Repressor of skeletal muscle differentiation, nucleic acid coding therefor and the use thereof in diagnosis and therapy

Abstract

Polypeptide sequences which play a part in the regulation of skeletal muscle differentiation, and nucleic acids coding therefor, and the use thereof in diagnosis and therapy are disclosed. Possible uses are also indicated for antibodies which are directed against corresponding epitopes of the GRIM1 polypeptide.

Description

[0001] The present invention relates to a novel corepressor whose function has been substantially elucidated. Various possible uses of this corepressor are disclosed.

[0002] The designation GRIM1 which has been chosen for the corepressor of the invention represents the abbreviation of the designation "Global Repressor Involved in Myogenic Differentiation". This name was chosen because the cloned factor plays an important part in particular during skeletal muscle differentiation.

[0003] In recent years a large number of molecules which prove to be responsible for direct transcriptional regulation has been found. Besides the DNA-binding factors (in the narrower sense referred to as transcription factors), there are regulatory molecules which, as coactivators, facilitate gene expression or which, as corepressors, actively bring about transcriptional repression. Cofactors are promiscuous and can be recruited in various combinations by various DNA-binding partners. The alternation between associated coactivator and corepressor complexes is an important regulatory step within cellular differentiation processes.

[0004] For the example of skeletal muscle differentiation, over the course of years a cascade of transcription factors which are necessary for myogenesis has been found. Besides other transcription factors, the bHLH proteins MyoD, myf5, MRF4 and myogenin bring about execution, in a proliferating myoblast precursor cell, of the genetic program which results in a terminally differentiated functional skeletal muscle cell. During the course of this phylogenesis, the cell passes through a cell cycle arrest, and it fuses with other committed muscle cells to give multinuclear myoblasts and expresses skeletal muscle-specific structural and metabolic enzymes.

[0005] The associated cofactors acting within this process have been investigated only in recent years. Positively regulated expression of myogenically specific genes takes place for example through a functional association of MyoD with the coactivator protein p300. Acetylated MyoD has far greater transcriptional activity than unmodified protein.

[0006] On the other hand, there are certain corepressors which actively prevent targeted repression of the expression of muscle-specific genes in proliferating cells. Thus, for example, a functional association between MyoD and the corepressor N-CoR has been described, and in this way revealed a new field of activity for the cofactors which had previously been assigned only to nuclear hormone receptors. In addition, members of the histone deacetylase (HDAC) family have been described as being involved in skeletal muscle differentiation (C2C12 cell culture model) and showing a subcellular relocalization during the first steps of differentiation.

[0007] Association of HDACs with active repression is regarded as one of the basic requirements for negative transcriptional regulation, because the reaction catalyzed by the HDACs is transmitted directly to the chromatin and provides a mechanistic explanation for the observed effects.

[0008] The data shown in this application describe GRIM1 as a novel "bona fide" transcriptional repressor with a novel mechanism of repression. GRIM1 is not associated with an HDAC activity, and the GRIM1-mediated repression cannot be influenced by specific HDAC inhibitors. GRIM1 is able to inhibit directly, via acidic domains in the N and C termini, the acetylation of histone N termini (one of the preconditions for directed transcriptional activation). Repression domains which have not to date been characterized in detail within GRIM1 likewise have high potential for repression of transcription activity. GRIM1 has the potential in transient transfections for dose-dependent repression of both complex and synthetic minimal promoters. Based on these data, the newly cloned factor has been designated GRIM1 (Global Repressor Involved in Myogenic differentiation).

[0009] GRIM1 shows relocalization during skeletal muscle differentiation, although at a distinctly later time than previously described for the HDACs 4, 5 and 7 in connection with MEF2-specific transcription. The available data suggest that the repression potential of GRIM1 must not be switched off until later times during skeletal muscle differentiation so that terminal differentiation of the myotubes is possible. In addition, GRIM1 likewise shows a subcellular change of localization during preadipocyte differentiation.

[0010] One function of GRIM1 is involvement as repressor in regulating the differentiation of skeletal muscle cells. The data show that GRIM1 shows a distinctly different role than the involved factors previously described. Elucidation of the in vivo role of GRIM1 takes place via GRIM1 knockout animals and transgenic mice. In the transgenic animals, GRIM1 mutants will express under skeletal muscle-specific promoters which can no longer reach the nucleus or which can no longer be transported out of the nucleus. The phenotype of the mice which results from the generated atypical localization of GRIM1 will contribute further to understanding the function of GRIM1 within differentiation processes.

[0011] Application of the present invention is particularly in the area of degenerative muscle disorders or in controlling muscle atrophy in the aging man. The therapeutic modification of the function of GRIM1 may in such cases counteract premature muscle degeneration or actively intervene in processes which build up muscle.

[0012] The present invention therefore relates firstly to polypeptides having a sequence of at least 20 consecutive amino acids from the sequence of hsGRIM1 (hs=homo sapiens) having the sequence ID No. 3.

[0013] The polypeptides of the invention preferably have a sequence of at least 40 consecutive amino acids. The polypeptides more preferably have a sequence of at least 80 consecutive amino acids, even more preferably a sequence of at least 120 consecutive amino acids and most preferably the polypeptides of the invention have a sequence of at least 200 consecutive amino acids from Seq. ID No. 3.

[0014] The invention further relates to polypeptides which have a sequence of at least 20 consecutive amino acids from the mmGRIM sequence (mm=mus musculus) having the Seq. ID No. 4.

[0015] These polypeptides more preferably have a sequence of at least 40 consecutive amino acids, even more preferably a sequence of at least 120 consecutive amino acids and very particularly preferably a sequence of at least 200 consecutive amino acids of Seq. ID No. 4.

[0016] The present invention further relates to antibodies which bind specifically to an epitope of a polypeptide of the invention. The antibodies of the invention can be prepared by customary standard methods. Either the polypeptides coding for GRIM1 can be used to immunize suitable laboratory animals such as, for example, rabbits or goats, and it is possible in this way to prepare suitable polyclonal antibodies. The antibodies of the invention may be directed against epitopes such as conformational epitopes, but also against peptides of the invention.

[0017] As an alternative to this it is possible to prepare suitable monoclonal antibodies by methods which have been well known since the publication by Kohler and Milstein in 1975 and belong to the standard repertoire of an average molecular biologist.

[0018] Antibodies of this type can be employed in therapy. However, to do this, it is usually necessary to prepare humanized antibodies, because antibodies having constituents derived from animals may cause unwanted side effects. If the binding regions of a suitable antibody have been sequenced, these sequences can be incorporated into a human basic antibody structure, and an antibody of this type can be used in therapy.

[0019] An alternative area of use of antibodies of this type is in diagnosis. It is perfectly possible for simple polyclonal antibodies, with which qualitative and/or quantitative detection of the presence of GRIM1 polypeptides in the cells or cell compartments of interest is possible, to suffice for this purpose. The average skilled worker can carry out suitable diagnostic methods with antibodies of this type. The present invention further relates to medicaments which comprise a polypeptide of the invention.

[0020] These medicaments are preferably employed for the treatment of disturbances of skeletal muscle differentiation and for the treatment of disturbances of fat cell differentiation.

[0021] The medicaments of the invention may comprise a polypeptide having the complete GRIM1 sequence or a suitable part of the sequence. The medicaments are administered in a suitable form to the patients to be treated. Oral or preferably parenteral pharmaceutical formulations are suitable in this connection.

[0022] The mechanism of action of the medicaments of the invention relies on modulation of the function of GRIM1. During skeletal muscle differentiation, GRIM1 is transported from the nucleus into the cytoplasm. The medicaments of the invention can intervene in this differentiation process by either specifically blocking importation or else blocking exportation. This can be achieved by incorporating the suitable segments of the polypeptides into appropriate formulations so that the target site of interest can be reached.

[0023] It has also been found within the scope of the present invention that GRIM1 is involved in further differentiation processes. GRIM1 shows a subnuclear relocalization during adipocyte differentiation. Involvement of GRIM1 in various other regulatory processes emerges from the findings of the present invention, in particular from the experimental data which show that GRIM1 shows a repression of a wide variety of complexes and synthetic promoters, and expression of GRIM1 is ubiquitous.

[0024] The present invention further relates to a method for identifying substances which influence the biological function of a polypeptide of the invention. This entails the substance to be identified being brought into contact with the polypeptide in a test system. It is possible with this test system to identify substances which interact with the GRIM1 polypeptide and inhibit or enhance the biological function of GRIM1.

[0025] The systems used according to the invention are preferably cellular differentiation systems which make use in particular of myogenic C2C12 cells or rapidly and inducibly differentiating 10T.sub.1/2 MyoD-ER cells. Detection of GRIM1 or the GRIM1 localization is possible for example immunohistologically. Suitable methods are, inter alia, the use of fluorescence-coupled or enzyme-coupled antibodies in a direct coloring step.

[0026] It is also possible to provide suitable cell lines which comprise an appropriate genetic construct with a GRIM1 gene. The substances to be investigated can then be brought into contact with the cells, and the effect of the substance to be investigated can be determined by appropriate comparative tests.

[0027] It is possible in a further embodiment to introduce suitable genetic constructs into laboratory animals and to use a transgenic animal for the test methods.

[0028] It is possible in a further embodiment of the invention to incubate recombinantly produced GRIM1 fragments in cell-free test systems with substances to be tested, and to obtain the desired result of the test by use of a suitable reporter molecule.

[0029] A further aspect of the invention is represented by the cDNA which codes for hsGRIM1 (homo sapiens) and which has a sequence of at least 1200 consecutive nucleotides from Seq. ID No. 1.

[0030] Another aspect of the invention is represented by the cDNA which codes for mmGRIM1 (mus musculus) and which has a sequence of at least 1200 consecutive nucleotides from Seq. ID No. 2.

[0031] The cDNAs of the invention can be employed in transfection vectors which have a cDNA of the invention. A preferred possibility in this connection is an adenoviral vector.

[0032] These transfection vectors are used to produce cells which have been transfected with such a vector.

[0033] In the examples which are described in detail below, besides customary standard methods, the following methods were preferably used, and the underlined sections of the methods are explained in more detail hereinafter:

[0034] Method 1 (Transient and Stable Transfection of Cell Culture Cells):

[0035] The cells were cultured in the media recommended by the ATCC on 15 cm cell culture dishes under sterile conditions. Cells are subcultured in a ratio of 1:5 to 1:20 every two to three days and kept at a maximum of 60% confluence. Old medium is removed, and the cells are washed with PBS buffer and treated with trypsin/EDTA solution (0.25% trypsin, 0.04% EDTA in PBS buffer) until they are detached from the culture dish. The cells are isolated in fresh medium and subcultured on new subculture dishes. TABLE-US-00001 PBS buffer: 137 mM NaCl; 2.7 mM KCl; 8 mM Na.sub.2HPO.sub.4; 1.8 mM KH.sub.2PO.sub.4 Media used: DMEM (Gibco BRL), 100 IU penicillin G/ml, 100 .mu.g/ml streptomycin, 2 mM glutamine, 10% fetal calf serum. As differentiation medium for C2C12 and L6 cells, 10% FCS was replaced by 2% horse serum. 10T1/2 MyoD-ER cells were cultured in hormone-free serum, and 1 .mu.M estradiol, 10 .mu.g/ml insulin and 10 .mu.g/ml transferrin were supplemented as differentiation medium.

[0036] To determine the transcriptional properties of hsGRIM1 proteins in eukaryotic cells, pCMX-hsGRIM1 expression plasmids were cotransfected with luciferase (LUC) reporter plasmids. Expression of the particular hsGRIM1 protein in a pCMX expression plasmid is under the control of the cytomegalovirus promoter. Luciferase expression in the LUC reporter plasmids used is controlled either by the thymidine kinase (TK) promoter, an E1b minimal promoter (TATA box), or by the respectively indicated complex promoters.

[0037] Transfection takes place by the calcium phosphate method or by the DOTAP liposomes method (Roche Diagnostics, product information). Unless otherwise indicated, 10.sup.5 cells/well were seeded in 1 ml of culture medium in 12-well dishes. In the CaPO.sub.4 method, the plasmid DNA to be transfected is coprecipitated with calcium phosphate, and the precipitate is distributed to the cells capable of division. The calcium phosphate precipitate is prepared by preparing the mixture indicated below for duplicates in 12-well dishes: TABLE-US-00002 Reporter plasmid 1 .mu.g hsGRIM1 expression plasmid 20-400 ng Carrier DNA (pUC18) ad 8 .mu.g CaCl.sub.2 solution (2.5 M) 20 .mu.l ddH.sub.2O ad 80 .mu.l

[0038] While agitating (vortexing), 80 .mu.l of 2.times. BES buffer are slowly and uniformly added dropwise to this mixture. The precipitate is incubated at RT for 10 min and distributed uniformly to the cells (80 .mu.l per well) and, after 8 hours, removed by washing with PBS. The cells are incubated in fresh culture medium for a further 18 to 24 h. TABLE-US-00003 BES buffer (2x): 50 mM N,N-bis[2hydroxyethyl]-2- aminoethanesulfonic acid; 280 mM NaCl; 1.5 mM Na.sub.2HPO.sub.4.2H.sub.2O; pH 6.95

[0039] Stable transfection of cell culture cells is carried out with the pcDNA6-His system supplied by Invitrogen. Cells are transfected as described above, and stable integration of the plasmids used is detected by the selection antibiotic blasticidin S. Individual clones are picked, expanded and tested by Western blotting and immunodetection for stable expression.

[0040] Method 2 (Protein-Protein Interactions):

[0041] Yeast two-hybrid interaction assays, mammalian THS, pull-down analyses, immunoprecipitation of cellular proteins followed by immunodetection or in combination with in vitro translated .sup.35S-methionine-labeled interactants, Phast gel system.

[0042] Glutathione S-transferase Pulldown

[0043] A protein is expressed as GST fusion protein in E. coli, immobilized on a GST-binding carrier material, and incubated with the potential interactant which is usually radiolabeled. In the subsequent washing steps, proteins which interact only weakly or not at all are washed off the carrier material or the carrier material-bound GST fusion protein. The amount of associated interactant can be determined by fractionation of the proteins by SDS-PAGE and subsequent autoradiography. To check the specificity of interaction between potential interactant and GST fusion protein, the interaction with GST alone is examined.

[0044] Aliquots of GST-GRIM1 fragments or GST-containing E. coli crude lysate are mixed in each case with 30 .mu.l of glutathione(GSH)-Sepharose and incubated on a bohemian wheel at 4.degree. C. for 1 h. The GSH-Sepharose is pelleted by centrifugation (1' at 2000 rpm) and washed twice in pulldown buffer. The pellet is then resuspended in 500 .mu.l of pulldown buffer and incubated with the in vitro translated, .sup.35S-methionine-labeled potential interactant at 4.degree. C. on the bohemian wheel for 1 h. After washing three times with pulldown buffer, the GSH-Sepharose pellet is boiled in 30 .mu.l of SDS sample buffer, the proteins are fractionated by SDS-PAGE, and the gel is dried and then subjected to autoradiography. TABLE-US-00004 Pulldown buffer: 20 mM HEPES (pH 7.7); 150 mM KCl; 0.1 mM EDTA; 25 mM MgCl.sub.2; 10 mM DTT; 0.15% (v/v) NP40.

[0045] Immunoprecipitation

[0046] If two proteins interact in solution, the stable complex of the two proteins can be precipitated with an antibody which is directed against only one of the two interactants. In this case, the protein-antibody complex is isolated from the solution with the aid of an antibody-binding carrier material (.gamma.-bind G Sepharose, Pharmacia). To investigate interactions between GRIM1 and putative interactants, either native cell extracts or GRIM1-containing cell extracts are mixed with in vitro translated, .sup.35S-methionine-labeled target proteins in 500 .mu.l of IP buffer. 5 .mu.g portions of anti-GRIM1 or nonspecific antibodies (rabbit IgG.sub.1) are mixed with 30 .mu.l of preequilibrated .gamma.-bind G Sepharose, and the mixture is incubated on a bohemian wheel at 4.degree. C. for 60 min. The G-Sepharose is then pelleted at 2000 rpm for 1' and washed three times with 300 .mu.l each time of ice-cold IP buffer and finally the pellet is boiled in 30 .mu.l of SDS sample buffer. The samples were finally separated by SDS-PAGE, and interaction was detected either by Western blotting or, in the case of the proteins translated in vitro in the presence of .sup.35S-methionine, by autoradiography. TABLE-US-00005 IP buffer: 20 mM TrisHCl (pH 8.0), 300 mM NaCl, 5 mM EDTA, 0.3% (v/v) NP40, 0.5 mM Pefablock Cell lysis buffer: 50 mM TrisHCl (pH 8.0), 170 mM NaCl, 0.1% NP40, 50 mM NaF, 2 mM NaO.sub.3V.sub.4, 0.2 mM DTT, 0.1 mM Pefablock, 1 .mu.g/ml aprotinin, 10% glycerol

[0047] Method 3 (Detection of Intracellular Localization):

[0048] Indirect immunofluorescence, cell fractionation followed by immunodetection.

[0049] Indirect Immunofluorescence (IIF)

[0050] Cells are seeded in the desired cell density on autoclaved slides and fixed on the support with 5% paraformaldehyde for 10'. Cell membranes are made permeable for antibodies by incubation in 0.2% TritonX100 for 10'. Before the incubation with the primary antibodies, the cells are blocked in 0.2% gelatin (in PBS) at RT for 1 h. Anti-hsGRIM1 antibodies are diluted 1:500 in 0.2% strength gelatin solution and incubated at RT for 1 h. After washing three times with PBS for 5' each time, the respective fluorochrome-coupled secondary antibody is added 1:2000 in 0.2% gelatin solution and incubated in the dark at RT for 30'. After the washing steps, the nucleus is counterstained with DAPI (1 .mu.g/ml PBS, Roche Diagnostics) and the cells are finally washed twice in 0.1% TritonX100 in order to minimize autofluorescences. The slides are preserved in Fluoromount M (Southern Biotechnology Associates) and analyzed using a fluorescence microscope.

[0051] Cell Fractionation

[0052] Intact nuclei can be isolated and lyzed separately by hypotonic lysis of the cell membrane. Cells are harvested in ice-cold PBS, pelleted and swollen in hypotonic lysis buffer (LB). The cell membrane is permeated by adding NP40 ad 0.1%, and the cytoplasmic constituents are released by gentle shaking and incubation on ice for 15'. Intact nuclei are pelleted by centrifugation at 14 000 rpm for 15', the CP fraction is removed quantitatively, and the protein concentration is determined. Nuclei are lyzed by incubation in nuclear lysis buffer (NLB) while vortexing for 15'. Debris is pelleted and the soluble nuclear preparation is removed. The quality of the preparation is examined by Western blotting and immunodetection with nucleus-specific (e.g. TIF2) and cytoplasmic (e.g. RasGAP) markers. TABLE-US-00006 LB: 10 mM HEPES (pH 7.9), 1.5 mM MgCl.sub.2, 10 mM KCl, 0.5 mM DTT, 0.5 mM Pefablock NLB: 20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM Pefablock, 10% glycerol

[0053] Method 4 (Detection of Enzymatic Activities):

[0054] HDAC assay, INHAT assay, luciferase assays.

[0055] HDAC Assay

[0056] The histone deacetylation reactions which were carried out were carried out within the framework of a kit obtained from Upstate Biotechnology (.sup.3H-labeled histone H3 N-terminus as substrate). Alternative substrates used were histones .sup.14C-acetyl-labeled with p300 or in vivo labeled histones (kindly provided by Dr Martin Gottlicher, Kernforschungszentrum Karlsruhe).

[0057] INHAT Assays

[0058] INHAT assays were carried out as described by Eckner et al. and by Seo et al. (Eckner et al., 1996, Genes and Development 10 (19), pp 2478-2490, Seo et al., 2001, Cell 104 (1), pp 119-130). Histones or histone mixtures are preincubated with the appropriate peptides in a total of 50 .mu.l of HAT buffer, then mixed with bacterially expressed p300 HAT domains and 0.5 .mu.Ci of .sup.14C-acetyl-coenzyme A, and incubated at 30.degree. C. and 1000 rpm for 2 h. Reactions are stopped by adding SDS Laemmli buffer. Proteins are fractionated in denaturing SDS-PAGE, and the gels are incubated in Amplify (NEN) for 1 h. Acetylations are detected in autoradiographies.

[0059] Luciferase Assays

[0060] Transiently transfected cells are washed once with PBS and lyzed by adding 50 .mu.l of reporter lysis buffer (Promega) and further disrupted by freezing at -80.degree. C. Cell lysates are transferred into Eppendorf reaction vessels, and cell detritus is pelleted at 14 000 rpm for 2'. 10 .mu.l portions of the lysates are pipetted into 96-well microtiter plates to determine the luciferase activity. The luciferase assay took place in an EG&G Berthold Microluminomat. 40 .mu.l of luciferase assay buffer are injected into the 10 .mu.l of cell lysate, and the resulting chemiluminescence is determined in a time integral of 10''. The measured values for the luciferase activity are related to the amount of protein present in the cell lysate. For this purpose, 3 .mu.l portions of the lysates are mixed in 96-well microtiter plates with 100 .mu.l of Bradford assay reagent (BioRad) diluted 1:5 with H.sub.2O, and the absorption of the solution is determined at 595 nm. Relative luciferase activities are found from the ratio of the luciferase activity to the protein absorption.

[0061] Luciferase assay buffer: 200 mM tricine; 1.07 mM (MgCO.sub.3).4Mg(OH) .sub.2; 0.1 M MgSO.sub.4, 10 mM EDTA (pH 8.0); 33 mM DTT; 0.5 M ATP; 270 mM acetyl-coenzyme A; 470 mM glowworm luciferin.

[0062] The invention is explained in more detail by the following examples:

EXAMPLE 1

[0063] The cloned human GRIM1 gene includes 2250 base pairs and codes for 749 amino acids. The cloned human GRIM1 sequence obtained by sequencing was deposited for the first time as unknown cDNA in 1999 under the accession number AL050019 (protein DKFZp564C186) as part of a "Random EST sequencing" project of the DKFZ in Heidelberg. A sequence which had been revised further was deposited in March 2001 (Wiemann et al., 2001). The function of the protein DKFZp564c186 has not previously been described. The cDNA of the invention is depicted in FIG. 1. The sequence has Seq. ID No. 1.

EXAMPLE 2

[0064] It was possible to calculate from mouse EST databases by comparative analysis with the hsGRIM1 aa sequence from independent ESTs a full-length protein which has a total of 750 amino acids and a total of 60% identical and more than 85% homologous residues to the known hsGRIM1 sequence. Analysis of mouse cDNA libraries and mouse genomic libraries (Celera) have confirmed the calculated and expected mmGRIM1 aa sequence. It was possible to confirm the putative cDNA by RT-PCR analyses. The full-length cDNA of mmGRIM1 has not yet been described or deposited. The sequence having Seq. ID No. 2 is depicted in FIG. 2.

[0065] Seq. ID No. 2 was obtained by screening mmEST dbs with the hsGRIM1 aa sequence. The resulting data were compared with mm cDNA dbs and mouse genomic dbs and the virtual mmGRIM1 cDNA was constructed. The sequence was confirmed by RT-PCR and genomic sequencing, and by a direct sequence comparison with the mouse genomic database of Celera Inc.

EXAMPLE 3

[0066] Demonstration of homology between hs and mmGRIM1 at the cDNA and at the protein level is shown in FIG. 3. FIG. 3 depicts a comparative demonstration of the homology of the mmGRIM1 and hsGRIM1 amino acid sequences including conserved sequence motifs.

[0067] The amino acid sequence of mmGRIM1 (mouse) is Seq. ID No. 4 and the amino acid sequence of hsGRIM1 (human) is Seq. ID No. 3.

[0068] All the functional and potentially interesting sequence motifs described in detail below are sequence-identically conserved in the mouse and the human protein (nuclear localization sequence NLS, nuclear export signal NES, so-called CoRNR box with which corepressors with their associated transcription factors are able to interact). Further motifs are HMG box-like domains within the C terminus and N terminus of GRIM1 of both species, which may possibly bring about contact with DNA and/or histones. In addition, in total 5 so-called LXXLL or related motifs are present within the primary hsGRIM1 aa sequence, an alpha-helical structure which has been reported to be responsible solely for contact of cofactors with their associated transcription factors. Within the mmGRIM1 primary sequence, only 2 of these motifs are conserved vis-a-vis the human sequence. No functional data are available on these motifs as yet. According to calculations, hsGRIM1 mainly has an alpha-helical structure (Columbia University PHD predict program) and is non-globular (GLOBE of the Columbia University PredictProtein server). GRIM1 is localized in the nuclei of proliferating cells, while GRIM1 is relocated from the nucleus to the cytoplasm within cellular differentiation systems (e.g. in C2C12, L6, 10T1/2 and 3T3-L1 cells).

EXAMPLE 4

[0069] For the purposes of the present invention, only hsGRIM1 was used on the basis of cDNA. However, it is assumed that corresponding results will be obtained on use of mouse cDNA. Various constructs were produced by deletion- and sequence-specific point mutagenesis from the human GRIM1 for detailed functional characterization and are compiled in the following tables. TABLE-US-00007 TABLE 1 hsGRIM1 point mutants used Name aa positions Constructs used .DELTA.NES I L309K, L312A -. FLAG, His, Xpress, GFP .DELTA.NESII L309R, L312A, L315Q, -. FLAG, His, Xpress, GFP L317A .DELTA.NLS I K649A, R650L, R651E, -. FLAG, His, Xpress, GFP K652A .DELTA.NLS II K649A, R650L, R651E, -. FLAG, His, Xpress, GFP K652A, R661D, K662I, K665E .DELTA.CoRNR I439A, I440S, I443A -. FLAG

[0070] TABLE-US-00008 TABLE 2 hsGRIM1 deletion mutants used Name aa positions Constructs used GRIM1 1-749 jcdc5 mut A 3-609 Gal4-DBD, FLAG, GFP, GST, His mut B 3-487 Gal4-DBD, GST, His mut C 3-423 Gal4-DBD, GST, His mut D 3-245 Gal4-DBD, GST, His mut E 3-147 Gal4-DBD, GST, His mut F 145-749 Gal4-DBD, GST, His mut G 246-749 Gal4-DBD, GST, His mut H 485-749 Gal4-DBD, GST, His mut I 609-749 Gal4-DBD, FLAG, GFP, GST, His mut J 145-609 Gal4-DBD, His mut M 468-749 GST mut N 246-609 Gal4-DBD mut O 246-485 Gal4-DBD mut P 485-609 Gal4-DBD mut Q 246-423 Gal4-DBD mut R 423-485 Gal4-DBD, GST mut S 145-245 Gal4-DBD 246-334 246-334 FLAG, GFP .DELTA.246-334 3-245 FLAG, GFP 335-749

[0071] Many of the mutants used carry specific tags such as the Gal4 DNA-binding domain of the yeast GAL4 transcription factor [Gal4-DBD], green-fluorescent protein [GFP], herpes simplex virus transactivator protein 16 [VP16], the antibody-specific signal sequences FLAG tag [FLAG], Xpress tag [XPRESS], c-myc tag [MYC], nickel-binding epitope His tag [His], maltose-binding epitopes [MBP], glutathione-binding portions of glutathione S-transferase [GST]. The use of proteins modified in this way is identified through use of the epitope name in the GRIM1 construct employed. The function of the individual regions of the polypeptides of the invention was investigated by these experiments.

EXAMPLE 5

[0072] Database analyses have shown that hsGRIM1-homologous proteins are to be found in a large number of species. Homologous sequences are to be found in Shizosaccharomyces pombe, Saccharomyces cerevisiae, Rattus spec., Bos bovis, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Danio rerio and also in Arabidopsis thaliana. A summarizing overview is obtained from the databases UNIGENE (National Center for Biotechnological Information, NCBI) and of the PredictProtein server of Columbia University and is shown in Table 3 below:

[0073] Selected Model Organisms in a Protein-Similarity Representation

[0074] Organism, protein, percent identity and length of the compared region. TABLE-US-00009 H. sapiens: sp: Q9Y3T9 - YU20_HUMAN HYPOTHETICAL 84.9 KDA PROTEIN DKFZP564C186 100% over 753 amino acids (according to the invention) M. musculus: sp: Q9WV70 - YU20 MOUSE HYPOTHETICAL PROTEIN 73% over 267 Aa R. norvegicus: sp:- - |Segment 1 of 2| COLLAGEN ALPHA 1 (I) CHAIN 29% over 328 AA A. thaliana: sp: Q9ZPV5 - YU20 ARATH HYPOTHETICAL PROTEIN T30D6.27 IN CHROMOSOME II 27% over 722 AA C. elegans: sp: O17580 - YTB2_CAEEL HYPOTHETICAL 82.0 KDA PROTEIN C07E3.2 IN CHROMOSOME II 27% over 656 AA D. melanogaster: sp: Q9VIF0 - YU20 DROME HYPOTHETICAL PROTEIN CG9246 37% over 681 AA S. cerevisiae: sp. P39744 - YO26_YEAST HYPOTHETICAL 81.6 KDA PROTEIN IN DEDI-RETI INTERGENIC REGION 29% over 674 AA

[0075] Table 3: Representation of the UNIGENE maximally homologous proteins from mouse, rat, Arabidopsis, threadworm, fruit fly and baker's yeast. The Swissprot accession numbers, UNIGENE accession numbers, investigated region and maximum homology are indicated (mouse Q9WV70 is not the full-length clone; compare with the described mouse clone described under Seq. ID No. 4).

[0076] FIG. 4 shows a comparison of the amino acids and establishment of a consensus sequence.

EXAMPLE 6

[0077] Genomic Organization

[0078] The characteristics of the genomic sequences of mmGRIM1 and hsGRIM1 are compared in Table 4 below. The genomic sequence of both species, including the detailed exon-intron boundaries, is indicated in FIGS. 5 and 6. TABLE-US-00010 TABLE 4 Genomic organization comparison of mm (mouse) versus hsGRIM1 (human). hsGRIM1 mmGRIM1 Number of exons 19 19 Location Chr. 1p33.36 Chr. 4p Pseudogenes Chr. 2 NT_022127.3 not known Chr. 2 NT_022140.3 Chr. 11 STS WI-15347 not available Accession # GenBank AF276983.1 Accession to the revised version under complete sequence is NCBI not available. NT_021903 Individual exons are UniGene Hs.134200 present in EST dbs. Swissprot YU_20 human ENSG00000077716 LokusLink 26155 GenBank AL050019.1 OMIM entries No muscle- or fat-related No muscle- or fat- phenotype. related phenotype. 1p33 is a highly mutated locus and is associated with breast, prostate and brain tumors Spliced isoforms nothing known nothing known fragmentary ESTs present in various dbs. Northern analyses show only one isoform (exception: heart, 3 isoforms). Size of the 14.24 kb (Ensembl) 12.36 kb (Celera) locus Quality of the Chromosome 1 build 27 no Ordered up to 5.5 kb contigs longer has any gaps; 5'- and 3'-flanking, ordered structure only a small gap between exons 2 and 3 (see annex 2).

[0079] FIG. 5 depicts the genomic sequence of hsGRIM1 (NCBI Human Genome Server, Chromosome 1 map view, build 27, December 2001). Exons have a shaded background, and the putative poly-A site is underlined. The sequence corresponds to Seq. ID No. 5.

[0080] FIG. 6 depicts the genomic sequence of mmGRIM1 including 5.5 kb flanking sequences (established from the public NCBI mouse genome db and the commercial Celera mouse genome gb). The exons have a shaded background, and intronic sequences are depicted in pale. Exon/intron junctions are depicted in bold. The putative poly-A site and the putative transcription start are underlined. As yet incompletely assembled sequence fragments are depicted by "N". The genomic mouse sequence has the Seq. ID No. 6.

EXAMPLE 7

[0081] Expression of Recombinant GRIM1 Protein

[0082] Many different strategies and expression systems were tested for expression of recombinant hsGRIM1 protein, but only expression of short protein fragments either in the HiS.sub.6.times. or in the GST context was successful. The following lists briefly summarize the results and the methods used.

[0083] a) Expression Systems and Brief Description of the Characteristics for Expression of Full-Length hsGRIM1

[0084] Bacterial systems: all the systems used employ Escherichia coli safety strains which were purchased from Stratagene.

[0085] BL21 (DE3)lysGOLD His-fused: Expression in the pRSET system or in a modified pRSET vector with unique hexahistidine tag, purification on a nickel affinity matrix (TALON, Clontech). Coexpression with GroESL and hspHJK chaperones.

[0086] BL21(DE3)lysGOLD GST-fused: Expression in the pGEX4 or 6 systems from Pharmacia, purification on GSH affinity columns, expression inducible by IPTG. Coexpression with GroESL and hspHJK chaperones.

[0087] BL21(DE3)lysGOLD NusA-fused: Increase in solubility of the target protein via the NusA content, purification by GPC (Stratagene, Novagen)

[0088] BL21(DE3)lysGOLD MBP-fused: periplasmic expression, and thus reduced cytotoxicity (Stratagene, New England Biolabs pMAL system)

[0089] BL21(DE3)lysRIL: Codon-optimized BL21 strain (Stratagene). Both hexahistidine- and GST-fused protein expression.

[0090] ABLE C: Safety strain which downregulates endogenous plasmid copy numbers by a factor of 4.times. (Stratagene). Both hexahistidine- and GST-fused protein expression.

[0091] ABLE K: Safety strain which downregulates endogenous plasmid copy numbers by a factor of 10.times. (Stratagene). Both hexahistidine- and GST-fused protein expression.

[0092] Plant Systems:

[0093] Physcomitrella patens moss: stable integration of a CaMV-driven expression plasmid for GST-hsGRIM1 (pRT99/35S-GST-hsGRIM1) via non-homologous recombination, triple selection via two different selection antibiotics and multiple seedings (in cooperation with Prof. Ralf Reski, Plant Biotechnology Institute of Freiburg University).

[0094] Cellular Systems:

[0095] Expression of GST fusion protein in eukaryotic cell culture cells under the control of a constitutively active CMV promoter. [0096] b) Expression of hsGRIM1 peptide Fragments

[0097] Short hsGRIM1 protein fragments were obtained both as hexahistidine- and as GST-fusion proteins only in BL21(DE3)lysGOLD without coexpression of chaperones. The following table summarizes the expression results obtained; unlisted fragments (compare table of the hsGRIM1 constructs used) could not be expressed: TABLE-US-00011 TABLE 5 Bacterially expressed hsGRIM1 fragments His-E (aa 3-147) +++ His-I (aa 609-749) +++ His-H (aa 485-749) ++ GST-E (aa 3-147) +++ GST-I (aa 609-749) +++ GST-H (aa 485-749) ++ GST-M (aa 468-749) + GST-R (aa 423-485) ++ +++ very soluble, readily inducible ++ readily inducible, low yield + solubility poor in some cases, low yield

EXAMPLE 8

[0098] Generation and Description of the .alpha.-GRIM1 Antibodies Used

[0099] To produce hsGRIM1-specific antibodies, two hexahistidine-fused hsGRIM1 protein fragments were expressed in E. coli, purified and used to immunize rabbits. Amino acids 3-147 and 609-749 of hsGRIM1 served as epitopes for the antibody. TABLE-US-00012 TABLE 6 N-terminal His.sub.6x-hsGRIM1 peptide (aa 3-147) which was used to immuniz rabbits, peptide "E" (S q. ID No. 7). The antibodies 2719 and 2720 resulted therefrom. N terminus MHHHHHHGMASEFGSAGSRKRRLAFLTVDEFLASGFDSESESESENSPQAETREAREAAPEPDKPGGS PSASRRKGRASEHKDQLSRLKDRDPEFYKFLQENDQSLLNFSDSDSSEEEEGPFHSLFDVLSEASSEE DGAEEGEDGDRVPRGLKGDDNSVPSTI* C-terminus

[0100] TABLE-US-00013 TABLE 7 C-terminal His.sub.6x-hsGRIM1 peptide (aa 609-749) which was used to immunize rabbits, peptide "I" (Seq. ID No. 8). The antibodies 2910 and 2911 resulted therefrom. N terminus MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSEEGTPLTLYYSHWRKLRDREIQEEISGKERLE (Seq. DLNFFEIKRRKMADRKDEDRKQFKDLFDLNSSEEDDTEGFSERGILRPLSTRHGVEDDREDEEEGEED (Seq. SSNSEDGDPDAEAGLAFGELQQLAQGPEDELEDLQLSEDD* C-terminus

[0101] These regions correspond to the putative INHAT domains of hsGRIM1 and are distinguished in particular by acidic (polyE/polyD clusters). The corresponding regions of the hsGRIM1 cDNA sequence were generated by restriction digestion with endogenous EcoRI (aa147) and XmaI (aa609) cleavage sites, and the products were cloned into the vector pRSET-B (Invitrogen) (aa 609-749) or pRSET-N+/H based on the PRSET-B with the difference that further tags have been deleted and the protein is expressed only with hexahistidine tag) (aa 3-147). The hexahistidine-fused hsGRIM1 protein fragments (aa 3-147 "E", aa 609-749 "I ") were overexpressed in E. coli BL21(DE3)lysGOLD and, after disruption of the bacteria, purified by TALON affinity chromatography. After isolation and purification from E. coli, both polypeptides had an estimated purity of about 80-90%. Rabbits were immunized with the polypeptides, and the raised antiserum was purified. The purified monospecific antibodies against aa 3-147 (2719, 2720) and against aa 609-749 (2910 and 2911) were functionally characterized.

[0102] The antibodies obtained in this way were employed in various immunological test methods that made it possible to detect expression of GRIM1. The two antibodies directed against the N terminus show exclusively nuclear localization of GRIM1 in subnuclear compartments which are generally referred to as "speckles". The number and size of the nuclear speckles varies from cell line to cell line, only the exclusively nuclear localization of GRIM1 being common to proliferating cells. The two antibodies directed against the C terminus recognize exclusively nuclear GRIM1 in proliferating cells, but not in speckles, rather in a pan-nuclear localization in the entire nucleoplasm.

EXAMPLE 9

[0103] Expression of GRIM1 mRNA and Protein

[0104] cDNA/mRNA Expression of hsGRIM1 and mmGRIM1

[0105] hsGRIM1 cDNA expression is ubiquitous according to the report of the Unigene database (UniGene Cluster Hs. 134200, Homo sapiens DKFZP564C186/DKFZP564C186 protein with the cDNA library 2334BT0407 used). Expression was detected:

[0106] Cervix, Pancreas, amnion_normal, bone,bone marrow, brain, breast, cervix, colon, colon_est, colon_ins, denis_drash, ear, epid_tumor, esophagus, cyc, genitourinary tract, germ cell, head_neck, heart, kidney, kidney_tumor, liver, lung, lung_normal, lung_tumor, lymph, muscle, nervous_normal, ovary, pancreas, placenta, pool, pooled, pooled brain, lung, testis, pooled colon, kidney, stomach, pooled lung and spleen, prostate, prostate_tumor, salivary gland, skin, small intestine, stomach, testis, testis-cell line, thymus pooled tonsil, uterus, uterus_tumor, whole embryo

[0107] The expression pattern of hsGRIM1 was visualized by Northern blot analysis (CLONTECH hsMTN I and hsMTN II, probe: both full-length hGRIM1 cDNA and N- and C-terminal fragments). The hsGRIM1 transcript size is about 3.3 kb (consistent with the deposited full-length mRNA under Acc. # AL 050019):

[0108] heart (1), brain (2), placenta (3), lung (4), liver (5), skeletal muscle (6), kidney (7), pancreas (8), spleen (9), thymus (10), prostate (11), testis (12, ovaries (13), small bowel (14), large bowel (15), peripheral lympocytes (15

[0109] The expression of mmGRIM1 in various tissues of the adult mouse (4-6 weeks) was determined by RT-PCR. The following tissues and organs were classified as mmGRIM1-positive:

[0110] breast, breast of suckling animals, placenta, testis, ovaries, fat, skin, bone, cartilage, spleen, lung, adrenergic gland, kidney, liver, small bowel, stomach, pituitary, thymus, tongue, skeletal muscle, heart, eye, spinal cord, cerebellum, medulla, hypothalamus, cerebral cortex, whole brain

[0111] Expression of mmGRIM1 mRNA in embryonic development was determined via RT-PCR with complete embryo mRNA. A distinct mmGRIM1 signal is detected at all times, and no large change in mmGRIM1 mRNA expression during development is observable.

[0112] Table 8 depicts a UNIGENE database analysis on EST expression selected in homology to mm/hsGRIM1 protein. The accession numbers of the IMAGE clone or of the sequences which are unknown in some cases, and the tissue type from which the cDNAs were isolated, are indicated. GRIM1 appears to be expressed ubiquitously also at the protein level. In total, 308 ESTs were found, and those which show an unambiguous tissue assignment are listed below: TABLE-US-00014 TABLE 8 BI070012 cervix R50545 breast BI870713 liver BG335512 placenta H26449 breast BE253493 eye BE903516 placenta AI674272 uterus BF345524 brain BG699598 brain AL382825 esopitagus BE070314 breast BI828140 brain R10573 pool AW375280 colon BG163998 skin R10574 pool BG388020 prostate BG760638 skin AW058011 thymus pooled BG179766 prostate BE894669 skin AI660736 thymus pooled BE583040 brain BG767001 skin AI765077 kidney BG764320 skin BF207343 brain AI677761 pancreas BE263417 lung BG337689 uterus AI266034 lung BE266643 lung BG394462 eye AI948457 kidney BE273581 kidney BF212645 bone marrow AA281338 tonsil BG674251 skin BE278111 placenta AA213719 tonsil BE514600 ovary BF984755 small intestine AI93564 lung BE778203 eye BG332620 lung AI85822 lung BI258705 placenta BG767665 skin AI660558 colon BI006483 kidney_tumor BG764689 skin AI681255 lung AW675721 cervix BG390277 prostate AI272873 colon BE874785 colon BI489732 pooled brain AI935918 pancreas BE545263 placenta lung testis AI799833 prostate BE746375 ovary BG776759 lung AI655977 germ cell AI989734 colon BG029437 breast AI633340 germ cell AW373221 colon BG699275 brain AI971531 germ cell AW365265 head_neck BE883535 uterus AA026277 heart BE746427 ovary BI459847 testis cell line AA026278 heart AW575244 colon BI461880 testis cell line AI400930 prostate BI113120 muscle BG830390 pancreas AI420991 prostate AI471616 kidney BI760633 pooled colon AI682274 prostate AL529521 brain kidney AI744703 colon BE779713 eye stomach U810701 prostate BI356811 placenta BE732072 placenta BE869684 colon AW237492 kidney AI254710 colon BE900176 placenta AW179094 stomach AI611186 uterus AW572690 lymph AW512856 uterus AI990470 thymus pooled BF868966 lung_tumor BE793414 lung AW028378 stomach BF346952 brain AW366003 head_neck R73178 breast AW673372 cervix AW104543 pool R93124 breast BI085040 salivary gland BE257381 eye AI678640 stomach BE515327 uterus BI222365 placenta R50640 breast BE410145 placenta BE591125 uterus BE746352 ovary AW365268 head_neck AW083553 colon AW366001 head_neck BG328934 kidney BE783940 eye BF874087 lung_tumor BE892015 skin BG327104 kidney AA427964 whole embryo BE543377 placenta BE754506 normal BE740821 ovary AW674628 placenta AW071075 breast AL526148 brain AW551591 colon T16893 brain BF341363 brain BF883105 lung_tumor BF773461 epid tumor BE262764 brain AW151790 stomach BC284070 prostate BH15932 lung BE391861 uterus AW366039 head_neck BE391378 uterus BI254811 placenta BG436850 lung BG576365 breast BE301196 kidney BE26490 lung BI259365 placenta BE296203 muscle BG829981 pancreas BG330302 lung BE278652 placenta AA315693 ATCC 111476 BI772656 pooled lung BF797114 lymph T19249 testis and spleen BI857618 breast AW364797 denis_drash BG761400 skin BG256028 prostate BF223807 germ cell BG681166 skin AL526094 brain AA355948 ATCC 158121 AW365999 head_neck AW364751 denis_drash BI007523 kidney_tumor BE841836 stomach BF765941 colon_est AW351978 head_neck BE379093 uterus AW364764 denis drash AW582985 Pancreas BF768551 epid_tumor BE070313 breast BE727721 skin BE898791 ovary BF026345 skin AH366053 head_neck BE760929 brain AW364775 denis drash BF509301 muscle BI857073 breast AW673544 cervix BF830322 kidney tumor BF883094 lung_tumor AW375245 colon BG110646 bone BE882355 uterus AW375242 colon BI084623 salivary gland AW473561 uterus BF907757 uterus_tumor BI033036 nervous BG421457 kidney BG762371 skin normal BE901462 placenta AI632285 germ cell A356032 head neck AW351639 colon AA427857 whole embryo BE593548 germ cell BI859530 breast AW351647 colon BI860984 breast BG91171 brain BE332104 breast AW366056 head_neck AW375257 colon AW351638 colon AW564784 denis drash AA502771 colon AA448947 whole embryo BG25338 liver BE742897 ovary BE795961 lung BE858431 prostate tumor AW020647 ear T16894 brain BF955772 nervous AI672826 colon AW366050 head_neck normal BG330696 lung BE383442 brain AW366040 head neck BG421512 kidney BF817292 colon_mus BG423930 kidney BE536984 cervix AI346533 colon AA449675 whole embryo AW375211 colon BF000005 colon BF913827 uterus_tumor BF732410 ovary BI160700 pancreas AW074084 BE273057 kidney BG177975 prostate AW365264 head_neck BI857814 breast AW528624 Cervix AW365167 head_neck BI033534 nervous BG030810 breast AW375277 colon normal BG110635 bone AW575314 colon AW366007 head_neck AL563470 brain AW594248 germ cell AW375312 colon AW365161 head neck BE389202 uterus BI762663 pooled colon BI007446 kidney_tumor U636573 germ cell kidney BF338161 brain BE746451 ovary stomach AW387399 stomach BF868930 lung_tumor AW272743 colon AW601835 breast BE729701 skin AW675196 placenta AW601811 breast AW806654 stomach AW179097 stomach BF846203 lung_normal BG281880 skin BF338242 brain BG254482 prostate BI094540 cervix BE896763 skin

EXAMPLE 10

[0113] Protein Expression

[0114] Since various experiments cannot be carried out directly on humans, cell culture and animal experiments which, however, also have validity for the human situation were carried out.

[0115] a) The expression of mmGRIM1 protein in primary cells and tissues is indicated below (all the listed cells are classified as GRIM1-positive in a Western blot (86 kD), and for cells the expression was also checked in IIF). Signals are obtained with both antibodies (N- and C-terminal epitope):

[0116] Mouse embryonic fibroblasts

[0117] Human cardiac tissue (DCM patients and normal heart)

[0118] Mouse smooth muscle cells (differentiated, undifferentiated and proliferating) [0119] Mouse organs: testis, ovaries, spleen, thymus and in a small amount also in prostate and stomach (depicted in the following figure), brain, lung, liver.

[0120] b) It was possible to detect expression of mmGRIM1 protein by in situ immunohistochemistry on sagittal sections of E10.5 mouse embryos. mmGRIM1 is expressed in epithelial structures, in the dermomyotome of the rear extremities, in the inner epithelial regions of the neural tube and in other cell populations which are not determined in detail. Concentrated expression is detectable only in the dermomyotome; only a few mmGRIM1-positive cells are detectable in the remaining areas. Ubiquitous expression of GRIM1 was found in mRNA and EST analyses. It was possible to show by in situ immunohistochemical analyses of E10.5 mouse embryos that although there is mmGRIM1 expression in every tissue type, it is far from being in every cell, but rather in specialized cell populations. mmGRIM1 is not expressed in every cell type but distributed over the entire organism in specialized cell types.

[0121] c) The expression of rnGRIM1 (rn=rat) in the rat brain was detected by in situ immunohistochemistry in sagittal sections through the brain of a Sprague-Dawley rat (Rattus norvegicus ssp).

[0122] rnGRIM1 is expressed in Purkinje cells of the cerebellum (nuclear and in some cases also cytoplasmic; strong immunoreactivity in 1-2 subnuclear structures), in bipolar cells of the mitral layer of the olfactory lobe (nuclear and in some cases also cytoplasmic; strong immunoreactivity in 1-2 subnuclear structures), in all cells of the choroid plexus (nuclear and in some cases also cytoplasmic; strong immunoreactivity in 2 subnuclear structures in most cases), in cells which have not yet been determined in detail in the whole region of the cortex (nuclear and in some cases also cytoplasmic; strong immunoreactivity in 1-2 subnuclear structures). rnGRIM1 is not expressed in every cell type, but distributed over the entire brain area in specialized cell types.

[0123] d) Expression of GRIM1 protein in cell culture cells. All the stated lines are classified as GRIM1-positive in Western blot analyses (species-inclusive size of about 86 kD) and by indirect immunofluorescence (IIF). Both assays were carried out with all the available .alpha.-GRIM1 antibodies. In the proliferative state, GRIM1 is detectable in every cell line in so-called speckle structures (as also described in the literature for many corepressors): TABLE-US-00015 Mouse: NIH3T3 fibroblasts, NIH3T3-L1 preadipocytes, NIH3T3-L1 adipocytes, C2C12 undifferentiated skeletal muscle cells, C2C12 differentiated skeletal muscle cells, N2a neural cells, P19 teratocarcinoma cells, 10 T.sub.1/2 fibroblasts, Monc-1 neural crest cells Human: HL1 myocardial cells, 293 embryonic kidney cells, LnCaP prostate carcinoma cells, PC3 prostate carcinoma cells Monkey: COS-7 and CV1 kidney cell lines Rat: L6 skeletal muscle cells Hamster: BHK fetal kidney cells

[0124] e) Drosophila melanogaster GRIM1

[0125] Expression of an hsGRIM1-like protein in Drosophila melanogaster was investigated by Western blotting and whole-fly in situ hybridizations.

[0126] No specific signal was detected with the anti-hsGRIM1-specific antibodies (resolution from 36 to 210 kD) either in whole-larva lysate or in whole-cell extracts from fly heads. Likewise, no specific dmGRIM1 (dm=Drosophila melanogaster) signal was detectable with the available GRIM1 antibodies in in situ hybridizations with various larval stages.

EXAMPLE 11

[0127] hGRIM1 Interactants

[0128] In the protein-protein interaction studies carried out to date, the following potential mm/hsGRIM1 interactants were investigated for direct or indirect interaction with the following methods: TABLE-US-00016 METHOD for detecting protein-protein interaction Symbol Co-IP from cell extracts of transfected cells tfIP Co-IP from cell extracts of untransfected cells utfIP Co-IP with in vitro translated proteins ivIP Yeast THS yTHS Mammalian THS mTHS Pulldown analyses (direct interaction) PD Transcriptional studies (transient transfections) tT

[0129] The underlined factors showed an interaction with hs/mm/maGRIM1 and are described separately at the end of the list (ma=mesocricetus aureus, BHK cells).

[0130] Nuclear Hormone Receptors

[0131] hsAR (PD, utfIP, tfIP, YTHS, tT), hsER.alpha. (utfIP, tfIP), hsER.beta., rnGR (tT, tfIP), hsMR (tT), hsPR (tT), PPAR.gamma. (tT), hsRAR.alpha. (tfIP), rnTR.alpha. (tT), hsTR.beta.-2 (tT), mmERR1 (tfIP, tT), mmGCNF (PD, ivIP, tfIP, tT), hsRevErbA (tT), mmRVR (tT, tfIP), mmSF1 (tT).

[0132] Transcription Factors

[0133] Myogenin (ivIP, tT), MEF2A (ivIP), MEF2B (ivIP), MEF2C (ivIP, tT), eHand (ivIP), dHand (ivIP), E47 (ivIP), E12 (ivIP), GATA4 (ivIP), MyoD (ivIP, tT), SEF (ivIP, tT), MRF4 (ivIP, tT), Myf5 (ivIP), Sp1 (tT), CBF (tT).

[0134] Transcriptional Corepressors.

[0135] N-COR (ivIP, tfIP, utfIP, PD), Sin3A (ivIP, tfIP, utfIP), Sin3B (tfIP, utfIP), SuN-CoR (ivIP, tT), SMRT (ivIP, tfIP, utfIP), HDAC1 (tfIP, utfIp), HDAC2 (tfIP, utfIP), RIP140 (ivIP).

[0136] Transcriptional Coactivators:

[0137] P300 (tT), FHL2 (ivIP, PD), FHL3 (PD), TIF2 (ivIP), RIP140 (ivIP)

[0138] Further factors: Histones H3, H4, H2A, H2B (PD)

[0139] It has been possible to date to show a strong direct interaction between hsGRIM1/mmGRIM1 only with the androgen receptor (hsAR) (detected by tfIP in both directions, utflP in both directions, YTHS, PD and tT). The interaction with the androgen receptor is ligand-independent and cannot be influenced by the presence of antagonists (Casodex, hydroxyflutamide, cyproterone acetate). GRIM1 interacts both with the "normal" AR having 17 glutamines and with AR isoforms which are associated with Kennedy's disease and comprise polyglutamine-rich insertions (17, 44 and 77 Q), likewise ligand-independently. The PD analyses were carried out with the GRIM1 fragments GST-I/E/H/M, and all the tested fragments apart from GRIM1 "E" (aa 3-147) interacted with the AR, in the sequence M.gtoreq.H>>I. The physiological relevance of the AR-GRIM1 interaction is described in detail in a separate section.

[0140] Weak interactions were detected between hsGRIM1 and the nuclear orphan receptors Germ Cell Nuclear Factor (mmGCNF) and Estrogen receptor Related Receptor 1 (mmERR1) (only ivIP, tfIP and PD), and a weak interaction with the estrogen receptor .alpha. (hsER.alpha.; interaction was detected only in tfIP and proved to be ligand-independent both in the presence of agonists (estradiol), of antagonists (raloxifene, tamoxifen, ICI) and in the absence of a ligand). The physiological relevance, in vivo colocalization or in vivo interaction is very substantially unclear. It was not possible to show any direct association between GRIM1 and mmGCNF, mmERR1 or hsER.alpha. in transient transfections.

[0141] GRIM1 shows a strong interaction with the core histones H4, H2B, H2A and H3 in GST pulldown analyses. Recombinantly expressed GST-GRIM1 fusion proteins (GST-aa 3-147 and GST-aa 609-749) show a marked interaction with all four core histones, providing further support for the observed INHAT activity of the N- and C-terminal GRIM1 domains.

[0142] Potential mm/hsGRIM1 interactants can be identified by conventional methods. These methods include yeast two-hybrid screen with various cDNA libraries and direct protein biochemical methods such as Co-IP of GRIM1 and associated proteins, fractionation by 2-dimensional gel electrophoresis, mass spectrometric analysis and microspray sequencing of the potentially interacting proteins from various cell culture cell lines (C2C12 differentiated, C2C12 undifferentiated, 3T3L1 differentiated, 3T3L1 undifferentiated, BHK and many more).

EXAMPLE 12

[0143] Physiological Function of GRIML: Analysis of Repression Potential of hsGRIM1 in Transient Transfections

[0144] All the described data were obtained with the hsGRIM1 clone. GRIM1 is a potent transcriptional repressor when used as Gal4-DBD fusion protein in transient transfections. Gal-GRIM1 represses expression of reporter genes both of Gal4-RE-controlled complex and synthetic minimal reporter constructs. The repression potential of GRIM1 is distinctly greater for example compared with the corepressor Gal-SuN-CoR. In these experiments, the appropriate corepressors are transiently expressed as heterologous Ga14-DBD fusion proteins in cells. The repression capacity on various synthetic promoter-reporter constructs compared with equal quantities of transfected wild-type Gal4-DBD is determined by luciferase reporter gene expression.

[0145] Table 9: Gal-GRIM1 is a stronger repressor than Gal-SuN-COR. 293 cells were cotransfected with G5ElbTATA-LUC reporter and the stated amounts of corepressors. The same effect can be observed on testing complex promoters (Gal4 .sub.3.times.-tk-LUC).

[0146] Gal-GRIM1 is equally strong as a repressor as a large number of described corepressors such as, for example, Gal-HDAC1-10, Gal-CBF1, Gal-Alien and represses only insignificantly weaker than Gal-N-CoR-RD or Gal-SMRT-RD. The repression potential of Gal-GRIM1 was investigated in conjunction with the complex Gal4.sub.3.times.-tk-LUC and Gal-MMTV-LUC promoter/reporter constructs and in conjunction with the minimal promoter/reporter constructs Gal.sub.5.times.-E1b-TATA-box and Gal.sub.3.times.-.beta.globin-TATA-LUC. In addition, the repression capacity of Gal-GRIM1 was investigated on an autonomous VP16 transactivation domain bound to DNA by means of LexA-DBD, via a LexA.sub.8.times.-Gal.sub.5.times.-LUC reporter (provided by Dr S. Khochbhin). The data obtained on Gal-GRIM1 has been tested experimentally in the following cell lines: A7r5, C2C12, CV1, BHK, 293, N2a, COS-7. The following figure shows representative results for BHK and C2C12 cells.

[0147] The Gal-GRIM1 repression is not impaired by addition of the specific histone deacetylase (HDAC) inhibitors trichostatin A (TSA, 300 nM) and of sodium butyrate (Na-But, 5 mM), suggesting that the mechanism of repression is fundamentally different from that described for other corepressors. Comparable cases have been described for other repressors, although no explanation of the method is described. In addition, the described factors show no involvement in skeletal muscle differentiation or adipogenesis. Data on the independence of HDACs in transient transfections are depicted in the following figure for the example of BHK cells. The transcriptional activity of Gal-GRIM1 is uninfluenced by TSA in any cell line already tested.

[0148] To determine the GRIM1 domains involved in repression, a total of 11 deletion mutants of GRIM1 were constructed and all were tested in the Gal4-DBD context for their repression potential in all the reporter systems already described. All the GRIM1 domains used show transcriptional repression in the range from 2.times. (Gal-E and Gal-I) to 15-20.times. (full-length Gal-GRIM1). All the tested deletion mutants proved to be insensitive to HDAC inhibitors.

[0149] In summary, it was possible to show that GRIM1 has at least 5 independent repression domains, with the 3 main repression domains being located in the hydrophobic core region of the protein (mutQ, mutR, mutp). However, every analyzed deletion mutant shows a repression capacity which is significantly above the background in the Gal4-DBD context. TABLE-US-00017 TABLE 12 Summarizing list of all hsGRIM1 deletion mutants used as Ga14-DBD fusion in transient transfections to investigate their repression potential. C-terminal N-terminal Intermediate deletion mutants deletion mutants deletion mutants aa aa aa mut A 3-609 mut F 145-749 mut J 145-609 mut B 3-487 mut G 246-749 mut N 246-609 mut C 3-423 mut H 485-749 mut O 246-485 mut D 3-245 mut I 609-749 mut P 485-609 mut E 3-147 mut Q 246-423 mut R 423-485 mut S 145-245

[0150] The GRIM1 subdomains "E" and "I" described in the section "INHAT activity" show the smallest repressive effect as Gal4-DBD fusions in the transcriptional assays, but are involved in preventing histone acetylation by p300. No further information on the Q, P and R domains is available, and it was not possible to prove a direct involvement in the recruitment of additional corepressors by the interaction studies cited above. At the moment there is only speculation about the mechanism of repression used by GRIM1, although involvement of HDACs can be ruled out, and it appears that at least two different mechanisms are used (E and I in contrast to Q, R and P).

[0151] Results with the LexA.sub.8.times.Gal.sub.5.times. system confirm these results but show a different relative repression of the individual fragments investigated (data not shown).

[0152] Full-length GRIM1 is able to repress both complex naturally occurring and synthetic minimal promoters. The following systems have been tested in transient transfections: GRIM1 and complex promoters (p21 (-4542)LUC, SKA-LUC, calponin-LUC, myogenin-LUC, myogenin proximal promoter-LUC, thymidine kinase-LUC, probasin-LUC, MMTV-LUC and many more).

[0153] Cotransfections of GRIM1 and E1b-TATA-box-containing minimal promoters with synthetic TF binding sites (AR, Gal4 and SEF binding sites) or with the complex MMTV promoter leads to repression of basal transcriptional activity (all transfections shown were carried out in BHK cells).

[0154] Repression of GRIM1 with minimal promoters without synthetic TF binding sites cannot be demonstrated more accurately owing to the low basal value of the constructs used.

[0155] The repression potential of GRIM1 on promoter-luciferase-reporter luciferase-reporter constructs which have been stably integrated into the genome of the particular cell line (HRL+N with RARE.sub.3.times.-LUC, NIH3T3 with cycA-LUC, 293 with NFkB-RE.sub.4.times.-LUC) is summarized in the next figure. Transfection of the cells with GRIM1 shows no effect on reporter gene transcription in the induced and in the basal state.

[0156] In addition, direct effects of GRIM1 on various transcription factors were investigated in transient transfections (compare also Example 11). Besides the AR, in particular mmGCNF, mmERR1 and mmRVR were analyzed experimentally as full-length proteins with the corresponding reporter constructs for a functional interaction with GRIM1. No significant effect was observable in any of the transfections.

[0157] Interactions of GRIM1 with further transcription factors or cofactors was investigated in the Gal4-DBD-based context, with the respective Gal4-DBD-fused factor being cotransfected in constant amount with an increasing amount of wild-type GRIM1. Factors investigated in this system are: Gal4-p300, Gal4-DBD, Gal-Sp1-4, Gal4-CBF). Once again, no significant effect was observed in any of the transfections.

EXAMPLE 13

[0158] Mechanism of GRIM1 Repression

[0159] a) HDAC Activity

[0160] The mechanism of repression by recruitment of histone deacetylases (HDACs), which is the most widespread to date, appears not to be involved with GRIM1. The repression potential of Gal4-DBD-GRIM1 in transient transfections is not impaired by application of specific HDAC inhibitors in a concentration of up to 5-fold above the KD (final concentration of trichostatin A 300 nM, of sodium butyrate 5 mM). Immunoaffinity-purified and high-stringency-washed GRIM1 shows no endogenous HDAC activity. Nor do associated complexes which have been coimmunoprecipitated with GRIM1 by mild washing conditions show any HDAC activity. In addition, direct involvement in HDAC/N-CoR/SMRT complexes have been ruled out by Co-IP studies.

[0161] b) INHAT Activity

[0162] A further possibility for transcriptional repression consists of direct masking of the histone N termini to prevent acetylation and thus loss of cohesion of the chromatin in the affected region. It was found by database analysis that the N terminus (aa 25-135) and the absolute C terminus (aa 638-749) of hsGRIM1 has acidic domains which display homology with a described INHAT domain of the Set protein. INHAT is an acronym for protein domains/proteins able to bind lysines and thus prevent histone acetylation.

[0163] It was possible to show in GST pulldown analyses that the N- and C-terminal fragments of hsGRIM1 can associate directly with histones in vitro. In addition, it can be shown in in vitro acetylation assays that recombinantly expressed GRIM1 aa 3-147 and aa 609-749 efficiently blocks acetylation of all four core histones (H3, H2A, H2B and H4) by p300. It was possible thereby to show that a possible mechanism of the GRIM1-mediated repression might be ensured by histone masking.

[0164] There are differences in the efficiency of inhibition of acetylation of individual histones by GRIM1. As yet, only blockage of acetylation of H2A and H2B have been unambiguously demonstrable.

[0165] c) Further Mechanisms of Repression

[0166] Lysin Methylation of Histones

[0167] Methylation of specific lysines in the free histone N termini (H3K9, H3K4) ensure compaction of the chromatin structure by preventing acetylation of the lysines which are important for regulation and thus preventing a loss of cohesion of the chromatin. In addition, methylated lysines represent a high-affinity binding site for the HP1 protein (heterochromatic protein 1). HP1 may subsequently recruit DNA methylases, MeCPs and also HDACs and lead to a targeted formation of transient or constitutive heterochromatin and thus repress transcriptional processes. Examples of HMTases described to date are Suv39h1, Suv39h2 and PRMT1.

[0168] Homology analyses show that no conserved SET domain responsible for histone methyltransferase (HMTase) activity is present in the GRIM1 sequence. It is therefore assumed that GRIM1 has no endogenous HMTase activity.

[0169] Recruitment of Chromatin-Reorganizing Complexes

[0170] A further possibility for specific repression would be targeted recruitment of chromatin-reorganizing complexes (e.g. BRG, SWI/SNF, CHRAC, ARC, RSC etc.). Promoter regions of the genes to be repressed can be wrapped up in compact chromatin by active chromatin reorganization in such a way that transcriptional initiation is distinctly impeded.

[0171] It is not possible to rule out a functional interaction of GRIM1 with one of the components of one of the remodeling complexes.

[0172] Blocking PIC Formation/Impeding th Initiation Reaction/Impeding Elongation

[0173] A further form of repression is impeding or preventing the formation of the preinitiation complex (PIC) at the start of transcription. Successful initiation is possible only if the TATA box-binding portion of TFIID can recruit the polymerase holocomplex at the starting point of transcription and if all the basal transcription factors involved are available in sufficient quantity. A further prerequisite for successful transcription is the need for the C terminus of RNAPII to be phosphorylated so that leaving of the initiation site is possible.

[0174] It is possible in an experiment familiar to the skilled worker to find whether GRIM1 directly interacts with one of the basal transcription factors or elongation factors and thus blocks efficient transcription (yeast two-hybrid system, pulldown analyses). No phosphatase domain can be found within the GRIM1 structure by homology analyses, and it is yet to be proven experimentally that GRIM1 is able to dephosphorylate RNAPII.

[0175] Titration out of Coactivators by Binding to GRIM1

[0176] The potential of GRIM1 to inactivate basal or general coactivators by binding and thus to repress the initiation or elongation process is currently under investigation by searching for potential hsGRIM1 interactants.

[0177] A total of at least 5 independent repressor domains (RDs) have been found within the GRIM1 protein in transient transfections. The fragments E and I of GRIM1 showed the smallest repression potential in transient transfections, but their function in repression by GRIM1 was demonstrated in the INHAT analyses.

[0178] However, the deletion mutants P, Q, R and also S show a far stronger repression function in Gal-based transfections, suggesting further mechanisms beyond the known facts. It is possible to find the contribution to repression made by the other regions of GRIM1 by conventional experiments. These were intended to identify interacting GRIM1 partner proteins in order to find whether there is an association with non-HDAC components of transcriptional corepressor complexes.

EXAMPLE 14

[0179] Analysis of Functional Motifs in hsGRIM1

[0180] Functional motifs within the hsGRIM1 structure were found by database homology analyses. A summary of the motifs found, their localization within the complete protein and the experimental strategy used is shown in the following table. TABLE-US-00018 TABLE 19 Summary of the functional motifs in hsGRIM1. Name Sequence aa position Detection LxxLL I.sub.RDLI 212-216 Significance not yet confirmed L.sub.QNLL 219-223 experimentally L.sub.AFLV 312-316 L.sub.TELL 362-366 II.sub.GCI 339-443 L.sub.FIL 476-480 IL.sub.RPL 686-690 NES L.sub.RVL.sub.AL.sub.VL 309-317 Detected by GFP relocation experiments and directed point mutagenesis NLS KRRK.sub.MADRK.sub.DEDRK.sub.QEK 649-666 Detected by directed point mutagenesis and GFP-GRIMI localization CoRNR Box .sub.PL.sub.AQVIIGCI.sub.KLI.sub.P 435-446 Not yet confirmed experimentally. Has no influence on AR-dependent interactions HMG Box poly E/poly D 25-51 INHAT activity of the investigated like 68-134 fragments. Direct DNA and/or 658-683 histone binding not yet confirmed 697-749 experimentally. LxxLL: interaction motif by which coactivators interact with NHRs. NES: nuclear export signal. NLS: nuclear localization signal. CoRNR box: interation motif by which corepressors interact with NHRs. HMG box: contact of HMG proteins to DNA and to histones.

[0181] Detection of the NES within hsGRIM1 For direct analysis of the motif, all four relevant lysines were inactivated by directed point mutagenesis so that the .alpha.-helical structure of the NES was destroyed. The inactivating mutations are depicted in the following figure. A directed inactivation of the two 3'-located lysines on its own proved to be insufficient to destroy the functionality of the NES. TABLE-US-00019 TABLE 20 Directed mutagenesis to inactivate the four lysines involved in the NES. ##STR1##

[0182] To demonstrate the functionality of the export signal, hsGRIM1 protein fragments were fused to GFP (green fluorescent protein) (depicted in the following figure) and expressed in cells by transient transfection. The localization of the respective fragments was detected by fluorescence microscopy. TABLE-US-00020 TABLE 21 GFP fusion proteins used to demonstrate the functionality of the nuclear export signal (NES). .DELTA.NES characterizes the point mutations described above. GFP*hsGRIM1 ##STR2## GFP* hsGRIM1.DELTA.NES ##STR3## GFP*hsGRIM1 .LAMBDA. aa 246-334 ##STR4## GFP*hsGRIM1 aa 246-334 ##STR5##

[0183] Only GFP showed a pan-cellular localization. Full-length GFP-hsGRIM1 was located exclusively in the nucleus through the dominance of the NLS, as were the NES deletion mutant and the fragment from which a region flanking the NES had been deleted (hsGRIM1 .DELTA.aa246-334). On fusion of the hsGRIM1 fragment which contains the NES to GFP (hsGRIM1 aa246-334), the resulting protein was located exclusively in the cytoplasm. On coupling of the point-mutated fragment of aa 246-334 to GFP, the pan-cellular localization was restored so that it was possible to demonstrate the functionality of the motif.

[0184] Detection of the NLS within hsGRIM1

[0185] To analyze the motif, besides the deletion mutants detailed hereinafter, all seven relevant basic residues were inactivated by directed point mutagenesis. Inactivation of the 3'-located 3 basic residues alone proved to be insufficient to inactivate the NLS. TABLE-US-00021 TABLE 22 Directed mutagenesis for inactivation of the seven basic residues involved in the NLS. ##STR6##

[0186] To demonstrate the functionality of the import signal, hsGRIM1 protein fragments were fused to GFP (depicted in the following figure) and expressed in cells by transient transfection. The localization of the respective fragments was detected by fluorescence microscopy. TABLE-US-00022 TABLE 23 GFP fusion proteins used to demonstrate the functionality of the nuclear localization signal (NLS). GFP*hsGRIM1 ##STR7## GFP* hsGRIM1 .DELTA.NLS ##STR8## GFP*hsGRIM1 aa 1-609 ##STR9## GFP*hsGRIM1 aa 609-749 ##STR10## and .DELTA.NLSII

[0187] Only GFP showed a pan-cellular localization. Full-length GFP-hsGRIM1 was located exclusively in the nucleus. On deletion of the last 140 amino acids (hsGRIM1 aa1-609), the GFP fusion protein showed both a cytoplasmic and a weak nuclear localization, while the corresponding counterpart fused to GFP (GFP-hsGRIM1 aa609-749) shows an exclusively nuclear localization. The full-length hsGRIM1 with the seven-fold point mutation within the NLS showed the same distribution pattern as the hsGRIM1 aa1-609 deletion mutant and confirmed the functionality of the NLS. On coupling of the point-mutated fragment of aa609-749 to GFP, the fusion protein was likewise located in the cytoplasm.

[0188] Detection of the CoRNR Box within hsGRIM1

[0189] To analyze the CoRNR box, all three highly conserved and necessary isoleucines in the hydrophobic core of the helix were replaced by alanines or by a serine: I436A, I437S, I444A. Further functional characterization of the point mutations made have revealed to date only that the repression potential of hsGRIM1 is not impaired, and that the interaction with the AR is not impaired by destroying the CoRNR box.

[0190] Intracellular Relocalization of mmGRIM1 in Cell Culture-Based Differentiation Models

[0191] GRIM1 shows a change in its subcellular localization during specific differentiation processes. In cell culture systems, GRIM1 is transported from the nucleus into the surrounding cytoplasm during skeletal muscle differentiation of mouse C2C12 cells. The timeframe for the observed translocation of GRIM1 is in the range for the expression of the myogenic bHLH transcription factor myogenin (My14) and of the structural protein skeletal muscle myosin heavy chain (MHC), i.e. at a more advanced time during muscle differentiation than previously described factors such as HDAC4, HDAC5 and HDAC7. C2C12 cells differentiate under low-serum conditions within 6-8 days from proliferating myoblasts to fused, multinuclear myotubes. During this, the cells undergo a G.sub.0 arrest, the CDK inhibitor p21 is upregulated, and an ordered cascade of myogenic transcription factors such as Myf5, MyoD and myogenin provide the transcriptional precondition for myogenesis. In differentiating C2C12 skeletal muscle cells, GRIM1 leaves the nuclei of fused myotubes after about 6 to 8 days, whereas GRIM1 is always located in the nucleus in mononuclear myoblasts. Expression of the structural protein of muscle MHC takes place at the time when the first GRIM1-free nuclei are to be observed, and was used in IIFs as temporal marker for GRIM relocalization.

[0192] The data determined for C2C12 also apply to other cellular skeletal muscle differentiation systems. The same effect was also detectable in rat L6 skeletal muscle cells. The cells have different differentiation kinetics and do not fuse until day 10-12 in differentiation medium (DM). GRIM1 also relocalizes in L6 cells, and GRIM1-free nuclei are detectable only from day 16 onwards in DM. In a further differentiation system, mouse 10T1/2 fibroblasts were stably transfected with an estrogen-inducible MyoD expression cassette (cells kindly made available by Dr. D. Bergstrom). Addition of 1 .mu.M estradiol enables skeletal muscle differentiation via MyoD expression. After only about 3-4 days in DM, many cells can be detected as multinuclear myotubes.

[0193] During skeletal muscle differentiation, the GRIM1 protein is not degraded and/or broken down but is present to the full extent also in the cytoplasm. GRIM1 immunoreactivity can be detected unchanged in Western blot analyses with whole cell extracts of C2C12 cell extracts which have been differentiated to myotubes in DM for 8 days (on day 8 after addition of DM, .gtoreq.60% of all the cells present were myotubes). The results can also be further confirmed by cell fractionations. In addition, RT-PCR analyses have confirmed that the amount of mmGRIM1-mRNA is not changed during C2C12 differentiation.

[0194] As a further check, it was investigated whether GRIM1 also experiences subcellular relocalization during smooth muscle differentiation. For this purpose, mouse smooth muscle cells were obtained ex vivo and dedifferentiated by growth factor addition. Cells were additionally differentiated again further to smooth muscle cells via differentiation medium in vitro. The results show that GRIM1 does not leave the nucleus during smooth muscle differentiation. In addition, a cell culture smooth muscle differentiation system (Monc-1 cells) was used in order to obtain in vitro data about possible GRIM1 relocalization. As already shown for the ex vivo cells, no GRIM1 relocalization in smooth muscle cells was observable during the investigated differentiation times.

[0195] A further in vitro differentiation model used for GRIM1 relocalization analyses is differentiation of 3T3-L1 preadipocytes to mature fat cells. In the total of three differentiation protocols carried out, GRIM1 twice showed a detectable relocalization from the core into the cytoplasm, while in one case no change in the localization of the GRIM1 immunoreactivity was detectable. Western blot analyses have shown that no decrease in GRIM1 immunoreactivity in whole-cell extracts was detectable in any of the three protocols.

EXAMPLE 15

[0196] Regulation of GRIM1 Function

[0197] Complete experimental results are not yet available on the regulation of GRIM1 function and on signal pathways which might be involved in the subcellular relocalization of GRIM1. No signal pathway modulating the transcriptional activity of GRIM1 has yet been unambiguously identifiable. Nor is there any experimental evidence of involvement of a signal cascade intervening in the GRIM1 relocalization process. The data available on this topic can be briefly summarized below.

[0198] Transcriptional Activity:

[0199] In the direct vicinity of the NES within the GRIM1 sequence there is a consensus PKC phosphorylation site (serine 308). The transcriptional activity of Gal-GRIM1 was investigated in transient transfections in various cell lines. Neither the treatment of the cells with the specific PKC inhibitor Go6850 (Parke-Davis, formerly Godecke AG) nor a stimulation with the PKC-activating agent TPA brought about a change in the transcriptional activity.

[0200] The sole experimental evidence for modification of GRIM1 is the detection of direct acetylation of GRIM1 (aa3-147 and of full-length GRIM1) by the HAT domain of p300.

[0201] Cotransfection of full-length p300 and Gal-GRIM1 shows no change in the repression pattern, and addition of trichostatin A (TSA) enhances general transcription but relative repression by Gal-GRIM1 remains unchanged.

[0202] Cotransfection of Gal-GRIM1 in combination with a constitutively active version of Rho GTPase (RhoAV14) likewise shows no change in the activity.

[0203] Relocalization:

[0204] Nuclear export of GFP-GRIM1 or of the mutant GFP-GRIM1 aa234-336 is not influenced by treatment of the cells with the bacterial toxin leptomycin B (LMB, 20 nM for 6 h before cell harvesting and analysis by IIF). LMB is a specific inhibitor of the export factor exportin1/Crm1, so that it can be concluded from the experiment that there must be an exportin-independent transport mechanism.

[0205] 14-3-3 proteins are responsible for active removal of the HDACs 4, 5 and 7 from the nucleus and retention in the cytoplasm during muscle differentiation. It was possible by Co-IP studies to rule out an interaction between GRIM1 and 14-3-3 proteins (family members zeta, beta, tau).

[0206] Treatment of differentiating C2C12 skeletal muscle cells with various inhibitors of central signal transduction pathways have no effect on relocalization or the number of nuclear speckles of GRIM1 within the investigated differentiation from day 0 to day 6 after addition of the differentiation medium. Tested inhibitors: pan-HDAC inhibitors TSA and sodium butyrate, protein kinase inhibitors Go6850 (specific PKC inhibitor, 1 .mu.M), LY294002 (specific P13kinase inhibitor, 10 .mu.M), Ro3l-8220 (PKC broad-spectrum inhibitor, 0.5 .mu.M), PD98059 (MEK1 inhibitor, 10 .mu.M), fasudil (Rock kinase inhibitor, 10 .mu.M), SB203580 (p38.alpha./.beta. inhibitor). Overacetylation and blockade of central signal cascades had drastic morphologically visible consequences for some of the cells, but there was no effect on relocalization or the number of detectable GRIM1 speckles.

EXAMPLE 16

[0207] Interaction of hsGRIM1 and the Androgen Receptor (AR) and Functional Consequences Resulting therefrom

[0208] It was possible to demonstrate in a yeast two-hybrid interaction assay that GRIM1 interacts with the nuclear hormone receptor androgen receptor (AR). The GRIM1 interaction domain in the yeast-based assay is located in the C terminus between aa 606 to 749. Further functional characterization of the interaction domains within GRIM1 was carried out by pulldown analyses with the expressible fragments of GRIM1. The interaction of GRIM1 with the AR is independent of the type of ligand used (agonists, antagonists or no ligands). The available data are summarized in the following picture: TABLE-US-00023 TABLE 24 The GRIM1 AR interaction domain is located between aa460-609. ##STR11## Regions of the protein depicted in white have not yet been expressed recombinantly.

[0209] The C terminus of GRIM1 shows, in contrast to the data obtained in yeast, no in vitro interaction with the AR, but the fragment from aa 460 to aa 609 shows a strong interaction independent of the ligand used.

[0210] The interaction between the AR and GRIM1 was likewise detectable in in vivo interaction assays. In co-immunoprecipitations, the interaction between GRIM1 and the AR was likewise ligand-independent and could not be significantly blocked even in washing buffer with 400 mM NaCl. It was possible to show the interaction with IP studies using antibodies both against the AR and against GRIM1.

[0211] The AR is a member of the superfamily of ligand-activatable nuclear hormone receptors. After binding of the natural AR ligand 5.alpha.-dihydrotestosterone, the AR is transported from the cytoplasm into the nucleus and brings about transcriptional activation of its target genes. Besides the activating ligands, each receptor has specific antagonists which likewise bring about translocation into the nucleus, but bring about a repressive conformation of the receptor ligand binding domain. Antagonist-bound receptor is able to recruit corepressor complexes and actively repress transcription. However, in some cell lines or in certain pathological situations, antagonists may become partial agonists and unnaturally bring about a transactivation by the receptor. It is known in the case of the AR that substances employed therapeutically for prostate carcinomas, such as cyproterone acetate, Casodex or hydroxyflutamide, may have partial agonistic effects in hormone-refractory tumors.

[0212] It was possible to show in the cell culture system that GRIM1 is able specifically to repress an AR transactivation brought about by CPA, Casodex or OH-flutamide. Non-liganded or DHT-loaded AR is not transcriptionally affected by GRIM1. The observed effect varies in the range between 2-3-fold repression of the CPA-induced AR transactivation. TABLE-US-00024 TABLE 25 GRIM1 selectively represses antagonist- activated AR. ##STR12## Transient transfections were carried out in BHK cells in which the antagonists used act as partial agonists. A minimal promoter with synthetic AR binding sites was used as reporter (ARE.sub.2x-(tk)TATA-LUC). The same effect was obtained on use of 1 .mu.M Casodex or OH-flutamide instead of 1 .mu.M CPA.

[0213] A further indication of the functionality of the effect is that the superactivation of the CPA-loaded AR by the coactivator TIF2 in BHK cells can be repressed or at least markedly restricted by cotransfection of GRIM1. TABLE-US-00025 TABLE 26 GRIM1 represses CPA-loaded and TIF2 superactivated androgen receptor. ##STR13## Transient transfections were carried out in BHK cells in which CPA (1 .mu.M) acts as partial agonist. ARE.sub.2x-(tk)TATA-LUC was used as reporter.

[0214]

Sequence CWU 1

1

8 1 2250 DNA Homo sapiens 1 atggcagctg cggggagccg caagaggcgc ctggcggagc tgacggtgga cgagttccta 60 gcttcgggct ttgactccga gtccgaatcc gagtccgaaa attctccaca agcggagaca 120 cgggaagcac gcgaggctgc ccggagtccg gataagccgg gcgggagccc ctcggccagc 180 cggcgtaaag gccgtgcctc tgagcacaaa gaccagctct ctcggctgaa ggacagagac 240 cccgagttct acaagttcct gcaggagaat gaccagagcc tgctaaactt cagcgactcg 300 gacagctctg aggaggaaga ggggccgttc cactccctgc cagatgtgct ggaggaagcc 360 agtgaggagg aggatggagc ggaggaagga gaagatgggg acagagtccc cagagggctg 420 aaggggaaga agaattctgt tcctgtgacc gtcgccatgg ttgagagatg gaagcaggca 480 gcaaagcaac gcctcactcc aaagctgttc catgaagtgg tacaggcgtt ccgagcagct 540 gtggccacca cccgagggga ccaggaaagt gctgaggcca acaaattcca ggtcacggac 600 agtgctgcat tcaatgctct ggttaccttc tgcatcagag acctcattgg ctgtctccag 660 aagctgctgt ttggaaaggt ggcaaaggat agcagcagga tgctgcagcc gtccagcagc 720 ccgctctggg ggaagcttcg tgtggacatc aaggcttacc tgggctcggc catacagctg 780 gtgtcctgtc tgtcggagac gacggtgttg gcggccgtgc tgcggcacat cagcgtgctg 840 gtgccctgct tcctgacctt ccccaagcag tgccgcatgc tgctcaagag aatggtggtc 900 gtatggagca ctggggagga gtctctgcgg gtgctggctt tcctggtcct cagcagagtc 960 tgccggcaca agaaggacac tttccttggc cccgtcctca agcaaatgta catcacgtat 1020 gtgaggaact gcaagttcac ctcgcctggt gccctcccct tcatcagttt catgcagtgg 1080 accttgacgg agctgctggc cctggagccg ggtgtggcct accagcacgc cttcctctac 1140 atccgccagc tcgccataca cctgcgcaac gccatgacca cccgcaagaa ggaaacatac 1200 cagtctgtgt acaactggca gtatgtgcac tgcctcttcc tgtggtgccg ggtcctgagc 1260 actgcgggcc ccagcgaagc cctccagccc ttggtctacc cccttgccca agtcatcatt 1320 ggctgtatca agctcatccc cactgcccgc ttctacccgc tgcgaatgca ctgcatccgt 1380 gccctgacgc tgctctcggg gagctcgggg gccttcatcc cggtgctgcc tttcatcctg 1440 gagatgttcc agcaggtcga cttcaacagg aagccagggc gcatgagctc caagcccatc 1500 aacttctccg tgatcctgaa gctgtccaat gtcaacctgc aggagaaggc gtaccgggac 1560 ggcctggtgg agcagctgta cgacctcacc ctggagtacc tgcacagcca ggcacactgc 1620 atcggcttcc cggagctggt gctgcctgtg gtcctgcagc tgaagtcgtt cctccgggag 1680 tgcaaggtgg ccaactactg ccggcaggtg cagcagctgc ttgggaaggt tcaggagaac 1740 tcggcataca tctgcagccg ccgccagagg gtttccttcg gcgtctctga gcagcaggca 1800 gtggaagcct gggagaagct gacccgggaa gaggggacac ccttgacctt gtactacagc 1860 cactggcgca agctgcgtga ccgggagatc cagctggaga tcagtggcaa agagcggctg 1920 gaagacctga acttccctga gatcaaacga aggaagatgg ctgacaggaa ggatgaggac 1980 aggaagcaat ttaaagacct ctttgacctg aacagctctg aagaggacga caccgaggga 2040 ttctcggaga gagggatact gaggcccctg agcactcggc atggggtgga agacgatgaa 2100 gaggacgagg aggagggcga ggaggacagc agcaactcgg aggatggaga cccagacgca 2160 gaggcggggc tggcccctgg ggagctgcag cagctggccc aggggccgga ggacgagctg 2220 gaggatctgc agctctcaga ggacgactga 2250 2 2253 DNA Mus musculus 2 atggctgcgt ctcgcgctcc ccgcaggcgc ctggaggacc tcagtgtgga cgagttcctg 60 gcttccggct tcgagtccgg atccgagtcg gagctggagg gcgccgcgga ggcggcggcg 120 gaggagcgca gggcgcgagg agccgcgtgg aaccgggagc ggcggggcgc gcgcacctcc 180 ccgggccccg caggacgccc gcgtaagggc cgcgcctctg agcacaaaga ccagctctct 240 cggctgaagg acagagaccc cgagttctac aagttcctgc aggagaatga ccggagccta 300 ctggacttca gtgactcgga cagctctgcg gaagaagaag agccattcca ctccctgcca 360 gacacgctgg aggaagcgag cgaaacagag gaagacggag gagaggacag tgacgcgttg 420 cccagagggc tgaggagcaa gaagaatgag cctgtacccg tgaccctcgc catggtggaa 480 aggtggaggc agggctccag gcaccacctt agtcccaggc tgttccatga agttgtacag 540 gcgttccgag cagctgtagc caccacccaa ggagagcagg aagctgctga gacttgcagg 600 ttccaggttg cagatagtgc tgtgttcaat gctctggtta ctttctgcat tcgagacctc 660 tgtggttgcc ttcagaagct gctgtttgga aagacaccaa aggatagcaa taggctgctg 720 ccatccagta gcccactgtg ggggaagctc cgtgtggatg tcaagacata cctaagtgcg 780 gtkgtgcagc tggtagcctg tctagcggaa gccacagtgt ctgcagctgt cctgcagcat 840 atcagcagct tggttcctta cttcctgact ttcccgaagc agtgccgaat gctgctcaag 900 aggatggtgg ttctgtggag cacgggtgaa gagtctctgc gggtcctggc cttcctggta 960 ctcatcagag tctgtcggca caagaaggaa gccttccttg gtcccattct gaagcaaatg 1020 tacatcatgt atgtgagaaa ctgcaagttc acctccccca gtaccctccc cctcataagc 1080 ttcatgcagc ggacactgac tgaaatgctt gccttggacc ccagcgtctc ctatcagcac 1140 gccttcctct acatccgcca gcttgccgtc cacctgcgga atgctatgac cacaggcaag 1200 aaggagacac accagtctgt gtacaactgg cagtatgtgc actgcctcta cctgtggtgt 1260 cgwgtcctga gtacccttgg ttccagtgag atcctgcagc cgctactcta ccctctctca 1320 cagatcatca ttggctgtat caagttgttg cccactgctc gattttatcc attgcgcatg 1380 cattgtgtac gtgccctgac actgctgtcc cagaccatcg gcaccttcat acctgtcctg 1440 cccttcattc tygagatttt ccagcaggtg gacttcaata ggcggccagg tcgcatgagc 1500 tccaagccca tcaacttctc tgtgatcttg aagctgtcca gcaccaacct gcaggagaag 1560 gcgtaccggg acgggctgct ggaacagctg tgtgacctta ctctggaata cctgcacagc 1620 caggcccaca gcatcgcttt cccagagttg gtgttgccta ctgttctaca gctgaaatct 1680 tttctccggg agtgcaaagt ggctaactac tgccggcagg tgcgccactm gctggagaaa 1740 gtgcaagaga atgcacaaca tatccaaagt cttcgacaga gcgcgacctt cagcgtgtct 1800 gaccggacgg cagtggatgc gtgggagaag caggttygtg aagaggggac cccactcacc 1860 agatactacg gccactggaa gaagctgagg gaccgtgaaa tccagctgga aatcagtggc 1920 aaagagcggc tagaagacct gaacttccca gagatcaaaa ggcggaaggt ggaagacagg 1980 aaggatgaag acaggaaaga attaaaggac ctgtttgagt tggacagttc tgagggcgag 2040 gacagcaccg acttctttga gagaggagta cctaggctcc cagaagctca ccaaggactg 2100 aaagaagatc aggaagaaga agataaagaa gaaggtgaca gcgattcaga ggatggagac 2160 acagacacgg gagtggatct gagcgaactg tggcagctgg ctcagggacc acaagatgag 2220 ctggaggatc ttcagctctc agaagaggac tga 2253 3 749 PRT Homo sapiens 3 Met Ala Ala Ala Gly Ser Arg Lys Arg Arg Leu Ala Glu Leu Thr Val 1 5 10 15 Asp Glu Phe Leu Ala Ser Gly Phe Asp Ser Glu Ser Glu Ser Glu Ser 20 25 30 Glu Asn Ser Pro Gln Ala Glu Thr Arg Glu Ala Arg Glu Ala Ala Arg 35 40 45 Ser Pro Asp Lys Pro Gly Gly Ser Pro Ser Ala Ser Arg Arg Lys Gly 50 55 60 Arg Ala Ser Glu His Lys Asp Gln Leu Ser Arg Leu Lys Asp Arg Asp 65 70 75 80 Pro Glu Phe Tyr Lys Phe Leu Gln Glu Asn Asp Gln Ser Leu Leu Asn 85 90 95 Phe Ser Asp Ser Asp Ser Ser Glu Glu Glu Glu Gly Pro Phe His Ser 100 105 110 Leu Pro Asp Val Leu Glu Glu Ala Ser Glu Glu Glu Asp Gly Ala Glu 115 120 125 Glu Gly Glu Asp Gly Asp Arg Val Pro Arg Gly Leu Lys Gly Lys Lys 130 135 140 Asn Ser Val Pro Val Thr Val Ala Met Val Glu Arg Trp Lys Gln Ala 145 150 155 160 Ala Lys Gln Arg Leu Thr Pro Lys Leu Phe His Glu Val Val Gln Ala 165 170 175 Phe Arg Ala Ala Val Ala Thr Thr Arg Gly Asp Gln Glu Ser Ala Glu 180 185 190 Ala Asn Lys Phe Gln Val Thr Asp Ser Ala Ala Phe Asn Ala Leu Val 195 200 205 Thr Phe Cys Ile Arg Asp Leu Ile Gly Cys Leu Gln Lys Leu Leu Phe 210 215 220 Gly Lys Val Ala Lys Asp Ser Ser Arg Met Leu Gln Pro Ser Ser Ser 225 230 235 240 Pro Leu Trp Gly Lys Leu Arg Val Asp Ile Lys Ala Tyr Leu Gly Ser 245 250 255 Ala Ile Gln Leu Val Ser Cys Leu Ser Glu Thr Thr Val Leu Ala Ala 260 265 270 Val Leu Arg His Ile Ser Val Leu Val Pro Cys Phe Leu Thr Phe Pro 275 280 285 Lys Gln Cys Arg Met Leu Leu Lys Arg Met Val Val Val Trp Ser Thr 290 295 300 Gly Glu Glu Ser Leu Arg Val Leu Ala Phe Leu Val Leu Ser Arg Val 305 310 315 320 Cys Arg His Lys Lys Asp Thr Phe Leu Gly Pro Val Leu Lys Gln Met 325 330 335 Tyr Ile Thr Tyr Val Arg Asn Cys Lys Phe Thr Ser Pro Gly Ala Leu 340 345 350 Pro Phe Ile Ser Phe Met Gln Trp Thr Leu Thr Glu Leu Leu Ala Leu 355 360 365 Glu Pro Gly Val Ala Tyr Gln His Ala Phe Leu Tyr Ile Arg Gln Leu 370 375 380 Ala Ile His Leu Arg Asn Ala Met Thr Thr Arg Lys Lys Glu Thr Tyr 385 390 395 400 Gln Ser Val Tyr Asn Trp Gln Tyr Val His Cys Leu Phe Leu Trp Cys 405 410 415 Arg Val Leu Ser Thr Ala Gly Pro Ser Glu Ala Leu Gln Pro Leu Val 420 425 430 Tyr Pro Leu Ala Gln Val Ile Ile Gly Cys Ile Lys Leu Ile Pro Thr 435 440 445 Ala Arg Phe Tyr Pro Leu Arg Met His Cys Ile Arg Ala Leu Thr Leu 450 455 460 Leu Ser Gly Ser Ser Gly Ala Phe Ile Pro Val Leu Pro Phe Ile Leu 465 470 475 480 Glu Met Phe Gln Gln Val Asp Phe Asn Arg Lys Pro Gly Arg Met Ser 485 490 495 Ser Lys Pro Ile Asn Phe Ser Val Ile Leu Lys Leu Ser Asn Val Asn 500 505 510 Leu Gln Glu Lys Ala Tyr Arg Asp Gly Leu Val Glu Gln Leu Tyr Asp 515 520 525 Leu Thr Leu Glu Tyr Leu His Ser Gln Ala His Cys Ile Gly Phe Pro 530 535 540 Glu Leu Val Leu Pro Val Val Leu Gln Leu Lys Ser Phe Leu Arg Glu 545 550 555 560 Cys Lys Val Ala Asn Tyr Cys Arg Gln Val Gln Gln Leu Leu Gly Lys 565 570 575 Val Gln Glu Asn Ser Ala Tyr Ile Cys Ser Arg Arg Gln Arg Val Ser 580 585 590 Phe Gly Val Ser Glu Gln Gln Ala Val Glu Ala Trp Glu Lys Leu Thr 595 600 605 Arg Glu Glu Gly Thr Pro Leu Thr Leu Tyr Tyr Ser His Trp Arg Lys 610 615 620 Leu Arg Asp Arg Glu Ile Gln Leu Glu Ile Ser Gly Lys Glu Arg Leu 625 630 635 640 Glu Asp Leu Asn Phe Pro Glu Ile Lys Arg Arg Lys Met Ala Asp Arg 645 650 655 Lys Asp Glu Asp Arg Lys Gln Phe Lys Asp Leu Phe Asp Leu Asn Ser 660 665 670 Ser Glu Glu Asp Asp Thr Glu Gly Phe Ser Glu Arg Gly Ile Leu Arg 675 680 685 Pro Leu Ser Thr Arg His Gly Val Glu Asp Asp Glu Glu Asp Glu Glu 690 695 700 Glu Gly Glu Glu Asp Ser Ser Asn Ser Glu Asp Gly Asp Pro Asp Ala 705 710 715 720 Glu Ala Gly Leu Ala Pro Gly Glu Leu Gln Gln Leu Ala Gln Gly Pro 725 730 735 Glu Asp Glu Leu Glu Asp Leu Gln Leu Ser Glu Asp Asp 740 745 4 750 PRT Mus musculus MISC_FEATURE (577)..(577) The 'Xaa' at location 577 stands for Ser. MISC_FEATURE (613)..(613) The 'Xaa' at location 613 stands for Arg, or Cys. 4 Met Ala Ala Ser Arg Ala Pro Arg Arg Arg Leu Glu Asp Leu Ser Val 1 5 10 15 Asp Glu Phe Leu Ala Ser Gly Phe Glu Ser Gly Ser Glu Ser Glu Leu 20 25 30 Glu Gly Ala Ala Glu Ala Ala Ala Glu Glu Arg Arg Ala Arg Gly Ala 35 40 45 Ala Trp Asn Arg Glu Arg Arg Gly Ala Arg Thr Ser Pro Gly Pro Ala 50 55 60 Gly Arg Pro Arg Lys Gly Arg Ala Ser Glu His Lys Asp Gln Leu Ser 65 70 75 80 Arg Leu Lys Asp Arg Asp Pro Glu Phe Tyr Lys Phe Leu Gln Glu Asn 85 90 95 Asp Arg Ser Leu Leu Asp Phe Ser Asp Ser Asp Ser Ser Ala Glu Glu 100 105 110 Glu Glu Pro Phe His Ser Leu Pro Asp Thr Leu Glu Glu Ala Ser Glu 115 120 125 Thr Glu Glu Asp Gly Gly Glu Asp Ser Asp Ala Leu Pro Arg Gly Leu 130 135 140 Arg Ser Lys Lys Asn Glu Pro Val Pro Val Thr Leu Ala Met Val Glu 145 150 155 160 Arg Trp Arg Gln Gly Ser Arg His His Leu Ser Pro Arg Leu Phe His 165 170 175 Glu Val Val Gln Ala Phe Arg Ala Ala Val Ala Thr Thr Gln Gly Glu 180 185 190 Gln Glu Ala Ala Glu Thr Cys Arg Phe Gln Val Ala Asp Ser Ala Val 195 200 205 Phe Asn Ala Leu Val Thr Phe Cys Ile Arg Asp Leu Cys Gly Cys Leu 210 215 220 Gln Lys Leu Leu Phe Gly Lys Thr Pro Lys Asp Ser Asn Arg Leu Leu 225 230 235 240 Pro Ser Ser Ser Pro Leu Trp Gly Lys Leu Arg Val Asp Val Lys Thr 245 250 255 Tyr Leu Ser Ala Val Val Gln Leu Val Ala Cys Leu Ala Glu Ala Thr 260 265 270 Val Ser Ala Ala Val Leu Gln His Ile Ser Ser Leu Val Pro Tyr Phe 275 280 285 Leu Thr Phe Pro Lys Gln Cys Arg Met Leu Leu Lys Arg Met Val Val 290 295 300 Leu Trp Ser Thr Gly Glu Glu Ser Leu Arg Val Leu Ala Phe Leu Val 305 310 315 320 Leu Ile Arg Val Cys Arg His Lys Lys Glu Ala Phe Leu Gly Pro Ile 325 330 335 Leu Lys Gln Met Tyr Ile Met Tyr Val Arg Asn Cys Lys Phe Thr Ser 340 345 350 Pro Ser Thr Leu Pro Leu Ile Ser Phe Met Gln Arg Thr Leu Thr Glu 355 360 365 Met Leu Ala Leu Asp Pro Ser Val Ser Tyr Gln His Ala Phe Leu Tyr 370 375 380 Ile Arg Gln Leu Ala Val His Leu Arg Asn Ala Met Thr Thr Gly Lys 385 390 395 400 Lys Glu Thr His Gln Ser Val Tyr Asn Trp Gln Tyr Val His Cys Leu 405 410 415 Tyr Leu Trp Cys Arg Val Leu Ser Thr Leu Gly Ser Ser Glu Ile Leu 420 425 430 Gln Pro Leu Leu Tyr Pro Leu Ser Gln Ile Ile Ile Gly Cys Ile Lys 435 440 445 Leu Leu Pro Thr Ala Arg Phe Tyr Pro Leu Arg Met His Cys Val Arg 450 455 460 Ala Leu Thr Leu Leu Ser Gln Thr Ile Gly Thr Phe Ile Pro Val Leu 465 470 475 480 Pro Phe Ile Leu Glu Ile Phe Gln Gln Val Asp Phe Asn Arg Arg Pro 485 490 495 Gly Arg Met Ser Ser Lys Pro Ile Asn Phe Ser Val Ile Leu Lys Leu 500 505 510 Ser Ser Thr Asn Leu Gln Glu Lys Ala Tyr Arg Asp Gly Leu Leu Glu 515 520 525 Gln Leu Cys Asp Leu Thr Leu Glu Tyr Leu His Ser Gln Ala His Ser 530 535 540 Ile Ala Phe Pro Glu Leu Val Leu Pro Thr Val Leu Gln Leu Lys Ser 545 550 555 560 Phe Leu Arg Glu Cys Lys Val Ala Asn Tyr Cys Arg Gln Val Arg His 565 570 575 Xaa Leu Glu Lys Val Gln Glu Asn Ala Gln His Ile Gln Ser Leu Arg 580 585 590 Gln Ser Ala Thr Phe Ser Val Ser Asp Arg Thr Ala Val Asp Ala Trp 595 600 605 Glu Lys Gln Val Xaa Glu Glu Gly Thr Pro Leu Thr Arg Tyr Tyr Gly 610 615 620 His Trp Lys Lys Leu Arg Asp Arg Glu Ile Gln Leu Glu Ile Ser Gly 625 630 635 640 Lys Glu Arg Leu Glu Asp Leu Asn Phe Pro Glu Ile Lys Arg Arg Lys 645 650 655 Val Glu Asp Arg Lys Asp Glu Asp Arg Lys Glu Leu Lys Asp Leu Phe 660 665 670 Glu Leu Asp Ser Ser Glu Gly Glu Asp Ser Thr Asp Phe Phe Glu Arg 675 680 685 Gly Val Pro Arg Leu Pro Glu Ala His Gln Gly Leu Lys Glu Asp Gln 690 695 700 Glu Glu Glu Asp Lys Glu Glu Gly Asp Ser Asp Ser Glu Asp Gly Asp 705 710 715 720 Thr Asp Thr Gly Val Asp Leu Ser Glu Leu Trp Gln Leu Ala Gln Gly 725 730 735 Pro Gln Asp Glu Leu Glu Asp Leu Gln Leu Ser Glu Glu Asp 740 745 750 5 15105 DNA Homo sapiens 5 ggcggcggaa gtgcgcagcc gcgcggcatt ctggggccgg aagtggggtg cacgcttcgg 60 gttggtgtca tggcagctgc ggggagccgc aagaggtaag ccgcgggtcc gagggccgat 120 ttggcctccc ggtgggtgtc tgtatccaag ggggcttttc ttgctcctct caacggggct 180 tgggccaact tggccttccg gccactttga cttcttcctt aaccgcaggc gcctggcgga 240 gctgacggtg gacgagttcc tagcttcggg ctttgactcc gagtccgaat ccgagtccga 300 aaattctcca caagcggaga cacgggaagc acgcgaggct gcccggagtc cggataagcc 360 gggcgggagc ccctcggcca ggttagtggg gacatgcgtg agcagaacct cttgctcgtc 420 ctgtcccacc ccggttgggt tgctgacctc gcctggggtc tcagggccaa ggcctaagga 480 tcgccgacct ctgcccccca gtcctttcag cttaacttac tctttcatcc gatgtttact 540 gagctcctgt tccgtgccaa actctgtcct aggcagggag cgtatgttgg tgagttgtgc 600 aaaaatctct tcccgatcta ggagaactct cacgttcttg ggtgaaatca ccgcctgcag 660 ttcggtagaa gagagataag tgctggggag ggaaaggaag gacagagggg ctgaagagag 720 agttgcaggg gagccgagtt gctctgttag aaggagcgga ggctgtgctc ggtggctcac 780 gcctgtaatg ccagcacttt gggaggccga ggcgggcaga ttacctgagg tcaggaattc 840 gagaccagcc tgaccaacac ggaaaaatca tgtctctact aaaaatataa aaattagccg 900 ggcgtggtgg gcacctgtaa tcccaactac tcgggaggct gaggcaggag aatcgcttga 960 acttgggagg cggaggttgc agtgagccga gatggcacca ttgcattcca gcctgggtga 1020 gagagtgaga

ctccatctca aaaagaaaaa gaaagaaatc aaaagataca aaaattagcc 1080 tggcgtggtg acacacacct gtcgtcccag ctactcggga ggctgaggca ggagaatcgc 1140 ttgaacccgg gaggcggagg ttgcggtgag tggagattgc gccactgcac tccagcctgg 1200 gcgacagagt gagattccgc ctcaggagaa aaaaaaaaaa aaaaaaaagc ggaggtggtt 1260 gtttgagaag atgacgtctg aataaagacc tcgaacaagg aggccacaca gacagtaggg 1320 gaaacgtgtc ttaggatttc aggcagcaga aaacagcaaa agccttgcag tgggagcatt 1380 tggggagagt acagaggtaa acgtggctac ggcaggagtg agggggtgaa taggggtaga 1440 tgagcccaga agggtgatga agagccaggt gccgtagacc gtcataagat tgcctcgtaa 1500 aacagggagt tacttctggg tttgagcggt ggagtgcctt gtaggtgaca agggtggaaa 1560 cagagagacc tttaggttaa ctgagaattg aactatgtgg tggtttgaga ggaggcacga 1620 acggttttga aggtgttgct gatgagatct gatgacaagt tgcgtgctgg gtattagaga 1680 cagagatttg cctttagtct gagcctctgg aagggtgaag ccaccatcag gcaaaagtag 1740 cgcagggcac aggcacagca agtccaggga agaccgcggc tgggggcctg tgtagcgcag 1800 ggcgcaggca cagcaagtcc agggaagacc gcggctgggg ggcctgtgta gtgtgtttga 1860 gggtatgttg tagaggttgt tgggtaggag acgtgagtct gccaaaaacg tacctcctgg 1920 caatcctgtc ttgctagttg gtaacccttg tgaagggagt agactgaccc tgtaggccac 1980 tccccgcccc ctctacttcc acttcccact ggggtcgctg acttctgcct cctcagccgg 2040 cgtaaaggcc gtgcctctga gcacaaagac cagctctctc ggctgaagga cagagacccc 2100 gagttctaca agttcctgca ggagaatgac cagagcctgc taaacttcag cgactcggac 2160 agctctgagg aggaagaggg gccgttccac tccctgccag atgtgctgga ggtgagggcg 2220 tgggccaaac cagaaggggg gcacttgtct ctacactctc cttcagctca gcctttctgt 2280 gcaggaagcc agtgaggagg aggatggagc ggaggaagga gaagatgggg acagagtccc 2340 cagagggctg aaggggaaga agaattctgt tcctgtgacc gtcgccatgg ttgagagatg 2400 gaagcaggca gcaaaggtga gcagcagcca ggggcgggca gctgggtgcc caggcagaaa 2460 tctggccttg cctcacctga ggtgagcagc agcgaggggc gggcagctgg gtgcccaggc 2520 agagatctgg ccttgcctca cctgaggcgc ctgaggctgt gctggtggga ggggctgttc 2580 tccacgcagg ggacgcttgg agccttcttc agcgcaagtg acaacacgtt gctcctcctg 2640 cttgcttggt tgccccagga ctgtgtgttc acttttgggt agattccagg ggctctgttg 2700 gcgccccact ccccaaagtc agtcccgctg tgggtgggac ggactgtgcc tttgttggtt 2760 ggtggagctg gggtccttct gagcctctca cagtgttttt ttccagggac aaggatacgg 2820 agagctccag ataccacctg gaggtggcca tagtccagga tctggaactc ccagtccttt 2880 tcctggggct cctgagccag actccctcct cttcccagag aattctagac tttgtttcca 2940 tttttgttat cagtatgggg ctctgtgcct ccccccaacc tctgccctat gtctgagggt 3000 gagggtgagg gtgcctttct ctggggctgc cgtttccact ctgccaagtg cagtctcagc 3060 tcccctgacg cccctggtac tcttgctcct tcagcaacgc ctcactccaa agctgttcca 3120 tgaagtggta caggcgttcc gagcagctgt ggccaccacc cgaggggacc aggaaagtgc 3180 tgaggccaac aaattccagg tcacggacag tgctggtgag cttgggggga gcctggcatc 3240 caggctgtct gttgcgttct ctgtcccgtg agtacatcca ggccttttcg ttgcagcatt 3300 caatgctctg gttaccttct gcatcagaga cctcattggc tgtctccaga agctgctgtt 3360 tggaaaggtg gcaaaggata gcagcaggta agaggggagg gggtgaaggg gggtagggtg 3420 gaaggtgggt cgggaccaca cacaggagaa gccagaggcc ttgtggctag gacagagaca 3480 tggcaacaga gccagcgtct tcttggggac cctgagaagg cagcggggca cgagggaccg 3540 ttgggagagg ctgggcactt ggggccagtt ggagcggccc caccttcttt ccttcctgaa 3600 aagggaagtt gtctgctcag agtctcagaa ggtgtggcgt gtgcagctgc ctttgctctc 3660 agattttccc aggttagggt tcttgctcta tttccctttg taagaacttg taagtcctgt 3720 ccatactcct tgatctctgc actatgactg gcctgtttcc tggagctgtg tgtgtctgta 3780 gacaggggcc tcatcaatgc tgagaacctg agggcctcaa cttccccttt agagaaagat 3840 tgggcagaga aggccgtgga aggttttcag agaagagaga cgcacggcca agcatttggt 3900 agagcatgga gcacgttggg gttccggggc tgtgcagcca tgtgaccttg aggcagtgga 3960 ggcctagagt tggaactgcc ccaggacacc ttcaaaggaa gaaaggagac ccacaggaga 4020 ggtctcgccg acagcggctc aggagacagg ggcactgttg ctggtggtag tggtcggggc 4080 tggacggata gaacagagcc atgtccacat ggaggcagtg ctgtccgaag gggacaagga 4140 agctggcaga tgggggtgga gggacaggca gcgattggga ggcagcacgc actgcagcct 4200 gagccaccag gtcagggacc cctgtcgagg cagcagctgt accggaggga gtcaggccgt 4260 ggggagcaga gctgcctcct gggtctgggt gggcagctgc aggtgcccag cagccagtgt 4320 cagcacaggt gtgaggaggg tcgaggacgg ggggcctttc tggggccctg ccctttgtcc 4380 ttcccatgct tcagcagatt ttagtgagcg cctactgcgt gggctctggt aaatgttttg 4440 ttttgttttg ttttgtttgt ttggtttttg gttttttttt ttccttacga gataggatct 4500 tgctctgtcg cccaggctgg aatgcagtga tcatagctca ctgcgacacg taccttctgg 4560 attcaagtga ttctctcacc ttagcctccc aagtaactgg gactccgcca ccaggcctgg 4620 ataatatttt gtatattttt gtagagacgg ggtctcactg tgttgttcag gctggtctca 4680 aactcctggg cttggagttt gatcctcctg ccttggcctc ccaaagtgca gtgagtactg 4740 gcatgagtct ccacacctgc cctctgattt tcttttcttt gtgcagcaaa tgtgggccaa 4800 aggaaaccag tctgcggcag ttggtggtgc ctgggcgacc ccagaggtgc tgagtaccag 4860 ctgctgggct tagggacctc gtgtggtctc acggtgggga tgtgatcaag gggacctaga 4920 aaaggtttat gtctgagagg gagttggagg ctgggacttc cggggactct tagggcggtg 4980 gccaaccctg ggcagggcag acaggagttc aaggccactg aaggaggagc taatgcactt 5040 gagagggtcc tcctaagccc ctgtgtctgt ccagctgtaa ggggccctga gcttttttga 5100 gtggagagac ggggggtctc tgcagagtcg tagaggctat gctggccagg gcacgcgcca 5160 acatgctgag cagcctcgcg tccgagccgt ggggcctccc agccaggggt gggtggtctc 5220 tctgcaggat gctgcagccg tccagcagcc cgctctgggg gaagcttcgt gtggacatca 5280 aggcttacct gggctcggcc atacaggtgc tattctggtg gggagggcac gggggcctgg 5340 ctgtatctgg tggtcggtcc ttttttgtat cccagaatac atgggtttgg ggcttcactg 5400 tcccctcctg cccccagctg gtgtcctgtc tgtcggagac gacggtgttg gcggccgtgc 5460 tgcggcacat cagcgtgctg gtgccctgct tcctgacctt ccccaagcag tgccgcatgc 5520 tgctcaaggt tcgtggccca gtcccctccc tgtgtctgtc atggggtcgg ggggccacac 5580 ggctaccccc accacatccc actcctggcc agggcacagg tggtgcccac actccactgg 5640 ctccgcttgg ctagagaggc cacagaagca cctggccccc acccccacct ggggtttctg 5700 tctcaggcag tccctgcctg cccgggcagc gcggctcagt ccgtgagcaa agccacggtg 5760 aggtatcttc ttcccggtgt gatctcacat acgtcggcgt gtctgacgtg gtaaactttt 5820 gtggtcctct tctcacaagg agcaacacac ctgttctgtt cacttctgta actgagcacc 5880 taactcacgg ctcccccgag gtgctttggg aagagctggt ggccatggga gcctttgcct 5940 ggctggggag ggctgtgctg gccctggggc atccctgctg aggaggctgg ggggccacca 6000 gtgacgtctg accttctgca gagaatggtg atcgtatgga gcactgggga agagtctctg 6060 cgggtgctgg ctttcctggt cctcagcaga gtctgccggc acaagaagga cactttcctt 6120 ggccccgtcc tcaaggtagt ggtgggccct gcgtctgtgt ccctcagcat ctgcattgga 6180 aatctcggcc taagggcagg gcgggctgcc tttgtggttg gtgcccctca ctggaccctc 6240 actgcagctc tgagcagacc tgggcccttg gatcacgaat gtctcacaga gcacctgggg 6300 gtggtgggca gggaagagag ccctcggcct tctcagggcc ccacctgacc ctgcttcaca 6360 cagcttcccc aggggagggc ctctctggct ggaggaggac actgggtgtt gggacctgag 6420 gccatggcca gggtacagtc ctactgcccg tccccaagcc atgggtggcc tgcatgtggg 6480 gaccctcagc ccccagaggg gcccagtgtg cagcaggagc ttctgcccca gcttctccca 6540 ggcctgaggc tggtgggcac ttgggggtgg gggctgtgca aggggctacg gctcttcctc 6600 gaggcccagc tctgagggaa aggcccaggt gttcacaggg gccctggagt gggcggtgga 6660 ggtgcatggc cctgatccca ggtggctctg acccgggtct ctccgcagca aatgtacatc 6720 acgtatgtga ggaactgcaa gttcacctcg cctggtgccc tccccttcat cagtttcatg 6780 cagtggacct tgacggagct gctggccctg gagccgggtg tggcctacca gcacgccttc 6840 ctctacatcc gccagctcgc catacacctg cgcaacgcca tgaccactcg caagaaggtg 6900 tgtggtgggg cccttccagg ctcatgctgg gcatggggtg gggcagccca ggtgcccgac 6960 ccaaggcagg gcctggggcc tccccgaagc ccctgtctgg agacagccca gcaccctggt 7020 gcagtcggtc cttgcaggtg ggggaagggt ggatgggttg agaccccgtg tgcaagatga 7080 ggaaatgatt cctgtgccgg cccaggagga acgtgcatca gcctgacttg tcagcctggc 7140 cagtagctga cgtggttctc tctgaccagg aaacatacca gtctgtgtac aactggcagt 7200 atgtgcactg cctcttcctg tggtgccggg tcctgagcac tgcgggcccc agcgaagccc 7260 tccagccctt ggtctacccc cttgcccaag tcatcattgg ctgtatcaag tgagttgtgg 7320 ggtgggcagg gttgtgcggg agggtcagga gaagcaaatt tgctgggact gtgtgggcgg 7380 tgcctctggc gttcagggcc ttggggcctg agtctgtgtc ctggccgtcc ctgaggaagg 7440 gctggggtcc ctgtacctgc tacggggaga tgctctggat tctggagagc tagggctggt 7500 gggcacctgt gacatgagct cctccaacag cggtttagcc gcctcgggtg ccacccagcg 7560 tgtgttctgg gggcttgtgt gcagtttgca gtgagtttgg ttcattacgt ggggttcttg 7620 ggtggagcac atctgatgca gtgaactgca ttttgggtgt gagcgcttag gagggtccag 7680 gcacggtagg ggctgcagca ggaaaaggtg ggagcagtac tgtggcctct ttggccagga 7740 gggggcatct gtctggctga gccttagaaa ctcaaggctg ggaagggagg tgggaagtcc 7800 aagggaagaa gtaggaaagg cgggaacagg gaggagagca ggcaggaaca gatgacccct 7860 gccaccgtgt tgaaataaag ctgaaagctg ggtaaggtac ctgcagcccc atagctgggc 7920 aaggggtgca ggtccctgcg gttcagacgt gccttgtcct gctttagggg tctgatgtcg 7980 gtgagtgggg gaggaggtcc aagacagcag ggggaggggc aggggctgcc agagccgggg 8040 cctctgctca ctcggccttc ccacccccag gctcatcccc actgcccgct tctacccgct 8100 gcgaatgcac tgcatccgtg ccctgacgct gctctcgggg agctcggggg ccttcatccc 8160 ggtgctgcct ttcatcctgg aggtgagtga ggctgtggtg ggcgtgtggc acctctgcct 8220 gctcctgtag ggagcatctg ctgctccggg cgtctgtgct gagttgtccg gcgacttccc 8280 ggagccctgg ccgcctcctt gtcacgggtg tcacgaggac agtgctcctc cgtgtgctgg 8340 gggcagtgtt ggggaacgtg ggaccactgg ggatgaaggc ggctgctgct agggctgtgc 8400 ttgaggatgc cgggacctga ccctgtaggt gcttgcccaa gactggagta ggcaggaggc 8460 gagatgggcc aaaacccaag tctgattgct gaactgtaca ctgaacagtg ccctgccctg 8520 acagttgtgt gcatgtgtat gcatttgtat ggaggatgtg tgtacatctg ggtgggtgcg 8580 tttgtctgtg catgcatgcg tgtgtgtgca ccatctgggt gcatgggtct gtaaatctgt 8640 gtgtgtgtgc atatcagttc atgtgtgcat ctgtatgtgt gtatgcacgt gtatccatga 8700 atgcctgtgt gcctgcaggt gtgtgcatct gtgcgtgtgt acacctgtgt gtatgcatgt 8760 gtgtaccttt gcgtgtacct ttgcgtgtgt gcacctgtgc atgtgtcttt gtataccagt 8820 gtgtacctgt gtgtacctgt atgcatgcac atgcgtgtgt acctgtgtgc acctgtctgc 8880 atgtgtgtac ctgtgcgtgt gtgcacctgt gtgcatgcat ttgcgtctgc atgtgtctac 8940 ctgtgcatgc atgaaccttt gcatgtgtgc atctgtgtgc atgcatgtga tgtgtgtctg 9000 cgtgcatcta tgtacctgtg tgcacctatg tgcatacaca cgtgtctgtg tgcacttgtg 9060 tgcatgcatt tgcatctgca tgtgtgtact gtgcgtgtgt acctgtgcgt gtacctgtac 9120 acttgggtgc atgcatgcac gtctgcttgt gtgtgcctgt gcgtgtgtgc acctgtgtgc 9180 atgtacgtgc gtctacgtgt gtgcctgtgc atgtgtgcgc ctgtgtgtac ctgtgtgtat 9240 gcatctttgc acgtgcacat gccactcagg tagggaaaga ttggagtccg aagtttcaac 9300 tttcagtggg tgggttaggc cccaccccgc tgtaaattta ggaattcacg atctccaccc 9360 tgtttatcta atgagttctc agcctcatga aggcccagag tcgtgtcaca gctgtccttg 9420 gggctgggtc ccaggttgct gggtccagaa ggtatggaag ccccaggcac gttctgattc 9480 cccttccact gaggcaggga tgctgaacat ctttaggaag ccatgttcac tcccatggca 9540 gccagcagtg gtctctgatt gcccagccct tggcctggcc cctgtgtctg tgggcctcca 9600 gctctgctgc ccagctccag gcatgctttg tgtctgtttc ccttgtccaa tctccttggc 9660 tacgtgcttt cttactctct tgcagtgtct gtttcttcac ttgtgcactg ccctggttca 9720 ctgcagccgc accctgtagg cccctctcac gcagggatgc aggcctctcc tctccggaaa 9780 aagcaaaccc taaaagctaa aacaaagccc tcagctgtag gccgtgcctg cccttccccg 9840 gtgcctggac aggaagccag tcgcctgccc atacttttgg cccaggctag agaagggcag 9900 tgtcctccca gaggttcatc agtaccaggg cgttttccca tctggacctg agctcagctg 9960 tctggcagcc acccctgctg agtggggtgt cttgctgggg cctccaccct tgggcccccc 10020 ataatctgct tctgtcctct ggtgccccag catgtaccct ggatctctct ggttcacagc 10080 ctgagggctc ctagtggttg gggaggggtc acaagactga gaggccaggc tgactctttc 10140 tctgctcctc ctggcatgtc ctacggaggt gcatggcctg tggcttctgt ggagggtgtg 10200 ggaggggccc cccaggcctc ccgtgacctc catctgtccc gtcctgtgtc tggcactctt 10260 tgctgttgct gctgcgtctt ctggttgctc gggacggagc cccatgtggc attgctgtgc 10320 tgagggccag gatgggcctc agtgccatgt tgtcaggaat gggggctgtc ctggtactct 10380 gtgtggcagg gacctctagg tctccagacg tgggtcctta gtgcttccca ggattttggg 10440 agagggcccg tgttcctgat ccttccctgc tgatcagagc cccactcggg gacacgccag 10500 gctgtgtggg gccatggggc tgggaccgtg cctagctgct tatctcttgt ttcgggttgg 10560 gtctcctcgt gctgaagcct gaggaccagg gtgaccaggg tgcagccagg tgcagggcca 10620 aagggaccag ggggaccagg gtgcagccag ggtgaagcca gggtgaccag gcatggggcc 10680 gagagagcct gacactggcc cttggggcag atgttccagc aggtcgactt caacaggaag 10740 ccagggcgca tgagctccaa gcccatcaac ttctccgtga tcctgaagct gtccaatgtc 10800 aacctgcagg agaaggcgta ccgggtgagg ctggctcctg gggagggcct gggcagttcc 10860 cagggtgggg ttgggggtgc tgaggtggat gggagggggc tggcatcctc caagttcaag 10920 catggacctt catggtctcc cagggctggg gcatggagcc ccttccttgc agtccgtgcc 10980 ctggagatgg cgctgccctg acagcctgag gggaaggggc ctgagtgcag ccccagcctc 11040 tccctgatgc actcggccct ctctcctgct cttcaggacg gcctggtgga gcagctgtac 11100 gacctcaccc tggagtacct gcacagccag gcacactgca tcggcttccc ggagctggtg 11160 ctgcctgtgg tcctgcaggt gtgtgtcctg cccacaccgg ctcgtggccg actcagactg 11220 tcttataacg ggcttggctg ccccaggctg gtaggaggtg cccttgtccc gggtacctgg 11280 gactggggtg gaacctgact gctggcaacg cgccccgtga ggcccccttg gaggggctga 11340 tgaggggttc taagccattc aggaggtttg ggggcaggcc tgggcagtgg gctgaggacc 11400 ctgggagcac aacagcctcc tccccaggtg ggaaccacaa ggctgatctt tgcttcgggg 11460 ttgggactga gcatgccgac cctggctcag gctggtacgc tgatcgcaca cttccccagg 11520 ccatcccggg tgtggggagt gggtggagtg gcttctcagg tggcaggaag gcctggcctg 11580 ccccgccaaa taccccacat cagcctcata ggaaggccca gcctgccccg ctaaataccc 11640 cacgtcagcc tcatcttagg caggagtggg gtggaggagg gggttctcct tatcctcaga 11700 aggtccttct gggcccccac gggaggtctg tttgctctca gccgtgatgt ttccagcctc 11760 agggagcgct gtgctagtgg aaggggtggg ggcctgctgt ctggccctgg tgctggaatc 11820 agatgtgccg actataggtc tgtgcagtgt ggggaggagg gatctgctca catgagccac 11880 aattggtcag aggcttatcc agatagaggt gtgtgcatgt gtgtgtactc acacacggcc 11940 acacatgtca catgcacaga gccgggaccc cctttctggg gcactcacag catgggccac 12000 agcctctgtt cctgcccatg tcctacttgg gtggtgatac ctggcattgg ggcatctctg 12060 cttctggact caagggccca gggtcgggtt ctggggtagg ggtcagaaaa tgtttttggt 12120 gaggggccaa atggtaaacg tggctttcta cggatgttca gctgcacagg caactgcaga 12180 accatgcaat gcacacgtgt ggctgcacgt cagcgagact gtattttatt agtagtagta 12240 gtattgtttg agatggagtc tcgctgtgtt gcccaggctg gagtgcaatg gtgcgatctc 12300 ggctcactgc aatctccatc tcctgggctc aagcgattct ccagcctcag cctcccaagg 12360 agctgggttt acaggcgccc accaccacac ccagctaatt ttggtatttt agtagagacg 12420 gggttttgcc acgttggcca ggctggtctt gaactcctga cctcaggtga tccacccgcc 12480 tcagcctccc aaagtgctgg gattacaggc gagagccacc acgcctggca taaaacttta 12540 tttccagagg tgagtagtgg gcagggttgg cccagagggt gggctgattc tgcctctgcc 12600 atcccagctg caacagctat gcacttgagc cctgagatgg gatccatgtc ccctcctggg 12660 gtatccccgt ggccaccacg cgtggttttg cacaggacct gggccagctg tgcacatgga 12720 gcggtcctgg gcttcagtgg ctgacccctc ccttccgcag ctgaagtcgt tcctccggga 12780 gtgcaaggtg gccaactact gccggcaggt gcagcagctg cttgggaagg ttcaggagaa 12840 ctcggcatac atctgcagcc gccgccagag ggtttccttc ggcgtctctg agcagcaggc 12900 agtggttagt gggccctggg ggtagtgcca cctgagggca cctgccaggg tatagcccca 12960 gctacatgtg ggggtttgcc cagggtgagg catgaccctg aactccccca accccccagg 13020 aagcctggga gaagctgacc cgggaagagg ggacacccct gaccttgtac tacagccact 13080 ggcgcaagct gcgtgaccgg gagatccagc tggagatcag tggcaaagag cgggtgcggc 13140 tcggcgaggg gacctggggg tgtgttgtga cttcctgggt ttcagatcta gcgcactatg 13200 acttgagacc agggcgaggg tttggaaaca gtgccaggcg gccagggccg tgcccggatg 13260 attcgacttg gagagggggt aggtgttgga gaactggcca gaaccaggcg tttccaggga 13320 ggggaagccc caggctgcac taggttgggg aggccatgcc ccctcaggcc tgatgggctg 13380 gaggctccgg gcaggtggag tggctggact gacctcgtca cccaggccag tatgtgggca 13440 ccaggggccc gtgaggagaa gcaggaaggg ctctgccttt gaccttggac atgggatgga 13500 caacttggag gatggctttg tgatttggga acagagggga ctagaaattg gccacatggg 13560 gccctggtgg tgggtctggc gatgcctggc cctgctgtgg ccgccagccc ctgccctctc 13620 tcacctgagc ccctggttct ttggccttcc agctggaaga cctgaacttc cctgagatca 13680 aacgaaggaa gatggctgac aggaaggatg aggacaggaa gcaatttaaa gacctctttg 13740 acctgaacag ctctgaagag gacgacaccg agggattctc ggagagaggt ggggcctgcg 13800 tggtgctccc aggggaaggg tgggcctgga gggctctgct ggacttccca gagccacgag 13860 ggccacctgt acccatcctg cagggggctc accagtctct ggcccagctg gggccaacct 13920 cagtgttgcc aggcttctgg tgccagcgcc ttccctcctt gaagtgaagg cctactggga 13980 ttggtaactc tgtccccagg cctgtgacct cccagttcct ccccagggct cctctccacc 14040 tgctggaagt cagcggaggg aagggtgttg ggagcctggc caccctcctg cccccactgt 14100 gactttgctg gtggaccctg tgggtgggag tcatatggac tctgcttctt gttcctcagg 14160 gatactgagg cccctgagca ctcggcatgg ggtggaagac gatgaagagg acgaggagga 14220 gggcgaggag gacagcagca actcggaggg tgaatggtct tggggtgaga gggtgtggcc 14280 ctgtgagccc atctggcggg agggcagagc cacgtgggcg gggggcgtgg ggctctgggc 14340 caggcttttc cctccctggg aaggccaggc caaatgctct gttctctggc agccagcaac 14400 agggataaat taattagtgc cgtgattaat tagtgatgag taacctctaa ggctggcttc 14460 ttcctgataa agcaaaattt atgtagcctc catctctccc cgcagatgga gacccagacg 14520 cagaggcggg gctggcccct ggggagctgc agcagctggc ccaggggccg gaggacgagc 14580 tggaggatct gcagctctca gaggacgact gaggcagccc atctgggggg cctgtagggg 14640 ctgccgggct ggtggccagt gtttccacct ccctggcagt caggcctaga ggctggcgtc 14700 tgtgcagttg ggggaggcag tagacacggg acaggcttta ttatttattt ttcagcatga 14760 aagaccaaac gtatcgagag ctgggctggg ctgggctggt gtggctgctg aagccccaca 14820 actgtgggct gctgaagtca actccgcggg ggaaacttac ccttgacgtc agcagaccga 14880 gaccagttcc cagttccagg ggaaggcttc aaggcccctg gcccttccac ccacctttgc 14940 cctcagtatg cagacctcgt ccatttgcac caggttctgc cttcactcca ccaagtcttt 15000 gaaatttgtt tcctttcctt tgaagtcaca ttttctttta aaattttttg ttttgcatcc 15060 gaaaccgaaa gaaataaagc ggggggaggc agggccattg tgttg 15105 6 23806 DNA Mus musculus misc_feature (4293)..(4312) n is a, c, g, or t misc_feature (6219)..(7065) n is a, c, g, or t 6 aacatgcagg atgccgccac ccacttgttg gtcctcgggt tgtacttctc tatggagttg 60 aggctggagc tgccgtcatt gccccccacg gcgtagagcc agccgtccat ggccaccagg 120 tcatgtgtgc tcctggggag ttggataggc aagagtaagc ccaaggggca agccaagaga 180 caggccaaat gagtggggca cagagggagt aaccataggg acagggcaac gaaaggagcc 240 ccctatgccc ggggggaacc caaggacctg ccgtactgtt gaggtgctgt gtgcacaacc 300 cctggggcta cactcctgga ataaaccaga tgtagtggca gaaccactca ccacccgccc 360 ctggccttat acacctgcgg atattcatgg gtgccacact ctcccaggca ccagccttgg 420 tgctatatct ctccactgag ttgaggcagc ttgtgccatc attgccccct gcgacataca 480 gggcaccctc cagcactgct actcctgctg agctgcgccg gctcagcatg gaagctacag 540 gtgtccatga attcacctgt agagacaata ggacaggctg gaggcagacc caccaagggc 600 cccccaaccc tgccaccaca tagactctac tcttggttgg gtatctgagg gtcaggggtg 660 ggatatagaa agcagacccc ttgggtggca cagatggcca gaatctggga ttatgcccca 720 tcccccccag cactttagag gaggacattg tgatggggtg cgctatgcac ctggggctca 780 tacttctcca cggtggccag gtgtgacgag ctgtcataac cacccacggc gtacaggttc 840 ccatctgtgg gaaggcaata tacccacgaa

gctcctgcag ggtgtaggag ttttgggggt 900 gggtgggaag gggagaggca ggaaagcggg taccacggga agcctgtcac ccaccaagtg 960 tggccacacg cacatatcgc ctccgggtgc tcatggcagc aatggatgtc catgttcccg 1020 tcagtgggtc atatcgttca gcactgtgga caccagaatg atgccgacgt tcagttacaa 1080 cagttaagtc atccttgccc aggctctgtc ccctcactcc ccccaaagct acggctgaaa 1140 tcccgggcat cacagactct atgaatagag agcaaggtgt gctgggcgga tactggctgc 1200 tggctgcttg gactggaggg agggcccctc caggaggaga atggtgccaa ggccctgcgg 1260 atgctgtgcc ctgaggtaag gaagccttga ggacccagta gcctggatta ggccctcatt 1320 ctcttagcca gatgtgactt catagttaag ccagccttag ctgctgctgc tgagccgacc 1380 ctgcaaagcc actggggccc taacccaaac tactacctgt tgaggcagga agccccatcg 1440 tagccaccag ctgcatacag gagcccatgc agagcagcta cacctaggca gctccgcctt 1500 gtgcccatgg agacctcggg ctgccatgtg tttgtcacag ggtcatagga ctccaccgta 1560 gccaggtctg aggttccatc gtagctagga gaagagtccg ctactgacca aggatggccc 1620 ggcaggcttg tgacaaacct ttcagcctac atggccagct tctatgctta cccgcccaca 1680 gcatacagtc ggttcccgac ggcagcaact cctacacgag cccgacgtgt agacatggag 1740 gccaccacat gccaacggtc agtccgcgtg tcatacgctt cacagtctcc gtggatcgcg 1800 aacaggctcc caccacctag agatgggtaa ctataaataa aggggctcaa ggctgggcag 1860 aggactctag tgtgacccaa gtacaaagac atgaaggctc caggggtggg aagagataga 1920 gactgggaca cactagtggg ccccatccta tagggtctct gagacacaca ggtctaagta 1980 taatgggttg ggtttatagt ctgagttggg gtggatggcc tagggcaaaa tgggaaggag 2040 aaggggttta gcataccgac agcaaagagc acagggccag cgccctcaca tcgacggggt 2100 ctcgtgcggc tggttcccag aacacctctc tgctcaggca gcaggtggaa cttaagggcc 2160 tcgatgagca gatccttgca gtcagggtga tgcctcacaa ggctttcggc atccacgtgg 2220 cccaggagga agtctcggct cagcagaggc agccgtacac acttcatcaa ctagggcagg 2280 aagcgccatg taaggcaaaa ccctgggcgt acttggccca gggctaagac ccactcaggt 2340 gcctatagca cctgtgagat tgggtcttga gacaggtggc tcaaccactc caaaggagtc 2400 cctggatgga agatgctaag gacttgaaga agtaagctta taccgcagcc acctgcattc 2460 ccttctgaca gcacgggagc tccaggacct ggcctcaccc ttggtacatg ctgcctccga 2520 gtgtctacgt catgtttgac ccagctcagg actgcacggt acacatcttc ctctgagggc 2580 acgttcaggc tatcactaga gaccaattcc agcacctggg ggcgagaggt cctcagacct 2640 gacatctggg taggggaggg cccagagaac cctgaacgtg tctgagagta tcagacagag 2700 ggcatgtgcg tgtccatgca caaacacgca cacttaacgt tcttagagtg aagaaccaag 2760 cagagtcaag attcacagat tttgttccca gtagaggggt gaggggtgtc caaggtccta 2820 agtagtctcg ggtacataga atcagagact gggaaatcat ggccctagaa tgcggtaaag 2880 acaggcatct aagacaaatg attgggtgac atgagaagat ggaaacaggg ttgggaggag 2940 atggccaggg aatgaatcat agggtgaaac tcgcaccaga aacttgaaat gaggattgca 3000 gaacactggc tgcaagggtc tgggcttggg caatacatgg tgggggctgg gcccaaactg 3060 gcatgttacc tgcttcagtg gttacctgct tcagtggcaa cagcatgaac tcctcagtct 3120 tggccacatc cacgaagtgc tgcagcacgt acctgtgtgc tgccttgagc aggtcactgc 3180 atgagtgtgt gtctgcaaat cctcgaatgc ccaggcagtt ggaagggtcc agctggctca 3240 ggaggaactt gcagcaggca tctcgaacac cattcaactg tagaaggctg gcagctggga 3300 gcagagtctg gggcatgaag atgggtgagg caggagggtt ccagaagagc acagagtgac 3360 tggatattgg ggagaggcat gggggggggg cagggcacac agatctgggc tggagatctc 3420 aggggacgtg ggtatgggaa tggtaggcct cacctgaacg ttgccctcgc ccaccacgat 3480 ctcagctgtg tatgcaaact gcaccagctg gtctaaggct tgcgggtcaa tgtcatgcag 3540 tgtcacatgt gtctggcggc tctcactcat ctcatctgtg aaaacagtgg attaggcggc 3600 ctacccgggg agatctgcgg agattccccc tagggctgcc cagactgttg tatgtggtac 3660 ttgcttgtga acatggcgtg gaagtagggg ctacaggagg ccaacaccac cttatgagcc 3720 cggatctcct tggcagccac atgcaggacg atgtcgcaca ggaggccacg ctgccgcatg 3780 cggctcatag ccacaaaagc atcatggtag tgccgctttg agttgtgtgc cacactgtga 3840 ccctctctgc tcaacagctg catcgcacct tccatcgggg ctgcaggccg ggcctgccgg 3900 ggccgagctc gctctgcctc ggggctgcag gccgcaggac taggtcaggg tttgtaccct 3960 gtagagggcc actcacccgc ctttgagaac tcccagcaca gccaaggcag ctgcggtcta 4020 tgcccaggag ttcgttcagt catcaggcac caaagcccat ggaggaccag cctgtcactc 4080 actgccactt tccagggctc tagatctctc tccggtccag gaacctcgct ggagccggag 4140 cagattcttt cataaatgta agcctgctct acctgtcccc cttcctctgc tgactacctc 4200 tcctggggct ccgagcccct catctagtgc ccaggccact catggggagg aagttggcaa 4260 acgttgtcct ctgaatcccc agatcccagt cannnnnnnn nnnnnnnnnn nncccgccct 4320 cccgcccgcc ctcccactca cgccggcggc tggggcggcg gtggcgcctc aggcccgggc 4380 cccgggctgc tgtgctcggg actctgcgtc ctgcccgccg gccgctcgcc acggggctgc 4440 ataactccag ccgccaaagc cgcgaccctg atggacggat tgggacgcgg gtgcccgccc 4500 ggccgctcgc tccactcggg cgctcgcccg tcgccgcaga ccctgtttct cggcgccgcc 4560 cgccctgccg cacgcaaggt tgcaggagcc gctgccaccc gcgtccccgc tctcctccca 4620 ggagcgcctt ccttcgtgtg gcgcctaggt tagcttcaca tcccagcgcc ccggttctca 4680 cctgctagag cttgctgtga ctgacaaagc tccatagcac gggggtagag cgggacactt 4740 gcaagtgtgt gtcccccccc ttaccctcag gcctcctgtg cacaagcgcg tgcaaaggtt 4800 cagcgcccct atacatgcat atttgagcat gaggcggacg ggggctgcta gcgtctgaga 4860 ggtgcaatta tctattgaca tggacgcagc attcttgtca cacccaggct cgccaaggaa 4920 ttgggtgtcc tttgacttaa aaaaaaaaat atatatatat gtttatatat atatattata 4980 tatatatata cccgtacaca taatatatat atatatatat atacatacac acatatatat 5040 atatatatat tttttttatg tgtacgggtg ttttgcctac ttgtgtacta tataaattac 5100 gtgtgtgcag tgcctacgga aaccagaaga gagcatcaga cctcctggga caacattggt 5160 gatccacttc gtagaccctg ggaattggac cccagtcctc tggaagagca gcttagtgct 5220 tttaagtccc ggccatctct atgcagccct tgcttgctcg gtcggtcggt ctgctgcctg 5280 cctacaattt atcatctatc aatccatcca tctatccctt tatctgcttt tttcctggaa 5340 tttgctctgt atggactgat ctcagagaca gcacagagcg agctcgcttg tctctgagcg 5400 cggagataaa aggcgtgcgg tcgggacagc cctttacttt ttaaaaaaaa aaaagatatt 5460 tttccccctc caccctccgg gcgtggtagg caggctgttc agatttccag ccttgtttcc 5520 cgggtgcgag cggtcgttcc aggcggggct ttgagctgtg agagcctgag ccgagctcgc 5580 cgccctcccg cccggggacc gcggccgcgg cggctcgaga acccgaaagc cagcagcagg 5640 ggtggcatgg gcggggctcg cgggaggaag tcggagggcg caaggcattc gtgggcccgg 5700 aagtgcggcg cacgcggctg ggcgcgccat ggctgcgtct cgcgctcccc gcaggtgagt 5760 ggcctgggcg ggcgcgcgga cccgggcggg cgcctggtcg cggggtccgt tccgatcctg 5820 acgcggtgtc actggcaggc gcctggagga cctcagtgtg gacgagttcc tggcttccgg 5880 cttcgagtcc ggatccgagt cggagctgga gggcgccgcg gaggcggcgg cggaggagcg 5940 cagggcgcga ggagccgcgt ggaaccggga gcggcggggc gcgcgcacct ccccgggccc 6000 cgcaggacgg tacgcggggc tccagaaagt ctttttgcaa aacgggcagc tgtcgtgggc 6060 tggagcagcg ggttgagctt aaggaacaca cgacgggggc ctcggggtcc gagcgatggg 6120 aagggttgca aggttggaaa tggaggagag cttggagggg gaagcgcacg cgtggttggg 6180 agggggaagc gcacgcgtgg ttgggagggg gaagcgcann nnnnnnnnnn nnnnnnnnnn 6240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnagggg gaagcgcacg 7080 cgtggttggg agggggaagc gcacgcgtgg cttggagggg gaagtgcacg cgtggttggg 7140 agggggaagc gcacgcgtgg ttgggagggg gaagtccacg cgtggttggg agggggaagc 7200 gcacgcgtgg ttgggagggg gaagcgcacg cgtggcttgg agggggaagt ccacgcgtgg 7260 ttgggagggg gaagcgcacg cgtggcttgg agggggaagt gcacgcgtgg tttggaggtg 7320 ctgttggagg agagctgagg acggtctgtt ttacagcaat gaggcgagca tggtgggtgg 7380 ggtcacagct gctctggggc cgggagggcg ctcagcgggt gctgagtttg agatggttgt 7440 gttggaacaa tgtcagaggt gctgtgcgtg tgacgatgga ggagaggcaa gcccgatggt 7500 catcgatccc acagtgaagg gaataaacta acatggcctc acggaggcga caaccagatt 7560 ggctgtgtag ggtcgaaaaa ctccgtcccc cgacccgaca cccgttttct cagcccgcgt 7620 aagggccgcg cctctgagca caaagaccag ctctctcggc tgaaggacag agaccccgag 7680 ttctacaagt tcctgcagga gaatgaccgg agcctactgg acttcagtga ctcggacagc 7740 tctgcggaag aagaagagcc attccactcc ctgccagaca cgctggaggt gaaacccggc 7800 agacctgggc gtgggggcac ctgtggtttc tatacaccgt cccagccttg tccttctgta 7860 tctggtccag gaagctagcg aaacagagga agacggagga gaggacagtg acgcgttgcc 7920 cagagggctg aggagcaaga agaatgagcc tgtacccgtg accctcgcca tggtggaaag 7980 gtggaggcag ggctccaggg taagaagcag tataagctgg gtggctgcat ctccacaggc 8040 gggagactct ggcctcgcct cgtttgaggc gtctgagaca gcaggtgaaa agactgtgtt 8100 tctgggaagc gcacaatgga ggtctttcca agtgccaggg tcctccagct acgtctcctt 8160 atttgtagtg catagtcctg cctgcttcct cacactctct atcagggttc tgacccttgt 8220 ggggtggttg tagagtttgt ttcatttctg ttaacaggat accacagacg gggcaatttg 8280 ttttttgttg ttgacttact cacctgcata taggttggtg taccgtatgc aagttcatag 8340 ttaaccgatg gttgtgagcc actgtgtggg tgctgggagt caaacctgga tcgtctggaa 8400 gaacagtcag tactcttaac taccaagcca tctttccagc tccagactag ttaatttgga 8460 aagaaattta actggctcac attgctggag gtaggcaggt caaaaagtac ggtgatgtct 8520 ttgttaaagg ccttgtgttg tgatccttgt tcagttacat cttttgaatt gttagaaatc 8580 aagcccagga cttgtacatt gtaggtaatc acactgtcct gagccctgag ccttttcgtt 8640 tggtttagtt tggtttggtt tttgagacag ggtttctctg tgcagccctg gctgtcctgg 8700 aactcactct gtagaccagg ctggcctcga actcagaaat ctgcctgcct ctgccttcca 8760 agtgctggga ttaaaggcat gtgccaccac tgcccggatg tttcatgtga tcttcattac 8820 tttccaaagg ctttatttcc aaataccacc aaataaactt ggatattaaa tttctactac 8880 ataagccttt gggctatagt caggcttagc attatttctt cactggggag tgggaaatga 8940 agtcagtgtc ctttgaataa tttcacactt aacttagagg ttttgggatt tttttttttt 9000 tttttttaca gttggagatc actatgtaaa tcaccctggt ggctaacttt gaactctatg 9060 tagagtagtt tggcctcaaa acttgctgta gtctccttgc ctttgctttc ccagtgctgg 9120 cgtagccatc agctatcatg cttggctttc actttggttt ctttagggaa gggagtgaaa 9180 agggagattc ccatacactt tctggagcaa gtttgtaatg cagtattctc tcccctttct 9240 tctgggactc ttgacacagg ctctgcctgt ccaggtgtgc gcaataccta ttttgttgtt 9300 ggctcatggt ccagtgatct tagccccaca gttgctctgg ccctgagatt gtgggttaga 9360 ttagcacctt ttcaacttgg agcaggttcc tagagccagg tactgtgaac tactctttta 9420 gtgatcttga tctcttgccc cttcagcacc accttagtcc caggctgttc catgaagttg 9480 tacaggcgtt ccgagcagct gtagccacca cccaaggaga gcaggaagct gctgagactt 9540 gcaggttcca ggttgcagat agtgctggtg agctggggac tggggacctg tgccaggctt 9600 cagtgtgaca cctgtgtgcc ctatataggt gaatgacact ttggttcctt gcagtgttca 9660 atgctctggt tactttctgc attcgagacc tctgtggttg ccttcagaag ctgctgtttg 9720 gaaagacacc aaaggatagc aataggtaag gtatggtcag gggttttgtc attaaggtag 9780 aggaggtcac aaagtcttaa ggaagcacag aaagcagcta ataaatacag atgagtcatg 9840 gtggggctgg gagtagacat agccatgtgt cttgagatag cagatgcaga agagcaaccc 9900 taaccctaac ctgaaggaga agatggtggc agttggttga tgcactgaca ggatgagtac 9960 atctagaggc agcagagagt gaggagagca agaaaaccca acaggtggag agggcagaaa 10020 ataagccagg tgatatgaca gaagcctaag tgaatggcta acacacccag aatctgggga 10080 gcagttgtca tgaagggaca agtgagattt tagaaacagg gttgccctct gaattgcata 10140 caatgcttga ggtgtcacac agatgagata gtgttggtct aggtatctgg aaggttagac 10200 agtggggtgc cttaaatggg ctttggtttt tgttcttggt acagttcaat agatcttggt 10260 aaacacctac tatgttgcca ggtgttcatg gttatacggc ttgtgctcgt gcaggcttgt 10320 gtctgaattt cggaatgggg tgggtgtatt ccctgcaggc tgctgccatc cagtagccca 10380 ctgtggggga agctccgtgt ggatgtcaag acatacctaa gtgcggtggt gcaggtacag 10440 aatcccagga gacctggtca tgtctgatag tctggtgctc tcttacaccc cagattccag 10500 tgactttctc ttcttcttta gctggtagcc tgtctagcgg aagccacggt gtctgcagct 10560 gtcctgcagc atatcagcag cttggttcct tactacctga cattccctaa gcagtgccga 10620 atgttgctca aggtacgtta gcttgtcctt gtcctactcc tggctagaat agaggcagca 10680 tttgtgccta tacttgctct gctcctactg tgtcctctac tcagcaaggg tccccagtct 10740 cagatggtcc cccgtccttt caggacagcg tggcttagta atgtgagact aggctccact 10800 gaggcatgcg tgctagccat ggagggttct aggccaggaa aggcagcgat gtatcttttt 10860 tcagaggatg gtggttctgt ggagcacggg tgaagaatct ctgcgggtcc tggccttcct 10920 ggtactcatc agagtctgcc ggcacaagaa ggaagccttc cttggtccca ttctgaaggt 10980 agcgtgagtc ttctatttgt tcattaaggt ctgtgttggg aattgtggtt gatggtcctt 11040 acgaggactt gacaccaaac tctgatcata gttttcttac caagaaaatt gggtgatggt 11100 gggaacagga acccatggtc ttgtgggtat tggtggtgat ggtgggaggg gctcttttcc 11160 tctgggtacg agaacgttat acctagaaaa taggaacgtg tctgaggcca ccaccaagat 11220 cagatcctgc tgtgtagctc caggcctgga gtggcatcac ctggactcct gctgtacaga 11280 cctagctcga agtagagcgt ctctacttcc ttcctcggcc ttcggtccag tccctgccta 11340 gtcctcatct tacccttctc ttgtacacct tcctctcttc tgcatatctt agattatttg 11400 tggtgggtac aagtgtgtag gctgagaaac atcagagttc ttgagtgctg ggaaccattc 11460 aggaattgag tctgcagctc tttcagacgg cagttctaat ggaaaaccct aggtgtgggc 11520 aggtggcttg gaactgaaca gtcgttgctc aggtggctct taattttggt ttgtcagcaa 11580 atgtacatca tgtatgtgag aaactgcaag ttcacctccc ccagtaccct ccccctcata 11640 agcttcatgc aacggacact gactgaaatg cttgccttgg accccagcgt ctcctatcag 11700 cacgccttcc tctacatccg acagcttgcc gtccacctgc ggaatgctat gaccgcaggc 11760 aagaaggtag gaggaggtca gacccttcga agccagtagc cccacccggg ccttagggtg 11820 gctagaagcc actctttaat gggggaggct gctcagcgct gagggaggct ttgggtggtt 11880 ctggcaggga agaacgggcc taggatgtgg actcactctc cccaggagac acaccagtct 11940 gtgtacaact ggcagtatgt gcactgcctc tacctgtggt gtcgagtcct gagtaccctt 12000 ggttccagtg agatcctgca gccgctactc taccctctct cacagatcat cattggctgt 12060 atcaagtgag ttcagggaca gggctatgtt agattagcct aggggttggg ggcagcaagt 12120 tcctcttggt atagggcaga agttcctgat ttgctagctt ctgtgaatta cattatacaa 12180 agaattttct cgggtggcca caccttaaat cttagctttt gtggctatag tctctgagga 12240 agggcctgag aagatgaact ggatgtggat ttcaggcagc tgggcttcgc agtcagcctc 12300 agcttaggta aataggttag gtgtccacag tgtagcacct atatggaccc ggctcgggcc 12360 cttgtgctat tggagggcaa tcagatgtag ttctaagtgc ttttgatagt ctgcgaatgt 12420 gctgtgccta cctctttggc taggaggggc ctctgtgtcc tctcgagctt tctcctgaca 12480 ctggagactg agctaggaag gggaagaggc aggtggtaca agaaaacaag gcagcaggtg 12540 gtgaggaaag gaggcaggag tgaatgaact aagctaaagt tggatgagcg cctttcagtc 12600 tcgcagctga gcagtggggc accaaggcca agcattttgg ttgtccctgt tggttttgct 12660 gactgtttaa attactattc cccatgggtg ctatgagtac ccaccctgta ggagtggcca 12720 gggtctcctg gagttggagc ttgtgaacgg ggaggagtca gaggcagcgg gcagttaact 12780 gatgtttggg cctggctacc tgactgggct ccgctcctct tttctccagg ttgttgccca 12840 ctgctcgatt ttatccattg cgcatgcatt gtgtacgtgc cctgacactg ctgtcccaga 12900 ccatcggcac cttcatacct gtcctgccct tcattctcga ggtgactgtg ctgggcacaa 12960 ctcgtgttgg acttagaaat gtctttagga aaccacacct ccgtctctgc gtctgccagt 13020 gctaacttct aacaatgacc ccgtgacccc cctctctttg cagtccttac agatctgcat 13080 tgagtctctc caagtctcac agttactatg acacctttct cgttttcctt attgtacctc 13140 taccttgagt cgctgtccct gccctcgggc ttcctatcag gtgcagattc cttgagaaag 13200 tgaagcacaa gggtgaatcc aagcccctgc taacaccgac accgcccttt ctttagtcta 13260 tggcgaggat aaaggtggat attgataacc agctctatat attagttagc atcctttgct 13320 cagagaaagg actttcccta cctccaattt attaatcact gtagctggtt caccttctag 13380 caacatgtta cgtaagttct cttatcaggg cttccctata gaacctcccc tctctatgct 13440 tctgtgtgta ccttgtgcct ccttggttta cctgttctgt gtggggcatc ctcggaccaa 13500 gaggttaggc taggctccga tttccctctt aggaaggcca aagtgtatat agagcctgca 13560 aggtttgttg ggtgtggaaa acacagtctc ccttctgtct ttctttattg gtggttttgt 13620 ccatagtgcc tgggatgcgt gaggcccaga tccgtatcta aaggcggtta agtgtatgca 13680 ggcgtcctta tggttatctg tcaaggggct cagggctctg gaaggcctgg tcttggggga 13740 tctctttggc tcgggcttac actctgtggg tagattttcc agcaggtgga cttcaatagg 13800 cggccaggtc gcatgagctc caagcccatc aacttctctg tgatcttgaa gctgtccagc 13860 accaacctgc aggagaaggc gtaccgggtg aggctgaact caaggccttc tgggaaaggc 13920 ctggggctat gttttagttg gtttacaagg acttaataag gccgttgata tacaagggag 13980 ggattcccat tgtggaccat ttgtggtttc ttttgagaca agatctctct attatattgc 14040 tcttgctgtc ctggaacttg ctatattgat caggctagcc ttgaactcac agagttccac 14100 ttttcaaatg ctaagattga ggtttttgta gtcatggagc aatgactcct taatctcagt 14160 tcccgtgaag ctgggtgtgg cttggggggg tctgtgcgta ctgttttata gccctggtct 14220 tggagattga gcagagttgg ggtgaaatgt aagaaaatgg gctactgggc ttcatcatct 14280 gatgaggttg ggcctgcccc ccaggacggg ctgctggaac agctgtgtga ccttactctg 14340 gaatacctgc acagccaggc ccacagcatc gctttcccag agttggtgtt gcctactgtt 14400 ctacaggtat gtactcatca tctgacttcc tttccctggc cagaccttgg cgtcctcctt 14460 gtggctatct tggacaatct tttagtaggt ttgtcgtccc cttatgttct agtcctggac 14520 cagtagcaac accgtgttta tccagttacc aatgggctgc cagatccatt ggcgccagtg 14580 cagggagaga gcgatcattt gccttgatag cttaaggaac tttctctagg gactgtggaa 14640 ttaacttgta aaggattatc ggcctctgct gaagttgggc tggggctggg taacttaatc 14700 aggatctcag gaatttgcag acccttcccc agtctgactg gagcctacca tcaactatgg 14760 aagtctaaag cctgggttgg tctggtgccc gttttgtttt tccgtgtggt catggtctct 14820 ctgtgcccct tctgagaatc tgtttgctct cagctcagct attttccatc taggtccttc 14880 tcataggagg gggctggtgc tgtctctttt ctggtactgg taggggatta gatccattgc 14940 tcacagactt gtcgtggtgt tgctcatacc tgagctatgc ctgggtcagg ctcctatggg 15000 cacaggtgta tgtgtgacgt tacatgtata gctgtgcaca cggctgtgtc gcggttggtt 15060 gcataatgtg gcattggctt ccccctgtgt cctcttggct gatgatgtca ggtagtcagg 15120 atacctttag ttttaggctc agcattttag gtgtatggat gtgtggatct agagtagaag 15180 ttagcagatg tgtttcagtt aaaataaaag tagcaaaaaa aaaaaaagaa acttttaagg 15240 tttatgtctt gtcacagctc ttcatctgct tacctctcgg gctgcgggga caagcatggt 15300 cttatgcctg tgagacctaa gagattgaca gcaggtcctt gtgattgtca catggctttt 15360 cactgcttgg acccctcgag ggtgaggaat ggaactgtca ctggagctgt actgagtctt 15420 tcctttctgc agctgaaatc ttttctccgg gagtgcaaag tggctaacta ctgccggcag 15480 gtgcgccagc tgctggagaa agtgcaagag aatgcacgac acatccaaag tcttcgacag 15540 agcgcgacct tcagcgtgtc tgaccggacg gcagtggtgg gttggacttg gcatctgcac 15600 tggaatggtg ctgcctgggg tcacctgtta gggtgtggtg gatgtttaag ggttttccag 15660 aatgatcatg gtcctgaatt tacttcaatc cttaggatgc gtgggagaag caggttcgtg 15720 aagaggggac cccactcacc agatactacg gccactggaa gaagctgagg gaccgtgaga 15780 tccagctgga aatcagtggc aaagagcggg tatggctggg gtctgtgaag atggcctagt 15840 agaaagtggc caagcaggtt atttgtaaag gggttggggc tgagggggag aagttggaaa 15900 aggaaacgaa atcaggaggg aagaaagaaa

ggtgagtttg agtgaaccct gtaagtttct 15960 gtttacctgc tgggcagctg gtccccggta ttgatataga tggttcccga ggcgcagctg 16020 ggccagttct ctcggcctct ccctaccctc acttgttgtt ctgtttcttg gtccttgaag 16080 ctagaagacc tgaacttccc agagatcaaa aggcggaagg tggaagacag gaaggatgaa 16140 gacaggaaag aattaaagga cctgtttgag ttggacagtt ctgagggcga ggacagcacc 16200 gacttctttg agagaggtga ggcctgagct ggagtggttg agagctgtgt gtgtctgggg 16260 cattcatgtc tggctagtgg gcagacctgg tttgttgttt ttaattttta tgtctgtgtg 16320 ttttggctac atgtctgtct gtatccagat cccctgagac tggagttaga gacacttgtg 16380 agcctcagtg tgggcactgg gatcaaaccc atgtccttga accactgagc catctctcta 16440 gcctccagat ctgttttttt tttttttttt taaagattta tttatttatt atatgtaagt 16500 acactgtagc tgtcttcaga cactccagaa gagggcgtca gatcttgtta cagatggttg 16560 tgacccacca tgtggttgct gggatttgaa ctcaggacct ttggaagagc agtcgggtgc 16620 tcttacccac tgagccatct caccagccct agatctgttt cttataccac tcattggccc 16680 cttatgtttg agctggggac tcagtattgg taattgtcac catgctgggg cctctatagg 16740 tggcagagag ggctagtggc cctgttgcta ctgctgagac ctggggccct gttgcatgtc 16800 cagagccagc cctcctgtgt agctactcat tgctttgtta tttcacccgg tgtgtgtgtg 16860 tgtctgtgtg tgtgtctgtg tgtgtctgtg tgtgtgtgtg acgaccgttc ttcatttgca 16920 ggagtaccta ggctcccgga agctcaccaa ggactgaaag aagatcagga agaagaagat 16980 aaagaagaag gtgacagcga ttcagagggt aagtggtttc tatatgaaat agattggccc 17040 tggagtctgt ctggaggcca gaccatggga agacagagct ctacgccagg cttctgcccc 17100 tccctggggt gccaggccag ctgctctgta ctctttagca accagctata gcatgctgat 17160 aaatttaatt aatgctgcaa ttaattagtg atgagtaact tctaaagctg gctttttcat 17220 gatatagcag aatttattta tgtggcctct tccttctctg tagatggaga cacagacacg 17280 ggagtggatc tgagcgaact gtggcagctg gctcagggac cacaagatga gctggaggat 17340 cttcagctct cagaagagga ctgagggcct cctgctgcct cctggaggcc tcgacagcaa 17400 ctgtgcggcg agaggacagc agagcgtaaa caggctttta ttgttacagc acagcagaac 17460 aagtgtacta ggagccattt tgaccctgaa agggagggaa ctgtttctgc agagccttct 17520 gctgccggaa gccgaaggct gaccctgatg ttgtcccagt tcagagtggg gagggcggct 17580 ccatggcctc tcctctccac tttgtctttg gtttgtctgc agagcactgc agttacctct 17640 agaagataaa cccattactt ttgtttgttt gtttgtttgt ttgtttgttt gtttgttttt 17700 tcgagacagg gtttctctgt gtagccctgg ctgtcctgga actcactctg tagaccaggc 17760 tggcctcgaa ctcagaaatc cgcctgcctc tgcctcccga gtgctgggat taaaggtgtg 17820 cgccacctct gcccagctca ttgcttttct tcttaagcct tatgttttcc ttttaaaaat 17880 actctcggca tttttgcatc taaaaagggg aaataccctg ggggcggagt gaggttagca 17940 atttgactgg gaatcacgga gtatgacggt tgcctgccgg atcattggag aagctgggaa 18000 gggtctgggg cacctgaaag cagagctatg gtccctgacc cattctcctg cttgggtgag 18060 gcttgtcctg tgggcaggtg ggtcccttca tacggggagg tggtggttgc tggcgacaga 18120 ggttgatgtc ctgggctgag ctcaggtgct tgcaggcttg gtggctgcag cggcagggcc 18180 acagggaagc tggccatgta gaagacacgg cccaggcgct tggccaccta caaagagacg 18240 aagctgggtg aatacctgct aggtaaaggc gcctagggaa acaactccag gtacagagaa 18300 tcctaaaacc ctctctagag tggtgaaaac actggacaaa cttacttgtg ccctgatctt 18360 gagggcaggc cctagcttca gtcccatggt gttcagaagg tgctcttctg tcagtagagg 18420 caaagtctct ccgtctattc cttgttctct gaatacctgg ggacaaggtg ggcccataga 18480 gtagagttgt agagcttgca tggaaaaata acaaaaggaa agttcttttt tgtgggagac 18540 cgggacattg tccagaactg ggccagaacc ctagggacct gccactcccc tcaccctggc 18600 atactcgcca cagccagaaa ggccccccac gaagttgcag acatcatcta cagtccactt 18660 gttgacatcc tcaagggttg tggtctcctc atcagtgaag agccccccca tggtacctgg 18720 agatagaaag gatgtcacta gggcttgggc tttcccattt ctttgcctcc ttccccactg 18780 gctgaccttt ctctgaattg gtaaagtctt tggcttaatg acaaatgatt tttctcccca 18840 cgggatcccc tatcagtggg tgactaaggc tgcccacctg tgtggaagta ggggctcact 18900 gccccgcaag ggaatcccag gggcagtgga gggggcagtg tggaccctga tagaagcccc 18960 cttccttctg ctctggtccc tcctgctggg acttggctgg gagtggaggt cccttccctg 19020 gcagctgctg tctcggggtc ctctccatct gagtccttgg atggctcctc agagacttct 19080 tgagcccaga ggccagcccc tgtcgtctcc ttgggttggc tgggctggac accaaggctc 19140 cccttttcgg accggcttcg agcagactcc ttgggtggga tagggggtcc tgggcctggg 19200 ggtccctggg gtggcagggc cagtagtggt gctgagctgt gtttcaaaac cagcatggag 19260 ccacgtcgct gcagttcgtc gggaccctcg gggatatgca aggccacctc tggtgccggt 19320 agcagtggcc ggtgaacact gcccagttct ttctgcctaa gcaactctga catttccagc 19380 ctggaccaaa aggacagacg agtcagcctg ggcacgctta gaactgctcc cgctggcctt 19440 gcagtcgctt accgagccag gctctgcttc cgtagaagtt cctgctgccg agctaacatc 19500 tcagcctggg cggagggtag gaagctgaaa cctgggatag aagaaaaggc tgctgaagcc 19560 ggatgctctt cactactttt atctatctgc ctaaagagag cctcatagga acacaaacgg 19620 cttacatacc cgactttctc acctggggtc tgacacacag ctgtgggcat ccccagaaaa 19680 ggaggcggga gatgggagct cattgtgatg tgggcggcat tctggggtga cagcaaaggg 19740 ggtggctgtg acatttccct gtggacagca gaatagtgag cacatgccta ggtccgtctg 19800 tgcacacccc ctctttcctg tcatcacctc tgggtgcacc cctgctaacc cttctagttg 19860 ccgacccctc aagtggccgt tggtttcctg attatggctt gcttgtgtac acacacacac 19920 acacacacac accccgcgcg caagtctcct tgtcagcaca gaggcaatgg ccccatgggc 19980 cacactgcgt cagtaattac cggggtgggg gatacctctc atcctgagca gtgaggggag 20040 gggtgcagga cagggcaatt agccccaatt tgtagatgtg gcagatgctg cagtctgcca 20100 ggatcagggt gagataggat gtgtttggga aacccgggga ccctgcctaa tgctctcctt 20160 ggcttaactc gtctgccgcg gagactactg tgagcatgat gtctctggag tttatagcag 20220 ggtagggatg gagggggcgt atgtccttca ctctgaatcc tctgtggttg ctttctcctt 20280 gccttcctca acatcccacc accaccacct gtgaggggag ggcctctgct ctcggggcag 20340 ctctactagg ctggaatagt cagagttgct tgacaagaaa attgtatttg gcattcttgc 20400 cagggagtgg gcatttctgc atattttgcc ccccaaattg agtctttgca gtagttttag 20460 gagcttcctg cagtggacct tgaacgtggt gaatgcatta tgcatatatg tgatggaaga 20520 ctcaagggaa agaaagaatt ggccttctca agtaacaagc atagcatgtg gggccttgct 20580 gaagcacgtc ttaatacctt tcagagaaag atgtggcagc agctggaacc gccccttccc 20640 ggcgctgaac taggccttgc ttccgtcgat ggtctgctgt agacgagggc aggtgaactt 20700 ccaagccact gggacccctc agtgttgcag ttgctacttc ctggcgtacc ctaagcagat 20760 ctgagaaggg aggtgatagc gggcccccct gataagctcc cgtaggattt cccctttcca 20820 tcttggtgag cagggcattg gcccggtgaa ggtatgaaca gagtgggagg aggagactct 20880 cacatgtcct gagcctgctt ttgtgtcaca ggctagagga tcagcaggga aggcactttg 20940 ctttaacacc tacatgctgt agccaggcaa gtgaacttga aaaggaatgt gaataagtag 21000 gtcatcgaaa ctggagaaga aaggctccaa catgtagagt attagtgcta atttggtttg 21060 aatattcaca aggtcagagg tggaagggca tagggctgct ccaagtaggt gagtgggaca 21120 agagtctatg gattaggagg ggacctcaaa cagaagcctg cccaggtaac tcctttctag 21180 acaggagaca agaacaggaa ggacttgccg tccctataaa gcgattcata gcatacatgg 21240 ttataatagc agataccact gactagcagc aacacacaat cacagacctg catggttgtg 21300 cccccattgc atttaggaag agcggaggct ccgagcccct ttaatccacc tgctcaagct 21360 ccacttctct accgctctgc ctgtttataa atcacgtagc tgaaaaaggg acgtgggcct 21420 agctagggct tgaagccaaa tcccagaccc taataagccc aggccacccc agtcatgcgc 21480 cacgcaatgg tgattgacag ccagcttgct tgcacagtct cctgcacagg ccagcagagc 21540 tgggtaagca attaaaaaga aagattttta aattattaac atttagtgaa ttattcaggc 21600 tctcggtgcc tgagctgctg ccctagggaa cagcgccttc tgcaagaatc aggaatcctt 21660 ttcccttctc tgagcccccg tccacttctg ccagaaaaga caagatttgt ccggtgctgt 21720 agcagcacag ggtgtctcac aagtcctcgg tagcaaggac ggtgcctcaa ggcttggctt 21780 ccctcaagca acctggggtg catcctgact gtgacgcagg ttcctatttt tgccatgccc 21840 tcctccctgt gtatcaacaa aacctgaaaa gtctgacaca gcatcaacac gaccacagat 21900 tcacgtattg cactgaccac tctgggagaa actagcacag gccagaggcc acatctcttc 21960 aacaaagaac aggtgaacaa actcttctgt cccaccctca cctgggctcc tcggggacgc 22020 acagtagagg actgaggcca gtgaggctgc atcactaccc ggaagccctt ctatgtattc 22080 aatcctggtc ttggtgttga cctccaagca tccagatctt tttcttgggg ttggcttagt 22140 ggggtggaaa ggtaagagag atttggctca cttgaagtct gagcgccggc cgtgggggtg 22200 gcacataccg tggctgggga tacccaagtg gtagagacgt tgagtttcct caaaggaact 22260 ggcctcactc agggccgaca taagctgatg gtagtggtcc tcaggggcca ttgtagactc 22320 tagcttggga agcagcgaag tcgctgttgg acagagcaca tggtgaggac cacggggaaa 22380 gtcaaggctt cttatggttt ccacccaagg tctggggagc ccttcaggac ccagttgctc 22440 ccccccttcc ctgtgacctg catccttttc cagagtcggg ctttcacctt gggggtcctt 22500 gcttctggcc ttcttctctg aagagcagtg actggtgatg caggggcagc tcagcctctt 22560 gcccaacagg tcgcctgtag aggatagagt gagccccacc cagtgtctca tggcttcgag 22620 agaaagccat ctctgcctta gctcagtctg gagccccaat caccactatg ttctccctgg 22680 cagttaaaga ccttgtccga tctggtgtcc caggctagag agcacgatga caggtagatg 22740 gagagtgcag taaagacaaa gcgatcctga cgctggcctt gggcacgtaa gtaacagtga 22800 gagctctgca tcagtgtttc cccagtcagt ctcctgtaag ctccttgcct ctcccctcgg 22860 tgcatttgcc catctgcata tccaggtctg ggttcttaat gggactccta agcatttcac 22920 tccatagtgc acagtctata tgtgctatgc ttttatgagg ccaggccaga cactgcttac 22980 aatatcccca gagataatac atgccgaatc tcaggcatta attgacccaa ttccagactg 23040 gcctgaacac tgctgcctaa cttcctcctg agctcctgcc ctgaggctag ggcctttgct 23100 gaacagccca tgacatcgcc atcatggctt caagctaagg ttcctgtctc tgccctcctt 23160 atcctcacca gctaggggag tgtggaggaa caattccgtg ggtgattcaa aatctccata 23220 atgcccaata aggggctgct tctacttgtg tgcccaccct ccctcaaagc tggtggtgtc 23280 agcgccagga ctgaattcca tttaaaaaaa attccttaat ttgtcatttt tatcagattg 23340 gggaagtgtg gcagcaaaaa cgctgatcaa tctccacgtt atggctgaca gaatggtgct 23400 aataacttat tgatctcaac tacctcactg ctgttagtct agcaggccat ctcagcctcc 23460 gccccagaga aagagacagc gcctactgtc tggacaaggg ctccacccat aatatctcaa 23520 ggcagatggg tccagcagac agataatggg tatctggctt cacttggtgc caccaatagt 23580 ttatcagaga ccagtaggca gaagaggcag cagctggagt agacaggaag gagggtccct 23640 caggtctgtg tggcctggtc tcactcactt tgtacctgtt attcttgtcc caacccacag 23700 atccccttta aggactaggc aagggctctt ttcacagcag tctattctca ctttaaaagt 23760 ggctaccccc aaaggagagg gggagggaga gagggagaaa aggagg 23806 7 163 PRT Homo sapiens 7 Met His His His His His His Gly Met Ala Ser Glu Phe Gly Ser Ala 1 5 10 15 Gly Ser Arg Lys Arg Arg Leu Ala Glu Leu Thr Val Asp Glu Phe Leu 20 25 30 Ala Ser Gly Phe Asp Ser Glu Ser Glu Ser Glu Ser Glu Asn Ser Pro 35 40 45 Gln Ala Glu Thr Arg Glu Ala Arg Glu Ala Ala Arg Ser Pro Asp Lys 50 55 60 Pro Gly Gly Ser Pro Ser Ala Ser Arg Arg Lys Gly Arg Ala Ser Glu 65 70 75 80 His Lys Asp Gln Leu Ser Arg Leu Lys Asp Arg Asp Pro Glu Phe Tyr 85 90 95 Lys Phe Leu Gln Glu Asn Asp Gln Ser Leu Leu Asn Phe Ser Asp Ser 100 105 110 Asp Ser Ser Glu Glu Glu Glu Gly Pro Phe His Ser Leu Pro Asp Val 115 120 125 Leu Glu Glu Ala Ser Glu Glu Glu Asp Gly Ala Glu Glu Gly Glu Asp 130 135 140 Gly Asp Arg Val Pro Arg Gly Leu Lys Gly Lys Lys Asn Ser Val Pro 145 150 155 160 Ser Thr Ile 8 176 PRT Homo sapiens 8 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Arg Trp Gly Ser Glu Glu Gly Thr Pro Leu Thr Leu Tyr Tyr Ser His 35 40 45 Trp Arg Lys Leu Arg Asp Arg Glu Ile Gln Leu Glu Ile Ser Gly Lys 50 55 60 Glu Arg Leu Glu Asp Leu Asn Phe Pro Glu Ile Lys Arg Arg Lys Met 65 70 75 80 Ala Asp Arg Lys Asp Glu Asp Arg Lys Gln Phe Lys Asp Leu Phe Asp 85 90 95 Leu Asn Ser Ser Glu Glu Asp Asp Thr Glu Gly Phe Ser Glu Arg Gly 100 105 110 Ile Leu Arg Pro Leu Ser Thr Arg His Gly Val Glu Asp Asp Glu Glu 115 120 125 Asp Glu Glu Glu Gly Glu Glu Asp Ser Ser Asn Ser Glu Asp Gly Asp 130 135 140 Pro Asp Ala Glu Ala Gly Leu Ala Pro Gly Glu Leu Gln Gln Leu Ala 145 150 155 160 Gln Gly Pro Glu Asp Glu Leu Glu Asp Leu Gln Leu Ser Glu Asp Asp 165 170 175

Claims

1. A polypeptide comprising at least 20 consecutive amino acids from the sequence of hsGRIM1 having the sequence ID No. 3.

2. The polypeptide as claimed in claim 1, comprising at least 40 consecutive amino acids.

3. The polypeptide as claimed in claim 1, comprising at least 80 consecutive amino acids.

4. The polypeptide as claimed in claim 1, comprising at least 120 consecutive amino acids.

5. The polypeptide as claimed in claim 1, comprising at least 200 consecutive amino acids.

6. A polypeptide comprising at least 20 consecutive amino acids from the mmGRIM sequence having the sequence ID No. 4.

7. The polypeptide comprising at least 40 consecutive amino acids.

8. The polypeptide as claimed in claim 6, comprising at least 120 consecutive amino acids.

9. The polypeptide as claimed in claim 6, comprising at least 200 consecutive amino acids.

10. An antibody that binds specifically to an epitope of a polypeptide as claimed in any of claims 1 to 9.

11. A medicament comprising a polypeptide as claimed in any of claims 1 to 9, and a pharmaceutically acceptable excipient.

12. The medicament as claimed in claim 11, that is employed for the treatment of disturbances of skeletal muscle differentiation.

13. The medicament as claimed in claim 11, characterized that is employed for the treatment of disturbances of fat cell differentiation.

14. A method for identifying substances which influence the biological function of a polypeptide as claimed in any of claims 1 to 9, comprising contacting the substance to be identified with said polypeptide in a test system.

15. A cDNA which codes for hsGRIM1, comprising at least 1200 consecutive nucleotides from the sequence ID No.1.

16. A cDNA which codes for mmGRIM, comprising at least 1200 consecutive nucleotides from the sequence ID No.2.

17. A transfection vector comprising a cDNA as claimed in claim 15 or 16.

18. The transfection vector as claimed in claim 17, which is an adenoviral vector.

19. A cell transfected with a vector as claimed in claim 17.

20. The cell of claim 19, wherein said vector is an adenovector.

21. A method of treating a disturbance of skeletal muscle differentiation comprising administering to a subject in need thereof an effective amount of the medicament of claim 12.

22. A method of treating disturbances of fat cell differentiation comprising administering to a subject in need thereof an effective amount of the medicament of claim 13.