATT 406 (2) INFORMATION ABOUT SEQ ID NO: 36: (i) SEQUENCE IDENTIFICATION: (A) LENGTH: 167 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): AML28

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

CTCCATAGAA GAGGAAGGCA AAGGCTGTGA CAGGCATCTT TCTCATTGTG AGCTGCTGCC 60

AAAGCCAGTG CTCCTGTGTA TCTCCTGGGT GAGGGTTTCC CAAGGGGCCT TTTCACTCCT 120

GAGGCTCAAG ATGAATTCTT CCAGTTACTG TCTCAAGCTC TGAATCC 167 (2) INFORMATION ABOUT SEQ ID NO: 37:

(i) SEQUENCE LABEL:

(A) LENGTH: 372 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): AML29

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

CACATTTTCT GTATATATGA ATTTGCATGG TTGGATTGTT AATGCTGTAA TCAACCATGA 60

TCATGTTATG ATTTAGTACA ATTCGGTCTC ATTTAACTGA ACTATATATG TCTATCACCA 120

CTTATGCTTT ATTGTCTTCT TATTGTCAAA TCTGAAAGAG AAGTAATTAA TTTGATGCTT 180

GCTGTATAGA CCAATGCAAT GACCTTGTAA TTCAGACAAC AAATGGGCCA CTTTCTCTGT 240

TGAAACTTTT TTTTTACGGC TGCCTGCACT CCTCAGGGCA TTCCAATACA TAGCACTCTG 300

TTTCATTCAC ATAAAACTTA GACTTGACAT TCAATTCTAC ATCCTTCTGG CTTTTCTGCA 360

GAATATTTTA TC 372 (2) INFORMATION ABOUT SEQ ID NO: 38:

(i) SEQUENCE LABEL:

(A) LENGTH: 566 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): AML31 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

CCTCCTACAC AATCGGATGC CCAAAGTCCA CACCAGAGGT TCATTGAAAA ATGATCAGGC 60

TGATTTTTTA AAGAGCTTTT CCTTCAGATT GAGTGAAGAA ATTGACCAAC CTTAATCTGA 120

ATAAATATAA AGTCTAAATA AACTAAGCAT ATGCTTTCAG AAAAATGCCA CTTGCTATGA 180

CAGCATACAT ACAAGAAAAA CGATAAGGAG GTCATACAGC TTAAAACGAT AAGCAAGAGC 240

TGACCAGGGT TAAATCAGGT CACAGGGGTA GGGCCTATTC TGATAGGGAT GGTGGCCTTA 300

TAGGAAGAGG GAAGCCAGCA CGTGCCCCTC TCTCTCCCTC TCCCAATCCC TCTCTCTTTT 360

CTCTCTCCAT GTAAGGACAC AATGAGAAGG AAGCCATAAA CCAGAGAGAG TTCTTTTCAG 420

GGAACCAAAT CCAGACCCTT GATCTTGAAC TTCTCAGCTC CCAGAATTGT GTGAAAATAA 480

ATTTCTATTA TTTAAAGCCT CCCAGTCTAT GGTATTTTGT TATGACAGGC TGACCTGAGT 540

AATCATTATT ACTCTTGCTA AATATG 566 (2) INFORMATION ABOUT SEQ ID NO: 39:

(i) SEQUENCE LABEL:

(A) LENGTH: 680 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): AML35

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

CACAGTGGAC ATGACTTCAA GTGGTAAATT TAGTTGTATA CTTTGGTCCA GGGGATGGTT 60

AGCACTACAT TGATTCATGA GCAGTTGCTA ATGGTTTAGC TGGATGATCA GGGACTTGGA 120

AAGAACAAAA TAGCAAGACT GGTGATAAAG AGGTCTGGGG AAAAGGTAGG TGGATGGACC 180

TCTTGGGATA GGTACAGAGT AAGAGGATAT TTGTGCCCCA TGTGAGAGTT TATCAAAGGA 240

CATGATGAAA ACAGTCTTTC AATAATCAGG TGGACAAAAT GGCATGACCT ATGGTATGTT 300

AGCTTGTTTT CTTTAATAAT TGGAAATAGA AACTTAAGAA TTAGTAGAGA AACTGATTTA 360

AAACAACAAA GGAAGGTGGA AGGGTCCCAC GTGGAATGAA GGTAATTCCC TCTTGTCAAA 420

TTGGGTTTCA GGTGCCTAAG GCACAGGGAA ACAGGAATGT TTGTGTTATT TCTACAAGTA 480

CTTGACAGTC CCTAGGCAAA GTGGGATTGA AGGAGGGAAA CAGCAGGCCA CCATCTCTCT 540

GCCCTTAGAC ATGTAAAAAT GCACCTGCTT AATTCAAGGC ATGAGGATGC ATATCTCATG 600

GAGTCATGAG TATTAACACT TGCAGTCTCA GAACTTTTCT AGGAAACACA CTTTATGAAG 660

CAGAACTGTA GATTTATCCC 680 (2) INFORMATION ABOUT SEQ ID NO: 40:

(i) SEQUENCE LABEL:

(A) LENGTH: 543 base pairs

(B) TYPE: nucleotide (C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): AML38

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

CTCTCAAGTC ACAAAGTCAA GATTTTTTAA CCGTGATTTT AGTTATCTCC ATGACTCCCA 60

GTGCAGTCCA TACACATACG CAAACACACA CAGAAAACTC ACCAGTGCCA TTCTCTTCTC 120

CCAAGAATTG ACAACCCTAT GATAGAATCT TCCACTGGTT ATATTTCAGT GCTTTTGAGC 180

AGTTATTTCT TAAATTTTTT CATGTGTTAT AGTTATCTGT GAGAGGATCA GTACAATAAG 240

AGCTTTTCTA GTCTTTACTA ATGGTGAAAA CTAAACTCTG CAAATATATT TTAGCCACTT 300

TGTATACCTT TATTATACTT AAAGAGATAA TAAAAAGAAA AGGTGTGGAG AAGAAATCTC 360

AAGATTGTGA TTTTTTTGGA AATAAATATT TTACGGGAGG CATGAATGAT GATTTAGAGT 420

TTTCTGAGCT TATAATAAAT AACCTCAGAT TGGTTTGGTT TTTAAGTTCT AGAATTAACA 480

TGTATAACTT TCCTAGTGCC ACTTATCACC CTCCTATTTT ATTGGTGTAT CATCAAGCCA 540

GTC 543 (2) INFORMATION ABOUT SEQ ID NO: 41:

(i) SEQUENCE LABEL:

(A) LENGTH: 580 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): AML40

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:

CACTTTTTGC CGACTTGGTC CCCAAACCTT TGATTAATCT AAAACATGGT GGTCACTAAA 60

GTCTTACATC TAAAAGATTT TTCCTTTGTT ATTAAAAATC AGAATAAATG ATGGAGATAT 120

CTAAAAGGAA AGGTGTAGAC AAAATAACAT TTATCAGTTC CATTATCAAA AAAAAAACAG 180

ATCTGAGTGT TTGAAATTCT AAATTACAAG TCAGCCCACT TTCTTTTGCA CAGCATTACT 240

TCTTTTCCTA AATGACAACA TTTCATCTGT AAAACACCCT TAGGTCTCTC AATTGCACAT 300

TACAATATAA ATCCTCTGTG GTGTAAGAGA CAGTACATTC TGAACTGAGA GATACTCCAT 360

TTTGTCACTT GGCCTGCTCC CCAGTAATGT GTGGTCTGCT GCAACTCCAC ATGTGAATGA 420

CAGGAAAGAC AAAGGCAAAC CAGGGGGTTG AATGGGAAGT ACAGCTAAAT AGTTGTGTTA 480

TTTGTTTTCA CTTGATGTCC ATAGTTAACA GAAAATATAG CTGTCCTATC TCTAGGATTT 540 AACTCTTTCA CAACTGAAGT GTTCCTTCAC TCCATAATCC 580

(2) INFORMATION ON SEQ ID NO: 42:

(i) SEQUENCE LABEL:

(A) LENGTH: 557 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): AML41

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

CAGCAACAAC CAGATTAACA GTCCAAACCT GCTCTCTAGA TTCCAGATAA AACAGCTTAT 60

TTGACATCAC CACTTGGCTG CCTAATAGAA ATCTCAAATT CAGCATGTCA AAAACTCAAT 120

CCTTTATTTC TAGCAGGGAG GACAGAGGAC AAAGAAGCTG GCTGGGCCTT GCAACTCAGA 180

GCATCTGGGC TGGGAAATGC AGCAAGACAT GGAATTGGTG AAGAGTAATT AAAGAACTAT 240

GGGAACTCCA TTTTAGACCT GGTAGGTATT AGTAGTAAAA GTTGTCATTC GGTTTTATTT 300

TTGGCATTTA TAATAGGTTC AAATATTAAA GAAGAGACAA CACCCAATAA ATTGATCCAC 360

AGGGGCAGTT AATTCTTCCA ATGGTCTCAA CTTCCTCAGT TATTACTTGA TAGTTCCAAT 420

AGTAACTGCA TCTATTGCCA TGCATATTTC CCATTTGAGG GAAATCTCAT CCCTTGTCTA 480

CCTCTAAGAT CCATCTTCAC CTTATAGAGG CCATGGGAAA ACTTCCTTTT TGCTCTCTGA 540

AAGTTTGCTC AAAAATC 557 (2) INFORMATION ABOUT SEQ ID NO: 43:

(i) SEQUENCE LABEL:

(A) LENGTH: 593 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): AML47

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:

CGCCACATTT TTAAATTGTG CAAACTGGTG TCAGGTTAAC CCAGGCAAAA GGCGCTGCCC 60

TGAGGCCCAC ACTTTGAAGG GCCCTGCTCT GACTCTCCTA TGGTTGCAAC TCTCCCCCCA 120

AAGTGGGCCA TGGGGCCTCA GGGACATGCA CATTTTGAGT TCAGATCCTC AGCTTCTATA 180

AACAAAATTT AAGTGAATGA AACCCCAGAA ATTTTCTGCT CAAACACCAT GAACACTACA 240

GATGGGAAGG TAATTGTGCA GGACTCTTGA TGGGAGTTAT CCCTCTCGGG GTACGCATTG 300

CCACAGGTGT ACAAAGCCCT ACCTTGTTGG GTGACATATG GCCAGTTGTT TTGGGAATTA 360 GAGTGCTAGA TCAAACTTGG ACGTGTATGC TGGCATGTCC ATGTGCTTGA ACCCTCTTGT 420

TAATGTGGAA AAGAGAGCCC AGGGATAAGA ATAAAAAATT CCAGGGGTAA GAATATGAAA 480

AAGGGCGTGG GGTTTAAGTG CCCAGGGCCA AATCCTCCCT TTGTTAAAAT GACTGTGGCC 540

CGCAGATATT AGATGGAAGA TTGCCTGATG GTTATGCCTT GTTACAACGA ATA 593 (2) INFORMATION ON SEQ ID NO: 44:

(i) SEQUENCE LABEL:

(A) LENGTH: 196 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-1

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:

TAAATATCAC GTGGCAACCT ACAGAATGGG AGAAAATTTT TGCAATCTAT CATCTGACAG 60

AGGTCTAATA TCAGAATCTA AGAAAAACTT AAACAAGTTT ACAAGAAAAA AACAAACAAC 120

CCCATAAAAA AGTGGGTGAA GGATATGAAC AGACAGTTCT CAAAAGAAGA CATTTATGTG 180

GTCAACGAAC ATATGA 196 (2) INFORMATION ON SEQ ID NO: 45:

(i) SEQUENCE LABEL:

(A) LENGTH: 78 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-101

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45: GATCAAAACG CAGAACCATT TGTGTTCTAT ACAATTTATC ACATCAATTA GTAGATTTGT 60 GCAATGACCA GCATGATC 78

(2) INFORMATION ON SEQ ID NO: 46:

(i) SEQUENCE LABEL:

(A) LENGTH: 122 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-103 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: GATCGATTCC ATACCTTGTC TATTGTGAAT AATGCAATAA TAAACATGAG AGTGCAGGTA 60 TCCCTTTGAC ATACTGATTT CTCGTGCTTT GGATAAATGC CAATTAGTGA CATTTTTGGA 120 TC

(2) INFORMATION ON SEQ ID NO: 47:

(i) SEQUENCE LABEL:

(A) LENGTH: 80 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-104

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: GATCCTGTCC ATATCCTGTG CAGTGATAAC TCCAGGAACT TGAACTACTG CCAAGAATTT 60 TCTACTGATA CTCATGGATC 80

(2) INFORMATION ON SEQ ID NO: 48:

(i) SEQUENCE LABEL:

(A) LENGTH: 63 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-105

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:

GATCAAGTCG TCAGCTCCGT AACCAGCTTA TGCCAGGCTA GAACCTCATG GCCCCCAGTG 60

ATC 63 (2) INFORMATION ABOUT SEQ ID NO: 49:

(i) SEQUENCE LABEL:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-106 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:

GATCAATTGC AATGTAAACT CACTCTTTGT GTGAAAGGAA AAAAAATGCT TAATATCTTT 60 TTGCTGGTGG AGGGCCTTGC CTCAGTGTGG ATGGCTGCTG ACTGATC 107

(2) INFORMATION ON SEQ ID NO: 50:

(i) SEQUENCE LABEL:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-107

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: GATCTGGCTT TCAAATGCCT GGGAGGCCAA CAGATC 36

(2) INFORMATION ON SEQ ID NO: 51:

(i) SEQUENCE LABEL:

(A) LENGTH: 231 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-11

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:

TATATAACAC GTCAGACATG AGCCACCACG CCCGGCCAGG AATGAATGTT CTATGCATGA 60

AACTGTAAAC GGCTTATTGC TATAATTGTC CTAGTTTATT ATTGTTAATT TCTTACTGTA 120

CCTAATTTAT AAATTAAGCT TTATCAGAGG TATGTATGTA TTAGAAAAAA TATAACATAT 180

ATAGGATACA GTACTATCCA GGATCAGGAA TCTACTGGGC ATACTAGAAA G 231 (2) INFORMATION ON SEQ ID NO: 52:

(i) SEQUENCE LABEL:

(A) LENGTH: 191 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-12

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: TATATATCAC GTAACTGTAA ACGGCTTAT GCTÄTAATTA TCCTAGTTTA TTATTGTTAA 60

TTTCTTACTG TACCTAATTT ATAAATTAAA CTTTATCAGA GGTATGTATG TATTAGAAAA 120

AATATAACAT ATATAGGATA CAGTACTATC CATGGTTTCA GGAATCTACT GGGCATACTA 180 GAAAGATCCC C 191

(2) INFORMATION ON SEQ ID NO: 53:

(i) SEQUENCE LABEL:

(A) LENGTH: 246 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-14

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:

TATATANCAC GTGGAGCTAC AATTCAAGAT GAGATTTGGA TGGGGACACT GCCAAACCAT 60

ATCATGGGGT TTGTCACTGG CCTGGATGGA ACATCCTTGT CAAGAGCACA CACACACACA 120

CACACACACA CACACACTTA GAGAGACAGA CAAAGAAGAG AGAGAGGAGA GGCACGGTTA 180

TAATGAGGAT GGACAGGTGA TTTTGGAAGC TGAGAGTCAT TACTGCCACT GCCAGCTAGG 240

TGTAAT 246 (2) INFORMATION ABOUT SEQ ID NO: 54:

(i) SEQUENCE LABEL:

(A) LENGTH: 281 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-16

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:

TATATATCAC GTTGTTATTA TAATTATCAG TCCTGAAACC TGTCTCATCT TAGAAACATA 60

GATTCTATAC AAGTGGCCCT GGAGTGCCTT TTAAGATGTT CTCAGGAGAT TTTGGTTGTC 120

TTCTAAACTT TCTGGAAACA TCTTGTTCAT TTCGTATTCT GGTGGAAACA TCTTGTTCAT 180

TTCGTATTCT GGATTTAAAA TTAGCTATTT CTCCAAGGAG ATGTCAGCCA TTGTGTTTAT 240

ACAAACTTGA GTTCCTTTAA GGTTTAACCT TCATTCAAGA A 281 (2) INFORMATION ABOUT SEQ ID NO: 55:

(i) SEQUENCE LABEL:

(A) LENGTH: 268 base pairs

(B) TYPE: nucleotide (C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-17

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:

TATATATCAC GTGAAACTGA TAAGTTGTTT CTCTAGCTTT TAAAAATACA TGGAGTGAGT 60

TCTATAATTC CTCAGGAAGT AAGAGCTGTT CCTTTTGAGC TGAAGAACAT ATTGCAATTC 120

CTATTTCATT GCAGAGATAT TTCTCTACTT TAAAATGCTA CCCGCTTCTC ATCCACACAA 180

AAATTGTGAA ACAGAGGGGG CTGGGGCAGT AAGGAAGTTA TGAAGAAAGT AATTCACATT 240

AGGTGAGCTG GAGCCAATAG CTTAATTT 268 (2) INFORMATION ABOUT SEQ ID NO: 56:

(i) SEQUENCE LABEL:

(A) LENGTH: 307 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-18

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:

TATATATCAC GTTAGGTGGT GCGTGCCTGT AGTCCCAGCT ACTCGGGAGG CTGAGGCACG 60

AGAATCACTT GAACCCGTGA AGCAGAGGTT GCAGTGAGCC GAGATCACCC ACTGCACTTC 120

AGCCTGGGCA ACAGAGCAAG ACTCCGTCTC AAAAAAAAAA AACAAAAAAC CAAAAACTTC 180

TTTATATGTA AGAAGAGATG TTCCTGACAT GAAAAATAGC ACCTTTACAG CACTTTTCTC 240

TAGTGTGGAC ACTGGCGGAT TACCAAGGTT GTCATTTAAA TACAGATGAC AGTTCCACTC 300

AGAGGTT 307 (2) INFORMATION ABOUT SEQ ID NO: 57:

(i) SEQUENCE LABEL:

(A) LENGTH: 245 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vi i) DIRECT ORIGIN: (B) CLON (E): HT29-2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:

TGCAGGCATG CAAGCTTGGC GTAATCATGG TCATAGCTGT TTCCTGTGTG AAATTGTTAT 60

CCGCTCACAA TTCCACACAA CATACGAGCC GGAAGCATAA AGTGTAAAGC CTGGGGTGCC 120

TAATGAGTGA GCTAACTCAC ATTAATTGCG TTGCGCTCAC TGCCCGCTTT CAGTCGGGAA 180

ACCTGTCGTG CCAGCTGCAT TAATGAATCG GCCAACGCGC GGGGAGAGGC GGTTTGCGTA 240

TTGGG 245 (2) INFORMATION ABOUT SEQ ID NO: 58:

(i) SEQUENCE LABEL:

(A) LENGTH: 256 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-20

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: TATATATCAC GTAGACGCCA TCTCAAAAAA AAAAAAAGCT ATGATTGGGT AAGATTATAA 60

AAAGGATAGT TAAAAATAAA TACCACCTAA ATATGAACCT AAAGAAAGCG AGTATAAAAA 120

TGTAAATATG TGATATGATA AACTTAGGTT TAAAAATATC AATAAGGGCT ACTCTGTATC 180

ATAGTATACA AGTGGACATG GCTGGGCCAG TGGCTCACGC CTGTAATCCA ACACTTTGGG 240

AGGCAGAGGC AGGCGT 256 (2) INFORMATION ON SEQ ID NO: 59:

(i) SEQUENCE LABEL:

(A) LENGTH: 122 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-202

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: TATATATCAC GTAGACCTAA AACCATAAAA ACCCTAGAGA AAACCTAAGG CATTAACCAA 60 TTCAGGACAT AGGTGGGCAG GATTAAGTCA AAACACCAAA ATAATGGCAA CAAAAGCCAA 120 AA 122

(2) INFORMATION ON SEQ ID NO: 60:

(i) SEQUENCE LABEL:

(A) LENGTH: 132 base pairs

(B) TYPE: nucleotide (C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-203

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: TATATATCAC GTATATGTCT TGAAGATGCA CTTCCAAGGT CATGAATGGA ACTCCACCAG 60 CCACAAAAAG ACCTGCCTAT ACCTGGAGTA TCGTGCAGTC AGAGTGCTCC GACTCCTGTG 120 GTGGAGG

(2) INFORMATION ON SEQ ID NO: 61:

(i) SEQUENCE LABEL:

(A) LENGTH: 230 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-206

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:

TATATATCAC GTAAGACCAA TGAGAGACAA TGCAGTCGCT GGGACAACCA GATTGGACCA 60

GGCAGTCAAG GTGGCTTGGA GGCCACAGCG AGAATTAAAA TACACTGAGA GATTATACTT 120

CAGGATCCTC TAGAGTCGAC CTGCAGGCAT GCAAGCTTGC TAATGCATGG TCTCATAGCT 180

GTTTCCTGTG TGAAATTGTT ATTCCTCCTC ACAATTCCAC ACAACATACC 230 (2) INFORMATION ON SEQ ID NO: 62:

(i) SEQUENCE LABEL:

(A) LENGTH: 335 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-207

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:

TATATATCAC GTGTCAATCA ATTTTATGAG AATAGCATTG AATCTATAAA TTACTTTGGG 60

CAGTATGGCC ATTTTCATGA TATTGATTCT TCCTATCCAT GAGGATGGAA TGTTTTTCCA 120

TTTGTTTGTG TCCTCTCTTA TTTCCTTGAG CAGTGGTTTG TAGTTCTCCT TGAAGAGGTC 180 TTTCACATCT CTTCTTACCT GTGTTGTTAG GTATTTATTC TCTTTGTAGC AGTGGCAAAT 240

GGGAGTTTAT TCATGATTGG CTCTCTGCTT GTTATTGTTG AGTAAGGAGT ACTGTGATTT 300

GCACATGCTT GCATCTAGAC TTATGAGTGC TACAG 335 (2) INFORMATION ABOUT SEQ ID NO: 63:

(i) SEQUENCE LABEL:

(A) LENGTH: 192 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-22

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:

TATATATCAC GTGTCAATCT CACTCTCTCC CCCAGGCTGA AGAGCAGTGG TGCGATCCTC 60

TAGAGTCGAC CTGCAGGCAT GCAAGCTTGG CGTAATCATG GTCATAGCTG TTTCCTGTGT 120

GAAATTGTTA TCGCTCACAA TTCCACACAA CATACGAGCG GAAGCATAAG TGTAAAGGCC 180

TGGGTGCCTT AA 192 (2) INFORMATION ABOUT SEQ ID NO: 64:

(i) SEQUENCE LABEL:

(A) LENGTH: 188 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-24

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:

TATATATCAC GTAAAATTTT TGGGCATTTC TATATCATAT TANCCAGAAA CTCAGCATAC 60

TTTAAAAAGT TACCACATAC ACAAAAAAGC AAGAAATACA GACTCATAAA AAATAAATCA 120

ATTATTAAAA ACAAATTCAC AGATAATCCA GATATTGGAG TTAATAGATA GGGCATCATA 180

ATATCTAT 188 (2) INFORMATION ABOUT SEQ ID NO: 65:

(i) SEQUENCE LABEL:

(A) LENGTH: 232 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (vii) DIRECT ORIGIN: (B) CLON (E): HT29-24

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:

TATATATCAC GTGGCACATG TCACCATACT GCACTATTTT AAATTTTTTG TAGGTCTTGC 60

TATGTTGCCC AGTCTGGTCT CGAACTCCTG ACCTCAAGCA ATCCTCCCAC CTTGGCCTCC 120

CAAAGTGCTT GCATTACAGG CATGAGCACT GCCTGGCCCA CCCAGGCAGT TCAAAAGCAT 180

CTTGAGAGTT CAAAAGCATA TTGGTGAGAC TCATTCCATG AGGCTGGTGA GG 232 (2) INFORMATION ON SEQ ID NO: 66:

(i) SEQUENCE LABEL:

(A) LENGTH: 204 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-27

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:

TATATATCAC GTAGATGTGA GCACCCTACT TGGCCAGCCA CCTGAAATTC TATCAGTGAC 60

AGATTCCTTT AGCTGAAGTC ATGTTCAAAT GTAATCCTCC CAAGAATGGT AACATTATAC 120

ATCAATGCAT TAAACAAAAC AACCTGTGCT TATGAAAAAG TCATATGTGC ACACGGTAAA 180

ACAAAATTCA AATAGTACAA AGGG 204 (2) INFORMATION ABOUT SEQ ID NO: 67:

(i) SEQUENCE LABEL:

(A) LENGTH: 192 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:

TAGCGTTTCC TGTGTGAAAT AGAATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG 60

CATAAAGTGT AAAGCCTGGG GTGCCTAATG AGTGAGCTAA CTCACATTAA TTGCGTTGCG 120

CTCACTGCCC GCTTTCAGTG GAAACCTGTC TGGCCAGCTG CATTAATGAA TCGGCCAACG 180

CGCGGGAGAG GC 192 (2) INFORMATION ON SEQ ID NO: 68: (i) SEQUENCE LABEL:

(A) LENGTH: 227 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-32

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:

TATATATCAC GTACAGAGCA AGACCCCATC TCAAAAAAAA AAAAAGCTAT GATTGGGTAA 60

GATTATAAGA GGGATAGTTA AAAATAAATA CCACCTAAAT ATGAACCTAA AGAAAGCGAG 120

ATAAAAAGTA AATATGTGAT ATGATAAACT TAGGTTTAAA AATACAATAA GGGCTACTCT 180

GTATGTATAG TATACAGTGG TACATGGCGG GCCAGTGGCT CACGCTG 227 (2) INFORMATION ON SEQ ID NO: 69:

(i) SEQUENCE LABEL:

(A) LENGTH: 234 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-34

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:

TATATATCAC GTTTTTTCAC ATTCCAATGG GAAAATAACA TGGAGCACGG CGTGTTCCCC 60

TCAAGGATAG CTGACAAAGG AAAAGGCCCT TCCTTTATCT CCACTGGGAG TTTTGTTTTC 120

CTTTTATCTA AAAGGGGTTA TCAGTGCTGC TCAAATAAAG AAGAAATGTG TGATTGAAAT 180

GGGTGGAGGA TATGCTAGGG AGAAAAAGAA AGCCTGAACA AATGAAAATT GTAT 234 (2) INFORMATION ON SEQ ID NO: 70:

(i) SEQUENCE LABEL:

(A) LENGTH: 115 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-36

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: TATATATCAC GTAACCCCCA ACCCCCCCTC TCCTCCTTCT TACTCAACGC CTTCGGGCTA 60 CTCAACGCCA CTCCCTTCTC ACCCCTCAGC ACCCTCCAAA CAGACACATC GAACA 115

(2) INFORMATION ON SEQ ID NO: 71:

(i) SEQUENCE LABEL:

(A) LENGTH: 265 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-37

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:

TATATANCAC GTAAGAGTCT GTTGGATTTA ATATTTTTTA GAAGGTTTGG TGAATTGGTG 60

TTTGCAAAAG CCAATTGCTG TGCATTGAGG AATATGTTTG ATTATTCATT CAAGTAATAA 120

AAAACAAAAA ATCAAGCTGA CTCTTGAAGA GGCCCCCCCA CACACACACA AAAGAAAGAA 180

ATGTTTGTCT TATGTGCTAA GCCTGTTCTA GGTTGTTGAG GATGTACTGG TAAACCAAAT 240

GTCCTGTGTT ATGACACTTA GATCT 265 (2) INFORMATION ABOUT SEQ ID NO: 72:

(i) SEQUENCE LABEL:

(A) LENGTH: 256 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-39

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:

TATATATCAC GTGCCAGTTC CTTTAGGTGC ATCATTAGGC TGTTTATTTG AAGTCTTTCT 60

ATCTTTTTGA TGAAAGCATT TATTGCTATA ATCTTCCCTC TTAGAACAGT TTTGCTGTAT 120

CCCATAGGTT TTGATATATT GTGTTTCCAT TGCCATTTGT CTTAAATTTT TAAATTTTCT 180

TTTTAATTTC TTCACTGGCT CATTCATTGT CCAGGAGCAT TGCTGTTTAA TTTCATGAGT 240

TTGTGCAGTT TCAACA 256 (2) INFORMATION ON SEQ ID NO: 73:

(i) SEQUENCE LABEL:

(A) LENGTH: 110 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (vii) DIRECT ORIGIN: (B) CLON (E): HT29-40

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: TATATATCAC GTTTATCAGA AGACAGGTAA TGTTGACACA TGGAGCTCTG AGATAAATGG 60 TCTCAAGAGA AGGGATCCTC TAGAGTCGAC CTGCAGGCAT GCAAGCTTGG 110

(2) INFORMATION ON SEQ ID NO: 74:

(i) SEQUENCE LABEL:

(A) LENGTH: 255 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-41

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:

TATATATCAC GTAGAAGGTT TGGTGAATTG GTGTTTGCAA AAGCCAATTG CTGTGCATTG 60

AGGAATATGT TTGATTATTC ATTCAAGTAA TAAAAAAGAA AAAAGCAAGC TGACTCTTGA 120

AGAGGCCCCC CCACACACAC ACAAAAGAAA GAAATGTTTG TCTTATGTGC TAAGCACTGT 180

TCTAGGTTGT TGAGGATGTA CTGGTAAACC AAAATGTCCT GTGTTTATGA CACTTAGATT 240

CTAATAGTTG GAGAT 255 (2) INFORMATION ABOUT SEQ ID NO: 75:

(i) SEQUENCE LABEL:

(A) LENGTH: 318 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-43

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:

TATATATCAC GTCCCTGTCT CTATTAAAAA TACAAAATTA GCTGTGTGTG GTGGCACACG 60

CCTGTAGTCC CAGCTACTTA GGAGGGGAGG GAGGTGCCAG GCTCTTTTTA ACAATCAGCT 120

CCCACAGGAA CTAATAGAGG AAGAACTCAC TCATTACCAC AAGGACAGCA CCAAGCCATT 180

CATGACGGAT GTACCTGCAT GACCCCAACA CCTCCAGTTA GGCCCAACGT TAGGGATCAT 240

GATAACTATG GGTGTGGTTA TGACTTTTTA GATGGTACAC CAAAGGCACA TAAATGAAAA 300

CATAACTGAA TCCTAGAC 318 (2) INFORMATION ON SEQ ID NO: 76:

(i) SEQUENCE LABEL:

(A) LENGTH: 249 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-44

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:

TATATATCAC GTGGAAAAGC ATGGATGGGA TTTAAGCTAC ACATACATGC TCCAAGGTTA 60

TTTTAATTTC TGCAAATAAC AATGAAATAA GGTATTTCTT TAGCTGTAAT AAACATCCTT 120

TGCAAATCAT TATTAGATGT TTTATCTTAA GATAAATTTT TCAAACCAAA GAAAGTTTCA 180

AACTTTGAAC TTTAAATGAA ATATCAAATT CCTTAATATC ACTTTTAATA TTCAAATTAA 240

ATCCATGCA 249 (2) INFORMATION ABOUT SEQ ID NO: 77:

(i) SEQUENCE LABEL:

(A) LENGTH: 224 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-45

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:

TATATATCAC GTGAGAATTC TTCTGTCTAG CAGAATATGA AGAAATCCCG AAACCAACGA 60

AGGCCTCAAG GAGGTCTGAA TATCCACTTG CAGACTTTAC AAACAGAGTG TTTCCTAACT 120

GCTCTATGAA AAAGAAAGGT TAAACTCTGT GAGTTGAACG CACACAAACA CAAAAGGAGT 180

TTATGAGAAT ACTTACTGTA CTAGTTTCTA TAAAGAAGAT ATTT 224 (2) INFORMATION ON SEQ ID NO: 78:

(i) SEQUENCE LABEL:

(A) LENGTH: 143 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78: TATATATCAC GTAAGGTAAA TTAACTGAAA GTAAAATTGG AAAATGTGGC TAAAAGGGGG 60 GAAAATAATC AAGTTTATAC CTCTAGAGTC GACCTGCAGG CATGACAACG TTGGCTGTACCT GTGGGTGGTAAT 120 GT

(2) INFORMATION ON SEQ ID NO: 79:

(i) SEQUENCE LABEL:

(A) LENGTH: 76 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-6

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: GATCTGCCCA CCTCGGCCTC CCAAAGTGCT GGGATTACAG GTGTGAGCCA CCACGGCTGG 60 CTGCATCAGT GTGATC 76

(2) INFORMATION ON SEQ ID NO: 80:

(i) SEQUENCE LABEL:

(A) LENGTH: 106 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-7

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80: GATCAATTGC AATGTAAACT CACTCTTTGT GTGAAAGGAA AAAAAATGCT TAATATCTTT 60 TTGCTGGTGG AGGGCCTTGC TCAGTGTGGA TGGCTGCTGA CTGATC 106

(2) INFORMATION ON SEQ ID NO: 81:

(i) SEQUENCE LABEL:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HT29-9

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81: TATATATCAC GTACTGTCAC TCAGGCTGGA GGGTAGTT 38

(2) INFORMATION ON SEQ ID NO: 82:

(i) SEQUENCE LABEL:

(A) LENGTH: 78 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): BH1

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82: GATCAAAACG CAGAACCATT TGTGTTCTAT ACAATTTATC ACATCAATTA GTAGATTTGT 60 GCAATGACCA GCATGATC 78

(2) INFORMATION ON SEQ ID NO: 83:

(i) SEQUENCE LABEL:

(A) LENGTH: 122 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): BH3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:

GATCGATTCC ATACCTTGTC TATTGTGAAT AATGCAATAA TAAACATGAG AGTGCAGGTA 60

TCCCTTTGAC ATACTGATTT CTCGTGCTTT GGATAAATGC CAATTAGTGA CATTTTTGGA 120

TC 122 (2) INFORMATION ON SEQ ID NO: 84:

(i) SEQUENCE LABEL:

(A) LENGTH: 80 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): BH4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: GATCCTGTCC ATATCCTGTG CAGTGATAAC TCCAGGAACT TGAACTACTG CCAAGAATTT 60 TCTACTGATA CTCATGGATC 80 (2) INFORMATION ON SEQ ID NO: 85:

(i) SEQUENCE LABEL:

(A) LENGTH: 63 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): BH5

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85: GATCAAGTCG TCAGCTCCGT AACCAGCTTA TGCCAGGCTA GAACCTCATG GCCCCCAGTG 60 ATC 63

(2) INFORMATION ON SEQ ID NO: 86:

(i) SEQUENCE LABEL:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): BH6

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86: GATCAATTGC AATGTAAACT CACTCTTTGT GTGAAAGGAA AAAAAATGCT TAATATCTTT 60 TTGCTGGTGG AGGGCCTTGC CTCAGTGTGG ATGGCTGCTG ACTGATC 107

(2) INFORMATION ON SEQ ID NO: 87:

(i) SEQUENCE LABEL:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): BH7

(xi) QU SEQUENCE DESCRIPTION: SEQ ID NO: 87: GATCTGGCTT TCAAATGCCT GGGAGGCCAA CAGATC 36

(2) INFORMATION ON SEQ ID NO: 88:

(i) SEQUENCE LABEL:

(A) LENGTH: 184 base pairs

(B) TYPE: nucleotide (C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB1

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:

GGCAACCTAC AGAATGGGAG AAAATTTTTG CAATCTATCA TCTGACAGAG GTCTAATATC 60

AGAATCTAAG AAAAACTTAA ACAAGTTTAC AAGAAAAAAA CAAACAACCC CATAAAAAAG 120

TGGGTGAAGG ATATGAACAG ACAGTTCTCA AAAGAAGACA TTTATGTGGT CAACGAACAT 180

ATGA 184 (2) INFORMATION ON SEQ ID NO: 89:

(i) SEQUENCE LABEL:

(A) LENGTH: 219 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB11

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:

CAGACATGAG CCACCACGCC CGGCCAGGAA TGAATGTTCT ATGCATGAAA CTGTAAACGG 60

CTTATTGCTA TAATTGTCCT AGTTTATTAT TGTTAATTTC TTACTGTACC TAATTTATAA 120

ATTAAGCTTT ATCAGAGGTA TGTATGTATT AGAAAAAATA TAACATATAT AGGATACAGT 180

ACTATCCAGG ATCAGGAATC TACTGGGCAT ACTAGAAAG 219 (2) INFORMATION ABOUT SEQ ID NO: 90:

(i) SEQUENCE LABEL:

(A) LENGTH: 179 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB12

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90: AACTGTAAAC GGCTTATTGC TATAATTNTC CTAGTTTATT ATTGTTAATT TCTTACTGTA 60 CCTAATTTAT AAATTAANCT TTATCAGAGG TATGTATGTA TTAGAAAAAA TATAACATAT 120 ATAGGATACA GTACTATCCA TGGTTTCAGG AATCTACTGG GCATACTAGA AAGATCCCC 179 (2) INFORMATION ON SEQ ID NO: 91:

(i) SEQUENCE LABEL:

(A) LENGTH: 234 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB14

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:

GGAGCTACAA TTCAAGATGA GATTTGGATG GGGACACTGC CAAACCATAT CATGGGGTTT 60

GTCACTGGCC TGGATGGAAC ATCCTTGTCA AGAGCACACA CACACACACA CACACACACA 120

CACACTTAGA GAGACAGACA AAGAAGAGAG AGAGGAGAGG CACGGTTATA ATGAGGATGG 180

ACAGGTGATT TTGGAAGCTG AGAGTCATTA CTGCCACTGC CAGCTAGGTG TAAT 234 (2) INFORMATION ON SEQ ID NO: 92:

(i) SEQUENCE LABEL:

(A) LENGTH: 269 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB16

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:

TGTTATTATA ATTATCAGTC CTGAAACCTG TCTCATCTTA GAAACATAGA TTCTATACAA 60

GTGGCCCTGG AGTGCCTTTT AAGATGTTCT CAGGAGATTT TGGTTGTCTT CTAAACTTTC 120

TGGAAACATC TTGTTCATTT CGTATTCTGG TGGAAACATC TTGTTCATTT CGTATTCTGG 180

ATTTAAAATT AGCTATTTCT CCAAGGAGAT GTCAGCCATT GTGTTTATAC AAACTTGAGT 240

TCCTTTAAGG TTTAACCTTC ATTCAAGAA 269 (2) INFORMATION ABOUT SEQ ID NO: 93:

(i) SEQUENCE LABEL:

(A) LENGTH: 256 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB17 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:

GAAACTGATA AGTTGTTTCT CTAGCTTTTA AAAATACATG GAGTGAGTTC TATAATTCCT 60

CAGGAAGTAA GAGCTGTTCC TTTTGAGCTG AAGAACATAT TGCAATTCCT ATTTCATTGC 120

AGAGATATTT CTCTACTTTA AAATGCTACC CGCTTCTCAT CCACACAAAA ATTGTGAAAC 180

AGAGGGGGCT GGGGCAGTAA GGAAGTTATG AAGAAAGTAA TTCACATTAG GTGAGCTGGA 240

GCCAATAGCT TAATTT 256 (2) INFORMATION ON SEQ ID NO: 94:

(i) SEQUENCE LABEL:

(A) LENGTH: 295 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB18

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:

TAGGTGGTGC GTGCCTGTAG TCCCAGCTAC TCGGGAGGCT GAGGCACGAG AATCACTTGA 60

ACCCGTGAAG CAGAGGTTGC AGTGAGCCGA GATCACCCAC TGCACTTCAG CCTGGGCAAC 120

AGAGCAAGAC TCCGTCTCAA AAAAAAAAAA CAAAAAACCA AAAACTTCTT TATATGTAAG 180

AAGAGATGTT CCTGACATGA AAAATAGCAC CTTTACAGCA CTTTTCTCTA GTGTGGACAC 240

TGGCGGATTA CCAAGGTTGT CATTTAAATA CAGATGACAG TTCCACTCAG AGGTT 295 (2) INFORMATION ON SEQ ID NO: 95:

(i) SEQUENCE LABEL:

(A) LENGTH: 166 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB19

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95: CCGTCACCAT GCCCAGCTAA TTTTTTTTGT ATTTTTAGTA GAGATGGGGT TTCACTATGT 60 TGGCCAGGTG GTCTTGAACT CCTGAACTTG TGATCCTCTA GAGTCGACCT GCAGCTTGGTCTG CATTATGGGTCG

(2) INFORMATION ABOUT SEQ ID NO: 96: (i) SEQUENCE LABEL: (A) LONG: 110 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96: AGACCTAAAA CCATAAAAAC CCTAGAGAAA ACCTAAGGCA TTAACCAATT CAGGACATAG 60 GTGGGCAGGA TTAAGTCAAA ACACCAAAAT AATGGCAACA AAAGCCAAAA 110

(2) INFORMATION ON SEQ ID NO: 97:

(i) SEQUENCE LABEL:

(A) LENGTH: 244 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB20

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97:

AGACGCCATC TCAAAAAAAA AAAAAGCTAT GATTGGGTAA GATTATAAGA AGGATAGTTA 60

AAAATAAATA CCACCTAAAT ATGAACCTAA AGAAAGCGAG TATAAAAATG TAAATATGTG 120

ATATGATAAA CTTAGGTTTA AAAATATCAA TAAGGGCTAC TCTGTATCAT AGTATACAAG 180

TGGACATGGC TGGGCCAGTG GCTCACGCCT GTAATCCAAC ACTTTGGGAG GCAGAGGCAG 240

GCGT 244 (2) INFORMATION ABOUT SEQ ID NO: 98:

(i) SEQUENCE LABEL:

(A) LENGTH: 180 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB22

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98: GTCAATCTCA CTCTCTCCCC CAGGCTGAAG AGCAGTGGTG CGATCCTCTA GAGTCGACCT 60 GCAGGCATGC AAGCTTGGCG TAATCATGGT CATAGCTGTT TCCTGTGTGA AATTGTTATC GCTCACAATT CCACACAACA TACGAGCGGA AGCATAAGTG TAAAGGCCTG GGTGCCTTAA 180

(2) INFORMATION ON SEQ ID NO: 99:

(i) SEQUENCE LABEL:

(A) LENGTH: 176 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB24

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99: AAAATTTTTG GGCATTTCTA TATCATATTA NCCAGAAACT CAGCATACTT TAAAAAGTTA 60 CCACATACAC AAAAAAGCAA GAAATACAGA CTCATAAAAA ATAAATCAAT TATTAAAAAC 120 AAATTCACATT ATAGATATAGA TATATGATATAGAT

(2) INFORMATION ON SEQ ID NO: 100:

(i) SEQUENCE LABEL:

(A) LENGTH: 220 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB25

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100:

GGCACATGTC ACCATACTGC ACTATTTTAA ATTTTTTGTA GGTCTTGCTA TGTTGCCCAG 60

TCTGGTCTCG AACTCCTGAC CTCAAGCAAT CCTCCCACCT TGGCCTCCCA AAGTGCTTGC 120

ATTACAGGCA TGAGCACTGC CTGGCCCACC CAGGCAGTTC AAAAGCATCT TGAGAGTTCA 180

AAAGCATATT GGTGAGACTC ATTCCATGAG GCTGGTGAGG 220 (2) INFORMATION ON SEQ ID NO: 101:

(i) SEQUENCE LABEL:

(A) LENGTH: 192 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB27 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:

AGATGTGAGC ACCCTACTTG GCCAGCCACC TGAAATTCTA TCAGTGACAG ATTCCTTTAG 60

CTGAAGTCAT GTTCAAATGT AATCCTCCCA AGAATGGTAA CATTATACAT CAATGCATTA 120

AACAAAACAA CCTGTGCTTA TGAAAAAGTC ATATGTGCAC ACGGTAAAAC AAAATTCAAA 180

TAGTACAAAG GG 192 (2) INFORMATION ABOUT SEQ ID NO: 102:

(i) SEQUENCE LABEL:

(A) LENGTH: 120 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102: ATATGTCTTG ANGATGCACT TCCAAGGTCA TGAATGGAAC TCCACCAGCC ACAAAAAGAC 60 CTGCCTATAC CTGGAGTATC GTGCAGTCAG AGTGCTCCGN CTCCTGTGGT 120 GGAGGTAGAT

(2) INFORMATION ON SEQ ID NO: 103:

(i) SEQUENCE LABEL:

(A) LENGTH: 215 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB32

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103:

ACAGAGCAAG ACCCCATCTC AAAAAAAAAA AAAGCTATGA TTGGGTAAGA TTATAAGAGG 60

GATAGTTAAA AATAAATACC ACCTAAATAT GAACCTAAAG AAAGCGAGAT AAAAAGTAAA 120

TATGTGATAT GATAAACTTA GGTTTAAAAA TACAATAAGG GCTACTCTGT ATGTATAGTA 180

TACAGTGGTA CATGGCGGGC CAGTGGCTCA CGCTG 215 (2) INFORMATION ON SEQ ID NO: 104:

(i) SEQUENCE LABEL:

(A) LENGTH: 222 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (vii) DIRECT ORIGIN: (B) CLON (E): HB34

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104:

TTTTTCACAT TCCAATGGGA AAATAACATG GAGCACGGCG TGTTCCCCTC AAGGATAGCT 60

GACAAAGGAA AAGGCCCTTC CTTTATCTCC ACTGGGAGTT TTGTTTTCCT TTTATCTAAA 120

AGGGGTTATC AGTGCTGCTC AAATAAAGAA GAAATGTGTG ATTGAAATGG GTGGAGGATA 180

TGCTAGGGAG AAAAAGAAAG CCTGAACAAA TGAAAATTGT AT 222 (2) INFORMATION ABOUT SEQ ID NO: 105:

(i) SEQUENCE LABEL:

(A) LENGTH: 103 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB36

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105: AACCCCCAAC CCCCCCTCTC CTCCTTCTTA CTCAACGCCT TCGGGCTACT CAACGCCACT 60 CCCTTCTCAC CCCTCAGCAC CCTCCAAACA GACACATCGA ACA 103

(2) INFORMATION ON SEQ ID NO: 106:

(i) SEQUENCE LABEL:

(A) LENGTH: 253 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB37

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106:

AAGAGTCTGT TGGATTTAAT ATTTTTTAGA AGGTTTGGTG AATTGGTGTT TGCAAAAGCC 60

AATTGCTGTG CATTGAGGAA TATGTTTGAT TATTCATTCA AGTAATAAAA AACAAAAAAT 120

CAAGCTGACT CTTGAAGAGG CCCCCCCACA CACACACAAA AGAAAGAAAT GTTTGTCTTA 180

TGTGCTAAGC CTGTTCTAGG TTGTTGAGGA TGTACTGGTA AACCAAATGT CCTGTGTTAT 240

GACACTTAGA TCT 253

(2) INFORMATION ON SEQ ID NO: 107:

(i) SEQUENCE LABEL:

(A) LENGTH: 245 base pairs (B) TYPE: Nucleotide 87

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vi-.) DIRECT ORIGIN: (B) CLON (E): HB39

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107:

GCCAGTTCCT TTAGGTGCAT CATTAGGCTG TTTATTTGAA GTCTTTCTAT CTTTTTGATG 60

AAAGCATTTA TTGCTATAAT CTTCCCTCTT AGAACAGTTT TTGCTGTATC CCATAGGTTT 120

TGATATATTG TGTTTCCATT GCCATTTGTC TTAAATTTTT AAATTTTCTT TTTAATTTCT 180

TCACTGGCTC ATTCATTGTC CAGGAGCATT GCTGTTTAAT TTCATGAGTT TGTGCAGTTT 240

CAACA 245 (2) INFORMATION ON SEQ ID NO: 108:

(i) SEQUENCE LABEL:

(A) LENGTH: 98 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB40

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108: TTATCAGAAG ACAGGTAATG TTGACACATG GAGCTCTGAG ATAAATGGTC TCAAGAGAAG 60 GGATCCTCTA GAGTCGACCT GCAGGCATGC AAGCTTGG 98

(2) INFORMATION ON SEQ ID NO: 109:

(i) SEQUENCE LABEL:

(A) LENGTH: 243 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB41

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109:

AGAAGGTTTG GTGAATTGGT GTTTGCAAAA GCCAATTGCT GTGCATTGAG GAATATGTTT 60

GATTATTCAT TCAAGTAATA AAAAAGAAAA AAGCAAGCTG ACTCTTGAAG AGGCCCCCCC 120

ACACACACAC AAAAGAAAGA AATGTTTGTC TTATGTGCTA AGCACTGTTC TAGGTTGTTG 180 AGGATGTACT GGTAAACCAA AATGTCCTGT GTTTATGACA CTTAGATTCT AATAGTTGGA 240 GAT 243

(2) INFORMATION ON SEQ ID NO: 110:

(i) SEQUENCE LABEL:

(A) LENGTH: 306 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB43

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110:

CCCTGTCTCT ATTAAAAATA CAAAATTAGC TGTGTGTGGT GGCACACGCC TGTAGTCCCA 60

GCTACTTAGG AGGGGAGGGA GGTGCCAGGC TCTTTTTAAC AATCAGCTCC CACAGGAACT 120

AATAGAGGAA GAACTCACTC ATTACCACAA GGACAGCACC AAGCCATTCA TGACGGATGT 180

ACCTGCATGA CCCCAACACC TCCAGTTAGG CCCAACGTTA GGGATCATGA TAACTATGGG 240

TGTGGTTATG ACTTTTTAGA TGGTACACCA AAGGCACATA AATGAAAACA TAACTGAATC 300

CTAGAC 306 (2) INFORMATION ABOUT SEQ ID NO: 111:

(i) SEQUENCE LABEL:

(A) LENGTH: 237 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB44

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111:

GGAAAAGCAT GGATGGGATT TAAGCTACAC ATACATGCTC CAAGGTTATT TTAATTTCTG 60

CAAATAACAA TGAAATAAGG TATTTCTTTA GCTGTAATAA ACATCCTTTG CAAATCATTA 120

TTAGATGTTT TATCTTAAGA TAAATTTTTC AAACCAAAGA AAGTTTCAAA CTTTGAACTT 180

TAAATGAAAT ATCAAATTCC TTAATATCAC TTTTAATATT CAAATTAAAT CCATGCA 237 (2) INFORMATION ON SEQ ID NO: 112:

(i) SEQUENCE LABEL:

(A) LENGTH: 212 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (vii) DIRECT ORIGIN: (B) CLON (E): HB45

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112:

GAGAATTCTT CTGTCTAGCA GAATATGAAG AAATCCCGAA ACCAACGAAG GCCTCAAGGA 60

GGTCTGAATA TCCACTTGCA GACTTTACAA ACAGAGTGTT TCCTAACTGC TCTATGAAAA 120

AGAAAGGTTA AACTCTGTGA GTTGAACGCA CACAAACACA AAAGGAGTTT ATGAGAATAC 180

TTACTGTACT AGTTTCTATA AAGAAGATAT TT 212 (2) INFORMATION ABOUT SEQ ID NO: 113:

(i) SEQUENCE LABEL:

(A) LENGTH: 131 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB5

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113: AAGGTAAATT ATCTGAAAGT AAAATTGGAA AATGTGGCTA AAAGGGGGGA AAATAATCAA 60 GTTTATACCT CTAGAGTCGA CCTGCAGGCA TGACAACGTT GGCGTAATCA TGTGTTAGCG. 120

(2) INFORMATION ON SEQ ID NO: 114:

(i) SEQUENCE LABEL:

(A) LENGTH: 218 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB6

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114: AAGACCANTG AGAGACAATG CAGTCGCTGG GACAACCAGA TTGGACCAGG CAGTCAAGGT 60 GGCTTGGAGG CCACAGCGAG AATTAAAATA CACTGAGAGA TTATACTTCA GGATCCTCTA 120 GAGTCGACCT GCAGGCATGC AAGCTTGCTA ATGCATGGTC TCATAGCTGT TTCCTGTGTG 180 AAATTGTTAT TCCTCCTCAC AATTCCACAC AACATACC 218

(2) INFORMATION ON SEQ ID NO: 115: (i) SEQUENCE LABEL: (A) LENGTH: 323 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB7

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115:

GTCAATCAAT TTTATGAGAA TAGCATTGAA TCTATAAATT ACTTTGGGCA GTATGGCCAT 60

TTTCATGATA TTGATTCTTC CTATCCATGA GGATGGAATG TTTTTCCATT TGTTTGTGTC 120

CTCTCTTATT TCCTTGAGCA GTGGTTTGTA GTTCTCCTTG AAGAGGTCTT TCACATCTCT 180

TCTTACCTGT GTTGTTAGGT ATTTATTCTC TTTGTAGCAG TGGCAAATGG GAGTTTATTC 240

ATGATTGGCT CTCTGCTTGT TATTGTTGAG TAAGGAGTAC TGTGATTTGC ACATGCTTGC 300

ATCTAGACTT ATGAGTGCTA CAG 323 (2) INFORMATION ABOUT SEQ ID NO: 116:

(i) SEQUENCE LABEL:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(vii) DIRECT ORIGIN: (B) CLON (E): HB9

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116: ACTGTCACTC AGGCTGGAGG GTAGTT 26

(2) INFORMATION ON SEQ ID NO: 117:

(i) SEQUENCE LABEL:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117:

Cys Gin Arg Lys Leu Lys Glu Leu Glu Val Ala Glu Ser Asp Gly Gin

1 5 10 15

Val Glu Leu Glu Arg Leu Arg Ala Glu Cys

20 25

(2) INFORMATION ON SEQ ID NO: 118: (i) SEQUENCE LABEL:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118:

Cys Glu Leu Glu Arg Leu Arg Ala Glu Ala Gin Gin Leu Arg Lys Glu 1 5 10 15

Glu Arg Ser Trp Glu Gin Lys Leu Glu Cys 20 25

(2) INFORMATION ON SEQ ID NO: 119:

(i) SEQUENCE LABEL:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119:

Cys Glu Ala Gin Val Leu Asp Ala Leu Val Glu Glu Lys Arg Gin Val 1 5 10 15

Ser Glu Glu Lys Arg Gin Lys Glu Ala Cys 20 25

(2) INFORMATION ON SEQ ID NO: 120:

(i) SEQUENCE LABEL:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120: ACCGCCTCTC CCCGCGCG 18

(2) INFORMATION ON SEQ ID NO: 121:

(i) SEQUENCE LABEL:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121: GCGTTGCTGG CGTTTTTCAT A 21

Claims

claims

1. Method for the detection, identification, extraction or removal of human or animal cells with disrupted

Cell cycle control or with the ability for unlimited proliferation or for the formation of tumors, so that the presence of an association from the cdc37 protein with extrachromosomal nucleic acid in cells or tissue fluids is proven.

2. The method according to claim 1, d a d u rc h g e k e n n z e i c h n et that the detection of the associate is carried out with a detectable substance specifically bindable with the associate.

3. The method of claim 2, d a d u rc h g e k e n n z e i c h n et that one as a specifically bindable substance with the

Associate bindable antibody used.

4. The method according to claim 3, which also means that polyclonal antibodies, a monoclonal antibody, a recombinant antibody or a bindable fragment of one of these antibodies is used as the antibody.

5. The method according to claim 4, characterized by the fact that antibodies are used which are directed against the cdc37 protein.

6. The method according to claim 4, d a d u rc h g e k e n n z e i c h n e t that one uses antibodies that are directed against the extrachromosomal nucleic acid.

7. The method according to any one of claims 3 to 6, d a d u rc h g e k e n n z e i c h n e t that a labeled antibody is used.

8. The method according to claim 7, which also means that an antibody is used which is coupled to an enzyme, a radionuclide, a dye or a toxic substance.

9. The method according to claim 2, which also means that a nucleic acid or oligonucleotide hybridizing with the nucleic acid of the associate is used as the bindable substance.

10. The method according to any one of the preceding claims, that the detection of the associate is carried out in a tissue biopsy, a tissue smear, a body fluid or a cell disruption.

11. The method according to claim 10, d a d u rc h g e k e n n z e i c h n et that the detection of the associate in a mitochondria-free

Cytoplasmic fraction or a body fluid is performed.

12. The method according to claim 10, d a d u rc h g e k e n n z e i c h n et that the detection of the associate is carried out on a cell surface.

13. The method according to claim 12, so that the association is detected on cell surfaces with bindable substances which are bound to a solid support and the cells bound thereby are separated from the other cells.

14. Application of the method of claim 13 for the removal of cells with disturbed cell cycle control, with the ability to unlimited proliferation or for tumor formation from blood, bone marrow or other body fluids.

15. The method according to any one of claims 3 to 8 and 10 to 14, d a d u rc h g e k e n n z e i c h n et that Fc-carrying antibodies are used and incubated with the cell-containing substrate until the associate-carrying cells are lysed.

16. cdc37 protein as an associate with extrachromosomal nucleic acid.

17. An associate according to claim 16, that the extrachromosomal nucleic acid represents DNA of a tumor cell or an RNA copy of such DNA.

18. A method for obtaining an association according to one of claims 16 and 17, In addition, the cytoplasmic fraction of permanently divisible human or animal cells is treated by adding detergents and / or by repeated freezing and thawing of the cells, the mitochondria are separated from the treated cytoplasmic fraction and the association is obtained by selective precipitation, electrophoretically or chromatographically .

19. The method according to claim 18, so that the mitochondrial-free cytoplasmic fraction is obtained by adding NaCl to 1.5 M, removing the precipitate formed and precipitating the associate from the resulting supernatant with ethanol or ammonium sulfate.

20. The method according to claim 18, which means that a cytoplasmic fraction containing organelles and / or membranes is incubated with a proteolytic enzyme and then the association is obtained from the lysate.

21. The method according to claim 20, d a d u rc h g e k e n n z e i c h n et that the associate falls from the lysate with ethanol or ammonium sulfate or purified by chromatography.

22. The method according to claim 20 or 21, so that the proteolytic enzyme Proteinase K is incubated at a temperature of 50 to 70 ° C for 0.5 to 3 hours.

23. The method according to any one of claims 20 to 23, dadu rc hgekennzeichn et that the cytoplasmic lysate is extracted with an organic solvent or solvent mixture and the associate is obtained from the organic aqueous interphase.

24. The method according to claim 23, d a d u r c h g e k e n n z e i c h n et that the interphase is extracted with phenol and chloroform.

25. The method of claim 24, d a d u r c h g e k e n n z e i c h n et that the associate from the organic aqueous interphase with

Ethanol falls.

26. Antibodies specific for cdc37 protein nucleic acid heteromers in labeled or unlabeled form.

27. Antibody according to claim 26, which is coupled with an enzyme, a radio nuclide, a dye or a toxic substance.

28. Antibody according to claim 26, which also is coupled with an enzyme, radionuclide, dye or toxin.

29. Pharmaceutical preparation, containing an antibody according to claim 26.

30. A preparation according to claim 29, containing antibodies according to claim 27.

31. A pharmaceutical preparation containing a content of nucleic acid or peptide which bind to the nucleic acid according to claim 17.