ANTI-MITOTIC AGENTS AND PROCESSES

Abstract: Provided is a genetic identification and characterization of a gene which encodes an essential yeast mitotic spindle protein. The protein functions in anaphase spindle elongation. The invention also provides an identification of a protein which interacts with this mitotic spindle protein. The proteins identified and characterized by the present invention are useful as development candidates for cancer chemotherapeutic agents, anti-fungal compounds, and other anti-mitotic agents.

Description: ANTI-MITOTIC AGENTS AND PROCESSES BACKGROUND OF THE INVENTION This invention was made with Government support under Grant No. GM- 50399 awarded by the National Institute of General Medical Sciences of the National Institutes of Health. The Government has certain rights to this invention. This invention relates generally to the identification and characterization of fungal genes and proteins, and in particular to mitotic spindle proteins useful as development candidates for anti-mitotic agents and processes using such agents. The mitotic spindle undergoes a remarkable series of transitions in response to cell cycle control signals. At each mitotic cell division, the spindle assembles, it forms attachments to each chromosome, it orients itself properly within the cell, and then, with extraordinarily high fidelity, it carries out chromosome segregation. Then it disassembles. Proper spindle assembly and function involves coordination of many events and processes including modulation of microtubule dynamics and creation of at least three distinct microtubule populations (kinetochore, polar, and astral microtubules). In addition, connections must be established between different spindle microtubule subpopulations, between spindle microtubules and chromosomes, between spindle microtubules and microtubule-associated proteins and motor proteins, and between spindle microtubules and the cell cortex (reviewed by Waters and Salmon, 1997). Proper spindle assembly is monitored by a cellular surveillance system which activates a mitotic checkpoint if the spindle is not assembled correctly (reviewed by Hardwick, 1998; Rudner and Murray, 1996). Once the spindle is assembled, a carefully orchestrated set of molecular events results in chromosome to pole movement (anaphase A) and separation of spindle poles (anaphase B). Genetic approaches to the study of spindle mechanics and regulation in S. cerevisiae, S. pombe, A. nidulans, and in a variety of other organisms have complemented studies m Xenopus extracts and mammalian and plant cells (reviewed by Nicklas, 1997; Sobel, 1997). Each different approach has provided an extremely powerful and unique avenue toward identification of mitotic spindle components and elucidation of their functions. Budding yeast contains five kinesin-related motor proteins and one dynein (reviewed by Winsor and Schiebel, 1997). Elegant genetic studies in yeast have revealed how the forces generated by these proteins work both synergistically and antagonistically to assemble and orient spindles, and to separate chromosomes (Cottingham and Hoyt, 1997; Gambino et al., 1984; Oakley and Morris, 1980; Oakley and Rinehart, 1985; Saunders and Hoyt, 1992). It is believed that a large number of proteins in the spindle function in concert with tubulin, the major spindle protein. Genetic studies have identified and provided functional tests of γ-tubulin and many other proteins associated with spindle pole bodies (Marschall et al, 1996; Oakley, 1994; Rout and Kilmartin, 1990; Sobel and Snyder, 1995; Spang et al, 1995). Also, a number of spindle accessory proteins have been found and studied functionally (Berlin et al., 1990; Machin et al., 1995; Pasqualone and Huffaker, 1994; Pellman et al., 1995; Wang and Huffaker, 1997). These genetic studies have been particularly valuable both because non-tubulin spindle components are typically low in abundance, making their discovery difficult by other means, and because genetic analysis facilitates tests of function in vivo. As indicated above, the mitotic spindle has been the subject of considerable research. The study of mitotic spindle proteins has yielded anti-mitotic compounds with important applications in cancer chemotherapy, and therapeutic agents targeted against fungal pathogens. For example, several plant and fungal secondary metabolites such as colchicine, vinblastine and taxol have been shown to interfere with mitotic spindle function in a wide variety of eukaryotes. The demonstrated effectiveness of these anti-mitotic compounds in important medical and agricultural applications demonstrates the desirability of identifying and characterizing anti-mitotic compound development candidates. SUMMARY OF THE INVENTION To achieve the foregoing, the present invention provides a genetic identification and characterization of a gene which encodes an essential yeast mitotic spindle protein. The protein functions in proper spindle assembly and anaphase spindle elongation. The invention also provides an identification of a protein which interacts with this mitotic spindle protein. The proteins identified and characterized by the present invention are useful as development candidates for cancer chemotherapeutic agents, anti-fungal compounds, and other anti-mitotic agents. In one aspect, the present invention provides a nucleic acid vector including a gene which encodes an essential mitotic spindle protein, and a plasmid capable of incorporating that gene. In preferred embodiments, the gene is the yeast gene YGLOόlc or the yeast gene YGR113w, and the plasmid is Bluescrpit SK + vector (pDD478). In another aspect, the invention provides a substantially pure protein essential to mitotic spindle formation. In preferred embodiments, the protein includes an amino acid sequence coded for by the yeast gene YGL061c or the yeast gene YGR113w. In still another aspect, the present invention provides a binding protein for a protein essential to mitotic spindle formation. The binding protein includes an amino acid sequence coded for by the yeast gene YGR113 w. In yet another aspect, the present invention provides a composition useful as a development candidate for an anti-mitotic agent. The development candidate includes an amino acid sequence selected from at least one of an amino acid sequence coded for by the yeast gene YGLOόlc, and an amino acid sequence coded for by the yeast gene YGR113w. In an additional aspect, the invention also provides an anti-mitotic agent identified by a screening method using one or more proteins essential to mitotic spindle formation. In preferred embodiments, the one or more proteins include an amino acid sequence selected from at least one of an amino acid sequence coded for by the yeast gene YGLOόlc, and an amino acid sequence coded for by the yeast gene YGR113w. In a further aspect, the invention provides a method of disrupting mitotic spindle formation in a cell. The method involves administering to the cell an anti- mitotic agent that disrupts the activity of one or more proteins essential to mitotic spindle formation. The one or more proteins include an amino acid sequence selected from at least one of the amino acid sequence coded for by the yeast gene YGLOόlc, and the amino acid sequence coded for by the yeast gene YGR113w. These and other features and advantages of the present invention will be presented in more detail in the following description of the invention and the accompanying figures which illustrate by way of example the principles of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 A and IB depict process flow diagrams illustrating anti-mitotic agent screening protocols using proteins in accordance with preferred embodiments of the present invention. Figure 2 shows the yeast strains used in the identification and characterization of genes and proteins in accordance with the present invention, listed in Table 1. Figure 3 shows the results of experiments undertaken to examine the temperature-sensitivity of the phenotype of a gene coding for an essential mitotic spindle protein in accordance with the present invention, noted in Table 2. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present invention will now be described with reference to preferred embodiments. Important properties and characteristics of the preferred embodiments are described in the text and illustrated in the accompanying drawings. While the invention will be described in conjunction with these preferred embodiments, it should be understood that the invention is not intended to be limited to these preferred embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be practiced without some or all of these specific details. In other instances, well known process operations have not been described in detail in order not to unnecessarily obscure the present invention. The present invention provides a genetic identification and characterization of a gene which encodes an essential yeast mitotic spindle protein. The protein functions in proper spindle assembly and anaphase spindle elongation. The invention also provides an identification of a protein which interacts with this mitotic spindle protein. The proteins identified and characterized by the present invention are useful as development candidates for cancer chemotherapeutic agents, anti-fungal compounds, and other anti-mitotic agents. The identification and characterization of the two novel and essential mitotic spindle proteins according to one aspect of the present invention, Duolp and Damlp, is described below. Duolp was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duolp was localized by immunofluorescence, by immuno-electron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies (SPBs). Temperature- sensitive duol mutants showed defects in proper spindle assembly and elongation. Damlp was identified by two-hybrid analysis as a protein which binds to Duolp. No homologues of Duolp or Damlp were identified in the sequence databases. By expressing a GFP-Damlp fusion protein in yeast, Damlp was shown to be associated with intranuclear spindle microtubules in vivo. Overproduction of Damlp caused mitotic defects similar to those caused by Duolp overproduction. Biochemical experiments demonstrated that Damlp is a microtubule-binding protein. It is believed that Damlp localizes Duolp to intranuclear microtubules to provide a previously unrecognized function (or functions) required for proper spindle assembly and elongation. Both Duolp and Damlp proteins are promising as anti-mitotic compound development candidates. Knowing that these proteins are each essential to proper mitotic cell division, screening studies may be performed using these proteins as targets in order to identify compounds which bind to the proteins and prevent them from carrying-out their intended functions. Such compounds would then have anti- mitotic activity and may be useful in a variety of applications. For example, since cancer cells undergo mitotic division at a faster rate than normal cells, an anti-mitotic compound identified by screening studies using the proteins of the present invention may be useful as a cancer chemotherapeutic agent. The anti-mitotic action of the agent would have a disproportionately greater effect on rapidly dividing cancer cells than on normal cells. In addition, fungal infections are a common affliction of both humans and animals, and agricultural crops and products. Anti-mitotic agents developed using the proteins of the present invention may be useful as drugs and pesticides to prevent or treat such fungal infections. Application of these proteins to the development of anti- fungal agents for use as pesticides on agricultural crops and for pharmaceutical applications may be particularly useful, since the lack of homology (with mammalian and, particularly, human sequences known to date) between these proteins and human proteins suggests that anti-mitotic agents effective against these fungal proteins would not have anti-mitotic activity against human cells thereby preventing any adverse health effects for humans contacting these agents. A variety of screening procedures may be used to identify anti-mitotic compounds using proteins in accordance with the present invention. Such screening procedures may exploit either of the two molecular interactions which have been established: the binding of Damlp directly to microtubules, polymers of the protein tubulin; and the binding of Damlp and Duolp directly to each other. Moreover, screening protocols focussing on gene expression may also be used, as discussed below. One preferred screening procedure using the Damlp-microtubule interaction is illustrated in the process flow of Figure 1 A. The process begins at a step 101, and at a step 102, Damlp is expressed and purified, preferably to 90% or greater purity, using a bacterial or yeast expression system. At a step 104, bovine brain microtubules are assembled under standard conditions, then stabilized with taxol. Molecules that are candidates for the inhibition of the Damlp-microtubule interaction by binding to Damlp are then screened for as follows: The Damlp, microtubules, for example about lOμM, and candidate molecules (for example, small organic molecules having a molecular weight of from about 300 to 800 Daltons, or peptides) are combined and incubated, at a step 106. Then, at a step 108, a low-throughput procedure is used to pellet microtubules by ultracentrifugation. The pellet is then assayed by polyacrylamide gel electrophoresis for presence of Damlp at a step 110. Absence of Damlp in the microtubule pellet indicates that the candidate molecule inhibits the Damlp-microtubule interaction and is therefore a promising anti-mitotic agent development candidate. The process ends at 112. For high-throughput assays, a fluorescent probe may be attached to Damlp and an assay for binding by Fluorescence Resonance Energy Transfer (FRET) of the fluorescent probe on the microtubule may be conducted. Alternatively, binding may be assayed by fluorescence anisotropy. Another approach is to make Damlp radioactive, attach microtubules to a solid matrix containing a scintilant, and use a scintillation proximity assay. A preferred screening procedure using the Damlp-Duolp interaction is illustrated in the process flow of Figure IB. The process begins at 151, and at steps 152 and 154 Duolp is produced and purified, preferably to 90% or greater purity, using a bacterial or yeast expression system, and bovine brain microtubules are assembled and stabilized, as described above. Several assays may be used. In one permutation, since Duolp binds to Damlp which in turn binds to microtubules, Damlp and Duolp are incubated with microtubules and small molecule drug development candidates at a step 156, as described above, only this time a fluorescent probe or radioactive moiety is attached to Duolp. Damlp-mediated interaction of Duolp with microtubules is then assayed for as described above for Damlp and microtubules. Briefly, at a step 158, microtubules are pelleted, and the pellet is then assayed for presence of the Duolp-Damlp complex at a step 160. Absence of Duol p- Damlp in the microtubule pellet indicates that the candidate small molecule inhibits the Damlp-microtubule interaction and is a promising anti-mitotic agent development candidate. The process ends at 162. In a second permutation, the Damlp-Duolp interaction may be directly assayed by immobilizing one of these two proteins on a solid matrix, labeling the other protein with a fluorescent or radioactive tag, and using a FRET or scintillation assay. In a third permutation, the technology that first demonstrated the Duolp- Damlp interaction, the two-hybrid interaction, may be applied. Both genes (DUOl and DAM1) are cloned in two-hybrid plasmids and show a robust interaction in living yeast cells. Both plasmids may be transformed into a yeast strain and the interaction scored by the production of β-galactosidase. Screening may then be conducted in living yeast cells for compounds that inhibit this interaction by identifying compounds that produce lower β-galactosidase levels. Expression of the DUOl or DAM1 genes may also be monitored by fusing the promoter sequences for these genes to reporter genes, such as the gene encoding Green Fluorescent Protein (GFP) or β-galactosidase. Inhibitors that block the expression of these genes but not other yeast genes may be screened by scoring for specific loss of expression of the reporter gene fused to the appropriate promoter sequence. Further details regarding screening techniques appropriate for use in accordance with the present invention may be found in the review article Nolan, J.P. and Sklar, LA., The Emergence of flow cytometry for sensitive, real-time measurements of molecular interactions. (1998) Nature Biotechnology 16, 633, and the references cited therein, which are incorporated by reference herein for all purposes. Of course, other effective screening methods may be used to identify candidates for drug development using proteins in accordance with the present invention. EXAMPLE The following example provides details concerning the identification and characterization of a gene coding for a mitotic spindle protein, DUOl, its corresponding protein Duolp, and an associated protein, Damlp, in accordance with the present invention. It should be understood the following is representative only, and that the invention is not necessarily limited by the details set forth in these examples. MATERIALS AND METHODS Strains and media The yeast strains used in this study are listed in Table 1 (Figure 2). Media were prepared and standard genetic techniques were carried out according to Rose et al, 1990. YPD is yeast rich medium and SM is synthetic minimal medium which was supplemented with the appropriate nutrients. The carbon source was 2% glucose, 2% raffmose, or 2% galactose and 2% raffinose. Plasmid construction and other DNA manipulations All DNA manipulations were carried out by standard methods (Maniatis et al., 1982). Restriction endonucleases and other enzymes were purchased from either New England Biolabs (Beverly, MA) or Boehringer Mannheim Corp. (Indianapolis, IN). Taq DNA Polymerase was obtained from Perkin-Elmer/Cetus (Norwalk, CT). DNA sequencing was performed by the UC Berkeley Sequencing Facility (Berkeley, CA) through the use of an Applied Biosystems sequencing machine. Primers used for both PCR and sequencing were purchased from either the Berkeley Oligo Synthesis Facility (Berkeley, CA) or Operon (Alameda, CA). Plasmid isolation and sequencing Plasmids were recovered from strains grown on glucose minimal plates under conditions which selected for an auxotrophic marker carried on the desired plasmid. Strains were grown to saturation in 5 ml of glucose minimal medium, pelleted, and washed once with water. Plasmids were isolated from these cells using the Qiagen Plasmid Miniprep Kit (Santa Clara, CA). Cell lysis was achieved by resuspending yeast in the cell lysis buffer provided with the kit, adding about lOOμl of glass beads and vortexing for 5 to 10 minutes. Following lysis, the steps in the Qiagen protocol were followed. The isolated plasmids were then transformed into E. coli and plasmid DNA was isolated using the Qiagen Plasmid Miniprep Kit. This DNA was used for sequencing and subcloning. General immunofluorescence and immunoblot procedures Yeast cells were grown to early log phase in minimal or rich medium. For galactose induction, cells were grown to log phase in medium containing either raffinose or glucose. Cells grown on glucose were washed twice with water and then diluted into medium containing galactose/raffinose. Galactose was added directly to medium containing raffinose. Fixation and immunofluorescence procedures were carried out as described by Drubin et al. (1988). The YOL134 anti-tubulin antibody was used at 1:200 and the anti-Duo lp antibody (preparation described below) at 1:2000. Fluorescein-conjugated anti-heavy chain secondary antisera were obtained from Cappel/Organon Teknika (Malvern, PA). Immunoblot analysis was performed using standard SDS-polyacrylamide and immunoblot transfer methods (Maniatis et al., 1982). The anti-Duolp antibody was used at a dilution of 1 :2000 for immunoblot analysis. Deletion of DUOl A DUOl disruption plasmid was constructed in three steps. A 1.2 kb PCR fragment amplified from pDD465 (contains genomic DUOl fragment) using M13Reverse and 0CHI8 (CCA TCG ATA TTG AAG ACT TGT TCA) (SEQ ID NO: 1) was digested with Clal and Xhol and ligated into Bluescript SK + . A 0.7 kb Nhel- Hindlll fragment (Hindlll site Klenowed) from pDD465 was then inserted into Xbal and Eagl site of the above plasmid (Eagl site Klenowed) resulting in vector pDD468. The H7S3 auxotrophic marker of plasmid LVl was cloned into the BamΗI site of pDD468 creating pDD469. URA3 was cloned from LV4 into the ΗinDIII site of pDD468 creating pDD470. A linear fragment was isolated from pDD469 or pDD470 after digestion with Clal and Sad. This fragment was transformed into the diploid strain DDY1445 by the Li-acetate method. Colonies were selected for the integration of the HIS3 marker by plating on minimal medium. Gene disruption was confirmed by Southern analysis using the ECL Southern Analysis Kit (Amersham, Arlington Heights, IL). Generation of temperature-sensitive duol mutants The plasmid pDD467 (containing a genomic copy of DUOl in pRS315) was mutagenized in vitro using hydroxylamine (HA). Plasmid DNA (20 μg), carrying the selectable marker LEU2 and the DUOl gene, was incubated at 75°C in a solution containing the mutagen (0.5M Hydroxylamine hydrochloride, 50 mM sodium pyrophosphate, pH 7.0, 100 mM NaCl, 2 mM EDTA). Reactions were stopped by placing them on ice at the 0, 20, 40, 60, 80 and 100 minute time points. The mutagen was removed by spinning the reactions through two 1 ml G-25 syringe columns. To determine the extent of mutagenesis, leuB" bacteria were transformed with the mutagenized plasmids to monitor mutations in the LEU2 gene. The mutation rate was defined as the percentage of bacterial transformants that were unable to grow on M9-leucine medium. Plasmids from the 20 minute and 40 minute time points, that gave 8% and 11% LEU2 mutagenesis respectively, were used for further studies. The mutagenized plasmids were transformed into a diploid strain that had both DUOl loci deleted and carried a genomic copy of DUOl on a URA3 marked CEN plasmid (pDD466). Transformants were plated on SC-Leu plates (plates containing all amino acids necessary for growth with the exception of leucine) at a density of about 300 colonies per plate. A total of 11,200 colonies were screened for the 20 minute time point, and 3,300 colonies were screened for the 40 minute time point. After 2 to 3 days of growth at 25°C, the colonies were replica plated onto 5-fluoroorotic acid (5- FOA) plates and incubated at 25°C and 37°C. Strains containing a URA3 plasmid are not able to grow on medium containing 5-FOA. This step was carried out to select colonies that had lost the unmutagenized genomic copy of DUOl marked with URA3 and only carried the mutagenized form marked with LEU2. Cells that did not grow at 37°C but did grow at 25°C on 5-FOA after three days were restreaked onto SC-Leu (synthetic complete medium lacking leucine) plates from the SC-Leu plates with the original transformants. These isolates were retested for growth on 5-FOA. The plasmids were recovered from all the 5-FOA selected clones and genomic inserts were cloned into an unmutagenized pRS315 vector as a Clal-HinDIII fragment. These constructs were retested. The open reading frames of the temperature-sensitive isolates duol-1 (pDD476) and duol-2 (pDD477) were sequenced to identify the mutations. Duolp antibody The DUOl coding sequence was amplified by PCR using primers oCH35 (GGA CTA GTG AGC AAA GCC AAT TAG ATG) (SEQ ID NO: 2) and oCH38 (GCG CGT CTA GAC CCG AAT CTT AAT TAT TTA CC) (SEQ ID NO: 3). The product was cloned into pHAT2 in frame with a six histidine tag (pDD471) using the Spel site in oCH35 and the HinDIII site in oCH38. The construct was transformed into BL21 cells and expression of the fusion protein was induced by addition of 0.4 mM IPTG for 4 hrs at 37°C. Inclusion bodies were harvested from 6 liters of log phase cells and solubilized by the addition of 8 M urea. The denatured fusion protein was purified on a Qiagen NTA-column as described in the product protocol. The protein was refolded by stepwise dialysis from 8 M urea into PBS (137 mM NaCl, 2.7 mM KC1, 4.5 mM Na2HPO4 7H20, 1.2 mM KH2PO4, pH 8.0) using a 2 M urea reduction per step. Antibodies were generated by injecting rabbits with the purified protein. An affinity matrix was created by immobilizing the fusion protein on Reacti-gel resin (Pierce, Rockford, IL). The protein was attached to the resin as described in the manual. Sera was circulated over the column and specific antibodies were purified by MgC12 elution (Harlow and Lane, 1988). The eluted antibody recognized the injected recombinant protein, a single 32 kD protein in whole yeast cell extracts and a GST- Duolp fusion protein purified from yeast. Cells overexpressing Duolp showed a clear increase in immunoreactivity in the 32 kD polypeptide in whole cell extracts, strongly supporting the conclusion that the 32 kD band was Duolp. Duolp immunofluorescence and immuno electron microscopy Yeast cells were grown to early log phase in either YPD or minimal medium. DUOl overexpressing cells were grown in minimal medium containing raffinose to early log phase and induced for 6-18 hrs by the addition of galactose. Cells were processed for immunofluorescence microscopy as described previously (Pringle et al., 1991). For Duolp staining, the cold methanol/acetone treatment was replaced by incubating cells with 0.1% SDS in PBS/BSA for 2 min. The monoclonal anti-α- tubulin antibody YOL134 was used at a 1 :200 dilution, and the anti-DUOl antibody was used at a 1 :2000 dilution. Detection of the primary antibody was accomplished by applying fluorescein labeled anti-rat or anti-rabbit secondary antibodies (Cappel/Organon Teknika, Malvern, PA) at a dilution of 1:1000. Cy3 secondary anti- rabbit antibodies (Sigma, St. Louis, MO) were used at a 1:500 dilution. Cells were cryofixed in a Bal-Tec HPM 010 high pressure freezer, freeze substituted in 0.2% glutaraldehyde plus 0.1% uranyl acetate in acetone for 2 days at - 78 °C, then warmed to -50 °C over a 12 hour period. Cells were infiltrated with Lowicryl HM20 at -50 °C and polymerized in BEEM capsules at -35 °C by UV irradiation. Blocks were UV irradiated at room temperature for an additional 48 hours to complete the resin curing. Thin (50 nm) sections were cut on a Reichert UltracutE microtome and picked up on Formvar and carbon coated nickel grids. The sections were incubated in primary antibody diluted 1:50 for 1 hour, rinsed with PBS , incubated in 10 nm goat anti-rabbit secondary diluted 1:20 for 1 hour, rinsed with PBS, fixed in 0.5% glutaraldehyde for 5 minutes, rinsed in dH2θ, and post-stained with uranyl acetate and lead citrate. Sections were examined in a JEOL 100CX electron microscope. Purification of GST-Duolp from yeast The entire coding sequence of DUOl was cloned into a galactose-inducible yeast GST fusion vector and tested for expression. A PCR product using primers oCH35 (GGA CTA GTG AGC AAA GCC AAT TAG ATG) (SEQ ID NO: 2) and oCH38 (GCG CGT CTA GAC CCG AAT CTT AAT TAT TTA CC) (SEQ ID NO: 3) was cloned into the pEG-KT vector resulting in pDD475. Transformants were grown in medium containing raffinose and expression was monitored after galactose induction. Expression reached a maximum level after 2 hours of induction. A total of 150 liters of cells were grown to log phase in raffinose, induced with galactose for 4 hours, and then harvested. Cells were washed once in ice cold water, frozen in liquid nitrogen, and then stored at -80°C. Cells were lysed in a Waring blender and resuspended in lysis buffer (50 mM Tris-Cl, pH 7.5, 1% Triton X-100, 150 mM NaCl, 2 mM MgC12, 1 mM EDTA, 0.5 mM PMSF and aqueous protease inhibitors (0.5 μg/ml each of antipain, leupeptin, pepstatin A, chymostatin and aprotinin). The extract was spun at 10,000 g for 10 min. The supernatant was recovered, pre-swollen GST-beads were added, and the mixture was incubated at 4°C for 4 to 14 hours. Beads were harvested by centrifugation at lOOOg for one minute, washed three times in wash buffer (50 mM Tris-Cl, pH 7.5, 1% Triton X-100, 300 mM NaCl, 2 mM MgC12, 1 mM EDTA, 0.5 mM PMSF and 0.5 μg/ml aqueous protease inhibitors), and then washed with elution buffer. GST-Duolp was eluted from beads by the addition of 15 mM glutathione in PME (80 mM Pipes, pH 6.8, 1 mM EGTA, 1 mM MgC12) at room temperature for 30 to 60 minutes. Beads were collected by centrifugation at lOOOg for one minute. The supernatant containing the fusion protein was recovered, flash frozen, and stored at -80°C. Microtubule binding experiments Bovine tubulin at a concentration of 8 mg/ml in PME (80 mM Pipes, pH 6.8, 1 mM EGTA, 1 mM MgC12) was thawed and prespun in a microfuge for 5 minutes at 4°C. GTP was added to a final concentration of 1 mM and tubulin was assembled into microtubules at 34°C for 30 min. After assembly, taxol was added to a concentration of 20 μM and the microtubules were incubated for 15 minutes at room temperature. Microtubules were diluted in PME containing 1 mM GTP and 10 μM taxol. For co-sedimentation experiments, in vitro translated protein using the TNT T7 Quick kit (Promega, Madison, WI)) was added to each 40 μl sedimentation reaction. The mixture was incubated for 20 minutes at room temperature to allow binding to occur. The mixture was then spun at room temperature in a TLA 100 at 60,000 rpm for 10 minutes. Binding was evaluated by analyzing audioradiographs using an IS-1000 densitometer (Alpha Innotech Corporation, San Leandro, CA). Two-hybrid screen A two-hybrid screen was carried out as described by Fields and Song (1989). Primers oCH36 (GCG CCC ATG GAG CAA AGC CAA TAA GAT GAT TCG) (SEQ ID NO: 4) and oCH37 (GCG GAT CCT AGA TAC ATT CCC G) (SEQ ID NO: 5) were used to PCR amplify DUOl from a plasmid and the PCR product was cloned into the AS1-CHY2 DNA binding domain vector creating pDD473, which was used as a bait to find binding partners in a cDNA library fused to the GAL4 activation domain. After the initial screen using both HIS3 to select for activation, and LacZ expression as reporter of activation, the plasmids of all positive clones were recovered as described above. Over 800,000 transformants were screened using full length Duolp as bait. A total of 174 positive clones remained positive after retesting. These clones were sequenced in batches of 40. Presence of plasmid inserts was confirmed by Xhol digestion before sequencing. Every clone that was identified at least twice by sequencing was used to probe all remaining unsequenced isolates by DNA hybridization using an Amersham ECL Southern Analysis Kit. This scheme allowed us to reduce the number of clones which required sequencing. For hybridization analysis, inserts were PCR-amplified directly from the plasmid recovered from yeast using the primers SY25 (GAG ATC TGG AAT TCG GAT CC) (SEQ ID NO: 6) and oCΗ41 (GGC ATG CCG GTA GAG GTG TGG) (SEQ ID NO: 7). Two microliters of each PCR reaction were spotted onto a 12 by 12 grid drawn onto nitrocellulose filters. Filters spotted with the PCR-amplified sequences were probed using the Amersham ECL Southern Analysis kit. Subcloning of DAMl The clones that were isolated in the two-hybrid screen were PCR-amplified from genomic DNA isolated from the yeast strain DDY1102: DAMl was amplified using primers oCH52 (GCG GGA TCC ATG AGC GAA GAT AAA GCT AAA TTA GGG) (SEQ ID NO: 8) and oCH56 (CTA GTC TAG AAT CAG TCA GCT CAT C) (SEQ ID NO: 9). All 5' primers contained the sequence for a BamHI restriction site and the original ATG of the individual clone. The 3' primers were located 100-200 bp downstream of the stop codon and contained a Xbal restriction site. The PCR products were subcloned into the Bluescript SK + vector (pDD478) and into the galactose-inducible GFP (Green Fluorescent Protein) construct pDDl 13 (pDD480). A DAMl deletion construct was created by PCR using primers oCH46 (CTG ATA AGC TCA GCA ATT GCA CCA AAA CAA TAT GAG AAA AGG CTT GTA TTG CCA CTT TCA CCG ATT GTA CTG AGA GTG CAC C) (SEQ ID NO: 10) and oCH47 (TTG TGA GGA GGA TAA TTC TTT GGT TGG GTT GGG CGT AGT CAT CTG AAG GGG GGC CTT GTA CTG TGC GGT ATT TCA CAC CGC) (SEQ ID NO: 11) and vector pRS313 as a template. The construct was purified by agarose gel electrophoresis and transformed into DDY1102. Correct integration was tested by genomic PCR using primers oCH48 (GCG TTG CCC GGA CAA TAT CG) (SEQ ID NO: 12) and oCH49 (CTG CCT TCC TCC CTA TTG C) (SEQ ID NO: 13). Localization and overexpression of Damlp To localize Damlp using the GFP -fusion constructs, the plasmids were transformed into strain DDY757 or DDY759. The strains were grown in minimal medium containing glucose into early log phase, washed in water and induced by resuspension in minimal medium containing 2% galactose/raffinose. After at least 6 hours of fusion protein overexpression at 30°C, GFP-fusion protein localization was observed directly by fluorescence microscopy. Alternatively, cells were fixed, stained with the YOL134 anti-tubulin antibody, and examined by fluorescence microscopy. To determine whether Damlp overexpression was lethal, its open reading frame was cloned behind the galacotse promoter in pDD42 (pDD482 and then transformed into yeast strain DDY759. These yeast strains were streaked onto glucose and galactose plates and incubated at 30°C. RESULTS Duolp overexpression causes spindle abnormalities DUOl (Death Upon Overproduction) (YGLOόlc) was isolated in a screen for genes which are toxic when overexpressed and cause morphlogical arrest phenotypes suggestive of defects in cytoskeleton function (Hofmann et al., in preparation). Cells overexpressing DUOl arrested at the large-budded cell cycle stage. DAPI staining revealed that these cells arrested with a single undivided nucleus. This phenotype suggested a defect in mitotic spindle function because the mitotic checkpoint arrests yeast at the large-budded stage and because large budded yeast typically contain a divided nucleus (Hoyt et al, 1990; Jacobs et al, 1988; Li et al, 1993; Reijo et al., 1994; Saunders and Hoyt, 1992). DUOl was also identified in a screen in which overexpression of CLN2, a gene which encodes a GI cyclin, could suppress the lethal phenotype of overexpressed genes. CLN2 overexpression suppressed the overexpression lethality of DUOl. The results presented below focus on the mitotic function of Duo 1 p. Consistent with the arrest phenotype described above, cells overexpressing DUOl contain abnormal microtubule arrangements. After 8 hours of DUOl overexpression induced from a galactose-regulated promoter, 91% of yeast cells contained a short mitotic spindle. After 16 hours of overexpression, no microtubules were observed by immunofluorescence in 99% of arrested cells. This block with short mitotic spindles can be seen well when the spindle pole bodies are stained with Tub4p antibodies. The spindle pole bodies remain separated by only about the diameter of the nucleus. Since essentially all of the arrested cells contained two distinct spindle pole bodies, we conclude that spindle pole body duplication is unaffected by DUOl overexpression. FACs analysis of haploid cells overexpressing Duolp revealed an average DNA content of about 4N. Thus, the arrest caused by DUOl overexpression appears not to have resulted from inhibition of DNA replication. The DUOl gene encodes a protein of 247 amino acid (MW = 27.5 kD) having the following amino acid sequence: 1 MSEQSQLDDS TIDKLIPQIF NEMRSNLNNT TNKFPKSTGG 41 GASDNISANS NSIRSFNSIT TQSLLKESES LDKITAMIKN 81 VTAALKNNLP VYVNQVHEVC KSTNSILDSW INIHSQAGYI 121 HKLMSDQTYL KLINDRLHNE NVNTNDEDGS TLHNVIALKK 161 KEILDLRQKL ENRKGEKDAA PAKPPNQGLN PRYGVQSGRR 201 PVPSAGISNN GRVRKTHVPA SKRPSGIPRV TNRWTKPTAS 241 SSRKMFR (SEQ ID NO: 14) Analysis of the protein sequence failed to reveal similarity to other proteins in the public databases or structural motifs. The DUOl gene and duolp protein sequences are also available online from the Saccharomyces Genome Database at http://genome- www.stanford.edu/Saccharomyces, the disclosure of which is incorporated by reference herein. Duolp co-localizes with spindle microtubules A rabbit antibody was raised against bacterially expressed Duolp containing a six His-tag. After affinity purification, the antibody recognized a single band at the predicted size for Duolp (32 kD) in yeast whole cell extracts. Indirect immunofluorescence microscopy experiments using this antibody showed that the protein is located along nuclear microtubules and appears concentrated at spindle pole bodies. Duolp does not appear to co-localize with cytoplasmic microtubules. Consistent with this observation, in Gl, "a cell cycle stage during which spindle assembly has not yet occurred, Duolp staining is only seen in the vicinity of the spindle pole body. Localization of a GFP-Duolp fusion protein yielded the same results. The spindle localization of Duolp was confirmed by immuno-electron microscopy. Staining of thin sections of yeast cells with Duolp antibody and gold- labeled secondary antibodies showed co-localization with spindle microtubules, but not with cytoplasmic microtubules. To determine whether the localization of Duolp was dependent on microtubules in vivo, microtubules of wild-type strain DDY898 were depolymerized by the addition of 20 μg/ml benomyl. After incubation for two hours at 25 °C, immunofluorescence revealed that essentially all of the microtubules were depolymerized. With the elimination of microtubules, Duolp staining also vanished. Temperature-sensitive duol mutants display a spindle elongation defect Deletion of DUOl showed that the gene is essential. Therefore, to examine the loss-of-function phenotype of DUOl in vivo, we created conditional-lethal alleles. A plasmid carrying the entire DUOl open reading frame was mutagenized using hydroxylamine. Plasmids conferring temperature-sensitivity in a duol deletion strain were isolated. We recovered two temperature-sensitive alleles. Each allele carries two mutations. pDD476 has the mutations E67K and A 157V and pDD477 has the mutations A117T and M124I. To examine the temperature-sensitive phenotype, strains carrying the mutated gene were grown at 25°C and then shifted to 37°C. The moφhologies of wild-type control cells and duol mutant cells were examined every 3 hours for 12 hours. Results are shown in Table 2, Figure 3. During this time, cells were maintained in log phase. The cell cycle distribution of wild-type cells as evaluated by cell morphology was not affected dramatically by the temperature shift. The mutant cultures, on the other hand, contained cells with short misaligned spindles, cells which appeared to have two unconnected half spindles and, after longer periods, cells which contained no microtubules at all. The primary defect seems to be the inability to elongate the spindle since this is the first of the defects to be observed. The duol-2 allele appears more severe than duol-1 since cells containing no microtubules appeared 18 hours after a shift to the non-permissive temperature for the former, and after about 24 hours for the latter. Identification of a protein, Damlp, which interacts with Duolp To identify a protein which might function with Duolp, a two-hybrid screen (Fields and Song, 1989) was carried out (see Materials and Methods). Thirteen genes were recovered at least four times (DAMl, LCP5, PUP1, AIP2, CIM5, VMA8, DEP1, YIR004w, YMR012w, YER049w, YDROlόc, YGR120c, YJL036w). Proteins encoded by genes of unknown function (DAMl, LCP5, CIM5, YIR004w, YMR012w, YER049w, YDROlόc, YGR120c), and therefore with the possibility of possessing a function related to Duolp, were localized as GFP-fusions. A protein showing spindle localization was characterized further (see below). The gene for this protein was designated DAMl (Duolp And Mpslp interacting) (YERl 13 w). The DAMl gene and damlp protein sequences are available online from the Saccharomyces Genome Database at http://genome-www.stanford.edu/SacchaiOmyces, the disclosure of which is incoφorated by reference herein. Precise genomic deletions of DAMl showed that the gene is essential for viability. Damlp is a protein of 335 aa (MW = 37.8 kD) having the following amino acid sequence: 1 MSEDKAKLGT TRSATEYRLS IGSAPTSRRS SMGESSSLMK 41 FADQEGLTSS VGEYNENTIQ QLLLPKIREL SDSIITLDSN 81 FTRLNFIHES LADLNESLGS LLYGIMSNSW CVEFSQAPHD 121 IQDDLIAIKQ LKSLEDEKNN LVMELSNMER GIKRKKDEQG 161 ENDLAKASQN KQFNQPLFPS SQVRKYRSYD NRDKRKPSKI 201 GNNLQVENEE DYEDDTSSEA SFVLNPTNIG MSKSSQGHVT 241 KTTRLNNNTN SKLRRKSILH TIRNSIASGA DLPIENDNVV 281 NLGDLHPNNR IYSEVVLQEW SMGPLRRTEI QCSQDVLKGN 321 PQKADILLQR KLKKK (SEQ ID NO: 15) Database searches and sequence analysis did not identify any homologues in the public databases or sequence motifs. To confirm that the two-hybrid interaction between Duolp and Damlp was the result of direct physical binding, in vitro translated Damlp was incubated with glutathione beads coated with GST or GST-Duolp. The beads were pelleted, washed with buffer containing 50 mM NaCl, and the presence of the in vitro translated products in the supernatant and pellet fractions was monitored by analysis on protein gels. Damlp interacts strongly with GST-Duolp, but not with GST alone. An in vitro translated fragment of mammalian tau protein (see below) served as a negative control, and did not interact with either GST or GST-Duolp coated beads. To determine the intracellular localization of Damlp, GFP fusion plasmids were constructed. GFP-Damlp was found exclusively associated with the intranuclear spindle throughout the cell cycle. Overexpression of Damp 1 is toxic (see below). In addition, overexpression of wild-type Damlp and GFP-Damlp had the exact same effect on cell growth, indicating some degree of functionality for the GFP- fusion protein. Damlp binds to microtubules Attempts to demonstrate direct interaction of Duolp with microtubules gave ambiguous results. GST-Duolp purified from yeast (see Materials and Methods) co- sedimented partially with yeast and bovine microtubules. Only a about 9% of the protein bound to microtubules and an increase in microtubule concentration did not result in increased Duolp pelleting. Furthermore, binding did not appear fully reversible. Dilution after binding did not reduce the amount of pelletable Duolp, and even washes with 1 M NaCl resleased only half of the bound GST-Duolp, raising concerns over whether this interaction was physiologically relevant, or resulted from trapping of GST-Duolp on the microtubules. Thus, these studies, while consistent with a direct Duolp-microtubule interaction, failed to demonstrate such an interaction uneqivocally. Therefore, the question of what interaction might account for co- localization of Duo 1 p with spindle microtubules remained open. The binding of Damlp to microtubules was then tested. It was found that Damlp co-sediments with bovine microtubules in a concentration-dependent manner. In co-sedimentation experiments at a microtubule concentration of 5 μM, approximately half of Damlp was found in the pellet and half in the supernatant, indicating that the KD of the interaction is approximately 5 μM. Furthermore, when binding reactions were diluted 10 fold after binding reached equilibrium, the amount of in vitro translated Damlp found in the pellet was markedly reduced. These observations show that Damlp binds microtubules specifically and reversibly. Damlp overexpression is lethal and causes spindle defects The overexpression phenotype for DAMl was determined. Overexpression of the wild-type gene gave the same phenotype as overexpression of the GFP-fusion. Overexpression of Damlp was lethal, resulting in a large budded cell cycle arrest in about 90% of the cells. The nuclei in these cells were undivided and the microtubules were abnormal. Approximately 20% of the cells had short spindles, with the rest of the cells having no microtubule structures or only a small spot that was detected by tubulin antibodies. After ten hours of DAMl overexpression, no microtubules were detected by immunofluorescence. This phenotype is reminiscent of that caused by Duolp oveφroduction. However, in contrast to the DUOl overexpression lethality, lethality due to DAMl overexpression is not suppressed by CLN2 overexpression. References The following documents, cited above, are incoφorated by reference herein for all puφoses: Berlin, V., Styles, C. A., and Fink, G. R. (1990): BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin. J. Cell Biol 111, 2573-86. Cottingham, F. R., and Hoyt, M. A. (1997): Mitotic spindle positioning in Saccharomyces cerevisiae is accomplished by antagonistically acting microtubule motor proteins [see comments]. J. Cell Biol 138, 1041-53. Drubin, D. G., Miller, K. G., and Botstein, D. (1988): Yeast actin-binding proteins: Evidence for a role in moφhogenesis. J. Cell Biol. 107, 2551-2561. Fields, S., and Song, O. (1989): A novel genetic system to detect protein-protein interactions. Nature 340, 245-6. Gambino, J., Bergen, L. G., and Morris, N. R. (1984): Effects of mitotic and tubulin mutations on microtubule architecture in actively growing protoplasts of Aspergillus nidulans. J Cell Biol. 99, 830-838. Hardwick, K. G. (1998): The spindle checkpoint. TIGS 14, 1-4. Harlow, E., and Lane, D. (\9%%)Antibodies-a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor. Hoyt, M. A., Stearns, T., and Botstein, D. (1990): Chromosome instability mutants of Saccharomyces cerevisiae that are defective in microtubule-mediated processes. Mol. Cel. Biol. 10, 223-34. Jacobs, C. W., Adams, A. E., Szaniszlo, P. J., and Pringle, J. R. (1988): Functions of microtubules in the Saccharomyces cerevisiae cell cycle. J. Cell BiollOl, 1409-26. Li, R., Havel, C, Watson, J. A., and Murray, A. W. (1993): The mitotic feedback control gene MAD2 encodes the alpha-subunit of a prenyltransferase [published erratum appears in Nature 1994 Sep 29;371(6496):438]. Nature 366, 82-4. Machin, Ν. A., Lee, J. M., and Barnes, G. (1995): Microtubule stability in budding yeast: characterization and dosage suppression of a benomyl-dependent tubulin mutant. Mol. Biol. Cell 6, 1241-59. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982): Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, Ν.Y. Marschall, L. G., Jeng, R. L., Mulholland, J., and Stearns, T. (1996): Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function. J. Cell Bioll34, 443-54. Masuda, H., and Cande, W. Z. (1987): The role of tubulin polymerization during spindle elongation in vitro. Cell 49, 193-202. Nicklas, R. B. (1997): How cells get the right chromosomes. Science 275, 632-7. Oakley, B., and Morris, N. R. (1980): Nuclear movement is b-tubulin dependent in Aspergillus nidulans. Cell 19, 255-262. Oakley, B. R. (1994): Gamma-tubulin. Wiley-Liss, Inc., New York. New York. Oakley, B. R., and Rinehart, J. E. (1985): Mitochondria and nuclei move by different mechanisms in Aspergillus nidulans. J. Cell. Biol. 101, 2392-2397. Pasqualone, D., and Huffaker, T. C. (1994): STUl, a suppressor of a beta-tubulin mutation, encodes a novel and essential component of the yeast mitotic spindle. J. Cell Biol 127, 1973-84. Pellman, D., Bagget, M., Tu, Y. H., Fink, G. R., and Tu, H. (1995): Two microtubule- associated proteins required for anaphase spindle movement in Saccharomyces cerevisiae [published erratum appears in J Cell Biol 1995 Oct;131(2):561]. J. Cell B/o/130, 1373-85. Pringle, J. R., Adams, A. E., Drubin, D. G., and Haarer, B. K. (1991): Immunofluorescence methods for yeast. Meth. Enzymol. 194, 565-602. Reijo, R. A., Cooper, E. M., Beagle, G. J., and Huffaker, T. C. (1994): Systematic mutational analysis of the yeast beta-tubulin gene. Mol. Biol. Cell 5, 29-43. Roof, D. M., Meluh, P. B., and Rose, M. D. (1992): Kinesin-related proteins required for assembly of the mitotic spindle. J. Cell BiolllS, 95-108. Rose, M. D., Winston, F., and Hieter, P. (1990): Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York. Rout, M. P., and Kilmartin, J. V. (1990): Components of the yeast spindle and spindle pole body. J. Cell Biol 111, 1913-27. Rudner, A. D., and Murray, A. W. (1996): The spindle assembly checkpoint. Current Opinion in Cell Biology 8, 773-80. Saunders, W. S., and Hoyt, M. A. (1992): Kinesin-related proteins required for structural integrity of the mitotic spindle. Cell 70, 451-8. Sobel, S. G. (1997): Mini review: mitosis and the spindle pole body in Saccharomyces cerevisiae. J. Exp. Zool. 277, 120-38. Sobel, S. G., and Snyder, M. (1995): A highly divergent gamma-tubulin gene is essential for cell growth and proper microtubule organization in Saccharomyces cerevisiae. J. Cell Bioll31, 1775-88. Spang, A., Courtney, I., Grein, K., Matzner, M., and Schiebel, E. (1995): The Cdc31p-binding protein Karlp is a component of the half bridge of the yeast spindle pole body. J. Cell Biol 128, 863-77. Vallen, E. A., Ho, W., Winey, M., and Rose, M. D. (1994): Genetic interactions between CDC31 and KAR1, two genes required for duplication of the microtubule organizing center in Saccharomyces cerevisiae. Genetics 137, 407-22. Wang, P. J., and Huffaker, T. (1997): Stu2p: A Microtubule-binding protein that is an essential component of the yeast spindle pole body. J. Cell Biol 139, 1271-1280. Waters, J. C, and Salmon, E. (1997): Pathways of spindle assembly. Current Opinion in Cell Biology 9, 3 '-43. Winey, M., Mamay, C. L., O'Toole, E. T., Mastronarde, D. N., Giddings, T. H., Jr., McDonald, K. L., and Mclntosh, J. R. (1995): Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle. J. Cell Biol 129, 1601-15. Winsor, B., and Schiebel, E. (1997): Review: an overview of the Saccharomyces cerevisiae microtubule and microfilament cytoskeleton. Yeast 13, 399-434. CONCLUSION Two mitotic spindle proteins, Duolp and Damlp have been described. Both proteins are essential for viability. Neither showed informative sequence homologies. Duolp appears to associate exclusively with intranuclear microtubules and spindle pole bodies, but not with cytoplasmic microtubules. Two-hybrid and biochemical data showed that Duolp interacts with the protein Damlp. This interaction appears to be physiologically relevant since Damlp is associated with intranuclear mitotic spindle microtubules. Furthermore, oveφroduction of Damlp, like oveφroduction of Duolp, causes mitotic defects. Co-sedimentation assays demonstrated specific binding of Damlp to microtubules. The localization data for both proteins suggest that Duolp may interact with Damlp along intranuclear spindle microtubules. Since Damlp binds to Duolp and to microtubules, it is believed that it mediates interaction of Duolp with microtubules. Both Duolp and Damlp have been linked functionally to the mitotic spindle. Both DUOl overexpression and DUOl loss of function caused spindle defects and a large-budded arrest phenotype. The identification of the essential gene DAMl, whose protein products interacts with Duolp may explain the observation that the loss of function and overexpression phenotypes of Duolp are similar. It is believed that DUOl overexpression mimics deletion of DAMl by titration of one or both of their gene products. The DUOl loss of function and overexpression phenotypes suggest an essential role in spindle elongation. SPBs were separated in both cells overexpressing DUOl, and in duol loss of function mutants. SPB separation is microtubule dependent. While short spindles formed, they did not elongate. Therefore, Duolp is critical for spindle elongation but does not seem to be involved in the initial step of spindle assembly. These proteins are promising candidates for anti-mitotic agent development. Application of these proteins to the development of anti-fungal agents for use as pesticides on agricultural crops and for pharmaceutical applications may be particularly useful, since the lack of homology between these proteins and human proteins suggests that anti-mitotic agents effective against these fungal proteins would not have anti-mitotic activity against human cells thereby preventing any adverse health effects for humans contacting these agents. Although the foregoing invention has been described in some detail for puφoses of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the invention. It should be noted that there are may alternative ways of implementing both the processes and compositions of the present invention. Accordingly, the present embodiments are to be considered as illustrative and not restrictive, and the invention is not to be limited to the details given herein, but may be modified within the scope and equivalents of the appended claims.

Claims: CLAIMS 1. A nucleic acid vector, comprising: a gene which encodes an essential mitotic spindle protein, and a plasmid capable of incoφorating that gene. 2. A vector according to claim 1, wherein the gene is yeast gene YGLOόlc (DUOl). 3. A vector according to claim 1, wherein the gene is yeast gene YGR113w (DAMl). 4. A vector according to claim 1 , wherein the plasmid is Bluescript SK + . 5. A substantially pure protein essential to mitotic spindle formation, comprising: an amino acid sequence coded for by the yeast gene YGLOόlc. 6. A substantially pure protein essential to mitotic spindle formation, comprising: an amino acid sequence coded for by the yeast gene YGR113w. 7. A substantially pure binding protein for a protein essential to mitotic spindle formation, comprising: an amino acid sequence coded for by the yeast gene YGR113w. 8. A composition useful as a development candidate for an anti-mitotic agent, comprising: an amino acid sequence selected from at least one of an amino acid sequence coded for by the yeast gene YGLOόlc, and an amino acid sequence coded for by the yeast gene YGR113w. 9. An anti-mitotic agent identified by a screening process, comprising: providing one or more proteins essential to mitotic spindle formation, the one or more proteins including an amino acid sequence selected from at least one of an amino acid sequence coded for by the yeast gene YGLOόlc, and an amino acid sequence coded for by the yeast gene YGR113w; combining said one or more proteins with one or more mitotic spindle components to which said one or more proteins is capable of binding, and one or more anti-mitotic agent candidate molecules; incubating said one or more proteins, one or more components, and one or more candidate molecules; and assaying the one or more components for presence of said one or more proteins. 10. An anti-mitotic agent according to claim 9, wherein said screening method further comprises identifying an anti-mitotic agent candidate which prevents binding of said one or more proteins to said one or more components as an anti- mitotic agent. 11. An anti-mitotic agent according to claim 9, wherein said screening method further comprises identifying an anti-mitotic agent candidate which prevents binding of said one or more proteins to another of said one or more proteins as an anti-mitotic agent. 12. An anti-mitotic agent according to claim 9, wherein said one or more mitotic spindle components comprises microtubules. 13. An anti-mitotic agent according to claim 11 , wherein said microtubules comprise bovine brain microtubules. 14. An anti-mitotic agent according to claim 9, wherein said anti-mitotic is agent is a anti-fungal agent. 15. An anti-mitotic agent according to claim 14, wherein said anti-mitotic agent is ineffective against mitotic spindle formation in mammalian cells. 16. An agricultural pesticide comprising an anti-mitotic agent according to claim 15. 17. An anti-mitotic agent according to claim 9, wherein said anti-mitotic agent is effective against mitotic spindle formation in mammalian cells. 18. An anti-mitotic agent according claim 17, wherein said anti-mitotic agent is effective as a cancer chemotheφeutic agent. 19. An anti-mitotic agent according to claim 18, wherein said anti-mitotic agent is effective against mitotic spindle formation in human cells. 20. A method of disrupting mitotic spindle protein formation in a cell, comprising: administering to the cell an anti-mitotic agent that disrupts the activity of one or more proteins essential to mitotic spindle formation, the one or more proteins including an amino acid sequence selected from at least one of an amino acid sequence coded for by the yeast gene YGLOόlc, and an amino acid sequence coded for by the yeast gene YGR113w. 21. A method according to claim 20, wherein the protein activity is binding. 22. A method according to claim 20, wherein the protein activity is expression. 23. A method according to claim 20, wherein said anti-mitotic agent is identified by a screening process, comprising: providing one or more proteins essential to mitotic spindle formation, the one or more proteins including an amino acid sequence selected from at least one of an amino acid sequence coded for by the yeast gene YGLOόlc, and an amino acid sequence coded for by the yeast gene YGR113w; combining said one or more proteins with one or more mitotic spindle components to which said one or more proteins is capable of binding, and one or more anti-mitotic agent candidate molecules; incubating said one or more proteins, one or more components, and one or more candidate molecules; and assaying the one or more components for presence of said one or more proteins. 24. A method according to claim 23, wherein said candidate molecules are at least one of organic molecules having a molecular weight of from about 300 to 800 Daltons and peptides. 25. A method according to claim 23, wherein the assaying is conducted by pelleting the one or more components by ultracentrifugation followed by poly aery lamide gel electrophoresis of the pellets. 26. A method according to claim 23, wherein the assaying is conducted by attaching a fluorescent probe to at least one of said one or more proteins and testing for binding of the fluorescent probe on the one or more components. 27. A method according to claim 26, wherein said testing is conducted by at least one of Fluorescence Resonance Energy Transfer (FRET) and fluorescence anisotropy.

Inventors: DRUBIN DAVID G, HOFMANN CHRISTIAN J, DRUBIN, DAVID, G., HOFMANN, CHRISTIAN, J.
Applicants: UNIV CALIFORNIA, THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Application year: 1999
Main classification: C07K14
IPC codes: A01N37, A01N63, C07K14