JP2001507226A - Proteins and compositions for regulating mitosis - Google Patents

Abstract

  (57) [Summary] The protein encoded by the human gene HEC (highly expressed in cancer) contains a long series of leucine heptad repeats and appears to be important for normal mitosis. HEC localizes to the nucleus of interphase cells and redistributes to centromeres during M phase. Ectopic expression of a mutated HEC containing only heptad repeats results in cells that cannot divide more than once. Inactivation of HEC results in disruption of sister chromatid alignment and separation, and the formation of non-viable cells with multiple fragmented micronuclei. HEC, via its leucine heptad repeat, interacts with several proteins involved in mitosis, including: nek2, sb1.8, and MSS1, two different regulatory subunits of the 26S proteasome. And p45. We note that the biochemical properties of these HECs may play a role in regulating proteins that are important for spindle attachment to the kinetochore, sister chromatid migration, and progression to M phase. Show.

Description

DETAILED DESCRIPTION OF THE INVENTION Proteins and Compositions for Regulating Mitosis 1. 0 BACKGROUND OF THE INVENTION The government owns rights in the present invention under grant numbers EY0 5758 and CA58318 from the United States National Institutes of Health. 1. FIELD OF THE INVENTION The present invention relates generally to the field of molecular biology, and more particularly to compounds and methods comprising novel DNA segments and polypeptides encoded by them, which are important in regulating cell growth. . The compounds can be adapted to control cellular malignancies and various other cell proliferative abnormalities at the cell mitosis stage. 1. 2 Description of Related Technologies In recent years, understanding of cell cycle progression at the molecular level has rapidly advanced. When a cell is stimulated to divide (eg, by a growth factor), a cascade of different but intersecting signals transmits the stimulus from the cell surface to the nucleus. The kinases are then activated, initiating an event in the early G1 phase, which causes the cell to rapidly enter the next phase and growth (Pines, 1995). The role of cyclins, cyclin-dependent kinases, and their inhibitors (CKIs) in contributing to cell cycle machinery has been reviewed (Harper and Elledge, 1996; Pines, 1995). The ultimate goal of cell division is to ensure high fidelity transmission of the replicated DNA to daughter cells. The physical separation of sister chromatid pairs into two daughter cells is precisely organized during the M phase, and many of the events involved are highly conserved in all eukaryotes (Yanagida , 1995). To ensure accurate progression through cell division, each step is matched by a group of structural and regulatory proteins that serve as checkpoints to monitor timing and accuracy of previous steps (Hartwell, 1992). Mitotic B-type cyclins, cyclin-dependent kinases, other kinases, and components of the centromere / kinetochore have all received considerable attention (reviewed by Harper and Elledge, 1996; He et al., 1995). . Under normal circumstances, incomplete cell cycle events are detected and cell cycle progression is prolonged until problems in previous steps can be resolved. In addition to transcriptional control of gene expression and phosphorylation-dephosphorylation, cell cycle progression involves targeted degradation of proteins that regulate key transition points. Two important checkpoints in mitosis, sister chromatid separation, and exit to G1 include, for example, the need for disruption of certain proteins (eg, late inhibitors and mitotic cyclins) (Glotzer et al., 1991; Holloway et al., 1993; Hunt et al., 1992; Irniger et al., 1995). Degradation is achieved primarily by a pathway involving ubiquitination (reviewed by Deshaises, 1995), a series of enzymatic modifications that mark proteins for destruction by multisubunit proteases (called proteasomes). . Many components of the ubiquitin pathway and proteasome have been cloned and characterized in yeast (Hilt and Wolf, 1995) and humans (Dubiel et al., 1995). Studies on mitosis In recent years, progress has been made by using fungi, Drosophila melanogaster, and Xenopus laevis to analyze molecular and cellular processes during mitosis in detail. In particular, yeast was utilized because of the relative ease with which those genes could be engineered to select for mutants defective in mitosis. Several genes leading to M-phase arrest have been isolated (reviewed in Hegemann and Fleig, 1993). Some encode proteins involved in proteolytic processes (eg, CIM3 (Sug1) and CIM5, both of which are subunits of the 26S proteasome in Saccharomyces cerevisiae (Ghislain et al., 1993; Swaffield et al., 1992)). Some (eg, fission yeast Nuc2 (Hirano et al., 1988) and Cut9 (Samejima and Yanagida, 1994)) are classified by sequences encoding the tetratricopeptide repeat (TPR) domain (Goebl and Yanagita, 1991). You. Nuc2 / CDC27Hs have recently been shown to associate with the centromere and mitotic spindle and function in the ubiquitin-mediated proteolytic pathway (King et al., 1995; Tugenreich et al., 1995). Kinases such as NimA in Aspergillus nidulans (Osmani et al., 1988) are homologous to human Nek2 (Schultz and Nigg, 1993) and have a protein phosphatase 1-α form, PP1a (Booher and Beach, 1989; Doonan and Morris). , 1989; Cyert and Thorner, 1989) also lead to mitotic arrest when inactivated. Other proteins, such as SMC1 and SMC2, are essential for chromosome segregation and condensation (Strunnikov et al., 1993; 1995) or tubulin (Weisenberg and Rosenfeld, 1975) and kinesin-like proteins (Walczak and Mitchison, 1996). (Reviewed in) are essential for spindle formation. Although the number of known proteins and genes required for chromatid separation is increasing rapidly, the exact mechanism responsible for mitotic molecular events remains elusive. Recent evidence suggests that metaphase arrest induced by primary structural abnormalities in kinetochores may also require interactions with proteins involved in spindle assembly and monitoring of mitotic checkpoints (Wang and Burke Wells and Murray, 1996). Proteins with properties similar to those of HEC have been characterized. For example, a nuclear protein (NuMA) that associates with the fission apparatus is also required for proper completion of mitosis (Compton and Cleveland, 1993). When NuMA is inactivated, either by a schematic mutation or by microinjection of pre-mitotic anti-NuMA antibodies, abnormalities in chromosome alignment and segregation result in the formation of daughter cells with micronuclei (Compton And Cleveland, 1993; Compton and Luo, 1995; Gaglio et al., 1995; Kallijoki et al., 1993; Yang et al., 1992; Zeng et al., 1994). In addition, some features of the abnormal mitotic phenotype (e.g., multiple spindle poles and disrupted metaphase chromosome alignment) may be caused by drugs that directly disrupt microtubule structure (e.g., taxol and vinca). (Alkaloids) in milk cells treated with the same (Jordon et al., 1992; 1993; Tinwell and Ashby, 1991). All of these drugs arrest cells in M phase, as well as injection of neutralizing antibodies against all known CENPs. Studies of molecules and checkpoints involved in chromatid segregation and checkpoint control are important for aneuploidy, ie, changes in chromosome number, are common in cancer cells, and are evident from incorrect chromosome segregation in M phase (Solo mon et al., 1991). The strong link between aneuploidy and cancer suggests that altered regulation of the mitotic process also contributes substantially to tumorigenesis and tumor progression. Furthermore, defects in the ubiquitin-mediated proteolytic pathway may enhance genomic instability or cause loss of control of cell growth and proliferation by affecting cyclin or CKI degradation (Bai et al., 1996; Zhang et al., 1995). For example, removal of mitotic checkpoints while retaining daughter cell viability confers clonal expansion advantages and may eventually lead to cancer. Therefore, understanding the molecular events of mitosis is important in learning that ways to control the chromosomal abnormalities observed in malignant cells can occur. This allows for the identification and development of drugs to control cell growth. Certain proteins important for mitotic progression must be identified, characterized, and linked to known pathways of mitotic protein regulation. 2. Summary of the Invention We describe the characterization of the human nucleoprotein HEC. HEC was isolated as clone 15 (C15) by its interaction with retinoblast lung protein (Rb) in the yeast two-hybrid system (Durfee et al., 1993). HEC proteins appear to play an important role in chromosome segregation during the M phase. Because HEC proteins are most abundantly expressed in rapidly dividing cells, they are localized in the centromere during mitosis, and when inactivated, prevent the next cell division, during chromosome association and segregation. It leads to serious abnormalities. The present inventors have characterized a novel gene, HEC, which plays a clearly important role in the M phase. Several lines of evidence indicate that HEC acts as a regulator to coordinate sister chromatid separations. First, HEC is most abundantly expressed in mitotic cells but not in terminally differentiated cells. Second, HEC redistributes into the dividing cell centromeres. Third, just as the expression of dominant negative HEC mutants containing only a long series of leucine heptad repeats interferes with M phase, inactivation of HEC by microinjection with specific antibodies delays M phase. Seriously interfere. Finally, HEC has several proteins important for mitosis (Nek2, sb1 .2) due to its leucine heptad repeat domain. 8, and two different regulatory subunits of the 26S proteasome, including MSS1 and p45). These results indicate that HEC can function to regulate proteins that mediate spindle attachment to kinetochores and to regulate the checkpoint of M-phase progression. The data suggest that HEC may function as an “adaptor molecule” due to its long leucine heptad repeat. In this regard, HEC may have properties similar to those of the S. budding Skp1 protein (Bai et al., 1996; Connelly and Heiter, 1996); alter the conformation of the multi-subunit complex, and (Including components of the mitotic spindle or kinetochore, components of the 26S proteasome, kinases or phosphatases, and checkpoint monitors). The dynamics of the spindle apparatus is regulated, at least in part, by the same kinases and components of the proteasome and the ubiquitin-dependent proteolytic pathway with which HEC may interact (Holloway et al., 1993; Irngriger et al., 1995; King et al. Tugenreich et al., 1995). The regulatory events during chromosome alignment and segregation are rapid and precisely timed, and they can be greatly disrupted in the absence of matching molecules such as HEC. 2. 1 Novel mitotic regulatory polypeptide Thus, in an important aspect, the present invention relates to the discovery of novel human nucleoproteins that have been found to be highly expressed in cancer cells. A novel protein, HEC, appears to be important in mitosis, presumably in regulating normal progression of M phase. The peptide sequence (SEQ ID NO: 2) has little homology to other Genbank database deposited protein sequences available at the time of the invention of the present invention. 2. 2 HEC Pharmaceutical Compositions Another aspect of the present invention includes novel compositions comprising isolated and purified HEC proteins, or nucleic acids encoding HEC proteins. Of course, it is understood that one or more than one HEC gene can be used in the methods and compositions of the present invention. Thus, nucleic acid delivery methods may require the administration of one, two, three, or more homologous genes. The maximum number of genes that can be applied is limited only by practical issues, such as efforts to co-prepare the majority of gene constructs, or even the potential to induce deleterious cytotoxic effects. Compositions that use the novel HEC proteins include a biologically effective amount of peptide (s). As used herein, a “biologically effective amount” of a peptide or composition refers to an amount effective to alter or modulate M phase mitosis. As disclosed herein, different peptide amounts (eg, amounts of about 6 mg / kg to about 11 mg / kg) can be effective as indicated in vitro and in vivo. The clinical dose will, of course, be determined by the nutritional status, age, weight, and health of the patient. The amount and volume of peptide composition administered will depend on the subject and the route of administration. The exact amount of active peptide required will depend on the judgment of the practitioner, and may be unique to each individual. However, in light of the data presented herein, determination of the proper dosage range for use in humans is straightforward. Compositions providing HEC according to the present invention comprise a full-length peptide having about 633 amino acid residues and a molecular weight of about 76 kDa, or a functional fragment and variant thereof (eg, the sequence set forth in SEQ ID NO: 2, or the sequence of SEQ ID NO: 2). (A region of amino acids 254-621). The term “peptide” or “polypeptide” in this sense means at least one peptide or polypeptide comprising the sequence of any of the above structures or variants thereof. The terms peptide and polypeptide are used interchangeably. In addition to including the amino acid sequence according to SEQ ID NO: 2, the peptide may include various other shorter or longer fragments, or other short peptidyl sequences of various amino acids. In certain embodiments, the peptide is a shorter sequence repeat (e.g., a leucine repeat heptad region between amino acids 254-621 of SEQ ID NO: 2), as long as the peptide still functions to regulate mitosis, or Additional sequences (eg, short targeting sequences, tags, labeled residues, amino acids intended to increase the half-life or stability of the peptide, or any additional residues for deliberate purposes) ). Such functionality can be readily determined by assays, such as the assays described herein. Any commonly occurring amino acid can be incorporated into the peptide (alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, Tryptophan, tyrosine, and valine). Similarly, any rare or modified amino acids, so-called, including the following, can also be incorporated into the peptides of the invention: 2-aminoadipic acid, 3-aminoadipic acid, β-alanine (β-aminopropionic acid) , 2-aminobutyric acid, 4-aminobutyric acid (piperidic acid), 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosin, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, Allo-isoleucine, N-methylglycine (sarcosine), N-methylisoleucine, N-methylvaline, norvaline, norleucine, and ornichi . The composition of the present invention may include a peptide that has been modified so that it is biologically protected. Biologically protected peptides have certain advantages over unprotected peptides when administered to a human subject and are described in US Pat. No. 5,028,592, which is incorporated herein by reference. As disclosed, protected peptides often exhibit increased pharmacological activity. Compositions for use in the present invention can also include peptides, including all L-amino acids, all D-amino acids, or mixtures thereof. The use of D-amino acids can confer additional resistance to proteases found naturally in the human body, and is expected to be less immunogenic and therefore have a longer biological half-life. Similarly, compositions utilizing the HEC encoding gene are also contemplated. A particular combination of genes may be two or more variants of the hec gene; or, where the HEC protein gene combines another gene and / or another protein (eg, Nuc2, Cut9, NimA, Nek2). Or even a phosphatase (eg, protein phosphatase 1-α or PP1) may be able to be combined with a gene encoding a cell surface receptor that can interact with the polypeptide product of the first gene. In the use of multiple genes, they can be combined in a single genetic construct under the control of one or more promoters, or they can be prepared as separate constructs of the same or different types. Thus, an almost endless combination of different genes and gene constructs can be used. Certain gene combinations may be designed to achieve a synergistic effect on stimulating cell proliferation and / or immune response, or their use may otherwise result in that achievement. Any and all such combinations are intended to be within the scope of the present invention. In fact, many synergistic effects have been described in the scientific literature, so that those skilled in the art can easily identify similar synergistic gene combinations, or even gene-protein combinations. If desired, a nucleic acid segment or gene encoding a HEC polypeptide can be administered in combination with an additional agent, such as a protein or polypeptide or various pharmaceutically active agents. As long as the composition contains the HEC gene, it is substantially limited to other components, which may also be included, provided that the additional agent does not cause a significant deleterious effect upon contact with the target cell or host tissue. There is no. Thus, nucleic acids can be delivered with a variety of other agents that are required in certain cases. Pharmaceutical compositions prepared according to the present invention find use in a number of applications, including inhibiting or modulating malignant cell growth, or regulating normal cell growth. Generally, such methods generally involve administering to a mammal an immunologically effective amount of a pharmaceutical composition, including the HEC composition. The composition may include an immunologically effective amount of either a HEC peptide or a HEC encoding nucleic acid composition. Such compositions can also be used to raise an immune response in a mammal. A therapeutic kit comprising a HEC peptide or HEC-encoding nucleic acid segment comprises another aspect of the invention. Such kits generally include a pharmaceutically acceptable formulation of the HEC peptide or HEC-encoding nucleic acid composition in suitable container means. The kit may have a single container means containing the HEC composition, or may have separate container means for the HEC composition and other reagents that may be included in such a kit. The components of the kit can be provided as a liquid solution or as a dry powder. When the components are provided in a liquid solution, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. If the reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. In a related embodiment, the invention contemplates the preparation of a diagnostic kit that can be used to detect the presence of a HEC protein or peptide and / or antibody in a sample. Generally, a kit according to the invention comprises a suitable HEC protein or peptide or an antibody against such a protein or peptide, together with an immunodetection reagent and a means for containing the antibody or antigen and reagent. The components of the diagnostic kit can be packaged either in an aqueous medium or in lyophilized form. Immunodetection reagents typically include a label conjugated to an antibody or antigen or conjugated to a secondary binding ligand. Exemplary ligands can include a secondary antibody to a first antibody or antigen, with a label attached, or a biotin or avidin (or streptavidin) ligand. Of course, as noted above, numerous exemplary labels are known in the art, and all such labels can be used with the present invention. The kit may include the antibody-label conjugate in either a fully conjugated form, in the form of an intermediate, or as a separate part bound by the kit user. The container means generally comprises at least one vial, test tube, flask, bottle, syringe, or other container means into which the antigen or antibody is placed, and preferably can be suitably aliquoted. Where a second binding ligand is provided, the kit will generally also include a second vial or other container into which the ligand or antibody may be placed. Kits of the invention also typically include means for restraining and including the antibody, antigen, and reagent containers for commercial sale. Such containers may include injection molded or blow molded plastic containers, which hold the desired vials. 2. 3 HEC Antibodies In another aspect, the invention includes one or more antibodies that are immunoreactive with a polypeptide of the invention. Antibodies can be polyclonal or monoclonal. In a preferred embodiment, the antibody is a monoclonal antibody, as exemplified by antibody 9G3 specific for HEC as shown in immunoprecipitation and immunoblotting studies. Polyclonal antisera (eg, polyclonal anti-C15 serum) are also part of the invention. This polyclonal antiserum recognizes the HEC protein having the amino acid sequence of SEQ ID NO: 2. Means for preparing and characterizing antibodies are well known in the art (see, eg, Howell and Lane, 1988). Briefly, polyclonal antibodies are prepared by immunizing an animal with an immunogen comprising a polypeptide of the invention and collecting antisera from the immunized animal. A wide variety of animal species can be used for the production of antisera. Typically, the animal used for the production of antisera is a rabbit, mouse, rat, hamster, or guinea pig. Rabbits are a preferred choice for polyclonal antibody production because of their relatively high blood volume. Antibodies (both polyclonal and monoclonal) specific for HEC or a selected epitope of HEC can be prepared using conventional immunization techniques, as generally known to those of skill in the art. One or more experimental animals (eg, rabbits or mice) can be immunized with a composition comprising an antigenic epitope of HEC, and the animals then begin to produce specific antibodies to HEC. Polyclonal antisera may be obtained by simply allowing the animal blood and preparing a serum sample from whole blood after allowing time for antibody production. To obtain a monoclonal antibody, a laboratory animal, often preferably a mouse, is first immunized with an LCRF composition. Then, after a period of time sufficient to allow antibody production, a population of spleen cells or lymph cells is obtained from the animal. The spleen cells or lymphocytes are then fused with a cell line (eg, a human or mouse myeloma line) to create an antibody-secreting hybridoma. These hybridomas are isolated to obtain individual clones, which can then be screened for antibody production against the desired HEC peptide. After immunization, spleen cells are removed and fused with plasmacytoma cells using standard fusion protocols to create hybridomas secreting monoclonal antibodies against HEC. Hybridomas producing monoclonal antibodies to the selected antigen are identified using standard techniques (eg, ELISA and Western blot). The hybridoma clones can then be cultured in liquid medium, and the culture supernatant can be purified to provide a HEC-specific monoclonal antibody. The monoclonal antibodies of the present invention are proposed to find useful applications in standard immunochemical procedures (eg, ELISA and Western blotting), as well as other procedures that can utilize antibodies specific for HEC epitopes. . It is further proposed that monoclonal antibodies specific for a particular mitotic regulatory protein can be utilized in other useful applications. For example, their use in immunosorbent protocols can be useful in the purification of native or recombinant HEC species or variants thereof. In general, both polyclonal and monoclonal antibodies to HEC can be used in various embodiments. For example, they can be used in antibody cloning protocols to obtain cDNA or genes encoding HEC or related proteins. They can also be used in inhibition studies to analyze the effects of HEC in cells or animals. Anti-HEC antibodies may also be used for immunolocalization studies to analyze HEC distribution during various cellular events (eg, to determine cellular or tissue-specific distribution of HEC peptides under different physiological conditions). It is useful in. A particularly useful application of such antibodies is in, for example, purifying native or recombinant HEC using an antibody affinity column. The operation of all such immunological techniques is known to those of skill in the art in light of the present disclosure. HEC mRNA is abundantly expressed in rapidly dividing cancer cells 3. BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood with reference to one or more of these figures, in conjunction with the detailed description of the specific embodiments set forth herein. FIG. HEC mRNA expression Figure 1A; HEC cDNA clone 1. Northern blot analysis of poly A selection RNA (2 μg each) from human brain (lane 1) and WERI-RB-27 cells (lane 2) probed with an 8 kb fragment. FIG. 1B; Northern blot analysis of total RNA from 12 different sources; 1, CV1 monkey kidney cells; 2, human brain; 3, C4-I cervical carcinoma; 4, C4-II cervical carcinoma; 6, MS751 cervical carcinoma; 6, SiHa cervical carcinoma; 7, Caski cervical carcinoma; 8, Molt4 acute lymphocytic leukemia; 9, T47D breast carcinoma; 10, HT-3 cervical carcinoma; , SW620 colon carcinoma; 12, WERI-RB-27 retinoblastoma. Blots were probed with C15 and Gβ-like cDNA, respectively. Gb-like mRNA is constitutively expressed and thus acts as an internal control. The amount of HEC mRNA compared to Gβ-like mRNA was determined by densitometer on RNA blot. FIG. 1C; HEC mRNA expression changes with cell cycle progression. CV1 monkey kidney cells were arrested at various stages of the cell cycle by serum deprivation or drug treatment. Lanes: 1, G1 (density arrest, time 0); 2, late G1 (8 hours after release from density arrest); 3, G1 / S border (aphidicolin arrest); 4, S (4 after release from aphidicolin arrest). Time); 5, M (nocodazole stopped). E2F-1 mRNA expression (peaking at G1 / S) and Gβ-like mRNA expression act as internal controls. FIG. HEC cDNA sequence and its encoded protein. FIG. 2A; complete nucleotide sequence of HEC cDNA. Potential NimA phosphorylation sites (Ser165) are underlined and long leucine heptad repeats are marked with a series of circled residues. FIG. 2B; a protein with an apparent molecular weight of 76 kD was specifically identified by a polyclonal anti-HEC serum. Using mouse serum raised against the GST-C15 fusion protein, ³⁵ S-methionine labeled protein (from either in vitro translated full-length HEC cDNA (lanes 1-3) or metabolically labeled T24 bladder carcinoma cells (lanes 4-6)) was immunoprecipitated. . For lanes 2 and 4, pre-immune serum was used rather than anti-C15 antibody. In lane 6, anti-C15 antibody was preabsorbed with GST-C15 antigen before immunoprecipitation. FIG. HEC distribution in organs, rapidly dividing cells, and differentiated cells. FIG. 3A; HEC protein expression in all mouse organs. HEC immunoprecipitated from organ lysates were detected in the thymus, spleen, testis, and ovaries and uterus. p84 served as a loading control. FIG. 3B, HEC expression peaks at M phase. T24 cells were either not synchronized (lane 1) or synchronized with G1 and were released over various time periods (such as G8 = 8 hours after release). Hypophosphorylated Rb protein (p110 ^RB ) And various phosphorylated forms (pp110 ^RB ) Sectioned the stages of the cell cycle: G1 (lanes 2-5); G1 / S border (lane 6); S (lane 7); and M (lane 8). P84 served as an internal control for protein loading. FIG. 3C, U937 lymphoma cells in exponential growth phase were induced and differentiated by the addition of phorbol ester (TPA). In cells that divide rapidly at time 0, the Rb protein is predominantly in the hyperphosphorylated state (pp110 ^RB After cell cycle arrest and terminal differentiation to monocytes / macrophages at 96 hours, Rb is predominantly hypophosphorylated (p110). ^RB ). In contrast, HEC is present in proliferating cells but not in terminally differentiated cells. FIG. 3D, unsynchronized (U) NIH 3T3-L1 preadipocytes, identical cells synchronized at G1 / G0 by density arrest (time 0), and terminally differentiated into adipocytes by hormonal treatment Cells induced (time 1-6 days after treatment) were analyzed in a manner similar to that used in C. FIG. Cell localization of HEC. Figure 4A. Biochemical fractionation of T24 cells (T) into nucleus (N), cytoplasm (C), and membrane (M) components. Each fraction was immunoprecipitated with either an anti-C15 antibody or an 11D7 anti-Rb mAb that detected Rb from the same cells as a marker for nucleoprotein. The same cell fraction was also incubated with glutathione agarose beads to identify glutathione-S-transferase, which acts as a marker for cytoplasmic proteins. Figure 4B. Immunocytochemical localization during different phases of the cell cycle. Panels: a, T24 cells fixed in late G1 phase show little staining in the nucleus (400 × initial magnification); b, G1 / S border cells stain more strongly in the nucleus and perinuclear cytoplasm C, S-phase cells; d, late cells (higher magnification, 1000 ×) showing staining surrounding the whole cell and more distinct staining in paired dots away from the center. FIG. 4C; Metaphase chromosomes were first stained with DAPI. The same microscopic field was then analyzed after indirect immunofluorescent antibody staining. Panels: a, anti-C15 polyclonal serum (1: 1000 dilution) and FITC-tagged anti-mouse IgG secondary antibody localize HEC to centromeres; b, human autoimmune (CREST) antiserum, which contains centromere protein And Texas Red tagged secondary antibodies also labeled centromeres; c, digital overwriting of anti-C15 and CREST antiserum images. FIG. Expression of HEC deletion mutants prevents mitosis. Figure 5A. Full length HEC, GFP-15PA containing only amino acids 1-250, and GFP-15Pst encoding amino acids 251-618 of the entire leucine heptad repeat domain. Figure 5B. Detection of GFP and GFP-HEC fusion proteins in transfected Saos-2 cells. After transient transfection, cell lysates were separated by SDS-PAGE. Expression of the GFP fusion protein was detected by immunoprecipitation with anti-Myc1-9E10 mAb (Evan et al., 1985), followed by blotting with anti-GFP antibody (Clontech. Palo. Alto, Calif.). Stars indicate GFP (lane 2), GFP-15PA (lane 3), and GFP-15Pst (lane 4) fusion proteins. Arrows indicate IgG heavy chains. FIG. 5C, Localization of GFP and GFP-HEC fusion proteins in Saos-2 cells. DAPI (blue, a, d, g) identifies DNA in the nucleus; GFP autofluorescence (green, b, e, h) indicates cellular localization of various GFP-HEC fusion proteins; Indirect immunofluorescence with -tubulin primary antibody and Texas Red labeled secondary antibody indicates the localization of α-tubulin (c, f, i). FIG. Division of Saos-2 cells expressing the GFP-HEC fusion protein out of place. Cells expressing GFP alone or GFP-15PA divide to form 2 and 4 cell colonies. However, cells expressing GFP-15Pst are not able to complete division at all times; they form few 2-cell colonies and no 4-cell colonies during the 99-hour observation period. FIG. Microinjection of anti-HEC results in abnormal mitosis. FIG. 7A; Characterization of mouse monoclonal antibody 9G3. Antibodies were raised against the same antigen used to generate polyclonal anti-C15 and 5 × 10 ^Five Were used for direct immunoblotting of protein lysates from CV1 (lane 1) or T24 cells (lane 2). FIG. 7B; T24 cells were released from density arrest and allowed to progress through the cell cycle. Twenty-four hours after release, most of the cells were in S phase, at which time they were microinjected with either non-specific mouse IgG (panels a, b) or mAb 9G3 (panels c, d). . Twenty-six hours later, after they had undergone mitosis, cells were fixed and analyzed by indirect immunofluorescence staining. Panels: a, c, DAPI fluorescence; b, d, staining with anti-mouse IgG antibody. Arrowheads in each panel successfully identify daughter cells of microinjected cells. Daughter cells marked by arrows in panels a and c were not microinjected. FIG. 7C; cells at different stages of mitosis. Panels a-f show normal mitosis in non-microinjected cells and cells microinjected with control mouse IgG; panels gl show the mitotic phase of cells injected with anti-HEC mAb 9G3. Show. Blue fluorescence is from DAPI and red fluorescence is from rabbit anti-tubulin primary antibody and Texas Red conjugated anti-rabbit IgG secondary antibody. Panels: a, b, above; c, d, metaphase; e, f, early terminal; g, h, abnormal spindle formation with at least 4 separate spindle poles; i, j, disrupted chromatids Alignment and absence of distinct metaphases; k, l, abnormal chromatid segregation: chromatids at k align along a nearly horizontal axis, but the corresponding spindles at 1 are 90 ° opposite To pull. 4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Abnormalities seen during metaphase and metaphase can result from primary problems early in the cell cycle. For example, it is known that microinjection of polyclonal CREST serum that recognizes several different centromeric proteins disrupts kinetochore assembly and blocks mitosis-mediated progression (Bernat et al., 1990; Simerly et al., 1990). ). The timing of the injection of antisera was important in these studies. When injected into the cytoplasm or nucleus during the S or G2 period, anti-centromere antibodies cause abnormalities in mitosis, the abnormalities described herein after injection of specific anti-HEC antibodies. And was very similar. In contrast, when injected into the nucleus after metaphase chromatid alignment was complete, anti-centromere antibodies had little effect on subsequent mitotic progression (Bernat et al., 1990). In this study, anti-HEC antibodies were microinjected into cells during S period and nuclear morphology was measured 26 hours after the expected time for all cells to complete mitosis. We cannot exclude the prolongation of the M phase of cells injected with mAb 9G3, but nevertheless, cells that stabilized after the completion of abnormal mitosis underwent nuclear division and cytokinesis. In normal cells, the “late anaphase” checkpoint detects tension and kinetochore attachment to microtubules (reviewed in Pluta et al., 1995). These checkpoints usually delay or prevent the completion of mitosis of cells with incorrect or incomplete division of chromosomes into daughter cells (Pluta et al., 1995; Rieder and Salmon 1994). In anti-HEC-injected cells, such checkpoints appear to be partially or completely avoided. This finding indicated that HEC may have a role not directly related to spindle attachment at the centromere. The abnormal state of the spindle organ, whose morphology probably dictates the location of the split neck during cytokinesis (Bernet et al., 1990), may explain the avoidance of a normal checkpoint. Conversely, inactivation of HEC does not leave cells in a mitotic state, but causes them to progress abnormally. This finding involves the problem of checkpoint control in HEC-inactivated cells. HEC can function as an adapter and regulate ubiquitin-dependent proteolytic mechanisms, centromere attachment, spindle motility and checkpoint proteins. Since the details of the mechanism by which HEC functions before and during mitosis have not been fully determined, the location of HEC at the centromere / kinetochore is associated with spindle attachment to chromosomes during prophase, And it shows that it may be indirectly associated with subsequent chromosome migration. However, the lack of a signature tubulin binding domain in the HEC molecule disproves direct microtubule attachment. HEC binding of mitosis-specific kinases to several proteasome subunits suggests another potential way in which HEC can affect chromosome assembly, segregation or segregation . This study shows that many HEC-related proteins isolated by the yeast two-hybrid screen bind in vitro to different and distinct regions within the long leucine heptad repeat domain of HEC proteins. Both MSS1 and Nek2 co-immunoprecipitate with HEC, especially in late S or M phase. These data suggest that the interaction between HEC and other mitotic proteins may be of biological significance in mammalian cells. 4.1 ELISA ELISA can be used in combination with the present invention. In an ELISA assay, a protein or peptide incorporating the HEC antigenic sequence is immobilized on a selected surface, preferably one that exhibits protein affinity, such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, the wells of the assay plate are treated with non-specific proteins known to be antigenically neutral for the test antiserum, such as bovine serum albumin (BSA), casein or milk powder solution And it is desirable to bond or coat. This allows for blocking of non-specific adsorption sites on the immobilized surface, thus reducing background due to non-specific binding of antisera to the surface. After binding of the antigenic material to the wells, coating with non-reactive material to reduce background, and washing to remove non-bound material, the immobilized surface was treated with antiserum, or immune complex ( (Antigen / antibody) is contacted with the clinical or biological extract to be tested in a manner to effect formation. Under such conditions, the antiserum It is preferable to include dilution with a diluent. Also, these additives tend to help reduce non-specific background. The layered antiserum is then incubated, preferably at a temperature on the order of about 25 to about 27 ° C, for about 2 to about 4 hours. After incubation, wash the antiserum coated surface and remove non-immunoconjugate Purification. After formation of a specific immune complex between the test sample and the bound antigen, and subsequent washing, the appearance and further amount of immune complex formation is similarly treated with a secondary antibody having specificity for the primary antibody Can be measured. To provide a means of detection, the secondary antibody preferably binds an enzyme that develops color upon incubation with a suitable chromogenic substrate. Thus, for example, the antiserum binding surface may be treated with urease or peroxidase conjugated anti-human IgG for a period of time, under favorable conditions for the progress of immune complex formation (eg, It is desirable to contact and incubate. After incubation with a secondary enzyme-tagged antibody and subsequent washing to remove unbound material, if the enzyme label is peroxidase, urea and bromocresol purple or 2,2′-azino-di- (3-ethyl -Benzthiazoline) -6-sulfonic acid (ABTS) and H _Two O _Two Incubate with a chromogenic substrate, such as, to quantify the amount of label. Quantitation is then achieved by measuring colorimetricity, for example, using a visible spectrum spectrophotometer. 4.2 Epitope core sequence The present invention also relates to whole cell and other peptide-free protein or peptide compositions. The composition has a purified protein or peptide incorporating an epitope that is immunologically cross-reactive with one or more anti-HEC antibodies. As used herein, the term `` incorporating an epitope that is immunologically cross-reactive with one or more anti-HEC antibodies '' refers to a primary, analogous to an epitope located within a HEC polypeptide. It is intended to refer to a peptide or protein antigen that contains a secondary or tertiary structure. The level of similarity is generally such that a monoclonal or polyclonal antibody directed against the HEC polypeptide binds, reacts, or otherwise recognizes the cross-reactive peptide or protein antigen. . Various immunoassays can be used for binding to antibodies, such as, for example, Western blotting, ELISA, RIA, etc., all of which are known to those of skill in the art. Identification of HEC epitopes suitable for use in vaccines, and / or their functional equivalents, is a relatively straightforward matter. For example, the method of Hopp can be used as taught in US Pat. No. 4,554,101, which teaches the identification and preparation of epitopes from amino acid sequences based on hydrophilicity, incorporated herein by reference. . Methods described in several other articles and software programs based thereon can also be used to identify epitope core sequences (see, for example, Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Patent No. 4,554,101). checking). The amino acid sequence of these "epitope core sequences" can then be readily incorporated into peptides by applying either peptide synthesis or recombinant techniques. Preferred peptides for use in accordance with the present invention are generally on the order of about 5 to about 25 amino acids in length, more preferably about 8 to about 20 amino acids in length. It is recommended that shorter antigenic HEC-inducing peptide sequences provide advantages in certain circumstances (eg, vaccine preparation or immunological detection assays). Exemplary advantages include ease of preparation and purification, relatively low cost and increased product reproducibility, and advantageous biodistribution. A particular advantage of the present invention is that synthetic peptides containing modified and / or extended epitopic / immunogenic core sequences are prepared, resulting in a "universal" epitope peptide directed to HEC and HEC-related sequences. It will be understood that this can be achieved by occurring. These regions are likely to represent regions that are likely to promote T-cell or B-cell stimulation in animals, and thus elicit specific antibody production in such animals. As used herein, an epitope core sequence is a relatively short stretch of amino acids that is “complementary” to, and thus binds to, the antigen binding site on a transfer binding protein antibody. Additionally or alternatively, the epitope core sequence is one of the sequences that elicits an antibody that is cross-reactive with an antibody directed against the peptide composition of the present invention. In the context of the present disclosure, the term "complementarity" refers to amino acids or peptides that exhibit traction to each other. Thus, a particular epitope core sequence of the invention can be defined by manipulation, either competing with the corresponding protein-directed antiserum, or possibly displacing its binding to the desired protein antigen. In general, the size of a polypeptide antigen is not considered to be particularly important as long as it is at least large enough to carry the identified core sequence. The minimum useful core sequence predicted by the present disclosure is generally about 5 amino acids in length, more preferably about 8 or 25 amino acids. Thus, this size generally corresponds to the smallest peptide antigen prepared according to the invention. However, the antigen size can be larger if desired, as long as it has the basic epitope core sequence. The identification of epitope core sequences can be performed, for example, by those skilled in the art, as described in US Pat. No. 4,554,101, which teaches the identification and preparation of epitopes from amino acid sequences based on hydrophilicity, which is incorporated herein by reference. It is known. In addition, a number of computer programs are available for predicting the antigenic portion of a protein (see, eg, Jameson and Wolf, 1988; Wolf et al., 1988). In addition, computerized peptide sequence analysis programs (eg, DNAStar It may be useful in the design of synthetic HEC peptides and peptide analogs according to the disclosure. Synthesis of epitope sequences or peptides having an antigenic epitope within the sequence is facilitated using conventional synthetic techniques, such as solid phase methods (eg, using a commercially available peptide synthesizer such as the Applied Biosystems Model 430A Peptide Synthesizer). Is achieved. The peptide antigen synthesized in this manner can then be aliquoted into aliquots and stored in a conventional manner, such as in an aqueous solution or, more preferably, in a powdered or lyophilized state, until use. In general, the peptides are relatively stable and, if desired, can be administered in substantially any aqueous solution, if desired, for an extended period of time, e.g., 6 months or more, without appreciable degradation or loss of antigenic activity. Can be easily stored. However, if long-term aqueous storage is intended, it is generally desirable to include a drug, including a buffer such as Tris or a phosphate buffer, and maintain the pH at about 7.0 to about 7.5. In addition, it may be desirable to include an agent that inhibits the growth of microorganisms, such as sodium azide or Merthiolate. For long-term storage in aqueous solution, it is desirable to store the solution at 4 ° C., more preferably, frozen. Of course, if the peptide is stored in lyophilized or powdered form, for example, in a measured aliquot that can be rehydrated with a predetermined amount of water (preferably distilled water) or buffer prior to use, , Can be stored indefinitely. 4.3 Immunoprecipitation The antibodies of the present invention are particularly useful for the isolation of antigens by immunoprecipitation. Immunoprecipitation involves the separation of a target antigen component from a complex mixture and is used to identify or isolate trace proteins. For isolation of membrane proteins, cells must be solubilized in detergent micelles. Non-ionic salts are preferred because other drugs, such as bile salts, precipitate at acidic pH or in the presence of divalent cations. In another embodiment, the antibodies of the invention are useful for juxtaposing two antigens. This is particularly useful, for example, for increasing the local concentration of an antigen, such as an enzyme-substrate pair. 4.4 Western Blot The compositions of the present invention find enormous utility in immunoblot or Western blot analysis. Anti-HEC antibodies can be used as high-affinity primary reagents to identify proteins immobilized on a solid support matrix, such as nitrocellulose, nylon, or a combination thereof. One-step reagents for use in the detection of antigens against secondary reagents, if the secondary reagents used for detection of the antigen in combination with immunoprecipitation and subsequent gel electrophoresis give rise to a harmful background Can be used as This may be the case if the antigen being studied is an immunoglobulin (excluding the use of immunoglobulins that bind to bacterial cell wall components), if the antigen being studied cross-reacts with the detection agent, or if the antigen is a cross-reaction signal. It is particularly useful when moving at the same relative molecular weight. Immunologically based detection methods used in conjunction with Western blots include enzymatic, radiolabeled, or fluorescently-tagged secondary antibodies to the toxin moiety that would be of particular use in this regard. The following antibodies may be mentioned. 4.5 Vaccines The present invention contemplates vaccines for use in active and receptive immunization embodiments. Immunogenic compositions deemed suitable for use as vaccines can be most easily prepared directly from immunogenic HEC peptides prepared in the manner disclosed herein. Preferably, the antigenic material is dialyzed extensively to remove unwanted low molecular weight molecules and / or lyophilized to most easily form the desired vehicle. The preparation of vaccines containing the HEC peptide sequence as an active ingredient is described in U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; , Which is generally well understood in the art. Typically, such vaccines are prepared for injection. As a liquid solution or suspension: Prior to injection, solid forms suitable for solution or suspension in liquid may also be prepared. The preparation can also be emulsified. The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as humidifying or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccine. The vaccine may be conventionally administered parenterally, for example, by subcutaneous or intramuscular injection. Formulations suitable for other modes of administration further include suppositories and optionally oral preparations. For suppositories, traditional binders and carriers include, for example, polyalkalene glycols or triglycerides, and such suppositories can be prepared from about 0.5 to about 10%, preferably from about 1 to about 2%. From the mixture containing the active ingredient in the range of Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders, and contain about 10 to about 95%, preferably about 25 to about 70%, of the active ingredient. Contains ingredients. The HEC-derivatized peptides of the present invention may be formulated into a vaccine in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino acids of the peptide) and inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids, such as acetic, oxalic, tartaric, mandelic, and the like. Addition salts with acids are mentioned. Also, salts formed with free carboxyl groups, for example, inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and isopropylamine, trimethylamine, 2-ethylamino It can be derivatized from organic bases such as ethanol, histidine, procaine. The vaccine is administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The dosage will depend on the subject to be treated, including, for example, the ability of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required for administration depend on the judgment of the attending physician. However, suitable dosage ranges are on the order of hundreds of micrograms of active ingredient per vaccination. Suitable methods for initial administration and booster administration also can vary, but are typified by the initial administration, followed by inoculation or other administration. The mode of application can vary widely. Conventional vaccine administration methods can also be applied. These are believed to include parenteral application such as by oral application injection with a solid physiologically acceptable base or physiologically acceptable dispersion. The dosage of the vaccine will depend on the route of administration, and will vary with the host size. Various methods for obtaining an adjuvant effect on the vaccine include aluminum hydroxide or aluminum phosphate (alum), typically used as about 0.05 to about 0.1% phosphate buffered saline solution, about 0.25% solution. Synthetic sugar polymer (C And the use of drugs such as protein aggregates in controlled vaccines. Aggregates by reactivation with pepsin-treated (Fab) antibodies to albumin, mixtures with bacterial cells such as endotoxin or lipopolysaccharide components of C. parvum or gram-negative bacteria, such as mannide monooleate (Aracel A) 20% of perfluorocarbon used as an emulsion of a physiologically acceptable oily vehicle or as a block substitute In many instances, it will be desirable to have multiple doses of the vaccine, usually no more than six vaccinations, more usually no more than four vaccinations, preferably one or more, usually at least about 3 vaccinations. Do vaccinations twice. Vaccinations are usually performed at intervals of 2 to 12 weeks, more usually at intervals of 3 to 5 weeks. Regular booster administration at 1-5 year intervals, usually at 3 year intervals, is desirable to maintain protective levels of the antibody. The course of the immunization can be followed by assays for antibodies to the supernatant antigen. Assays can be performed by labeling with conventional labels such as radionuclides, enzymes, fluorescence and the like. These techniques are well known and can be found in a wide variety of patents, such as US Pat. Nos. 3,791,932; 4,174,384 and 3,949,064, which exemplify these types of assays. 4.6 DNA Segments In another embodiment, it is contemplated that certain advantages may be obtained by placing the coding DNA segment under the control of a recombinant or heterologous promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with the DNA segment encoding the HEC peptide in its natural environment. Such promoters include other genes and usually associated promoters, and / or isolated from viruses, prokaryotic (eg, bacterial) cells, eukaryotic (eg, fungal yeast, plant or animal) cells. Promoters and especially mammalian cell promoters may be mentioned. Of course, it is important to use a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those skilled in the field of molecular biology. See, for example, Sambrook et al., 1989. The promoter used may be constitutive or inducible, and may be used under appropriate conditions to enhance the introduced DNA segment, which would benefit mass production of the recombinant protein or peptide. Level expression can be directed. Suitable promoter / expression systems contemplated for use in high level expression include the Pichia expression vector system (Pharmacia LKB Biotechnology), the baculovirus system for expression in insect cells, or any suitable yeast or bacterial expression system. But not limited to these. For expression embodiments for preparing recombinant proteins and peptides, longer DNA segments are intended to be used most frequently, with DNA segments encoding the entire peptide sequence being most preferred. However, it is understood that the use of shorter DNA segments that direct the expression of the HEC peptide or epitope core region, such as can be used to generate anti-HEC antibodies, is also within the scope of the invention. A DNA segment encoding a HEC peptide antigen that is about 10 to about 100 amino acids in length, or more preferably about 20 to about 80 amino acids in length, or even more preferably about 30 to about 70 amino acids in length, Are intended to be particularly useful. In addition to the use of the present invention in directing HEC peptide expression, the nucleic acid sequences contemplated herein also have various other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. Thus, a nucleic acid segment having the same sequence as a contiguous DNA segment having a length of about 14 nucleotides in SEQ ID NO: 2 or comprising a sequence region consisting of a contiguous sequence of at least about 14 nucleotides in length is complementary. It is intended to find particular utility. Longer contiguous identical or complementary sequences include, for example, sequences of about 20, 30, 40, 50, 100, 200 (including all intermediate lengths), and even up to about 220-bp (full length). Sequences (including) are also useful in certain embodiments. The ability of such nucleic acid probes to specifically hybridize to a HEC coding sequence may be useful in detecting the presence of a complementary sequence in a given sample. However, other uses are contemplated, including the use of sequence information to prepare mutant species primers or primers for use in preparing other genetic constructs. A nucleic acid molecule having a sequence region consisting of contiguous nucleotides ranging from about 14, 15 to 20, 30, 40, 50 or about 100 to about 200 or more nucleotides is identical or complementary to the DNA sequence of SEQ ID NO: 1. In particular, it is specifically contemplated as a hybridization probe for use in Southern and Northern blotting. Smaller fragments generally find use in hybridization embodiments. Here, the length of the contiguous complementarity region can vary, such as between about 10-14 and up to about 100 nucleotides, but a larger contiguous complementarity range is the complement of the length desired to be detected. Can be used according to the sex sequence. The use of a hybridization probe about 14 nucleotides in length allows for the formation of duplex molecules that are both stable and selective. However, molecules with complementary sequences that are contiguous over a range greater than 14 bases in length increase the stability and selectivity of the hybrid, and thereby improve the quality and extent of the resulting specific hybrid molecule To be generally preferred. Preferably, generally, nucleic acid molecules with about 15 to about 20 contiguous nucleotides or, if desired, even longer gene complementarity ranges are designed. Of course, fragments may also be obtained by other techniques such as, for example, mechanical shearing or restriction enzyme digestion. Small nucleic acid segments or fragments can be readily prepared, for example, by directly synthesizing the fragments by chemical means, such as usually performed using an automated oligonucleotide synthesizer. Fragments can also be obtained by application of nucleic acid reproduction techniques such as PCR, introduction of selected sequences into recombinant vectors for recombinant production, and other sets generally known to those skilled in the field of molecular biology. It can be obtained by recombinant DNA technology. Thus, the nucleotide sequences of the invention can be used for their ability to selectively form duplex molecules with the complementarity range of a DNA fragment. Depending on the intended application, it is desirable to use different hybridization conditions to achieve different degrees of selectivity of the probe for the target sequence. For applications requiring high selectivity, it is typically desirable to form hybrids using relatively stringent conditions. For example, high stringency conditions that select for relatively low salt and / or high temperature conditions, such as provided by a temperature of about 0.02M to about 0.15M NaCl, about 50 ° C to about 70 ° C. Such selective conditions allow little, if any, mismatch between the probe and the template or target strand, and are particularly suitable for the isolation of HEC-encoding DNA segments. The detection of DNA segments via hybridization is well known to those of skill in the art, and the teachings of US Patent Nos. 4,965,188 and 5,176,995, each of which is incorporated herein by reference, are examples of hybridization analysis methods. is there. Teachings such as those found in the text of Maloy et al., 1994; Segal, 1976; Prokop, 1991; and Kuby, 1994, are of particular relevance. Of course, for some applications, for example, where it is desired to prepare a mutant using a mutant primer strand hybridized to a base template, or to isolate the HEC coding sequence from related species, functional equivalents, etc. If attempted, lower stringency hybridization conditions are typically required to allow heteroduplex formation. In these circumstances, it may be desirable to use conditions such as about 0.15 to about 0.9M salt, a temperature range of about 20C to about 55C. Thereby, the cross-hybridizing species can be easily identified as a signal that hybridizes positively for control hybridization. In any event, it is generally understood that the conditions can be made more stringent by the addition of increasing amounts of formamide. Formamide serves to destabilize the hybrid duplex in the same way that the temperature increases. Thus, the hybridization conditions can be easily manipulated, and thus this is generally a method of selection depending on the desired result. In certain embodiments, it is advantageous to use the nucleic acid sequences of the invention in combination with appropriate means, such as labels for determining hybridization. A wide variety of suitable indicating means are known in the art, including fluorescence, radioactivity, enzymes or other ligands such as avidin / biotin. Avidin / biotin can give a detectable signal. In a preferred embodiment, it appears that the use of fluorescent or enzymatic tags such as urease, alkaline phosphatase or peroxidase instead of radioactive or other environmentally undesirable reagents is desired. In the case of an enzyme tag, the indicator substrate for colorimetry is known and is used to provide a means visible to the human naked eye or by spectrophotometry to provide specific means for the complementary nucleic acid containing sample. Hybridization can be identified. In general, the hybridization probes described herein are intended to be useful as reagents in both solution hybridization and solid phase embodiments. In embodiments that include a solid phase, the test DNA (or RNA) is adsorbed or otherwise attached to a selected matrix or surface. The immobilized single-stranded nucleic acid is then subjected to specific hybridization with the selected probe under desired conditions. The conditions selected will depend on the particular environment based on the particular criteria required. (Eg, depending on G + C content, target nucleic acid type, source of nucleic acid, size of hybridization probe, etc.). After washing the hybridization surface and removing non-specifically bound probe molecules, labeling detects or even quantifies specific hybridization. 4.7 Biological Functional Equivalents Modifications and alterations can be made in the structure of the peptides of the invention and the DNA segments encoding them, and still obtain functional molecules that encode proteins or peptides having the desired properties. The following are findings based on amino acid changes in the protein that make up the equivalent, or even an improved second generation molecule. Amino acid changes can be achieved by changing the codons of the DNA sequence according to the following codon table. For example, certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity for a structure, such as, for example, the antigen-binding region of an antibody or a binding site on a substrate molecule. . Because it is the interaction capacity and properties of the protein that define the biological functional activity of the protein, certain amino acid sequence substitutions can be made in the protein sequence and, of course, in the basic DNA coding sequence, and A protein with properties can be obtained. Accordingly, various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences encoding the above-described peptides, without appreciable loss of biological utility or activity. Is contemplated by the inventors. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, reference herein). Incorporated as). The relative hydrophobicity and hydrophilicity of amino acids contributes to the secondary structure of the resulting protein, which in turn allows the interaction of this protein with other molecules, such as, for example, enzymes, substrates, receptors, DNA, antibodies, and antigens. It is allowed to specify. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge properties (Kyte and Doolittle, 1982). They include isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1). Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6) Histidine (-3.2); Glutamic acid (-3.5); Glutamine (-3.5); Aspartic acid (-3.5); Asparagine (-3.5); Lysine (-3.9) And arginine (-4.5). In the art, it is possible to obtain a protein having a similar biological activity even when a specific amino acid is replaced with another amino acid having a similar hydrophobicity index or score, i.e., a protein having an equivalent biological function. Is known. In making such modifications, amino acid substitutions with a hydropathic index within ± 2 are preferred, substitutions within ± 1 are particularly preferred, and substitutions within ± 0.5 are even more particularly preferred. It is also understood in the art that substitution of similar amino acids can be made efficiently on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 states that the maximum localized average hydrophilicity of a protein, determined by the hydrophilicity of its adjacent amino acids, correlates with the biological properties of the protein. This patent is incorporated herein by reference. As described in detail in US Pat. No. 4,554,101, the following hydrophilicity values have been assigned for amino acid residues: arginine (+3.0); lysine (+3.0); asparagine. Glutamic acid (+ 3.0 ± 1): Serine (+0.3): Asparagine (+0.2): Glutamine (+0.2); Glycine (0); Threonine (−0.4) ); Proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); ); Leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that one amino acid can be replaced by another amino acid having a similar hydrophilicity value, and that a biologically equivalent, in particular an immunologically equivalent, protein can be obtained. In such changes, amino acid substitutions with hydrophilicity values within ± 2, substitutions within ± 1, are particularly preferred, and substitutions within ± 0.5 are even more particularly preferred. Thus, as outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. . Exemplary amino acid substitutions that take into account the various features discussed above are well known to those of skill in the art and include: arginine and lysine; glutamic and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and Isoleucine. 4.8 Site-directed mutagenesis Site-directed mutagenesis is effective for the preparation of individual peptides or biologically functional equivalent proteins or peptides by specifically mutating the underlying DNA. Technology. Furthermore, this technique provides an easy ability to prepare and test sequence variants, for example, by introducing one or more nucleotide sequence changes into DNA, taking into account one or more of the above considerations. Provided. Site-directed mutagenesis is by the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, and is large and small enough to form a stable duplex on both sides of the truncated deletion junction. Mutants are generated by using a sufficient number of adjacent nucleotides to provide a primer sequence having sequence complexity. Typically, a primer of about 17 to 25 nucleotides in length, with about 5 to 10 residues on each side of the junction of the sequence to be altered, is preferred. In general, techniques for site-directed mutagenesis, as exemplified by various publications, are well known in the art. As will be appreciated, this technique typically employs a phage vector that exists in both single-stranded and double-stranded form. Representative vectors useful in site-directed mutagenesis include vectors such as M13 phage. These phages are readily available commercially and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely used in site-directed mutagenesis. Site-directed mutagenesis eliminates the step of transferring the gene of interest from a plasmid to a phage. In general, site-directed mutagenesis according to the present invention involves first obtaining a single-stranded vector containing a DNA sequence encoding the desired peptide in its sequence or melting and separating the double-stranded of a double-stranded vector. It is implemented by doing. Oligonucleotide primers having the desired mutated sequence are generally prepared synthetically. The primer is then annealed to the single-stranded vector and subjected to a DNA polymerase such as E. coli polymerase I Klenow fragment to complete the synthesis of the mutated strand. In this way, a heteroduplex is formed, wherein the first strand encodes the original unmutated sequence and the second strand has the desired mutation. The heteroduplex vector is used to transform appropriate cells, such as E. coli cells, and a clone containing the recombinant vector with the mutated sequence arrangement is selected. The preparation of sequence variants of selected DNA segments encoding peptides using site-directed mutagenesis is provided as a means of generating potentially useful species of organisms, and the sequence variants of peptides and DNA encoding them It is not intended to be limited to this means, as there are other ways in which sequences can be obtained. For example, a sequence variant can be obtained by treating a recombinant vector encoding a desired peptide sequence with a mutagen such as hydroxylamine. 4.9 Monoclonal Antibodies Means for preparing and characterizing antibodies are well known in the art (see Harlow and Lane, 1988, incorporated herein by reference). Methods for producing monoclonal antibodies (mAbs) generally begin with the same system used to prepare the polyclonal antibodies. Briefly, polyclonal antibodies are prepared by immunizing an animal with an immunogenic composition according to the present invention and collecting antisera from the immunized animal. A wide range of animal species can be used to generate antisera. Typically, the animal used for the production of anti-antisera is a rabbit, mouse, rat, hamster, guinea pig, or goat. Rabbits are a preferred option for producing polyclonal antibodies because they have a relatively large blood volume. As is well known in the art, a given composition may vary in its immunogenicity. Therefore, it is often necessary to boost the host immune system, as can be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and suitable carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for attaching the polypeptide to the carrier protein are well known in the art and include, for example, glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-diazotized benzidine. Also, as is well known in the art, the immunogenicity of certain immunogenic compositions can be enhanced by the use of non-specific stimulators of the immune response known as adjuvants. Exemplary and suitable adjuvants include Freund's complete adjuvant (a non-specific stimulator of the immune response, including killed Mycobacterium tuberculosis), Freund's incomplete adjuvant, and aluminum hydroxide adjuvant. The amount of the immunogenic composition used to produce the polyclonal antibody will vary depending on the nature of the immunogen and the animal used for immunization. Various routes (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal) may be used for the administration of the immunogen. Polyclonal antibody production can be monitored at various times after immunization by sampling the blood of the immunized animal. A second (boost) injection may also be given. The process of boosting and titration is repeated until a stable titer is achieved. If the desired level of immunogenicity is obtained, the immunized animal can be bled, the serum separated and stored, and / or the animal can be used to prepare a mAb. mAbs can be readily prepared, for example, by using well-known techniques, such as illustrated in US Pat. No. 4,196,265, which is incorporated herein by reference. Typically, the method involves immunizing a suitable animal with a selected immunogenic composition (eg, a purified or partially purified LCRF protein, polypeptide or peptide). The immunizing composition is administered in a manner effective to stimulate the antibody-producing cells. Rodents such as mice and rats are preferred animals, but the use of rabbit, sheep, frog cells is also possible. Although the use of rats has certain advantages (Goding, 1986), mice are preferred, most commonly used, and generally yield a higher percentage of stable fused cells, and are therefore particularly useful in BALB / c mice. Use is most preferred. After immunization, somatic cells capable of producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generation protocol. These cells can be obtained from a biopsy spleen, tonsils, or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred because the former is a rich source of antibody-producing cells at the dividing plasmablast stage and the latter is because peripheral blood is readily available. Frequently, spleen lymphocytes are obtained by immunizing a group of animals and removing the spleen of the animal with the highest antibody titer and homogenizing the spleen with a syringe. Typically, spleens from immunized mice contain approximately 5 × 10 ⁷ From 2 × 10 ⁸ Lymphocytes. Next, the antibody-producing B lymphocytes of the immunized animal are fused with immortal myeloma cells, generally cells of an immortal myeloma cell of the same species as the immunized animal. Myeloma cell lines suitable for use in the hybridoma-producing fusion procedure are preferably non-antibody-producing, have high fusion efficiency, and are in certain selection media to support growth of only the desired fusion cells (hybridomas). Has enzyme deficiency that prevents growth. As is known to those of skill in the art, one of any of a number of myeloma cells can be used (Goding, 1986; Campbell, 1984). For example, if the animal to be immunized is a mouse, P3-X63 / Ag8, X63-Ag8.653, NS1 / 1.1. Ag41, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG1.7 and S194 / 5XX0 Bu1 can be used; for rats, R210. RCY3, Y3-Ag 1.2.3, IR983F, and 4B210 can be used; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusion It is. One preferred mouse myeloma cell is the NS-1 myeloma cell line (also referred to as P3-NS-1-Ag4-1), which is available from NIGMS Human Genetic Mutant Cell Repository under Cell Line Deposit No. It is readily available by requesting GM3573. Other mouse myeloma cell lines that can be used include 8-azaguanine-resistant mouse mouse myeloma SP2 / 0 non-producing cell lines. A method for producing a hybridoma of antibody-producing spleen or lymph node cells and myeloma cells is generally performed by somatic cells and myeloma cells at a ratio of 2: 1 (the ratio is about 20: 1 to about 1: 1). And mixing in the presence of (chemical or electrical) factors that promote cell membrane fusion. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and methods using PEG such as, for example, 37% (v / v) polyethylene glycol (PEG) are described in Getter et al. 1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986). Fusion procedures are usually infrequent (about 1 × 10 ^-6 From 1 × 10 ^-8 ) Only produce viable hybrids. However, this does not create a problem. This is because viable fusion hybrids are distinguished from unfused parent cells (particularly unfused myeloma cells, which usually forever divide) by culturing in a selective medium. The selection medium is generally a medium containing an agent that blocks new nucleotide synthesis in the tissue culture medium. Exemplary and suitable agents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block the de novo synthesis of both purines and pyrimidines, while azaserine blocks only the synthesis of purines. If aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as nucleotide sources (HAT medium). If azaserine is used, the medium is supplemented with hypoxanthine. A preferred selection medium is HAT. Only cells capable of functioning the nucleotide salvage pathway are viable in HAT medium. Myeloma cells lack vital enzymes of the salvage pathway, such as hypoxanthine phosphoribosyltransferase (HPRT), and cannot grow. Although B cells can function this pathway, B cells have a limited life span in culture and generally die within about two weeks. Thus, the only cells that can survive in the selection medium are hybrids formed from myeloma cells and B cells. This culture provides a hybridoma population from which a particular hybridoma is selected. Typically, selection of hybridomas involves culturing cells by dilution of a single clone in a microtiter plate and then testing the supernatants of the individual clones (after about 2-3 weeks) for the desired reactivity. It is performed by This assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays and the like. The selected hybridomas can then be serially diluted and cloned into individual antibody-producing cell lines, and the clones can then be grown semi-permanently to provide the mAb. Cell lines can be used in two basic ways in making mAbs. The hybridoma sample can be injected (often into the peritoneal cavity) into the histocompatible type of animal used to provide the somatic and myeloma cells used for the initial fusion. Injected animals will form tumors and secrete specific monoclonal antibodies produced by the hybrid fusion cells. Then, the body fluid of the animal such as serum or ascites is collected to obtain mAb at a high concentration. Also, individual cell lines are cultured in vitro, where the mAb is naturally secreted into the culture medium, from which the mAb can be readily obtained at high concentrations. If desired, the mAbs produced by any means can be further purified using various chromatographic methods, such as filtration, centrifugation, HPLC, or affinity chromatography. 4.10 Pharmaceutical Compositions The pharmaceutical compositions disclosed herein can be orally administered, for example, with an inert diluent or an assimilable edible carrier, or can be encapsulated in a hard or soft gel capsule. Or compressed into tablets, or incorporated directly into the diet of the diet. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be between about 2 to about 60% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Tablets, troches, pills, capsules and the like may also include: binders such as tragacanth gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid; Lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose or saccharin, or flavoring agents such as peppermint, wintergreen oil, cherry flavor may be added. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be presented as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac or sugar, or both. A syrup of elixir may contain the active compound sucrose as a sweetening agent, methyl and propylparabens as preservatives, for example, a cherry or orange flavor as a coloring and flavoring agent. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, active compounds can be incorporated with sustained release preparations and formulations. The active compounds can also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmaceutically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. In addition, dispersants can be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders as the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved by the contaminating action of microorganisms such as bacteria and mold. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oil. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In most cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of compositions in time delay agents such as, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compound in the desired amount in an appropriate solvent, as appropriate with various other ingredients as described above, followed by sterile filtration. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the ingredients as described above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is to dry the powder in a vacuum, freezing to yield the active ingredient, and any further desired ingredients from that solution, which have been previously filter sterilized. Drying. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents and the like. The use of such media and reagents for pharmaceutically active substances is well-known in the art. To the extent that any conventional media or reagent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not cause an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection are also prepared. obtain. The preparation can be emulsified. The composition may be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed at the free amino groups of proteins) and include, for example, inorganic acids such as hydrochloric acid or phosphoric acid, or acetic acid, oxalic acid, tartaric acid, It is formed with organic acids such as mandelic acid. Salts formed with free carboxyl groups can also be derived from, for example, inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, or organic bases such as isopropylamine, trimethylamine, histidine, procaine. . Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations can be readily administered in a variety of dosage forms, eg, injectable solutions, drug release capsules, and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary, and the diluted liquid should be first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those skilled in the art from the present disclosure. For example, one dose can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous injection, or can be injected into a given drip site (see, for example, “Remington's Pharmaceutical Sciences” 15). Editions, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. In addition, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Biologicals standards. I have to. 5.0 Examples The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art will appreciate that the techniques disclosed in the exemplary technique embodiments work well in the practice of the present invention and are thus likely to constitute the preferred mode of practice. Understand what you get. However, one of ordinary skill in the art can, from the disclosure of the present invention, make many changes in the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the invention. You should understand that. 5.1 Example 1-Isolation of full-length cDNA A 1.8 kb C15 cDNA fragment was first isolated from a human B cell cDNA library by interaction with the C-terminus of Rb in the yeast two-hybrid system (Durfey et al., 1993). Cloned from Next, using this fragment as a probe, a cDNA library was screened to obtain several overlapping cDNA clones. The longest clone of about 2.3 kb thus obtained was ligated into pBKS to generate pBKS-C15, from which the HEC cDNA was sequenced, and from which the longest open reading frame was deduced. Using the C15 fragment as a probe, a single 2. A 3 kb mRNA species was found (FIG. 1A). Most of the tumor cell lines expressed more C15 mRNA than normal tissues and non-transformed cells (FIG. 1B). According to this expression pattern, it was named HEC (high expression in cancer). To determine whether expression in cells alters cell cycle progression, RNA was prepared from synchronized CV1 monkey kidney cells at different times after G1 quiescence. Total RNA extraction, polyA mRNA isolation and RNA blotting analysis were performed according to standard methods (Chen et al., 1989). Gb-like and E2F-1 mRNA probes have been described previously (Shan et al., 1992). Gb-like mRNA is constitutively expressed and acts as an internal loading control (Gullemont et al., 1989). Monkey kidney CV1 cells treated with drugs to enrich for various cell cycle stages were used for RNA extraction as described (Shan et al., 1992). HEC mRNA expression changed with the cell cycle and increased in S and M phases (FIG. 1C); such an expression pattern peaked in G1 / S and decreased in M, a transcription factor E2F-1. (Shan et al., 1992). These results indicated that the protein encoded by HEC could play a normal role in cell proliferation. To obtain the full length HEC cDNA, a human B cell cDNA library was screened using the first 1.8 kb C15 cDNA fragment as a probe. Several clones containing different fragments about 2 kb long were sequenced. The longest clone was found to be an open reading frame encoding a 642 amino acid protein with a predicted molecular weight of 72 kD (FIG. 2A). This protein was acidic and had an isoelectric point of 5.5. A recent GenBank search showed no significant homology with any of the genes encoding the characterized proteins. 5.2 Example 2-Isolation and Identification of the HEC Protein The generation of a GST-C15 fusion protein containing amino acids 56-642 was performed using the unique XhoI-XhoI C15 cDNA fragment (nucleotides 264-2045) in pGEX-3X (Stratagene). (San Diego, Calif.) To generate pGST-C15. This protein was expressed in E. coli (induced by isopropyl-bD-thiogalactopyranoside (0.1 mM)) and purified on glutathione-Sepharose beads as described (Chen, P.-L. et al., 1995). did. The recovered protein (purity> 95%) was then used as an antigen in mice. Serum from immunized mice was pre-adsorbed on a GST column and used directly for immunoprecipitation, developing immunoblot and immunostaining. Pre-immune serum was obtained from the same mice and used at the same dilution (1: 1000). Monoclonal antibodies were prepared according to standard procedures (Harrow and Lane, 1988) and were characterized as described above. To identify the cellular protein encoded by HEC, a polyclonal antibody raised against the GST-C15 fusion protein was generated. T24 bladder cancer cells, ³⁵ Labeled metabolically with S-methionine and prepared cell lysates for immunoprecipitation. To label cellular proteins, T24 cells (5 × 10 in each lane) ⁶ ) To about 70% confluence, ³⁵ Incubated with S-methionine (300 mCi) for 2 hours. The cells were then lysed in Lysis 250 buffer (Chen, Y. et al., 1996) for immunoprecipitation. For in vitro translation, the full-length HEC cDNA was inserted into pBKS and using a TNT-coupled reticulocyte lysate system, ³⁵ Transcribed and translated in the presence of S-methionine (Promega, Madison, WI). The anti-C15 antibody specifically immunoprecipitated a migrating cellular protein with an apparent molecular weight of 76 kD on SDS-PAGE (FIG. 2B, lane 5). This protein was not detected in pre-immune serum (lane 4). Furthermore, the GST-C15 fusion protein competed for binding to the antibody in immunoprecipitation and completely inhibited the precipitation of cellular proteins (lane 6). An unknown 46 kD protein co-immunoprecipitated. This may be one of the HEC interacting proteins, as described below (FIG. 2B, lane 5). When the full-length HEC cDNA was used as a template for in vitro transcription and translation, the synthesized protein was also immunoprecipitated by the same antibody and moved to the same position as the cellular protein (FIG. 2B, lane 3). These results indicate that cellular HEC is a 76 kD protein, consistent with the size exactly estimated from full-length HEC cDNA. The salient feature of this protein is that it has a typical long series of leucine heptad repeats. These repeats span the region between amino acids 254 and 621 (approximately two thirds of the total protein). 5.3 Example 3-Expression of HEC protein in actively dividing cells To evaluate the HEC expression pattern, protein lysates prepared from different organs of adult mice were used for straight western blot analysis (Fig. 3A). HEC protein could only be detected in tissues with high mitotic index, such as testis, spleen and thymus (FIG. 3A, upper panel). The internal regulatory protein p84 (Durphy et al., 1994) was expressed in approximately the same amount in all these tissues. Expression of HEC in tissues with high mitotic index was consistent with the mRNA expression pattern, generally suggesting a possible role for HEC in proliferation or especially mitosis. To further confirm this view, the expression pattern of HEC was monitored during the course of the cell cycle. Human bladder cancer cells T24 were grown in DMEM / 10% FCS and synchronized to G1 by density arrest in DMEM / 0.5% serum before being placed in DMEM / 10% FCS at 2 per 10 cm plate. × 10 ⁶ Released at time 0 by replating at cell density. The cells were then harvested at various time points (18 hours for G1 / S, 22 hours for S, 33 hours for G2). Nocodazole (0.4 mg / ml) was added to the culture medium for 8 hours before harvest to obtain M-phase cells. Cell samples were fixed with ethanol and analyzed using fluorescence-activated cell sorting to determine cell cycle phase as described (Cheng, Y. et al., 1996). Cell lysates prepared from synchronized populations of T24 cells were prepared using three different antibodies, mAb 11D7 (Rb) (Shang et al., 1992), anti-C15 (HEC) and anti-N5 (p84) (Darphy et al., 1994). Was analyzed by Western blotting. HEC protein is expressed in detectable amounts in late SM (Figure 3B). Rb expression patterns at different phases of the cell cycle have been described previously (Chen et al., 1989) and serve as markers for phases in the cell cycle. p84 expression does not change with cell cycle progression and serves as an internal load control (Darphy et al., 1994). HEC was expressed and detectable in rapidly dividing U937 monocyte lymphoma cells. Differentiation of mouse fibroblast 3T3 / L1 was induced as previously described by Student et al. (1980). This induction involves first growing the cells to confluence and then exposing the cells to fresh DMEM containing 10% FBS, 1 mM dexamethasone, 10 mM forskolin and 10 mg / ml insulin for 48 hours on day 0 of the differentiation induction program. Initiating lipogenesis. The medium was then replaced with DMEM containing 10% FBS and 10 mg / ml insulin and the cells were fed every other day until day 8. Cells were fixed at specific time points with 3% glutaraldehyde / 100 mM sodium phosphate (pH 7.4) and stained with Oil Red EGN to confirm the appearance of an adipogenic phenotype, especially the accumulation of lipid droplets in the cytoplasm. did. Similarly, the initial density 5 × 10 ^Five / Ml U937 cells were incubated for 4 days in the presence of phorbol ester (TPA, 100 ng / ml). On day 4, macrophages were observed as previously described (Cheng et al., 1989). However, when these cells were induced by phorbol esters and eventually differentiated into macrophages (Chen, P.-L. et al., 1996: Sundstrum and Nilsson, 1976) (FIG. 3C, lane 4), HEC expression was reduced to undetectable amounts. Similarly, when mouse 3T3 / L1 cells were induced with appropriate hormones to differentiate into adipocytes (Chen et al., 1989; Student et al., 1980) (FIG. 3D), HEC expression was readily detected in dividing cells. The resulting (FIG. 3D, lanes U, 1) was not detectable in cells arrested at G0 / G1 (lane 0) or cells that had differentiated to the end (lanes 4-6). These results indicate that HEC is not expressed in terminally differentiated cells, further reinforcing the suggestion that HEC may function specifically in mitosis. 5.4 Example 4-Localization of HEC To assess the potential function of HEC in dividing cells, the subcellular location of HEC was determined. T24 cells were biochemically fractionated or fixed and immunostained with specific anti-HEC antibodies. The procedure for separating the membrane, nuclear and cytoplasmic fractions employed previously published procedures (Abrams et al. 1982). All three fractions were then assayed for Rb protein and HEC by immunoprecipitation as described above. Aliquots of each fraction were also incubated with glutathione-agarose beads, then separated by SDS-PAGE and stained with Coomassie blue. In this way, glutathione-S-transferase was identified as a cytoplasmic marker (Galmont et al., 1989). In cells biochemically fractionated into nucleus, cytoplasm and membrane components (Abrams et al., 1982), HEC is mainly distributed in the nuclear fraction (FIG. 4A). Rb, a nuclear protein, and glutathione transferase, a cytoplasmic protein, served to control the fractionation procedure. According to the immunocytochemical staining method, HECs are also spotted within the nucleus (FIGS. 4B, a, b and c). In mitotic cells, this protein was located as paired dots on the chromosome (FIG. 4B, panel d and C, panel a). Staining of the metaphase chromosomal spread revealed that it co-located with the centromere protein (CE NP) recognized by the sera of patients with autoimmune disease and CREST syndrome (Moroi et al., 1980) (FIG. 4C). The use of pre-immune serum resulted in a lack of fluorescent signal, indicating that the anti-HEC antibody was indeed specific. These results suggest that HEC is a nuclear centromere-associated protein and again has a role for HEC in M phase. 5.5 Example 5-Effect of truncated HEC on tubulin formation The long stretch of leucine heptad repeats of HEC indicates that the C-terminal portion of this protein may be important for binding to other proteins (Landschulz et al., 1988). We believe that ectopic expression of HEC variants containing only this heptad repeat may bind to endogenous HEC or compete with endogenous HEC for binding to other proteins, which may lead to mitosis. Has been affected. To test this hypothesis, two HEC variants under the control of the CMV immediate gene promoter fused to green fluorescent protein (GFP) were constructed. One (GFP-15PA) contains only the N-terminal region (amino acids 1-250); the other (GFP-15Pst) contains the entire series of leucine heptad repeats (amino acids 251-618) (FIG. 5A). The GFP plasmid construct alone served as a control. Transfection of these three constructs into Rb-negative Saos-2 cells resulted in the expression of the corresponding proteins, which were firstly performed by immunoprecipitation with an anti-myc tag antibody and then with the anti-GFP antibody. It could be detected by Western blotting used as a probe (FIG. 5B, lanes 2-4). Twenty-four hours after transfection, cells were directly observed under a fluorescence microscope. GFP expression was observed in the nucleus and cytoplasm (FIG. 5C, panel b), whereas GFP-15PA was observed only in the nucleus (FIG. 5C, panel e), and GFP-15Pst was observed only in the cytoplasm. (FIG. 5C, panel h). Cells grown on tissue culture dish coverslips were washed with phosphate buffered saline (PBS) and fixed with 4% formaldehyde in PBS (containing 0.5% Triton X-100) for 30 minutes. After treatment with 0.05% saponin in water for 30 minutes and extensive washing with PBS, cells were blocked in PBS containing 10% normal goat serum. Incubate for 1 hour with the appropriate antibody diluted in 10% goat serum, followed by 3 washes, then incubate for an additional hour with a fluorochrome-conjugated secondary antibody. Co-localization of HEC and CENP was performed using a mixture of polyclonal mouse anti-C15 antibody with human CREST autoimmune serum. Tubulin was localized using polyclonal (ICN, Costa Mesa, CA) rabbit or DM1A monoclonal (Sigma, St. Louis, MO) anti-tubulin antibodies. Each antigen was visualized with goat anti-human IgG conjugated to Texas Red or goat anti-mouse conjugated to goat anti-rabbit IgG and FITC. After extensive washing in an excess of PBS containing 0.5% Nonidet-P40, the cells were further stained with the DNA-specific dye 4 ', 6-diamidino-2phenylindole (DAPI), and Permafluor (Lipshaw-Immunonon, Inc., Pittsburgh, PA). If the pictures were taken from a standard fluorescence microscope (Axiophot Photomicroscope, Zeiss), Ektachrome P1600 film was used. When cells were immunostained with anti-tubulin mAb, tubulin localized almost exclusively to the nuclei of GFP-15Pst expressing cells (FIG. 5C, panel i). In contrast, tubulin was found predominantly in the cytoplasm of cells transfected with both GFP and GFP-15PA (FIG. 5C, panels c and f). Normally, spindle-associated tubulin should be completely degraded after mitosis, and tubulin present in interphase cells should be distributed only in the cytoplasm. However, tubulin was abnormally localized in the nucleus of cells expressing GFP-15Pst HEC mutant protein (FIG. 5C, panel i). This suggests that ectopic expression of the N-terminal truncated HEC mutant disrupted the mechanism by which tubulin is degraded or redistributed after mitosis. To further examine the effects of the HEC variants, individual cells expressing a fusion of GFP and the GFP-HEC variant were continuously tracked and evaluated for their ability to divide at 99 hours post-transfection. . Three constructs, CNPL-GFP, a plasmid derivative derived from a mammalian expression vector containing a myc tag mutant form of green fluorescent protein (S65T) (Heim et al., 1994); N-terminus of HEC (amino acids 1-250) And GFP-containing CNPL-GFP-15Pst fused to the C-terminus of HEC (amino acids 251-618) were used in the transient transfection assay. Transfection 1 × 10 at a time ⁶ The cells were performed by conventional calcium phosphate / DNA co-immunoprecipitation. The sediment was removed 12 hours after transfection and the culture was re-fed with fresh medium. The cells were observed under a fluorescence microscope. Substantial numbers of Saos-2 cells that expressed either GFP or GFP-15PA could divide into four cell colonies (FIGS. 6A, B). However, expression of GFP-15Pst in Saos-2 cells not only did not show 4-cell colonies, but also substantially reduced the number of 2-cell colonies (FIG. 6C). Also, human bladder cancer T24 and monkey kidney CV1 cells were used in the same study and similar results were obtained. Many cells expressing the GFP-15Pst HEC variant undergo first mitosis, but often fail to complete the next cycle of cell division. This observation is consistent with an abnormal distribution of tubulin within the same cell; tubulin in the nucleus suggests an abnormal mitotic organization. 5.6 Example 6 Effect of Anti-HEC Antibody on M Phase To directly test whether HEC is functionally important for M phase, another approach was used to inactivate HEC. First, a mouse monoclonal antibody 9G3 was prepared and performed using the same GST-C15 fusion protein immunogen used in the preparation of the polyclonal antiserum. This mAb is specific for HEC in immunoprecipitation and straight immunoblotting (FIG. 7A); it recognizes the same 76 kD protein as polyclonal anti-C15 serum. MAb 9G3 monoclonal antibody was microinjected into T24 human bladder cancer cells synchronized in S phase. For the cells, the antibody solution was added to a microinjection buffer [20 mM NaHPO _Four (PH 7.2), 0.1 mM EDTA, 10% glycerol] at a concentration of 2 mg / mL using the described Eppendorf microinjection device (Goodrich et al., 1991). Twenty-six hours later, at the time when all cells should have completed mitosis, most cells were injected with mAb 9G3 containing a large number of fragmented nuclei. Cells not injected and cells injected with a control antibody (whole mouse IgG) split into two normal daughter cells (FIG. 7B). Many of the cells injected with the anti-HEC antibody had disappeared and appeared to have died 26 hours after injection. The results of the three separate studies were consistent (Table 2). In cells injected with anti-HEC monoclonal antibody, progression of M phase was closely monitored to more clearly determine the appearance of abnormal nuclei and cell death and the events responsible for cell death. Chromosomes in anti-HEC-injected cells condensed but did not associate and dissociate correctly (FIG. 7C), and metaphase plates were not clearly observed, and spindles were perturbed to their centromeres (FIG. 7C, panels g-1). Numerous spindle poles were observed in many cells (FIG. 7C, panels g-h). Overprinting of images of the same cells stained with DAPI (for chromosome confirmation) and tubulin-recognizing antibody (for confirmation of the spindle apparatus) ensures that many spindles are correctly orthogonal to their chromatids. It was shown that the positional relationship could not be assumed (FIG. 7C, panel k, 1). Cells injected with anti-HEC were able to undergo cytokinesis, but the chromosomes eventually segregated randomly into nonviable grossly abnormal daughter cells. 5.7 Example 7-Interaction of HEC with Other Proteins The effect of overexpression of HEC mutants on mitosis indicated that the leucine heptad repeat of HEC is important in protein function. To examine the potential biochemical basis of abnormal mitosis following HEC inactivation, we examined proteins that interact with HEC. The C-terminal half of HEC (amino acids 251-2618), which contains a long range of leucine heptad repeats, was used as a bait for performing a yeast two-hybrid screen on a human lymphocyte cDNA library. Of the 16 clones that interacted strongly in this screen, 10 clones were identified as cDNA fragments encoding MSS1, a component of subunit 7 of the 26S proteasome (Dubiel et al., 1993; Shibuya et al., 1992). Others include the subunit p45 of the 26S proteasome complex (Akiyama et al., 1995); Sb1.8, a human homolog of yeast Smc1 / Smc2 (Rocques et al., 1995, Strunnikov et al., 1993 and 1995); and Aspergillus A human homologue of NimA from Nidulans (Osmani et al., 1988), encoding a kinase Nek2 (Schutz and Nigg, 1993) that is important for M-phase progression (Table 3). All four of these proteins interacted with HEC by either co-immunoprecipitation or GST pull-down assays (Table 3). All HEC-related proteins characterized were usually associated with M phase. These interacting proteins provided further evidence that HEC is involved in the control of events important for chromosome fidelity during M phase to daughter cells. 5.8 Example 8 Materials and Methods Immunoprecipitation analysis and Western blot analysis Cell lysates in Lysis 250 buffer are subjected to three cycles of freeze / thaw (liquid nitrogen / 37 ° C) and clarified by centrifugation (14,000 rpm, 2 minutes, room temperature). The supernatant was used for immunoprecipitation as described (Chen, P.-L. et al., 1996). Briefly, 1 mL of mouse polyclonal anti-C15 antiserum was added to each clarified supernatant. For competition studies, antigen and antibody were incubated together for 1 hour before adding to cell lysate. After 1 hour of incubation, Protein A Sepharose beads were added and running for another hour. The beads were then collected and washed five times with lysis buffer containing 250 mM NaCl and boiled in SDS loading buffer for the immunoblot analysis as described (Chen, Y. et al., 1996). Metaphase chromosome distribution Growing T24 cells were treated with nocodazole for 8 hours. Mitotic cells were removed from the culture dishes by shaking and lysed hypotonically in 75 mM KCL. The free chromosomes were then cytospinned onto cover slips and incubated with one drop of DAPI for 10 minutes. The same chromosomes were stained with anti-HEC and human autoimmune (CREST) antisera, then counterstained with a FITC-conjugated secondary antibody, Texas Red-conjugated secondary antibody. Digital images were obtained using a Zeiss microscope (400 × magnification) and a Hamamatsu Photonics camera. Images were overprinted using Photoshop for Power Macintosh software. Similar results were obtained when the confocal imaging system was applied. Materials: CREST antiserum was obtained from Dr. B. Brinkley (COMPANY, CITY, STATE). GFP plasmid was obtained from Dr. R. Tsien (COMPANY, CITY, STATE). A yeast two-hybrid system (Durfee et al., 1993) modified as described for the yeast two-hybrid screen pAS-15Pst, which contains amino acids 251-618 of HEC, was used as the bait. 6.0 REFERENCES The following references are specifically incorporated herein by reference to the extent that they provide supplemental exemplary procedures or other details of those described herein. All of the compositions and methods disclosed and claimed herein can be made and executed in light of the present disclosure without undue experimentation. While the compositions and methods of the present invention are described with respect to preferred embodiments, the compositions, methods and steps or steps of a series of methods described herein without departing from the spirit, scope, and concept of the present invention. It will be apparent to those skilled in the art that modifications may be applied to More specifically, certain agents, both chemically and physiologically, may replace the agents described herein, but achieve the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. Accordingly, the exclusive rights required to be granted a patent are set forth in the following claims.

[Procedure amendment] [Submission date] July 1, 1999 (1999.7.1) [Content of amendment] 6.1 The description will be amended as follows. 6.1.1 Amend "Figure 2A" on page 12, line 25 of the specification to "Figure 2A-1 and Figure 2A-2". 6.1.2 "Figure 4B" on page 14, line 5 of the specification is amended to "Figure 4B-Figure 4E". 6.1.3 Amend “Panel: a” on page 14, line 5 of the description to “Fig. 4B”. 6.1.4 Correct “b” on page 14, line 7 of the description to “Fig. 4C”. 6.1.5 Correct "c" on page 14, line 8 of the description to "Fig. 4D". 6.1.6 Correct "d" on page 14, line 8 of the specification to "Fig. 4E". 6.1.7 Correct "Figure 4C" on page 14, line 11 of the specification to "Figure 4F-Figure 4H". 6.1.8 Correct "Panel: a" on page 14, line 12 of the specification to "Fig. 4F". 6.1.9 Correct "b" on page 14, line 14 of the description to "Fig. 4G". 6.1.10 Correct "c" on page 14, line 16 of the description to "Fig. 4H". 6.1.11 Amend “Figure 5C” on page 14, line 26 of the description to “Figure 5C to Figure 5K”. 6.1.12 Correct the "(blue, a, d, g)" on page 14, lines 26-27 of the specification to "(blue, Fig. 5C, Fig. 5F, Fig. 5I)". 6.1.13 Correct the "(green, b, e, h)" on page 14, lines 27-28 to "(green, Fig. 5D, Fig. 5G, Fig. 5J). Correct “(c, f, i)” on page 15, line 1 to “(Fig. 5E, 5H, 5K).” 6.1.15 Write “Fig. 6” on page 15, line 2 6.1.16 Correct the "Figure 7B" on page 15, line 12 of the specification to "Figures 7B-7E" 6.1.17 Specification, page 15 Correct “(Panels a and b)” on line 14 to “(Figs. 7B and 7C).” 6.1.18 “(Panels c and d)” on page 15 and line 14 in the specification 6.1.19 Correct the “Panel: a, c” on page 15, line 16 of the specification to “Figure 7B, Figure 7D.” 6.1.20 Specification 15 Correct "b, d" on page 16 line to "Figure 7C, 7E" 6.1.21 "Panels a and c" on page 15, line 18 to "Figure 7B and 7D" Compensate 6.1.22 Correct "Figure 7C" on page 15, line 20 of the book to "Figures 7F to 7Q" 6.1.23 Replace "Panel a to f" on page 15, line 20 with "Figures 7F to 7K" 6.1.24 Correct "Panel gl" on page 15, line 22 of the specification to "Fig. 7L-Figure 7Q." 6.1.25 " Panel: a, b ”is corrected to“ Fig. 7F, 7G. ”6.1.26“ c, d ”on page 15, lines 24 to 25 of the specification is corrected to“ Fig. 7H, 7I ” 6.1.27 Correct "e, f" on page 15, line 25 of the specification to "Fig. 7J, Figure 7K" 6.1.28 Replace "g, h" on page 15, line 25 of the specification with " Fig. 7L, Fig. 7M. 6.1.29 Correct "i, j" on page 15, line 26 of the specification to "Fig. 7N, Fig. 70." 6.1.30 Specification, page 15 Correct "k, l" on line 27 to "Fig. 7P, Fig. 7Q" 6.1.31 Correct "k" on line 27, page 15 to "Fig. 7P" 6.1.32 "L" on page 28, line 28 7Q ”6.1.33 Amend“ (Fig. 2A) ”on page 41, lines 20-21 to“ (Fig. 2A-1 and 2A-2) ”. "(Fig. 4B, a, b, and c)" on page 45, line 15 is corrected to "(Fig. 4B, 4C, and 4D)." 6.1.35 Amend “(Fig. 4B, Panel d and C, Panel a)” on page 45, lines 16-17 to “(Fig. 4E and 4F)”. 6.1.36 Correct the "(Fig. 4C)" on page 45, lines 19-20 of the specification to "(Fig. 4F-Fig. 4H)". 6.1.37 Amend “(Fig. 5C, panel b)” on page 46, line 13 of the specification to “(Fig. 5F)”. 6.1.38 Amend “(Fig. 5C, panel e)” on page 46, line 14 of the specification to “(Fig. 5G)”. 6.1.39 Amend “(Fig. 5C, panel h)” on page 46, line 15 of the specification to “(Fig. 5J)”. 6.1.40 On page 47, line 4 of the description, “(Fig. 5C, Panel i)” is corrected to “(Fig. 5K)”. 6.1.41 Amend "(Fig. 5C, panels c and f)" on page 47, line 6 to "(Fig. 5E and Fig. 5H)". 6.1.42 Correct the "(Fig. 5C, Panel i)" on page 47, lines 9-10 to "(Fig. 5K)". 6.1.43 Amend "(Fig. 6A, B)" on page 47, line 23 of the specification to "(Fig. 6A, Fig. 6B)". 6.1.44 Amend “(Fig. 7B)” on page 48, line 18 of the specification to “(Fig. 7B to 7E)”. 6.1.45 Amend “(Fig.7C)” on page 49, line 18 of the specification to “(Fig.7F-Fig.7Q)”. 6.1.46 On page 49, line 19 of the description, “(Fig. 7C, panels g to l)” is corrected to “(Fig. 7L to 7Q)”. 6.1.47 On page 49, line 20 of the description, “(Fig. 7C, panels g to h)” is corrected to “(Fig. 7L to 7M)”. 6.1.48 Amend “(Fig. 7C, panels k, l)” on page 49, lines 23-24 of the specification to “(Fig. 7P, Fig. 7Q)”. 6.2 Correct the drawing as shown in the attachment. FIG. FIG. FIG. FIG. 2 FIG. 2 FIG. 2 FIG. 3 FIG. 3 FIG. 3 FIG. 3 FIG. 4 FIG. 4 FIG. 4 FIG. 5 FIG. 5 FIG. 5 FIG. 6 FIG. 7 FIG. 7 FIG. 7

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C07K 16/18 C12N 1/21 C12N 1/21 C12P 21/02 C 5/10 C12Q 1/68 A C12P 21 / 02 G01N 33/53 D C12Q 1/68 M G01N 33/53 C12N 5/00 B A61K 37/10 (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, A T, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, GW, HU, ID , IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Chen, Yumei United States Texas 78245, San Antonio, Omicron Drive Number 201 14825 (72) Inventor Lily, Daniel Jay. United States Texas 78229, San Antonio, Hamilton Wolfe Road Number 1407 5303 (72) Inventor Chen, Fan-Run The United States, Texas 78245, San Antonio, Omicron drive na members 201 14825

Claims (1)

[Claims] 1. Encodes a human nucleoprotein (HEC) comprising the amino acid sequence of SEQ ID NO: 1 , An isolated nucleic acid segment. 2. SEQ ID NO: 2 nucleic acid sequence or its complement, or highly stringent Further defined as containing a sequence that hybridizes to the sequence of SEQ ID NO: 2 under mild conditions. 2. The nucleic acid segment of claim 1, wherein the segment is defined. 3. 2. The nucleic acid segment of claim 1, further defined as an RNA segment. . 4. A human nuclear protein encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 An isolated DNA segment containing the cytoplasmic gene (HEC). 5. A recombinant host cell comprising the DNA segment of claim 4. 6. The set of claim 5, further defined as a bacterial or eukaryotic cell. Replacement cells. 7. 7. The recombinant cell according to claim 6, wherein said eukaryotic cell is a human cell. 8. A method using a DNA segment encoding human nuclear protein (HEC), , The following steps:   a) the DNA segment encoding the HEC protein or peptide is a promoter Preparing a recombinant vector under the control of   b) introducing the recombinant vector into a host cell;   c) effective to allow expression of the encoded HEC protein or peptide Culturing the host cell under conditions that are suitable;   d) recovering the expressed HEC protein or peptide; A method comprising: 9. Less than:   a) has the same sequence as 18 contiguous nucleotides of SEQ ID NO: 2, or At least 18 contiguous nucleotides complementary to 18 contiguous nucleotides of SEQ ID NO: 2 A nucleic acid segment comprising a sequence region consisting of leotide; or   b) Under stringent hybridization conditions, SEQ ID NO: 2 From about 18 to about 2090 nuclei that hybridize to the nucleic acid segment of Reotide length nucleic acid segments; An isolated nucleic acid segment, characterized as: 10. Nucleic acid sequences encoding human nuclear protein (HEC) proteins or peptides A method for detecting a sequence, comprising:   a) obtaining a sample nucleic acid suspected of encoding a HEC protein or peptide Step;   b) conditions effective to permit hybridization of substantially complementary nucleic acids. Subjecting the sample nucleic acid to an isolated nucleic acid segment encoding the HEC protein. Contacting with a cement; and   c) detecting the hybridized complementary nucleic acid so formed; How to include. 11. Encodes human nuclear protein (HEC) protein in a suitable container A nucleic acid detection kit comprising a nucleic acid segment and a detection agent. 12. A human nucleoprotein comprising eight consecutive amino acid sequences from SEQ ID NO: 1 (HE A peptide composition comprising C). 13. A purified antibody that binds to human nucleoprotein (HEC) or peptide. 14． Detect human nucleoprotein (HEC) or peptides in biological samples Comprising the steps of:   a) Obtain a biological sample suspected of containing the HEC protein or peptide Process;   b) combining the sample with an antibody that specifically binds to the protein or peptide; Contacting under conditions effective to permit formation of the complex; and   c) detecting the complex so formed; A method comprising: 15. In a suitable container means, specialize in human nucleoprotein (HEC) or peptides. An immunodetection kit comprising an antibody that binds differently and an immunodetection reagent. 16. A method for preparing a human nuclear protein (HEC), comprising the following steps:   a) transforming according to claim 5 under conditions effective to produce the HEC protein; Culturing the isolated host cells; and   b) obtaining the HEC protein from the cells; A method comprising: 17． A method that disrupts the alignment and separation of sister chromatids in interphase cells. Specifically binds to said cells in an inactivating amount of human nucleoprotein (HEC) Administering an antibody, wherein mitosis is disrupted. 18. An effective amount of human nuclear protein (HEC) to disrupt chromatid separation into cells A method of regulating cell cycle progression, comprising the step of administering.